CN1347453A - Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens - Google Patents
Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens Download PDFInfo
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Abstract
Chimeric parainfluenza viruses (PIVs) are provided that incorporate a PIV vector genome or antigenome and one or more antigenic determinant(s) of a heterologous PIV or non-PIV pathogen. These chimeric viruses are infectious and attenuated in humans and other mammals and are useful in vaccine formulations for eliciting an immune responses against one or more PIVs, or against a PIV and non-PIV pathogen. Also provided are isolated polynucleotide molecules and vectors incorporating a chimeric PIV genome or antigenome which includes a partial or complete PIV vector genome or antigenome combined or integrated with one or more heterologous gene(s) or genome segment(s) encoding antigenic determinant(s) of a heterologous PIV or non-PIV pathogen. In preferred aspects of the invention, chimeric PIV incorporate a partial or complete human, bovine, or human-bovine chimeric, PIV vector genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a heterologous PIV or non-PIV pathogen, wherein the chimeric virus is attenuated for use as a vaccine agent by any of a variety of mutations and nucleotide modifications introduced into the chimeric genome or antigenome.
Description
Background of invention
3 type human parainfluenza viruses (HPIV3) be one-year-old a kind of common disease with serious lower respiratory infection among interior baby and the children because of.As due to illness malicious lower respiratory illness in this age group and the major cause of hospital care, it is only second to respiratory syncytial virus (RSV) (people such as Collins, the 1205-1243 page or leaf, B.N.Fields (people such as Knipe writes), " wild virusology ", the third edition, the first roll, Lippincott-Raven Publishers, Philadelphia, 1996; People such as Crowe, vaccine (Vaccine) 13:415-421,1995; People such as Marx, transmissible disease magazine (J.Infect.Dis.) 176:1423-1427,1997).The infection of this virus caused higher incidence at 3 years old in interior children.HPIV1 and HPIV2 be laryngotracheobronchitis (croup) main diseases because of, also can cause serious pneumonia and bronchiolitis (people such as Collins, the third edition, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, the 1205-1243 page or leaf.Lippincott-RavenPublishers，Philadelphia，1996)。In 20 years study for a long period of time, HPIV-1, HPIV2 and HPIV3 be determined to be in respectively occupy altogether institute treatment 18% because of 6.0%, 3.2% and 11.5% the cause of disease in the respiratory tract disease hospital care, need a kind of effective vaccine (people such as Murphy for this reason, virus research (Virus Res.) 11:1-15,1988).The virus induction middle ear of significant proportion ooze out and have also identified parainfluenza virus (people such as Heikkinen, New England Journal of Medicine (N.Engl.J.Med.) 340:260-4,1999) in the case in the otitis media infant.Therefore, need production a kind of, can prevent serious lower respiratory illness and the otitis media of following these HPIV to infect at these viral vaccines.HPIV-1, HPIV2 and HPIV3 are the different serotypes that does not cause obvious cross protection immunity.
Although, also do not obtain the approved vaccine preparation that is used for any HPIV serotype or alleviates the HPIV relative disease for a large amount of effort of effective vaccine treatment having carried out of development at HPIV.Up to now, have only the strain of two kinds of attenuated live PIV vaccine candidates to be subjected to special concern.A kind of candidate's strain is ox PIV (BPIV3) strain, and it is relevant with the HPIV3 antigenicity, and shows the antagonism HPIV3 that can watch for animals.BPIV3 is attenuation, inheritance stability in baby and children and has immunogenicity (people such as Karron, transmissible disease magazine 171:1107-14,1995a; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).Second kind of a kind of cold-adapted mutant (people such as Karron, transmissible disease magazine 172:1445-1450,1995b that PIV3 vaccine candidate strain JS cp45 is JS wild-type (wt) strain of HPIV3; People such as Belshe, medical virology magazine (J.Med.Virol.) 10:235-42,1982).(ts), acclimatization to cold (ca) and attenuation (att) phenotype of this attenuation, cold going down to posterity (cp) PIV3 vaccine candidate alive strain displays temperature sensitivity are stable behind the virus replication in vivo.Cp45 virus is attacked for people PIV3 in laboratory animal protectiveness, and is attenuation, inheritance stability and immunogenic (people such as Hall, virus research 22:173-184,1992 in seronegative baby and children; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).So far most promising is attenuated live vaccine virus, because shown that they are still effective in non-human primate even in the presence of the antibody of passive transfer, the experimental state of this state that to be a kind of simulation exist in the extremely young baby of containing the antibody that parent obtains (people such as Crowe, vaccine 13:847-855,1995; People such as Durbin, transmissible disease magazine 179:1345-1351,1999).The strain of two kinds of attenuated live PIV3 vaccine candidates, a kind of is temperature sensitive (ts) derivative (called after PIV3cp45) of wild-type PIV3 JS strain, a kind of is ox PIV3 (BPIV3) strain, carry out clinical evaluation (people such as Karron, paediatrics transmissible disease magazine (Pediatr.Infect.Dis.J.) 15:650-654,1996; People such as Karron, transmissible disease magazine 171:1107-1114,1995a; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).The strain of attenuated live PIV3cp45 vaccine candidate by at low temperatures in cell culture series go down to posterity and produce by HPIV3 JS strain, and find that attack has protectiveness to HPIV3 in laboratory animal for it, and be attenuation, inheritance stability and immunogenic (people such as Belshe satisfactorily in seronegativity baby and children, medical virology magazine 10:235-242,1982; People such as Belshe are infected and immunity (Infect.Immun.) 37:160-5,1982; People such as Clements, clinical microbiology magazine (J.Clin.Microbiol.) 29:1175-82,1991; People such as Crookshanks, medical virology magazine 13:243-9,1984; People such as Hall, virus research 22:173-184,1992; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).Because these PIV3 candidate vaccine viruses are biology deutero-, do not find the method for verified adjusting attenuation level as yet, as find suitable method, in the clinical trial of carrying out, be essential then.
In order to promote the development of PIV vaccine candidate strain, recombinant DNA technology makes recently and can obtain the infectivity minus-stranded rna virus by cDNA (summary is seen Conzelmann, general virology magazine (J.Gen.Virol.) 77:381-89,1996; People such as Palese, newspaper (Proc.Natl.Acad.Sci.USA) 93:11354-58 of institute of NAS, 1996).In context, to infectious respiratory syncytial virus (RSV), rabies virus (RaV), simian virus 5 (SV5), rinderpest virus, Avian pneumo-encephalitis virus (NDV), vesicular stomatitis virus (VSV), Measles virus (MeV) and Sendai virus (SeV) reported in the presence of essential viral protein from the reorganization of anti-genome (antigenome) RNA of cDNA coding save (referring to, for example, people such as Garcin, EMBO J.14:6087-6094,1995; People such as Lawson, institute of NAS reports 92:4477-81, and 1995; People such as Radecke, EMBO J.14:5773-5784,1995; People such as Schnell, EMBOJ.13:4195-203,1994; People such as Whelan, institute of NAS reports 92:8388-92, and 1995; People such as Hoffman, Journal of Virology 71:4272-4277,1997; People such as Kato, gene is to cell (Genes to Cells) 1:569-579,1996; People such as Roberts, virusology 247:1-6,1998; People such as Baron, Journal of Virology 71:1265-1271,1997; International patent WO97/06270; People such as Collins, institute of NAS reports 92:11563-11567, and 1995; The Application No. 08/892 of application on July 15th, 1997,403 (the preferential U.S. Provisional Applications of the international application no WO 98/02530 that is equivalent to announce and on May 23rd, 1997 application number 60/047,634,60/046 of application on June 9th, 1997,141, with on July 15th, 1996 application 60/021,773); The Application No. 09/291,894 of application on April 13rd, 1999; The international application no PCT/US00/09695 of application on April 12nd, 2000 (requiring right of priority) to the U.S. Provisional Patent Application series number 60/129,006 of application on April 13rd, 1999; The international application no PCT/US00/17755 of application on June 23rd, 2000 (requiring the right of priority in the U.S. Provisional Patent Application series number 60/143,132 of application on July 9th, 1999) to people such as Bucholz; People such as Juhasz, Journal of Virology 71:5814-5819,1997; People such as He, virusology 237:249-260,1997; People such as Peters, Journal of Virology 73:5001-5009,1999; People such as Baron, Journal of Virology 71:1265-1271,1997; People such as Whitehead, virusology 247:232-9,1998a; People such as Whitehead, Journal of Virology 72:4467-4471,1998b; People such as Jin, virusology 251:206-214,1998; People such as Bucholz, Journal of Virology 73:251-259,1999; With people such as Whitehead, Journal of Virology 73:3438-3442,1999, be all purposes and be incorporated herein by reference).
More particularly for the present invention, latest developments a kind of method of producing HPIV with wt phenotype by cDNA, with recover infectivity reorganization HPIV3 JS strain (referring to, for example, people such as Durbin, virusology 235:323-332,1997; The U.S. Patent Application Serial 09/083,793 of on May 22nd, 1998 application (U.S. Provisional Application number 60/059,385 that is equivalent to application on September 19th, 1997); U.S. Provisional Application number 60/047,575 (being equivalent to international publication number WO 98/53078) with application on May 23rd, 1997, all be incorporated herein by reference).In addition, these disclosures make and can clone by genetic manipulation virus cDNA, to determine the genetics basis of phenotypic alternation in the biological mutant, for example, its ts, ca and att phenotype are determined in sudden change in the HPIV3 cp45 virus, and the gene of BPIV3 or genome section are determined its attenuation phenotype.In addition, these and relevant disclosure make make up the novel PIV vaccine candidate strain that contains extensive different sudden changes and estimate its attenuation, immunogenicity and phenotypic stability level feasible (referring to, the U. S. application of on June 1st, 2000 application number 09/586,479 (the U.S. Provisional Patent Application series numbers 60/143,134 that are equivalent to application on July 9th, 1999); With the U.S. Patent Application Serial 09/350,821 of people such as Durbin, all be incorporated herein by reference) in application on July 9th, 1999.
Therefore, recovered infectivity wild-type reorganization PIV3 by cDNA now, (r) PIV3, and a large amount of ts derivative, and produced and carried the infective virus of specifying the attenuation sudden change, and the genetics basis of the attenuation of the existing vaccine virus of research with the reverse genetic system.For example, ts and attenuation phenotype are determined in the 3 seed amino acids displacements of finding in the L gene that has been found that at cp45 (alone or in combination).In other zones of PIV3cp45, there are other ts and attenuation sudden change.In addition,, replace with the ORF of PIV1, produce the strain of chimeric PIV1 vaccine candidate by PIV3 HN and F open reading frame (ORF) being used in the PIV3 full-length cDNA that contains 3 attenuations sudden changes among the L those with PIV3 cDNA rescue system.Be named as rPIV3-1.cp45L (people such as Skiadopoulos, Journal of Virology 72:1762-8,1998 by this cDNA deutero-reorganization embedded virus; People such as Tao, Journal of Virology 72:2955-2961,1998; People such as Tao, vaccine 17:1100-1108,1999, be incorporated herein by reference).RPIV3-1.cp45L is attenuation in hamster, and induces the high-level resistance that PIV1 is attacked.Also produced another kind of reorganization embedded virus, be named as rPIV3-1.cp45, it contains in 15 cp45 sudden change 12,, is not included in the sudden change that takes place among HN and the F that is.This recombiant vaccine candidate strain is at the upper respiratory tract and the lower respiratory tract camber attenuation of hamster, and induces the infectious high level protection of HPIV1 people such as (, vaccine 18:503-510,1999) Skiadopoulos.
Recently, big quantity research concentrates on the possible purposes that virus vector is expressed exogenous antigen, and its purpose is to develop at its vaccine vaccine of not successful pathogenic agent as yet.In context, a large amount of reports propose, and foreign gene can successfully insert in the recombinant negative strand rna viral genome or anti-genome with same-action not (people such as Bukreyev, Journal of Virology 70:6634-41,1996; People such as Bukreyev, institute of NAS reports 96:2367-72, and 1999; People such as Finke, Journal of Virology 71:7281-8,1997; People such as Hasan, general virology magazine 78:2813-20,1997; People such as He, virusology 237:249-60,1997; People such as Jin, virusology 251:206-14,1998; People such as Johnson, Journal of Virology 71:5060-8,1997; People such as Kahn, virusology 254:81-91,1999; People such as Kretzschmar, Journal of Virology 71:5982-9,1997; People such as Mebatsion, institute of NAS reports 93:7310-4, and 1996; People such as Moriya, FEBS Lett425:105-11,1998; People such as Roberts, Journal of Virology 73:3723-32,1999; People such as Roberts, Journal of Virology 72:4704-11,1998; People such as Roberts, virusology 247:1-6,1998; People such as Sakai, FEBS Lett.456:221-226,1999; People such as Schnell, institute of NAS reports 93:11359-65,1996a; People such as Schnell, Journal of Virology 70:2318-23,1996b; People such as Schnell, cell 90:849-57,1997; People such as Singh, general virology magazine 80:101-6,1999; People such as Singh, Journal of Virology 73:4823-8,1999; People such as Spielhofer, Journal of Virology 72:2150-9,1998; People such as Yu, gene is to cell 2:457-66,1999; The U.S. Patent Application Serial 09/614,285 of application on July 12nd, 2000 (the U.S. Provisional Patent Application series number 60/143,425 that is equivalent to application on July 13rd, 1999) all is incorporated herein by reference).When being positioned at virus transcription gene-initial and gene-termination signal control in inserting viral genome, foreign gene can be transcribed into mRNA separately, and produces tangible protein expression.Astonishing, report that in some cases exogenous array is stable and can expressive function protein in subculture in vitro separately process repeatedly.
Yet,, prove that just stable high-caliber protein expression is not enough for successful development is used for the carrier of vaccine.For example, from the phase early 1980s to mid-term, be possible with vaccinia virus recombinant and adenovirus, but these carriers are disappointing in the vaccine for man in development.Similarly, most of non-segmented negative-strand viruses that developed into carrier do not have man-rated character or immunization strategy.Example in this specification sheets comprises vesicular stomatitis virus, and a kind of ungulate pathogenic agent is not used history to the people except the accident of a few experiments chamber; Sendai virus, a kind of mouse pathogenic agent is not used history to the people; Simian virus 5, a kind of dog disease substance is not used history to the people; With a kind of attenuated strain of Measles virus, it must systemic administration, and can be by because maternal antibody and be extensive use of the permission vaccine and the measles specific antibody neutralization that be existed in nearly all crowd.In addition, some in these existing carrier candidate strains have negative impact, and are as immunosuppression, inconsistent with its purposes as carrier.Therefore, must identify that its growth characteristics, tropism and other biological character make it to be suitable for the carrier as the human carrier.Further a kind of loose joint kind strategy be must develop, immunogenicity and effective route of administration comprised.
Currently think the negative strand virus (mononegavirus) of the three-type-person who can be used as vaccine carrier, i.e. measles, mumps and rabies virus have other restriction, combine to make it to become the weak candidate's strain that is further development of carrier.For example, considered Measles virus as the carrier of hepatitis B virus protective antigen people such as (, Journal of Virology 73:4823-8,1999) Singh.Yet this combination Measles virus-Hepatitis B virus vaccine can only be used after big at 9 months as the measles virus vaccines of permission, and current Hepatitis B virus vaccine is recommended in and uses in early days infancy.This is because the measles virus vaccines of current permission are parenteral administration, and extremely responsive to the neutralization and the immunosuppression of maternal antibody, therefore if used before big at 9-15 month then invalid.Therefore can not be used for load can cause in early days in infancy and the antigen of disease therefore can not be used for the virus as RSV and HPIV.The another kind of well-known distinctive effect of viral infection of measles is virus-mediated immunosuppression, and it can continue some months.Immunosuppression is not the feature of wishing for carrier.When using with the dosage that increases, the immunne response of attenuated measles virus vaccine and change is relevant with too high mortality ratio, and this may be the immunosuppression owing to virus induction at least in part, shows even attenuated measles virus also may be not suitable as carrier.In addition, Measles virus does not meet the global effort of eradicating this pathogenic agent as the application of carrier.In fact, owing to these reasons, as described here, stop using Measles virus alive and replace existing measles virus vaccines to wish with the PIV carrier of expressing the Measles virus protective antigen.
Rabies virus, human infection's a kind of rare reason, be considered as carrier (people such as Mebatsion, institute of NAS reports 93:7310-4,1996), but development is impossible to people's 100% lethal carrier as the attenuated live virus vector, particularly because of the rabies virus immunity that does not need for the general population not being general human pathogen.Although mumps and Measles virus are pathogenic relatively poor, the infection of its any virus has undesirable characteristic.Mumps virus infects the parotid gland, and can be diffused into testis, causes sterile sometimes.Measles virus causes viremia, and the illustration of the general character of this infection is the rash of the extensive diffusive of being correlated with.Slight encephalitis between mumps and measles period of infection is unrare.Measles virus is also relevant with a kind of rare carrying out property lethality sacred disease that is called as subacute sclerosis encephalitis.On the contrary, the PIV in the normal individual infects and disease only limits to respiratory tract, and this is the position that more helps immunity than parenteral route.Viremia can take place and to the diffusion at second position in the laboratory animal of severe non-responsiveness and human body, but this not the feature that typical PIV infects.Acute respiratory disease is the disease relevant with PIV just.Therefore,, use PIV and will avoid complication,, cause the infection of immunosuppression or secondary organ such as testis or central nervous system, cause other complication as the interaction of virus with periphery lymphocyte as carrier according to its biological property.
Desirable human pathogen host is a Measles virus based on the vaccine method of carrier.Attenuated live vaccine has been used and has been surpassed 30 years, and obtains immense success in the measles disease of eliminating the U.S..Yet the World Health Organization estimates the annual measles case that still takes place more than 4,500 ten thousand, and particularly in developing country, this virus causes about 1,000,000 people's death every year.
Measles virus is a member (people such as Griffin of Paramyxoviridae Morbillivirus (Morbillivirus), " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, the 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。It is known to one of communicable infective agent of tool of people, and from people to people, propagate (people such as Griffin, " wild virusology ", B.N.Fields by respiratory pathways, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。Measles virus has complicated pathogenesis, be included in (people such as Griffin, " wild virusology ", the B.N.Fields of duplicating at respiratory tract and different whole bodies position, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。
Although mucous membrane IgA and serum IgG Measles virus specific antibody can both participate in the control of Measles virus, in extremely young baby, there is not the Measles virus disease with Measles virus specific antibody that parent obtains, determine that serum antibody is the main medium (people such as Griffin of disease resistance, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, the 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。Two kinds of Measles virus glycoprotein, hemagglutinin (HA) and merge (F) albumen is main neutralization and protective antigen (people such as Griffin; " wild virusology "; B.N.Fields, D.M.Knipe, P.M.Howley; R.M.Chanock; J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile; the first roll, the 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。
Current available attenuated live Measles Vaccine is used (people such as Griffin by parenteral route, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, the 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。Wild-type Measles virus and vaccine virus are all very easily neutralized by antibody, even the Measles virus specificity neutralizing antibody that obtains of extremely low-level parent can both make measles virus vaccines not have infectivity (people such as Halsey, New England Journal of Medicine 313:544-9,1985; People such as Osterhaus, vaccine 16:1479-81,1998).Therefore, when reducing to undetectable level, the maternal antibody of passive acquisition just can use vaccine virus.In the U.S., just used measles virus vaccines when big up to 12-15 month, nearly all children this moment easy infection measles virus vaccines all.In developing country, Measles virus continues that high mortality is arranged, particularly half years old (people such as Gellin, transmissible disease magazine 170:S3-14,1994 in one-year-old children; People such as Taylor, U.S.'s epidemiology magazine (Am.J.Epidemiol.) 127:788-94,1988).It is because these area Measles viruss all the fashion can infect Measles virus specific antibody that parent obtains reduced to can not detection level baby's subgroup.Therefore, the measles virus vaccines that need even in the presence of the Measles virus neutralizing antibody, still can bring out protective immune response, its purpose is to eliminate 1 years old with Measles virus disease interior generation and that take place afterwards.Because this needs have carried out making great efforts to study half years old to 1 years old baby of a kind of protection in a large number and have exempted from the immunization strategy of Measles virus, are effective but still there is not a kind of strategy up to now.
First kind of strategy researching and developing early stage Measles Vaccine comprises with one of following two kinds of methods uses the live,attenuated measles virus vaccine (people such as Cutts, biology (Biologicals) 25:323-38,1997) of permission to about 6 months big babies.A kind of common solution is, dropwise intranasal administration attenuated live measles virus (people such as Black, New England Journal of Medicine 263:165-169,1960; People such as Kok, Trans.R.Soc.Trop.Med.Hyg.77:171-6,1983; People such as Simasathien, vaccine 15:329-34,1997), or in lower respiratory tract, use (people such as Sabin, transmissible disease magazine 152 by aerosol; 1231-7,1985), to cause respiratory tract infection.Second kind of scheme is, with the higher dosage parenteral administration Measles virus of using than current vaccine.For attenuated live poliovirus and Rotavirus Vaccine, successfully realized (people such as Melnick, " wild virusology ", the B.N.Fields of using of the vaccine that can duplicate at mucomembranous surface in early days in infancy, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 655-712 page or leaf.Lippincott-Raven Publishers, Philadelphia, 1996; People such as Perez-Schael, New England Journal of Medicine 337:1181-7,1997), this may be because the IgG antibody of passive acquisition is compared few near mucomembranous surface with the system position of virus replication.In this case, attenuated live poliovirus vaccine virus can infect infant's the gi tract or the mucomembranous surface of respiratory tract, comprises the surface of containing maternal antibody, causes inducing of protective immune response.
Therefore, a kind of reasonable method is with the respiratory immunity of live,attenuated measles virus vaccine by the infant, because this is the natural approach of viral infection of measles.Yet, by the infectious attenuated live measles virus of parenteral route by approach in the nose not necessarily be infectious (people such as Black, New England Journal of Medicine 263:165-169,1960; People such as Cutts, biology 25:323-38,1997; People such as Kok, Trans.R.Soc.Trop.Med.Hyg.77:171-6,1983; People such as Simasathien, vaccine 15:329-34,1997), and this communicable reduction for current be that the measles virus vaccines Schwartz strain of vaccine strain is obvious especially.Make in the process of viral attenuation by going down to posterity in bird source tissue culturing cell, virus may be lost a large amount of infectivity for people's upper respiratory tract.In fact, the biological hallmark of Measles virus is that when in growth in vitro, the biological property of virus has quick change.Because this simple relatively immunization route is not succeeded,, comprise by aerosol and in lower respiratory tract, use live-virus vaccine (people such as Cutts, biology 25:323-38,1997 so attempted second method; People such as Sabin, transmissible disease magazine 152:1231-7,1985).
Realized that in the experimental study of highly control using measles virus vaccines by aerosol infects the infant, but can repeatedly use live,attenuated measles virus vaccine to ill-matched infant in wild mode by aerosol still can not (people such as Cutts, biology 25:323-38,1997).In 6 months big babies' of immunity another kind trial, to improve 10 to 100 times dosage parenteral administration Measles Vaccine virus people such as (, New England Journal of Medicine 322:580-7,1990) Markowitz.Although high measles vaccination alives of tiring can improve 4-6 month baby's seroconversion, in tiring the vaccine acceptor, later stage infancy height has relevant mortality ratio raising (people such as Gellin, transmissible disease magazine 170:S3-14,1994; People such as Holt, transmissible disease magazine 168:1087-96,1993; People such as Markowitz, New England Journal of Medicine 322:580-7,1990), this immunization method is abandoned.
The second kind of strategy that was used to study measles virus vaccines in the past is to use the measles virus vaccines of deactivation, the complete Measles virus of formalin deactivation or particularly by the subgenomic viral vaccine of Measles virus preparation (people such as Griffin, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 1267-1312 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。Yet these vaccines have shown CR Critical complication in the clinical application of nineteen sixties, that is, the virus vaccines of deactivation has been strengthened disease rather than preventing disease (people such as Fulginiti, JAMA 202:1075-80,1967).This is (people such as Fulginiti, JAMA 202:1075-80,1967) found for the first time with the measles virus vaccines of formalin deactivation.During beginning, this vaccine prevention measles, but the inoculator has lost resistance to infecting after several years.When after when infecting natural round-robin Measles virus, the inoculator develops into has the constitutional symptom that increases the weight of and atypia disease (people such as Fulginiti, JAMA 202:1075-80,1967 of pneumonia; People such as Nader, paediatrics magazine (J.Pediatr.) 72:22-8,1968; People such as Rauh, U.S. children disease magazine (Am.J.Dis.Child) 109:232-7,1965).Retrospective analysis shows, the formalin deactivation has destroyed the protein induced haemolysis of measles fusion (F) and suppressed the ability of antibody, but do not destroy ability (people such as Norrby, transmissible disease magazine 132:262-9,1975 of the protein induced neutralizing antibody of HA (hemagglutinin or adhere to); People such as Norrby infect and immune 11:231-9 1975).When the protein induced immunity of HA fully reduces when causing the extensive infection of wild-type Measles virus, Measles virus duplicate the position see change, more serious disease (Bellanti, paediatrics (Pediatrics) 48:715-29,1971 sometimes; Buser, New England Journal of Medicine 277:250-1,1967).This atypia disease is considered to partly wherein preferentially to induce the Th-2 cell by the cell-mediated immune responses mediation that changes, the disease performance that causes at the virus replication position increasing the weight of people such as (, national medical science (Nat.Med.) 5:629-34,1999) Polack.Because this experience aspect the measles virus vaccines that do not survive also because the antibody capable of passive transfer reduces the immunogenicity of the vaccine of these parenteral administration, is estimated quite difficulty of these vaccines in the baby.Should be pointed out that disease increase the weight of as if only relevant with the vaccine of death.
The another kind of strategy that is used for developing the Measles Vaccine that is used for the baby be to use virus vector express the protective antigen of Measles virus (people such as Drillien, institute of NAS reports 85:1252-6,1988; People such as Fooks, general virology magazine 79:1027-31,1998; People such as Schnell, institute of NAS reports 93:11359-65,1996a; People such as Taylor, virusology 187:321-8,1992; People such as Wild, vaccine 8:441-2,1990; People such as Wild, general virology magazine 73:359-67,1992).After deliberation variety carrier, comprise that poxvirus such as replicative vaccinia virus or replication defect type modify vaccinia virus ankara (MVA) strain.The rf bovine vaccine recon of expressing Measles virus F or HA glycoprotein is effective in the inoculator of immunology nature.Yet when parenteral administration in the presence of the Measles virus passive antibody, its immunogenicity and protection usefulness are eliminated (people such as Galletti, vaccine 13:197-201,1995 basically; People such as Osterhaus, vaccine 16:1479-81,1998; People such as Siegrist, vaccine 16:1409-14,1998; People such as Siegrist, biologic criteria progress (Dev.Biol.Stand.) 95:133-9,1998).
The rf bovine vaccine recon of expressing the RSV protective antigen also shows; when parenteral administration in the presence of passive antibody, inducing invalid (people such as Murphy, Journal of Virology 62:3907-10 aspect the protective immune response; 1988a), but when intranasal administration, be easy to protect these hosts.Regrettably, the replicative vaccinia virus recon can not be by abundant attenuation being used for the non-responsiveness host, as the people that infects human immunodeficiency virus (HIV) (people such as Fenner, the World Health Organization, Geneva, 1988; People such as Redfield, New England Journal of Medicine 316:673-676,1987), and it will have problem by approach in the nose to using of immunocompetence individuality.Therefore can not be used for the baby as carrier, wherein some baby's PI HIV.
(people such as Mayr is infected 3:6-14,1975 to the MVA carrier that produces by going down to posterity in chick-embryo cell more than 500 times; People such as Meyer, general virology magazine 72:1031-1038,1991), also be considered to be used for potential vaccine carrier (people such as Durbin, transmissible disease magazine 179:1345-51, the 1999a of the protective antigen of several paramyxovirus; People such as Wyatt, vaccine 14:1451-1458,1996).MVA is a kind of highly attenuated host range mutant, can duplicate in the bird cell but at most of mammalian cells, comprises from the cell that monkey and people obtain and not duplicating (people such as Blanchard, general virology magazine 79:1159-1167,1998; People such as Carroll, virusology 238:198-211,1997; People such as Drexler, general virology magazine 79:347-352,1998; People such as Sutter, institute of NAS reports 89:10847-10851, and 1992).Also made up the fowlpox virus vaccine carrier with the host range restriction that is similar to MVA, it expresses Measles virus protective antigen people such as (, virusology 187:321-8,1992) Taylor.MVA is not pathogenic in the non-responsiveness host, and many people have been used and do not meet accident (people such as Mayr, Zentralbl.Bakteriol.[B] 167:375-90,1978; People such as Stickle, Dtsch.Med.Wochenschr.99:2386-92,1974; People such as Werner, virusology journal 64:247-256,1980).Regrettably, no matter be to use by parenteral or local approach, immunogenicity and the usefulness of expressing the MVA of paramyxovirus protective antigen all is eliminated people such as (, virusology 235:323-332,1999) Durbin in the rhesus monkey of passive immunization.The immunogenicity of the dna vaccination of the expression Measles virus protective antigen of parenteral administration also reduces people such as (, biologic criteria progress 95:133-9,1998) Siegrist in the host of passive immunization.The current replication-defective vector of expressing the Measles virus protective antigen of having estimated comprises adenovirus-Measles virus HA recon people such as (, general virology magazine 79:1027-31,1998) Fooks.In context; express the antigenic MVA recon of parainfluenza virus; be different from the replicative vaccinia virus recon; when the animal of the antibody that contains passive acquisition being used by mucosal route; lack protective capability, they or similar fowl pox carrier can not use in the baby of the Measles virus antibody that contains the parent acquisition.
As if according to above-mentioned report, the rf of expression Measles virus protective antigen or replication defect type fowl pox carrier or dna vaccination can not have gratifying immunogenicity or validity in the baby of the parent Measles virus specific antibody that contains passive acquisition.
Developed a kind of reproducible in animal host's respiratory tract, duplicating virus carrier of expressing Measles virus HA recently, be vesicular stomatitis virus (VSV), but a kind of natural infected cattle but rhabdovirus (people such as Roberts, Journal of Virology 73:3723-32,1999 of non-infected person; People such as Schnell, institute of NAS reports 93:11359-65,1996a).Because VSV is a kind of animal virus that can cause human diseases, produce this human recon and need at first identify in the baby VSV main chain of attenuation (people such as Roberts satisfactorily, Journal of Virology 73:3723-32,1999), but these clinical studyes do not begin as yet.
Although comprise that at PIV and other pathogenic agent the development of the effective vaccine agent of measles has had a large amount of progress, but still very need make up the tool and method of vaccine safely and effectively in the art, to alleviate the serious health problem that particularly in the baby, causes by these pathogenic agent.The challenge that still exists is in this manual, need other instruments produce suitable attenuation, immunogenic, inheritance stability, at the vaccine candidate strain of one or more pathogenic agent, to be used for different clinical settings.For ease of this purpose, must the existing authentication method of expansion and in recombinant vaccine strain, add the attenuation sudden change and development based on the method for the vaccine and the immunization method of carrier.Surprisingly, the present invention has satisfied these needs, and has other advantages as described below.
Summary of the invention
The invention provides chimeric parainfluenza virus (PIV), it is infectious in human and other animals, and can in multiple composition, use, with it being infected one or more PIV that create antagonism among the responsive host, perhaps resist the immunne response of the hope of a kind of PIV and one or more other pathogenic agent.Aspect preferred, the novel method that the invention provides design and produce attenuation, chimeric PIV, it can be used as vaccine to prevent and/or treat the infection that is caused by PIV and one or more other pathogenic agent and related disorders is arranged.That these aspects of the present invention comprise is novel, isolating polynucleotide molecule and be mixed with the carrier of these molecules, they contain a kind of chimeric PIV genome or anti-genome (antigenome), comprise combine or integrate with one or more heterologous genes of one or more antigenic determinants of one or more allos pathogenic agent of coding or genome section or partial or complete PIV vector gene group or anti-genome.The present invention also provides method and mixes the composition of chimeric PIV, is used to prevent and treats the infection that is caused by the PIV that selects and one or more allos pathogenic agent (as allos PIV) or non-PIV pathogenic agent (as Measles virus).
Therefore the present invention relates to be used to develop method and composition, wherein use the subviral particle or the parainfluenza virus of mixing one or more antigenic determinants of allos pathogenic agent through recombinant modified based on chimeric living vaccine candidate strain.Make up chimeric PIV of the present invention by viral recovery system based on cDNA.The chimeric PIV of reorganization by the cDNA acquisition can be independently duplicated, and breed in the mode that is similar to biologically-derived virus.Transform recombinant virus, mix carrier (i.e. " acceptor " or " background ") PIV genome or anti-genomic nucleotide sequence, with one or more allos " donor " sequence of one or more antigenic determinants of coding different PIV or allos pathogenic agent, to produce a kind of infectivity embedded virus or subviral particle.Like this, modified recombinant candidate vaccine virus it being infected the immunne response that causes in the responsive Mammals at one or more PIV, or is replied at the PIV that selects and the polyspecific of non-PIV pathogenic agent.Preferably, the PIV of heterologous sequence coding for antigens determinant and/or non-PIV pathogenic agent are human pathogens, and the host is a human host.Equally preferably, carrier PIV is people PIV, although inhuman PIV as ox PIV (BPIV), also can be used as carrier, mixes the antigenic determinant of people PIV and other people class pathogenic agent.Chimeric PIV according to the present invention can cause the immunne response at specificity PIV such as HPIV1, HPIV2, HPIV3, or at the polyspecific immunne response of multiple PIV such as HPIV1 and HPIV2.In addition, chimeric PIV of the present invention can cause the immunne response at one or more PIV and non-PIV pathogenic agent such as Measles virus.
The chimeric PIV of typical case of the present invention has mixed a kind of aforesaid chimeric PIV genome or anti-genome, and a kind of main nucleocapsid (N) albumen, a kind of nucleocapsid phosphorprotein (P) and a kind of big polymerase protein (L).Also can comprise other PIV albumen, producing multiple infectivity subviral particle, until complete virion or contain the virion of additional proteins, antigenic determinant or other compositions with multiple combination.
Chimeric PIV of the present invention comprises and derives from or according to partial or complete " carrier " PIV genome or the anti-genome of people PIV or inhuman PIV, it and one or more heterologous genes or the combination of genome section of different PIV or other pathogenic agent form chimeric PIV genome or anti-genome.Of the present invention preferred aspect, mix one or more heterologous genes or the partial or complete people PIV of genome section bonded vector gene group or anti-genome among the chimeric PIV with second kind of people PIV or inhuman PIV pathogenic agent such as Measles virus.
PIV " carrier " genome or anti-genome are generally as having added or having mixed one or more " donor " genes of allos pathogenic agent or the acceptor or the carrier of genome section.The polynucleotide of one or more antigenic determinants of coding allos pathogenic agent generally are added into or replace in vector gene group or the anti-genome, produce a kind of chimeric PIV, it has obtained to cause the ability at the immunne response of this allos pathogenic agent in selected host.In addition, compare with the allos pathogenic agent with carrier PIV, embedded virus can show the phenotypic characteristic that other are new.For example, compare, in carrier PIV strain, add or displacement heterologous gene or genome section can be in addition or cause the phenotypic alternation of attenuation, growth alteration or other hope independently with the vector virus of unmodified and/or the corresponding phenotype of donor.In one aspect of the invention,, make chimeric PIV attenuation, determined the attenuation level of the embedded virus of generation according to inserting segmental size by mixing big polynucleotide insertion fragment in order to have higher performance as the vaccine candidate strain.
Preferred chimeric PIV vaccine candidate of the present invention strain contains one or more major antigen determinants of people PIV such as HPIV1, HPIV2 or HPIV3, thereby can cause the effective immunne response at selected PIV in human host.Can be to the special antigenic determinant of selected people PIV by vector gene group or anti-genome encoding, or can be used as from the heterologous polynucleotide sequence of different PIV and insert in PIV vector gene group or the anti-genome or be attached thereto.The main protection antigen of people PIV is its HN and F glycoprotein, replys although other protein also can cause protectiveness or therapeutic immunization.In this manual, representative vaccine candidate strain of the present invention advantageously causes body fluid and cell-mediated immune responses.Therefore, in vector gene group or anti-genome, may exist, or the polynucleotide of the coding for antigens determinant of integrating with it as heterologous gene or genome section, codified derives from immunogenic fragments or its epi-position of one or more PIV N, P, C, D, V, M, F, HN and/or L albumen or the selection of anyone PIV.
Except one or more major antigen determinants with selected people PIV, the preferred chimeric PIV vaccine virus of the present invention contains one or more major antigen determinants of second kind of people PIV or non-PIV pathogenic agent.Aspect typical, chimeric PIV comprises a kind of vector gene group or anti-genome, it is partial or complete people PIV (HPIV) genome or anti-genome as HPIV3, further comprises one or more heterologous genes or the genome section of the antigenic determinant of at least a allos PIV of coding such as HPIV1 and/or HPIV2.Preferably, vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are one or more allos HPIV.In another embodiment, can in partial or complete HPIV3 genome or anti-genome, add or one or more genes or the genome section of one or more antigenic determinants of permutation encoding HPIV1.Preferably, the antigenic determinant of HPIV is selected from HPIV1 HN and F glycoprotein, or contains one or more antigenic domains, fragment or the epi-position of HN and/or F glycoprotein.In different typical embodiments, with the HPIV1 gene replacement HPIV3 vector gene group of coding HN and F glycoprotein or corresponding HPIV3HN and the F gene in the anti-genome.These constructs are created in and can cause in the human body the list of HPIV3 and/or HPIV1 or the chimeric PIV of polyspecific immunne response.
In other typical embodiments, in partial or complete HPIV genome or anti-genome, add or mix one or more genes or the genome section of one or more antigenic determinants of coding HPIV2, the mosaic that obtains is only had at HPIV2, or at the new or other immunologic opsonin of HPIV3 and HPIV2.Aspect more detailed, in partial or complete HPIV3 vector gene group or anti-genome, add or mix one or more HPIV2 genes or the genome section of one or more HN of coding and/or F glycoprotein or its antigenic domain, fragment or epi-position.
In another aspect of this invention, in partial or complete PIV vector gene group or anti-genome, preferably in HPIV vector gene group or anti-genome, add or mix multiple heterologous gene or the genome section of the antigenic determinant of the multiple allos PIV of coding.In a preferred embodiment, in partial or complete HPIV3 vector gene group or anti-genome, add or mix heterologous gene or the genome section of the antigenic determinant of coding HPIV1 and HPIV2.In detailed aspect more, in partial or complete HPIV3 vector gene group or anti-genome, add or mix one or more HPIV1 genes of one or more HN of coding and/or F glycoprotein (or its antigenic domain, fragment or epi-position) or one or more HPIV2 genes or the genome section of genome section and coding HN and/or F glycoprotein, antigenic domain, fragment or epi-position.In one embodiment, HPIV1 gene with coding HN and F glycoprotein is replaced corresponding HPIV3 HN and F gene, form chimeric HPIV3-1 vector gene group or anti-genome, further modify by one or more genes or the gene fragment that add or mix one or more antigenic determinants of coding HPIV2.This is easy to realize in the present invention, for example, adds or replace the transcriptional units of the open reading frame (ORF) of a kind of HPIV2 of containing HN in chimeric HPIV3-1 vector gene group or anti-genome.According to this method, can produce explanation special construct of the present invention, this generation contains the chimeric PIV of the antigenic determinant of HPIV1 and HPIV2, and example is the vaccine candidate strain rPIV3-1.2HN and the rPIV3-1cp45.2HN of the following stated.
In another aspect of this invention, chimeric PIV of the present invention is based on people PIV vector gene group or anti-genome, and the latter is as the acceptor of the major antigen determinant that mixes non-PIV pathogenic agent.The pathogenic agent that its one or more antigenic determinants can be absorbed in the strain of chimeric PIV vaccine candidate includes but not limited to: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.Only exemplified the assembling of the pathogenic agent that can be used for the method according to this invention development vaccine herein, but it will be appreciated by those skilled in the art that with the PIV carrier and carry a large amount of hosts that the antigenic determinant wide spread arrives other pathogenic agent.
At different aspect of the present invention, can choose PIV genome or anti-genome are used to mix one or more major antigen determinants of multiple non-PIV pathogenic agent as carrier.The representative major antigen that can mix among the chimeric PIV of the present invention includes but not limited to: Measles virus HA and F albumen; The F of subgroup A and subgroup B respiratory syncytial virus, G, SH and M2 albumen, mumps virus HN and F albumen, human mammilla tumor virus L 1 albumen, 1 type and 2 type human immunodeficiency virus gp160 albumen, the gB of hsv and cytomegalovirus, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM albumen, rabies virus G albumen, Epstein-Barr virus gp350 albumen; Filovirus G albumen, bunyavirus G albumen, flavivirus E and NS1 albumen and Alphavirus E albumen.
Can carry the heterologous antigen determinant of non-PIV pathogenic agent with different people PIV carrier, with one or more special body fluid or the cell-mediated immune responses at the entrained antigenic determinant of chimeric virus of initiation in susceptible host, thereby initiation is at effective immunne response of wild-type " donor " pathogenic agent.In preferred embodiments, one or more heterologous genes or the genome section that derive from the donor pathogenic agent are connected with partial or complete HPIV3 genome or anti-genome, or insert wherein.In addition, heterologous gene or genome section also can mix in chimeric HPIV vector gene group or the anti-genome, for example carry in the partial or complete HPIV3 genome or anti-genome of one or more genes of allos PIV or genome section.For example, gene of the antigenic determinant of the non-PIV pathogenic agent of encoding or genome section can combine with partial or complete HPIV3-1 vector gene group or anti-genome, for example, use a kind of or the corresponding HPIV3 HN of these two kinds of HPIV1 gene substitutions and the F gene of coding HN and F glycoprotein as mentioned above.In addition, gene of the antigenic determinant of the non-PIV pathogenic agent of encoding or genome section also can combine with partial or complete mosaic gene group or anti-genome, it is in HPIV1 or HPIV3 vector gene group or anti-genome, or one or more antigenic determinants of in aforesaid chimeric HPIV3-1 vector gene group or anti-genome, mixing HPIV2, as HPIV2 HN gene.Encoding heterologous gene or the genome section of one or more measles antigenic determinants can be with disclosed any PIV carrier or chimeric PIV carrier combines herein.Among the embodiment that provides herein, vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome, or chimeric HPIV genome or anti-genome, wherein contain and added or mix one or more genes of coding allos HPIV antigenic determinant or the partial or complete HPIV3 genome or the anti-genome of genome section.In a kind of such chimeric construct body, add the transcriptional units of the open reading frame (ORF) that contains Measles virus HA gene to HPIV3 vector gene group or anti-genomic a plurality of sites, produce typical chimeric PIV/ Measles Vaccine candidate strain rPIV3 (HAHN-L), rPIV3 (HA N-P), rcp45L (HA N-P), rPIV3 (HA P-M) or rcp45L (HAP-M).
In other typical embodiments, PIV vector gene group or anti-genome are a kind of chimeric HPIV genome or anti-genome, and it contains and adds or mix one or more genes of one or more antigenic determinants of coding HPIV1 or the partial or complete HPIV3 genome or the anti-genome of genome section.This construct can be used as carrier, and as the carrier of Measles virus, wherein the heterologous antigen determinant is selected from Measles virus HA and F albumen and antigenic domain, fragment and epi-position.In one embodiment, be replaced by to HPIV3 HN and F ORF and add in the HPIV3-1 vector gene group of the HN of HPIV1 and F ORF or the anti-gene or mix the transcriptional units of the open reading frame (ORF) that contains Measles virus HA gene.This class recon has the vaccine candidate strain that is accredited as rPIV3-1HAP-M or rPIV3-1 HAP-M cp45L herein.
In other detailed embodiment of the present invention, partial or complete PIV vector gene group or anti-genome and one or more " additionally " are (promptly, outside the full gene, no matter be or in mutant, exist at the wild-type carrier, as the chimeric vector main chain) heterologous gene or the combination of genome section, form chimeric PIV genome or anti-genome.Normally a kind of complete HPIV3 of vector gene group or anti-genome or HPIV3-1 mosaic gene group or anti-genome, extra heterologous gene or genome section are selected from HPIV1 HN, HPIV1 F, HPIV2 HN, HPIV2 F, Measles virus HA and/or translate reticent synthetic gene unit.In some typical embodiments, one of HPIV1 HN and/or HPIV2 HN ORF or both insert respectively in HPIV3 vector gene group or the anti-genome.In more detailed embodiment, HPIV1 HN, HPIV2 HN and Measles virus HA ORF insert respectively between N/P, P/M and the HN/L gene.In addition, HPIV1 HN and HPIV2 HN gene also can insert respectively between N/P and the P/M gene, insert fragment and add 3918-nt GU between HN and L gene.This class recon comprises the vaccine candidate strain that is accredited as rHPIV3 1HNN-P, rHPIV3 1HNP-M, rHPIV3 2HNN-P, rHPIV3 2HNP-M, rHPIV3 1HNN-P2HNP-M, rHPIV3 1HNN-P 2HNP-M HAHN-L and rHPIV3 1HNN-P2HNP-M 3918GUHN-L herein.
The chimeric PIV of such design of the present invention and structure can contain and derives from protective antigens a kind of, two kinds, three kinds, four kinds or more different pathogens.The vaccine candidate strain of the protective antigen that contains the 1-4 kind pathogenic agent that is selected from HPIV3, HPIV1, HPIV2 and Measles virus for example, is provided.For making up this class polyspecific vaccine candidate strain, can add one or more extra heterologous genes or genome section, this can add the extra exogenous array that length overall is 30%-50% or more (for example, comparing with the wild-type HPIV3 genome length of 15462 nt) in recombination group or anti-genome.In context, the interpolation of one or more extra heterologous genes or genome section produces the attenuation phenotype of chimeric PIV usually, this is presented at duplicating in the upper respiratory tract and/or the lower respiratory tract and reduces 10-100 at least doubly, and 100-1000 doubly can reach 1000-10000 times or higher usually.
In order to make up chimeric PIV clone of the present invention, can be at the heterologous gene or the genome section of vector gene group or anti-genomic any operable position adding or displacement donor PIV or non-PIV pathogenic agent.The position of gene or gene fragment is usually corresponding to the wild type gene ordinal position of corresponding gene or genome section in partial or complete PIV vector gene group or the anti-genome.In other embodiments, more approaching or with respect to the wild type gene ordinal position of corresponding gene or genome section in background genome or anti-genome away from the position of promotor, add or displacement heterologous gene or genome section, to improve or to reduce the expression of heterologous gene or genome section respectively.In more detailed aspect of the present invention, heterologous gene group section, as the genome section of the immunogenicity extracellular domain of encode allos PIV or non-PIV pathogenic agent, can replace the corresponding gene group section of corresponding gene in PIV vector gene group or the anti-genome, produce the construct of coding chimeric protein, for example contain the kytoplasm tail of a kind of PIV that merges with another kind of PIV or non-PIV pathogenic agent extracellular domain and/or the fusion rotein of membrane-spanning domain.In other embodiments, can transform chimeric PIV genome or anti-genome, the polyspecific chimeric glycoprotein albumen in make it to encode recombinant virus or the subviral particle, it contains immunogenicity glycoprotein territory or the epi-position that derives from two kinds of different pathogens.In other embodiments, can in PIV vector gene group or anti-genome, add heterologous gene or the genome section that (promptly need not replace) derives from a kind of PIV or non-PIV pathogenic agent, in the clone who obtains, produce new immunogenicity.In these situations, can add heterologous gene or extra gene of genome section conduct or genome section with complete PIV vector gene group or anti-genome, randomly be in order to make the purpose of the embedded virus attenuation that obtains.In addition, also can be in vector gene group or anti-genome in selected gene or the genome section disappearance, add heterologous gene or genome section.
In a preferred embodiment of the invention, heterologous gene or genome section are added in the position between partial or complete PIV vector gene group or anti-genomic gene.In addition, also can be at genomic other non-coding regions, as 5 ' or 3 ' non-coding region, or in vector gene group or anti-genome, there are other sites of non-coding nucleotide, insert gene or genome section.In some cases, may wish in vector gene group or anti-genome corresponding to or be overlapped in the non-coding site that cis acting is regulated sequence, for example efficiently duplicating, transcribing and/or translating in the required sequence, insert heterologous gene or genome section.Vector gene group or anti-genomic these Regional Representative are used to destroy or the target site of the regulatory function that change is relevant with the introducing of heterologous gene or genome section.
In order to make up the preferred purpose that is used for clinical candidate vaccine virus, wish other attenuation phenotypes of suddenling change and regulating chimeric PIV of the present invention that improve or reduce recombinant virus attenuation level by introducing usually.Therefore, in other aspects of the present invention, produce the chimeric PIV of attenuation, wherein by introducing one or more attenuations suddenly change further modified chimeric genome or anti-genome, these sudden changes make the virus or the subviral particle that obtain have the attenuation phenotype.From the beginning these attenuation sudden changes can produce according to the mutagenesis strategy of well-known appropriate design, and test its attenuation.In addition, also can in prior biological deutero-sudden change PIV or other virus, identify the attenuation sudden change, mix afterwards among the chimeric PIV of the present invention.
Preferably the attenuation sudden change is easy to identify and mix among the chimeric PIV hereinafter, and method is by clone or mutagenesis vector gene group or anti-genome makes it to contain the attenuation sudden change and insertion sudden change in vector gene group or anti-genome.Preferably, in vector gene group or anti-genome, make up the attenuation sudden change, and introduce or copy from biology deutero-attenuation PIV mutant.They are believed to comprise, for example, and (hr), little plaque (sp) and/or temperature sensitive (ts) PIV mutant of (ca) of cold (cp) that goes down to posterity, acclimatization to cold, host range restriction.In typical embodiments, in chimeric PIV of the present invention, mix one or more attenuation sudden changes that exist in the JS HPIV3 cp45 mutant strain of well-characterized, be preferably incorporated in one or more sudden changes of identifying in the polysaccharase L albumen, for example at Tyr corresponding to JS
942, Leu
992Or Thr
1558The position mix.In addition, also can introduce the attenuation sudden change that exists in the JS HPIV3 cp45 mutant strain in chimeric PIV clone's N albumen, for example it is coded in the residue Val corresponding to JS
96Or Ser
389The amino-acid substitution of position.Amino-acid substitution in other useful attenuations sudden change coding C albumen, for example in M albumen corresponding to the Ile of JS
96The position, for example corresponding to Pro
199Position (Pro for example
199Sport Thr).In F albumen, for example at Ile corresponding to JS
420Or Ala
450The position and in HN albumen, for example at residue Val corresponding to JS
384The position, other sudden changes of having found in PIV3 JS cp45, to identify, they can be used for regulating the attenuation of chimeric PIV of the present invention.
Be used for comprising also that to the attenuation sudden change that chimeric PIV of the present invention mixes from biology deutero-PIV mutant PIV genome or anti-genomic non-encoding part are as the sudden change in the 3 ' leader sequence.Can be in 3 ' leader of recombinant virus construct typical case's sudden change in this specification sheets corresponding to the position of the Nucleotide 23,24,28 of JS cp45 or 45.For example, by for example changing in the N gene homing sequence one or more Nucleotide, can in N gene homing sequence, construct other typical cases and suddenly change corresponding to the position of the Nucleotide 62 of JS cp45.
Learn deutero-PIV mutant by PIV3 JS cp45 and other biological and obtained a large amount of attenuation sudden changes, every kind of sudden change can both suddenly change with any other and combine, and accurately regulates the attenuation level of chimeric PIV vaccine candidate of the present invention strain.In typical embodiments, make up one or more that comprise HPIV3 JS cp45, the chimeric PIV of two or more sudden changes preferably.Therefore, use the of the present invention chimeric PIV that selects for vaccine and contain two, the three or more attenuation sudden changes that derive from biology deutero-PIV mutant or close copy source sometimes usually, for extensive clinical application reaches satisfied attenuation level.Preferably, a plurality of nucleotide subsitutions in the codon of determining sudden change can be stabilized in these attenuation sudden changes of mixing among the chimeric PIV of reorganization of the present invention.
To the PIV carrier of selecting, comprise the sudden change that imports the phenotype that causes attenuation and other hope in chimeric ox-people PIV carrier, can be by in PIV vector gene group or anti-genome, shifting heterologous gene or the genome section that contains this sudden change, the for example proteic gene of encoding mutant L, or its part and realizing.In addition, sudden change also can be present in the vector gene group or anti-genome of selection, and the heterologous gene or the genome section that import can not contain sudden change, maybe can contain one or more other different sudden change.
In certain embodiments; in allos " donor " virus (for example; allos ox or people PIV or non-PIV minus-stranded rna virus) in modify vector gene groups or anti-genome corresponding to the one or more site in mutational site; make it to contain or (for example encode identical with the sudden change of in donor virus, identifying or conservative relevant sudden change; conservative amino acid replacement) (sees; the PCT/US00/09695 of application on April 12nd, 2000; and the preferential U.S. Provisional Patent Application series number 60/129 of applying on April 13rd, 1999; 006, all be incorporated herein by reference).In a typical embodiments, as implied above, one or more site modification PIV vector gene groups or anti-genome corresponding to HPIV3 JS cp45 mutational site make it the identical or conservative relevant sudden change that contains or encode and identify in cp45 " donor ".
Be used for identifying and comprise (ca) of cold (cp) that goes down to posterity, acclimatization to cold, (hr), little plaque (sp) and/or temperature sensitive (ts) mutant, for example the JS HPIV3 cp45 mutant strain of host range restriction to the preferred sudden change PIV strain that PIV carrier of the present invention mixes the attenuation sudden change.Be used for comprising also that to the attenuation sudden change that human-bovine chimeric PIV of the present invention mixes PIV genome or anti-genomic non-encoding part are as the sudden change in the 3 ' leader sequence from biology deutero-PIV mutant.Typical case's sudden change in this specification sheets can be corresponding to the Nucleotide 23,24,28 of JS cp45 or 45 site structure in recombinant virus 3 ' leader.For example, by for example changing in the N gene homing sequence one or more Nucleotide, can in N gene homing sequence, construct other typical cases and suddenly change corresponding to the position of the Nucleotide 62 of JS cp45.
Other sudden changes that may be utilized or be transferred to PIV carrier of the present invention can be identified in the non-sections of non-PIV (nonsegment) minus-stranded rna virus, and be incorporated in the PIV mutant of the present invention.By the sudden change that will identify in the allos minus-stranded rna virus the corresponding homologous site in acceptor PIV genome or the anti-genome is mapped, and to make the existing series jump in the acceptor be mutator gene type (by identical or conservative sudden change), be easy to realize this point, as the PCT/US00/09695 of application on April 12nd, 2000 and the preferential U.S. Provisional Patent Application series number 60/129 of applying on April 13rd, 1999,006 is described, all is incorporated herein by reference.According to present disclosure, in chimeric PIV of the present invention, be easy to adopt or be configured in other attenuations sudden changes that other viruses are particularly identified in other non-segmented negative-strand RNA viruses.
Another aspect of the present invention, structure contain or do not contain the chimeric PIV of the attenuation sudden change of biology deutero-attenuation mutated viruses modeling afterwards, make it to contain other nucleotide modifications, with phenotype, structure or the changing function that causes hope.Generally in partial or complete PIV vector gene group, carry out selected nucleotide modification, but also can in any heterologous gene that produces chimerical clone or genome section, carry out these modifications.These modify the phenotypic alternation that preferably causes hope, for example growth characteristics, attenuation, temperature sensitivity, acclimatization to cold, plaque size, host range restriction or immunogenic change.Structural modification in this specification sheets comprises for easy handling and identifies importing or excision restriction site in the cDNA of coding PIV.
In preferred embodiments, the change of the Nucleotide in the genome of chimeric PIV or the anti-genome comprises by the partially or completely disappearance of gene or the reduction or elimination (knocking out) the modification virus gene of its expression.Sudden change target gene in this specification sheets comprises any PIV gene, comprises the product of nucleocapsid protein N, phosphorprotein P, big polymerase L, stromatin M, hemagglutinin-neuraminidase HN, fusion protein F and C, D, V open reading frame (ORF).In order to make recombinant virus survival and tool infectivity, each in these protein can be whole or partly, separately or with modification selectivity disappearance, displacement or the rearrangement in combination of other hope, to obtain novel disappearance or to knock out mutant.For example, one or more C, D and/or V gene can whole or excalations, or it express to reduce or eliminates (for example, by importing terminator codon, rna editing site mutation, change the coded amino acid whose sudden change of initiator codon, or the phase shift mutation among the target ORF).In one embodiment, can in the editor site, suddenly change, prevent to edit and eliminate the protein expression that its mRNA produces by rna editing (people such as Kato, EMBO J 16:578-587,1997 and people such as Schneider, virusology 227:314-322,1997, be incorporated herein by reference).In addition, one or more C, D and/or V ORF also can whole or excalations, to change the phenotype of the recombinant clone that produces, (see with the phenotypic characteristic that improves growth, attenuation, immunogenicity or other hope, people such as Durbin are in the U.S. Patent Application Serial 09/350 of application on July 9th, 1999,821, be incorporated herein by reference).
Other nucleotide modifications among the chimeric PIV of the present invention comprise disappearance, insertion, interpolation or the rearrangement of the cis acting adjusting sequence of selected gene in recombination group or the anti-genome.In one embodiment, the cis acting that changes a kind of PIV gene is regulated sequence, makes it to regulate sequence corresponding to allos, and it can be the corresponding cis acting adjusting sequence of homologous genes among the different PIV, or the cis acting of different PIV genes is regulated sequence.For example, can be by in identical PIV strain, transforming or replace heterogeneic gene termination signal modifying factor termination signal.In other embodiments, nucleotide modification can comprise insertion, disappearance, displacement or the rearrangement of translation initiation site in recombination group or the anti-genome, for example, and for the protein form of selecting is removed selective translation initiation site.
In addition, also can be separately or with one or more attenuations sudden changes that biology deutero-sudden change PIV adopts, in chimeric PIV genome or anti-genome, produce multiple other hereditary changes.For example, can whole or partly insert the gene or the genome section in non-PIV source.At this on the one hand, the invention provides the method that makes chimeric PIV vaccine candidate strain attenuation, this is based on owing to import as derive from one or more genes of inhuman PIV or the host range effect that the genome section causes in the embedded virus of people PIV carrier.For example, derive from the nucleotide sequence of ox PIV (BPIV) by importing, can give chimeric construct body host range attenuation (as the U. S. application series number 09/586 of application on June 1st, 2000 based on the HPIV carrier, 479 is disclosed, the U.S. Provisional Application series number 60/143 that is equivalent to application on July 9th, 1999,134, be incorporated herein by reference).These effects cause the 26S Proteasome Structure and Function difference between carrier and the donor virus, and provide stability fundamental for attenuation.For example, between HPIV3 and BPV3, the every kind of proteic percentage amino acid of N identity is 86%, and P is 65%, and M is 93%, and F is 83%, and HN is 77%, and L is 91%.Therefore all these protein all are to import the material standed for that is difficult for being replied the attenuated chimeric virus that changes to produce in the HPIV carrier.In typical embodiments, vector gene group or anti-genome are HPIV3 genome or anti-genome, and heterologous gene or genome section are the N ORF that derives from selected BPIV3 strain.
Therefore, provide in the present invention based on being human-bovine chimeric PIV genome or anti-genomic vector gene group or anti-genomic chimeric PIV.In certain embodiments, human-bovine chimeric vector gene group or anti-genome combine with one or more heterologous genes or the genome section of one or more antigenic determinants of coding allos pathogenic agent, and this allos pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
Another aspect of the present invention, human-bovine chimeric vector gene group or anti-genome contain one or more heterologous genes or partial or complete HPIV genome or the anti-genome of genome section bonded with BPIV.In a typical embodiments, in vector gene group or anti-genome, replace the corresponding N ORF of HPIV3 vector gene group with the transcriptional units of the open reading frame (ORF) that contains BPIV3 N ORF.Use this and similar construct, vector gene group or anti-genome combine with the selected antigenic determinant of Measles virus HA gene or another kind of pathogenic agent, insert fragment as extra gene, for example are accredited as the vaccine candidate strain of rHPIV3-NB HAP-M.
In other aspects of the present invention, human-bovine chimeric vector gene group or anti-genome contain one or more heterologous genes or partial or complete HPIV genome or the anti-genome of genome section bonded with BPIV.For example; can in partial or complete cow genome group or anti-genome, add or mix one or more HPIV genes or the genome section of coding HN and/or F glycoprotein; or its a kind of or panimmunity originality territory, fragment or epi-position, form vector gene group or anti-genome.In certain embodiments, with the corresponding BPIV3HN of HPIV3 gene substitution and the F gene of coding HN and/or F glycoprotein, form vector gene group or anti-genome.Use this and similar construct, vector gene group or anti-genome combine with the selected antigenic determinant of RSV F and/or G gene or another kind of pathogenic agent, insert fragment as extra gene, as be accredited as shown in the vaccine candidate strain of rBHPIV3-G1 or rB/HPIV3-F1.
In more detailed embodiment; mix one or more HPIV1 HN and/or the F gene or the genome section of coding one or more immunogenicity territories, fragment or epi-position among chimeric people-Niu Zaiti; and further one or more HPIV1 HN and/or F gene or the modification of genome section by mixing coding one or more immunogenicity territories, fragment or epi-position, formation can be expressed the mosaic gene group or the anti-genome of the protective antigen of HPIV1 and HPIV2.The example of this chimeric PIV is to be accredited as rB/HPIV3.1-2F, rB/HPIV3.1-2HN or rB/HPIV3.1-2F, the multiple vaccine candidate strain of 2HN.
In other aspects of the present invention, can change gene order to cause attenuation or reduction or the expression that improves specific gene.In addition, the PIV genomic promoter can replace with its anti-genome homologue, to cause the phenotypic alternation of other hope.For ease of operation, can carry out different or other modifications in recombination group or the anti-genome, as in the district between different genes or insert unique restriction site elsewhere.Can remove the untranslated gene order to improve the ability of inserting exogenous array.
In other respects; can modify the chimeric PIV genome of coding or anti-genomic polynucleotide molecule or carrier; make its non-PIV sequence of encoding; assist epi-position, restriction site mark as cytokine, T-, or can in the purpose host, cause the protein or the immunogenicity epi-position of the microbial pathogen (for example virus, bacterium or fungi) of protective immune response.In a kind of such embodiment, make up chimeric PIV, wherein mix a kind of gene of the Codocyte factor, make the mosaic that obtains produce novel phenotype and immunogenicity effect.
Provide the chimeric PIV except using for vaccine, the present invention also provides Related cDNAs clone and carrier, it has mixed a kind of PIV vector gene group or anti-genome, with the heterologous polynucleotide of one or more heterologous antigen determinants of coding, wherein these clones randomly mix the sudden change and relevant modification that causes aforesaid one or more attenuation sudden changes or other phenotypic alternations with carrier.According to method described herein coding for antigens determinant and/or the heterologous sequence that causes the phenotypic alternation of hope being imported with the combination of selecting for example is in recombinant cDNA vector gene group or the anti-genomic isolating polynucleotide, produces the infective virus or the subviral particle of suitable attenuation.These methods combine with the evaluation of conventional phenotype, produce a large amount of chimeric PIV, and it has the feature of hope, as the immunogenicity of attenuation, temperature sensitivity, change, acclimatization to cold, little plaque size, host range restriction, genetic stability etc.Preferred vaccine virus is an attenuation in these time strains, but has sufficient immunogenicity, to cause protective immune response in the mammalian hosts of inoculation.
In the parties concerned of the present invention, composition (for example mixing isolating polynucleotide and the carrier of the cDNA of a kind of chimeric PIV that encodes) and method are provided, be used for producing the chimeric PIV of isolating infectivity.That these aspects of the present invention comprise is novel, isolating polynucleotide molecule and carrier, wherein is mixed with and contains chimeric PIV genome or anti-genomic molecule.Identical or different expression vector also is provided, and it contains coding N, P and proteic one or more the isolating polynucleotide molecules of L.These protein also can directly be expressed by genome or anti-genome cDNA in addition.These carriers are preferably expressed or coexpression in cell or acellular lysate, thereby produce infectivity chimeric parainfluenza virus particle or subviral particle.
Above-mentioned method and composition generation infective virus or the subviral particle or derivatives thereof that is used to produce chimeric PIV.A kind of infective virus is equivalent to real PIV particle, and as its tool infectivity.It can the new fresh cell of direct infection.The infectivity subviral particle generally is the subcomponent of virion, and it can initial under proper condition infection.For example, containing the proteic nucleocapsid of genome or antigenomic RNA and N, P and L is an example of subviral particle, and it can initial infection in the tenuigenin if be imported into.Subviral particle provided by the invention comprises the virion that lacks non-infectious essential one or more protein, protein fragments or other virus compositions.
In other embodiments, the invention provides a kind of cell or acellular lysate, it contains a kind of expression vector that contains isolating polynucleotide molecule, this molecule contains aforesaid chimeric PIV genome or anti-genome, with N, the P and the proteic expression vector (identical or different carrier) that separates polynucleotide molecule of L that contain one or more codings PIV.In these albumen one or more also can be expressed by genome or anti-genome cDNA.In case express, genome or anti-genome and N, P and L protein binding produce a kind of infectivity chimeric parainfluenza virus or subviral particle.
In other embodiments of the present invention, a kind of cell or acellular expression system (for example acellular lysate) are provided, wherein be mixed with a kind of expression vector that contains the separation polynucleotide molecule of the chimeric PIV that encodes, with N, the P and the proteic expression vector that separates polynucleotide molecule of L that contain one or more codings PIV.In case express, genome or anti-genome and N, P and L protein binding produce a kind of infectivity PIV particle, as a kind of virus or subviral particle.
Chimeric PIV of the present invention can use in multiple composition, to produce among the responsive host at one or more PIV or at the immunne response of the hope of PIV and non-PIV pathogenic agent in that it is infected.Chimeric PIV recon can cause single or how special protective immune response in infected mammalian hosts, fully attenuation does not consequently cause unacceptable illness in by the host of immunity.The virus of attenuation or subviral particle can be present in the cell culture supernatant, from culture, separate, or purifying partially or completely.Virus also can be by freeze-drying, and as desired, can combine so that store or the host is used with multiple other compositions.
The present invention further provides the new generation vaccine that contains physiology acceptable carrier and/or adjuvant and aforesaid isolating attenuated chimeric parainfluenza virus or subviral particle.In preferred embodiments, vaccine is made up of chimeric PIV, its contain at least a, preferably two or more determine other sudden changes or other nucleotide modifications of proper equilibrium between attenuation and the immunogenicity.Vaccine can be with 10
3-10
7The dosage preparation of PFU attenuated virus.Vaccine can contain and causes at a kind of PIV strain or at the attenuated chimeric PIV of the immunne response of multiple PIV strain or group.In this, chimeric PIV can combine with other PIV vaccine strains in vaccine preparation, or combines as RSV with other virus vaccines viruses.
In the parties concerned, the invention provides a kind of method, be used for stimulating the individual immunity system to cause in mammalian object at one or more PIV or at the immunne response of PIV and non-PIV pathogenic agent.This method comprises uses a kind of preparation that contains the chimeric PIV of immunology capacity in physiology acceptable carrier and/or adjuvant.In one embodiment, the vaccine that immunogenic composition is made up of chimeric PIV, this chimeric PIV contain attenuation sudden change or other nucleotide modifications of phenotypes at least a, preferably two or more definite above-mentioned hope and/or attenuation level.Vaccine can be with 10
3-10
7The dosage preparation of PFU attenuated virus.Vaccine can contain and causes at a kind of PIV strain or at multiple PIV such as HPIV1 and HPIV3 or at the attenuated chimeric PIV of the immunne response of one or more PIV and non-PIV pathogenic agent such as measles or RSV.In this manual, chimeric PIV can cause at single specific immune of multiple PIV or one or more PIV and non-PIV pathogenic agent and replys or many specific immunes are replied.In addition; chimeric PIV with different immunogenicity features also can combination in vaccine mixture; or in the partner treatment scheme separate administration, to cause at a kind of PIV, at multiple PIV or at more effective protection of one or more PIV and non-PIV pathogenic agent such as measles or RSV.Preferably, for example the upper respiratory tract is used immunogenic composition of the present invention by spraying, droplet or aerosol.Preferably, for example the upper respiratory tract is used immunogenic composition by spraying, droplet or aerosol.
The present invention also provides novel combination-vaccine and the simultaneous inoculation scheme that comprises multiple PIV and/or PIV and non-PIV pathogenic agent for pathogenic agent.For example, the early stage vaccination target of selecting according to these compositions comprises RSV and PIV3, they can both cause considerable disease in preceding 4 middle of the month of life, and most of diseases that PIV1 and PIV2 cause 6 months big after the time (people such as Collins, " wild virusology ", the first roll take place, the 1205-1243 page or leaf, Lippincott-Raven Publishers, Philadelphia, 1996; People such as Reed, transmissible disease magazine 175:807-13,1997).A kind of preferred immune programme for children that uses attenuated live RSV and PIV vaccine is that (for example 1 and 2 months big) uses RSV and PIV3 as far back as 1 month when big, subsequently 4 with used two valency PIV1 and PIV2 vaccine in 6 months when big.Therefore wish with the co-administered multiple PIV vaccine of method of the present invention; comprise one or more chimeric PIV vaccines; for example in mixture, use simultaneously; or the time sequence separate administration (for example with every day or order weekly) to determine, wherein every kind of vaccine virus is preferably expressed different allos protective antigens.This associating/sequential immunization strategy, can induce the secondary antibodies of multiple viral respiratory disease substance is replied, strong and utmost point immunization protocol flexibly is provided, and this is needed with other pathogenic agent immunity at each of three kinds of PIV viruses in early days in infancy.
Importantly, for the PIV3 disease that takes place than more Zao age of PIV1 and PIV2 the time, the existence of multiple PIV serotype and unique epidemiology thereof use the different PIV carrier continuous immunity babies that express identical heterologous antigen determinant such as Measles virus HA to wish.This continuous immunity allows to induce secondary antibodies to reply the high-titer antibody of peculiar heterologous protein.In one embodiment, with attenuated chimeric virus of the present invention, for example express the proteic chimeric HPIV3 of Measles virus HA, the early stage baby of immunity (for example 2-4 month big baby), and also make it be suitable for the immunne response of initiation at HPIV3 such as rcp45L (HA P-M).Subsequently, for example at 4 months when big, the baby is immunity once more, but uses second kind of different vector construction bodies, as expressing Measles virus HA gene and the HPIV1 antigenic determinant rPIV3-1cp45L virus as the functional obligate glycoprotein of carrier.After inoculation for the first time, the vaccinate will cause PIV3 HN and F albumen and Measles virus HA albumen primary antibody will be replied, but PIV1 HN and F albumen will not be caused.After with the rPIV3-1 cp45L that expresses Measles virus HA immunity for the second time; the vaccinate is owing to not existing the proteic antibody of PIV1 HN and F to be easy to infect this vaccine; and will develop the primary antibody of PIV1 HN and F protective antigen is replied and the secondary antibodies that the proteic height of allos Measles virus HA is tired is replied.Also can develop a kind of similar continuous immunity scheme; wherein cause immunity continuously to HPIV3 and HPIV2 with one or more chimerics viruses disclosed herein, stimulate simultaneously at measles or another kind of non-PIV pathogenic agent the secondary then at first height protective response of tiring.This continuous immunity strategy, the different serotypes of preferably using PIV have been avoided the immunity of first grade carrier inductive effectively as the primary and secondary carrier, and this is the factor that final restriction only contains a kind of carrier validity of serotype.Verified success people such as (, vaccine 17:1100-8,1999) Tao of involving continuous immunity with aforesaid rPIV3 and rpIV3-1 virus vaccines candidate in a criminal case.
The accompanying drawing summary
Figure 1A and the insertion (note: shown here all figures and associated description all be just anti-genome about HPIV3,5 '-3 ') of 1B explanation Measles virus HA gene in the HPIV3 genome.
Figure 1A provides 1926nt to insert segmental figure (last figure; Not to scale (NTS)), wherein contain the complete open reading frame of hemagglutinin (HA) gene of Measles virus Edmonston wild strain, it is transformed the back and expresses Measles virus HA by outer transcriptional units.This insertion fragment contains with 5 '-3 ' direction: the AflII site; Contain the anti-genomic nts 3699-3731 of HPIV3 that the P/M gene connects, comprise downstream non-coding sequence, its gene termination signal, intergenic region and the M gene start signal of P gene; Other three kinds of nts (GCG); Complete Measles virus HA ORF; The HPIV3 nt 3594-3623 of P gene downstream non-coding region; With second AflII site.The complete anti-genome that Figure 1A divides Fig. 1 that HPIV3JS wild-type strain (rPIV3) is described, wherein before measles HA ORF inserts fragment (last figure) and afterwards (figure below) in 3 '-non-coding region of N gene, introduced the AflII site.The complete anti-genome that Figure 1A divides Fig. 2 that HPIV3JS wild-type strain (rPIV3) is described, wherein before measles HA ORF inserts fragment (last figure) and afterwards (figure below) in 3 '-non-coding region of P gene, introduced the AflII site.SEQ ID NO.1 and SEQ ID NO.2 are shown among Figure 1A.
Figure 1B provides 2028 nt to insert segmental figure (last figure; Not to scale (NTS)), the complete ORF that wherein contains Measles virus HA gene.This insertion fragment contains with 5 '-3 ' direction: the StuI site; The anti-genomic nts 8602-8620 of HPIV3 that forms by HN gene downstream non-coding sequence and gene termination signal thereof; Distinguish between conservative gene; The nts 6733-6805 that contains the HPIV3 of HN gene initial sum upstream non-coding region; Measles virus HA ORF; HPIV3 nts 8525-8597, it is the downstream non-coding sequence of HN gene; With second StuI site.Structure is used for regenerating after insertion and contains the HPIV3 HN gene in StuI site, and Measles virus ORF is placed thereafter, and flank is HPIV3 HN gene transcription signal and non-coding region.Next (in) the complete anti-genome of introducing the HPIV3 JS wild-type (rPIV3) in StuI site at 8600 places, nt site of HN gene 3 ' non-coding region has been described among the figure.Be the anti-genome that inserts the proteic HPIV3 of expression measles HA in the StuI site below.The HA cDNA that is used for this insertion derives from existing plasmid, rather than derives from Edmonston wild-type Measles virus, and it is used for inserting N/P and P/M district.The HA albumen that inserts among this cDNA and Fig. 1 has two amino acid whose differences, and its position in Measles virus HA gene marks with an asterisk in Figure 1B.SEQ ID NO.3 and SEQID NO.4 are shown in Figure 1B.
The HA albumen of Measles virus is expressed in Fig. 2 explanation with rHPIV3-Measles virus-HA embedded virus in the LLC-MK2 cell.This figure shows a kind of radioimmuno-precipitation assay (RIPA), its proof measles HA albumen is expressed by reorganization embedded virus rcp45L (HA P-M) and rcp45L (HA N-P), and express (measles) by the strain of Measles virus Edmonston wild-type, express and can't help rJS wild-type HPIV3 (rJS).Road A---with three kinds of HPIV3 HN albumen specific monoclonal antibody mixture immunoprecipitations
35The cells infected lysate of S mark.All exist in the cell lysate (road 3,5 and 7) that 3 kinds of HPIV3 infect corresponding to the proteic 64kD band of HN (hollow arrow), but in the cell lysate (road 9) of viral infection of measles, do not exist, confirm that rcp45L (HA P-M) and rcp45L (HA N-P) mosaic are HPIV3 really, and express the HN albumen of similar level.Road (b)---with monoclonal antibody mixture (79-XV-V17,80-III-B2,81-1-366) immunoprecipitation that can discern Measles virus HA glycoprotein
35The cells infected lysate of S mark (people such as Hummel, Journal of Virology 69:1913-6,1995; People such as Sheshberadaran, virusology journal 83:251-68,1985, be incorporated herein by reference).Exist in the cell lysate that infects rcp45L (HA) embedded virus (road 6,8) and Measles virus (road 10) corresponding to the proteic 76kD band of HA (filled arrows), but do not exist in the lysate (road 4) of the cell that rJS infects, rJS is the HPIV3 wild-type virus of Measles virus HA gene of not encoding.
Fig. 3 illustrate HPIV2 HN gene as transcribe outward/(note: rPIV3-1 is a kind of rPIV3 in the insertion of translation unit in the anti-genome cDNA of coding rPIV3-1 or rPIV3-1 cp45 embedded virus, wherein HN and F gene are replaced by HN and the F gene of HPIV1, and rPIV3-1cp45 contains 12 forms that derive from the sudden change of cp45 attenuated virus in addition).Utilize the HPIV2HN gene specific primer, with the vRNA amplification HPIV2 HN gene (road A) of RT-PCR from HPIV2.Contain NcoI site that primer introduces, contain the amplification cDNA in HindIII site at 5 ' end with NcoI-HindIII digestion, and is connected generation pLit.PIV32 HNhc (component B) with the pLit.PIV31HNhc of NcoI-HindIII digestion at 3 ' end.Produce a kind of PIV2 HN box (component C) of modification with the pLit.PIV32HNhc plasmid as template, it has a PpuMI site at 5 ' end, has the PpuMI site of an introducing at 3 ' end.This box from left to right contains: the PpuMI site of 5 ' end, part 5 ' the non-translational region (UTR) of PIV3 HN, PIV2HN ORF, 3 '-UTR of PIV3 HN, stop with the gene that exists during the L gene is connected at PIV3 HN, between gene, the gene homing sequence, the part of the 5 ' non-translational region of PIV3 L and the PpuMI site of introducing at 3 ' end.Digest this cDNA box with PpuMI, p38 ' the Δ PIV31hc with PpuMI digestion is connected then, produces p38 ' Δ PIV31hc.2HN (component D).The 8.5KbBspEI-SphI fragment is assembled in the BspEI-SphI window of pFLC.2G+.hc or pFLCcp45, produces the anti-genome cDNA of final total length respectively, pFLC.3-1hc.2HN (component E) or pFLC.3-1hc.cp45.2HN (component F).PFLC.2G+.hc and pFLCcp45 are the anti-genomic clones of total length of encoding wild type rPIV3-1 and rPIV3cp45 respectively, existing in the past description the (people such as Skiadopoulos, Journal of Virology 73:1374-81,1999a; People such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference).
Fig. 4 is detailed to be shown and confirmed to carry the structure that PIV2 HN ORF inserts segmental rPIV3-1.2HN embedded virus between PIV1 F and HN gene.Panel A shows the textural difference of rPIV3-1 and rPIV3-1.2HN, and the latter is contained PIV2 HN ORF and inserted fragment between the PIV1 of rPIV3-1 F and HN ORF.Arrow indicates the apparent position of in the component B-D segmental RT-PCR primer of analyzing of being used for increasing.Component B and C show the expection size of the restriction enzyme digestion fragment that produces from the RT-PCR product of rPIV3-1 and rPIV3-1.2HN amplification with PpuMI or NcoI restriction endonuclease, and clip size is with base pair (bp) expression, and the result is presented among the component D.The vRNA that extracts in the virus with the LLC-MK2 cell harvesting that infects from rPIV3-1.2HN or rPIV3-1 is as template, in the existence of ThermoScript II (RT) or not in the presence of, with the cDNA fragment that increases of the primer PCR shown in the panel A.In the RT-PCR reactant that lacks RT, do not contain the PCR fragment, show that the template that is used for the dna fragmentation amplification is RNA, rather than the cDNA (road A and the C of component D) that pollutes.When comprising the RT step, rPIV3-1.2HNvRNA (road B) produces the big fragment near 2kb than its rPIV3-1 parent (road D), shows the insertion fragment that has 2kb.In addition, digest this 3kb fragment with several different restriction endonucleases and show, for the every kind of restriction endonuclease that detects, the RT-PCR fragment (odd numbered trace) of rPIV3-1.2HN has the pattern that is different from rPIV3-1 parent (even number road).For each digestion, the forecasting sequence of the RT-PCR product of site quantity that is obtained and clip size and rPIV3-1 and rPIV3-1.2HN is in full accord.Listed representative example.At first, the RT-PCR product (road 1) of PpuMI digestion rPIV3-1.2HN produces three bands of expection size, show to have two PpuMI sites, and the RT-PCR product of PpuMI digestion rPIV3-1 produces two bands (road 2) of expection size, shows only to have a PpuMI site.Secondly, the RT-PCR product (road 5) of NcoI digestion rPIV3-1.2HN produces 4 bands, comprises the 0.5kb fragment of HPIV2 HN gene, and the RT-PCR product (road 6) of NcoI digestion rPIV3-1 produces two fragments of expection.M refers to contain the road as the 1kb dna ladder level (LifeTechnology) of the big tick marks of Nucleotide (nt).Similarly the result confirms to exist among the rPIV3-1cp45.2HN HPIV2 HN to insert fragment.
Fig. 5 proves that rPIV3-1.2HN expresses HPIV2 HN albumen.Infect the LLC-MK2 individual layer with MOI 5 usefulness rPIV3-1, rPIV3-1.2HN or PIV2/V94 wild-type virus.Infected individual layer is at 32 ℃ of following incubations, and after infection 18-36 hour with
35S-met with
35S-cys mixture mark.Results and lysing cell are with anti--HPIV2 HN mAb 150S1 immunoprecipitation protein (people such as Durbin, virusology 261:319-330,1999; People such as Tsurudome, virusology 171:38-48,1989, be incorporated herein by reference).The sample of sex change immunoprecipitation separates on the 4-12%SDSPAGE gel, and radioautograph (road: 1, rPIV3-1; 2, rPIV3-1.2HN; 3, PIV2/V9412-6).MAb precipitating proteins from the LLC-MK2 cell of rPIV3-1.2HN and PIV2/V94 infection that HPIV2 HN is special, but from the cell that rPIV3-1 infects, do not precipitate, has the proteic expection size of the 86kD Kd HN of HPIV2 (people such as Rydbeck, general virology magazine 69:931-5,1988, be incorporated herein by reference).
Fig. 6 shows location and the structure that genetic unit (GU) inserts or HN gene 3 ' non-coding region (NCR) extends.Nucleotide sequence and unique restriction enzyme cloning site that GU and NCR insert the site are presented at respectively among panel A and the B.Marked cis acting and transcribed signal sequence, promptly gene stops (IG) and gene initial (GS) signal sequence between (GE), gene.In Fig. 6 panel A, show to determine double-stranded (Nucleotide that underscore marks) in the StuI restriction site of introducing (, seeing Figure 1B and embodiment 1) that inserts of the oligonucleotide of HN GE, IG and GS signal sequence about the position in the StuI site of introducing.The restricted fragment of the anti-geneome plasmid of RSV is cloned in the HpaI site.In case of necessity, in the MluI site with the double-stranded insertion of short oligonucleotide multiple clone site, make the segmental length overall of insertion meet 6 principle.In Fig. 6 component B, HN gene 3 '-NCR is inserted the HpaI site of fragment cloning to indicated 32 nt multiple clone site, it has been cloned in the StuI restriction site described in Fig. 6 panel A.Make the sequence of insertion meet 6 principle by in the MluI site of multiple clone site, inserting the short oligonucleotide two strands.SEQ ID NO.5 and 6 is shown among Fig. 6.
Fig. 7 illustrates that 3079 bp RSV insert the open reading frame (ORF) in the fragment.(three with direction to have shown 6 possible reading frames in the 3079bp RSV fragment; 3,2,1 ,-1 ,-2 ,-3).Short-term is represented translation initiation codon.Long line is represented translation stop codon.The 3079bp fragment is inserted between HN 3 ' NCR (NCR ins) or HN and the L gene as genetic unit (GU ins), and direction of insertion is to make reading frame that the PIV3 machine translator runs into corresponding to-3 among the figure ,-2 and-1.These reading frames contain a large amount of terminator codons in the sequence of complete length, therefore should not produce any functional protein.
Fig. 8 proves the rPIV3 insertion and extends the insertion fragment that mutant contains suitable size.With flank is to insert the special primer of the PIV3 in site to carrying out RT-PCR, separates the RT-PCR product by agarose electrophoresis.For rPIV3wt (being also referred to as rJS), the segmental expection size of RT-PCR is 3497bp, and for other rPIV3s GU or NCR mutant, length increases with inserting size.Panel A shows that GU inserts (ins) mutant: 1.rPIV3 wt; 2.r168 nt GU ins; 2.r678 nt GU ins; 3.r996 nt GU ins; 4.r1428 nt GU ins; 5.r1908 nt GUins; 6.r3918 nt GU ins.M: the HindIII restriction enzyme digestion product of lambda bacteriophage dna.Marked the size of sizes related marker.Component B shows that NCR inserts mutant: 1.rPIV3wt; 2.r258 nt NCR ins; 3.r972 nt NCR ins; 4.r1404 nt NCR ins; 5.r3126nt NCR ins; 6.r3894 nt NCR ins.M: the HindIII restriction enzyme digestion product of lambda bacteriophage dna.Marked the size of sizes related marker.
Fig. 9 A-9C demonstration is compared with rcp45L with rHPIV3 wt, and GU and NCR insert the multistage growth curve of sudden change.In triplicate, the infection multiplicity with 0.01 (m.o.i.) infects LLC-MK2 individual layer in 6 orifice plates with HPIV3, and is removing virus removal supernatant liquor after scouring 4 times.With the interval of 0 hour and 24 hours, in back 6 days of infection,, and add the 0.5ml fresh culture to every hole from every hole results 0.5ml virus culture base.The sample of results is stored in-80 ℃.By titration on the LLC-MK2 in 96 orifice plates of 32 ℃ of incubations, the virus that exists in the quantitative sample.Virus titer is expressed as TCID
50/ ml.Three independent mean values that infect in the once experiment have been shown.Be limited to 0.7 log under detecting
10TCID
50/ ml.Fig. 9 A-GU inserts mutant; Fig. 9 B-NCR inserts mutant; Fig. 9 C-cp45L/GU inserts mutant.
Figure 10 illustrates that extra gene is inserted fragment places the P of rHPIV3 and the strategy between the M gene.Modify downstream (the 3 ') NCR of rHPIV3 P gene, make it to contain AflII restriction site (Durbin, Journal of Virology 74:6821-31 2000, are incorporated herein by reference) at 3693-3698 place, anti-genome sequence site.Insert a kind of oligonucleotide duplex (last figure shows) with this site then, it contains the HPIV3 cis acting transcribes signal sequence, and promptly gene stops (IG) and gene initial (GS) motif between (GE), gene.This duplex also contains a series of restriction enzyme recognition sequences that can be used for inserting external source ORF.For HPIV1 and HPIV2 HN ORF, cloning site is NcoI and HindIII.Be placed on one group of HPIV3 in the external source ORF insertion multiple clone site and transcribe under the signal control, make that this gene is transcribed into isolating mRNA by the HPIV3 polysaccharase in whole recombinant virus.In case of necessity, the short oligonucleotide duplex being inserted in the MluI site of multiple clone site, is 6 even multiple with the whole length of regulatory gene group, and this is considered to needs (people such as Calain, Journal of Virology 67:4822-30,1993 of efficient rna replicon; People such as Durbin, virusology 234:74-83,1997b).With a kind of similar strategy, use the AflII restriction site of introducing at site 1677-1682 9 places (SEQ ID NO.7), HPIV1 and HPIV2 gene insertion fragment are placed between the N and P gene of rHPIV3.
Figure 11 is the genomic figure (disproportionate) of a series of chimeric rHPIV3, and it contains a kind of, two or three extra gene insertion fragment, a kind of protective antigen of every kind of all encode PIV1, PIV2 or Measles virus.The synoptic diagram of rHPIV3 (not to scale (NTS)) shows the coding HPIV1 that inserts in the rHPIV3 main chain ()
Or HPIV2
HN (hemagglutinin-neuraminidase) glycoprotein or Measles virus HA (hemagglutinin) glycoprotein
The segmental relative position of insertion.RHPIV3 construct shown in figure below contains not, and the 3918-nt of coded protein inserts fragment (GU)
(people such as Skiadopoulos, virusology 272:225-34,2000, be incorporated herein by reference).Every kind of external source is inserted fragment and all is subjected to one group of HPIV3 gene initial sum gene to stop transcribing signal control, and is expressed as independent mRNA.A. divide with m.o.i.0.01 with specified every kind of virus in triplicate and open the LLC-MK2 individual layer that infects in 6 orifice plates (Costar).In the 5th, 6,7 day results supernatant liquor, quantitative virus people such as (, virusology 272:225-34,2000) Skiadopoulos as previously mentioned.The average peak titre of every kind of virus acquisition is shown as log
10TCID
50/ ml.B. the mean value of twice experiment.The virus of serial dilution 32 ℃ and 39 ℃ of incubations 7 days on LLC-MK2 monolayer culture thing utilize guinea-pig red blood cell to determine the existence of virus by hemocyte absorption.The average reduction of titre in the times of compared when having shown 39 ℃ with 32 ℃.
Figure 12 provides an explanation to rHPIV3 main chain rHPIV3-N
BThe extra gene of middle insertion inserts segmental figure (not to scale (NTS)), wherein HPIV3 N ORF has been replaced into its BPIV3 homologue, because host range restriction provides attenuation phenotype (people such as Bailly, Journal of Virology 74:3188-3195,2000a is incorporated herein by reference).Synoptic diagram shows rHPIV3 (last figure) and biology deutero-BPIV3 (figure below).Shown the N ORF sequence that derives from BPIV3 Kansas strain in the PIV3 main chain
With the Measles virus hemagglutinin gene
Relative position.In each case, exogenous array is subjected to one group of HPIV3 to transcribe signal control.A part that has shown the plasmid vector that contains the NgoMIV site
, for the recombinant virus (right side) of anti-genome cDNA clone (left side) and coding thereof provides name.
Figure 13 illustrates that RSV G or F are as the insertion of extra gene in the rB/HPIV3 genome in the promotor proximal position.RB/HPIV3 is a kind of recombinant forms of BPIV3, and wherein BPIV3 F and HN gene have been replaced by its HPIV3 homologue and (have been respectively F
HAnd HN
H).The direct downstream of the ATG initiator codon of N ORF produces the BlpI site in the B/HPIV3 main chain.Insert RSV G or F open reading frame (ORF) to this BlpI site.Arbitrary RSV inserts segmental downstream end and is designed to contain the termination of PIV3 gene (GE) and gene initial (GS) sequence of being separated by intergenic sequence CTT (be respectively AAGTAAGAAAAA (SEQ IDNO.8) and AGGATTAAAG, be justice).Every kind is inserted fragment and also contains the NheI site that can insert the site as other extra genes.AGGATTAAAGAACTTTACCGAAAGGTAAGGGGAAAGAAATCCTAAGAGCTTAGCGATG(SEQ?ID?NO.9)。GCTTAGCGATG(SEQ?IDNO.10)。AAGCTAGCGCTTAGC(SEQ?ID?NO.11)。GCTTAGCAAAAAGCTAGCACAATG(SEQ?ID?NO.12)。
Figure 14 illustrates rB/HPIV3-G1, rB/HPIV3-F1 and reorganization and biology parental virus thereof the many recursive copyings in ape LLC-MK2 cell.Use rB/HPIV3-G1, rB/HPIV3-F1, or following contrast virus, with each cell 0.01 TCID
50Input MOI infect three parts of monolayer culture things: rBPIV3 Ka, it is the recombinant forms of BPIV3 strain Ka; RB/HPIV3, it is the rBPIV3 form that BPIV3 F and HN glycoprotein gene are replaced into the HPIV3 counterpart; HPIV3 JS, it is biology deutero-HPIV3 strain JS; With BPIV3 Ka, it is the biologically-derived form of BPIV3 strain Ka.Virus titer is shown as the average log of three duplicate samples
10TCID
50/ ml.The detection lower limit of this test is 10
1.45TCID
50/ ml.
Figure 15 is the genomic figure (not to scale (NTS)) of rBPIV3 (#1) and a series of chimeric rB/PIV3 (#2-6), it contains BPIV3 F and the HN gene is inserted fragment (#4-6) by the F of the displacement of the F of HPIV3 (#2) or HPIV1 (#3-6) and HN gene and one or two HPIV2 that encodes and/or the extra gene of HN ORF.The relative position of the extra gene of the F of the synoptic diagram of rB/HPIV3.1 embedded virus (not to scale (NTS)) code displaying HPIV2 and HN glycoprotein (being respectively F2 and HN2).Every kind of external source is inserted fragment and is subjected to that one group of HPIV3 gene is initial to be stopped transcribing signal control with gene, and is expressed as mRNA separately.
Figure 16 insertion of Measles virus HA encoding sequence in several different rPIV3 main chains that furnish an explanation.Three kinds of main chain: wild-type rHPIV3 (uppermost construct) have been described; Wild-type rHPIV3-1 (from last second kind of construct) (people such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference), wherein HPIV3 F and HN glycoprotein gene are replaced by F and the HN glycoprotein gene of HPIV1; With rHPIV3-1 cp45L (the third construct), it is a kind of derivative of wild-type rHPIV3-1, contains the point mutation of three kinds of attenuation amino acid (people such as Skiadopoulos, Journal of Virology 72:1762-8 in deriving from the L gene of cp45 vaccine strain, 1998, be incorporated herein by reference).HPIV1 F and HNORF sequence in the rPIV3 main chain () have been shown
With Measles virus HA gene
Relative position.In each case, every kind of external source ORF is subjected to one group of HPIV3 to transcribe signal control.Indicate and (*) relative position of three kinds of cp45L amino acid point mutation in the L gene.A part that has shown the plasmid vector that contains unique NgoMIV site
Figure 17 illustrates the structure of coding total length PIV2 HN and the chimeric anti-genome cDNA pFLC.PIV32hc of the proteic PIV3-PIV2 of F.Right with RT-PCR and PIV2 F special primer, contain the cDNA fragment (A1) that total length PIV2 F ORF, flank are the restriction site pointed out by PIV2/V94 vRNA amplification.This fragment adds BamHI digestion (C1) with NcoI, and is connected with the NcoI-BamHI window of pLit.PIV31.fhc (B1), produces pLit.PIV32Fhc (D1).Simultaneously, to (3 in the table 22,4), contain the cDNA fragment that total length PIV2 HJN ORF, flank are the restriction site (A2) pointed out with RT-PCR and PIV2 HN special primer by PIV2/V94 vRNA amplification.This fragment adds HindIII digestion (C2) with NcoI, and is connected with the NcoI-HindIII window of pLit.PIV31.HNhc (B2), produces pLit.PIV32HNhc (D2).PLit.PIV32Fhc and pLit.PIV32HNhc digest with PpuM1 and SpeI, and the generation pLit.PIV32hc (E) that is assembled together.PLit.PIV32hc is further with BspEI and SpeI digestion, and the BspEI-SpeI window of importing p38 ' Δ PIV31hc (F), produces p38 ' Δ PIV32hc (G).BspEI-SpeI window with chimeric PIV3-PIV2 construct importing pFLC.2G+hc produces pFLC.PIC32hc (H).
Figure 18 has described the structure of chimeric anti-genome cDNA pFLC.PIV32TM of total length PIV3-PIV2 and pFLC.PIV32TMcp45, and its coding contains F and the HN albumen that PIV2 deutero-extracellular domain and PIV3 deutero-are striden film and cytoplasmic region.With PIV3 F special primer to (9 in the table 22,10) in PCR deletion pLit.PIV3.F3a (A1), the encode PIV3 F ORF zone of extracellular domain.With PCR and PIV2 F special primer to (5 in the table 22,6) PIV2 F ORF zone by pLit.PIV32Fhc (B1) amplification coding extracellular domain.The two kinds of fragments (C1 and D1) that produce are connected, and produce pLit.PIV32FTM (E1).Simultaneously, with PIV3 HN special primer to (11 in the table 22,12) in PCR deletion pLit.PIV3.HN4 (A2), the encode PIV3HN ORF zone of extracellular domain.By PCR and PIV2 HN special primer to (8 in the table 22,9) PIV2 HN ORF zone by pLit.PIV32HNhc (B2) amplification coding extracellular domain.These two kinds of dna fragmentations (C2 and D2) link together and produce pLit.PIV32HNTM (E2).PLit.PIV32FTM and pLit.PIV32HNTM digest with PpuMI and SpeI, and assembling produces pLit.PIV32TM (F).To be connected with the BspEI-SpeI window of p38 ' _ PIV31hc (G) from the BspEI-SpeI fragment of pLit.PIV32TM, produce p38 ' _ PIV32TM (H).The insertion fragment that will contain chimeric PIV3-PIV2 F and HN imports in the BspEI-SphI window of pFLC.2G+.hc and pFLCcp45 as 6.5kb BspEI-SphI fragment, produces pFLC.PIV32TM and pFLC.PIV32TMcp45 (I) respectively.
Figure 19 shows the structure of chimeric anti-genome cDNA pFLC.PIV32CT of total length PIV3-PIV2 and pFLC.PIV32Ctcp45, and its coding contains the F and the HN albumen of PIV2 deutero-extracellular domain, PIV2 deutero-membrane-spanning domain and PIV3 deutero-tenuigenin structural domain.With PIV3 F special primer to (17 in the table 22,18) in PCR deletion pLit.PIV3.F3a (A1), the encode PIV3 F ORF zone of this extracellular domain and membrane-spanning domain.The PIV2 F ORF zone that (13 in the table 22,14) is added membrane-spanning domain by this extracellular domain of pLit.PIV32Fhc (B1) amplification coding with PCR and PIV2 F special primer.The two kinds of fragments (C1 and D1) that produce are connected, and produce pLit.PIV32FCT (E1).Simultaneously, with PIV3 HN special primer to (19 in the table 22,20) in PCR deletion pLit.PIV3.HN4 (A2), the encode PIV3HN ORF zone of this extracellular domain and membrane-spanning domain.The PIV2 HN ORF zone that (15 in the table 22,16) is added membrane-spanning domain by pLit.PIV32HNhc (B2) pcr amplification coding extracellular domain with PIV2 HN special primer.These two kinds of dna fragmentations (C2 and D2) are connected generation pLit.PIV32HNCT (E2).PLit.PIV32FCT and pLit.PIV32HNCT digest with PpuMI and SpeI, and assembling produces pLit.PIV32CT (F).To be connected with the BspEI-SpeI window of p38 ' _ PIV31hc (G) from the BspEI-SpeI fragment of pLit.PIV32CT, produce p38 ' _ PIV32CT (H).The insertion fragment that will contain chimeric PIV3-PIV2 F and HN imports in the BspEI-SphI window of pFLC.2G+.hc and pFLCcp45 as 6.5kb BspEI-SphI fragment, produces pFLC.PIV32CT and pFLC.PIV32CTcp45 (I) respectively.
The detailed genetic construction of PIV3-PIV2 embedded virus and the gene catenation sequence of rPIV3-2CT and rPIV3-2TM of having shown of Figure 20.Panel A illustrates the genetic construction (middle three figure) of rPIV3-2 embedded virus, and compares with rPIV3-1 (base map) virus with rPIV3 (top figure).Cp45 derivative arrow mark shows the relative position that cp45 suddenlys change.For the cp45 derivative, have only F different, and other genes are still identical, all derive from PIV3 with the HN gene.From top to bottom, three kinds of chimeric PIV3-PIV2 virus contains the PIV3 glycoprotein gene of decrement.Notice that the rPIV3-2 that contains complete PIV2 HN and F ORF is irrecoverable.The nucleotide sequence that component B provides the chimeric F of rPIV3-2TM to be connected with the HN glycoprotein gene provides protein translation together.The dash area representative derives from the sequence of PIV2.Amino acid is according to its Position Number in corresponding wild-type glycoprotein.Other three kinds of Nucleotide are inserted among the PIV3-PIV2 HN TM, be used for making construct to meet 6 principle.Component C shows the nucleotide sequence that the chimeric F of rPIV3-2CT is connected with the HN glycoprotein gene, and protein translation is provided together.The dash area representative derives from the sequence of PIV2.Amino acid is according to its Position Number in corresponding wild-type glycoprotein.The GE=gene stops; Between the I=gene; The GS=gene is initial; The ORF=open reading frame; The TM=membrane-spanning domain; The CT=cytoplasm domain; *=terminator codon.
Figure 21 explanation is compared many recursive copyings of rPIV3-2 embedded virus with rPIV3/JS with PIV2/V94 wild-type parent virus.Panel A---rPIV3-2TM and rPIV3-2TMcp45 virus with rPIV3/JS and rPIV2/V94 wt parental virus, infect LLC-MK2 cell with MOI 0.01 with them in 6 orifice plates, all triplicate.All cultures are all at 32 ℃ of following incubations.After 1 hour adsorption cycle, remove inoculum, with serum-free OpitMEM washed cell three times.The every hole of culture covers the 2ml same medium.Culture plate for rPIV3-2TM and rPIV3-2TMcp45 infection adds 0.5mg/ml p-trypsinase in every hole.From every hole, take out the 0.5ml equal portions with 24 hours intervals, continue 6 days, on dry ice, freeze suddenly, be stored in-80 ℃.Each equal portions is replaced by and contains or do not contain the tryptic 0.5ml fresh culture of aforesaid p-.Under 32 ℃, the virus that exists in the titration equal portions on the LLC-MK2 plate that liquid covers totally 7 days, is determined terminal point by hemocyte absorption.Component B---rPIV3-2CT and rPIV3-2CTcp45 with rPIV3/JS and rPIV2/V94 wt parental virus, infect the LLC-MK2 cell with them in 6 orifice plates, all triplicate, as described in panel A.Take out equal portions, and to handle with the described identical method of panel A.For twice experiment that shows among panel A and the B, virus titer is expressed as log
10TCID
50/ ml ± standard error.
Specific embodiments is described
The invention provides and produce and the method and composition of using novel chimeric parainfluenza virus (PIV) and relevant vaccine.Embedded virus of the present invention has infectivity and immunogenicity in human and other Mammalss, and can be used for producing at one or more PIV, for example at the immunne response of one or more people PIV (HPIV).In addition, also provide chimeric PIV, it can cause at the PIV that selects and one or more other pathogenic agent, for example at the immunne response of HPIV and Measles virus.The immunne response that causes can comprise body fluid and/or cell-mediated one of replying or both.Preferably, make chimeric PIV attenuation of the present invention, use for vaccine and produce the attenuation and immunogenic the trading off of wishing.
The invention provides novel method, be used to design and produce and can be used as vaccine and prevent and/or treat the infection that is attributable to PIV and other pathogenic agent and the attenuated chimeric PIV of associated disease symptom.The method according to this invention, PIV " carrier " genome or the anti-genome that have mixed one or more antigenic determinants of allos pathogenic agent with recombinant modified make up chimeric parainfluenza virus or subviral particle.This vector gene group or anti-genome are made up of partial or complete PIV genome or anti-genome, and itself can contain nucleotide modification such as attenuation suddenlys change.Modify vector gene group or anti-genome, form embedded structure by mixing heterologous gene or genome section.More specifically, make up chimeric PIV of the present invention by viral recovery system based on cDNA, this produces recombinant virus, and it contains one or more partial or complete carriers of " donor " nucleotide sequence bonded or " background " PIV genome or anti-genome with coding heterologous antigen determinant.Preferably, the PIV carrier contains a kind of HPIV genome or anti-genome, although inhuman PIV such as ox PIV (BPIV) also can be used as carrier, in order to mix the antigenic determinant of people PIV and other people pathogenic agent.Herein in the typical embodiments of Miao Shuing, modify people PIV3 (HPIV3) vector gene group or anti-genome, with one or more genes or the genome section of the antigenic determinant of mixing coding one or more allos PIV (as HPIV1 and/or HPIV2) and/or non-PIV pathogenic agent (for example Measles virus).The of the present invention chimeric PIV of Gou Jianing can cause at special PIV (as HPIV1, HPIV2 and/or HPIV3) or at the immunne response of non-PIV pathogenic agent like this.In addition, also provide and to cause at multiple PIV (as HPIV1 and HPIV3) or at the composition and the method for the polyspecific immunne response of one or more HPIV and non-PIV pathogenic agent (as Measles virus).
The chimeric PIV of typical case of the present invention contains a kind of aforesaid chimeric PIV genome or anti-genome, and a kind of main nucleocapsid (N) albumen, nucleocapsid phosphorprotein (P) and big polymerase protein (L).Other PIV albumen that can comprise various combination are producing multiple infectivity subviral particle, until complete virion or contain the virion of additional proteins, antigenic determinant or other compositions.
Of the present invention preferred aspect, chimeric PIV contains partial or complete people PIV vector gene group or anti-genome, it combines with one or more heterologous genes or the genome section that derive from second kind of people PIV or non-PIV pathogenic agent such as Measles virus.PIV " carrier " genome or anti-genome generally are used as adding or mix one or more " donor " genes of allos pathogenic agent or the acceptor or the carrier of genome section.General in vector gene group or anti-genome, the adding or the polynucleotide of one or more antigenic determinants of permutation encoding allos pathogenic agent, produce a kind of chimeric PIV, thereby it has obtained to cause the ability at the immunne response of this allos pathogenic agent in the host who selects.In addition, compare with one of allos pathogenic agent or both with carrier PIV, embedded virus also can show other novel phenotypic characteristics.
Partial or complete vector gene group or anti-genome are usually as the main chain to heterologous gene that wherein mixes different pathogens or genome section.The normally different PIV of allos pathogenic agent derive from its one or more genes or the genome section combines with vector gene group or anti-genome or displacement therein.Except new immunogenicity feature is provided, in carrier PIV strain, add or displacement heterologous gene or genome section, compare with the carrier of unmodified and the corresponding phenotype of donor virus, can cause the phenotypic alternation of increase or reduction, growth alteration or other hope of attenuation.Can be selected at and insert among the chimeric PIV of the present invention or the heterologous gene that derives from other PIV of interpolation and gene or the genome section that the genome section comprises coding PIV N, P, C, D, V, M, F, HN and/or L albumen or its one or more antigenic determinants.
The heterologous gene of a kind of PIV or genome section can be used as extra genome element and join in the partial or complete genome or anti-genome of different PIV.In addition, one or more heterologous genes of a kind of PIV or genome section also can be in the position displacements corresponding to the wild type gene ordinal position of corresponding gene that lacks in PIV vector gene group or anti-genome or genome section.In other embodiments, comparing, more adding or displacement heterologous gene or genome section near promotor or away from the position of promotor with the wild type gene ordinal position of corresponding gene or genome section in vector gene group or the anti-genome.To improve or to reduce the expression of heterologous gene or genome section respectively.
Be particularly useful in by the host of immunity, producing immunne response for producing chimeric PIV importing alloimmunization originality protein, protein domain and immunogenicity epi-position.In acceptor PIV vector gene group or anti-genome, add or displacement derives from a kind of immunogenic gene of donor pathogenic agent or genome section and can produce at donor pathogenic agent PIV carrier or at the immunne response of donor pathogenic agent and carrier.
For reaching this purpose, can make up chimeric PIV, it expresses a kind of chimeric protein, immunogenicity glycoprotein for example, contain carrier special kytoplasm tail and/or membrane-spanning domain, it merges with the allos extracellular domain of different PIV or non-PIV pathogenic agent, and generation can cause the fusion rotein at the immunne response of allos pathogenic agent.For example, the heterologous gene group section that coding derives from the glycoprotein extracellular domain of people PIV1 HN or F glycoprotein can be connected with the genome section of the corresponding HPIV3 HN of coding or F glycoprotein kytoplasm and membrane-spanning domain, and formation can cause the HPIV3-1 chimeric glycoprotein albumen at the immunne response of HPIV1.
In brief, express the proteic PIV of the present invention of chimeric glycoprotein and contain a kind of main nucleocapsid (N) albumen, a kind of nucleocapsid phosphorprotein (P), a kind of big polymerase protein (L) and a kind of modification back coding chimeric glycoprotein proteic HPIV vector gene group or anti-genome.This chimeric glycoprotein albumen contains one or more heterologous antigen territories, fragment or the epi-position of the HPIV of second kind of antigenicity uniqueness.Preferably, implementation method is one or more heterologous gene groups of second kind of HPIV of one or more antigenic domains of permutation encoding, fragment or epi-position in HPIV vector gene group or anti-genome, thus this genome or the anti-genome coding chimeric glycoprotein albumen different with the parent vector virus antigenicity.
In detailed aspect more, heterologous gene group section preferably encode a kind of glycoprotein extracellular domain or its immunogenicity part or epi-position, other parts that randomly comprise allos or " donor " glycoprotein for example replace the extracellular domain of corresponding glycoprotein extracellular domain and membrane-spanning domain in vector gene group or the anti-genome and stride the film district.In this manual, preferred chimeric glycoprotein albumen can be selected from HPIV HN and/or F glycoprotein, and can modify vector gene group or the anti-genome multiple chimeric glycoprotein albumen that makes it to encode.In preferred embodiments, HPIV vector gene group or anti-genome are section H PIV3 genome or anti-genome, and second kind of different HPIV of antigenicity is HPIV1 or HPIV2.In a typical embodiments of the following stated, with the glycoprotein extracellular domain displacement HPIV3 vector gene group of HPIV2 HN and F glycoprotein or corresponding HN and the F glycoprotein extracellular domain in the anti-genome.In another typical embodiments, one of HN and F glycoprotein or both PIV2 extracellular domains and stride the corresponding PIV3 cytoplasmic tail in film district and merge formation chimeric glycoprotein albumen with one or more.Apply on December 10th, 1999 people such as Tao, and the title that is confirmed as proxy's recording mechanism 17634-000340 is incorporated herein by reference for the more detailed description about these aspects of the present invention is provided in the U.S. Patent application of " expressing the structure and the application of the proteic recombinant parainfluenza virus of a kind of chimeric glycoprotein ".
In order to make up the of the present invention chimeric PIV of the heterologous antigen determinant that contains non-PIV pathogenic agent, can add or replace the heterologous gene or the genome section of donor pathogenic agent by any operable position in vector gene group or anti-genome.In one embodiment, can in PIV vector gene group or anti-genome, add heterologous gene or the genome section that (promptly not replacing) derives from a kind of non-PIV pathogenic agent, make the clone of acquisition produce new immunogenicity.In these situations, randomly for the purpose of the embedded virus attenuation that makes generation, heterologous gene or genome section can be used as extra gene or the genome section adds with complete PIV vector gene group or anti-genome.In addition, also can in vector gene group or anti-genome, lack gene or the genome section selected and add heterologous gene or genome section simultaneously.
In a preferred embodiment of the invention, the position adds heterologous gene or genome section between the gene in partial or complete PIV vector gene group or anti-genome.In addition, also can as in 5 ' or 3 ' non-coding region, or in vector gene group or anti-genome, exist other positions of non-coding nucleotide to insert gene or genome section at genomic other non-coding regions.An aspect, with vector gene group or anti-genome in cis acting regulate the non-coding site of sequence eclipsed, for example efficiently duplicating, transcribing and/or translating in the required sequence, insert heterologous gene or genome section.Vector gene group or anti-genomic these Regional Representative are used for the target site that the regulatory function relevant with the importing of heterologous gene or genome section destroyed or changed.
When this used, term " gene " typically referred to a genome such as the genomic part of PIV of a kind of mRNA of coding, generally began at the upstream termination that contains gene initial (GS) signal, was containing the downstream end end that gene stops (GE) signal.The term gene also can exchange with term " translation open reading frame " or ORF, is particularly expressing a kind of protein such as the proteic situation of PIV C from other ORF rather than from unique mRNA.In the typical case of HPIV3, genome is that length is mononegavirale RNA (people such as Galinski, virusology 165:499-510,1988 of 15462 Nucleotide (nt); People such as Stokes, virus research 25:91-103,1992).At least 8 kinds of protein is by the HPIV3 genome encoding: the C of nucleocapsid protein N, phosphorprotein P, Unknown Function and D albumen, stromatin M, fusion glycoprotein F, hemagglutinin-neuraminidase glycoprotein h N and big polymerase protein L (people such as Collins, the third edition, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 1205-1243 page or leaf.Lippincott-RavenPublishers，Philadelphia，1996)。The viral genome of all PIV also contains outer leader of gene and tail region, and have virus replication and transcribe required all or part of promotor, and non-coding region and intergenic region.Therefore, the PIV genetic map is represented as 3 ' leader-N-P/C/D/V-M-F-HN-L-5 ' tail region.Transcribe 3 ' terminal initial, and undertaken by the continuous termination-beginning mechanism that instructs by the conservative motif of the weak point of finding at the gene boundary.The upstream termination of every kind of gene all contains a kind of guidance initial gene initial (GS) signal of mRNA separately.The downstream end of every kind of gene all contains a kind of polyadenylation and terminated gene of instructing and stops (GE) motif.People PIV3 strain JS (GenBank preserving number Z11575 has been described, be incorporated herein by reference) and Washington (Galinski M.S., at Kingsbury, D.W. (volume) " paramyxovirus " 537-568 page or leaf, Plenum press, New York, 1991, be incorporated herein by reference), and to the typical genome sequence of ox PIV3 strain 910N (GenBank preserving number D80487 is incorporated herein by reference).
In order to make up chimeric PIV of the present invention, one or more PIV genes or genome section can whole or excalation, insertion or displacements.This means that part or whole disappearance, insertion or displacement can comprise the open reading frame and/or the cis acting adjusting sequence of any one or more PIV gene or genome section." genome section " is meant the continuous nucleotide that derives from the genomic any length of PIV, and it can be part of O RF, gene or gene outskirt or its combination.When the genome section is encoded a kind of antigenic determinant, at least a immunogenicity epi-position that can in mammalian hosts, cause body fluid or cell-mediated immune responses of this genome section coding.The genome section is a kind of immunogenic fragments of codified or protein territory also.In other respects, donor gene group section codified panimmunity originality territory or epi-position comprise the sequence that is re-combined into that contains multiple, repetition or different, immunogenicity territory or epi-position.
The chimeric PIV of another kind of the present invention can contain the protective antigen determinant of HPIV1, HPIV2 and/or HPIV3.This preferably realizes by express one or more HN and/or F gene or genome section with carrier PIV, or as the alia gene or the alternative gene of allos donor pathogenic agent.In certain embodiments, can make up HPIV3-1 or HPIV3-2 embedded virus as vaccine or carrier strain, wherein HPIV1 or HPIV2 HN and/or F gene replace its PIV3 homologue (people such as Skiadopoulos, vaccine 18:503-510,1999; People such as Tao, vaccine 17:1100-1108,1999; The U.S. Patent Application Serial 09/083,793 (with corresponding International Application No. WO 98/53078) of application on May 22nd, 1998; The U.S. Patent Application Serial 09/458,813 of application on December 10th, 1999; The U.S. Patent Application Serial 09/459,062 of application on December 10th, 1999; All be incorporated herein by reference).In this manual, in the full-length cDNA by the sudden change of three kinds of attenuations in containing L, PIV3 HN and F open reading frame (ORF) are replaced with the ORF of PIV1, produced the strain of chimeric PIV1 vaccine candidate with PIV3 cDNA rescue system.The reorganization embedded virus that is produced by this cDNA is named as rPIV3-1.cp45L (people such as Skiadopoulos, Journal of Virology 72:1762-8,1998; People such as Tao, Journal of Virology 72:2955-2961,1998; People such as Tao, vaccine 17:1100-1108,1999, be incorporated herein by reference).RPIV3-1.cp45L is attenuation in hamster, and can induce the high-level resistance that PIV1 is attacked.Also produced the reorganization embedded virus of a kind of rPIV3-1.cp45 of being named as, it contains in 15 cp45 sudden change 12, promptly, do not comprise the sudden change among HN and the F, it is the upper respiratory tract and the lower respiratory tract camber attenuation (people such as Skiadopoulos of hamster, vaccine 18:503-510,1999, be incorporated herein by reference).
In a preferred embodiment of the invention, chimeric PIV contains a kind of people PIV, or various human PIV, comprises one or more major antigen determinants of HPIV1, HPIV2 or HPIV3.These preferred vaccines candidate strain can cause effective immunne response of selecting HPIV at one or more in human body.As mentioned above, causing can be by vector gene group or anti-genome encoding at the antigenic determinant of the immunne response of HPIV, or can be used as heterologous gene or gene fragment and insert in PIV vector gene group or the anti-genome or be attached thereto.The main protection antigen of people PIV is its HN and F glycoprotein.Yet all PIV genes all are the candidates of coding purpose antigenic determinant, comprise the interior protein gene of the determinant of codified such as CTL epi-position.
Preferred chimeric PIV vaccine virus of the present invention contain derive from multiple HPIV any or derive from a kind of HPIV and one or more major antigen determinants of non-PIV pathogenic agent.The chimeric PIV of Gou Jianing comprises partial or complete HPIV genome or anti-genome like this, for example one or more heterologous genes or the genome section of HPIV3's and coding allos PIV such as HPIV1 or HPIV2's antigenic determinant.In alternative embodiment, can in partial or complete HPIV3 genome or anti-genome, add or one or more genes or the genome section of one or more antigenic determinants of permutation encoding HPIV1 or HPIV2.In a plurality of typical embodiments of the following stated, with corresponding HPIV3 HN and the F gene in the chimeric PIV vaccine candidate of the HPIV1 gene substitution strain of coding HN and F glycoprotein.These and other construct produces and can cause the chimeric PIV that replys at the special or many specific immunes of the list of one or more HPIV in human body.Apply on December 10th, 1999 people such as Tao; and the title that is confirmed as proxy's recording mechanism 17634-000340 is in the U.S. Patent application of " expressing the structure and the application of the proteic recombinant parainfluenza virus of a kind of chimeric glycoprotein "; with people such as Murphy on December 10th, 1999 application; and be confirmed as in the U.S. Patent application of title of proxy's recording mechanism 17634-000330 for " recombinant parainfluenza virus is as the application of carrier protection antagonism PIV and respiratory syncytial virus (RSV) associated diseases "; more detailed aspect of the present invention is provided, has been incorporated herein by reference.
Of the present invention typical aspect, the heterologous gene of the antigenic determinant of coding HPIV1 and HPIV2 or genome section added or mix in partial or complete HPIV3 vector gene group or the anti-genome.For example, can in partial or complete HPIV2 vector gene group or anti-genome, add or mix one or more HPIV1 genes of coding HN and/or F glycoprotein or its antigenic determinant or one or more HPIV2 genes or the genome section of genome section and coding HN and/or F glycoprotein or antigenic determinant.In an embodiment of the following stated, with the corresponding HPIV3 HN of HPIV1 gene substitution and the F gene of coding HN and F glycoprotein, to form chimeric HPIV3-1 vector gene group or anti-genome.This vector construction physical efficiency is further modified by one or more genes or the gene fragment that add or mix coding HPIV2 antigenic determinant, therefore, special construct of the present invention is provided, its generation contains the chimeric PIV of the antigenic determinant of HPIV1 and HPIV2, as described below shown in vaccine candidate strain rPIV3-1.2HN and the rPIV3-1cp45.2HN.
Of the present invention other preferred aspect, chimeric PIV contains a kind of HPIV vector gene group or anti-genome that one or more major antigen determinants of non-PIV pathogenic agent such as Measles virus are expressed in the back of modifying.Method of the present invention is suitable for mixing the antigenic determinant from multiple other pathogenic agent usually in the strain of chimeric PIV vaccine candidate.In this, the present invention also is used for developing the vaccine candidate strain at subgroup A and subgroup B respiratory syncytial virus (RSV), mumps virus, human papillomavirus, 1 type and pathogenic agent such as 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.In this, can comprise virus and bacterial pathogens according to the pathogenic agent of method development vaccine of the present invention, and protozoon and many cells pathogenic agent.Known from the useful antigenic determinant of multiple important human pathogen in this manual, perhaps easily be identified in chimeric PIV of the present invention, mixing.Therefore, for above-mentioned typical pathogenic agent, Measles virus HA and F albumen; The F of subgroup A and subgroup B respiratory syncytial virus, G, SH and M2 albumen, mumps virus HN and F albumen, human mammilla tumor virus L 1 albumen, 1 type and 2 type human immunodeficiency virus gp160 albumen, the gB of hsv and cytomegalovirus, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM albumen, rabies virus G albumen, Epstein-Barr virus gp350 albumen; Filovirus G albumen, bunyavirus G albumen, flavivirus E and NS1 albumen and Alphavirus E albumen have been determined major antigen.For cited pathogenic agent etc., these major antigens, and other antigens known in the art characterize well, so that its many antigenic determinants comprise full length protein and form antigenic domain, fragment and the epi-position immunogen activity separately of having identified, mapped and characterized.
The epitope mapping research that a large amount of typical case's mapping researchs of identifying and characterizing the major antigen of the different pathogens of using in the present invention are hemagglutinin-neuraminidase (HN) genes to HPIV3 (people such as vanWyke Coelingh, Journal of Virology 61:1473-1477,1987, be incorporated herein by reference).This report provides the detailed antigenic structure analysis to 16 kinds of antigenic variant of HPIV3 variant, and these variants are selected with anti-neuraminidase, blood clotting or the two kinds of proteic monoclonal antibodies of active HN (MAb) of showing.Every kind of variant all has simple point mutation in the HN gene, the monamino acid displacement in the coding HN albumen.The proteic operation of HN is relevant with the metathetical relative position with topological diagram.Proteic computer-aided analysis has been predicted a kind of mainly by the secondary structure of forming by the interconnected hydrophobic β lamella of hydrophilic coiled structure at random to HN.The HN epi-position is arranged in the curling district of prediction.Can be arranged in as if in the conservative zone that also can represent the sialic acid binding site of HN molecule of several paramyxovirus HN albumen structures by the active epi-position of demonstration virus neuraminidase that MAb discerns.
Use conventional antigen drawing method this representative performance appraisal for the vital monamino acid of the integrity of HN epi-position.The C that great majority in these epi-positions are positioned at this molecule holds half one, as for the protein that is anchored on N end desired people such as (, Journal of Virology 57:481-489,1986) Elango.Operation and the topological diagram of the PIV3 HN that delivered in the past show, used MAb can discern 6 kinds of not on the same group epi-positions (I-VI), and they form two sites (A and B) that topology is separated, by the 3rd site (C) part bridge joint.The useful candidate of the antigenic determinant among the chimeric PIV of the present invention can separately or be mixed in these group epi-position representatives with various combination.(referring to, people such as Coelingh, virusology 143:569-582,1985; People such as Coelingh, virusology 162:137-143,1988; People such as Ray, virusology 148:232-236,1986; People such as Rydbeck, general virology 67:1531-1542,1986, be incorporated herein by reference).
Other researchs (Journal of Virology 63:375-382,1989) of people such as van Coelingh provide the further information of relevant selection for the PIV antigenic determinant of the present invention's use.In this research, check antigenic structure, biological property and the natural variation of fusion (F) glycoprotein of HPIV3 with 26 kinds of monoclonal antibodies (MAb) (14 kinds is neutralizing antibody, and 12 kinds is nonneutralizing antibody).To the antigenic variant of laboratory selection and the analysis revealed of PIV3 clinical analysis strain, MAb can discern at least 20 kinds of epi-positions, and wherein 14 kinds can be neutralized precipitation.Competition confirms that in conjunction with measuring 14 kinds of neutralizing epitopes are formed three non-overlapped major antigen districts (A, B and C) and a bridge joint site (AB), and 6 kinds of non-neutralizing epitopes form 4 sites (D, E, F and G).In the great majority and MAb participate in the non-association reaction of competition each other, show that they induce the conformational change of other neutralizing epitopes.
Other antigenic determinants of using have been identified and have characterized for the present invention for respiratory syncytial virus (RSV).For example, people such as Beeler, Journal of Virology 63:2941-2950,1989, be incorporated herein by reference, use the detailed topological sum operation collection of illustrative plates that makes up the epi-position that participates in the RSV neutralization and merge for the special 18 kinds of neutralizing monoclonal antibodies (MAb) of the fusion glycoprotein of RSV A2 strain.Competition identifies three non-overlapped antigenic regions (A, B and C) and a bridge joint site (AB) in conjunction with test.Select 13 kinds of MAb resistant mutants (MARM), identify minimum 16 epi-positions with pattern in MAb and MARM or the RSV clinical strain.The MARM with 6 site A and AB epi-position that selects with antibody shows little plaque phenotype, and this change with F molecular biological activity district is consistent.Analysis to MARM also shows, these neutralizing epitopes occupy the different but complementary zone of conformation of the topology with unique biology and immunological properties.Then with the cross neutralization test of 18 kinds of anti-F MAb in the antigen that detects in the F epi-position with 23 kinds of clinical analysis strains (18 kinds of subgroup A and 5 kinds of subgroup Bs) change.This Analysis and Identification constant, the variable and hypervariable region on the molecule, it is the result of non-cumulative bad genetic heterogeneity that the antigen that shows RSV F glycoprotein neutralizing epitope changes.In 16 kinds of epi-positions, 8 kinds are present on whole or the whole 23 kinds of subgroup A or subgroup B clinical strain except that a kind.These antigenic determinants comprise full length protein and form antigenic domain, fragment and epi-position that all representative is used for integrating to cause the useful candidate of aforesaid novel immunne response in chimeric PIV of the present invention.(referring to, people such as Anderson, transmissible disease magazine 151:626-633,1985; People such as Coelingh, Journal of Virology 63:375-382,1989; People such as Fenner, Scandinavia Journal of Immunology (Scand.J.Immunol.) 24:335-340,1986; People such as Fernie test biomedical association journal (Proc.Soc.Exp.Biol.Med.) 171:266-271,1982; People such as Sato, general virology magazine 66:1397-1409,1985; People such as Walsh, general virology magazine 67:505-513,1986; With people such as Olmsted, Journal of Virology 63:411-420,1989, be incorporated herein by reference).
For the antigenic determinant of expressing heterologous PIV and non-PIV pathogenic agent, the invention provides various human and inhuman PIV carrier, comprise ox PIV (BPIV) carrier.These carriers are easy to modify according to recombination method described herein, to have heterologous antigen determinant and initiation one or more special body fluid or cell-mediated immune responses at allos pathogenic agent and carrier PIV.In typical embodiments, combine with HPIV3 vector gene group or anti-genome from one or more heterologous genes or the genome section of donor pathogenic agent.In other typical embodiments, heterologous gene or genome section mix in chimeric HPIV vector gene group or the anti-genome, and for example one or both HPIV1 genes with coding HN and F glycoprotein replace in the chimeric HPIV3-1 vector gene group or anti-genome of corresponding HPIV3 HN and/or F gene.In more detailed embodiment, the transcriptional units that will contain the open reading frame (ORF) of Measles virus HA gene is added to HPIV3 vector gene group or anti-genomic different positions, produces typical chimeric PIV/ Measles Vaccine candidate strain rPIV3 (HA HN-L), rPIV3 (HA N-P), rcp45L (HA N-P), rPIV3 (HA P-M) or rcp45L (HA P-M).In addition, the chimeric PIV that uses for vaccine can mix HPIV2 in chimeric HPIV3-1 vector gene group or anti-genome one or more antigenic determinants, for example HPIV2 HN gene.
In other detailed embodiment of the present invention, make up the chimeric PIV of the heterologous nucleotide sequence of mixing coding respiratory syncytial virus (RSV) protective antigen, produce infectivity attenuated vaccine candidate strain.The clone of RSV cDNA and other disclosures provide in following document: the U.S. Provisional Patent Application of application on September 27 nineteen ninety-five number 60/007,083; The Application No. 08/720,132 of application on September 27th, 1996; The U.S. Provisional Patent Application of on July 15th, 1996 application number 60/021,773; The U.S. Provisional Patent Application of on June 9th, 1997 application number 60/046,141; The U.S. Provisional Patent Application of on June 23rd, 1997 application number 60/047,634; The Application No. 08/892,403 (being equivalent to international publication number WO98/02530) of application on July 15th, 1997; The Application No. 09/291,894 of application on April 13rd, 1999; The international application no PCT/US00/09696 of application on April 12nd, 2000 is equivalent to the U.S. Provisional Patent Application series number 60/129,006 of applying on April 13rd, 1999; People such as Collins, institute of NAS reports 92:11563-11567, and 1995; People such as Bukreyev, Journal of Virology 70:6634-41,1996; People such as Juhasz, Journal of Virology 71:5814-5819,1997; People such as Durbin, virusology 235:323-332,1997; People such as He, virusology 237:249-260,1997; People such as Baron, Journal of Virology 71:1265-1271,1997; People such as Whitehead, virusology 247:232-9,1998a; People such as Whitehead, Journal of Virology 72:4467-4471,1998b; People such as Jin, virusology 251:206-214,1998; With people such as Whitehead, Journal of Virology 73:3438-3442,1999; With people such as Bukreyev, institute of NAS reports 96:2367-72, and 1999, all be incorporated herein by reference.At these other reports of quoting or listing with discuss to determine and characterized the RSV antigenic determinant that to use in the present invention.
The PIV mosaic that mixes one or more RSV antigenic determinants preferably contains a kind of people PIV (for example HPIV1, HPIV2, HPIV3) vector gene group or anti-genome, wherein contains the heterologous gene or the genome section of coding for antigens rsv glycoprotein, protein structure domain (for example glycoprotein extracellular domain) or one or more immunogenicity epi-positions.In one embodiment, combine with vector gene group or anti-genome, form the strain of chimeric PIV vaccine candidate from one or more genes or the genome section of RSVF and/or G gene.In these constructs some will be expressed chimeric protein, and for example the fusion rotein of the extracellular domain of the kytoplasm tail of PIV and/or membrane-spanning domain and RSV fusion produces a kind of novel attenuated virus that causes at the multivalent immune responses of PIV and RSV.
As mentioned above, common hope is regulated the phenotype for the chimeric PIV of vaccine use by introducing other sudden changes that can improve or reduce attenuation or change the embedded virus phenotype in addition.Be used for producing reorganization PIV, and carry out providing as being described in detail in the following document of materials and methods of described different sudden changes of additional aspect of the present invention and nucleotide modification herein, for example: people such as Durbin, virusology 235:323-332,1997 with detection by cDNA; The U.S. Patent Application Serial 09/083,793 of application on May 22nd, 1998; The U.S. Patent Application Serial 09/458,813 of application on December 10th, 1999; The U.S. Patent Application Serial 09/459,062 of application on December 10th, 1999; U.S. Provisional Patent Application number 60/047,575 (being equivalent to international publication number WO 98/53078) of application on May 23rd, 1997; With the U.S. Provisional Application of on September 19th, 1997 application number 60/059,385; All be incorporated herein by reference.Particularly, these file descriptions mutagenesis, separation and sign PIV to obtain method and the step that attenuation mutant ((hr) mutant strain of for example temperature sensitive (ts), cold (cp) that goes down to posterity, (ca) of acclimatization to cold, little plaque (sp) and host range restriction) and evaluation cause the hereditary change of attenuation phenotype.With these methods, above-mentioned file has described in detail to be measured attenuation people PIV that biology deutero-and reorganization produce and comprises the method for duplicating in mouse and the non-human primate model system, immunogenicity, genetic stability and protection usefulness in the model system of generally acknowledging.In addition, these file descriptions development and detect immunogenic composition and comprise unit price and bivalent vaccine, the usual way that prevention and treatment PIV infect.In file cited above, also described by making up and express and the coding PIV genome of essential PIV albumen coexpression or the method for anti-genomic cDNA production infectivity reorganization PIV, comprised the description about the following typical plasmid that can be used to production infectivity PIV clone: p3/7 (131) (ATCC 97990); P3/7 (131) 2G (ATCC 97889); And p218 (131) (ATCC 97991); All by the clause of budapest treaty by US mode culture collection center (ATCC), 10801 Boulevard universities, Manassas, Virginia 20110-2209, USA preservation, and authorize above definite preserving number.
In reference cited above, also disclose the method that makes up and estimate infectivity reorganization PIV, be incorporated in the special sudden change of identifying in (ca) of for example cold (cp) that goes down to posterity of biology deutero-PIV mutant, acclimatization to cold, (hr), little plaque (sp) and/or temperature sensitive (ts) mutant that host range limits such as the JS HPIV3 cp45 mutant strain of phenotype after this reorganization PIV is modified.The sudden change of identifying in these mutant is easy to mix among the chimeric PIV of the present invention.In typical embodiments, in polysaccharase L albumen, for example at Tyr corresponding to JS cp45
942, Leu
992Or Thr
1558The position, the sudden change of one or more attenuations takes place.Preferably, the identical or conservative amino-acid substitution by as identifying in biological mutant mixes these sudden changes among the chimeric PIV of the present invention.In detailed aspect more, the chimeric PIV that is used for vaccine mixes one or more sudden changes, wherein Tyr
942Be replaced into His, Leu
992Be replaced into Phe, and/or Thr
1558Be replaced into Ile.Replace the conservative displacement of amino acid for these and also be used in the attenuation that reaches hope in the chimeric candidate strain.HPIV3 JS cp45 strain according to the clause of budapest treaty by US mode culture collection center (ATCC), 10801 Boulevard universities, Manassas, Virginia20110-2209, the USA preservation, the patent preserving number is PTA-2419.
Other the typical case sudden changes that can adopt in chimeric PIV that derive from biologically-derived PIV mutant comprise one or more sudden changes in the N albumen, are included in the residue Val corresponding to JS cp45
96Or Ser
389The special sudden change of position.Aspect more detailed, these sudden changes are expressed as Val
96To Ala or Ser
389To the sudden change of Ala, or its conservative substitution.Also can be used for the amino-acid substitution in the C albumen of having among the chimeric PIV of the present invention, for example at Ile corresponding to JS cp45
96The sudden change of position, represent preferably Ile
96Identical or conservative substitution to Thr.Can comprise proteic one or more sudden changes of F from other typical case's sudden changes that biology deutero-PIV mutant adopts, comprise from JS cp45 adopt at residue Ile corresponding to JS cp45
420Or Ala
450The sudden change of position, represent preferably Ile
420To Val or Ala
450To the acid displacement of Thr, or its conservative substitution.In addition, chimeric PIV of the present invention also can adopt one or more amino-acid substitutions in the HN albumen, as the residue Val corresponding to JS cp45
384The sudden change of position, represent preferably Val
384Displacement to Ala.
Other embodiments of the present invention are included in the PIV genome or anti-genomic non-encoding part is as mixing the chimeric PIV of one or more sudden changes in the 3 ' leader sequence, and this causes the phenotypic alternation such as the attenuation of hope.Can change the typical case's sudden change in this specification sheets of structure corresponding to the Nucleotide 23,34,28 of JS cp45 or 45 position in 3 ' leader of embedded virus.Also can in N gene homing sequence, change other typical case sudden changes of structure, for example by changing one or more Nucleotide in the N gene homing sequence, for example in position corresponding to the Nucleotide 62 of JS cp45.In detailed aspect more, chimeric PIV has the change of T to C at Nucleotide 23 places, at Nucleotide 24 places the change of C to T is arranged, and at Nucleotide 28 places the change of G to T is arranged, and/or at Nucleotide 45 places the change of T to A is arranged.The example of other sudden changes in the extragenic territorial sequences has in the N gene homing sequence corresponding to the position A of the Nucleotide 62 of the JS change to T.
Above-mentioned these typical case's sudden changes that can change structure in chimeric PIV of the present invention successfully make up, and recovering among the PIV in reorganization---representative has the reorganization PIV clone that is named as rcp45, rcp45 L, rcp45 F, rcp45M, rcp45 HN, rcp45C, rcp45F, rcp45 3 ' N, rcp3 ' NL and rcp45 3 ' NCMFHN (people such as Durbin, virusology 235:323-332,1997; People such as Skiadopoulos, Journal of Virology 72:1762-1768,1998; People such as Skiadopoulos, Journal of Virology 73:1374-1381,1999; The U.S. Patent Application Serial 09/083,793 of application on May 22nd, 1998; The U.S. Patent Application Serial 09/458,813 of application on December 10th, 1999; The U.S. Patent Application Serial 09/459,062 of application on December 10th, 1999; U.S. Provisional Application number 60/047,575 (being equivalent to international publication number WO98/53078) of application on May 23rd, 1997; With the U.S. Provisional Application of on September 19th, 1997 application number 60/059,385, all be incorporated herein by reference).In addition, reference cited above has also been described the structure of chimeric PIV recon, for example, the HN of HPIV1 and F gene are displaced in section H PIV3 main chain genome or the anti-genome, and it is further modified and have a sudden change of one or more attenuations of identifying in HPIV3 JS cp45.All attenuation sudden changes of identifying in the L gene that a kind of so chimeric recon has mixed at cp45.Show that all the cp45 sudden changes outside allos (HPIV1) HN and the F gene can both be mixed the HPIV3-1 recon, produce a kind of chimeric candidate strain of attenuation.
Learn deutero-PIV mutant by JS cp45 and other biological, a large amount of attenuation sudden changes are provided, each all can suddenly change with any other and combine, with attenuation, immunogenicity and the genetic stability level of regulating chimeric PIV of the present invention.In this manual, many chimeric PIV can comprise one or more, and preferably two or more are from the sudden change of biology deutero-PIV mutant, for example, and any sudden change or the combination in JS cp45, identified.Preferred chimeric PIV of the present invention will be incorporated in JS cp45 or other biological is learned exist in the deutero-sudden change PIV strain multiple until whole complementary sudden changes.Preferably, the multiple nucleotide subsitution in the codon of determining each sudden change can be stablized these sudden changes, avoids replying in chimeric PIV.
Other sudden changes that can mix among the chimeric PIV of the present invention are the sudden changes that suddenly change as the attenuation of identifying in allos PIV or other non-segmented negative-strand RNA viruses.Particularly, the sudden change of the attenuation of identifying in a kind of minus-stranded rna virus and other hope can be " transferred " as the genome that is copied to chimeric PIV or the corresponding position in the anti-genome.In brief, the sudden change of the hope in a kind of allos minus-stranded rna virus is transferred to chimeric PIV acceptor (in vector gene group or anti-genome, or in allos donor gene or genome section).This comprises maps to the sudden change in the allos mutated viruses, determine corresponding site among the acceptor PIV by conventional sequence parallelism, make the original series in the PIV acceptor sport mutator gene type (by identical or conservative sudden change), as described in the international application no PCT/US00/09695 of application on April 12nd, 2000, it is equivalent to the U.S. Provisional Patent Application series number 60/129 of application on April 13rd, 1999,006, be incorporated herein by reference.As described in present disclosure, preferably chimeric PIV genome of modified receptor or anti-genome make it to encode conservatively corresponding to the change at the place, mutational site of the change of identifying in the allos mutated viruses.For example, if in the amino-acid substitution mark mutated viruses with respect to the mutational site of corresponding wild-type sequence, then can make up similarly displacement in the corresponding residue place in recombinant virus.Preferably, this displacement will make identical or conserved amino acid becomes the alternative residue that exists in the mutated viruses albumen.Yet, also can be at the place, mutational site with respect to the non-natural amino acid residue (for example, utilizing any other amino acid to destroy or weaken the function of wild-type residue) that conservatively changes of the alternative residue in the mutein.Therefrom identify typical case's sudden change and its minus-stranded rna virus that is transferred to reorganization PIV of the present invention is especially comprised other PIV (for example HPIV1, HPIV2, HPIV3, BPIV and MPIV), RSV, Sendai virus (SeV), Avian pneumo-encephalitis virus (NDV), simian virus 5 (SV5), Measles virus (MeV), rinderpest virus, canine distemper virus (CDV), rabies virus (RaV) and vesicular stomatitis virus (VSV).Disclose multiple typical case sudden change, included but not limited to the amino-acid substitution of RSV L albumen position 521 place's phenylalanines, its corresponding to and therefore transferable be the displacement (or conservative amino acid of being correlated with) of the phenylalanine at 456 places, HPIV3 L albumen position.For lacking or insert the sudden change that is caused, their energy be incorporated in the recombinant virus as lacking accordingly or inserting, yet the specific size of the protein fragments that lacks or insert is different with the aminoacid sequence possibility.
The attenuation sudden change that is used for incorporation into biology deutero-PIV among the chimeric PIV of the present invention and other non-segmented negative-strand RNA viruses can spontaneous generation, maybe can be incorporated in the wild-type PIV strain by well-known mutafacient system.For example, not exclusively attenuation parent PIV strain can produce by the following method, virus growing period chemomorphosis in the cell culture that adds chemical mutagen, screening is in order to introduce the virus that the growth limitation sudden change has been gone down to posterity under inferior optimum temperuture, or the mutagenesis virus of screening little plaque of generation (sp) in cell culture, as described in reference cited above." biology deutero-PIV " is meant any PIV that is not by the recombination method generation.Therefore, biology deutero-PIV comprises all naturally occurring PIV, for example comprise, the naturally occurring PIV that contains wild type gene group sequence, with contain and PIV with reference to different allelotrope of wild-type PIV sequence or the variation of mutator gene group, for example, the PIV that contains the sudden change that causes the attenuation phenotype.Equally, biology deutero-PIV comprises especially by induced mutations and screening method by parent PIV deutero-PIV mutant.
As mentioned above, fully the generation of the biology deutero-PIV mutant of attenuation can realize by several known methods.A kind of such method comprises that the virus that makes the part attenuation goes down to posterity in cell culture under the attenuation temperature that reduces gradually.For example, the mutant of part attenuation can produce by going down to posterity in cell culture under inferior optimum temperuture.Therefore, the PIV strain of cp mutant or other part attenuations is suitable for fully growing at a lower temperature by going down to posterity in culture.During cold going down to posterity, the screening of sudden change PIV is compared any residual toxicity that greatly reduces in the strain of deriving with the parent of part attenuation.In addition, also can carry out chemomorphosis by parental virus to the part attenuation, special sudden change is introduced among the biology deutero-PIV, for example, introduce the ts sudden change, perhaps for the virus that itself is ts, introducing is enough to give the attenuation of raising and/or other ts of attenuation derivative ts phenotypic stability suddenly change.The method of introducing the ts sudden change in PIV comprises according to known method replication-competent virus in the presence of mutagenic compound such as 5 FU 5 fluorouracil.Also can use other chemical mutagens.Attenuation may be because ts sudden change in nearly all PIV gene, is polysaccharase (L) gene though found to be used for the specially suitable target of this purpose.The temperature sensitivity level of duplicating in the typical attenuation PIV that uses for the present invention is by relatively arbitrarily determining with duplicating under several limiting temperatures under the temperature.Virus replication is called as by temperature than 100 times of reductions under random temperature or more minimum temperature.In laboratory animal and human body, PIV duplicate with virulence all with mutant by temperature relevant.
Have been found that JS cp45 HPIV3 mutant is relatively stable on genetics, have hyperimmunization originality, and attenuation satisfactorily.Nucleotide sequence analysis to this biology deutero-virus and the recombinant virus of finding that mixes Different Individual and combinatorial mutagenesis herein shows that enhanced attenuation level is all relevant with specific nucleotide and amino-acid substitution.Reference cited above also discloses how to pass through separately and with various combination to import sudden change in infecting PIV cloned genes group or anti-genome the reticent accidental sudden change that suddenlys change and cause phenotypic difference of routine difference.This method combine the sudden change that can determine to be responsible for as the feature of hope such as attenuation, temperature sensitivity, acclimatization to cold, little plaque size, host range restriction with the phenotypic characteristic of estimating parent and derived virus.
The sudden change of Que Dinging is edited as a kind of " menu " like this, then as wish that the single or combination in ground imports, with achieve one's goal with chimeric PIV of the present invention be adjusted to suitable attenuation, immunogenicity, to levels such as the regressive genetic resistances of attenuation phenotype.According to above description, produce the change of ability permission special structure of introducing in chimeric PIV of infectivity PIV by cDNA.Particularly, infectivity reorganization PIV is used for the special sudden change of characterization of biological deutero-attenuation PIV strain, for example determines the sudden change of ts, ca, att and other phenotypes.Identify the sudden change of wishing like this, and import in the chimeric PIV vaccine strain.The ability that produces virus by cDNA allows these sudden changes is single or mix in the full length cDNA clone with the various combination routine of selecting, and can easily determine to contain the phenotype of rescue recombinant virus of the sudden change of introducing afterwards.
Be tested and appraised and mix and the phenotype of wishing such as cp or the relevant special sudden change of ts phenotype in the chimeric PIV clone of infectivity, the present invention can or be right after this place in the sudden change of being identified place other site-specific modification is provided.And the most of attenuation sudden changes that produce in biology deutero-PIV are single nucleotide alterations, and other " site-specific " sudden change also can be mixed among the chimeric PIV by recombinant technology.When this uses, the Nucleotide that site-specific sudden change comprises 1-3, can reach the individual or more changes of about 5-15 (for example, change by wild-type PIV sequence, sequence by the sudden change PIV strain of selecting changes, or is changed by the parent who the stands mutagenesis PIV clone that recombinates) insertion, displacement, disappearance or rearrangement.These site-specific sudden changes can be impregnated in biologically-derived point mutation place or zone of selection.In addition, these sudden changes also can be introduced in other different contents of PIV clone, for example are positioned at or regulate near cis acting the nucleotide sequence of sequence or coded protein avtive spot, binding site, immunogenicity epi-position etc.Site-specific PIV mutant generally keeps the attenuation phenotype of hope, but can show the phenotypic characteristic of the change that has nothing to do with attenuation in addition, for example, and the immunogenicity that strengthens or widen, and/or improved growth.Other examples of the site-specific sudden change of wishing comprise the reorganization PIV that is used for mixing other stable coding mutations in the codon that causes the attenuation point mutation.As possibility, the codon place of attenuation amino acid change introduces two or more nucleotide subsitutions in causing parent's mutant or reorganization PIV clone, produces a kind of to reply the PIV that higher genetic resistance is arranged from the attenuation phenotype.In other embodiments, (N extreme direction) or downstream (C extreme direction) introduces site-specific nucleotide subsitution, interpolation, disappearance or rearrangement in the upstream, for example 1-3,5-10, can reach 15 Nucleotide or more with respect to 5 ' or 3 ' of target nucleotide site, for example, be used for making up or eliminating the cis-acting regulatory element of existence.
Except single-point and multipoint mutation and site-specific sudden change, the change of chimeric PIV disclosed herein comprises disappearance, insertion, displacement or the rearrangement of one or more genes or genome section.The useful especially disappearance that relates to one or more genes or genome section, these disappearances show the phenotype effectiveness that produces other hope.Therefore, people such as Durbin are in the U.S. Patent Application Serial 09/350 of application on July 9th, 1999,821, be incorporated herein by reference, described following method and composition,, mixed sudden change that changes the initiator codon coding assignment or the sudden change of introducing one or more terminator codons by modifying PIV genome or anti-genome, reduce or remove one or more HPIV genes, as the expression of one or more C, D and/or V ORF.In addition, also can whole or partly lack one or more C, D and/or V ORF,, or destroy protein expression simultaneously so that corresponding proteins matter does not partially or completely have function.The chimeric PIV that contains these sudden changes in C, D and/or V or other dispensable genes has the phenotypic characteristic that the extremely that is used for vaccine development is wished.For example, these modifications can cause the phenotypic alternation of one or more hope, comprise the growth properties in cell culture that (i) changes, the (ii) attenuation in the Mammals upper respiratory tract and/or lower respiratory tract, the change of (iii) viral plaque size, the (iv) change of cytopathic effect and (v) immunogenic change.The typical case that a kind of C of shortage ORF expresses " knocks out " sudden change PIV, is named as rC-KO, can induce the protective immune response of attacking at wild-type HPIV3 in non-human primate model, though its useful attenuation phenotype.
Therefore, in more detailed aspect of the present invention, chimeric PIV mixes disappearance or knocks out sudden change in C, D and/or V ORF or other dispensable genes, and this can change or eliminate the gene of selection or the expression of genome section.Its implementation can be, for example, in the encoding sequence of selecting, introduce phase shift mutation or terminator codon, change translation initiation site, change gene locus or introduce the upstream initiator codon to change its expression rate, change GS and/or GE and transcribe signal changing phenotype, or modify rna editing site (for example, growth, to the temperature limitation of transcribing etc.).In more detailed aspect of the present invention, so chimeric PIV is provided, wherein being expressed on translation or the transcriptional level of one or more genes such as C, D and/or V ORF eliminated, and there are not its gene or its segmental disappearance, implementation method for example is, in translation open reading frame (ORF), introduce a plurality of translation stop codon, change initiator codon, or modify the editor site.The virus that knocks out of these forms is usually displayed on growth velocity and the little plaque size that reduces in the tissue culture.Therefore, these methods provide other novel attenuation sudden change, and it can eliminate the expression of the virogene that is not one of main viral protective antigen.In this manual, as state and to produce the viral phenotype that knocks out that does not have gene or genome section disappearance by deletion mutagenesis, can recover the more gain mutant of target protein synthetic with effective eliminating.Can with the design of known other in this area and method to C, D and/or VORF lack and knock out mutant carry out several other gene knockout (as people such as Kretschmer, virusology 216:309-316,1996; People such as Radecke, virusology 217:418-421; People such as Kato, EMBO J.16:578-587,1987; With people such as Schneider, virusology 277:314-322,1996 is described, is incorporated herein by reference).
The nucleotide modification that can import chimeric PIV construct of the present invention can change in vector gene group or anti-genome or allos donor gene or the genome section a small amount of base (for example, 15-30 base, can reach 35-50 base or more), a large amount of Nucleotide (for example, 50-100,100-300,300-500,500-1000 base), or near complete or whole gene (1000-1500 Nucleotide for example, 1500-2500 Nucleotide, 2500-5000 Nucleotide, 5000-65000 Nucleotide or more), this character that depends on change (promptly, can change a small amount of base, to insert or to eliminate the immunogenicity epi-position, or change minigene group section, and when adding, displacement, when disappearance or rearrangement gene or big genome section, relate to a large amount of bases).
In the parties concerned, replenish the sudden change of adopting among the chimeric PIV clone that the present invention can be used for being produced by biology deutero-PIV, for example cp and ts sudden change, and the other types sudden change that relates to the identical or different gene among the PIV clone of further modification.Can be on expression level every kind of PIV gene of selectively changing, or can be separately or with the modification of other hope, whole or partly add, lack, replace or reset, produce the chimeric PIV that shows the new generation vaccine feature.Therefore, except from the sudden change of the attenuation of biology deutero-PIV mutant, or simultaneously, the present invention also provides many additive methods, is used for modified recombinant according to infectivity PIV clone, weakens or otherwise change the phenotype of chimeric PIV.Can in the separation polynucleotide sequence of coding target gene or genome section, produce multiple change, comprise being used for the chimeric PIV genome or the donor in the anti-genome or acceptor gene or the genome section that mix to infectious clone.More specifically, the structure and the phenotypic alternation of wishing for the PIV that realizes recombinating, the present invention's permission introducing in chimeric PIV clone can lack, replaces, imports or reset the modification from the Nucleotide or the nucleotide sequence of parental gene group or anti-genomic selection, and the sudden change that can lack, replace, import or reset whole gene or genome section.
Therefore the modification of chimeric PIV of the present invention is provided, it just changes or eliminates selected expression of gene, for example pass through: in the PIV encoding sequence of selecting, import a terminator codon, or change its translation initiation site or rna editing site, change the position of PIV gene with respect to the promotor of effective connection, import the upstream initiator codon to change expression rate, modify and (for example change the position, change the sequence that exists, maybe will have sequence now and be replaced into heterologous sequence) GS and/or GE transcribe signal to change phenotype (for example growth, to the temperature limitation of transcribing etc.) and cause virus replication, the different disappearances of other of the quantitative or qualitative change of selected genetic transcription or selected protein translation, displacement, add and rearrangement.In this manual, can in reorganization PIV, remove or otherwise modify non-growth essential any PIV gene or genome section, to cause effect the hope of toxicity, pathogeny, immunogenicity and other phenotypic characteristics.For encoding sequence, can delete, replace or modify non-coding region, leader, tail region and intergenic region similarly, its phenotype effect can utilize as minimum replicon and reorganization PIV and easily analyze.
In addition, can in PIV genome or anti-genome, produce multiple other hereditary changes, be used for mixing chimeric PIV separately or with one or more attenuation sudden changes from biology deutero-sudden change PIV, so that for example regulate growth, attenuation, immunogenicity, genetic stability, or provide other favourable structures and/or phenotype effect.Also disclose the sudden change of these types in the reference of quoting in front, and can easily be building up among the chimeric PIV of the present invention.For example, the conventional restriction site mark of introducing in chimeric PIV is beneficial to cDNA and makes up and operate.
Except that these change, also can change the gene order in the chimeric PIV construct, the PIV genomic promoter is replaced with anti-genome counterpart, remove or the replacing section gene, even the deletion complete genome.Different or other modifications that can carry out in the sequence are beneficial to operation, as unique restriction site of in the district between different genes or other places insertion.Can remove the untranslated gene order to improve the ability of inserting exogenous array.
Be used for comprising sudden change to the cis acting signal to other sudden changes that chimeric PIV construct of the present invention mixes, this can easily identify by the mutation analysis of PIV minimal genome.For example, but to the insertion of leader and tail region and flanking sequence and deletion analysis identifying virus promotor with transcribe signal, and provide with in various degree rna replicon or transcribe the relevant a series of sudden changes of reduction.Saturation mutagenesis (so each position is modified to the Nucleotide alternatives) to these cis acting signals has also been identified many sudden changes that can influence rna replicon or transcribe.Any of these sudden change can both be inserted in chimeric as described here PIV genome or the anti-genome.As described in reference cited above, utilize the PIV minimal genome can help to use of evaluation and the operation of complete anti-genome cDNA to trans-acting albumen and cis acting RNA sequence.
Other sudden changes in the chimeric PIV of the present invention can comprise also genome 3 ' end is replaced into anti-genomic counterpart that this is relevant with the change of transcribing with rna replicon.In a typical embodiments, native sequences is replaced into synthetic preparation and meets the sequence that effective translation designs, this can improve special PIV albumen such as protectiveness HN and/or the antigenic expression level of F.In this manual, the sub-use of display password may be a principal element (people such as Haas, modern biology (Current Biol.) 6:315-324 1996, are incorporated herein by reference) of mammalian virus protein translation level.Codon use by recombination method optimization coding PIV HN and the proteic mRNA of F can improve these expression of gene.
In another typical embodiments, separately or with introducing the upstream initiator codon, modify the sequence (preferably including the Nucleotide of 3 positions) around the translation initiation site of selected PIV gene, with the rise by determining translation or under transfer to regulate PIV genetic expression.In addition, or with disclosed other recombinant modified herein, by changing the genetic expression that GS or GE signal can be regulated chimeric PIV of transcribing of any virogene of selecting.In alternative embodiment, on transcriptional level, change the gene expression dose of chimeric PIV vaccine candidate strain.An aspect can be changed into the position of selected gene in the PIV gene mapping more near promotor or further from the position of promotor, thereby more effective respectively or efficient is more expressed this gene in the lowland.According to this aspect, can realize adjusting to the expression of specific gene, compare with the wild-type level of following the corresponding reduction of mutual alignment metathetical gene expression dose usually, genetic expression reduced or improve 2 times, general 4 times, can reach 10 times or more than.These and other swivel bases change and produce novel chimeric PIV vector virus, and they have the attenuation phenotype because the expression of for example relevant with rna replicon selected viral protein reduces, or have the antigen presentation of the character of hope as raising.
In other embodiments, the conventional chimeric PIV that modifies in the vaccine preparation of energy is to adapt to the antigenic drift of circulating virus.This is modified generally in HN and/or F albumen.By the respective regions in the receptor cloning of replacing different PIV strains or group, or by adding the gene of one or more copies, make it show many antigen form, and will be from the complete HN or the F gene of a kind of PIV strain or group, or the genome section in its specific immunogenicity district of encoding, mix in a kind of chimeric PIV genome or the anti-genome cDNA.Can in vaccination regimen, use then by the progeny virus that the PIV clone who modifies produces at emerging PIV strain.
People PIV encoding sequence or non-coding sequence (for example promotor, gene stop, gene is initial, between gene or other cis-acting elements) replace with the allos counterpart and can produce and have the multiple possible attenuation and the chimeric PIV of other phenotype effectiveness.Particularly; the effectiveness of host range and other hope results from and replace ox PIV (BPIV) or mouse PIV (MPIV) albumen of introducing in people PIV background; protein structure domain; gene or genome section; wherein ox or musculus cdna can't useful effects in people's cell; for example since the people PIV sequence of heterologous sequence or protein and biological interaction or proteinic inadaptability (promptly; common sequence or protein of cooperating with replacement sequence or protein; be used for virus transcription; translation; assembling etc.); or host range restriction generally arranged, between random and imperious host different cell protein or cellular environment some other aspect.In typical embodiments,, select ox PIV sequence in people PIV, importing according to the known aspect of ox and people PIV 26S Proteasome Structure and Function.
In detailed aspect more, the invention provides according to the mosaic that further makes up HPIV and inhuman PIV such as HPIV3 and BPIV3 makes the method for chimeric PIV vaccine candidate strain attenuation (disclosed in the U.S. Patent Application Serial 09/586,479 of application on June 1st, 2000 as people such as Schmidt; People such as Schmidt, Journal of Virology 74:8922-9,2000, be incorporated herein by reference).This method of attenuating is based on by one or more genes of introducing inhuman PIV in based on the embedded virus of people PIV carrier or the host range effectiveness that the genome section causes.For example, a large amount of Nucleotide and aminoacid sequence difference are arranged between BPIV and HPIV, this is reflected in the host range difference.Between HPIV3 and BPIV3, following each proteinic percentage amino acid identity is: N (86%), P (65%), M (93%), F (83%), HN (77%), L (91%).The illustration of host range difference is, with two kinds of BPIV3 not limited duplicate of homophyletic in same animal compare HPIV3 growth very freely in rhesus monkey (people such as van Wyke Coelingh, transmissible disease magazine 157:655-662,1988, be incorporated herein by reference).Although the basis of host range difference still has to be determinedly between HPIV3 and the BPIV3, it may comprise more than one gene and a plurality of amino acid difference.A plurality of genes and may be the participation that cis acting is regulated sequence all relate to multiple amino acids or nucleotide difference, and utmost point attenuation basis widely is provided, and this can not reply change easily.This situation with other attenuated live HPIV3 virus of passing through one or several point mutation attenuation is different.In this case, the answer of any single sudden change all can cause obviously regaining toxicity, and when having only a residue to determine attenuation, toxicity regains fully.
In typical embodiments of the present invention, vector gene group or anti-genome are a kind of HPIV3 genome or anti-genome, and heterologous gene or genome section are the Ka of a kind of BPIV3 of deriving from or the N ORF of SF strain (aminoacid sequence 99% is relevant).The anti-genomic N ORF of HPV3 background is replaced into corresponding BPIV3 N ORF---produce the chimeric PIV clone of new reorganization.The counterpart that the HPIV3 N ORF of HPIV3 replaces with BPIV3 Ka or SF produces the have an appointment protein (depending on relevant strain) of 70 amino acid differences with HPIV3 N.N is one of more conservative protein, and the displacement alone or in combination of other protein such as P can cause more amino acid difference.The participation that causes the several genes of multiple amino acids or nucleotide difference and genome section is for providing basis widely to replying high stability attenuation.
This attenuation pattern is fundamentally different than the HPIV vaccine candidate strain by one or more point mutation attenuations, and wherein the answer of each sudden change can cause obviously toxic or regain completely.In addition, several known attenuation point mutation generally produce the temperature sensitive phenotype among the HPIV.A problem of the attenuation relevant with temperature sensitivity is, virus duplicating in lower respiratory tract too limited, and in the upper respiratory tract attenuation.This is because have thermograde in respiratory tract, temperature higher (restricted higher) in lower respiratory tract, lower in the upper respiratory tract (restricted lower).The ability that attenuated virus duplicates in the upper respiratory tract can lead to complications, and comprises hyperemia, rhinitis, heating and otitis media.Therefore, the attenuation of only realizing by temperature sensitive mutation may not be an ideal.On the contrary, the host-range mutant that exists among the PIV of the present invention does not cause temperature sensitivity in most applications.Therefore, compare with other known PIV vaccine candidate strains, the novel method that the PIV attenuation that provides is provided for this class is on the genetics and more stable, more impossible relevant with the residual toxicity in the upper respiratory tract on the phenotype.
Reference cited above discloses Ka and the chimeric recon of SF HPIV3/BPIV3 is all survived, and in cell culture the same efficient duplicating with HPIV3 or BPIV3 parent---show that chimeric recon does not show the gene uncompatibility that limits replication in vitro.This is important in external character of efficiently duplicating, because it allows this biological product of High-efficient Production.The chimeric recon of Ka and SFHPIV3/BPIV3 (being called cKA and cSF) only contains a kind of cow genome, also almost is equal to its BPIV3 parent in the rhesus monkey respiratory tract on the host range limited degree.Particularly, compare with duplicating of HPIV3, cKA and cSF virus duplicating in the rhesus monkey upper respiratory tract shows the reduction of about 60 times or 30 times respectively.According to this discovery, expect that other BPIV3 genes also will cause the host range restriction of level of hope in chimeric PIV of the present invention.Therefore, according to method herein, be easy to identify a series of attenuation determinants in the heterologous gene of BPIV and other inhuman PIV and genome section, they will give the host range restriction and the immunogenicity of the chimeric PIV level of hope of selecting into vaccine use with suitable combination.
Chimeric people-Niu PIV as carrier comprises partial or complete " background " PIV genome or anti-genome in the present invention, it derives from different PIV strains or subgroup virus, or make up formation human-bovine chimeric PIV genome or anti-genome in conjunction with the back with one or more heterologous genes or the genome section of different PIV strains or subgroup virus in people or ox PIV strain or subgroup virus.Of the present invention preferred aspect, chimeric PIV has mixed and one or more heterologous genes or partial or complete people PIV background genome or the anti-genome of genome section bonded from ox PIV.Partial or complete background genome or anti-genome have wherein added corresponding heterologous gene or the genome section of people or ox PIV generally as acceptor main chain or carrier.Corresponding heterologous gene or genome section representative " donor " gene or polynucleotide from people or ox PIV, they combine with background genome or anti-genome or displacement therein, produce one of PIV with obtaining or both compare the human-bovine chimeric PIV that shows new phenotypic characteristic.For example, compare with the acceptor of unmodified and/or the corresponding phenotype of donor, in the acceptor PIV strain of selecting the interpolation of heterologous gene or genome section or displacement can cause attenuation, growth alteration, immunogenicity to change or the increase of the phenotypic alternation of other hope or reduction (people such as Schmidt is in the U.S. Patent Application Serial 09/586,479 of application on June 1st, 2000; People such as Schmidt, Journal of Virology 74:8922-9,2000, be incorporated herein by reference).
Can be chosen in and be used for gene or the genome section that allos displacement or the gene that adds and genome section comprise coding PIV N, P, C, D, V, M, F, SH when suitable (), HN and/or L albumen or its part in the human-bovine chimeric PIV carrier.In addition, the non-PIV albumen of encoding also can mix among people of the present invention-Niu PIV as proteic gene of SH and the genome section of finding in mumps and SV5 virus.Regulatory region such as gene outer 3 ' leader or 5 ' tail region and gene is initial, gene termination, intergenic region or 3 ' or 5 ' non-coding region also can be used as the allos displacement or add.
Some human-bovine chimeric PIV carrier of Shi Yonging is in the displacement by one or more BPIV3 genes or genome section or interpolation and contain one or more major antigen determinants in the background of attenuation in the present invention.The main protection antigen of PIV is its HN and F glycoprotein, although other protein also can cause protective immune response.In certain embodiments, background genome or anti-genome are a kind of HPIV genome or anti-genome, for example HPIV3, HPIV2 or HPIV1 background genome or anti-genome wherein add or have replaced one or more BPIV genes or genome section, preferably from BPIV3.In a typical embodiments described below, replace the ORF of HPIV with the BPIV3N gene ORF.In addition, background genome or anti-genome also can be one or more genes or genome section bonded BPIV genome or anti-genomes a kind of and coding HPIV3, HPIV2 or HPIV1 glycoprotein, glycoprotein territory or other antigenic determinants.
The method according to this invention, any BPIV gene or genome section can be separately or with one or more BPIV genes, combine with the HPIV sequence, produce the strain of human-bovine chimeric PIV vaccine candidate.Any HPIV, the not homophyletic that comprises specific HPIV serotype such as HPIV3 will be the reasonable acceptors of attenuation BPIV gene.Usually, for being used as the vaccine at people PIV, HPIV3 gene or the genome section selected in order to comprise in human-bovine chimeric PIV comprise one or more HPIV protective antigens such as HN or F glycoprotein.
Of the present invention typical aspect, the human-bovine chimeric PIV that contains one or more cow genomes or genome section shows the host range restriction of height, for example, in the respiratory tract of Mammals model that people PIV infects such as non-human primate.In typical embodiments,, make people PIV attenuation by in partial or complete people such as HPIV3, PIV background genome or anti-genome, adding or replacing one or more cow genomes or genome section.In one embodiment, HPIV3 N gene is replaced into BPIV3 N gene, produces new human-bovine chimeric PIV carrier, and measles HA gene is replaced in this carrier, produces a kind of multivalence HPIV/ Measles Vaccine candidate strain, and rHPIV3-N as described below recombinates
BHA
P-M
Preferably, it is suitable to be used to develop the shown host range limited degree of the shown host range limited degree of the human-bovine chimeric PIV carrier of vaccine candidate strain of the present invention and each BPIV parent or " donor " strain.Preferably, restriction should have real host range phenotype, that is, should be special for described host, should not limit in suitable clone and duplicate and external vaccine production.In addition, the human-bovine chimeric PIV carrier that contains one or more cow genomes or genome section can cause high-caliber resistance in the responsive host of PIV infection.Therefore, the present invention is for developing the new basis that the attenuated live virus vector is provided at the vaccine of PIV and other pathogenic agent, and this depends on host range effectiveness.
In the parties concerned of the present invention, human-bovine chimeric PIV carrier contains a kind of BPIV acceptor or main chain virus, and it mixes the heterologous gene of one or more coding HPIV HN and/or F glycoprotein.In addition, chimeric PIV can mix one or more coding HPIV HN and/or the extracellular domain (cytoplasm domain and/or membrane-spanning domain in addition) of F glycoprotein or genome sections of immunogenicity epi-position.These immunogenic proteins, structural domain and epi-position are particularly useful among the human-bovine chimeric PIV, because they produce new immunne response in by the host of immunity.Particularly, HN and F albumen and immunogenicity territory and epi-position provide main protective antigen.
In certain embodiments of the invention, in ox back scape or acceptor gene group or anti-genome, add or replace one or more immunogenic gene or genome sections from people PIV subgroup or strain, generation can cause reorganization, embedded virus or the subviral particle that comprises the immunne response of one or more special people PIV subgroups or strain at people's donor virus, and the ox main chain provides a kind of attenuation phenotype, makes mosaic become a kind of candidate's strain that can be used for the vaccine development.In a typical embodiments; one or more people PIV glycoprotein genes as HN and/or F, add in partial or complete cow genome group or anti-genome or displacement; produce a kind of attenuation, communicable human-bovine chimeric body, it can cause anti-people PIV immunne response in susceptible host.In a kind of such typical carriers, (in the BPIV3 background, carry HPIV3 JS HN and F glycoprotein gene), RSV A glycoprotein gene G and F successfully insert as allos ORF, produce the strain of multivalence HPIV/RSV vaccine candidate, as described below recombinant virus rB/HPIV3-G1 and rB/HPIV3-F1.
In another embodiment, human-bovine chimeric PIV carrier mixes a kind of gene or genome section in addition, its coding is from various human PIV strain, for example from the two kinds of HN of different HPIV such as HPIV1 or HPIV2 or immunogenic protein, protein domain or the epi-position of F albumen or its immunogenicity part.In addition, also can be under the polynucleotide situation that in genome or anti-genome, does not add glycoprotein outside the coding or determinant, by displacement, provide a kind of glycoprotein or immunogenic determinant by first kind of HPIV, and provide second kind of glycoprotein or immunogenic determinant by second kind of HPIV.The displacement of HPIV glycoprotein and antigenic determinant or add also can be by making up the coding proteic genome of chimeric glycoprotein or anti-genome be realized in recombinant virus or subviral particle for example contains immunogenicity epi-position, antigenic region or the complete extracellular domain of first kind of HPIV that the cytoplasm domain with allos HPIV merges.For example, coding can be connected with the genome section of the corresponding HPIV3 HN of coding or F glycoprotein kytoplasm/interior functional zone in background genome or anti-genome from the heterologous gene group section of the glycoprotein extracellular domain of HPIV1 or HPIV2 HN or F glycoprotein.
In other embodiments, a kind of replacement in human-bovine chimeric PIV vector gene group or anti-genome codified recombinant virus or the subviral particle, outer or chimeric glycoprotein albumen or its antigenic determinant produce the viral recon that contains people and Niu glycoprotein, glycoprotein territory or immunogenicity epi-position.For example, the heterologous gene group section of the glycoprotein extracellular domain of coding people PIV HN or F glycoprotein can be connected with the genome section of the corresponding ox HN of coding or F glycoprotein kytoplasm/interior functional zone in background genome or anti-genome.In addition, people PIV HN or F glycoprotein or its part also can be connected with the encode another kind of PIV strain or the HN of serotype or the genome section of F glycoprotein or its part.
With the host range phenotypic effect that human-bovine chimeric PIV of the present invention provides, wish usually to regulate the attenuation phenotype by introducing other sudden changes that can improve or reduce the embedded virus attenuation.Therefore,, produce the human-bovine chimeric PIV carrier of attenuation, wherein by in virus that obtains or subviral particle, introducing one or more attenuations of being responsible for the attenuation phenotype suddenly change further modified chimeric genome or anti-genome in other aspects of the present invention.They comprise that RNA regulates the sudden change in sequence or the encoded protein matter.From the beginning these attenuation sudden changes can produce, and detect attenuation according to rational design mutagenesis strategy.In addition, also can in prior biological deutero-sudden change PIV, identify the attenuation sudden change, be incorporated into afterwards among the human-bovine chimeric PIV of the present invention.
In preferred chimeric candidate of the present invention strain, characteristics are the generally acknowledged animal model that PIV duplicates in human body, as the attenuation that duplicates in the lower respiratory tract of hamster or rhesus monkey and/or the upper respiratory tract, growth phase ratio with corresponding wild-type or sudden change parent PIV strain, can reduce at least about 2 times, more commonly about 5 times, 10 times or 20 times, preferably 50-100 doubly, can reach 1000 times or above (for example, in 3-8 after infection days, measuring).
The chimeric PIV carrier cloning of infectivity of the present invention also can be according to method and composition transformation disclosed herein, improving immunogenicity, and the stronger protection that causes of induction ratio wild-type parent (that is, carrier or allos donor) PIV or non-PIV pathogenic infection.For example, from allos PIV strain or type, or, can change by the suitable Nucleotide in mosaic gene group or the anti-genome and add among the chimeric PIV from one or more additional immunogenicity epi-positions, protein domain or the protein of non-PIV pathogenic agent such as measles or RSV.In addition, also can transform chimeric PIV of the present invention, adding or to remove (for example) immunogenic protein, protein domain by aminoacid insertion, displacement or disappearance, form with hope or the relevant specific protein of undesirable immunological response.
In the method for the invention, can be in chimeric PIV vector gene group or anti-genome or near other genes of insertion or genome section.These genes can be controlled by acceptor gene usually, maybe can be subjected to independently one group to transcribe signal control.Except the gene or genome section of coding for antigens determinant, goal gene in this specification sheets comprises the gene of the Codocyte factor, as interleukin-(interleukin II (IL-2), interleukin-4 (IL-4), t cell growth factor (IL-5), interleukin 6 (IL-6), interleukin-18 (IL-18)), tumor necrosis factor alpha (TNF α), (IFN γ), or granulocyte-macrophage colony stimutaing factor (GM-CSF), and IL2 is to IL-18, IL-2 particularly, IL-6 and IL-12, and IL-18, gamma-interferon (is seen, for example, the U. S. application of on July 13rd, 2000 application number 09/614,285, be equivalent to the U.S. Provisional Application series number 60/143,425 of application on July 13rd, 1999, be incorporated herein by reference).These co-expression of proteins provide quantitatively and have changed and improved ability at the immunne response of chimeric PIV of the present invention qualitatively.
The disappearance, insertion, displacement and other sudden changes that relate to interior whole virogene of chimeric PIV of the present invention or genome section produce high stability vaccine candidate strain, and they are for the immunosuppressed individuals particularly important.Many these changes will cause the vaccine strain attenuation that produces, and other the phenotypic alternation that will cause dissimilar hope.For example, attached (that is, not being that growth in vitro is necessary) gene is the proteinic outstanding candidate (seeing that for example, J.16:578-87 people such as Kato, EMBO 1997, are incorporated herein by reference) of the special interference host immune of coding.Removing these genes in vaccine virus estimates to reduce toxicity and pathogenesis and/or improves immunogenicity.
The introducing of said mutation in the chimeric PIV clone of infectivity can realize by multiple well-known method.For DNA, " infectivity " clone is meant cDNA or its synthetic or other products, and they can be transcribed into and can be used as genomic genome or the antigenomic RNA that template produces infective virus or subviral particle.Therefore, can pass through routine techniques (as site-directed mutagenesis) sudden change as described in the importing in genome or anti-genomic cDNA copy.The application that anti-genome or genome cDNA subfragment are assembled into complete anti-genome described herein or genome cDNA has each zone can both separate operation (less cDNA bigger be easier to operation) can easily be assembled into the advantage of global cDNA then.Therefore, complete anti-genome or genome or its subfragment can be used as the template of the mutagenesis of oligonucleotide guidance.This can be by the intermediate of strand phasmid form, as use Bio-Rad laboratory (Richmond, CA) Muta-gene test kit, or directly use the method for double-stranded plasmid as template, as Stratagene (La Jolla, CA) chameleon mutagenesis kit, or by using Oligonucleolide primers or containing the polymerase chain reaction of the template of purpose sudden change.The subfragment of sudden change can be assembled into complete anti-genome or genome cDNA then.Known multiple other induced-mutation technique is used in and produces the purpose sudden change in anti-genome of PIV or the genome cDNA.Sudden change may be different from single nucleotide alteration, and replaces the big cDNA sheet that contains one or more genes or genome district.
Therefore, in an illustrative embodiment, utilize available from the Muta-gene phasmid vitro mutagenesis test kit of Bio-Rad and introduce sudden change.In brief, the cDNA of coding PIV genome or an anti-genome part is cloned among the plasmid pTZ18U, and be used for transforming the CJ236 cell (LifeTechnologies, Gaithersburg, MD).Recommend preparation phasmid goods by manufacturer.By introducing the Nucleotide that changes, be designed for the oligonucleotide of mutagenesis at genome or anti-genomic desired location.Amplification contains the genome of genetics change or the plasmid of anti-genome section then, and then the fragment of will suddenling change is introduced in the genome or anti-genomic clone of total length.
The present invention also provides the method that is produced the chimeric PIV of infectivity by one or more isolating polynucleotide such as one or more cDNA.According to the present invention, for essential viral protein born of the same parents in or external coexpression, make up coding PIV genome or anti-genomic cDNA, to form infectivity PIV." the anti-genome of PIV " is meant isolating just polynucleotide molecule, and it is as the synthetic genomic template of offspring PIV.Preferably, be configured to the cDNA of PIV genome justice form, it is corresponding to RNA in the middle of the rf, or anti-genome, so that with the possibility minimum of the just transcript hybridization of complementary sequence, these sequence encodings produce transcribes, duplicates nucleocapsid promptly encode N, P and the required protein of the proteic sequence of L.
For the present invention, the genome of reorganization PIV of the present invention or anti-genome only need contain the virus or the subviral particle that make coding and have the necessary gene of infectivity or its part.In addition, also available more than one polynucleotide molecule provides these genes or its part, that is, complementation that can be by the separating nucleotide molecule etc. provides a kind of gene, or can directly be expressed by genome or anti-genome cDNA.
Reorganization PIV be meant directly or indirectly produce by recombinant expression system or from the virus of such generation or PIV or the PIV sample virus or the subviral particle of subviral particle breeding.Recombinant expression system uses a kind of recombinant expression vector, this carrier contains a kind of transcriptional units of effective connection, this unit contains at least a genetic elements that regulating effect is arranged in PIV genetic expression, promotor for example, can be transcribed into structure or encoding sequence and suitable transcription initiation and the terminator sequence of PIV RNA.
For the genome or the anti-genome of being expressed by cDNA produces infectivity PIV, genome or anti-genome with (i) produce nucleocapsid that can rna replicon and (ii) make offspring's nucleocapsid can rna replicon and transcribe necessary PIV albumen coexpression.Transcribing of genome nucleocapsid produces other PIV albumen, and initial productive infection.In addition, also can provide productivity to infect other required PIV albumen by coexpression.
By in the cell or one or more isolating polynucleotide molecules of acellular coexpression coding PIV genome or antigenomic RNA, produce the polynucleotide of transcribing, duplicate the necessary virus protein of nucleocapsid with one or more codings, can produce infectivity PIV of the present invention.Can be used for coexpression and main nucleocapsid protein (N), nucleocapsid phosphorprotein (P), big (L) polymerase protein, fusion rotein (F), hemagglutinin, neuraminidase glycoprotein (HN) and matrix (M) albumen are arranged with the virus protein that produces infectivity PIV.The product of the C of PIV, D and V ORF is also useful in this manual.
For with essential viral protein cell plastid in or external coexpression, make up coding PIV genome or anti-genomic cDNA, to form infectivity PIV." the anti-genome of PIV " is meant isolating just polynucleotide molecule, and it is as the synthetic genomic template of offspring PIV.Preferably, the PIV genome that makes up with respect to RNA in the middle of the rf is the cDNA of just form, or anti-genome so that with the possibility minimum of the just transcript hybridization of complementary sequence, these sequence encodings produce transcribes, duplicates the required protein of nucleocapsid.
In certain embodiments of the invention, the reorganization genome of PIV or anti-genome only need contain the virus or the subviral particle that make coding and have the necessary gene of infectivity or its part.In addition, also available more than one polynucleotide molecule provides these genes or its part, that is, complementation that can be by the separating nucleotide molecule etc. provides a kind of gene.In other embodiments, PIV genome or anti-genome encoding viral growth, duplicate and infect necessary all functions, do not comprise helper virus or viral function that plasmid or auxiliary cell line provide.
" reorganization PIV " be meant directly or indirectly produce by recombinant expression system or from the virus of such generation or PIV or the PIV sample virus or the subviral particle of subviral particle breeding.Recombinant expression system uses a kind of recombinant expression vector, this carrier contains a kind of transcriptional units of effective connection, this unit contains at least a genetic elements that regulating effect is arranged in PIV genetic expression, promotor for example, can be transcribed into structure or encoding sequence and suitable transcription initiation and the terminator sequence of PIV RNA.
For the genome or the anti-genome of being expressed by cDNA produces infectivity PIV, genome or anti-genome with (i) produce nucleocapsid that can rna replicon and (ii) make offspring's nucleocapsid can rna replicon and transcribe necessary PIV N, P and L albumen coexpression.Transcribing of genome nucleocapsid produces other PIV albumen, and initial productive infection.In addition, also can provide productivity to infect other required PIV albumen by coexpression.
PIV genome or anti-genome and above-mentioned virus protein synthetic also can behind transfectional cell, use make up transcribe-translation reaction finishes at external (acellular).These helper viruses can be wild-type or mutant.Preferably, helper virus can be different from the coded virus of PIV cDNA on phenotype.For example, wish to produce can be with helper virus but not with the monoclonal antibody of the virological immunology reaction of PIV cDNA coding.These antibody can be neutralizing antibodies.In certain embodiments, can in affinity chromatography, separate helper virus and recombinant virus with these antibody.For helping to obtain these antibody, can in PIV cDNA, introduce sudden change, so that the antigen diversity from helper virus to be provided, as the antigen diversity in HN or the F glycoprotein gene.
In other embodiments of the present invention, the PIV albumen of N, P, L and other hope is by one or more non-virus expression carrier codings, and they can be identical, or are different from encoding gene group or anti-genomic.Can comprise other protein when wishing, by himself carrier or coded by the proteic carrier of PIV of coding one or more N, P, L and other hope or complete genome group or anti-genome.For example can realize down by the plasmid expression genome of transfection or anti-genome and protein by cDNA being placed the control of T7 rna polymerase promoter, pass through subsequently with a kind of expression system for the T7 RNA polymerase, as the vaccinia virus MVA strain recon of expressing the T7 RNA polymerase, infection, transfection or transduction provide (people such as Wyatt, virusology 210:202-205,1995, be incorporated herein by reference).Virus protein and/or T7 RNA polymerase also can produce by the mammalian cell that transforms or by preformed mRNA of transfection or protein.
For using in the present invention, can make up the anti-genome of PIV, method is the cDNA fragment of for example assembling the clone, totally shows complete anti-genome, by (the PCR such as polymerase chain reaction to the reverse transcription copy of PIV mRNA or geneome RNA; For example at U.S. Patent number 4,683,195 and 4,683,202 and " PCR method: method and application guide ", people such as Innis compile, press of institute, San Diego, 1990 is described, is incorporated herein by reference).For example, produce the first kind of construct that contains cDNA, it contains anti-genomic left hand end, strides across suitable promotor (for example t7 rna polymerase promotor), is assemblied in suitable expression vector such as plasmid, clay, phage or the dna viral vector.Can contain the synthetic polylinker modification carrier that unique restriction site is beneficial to assemble by mutagenesis and/or insertion.For ease of preparation, the PIV albumen of N, P, L and other hope can be assemblied in one or more isolated vectors.When wishing, the right hand end of anti-geneome plasmid can contain other sequences, as flank ribozyme and series connection T7 transcription terminator.Ribozyme can be the tup type (for example, people such as Grosfeld, Journal of Virology 69:5677-5686,1995), generation is contained 3 ' of a kind of non-viral nucleotide holds, maybe can be any other suitable nucleic acid, ribozyme (people such as Perrotta, natural 350:434-436,1991 as hepatitis D virus, be incorporated herein by reference), it will produce the 3 ' end that does not contain non-PIV Nucleotide.Connect left hand end and right hand end by restriction site commonly used then.
Can or carry out multiple Nucleotide during cDNA makes up afterwards in PIV genome or anti-genome inserts, lacks and reset.For example, can synthesize special nucleotide sequence of wishing, and be inserted into suitable restriction site in the appropriate area of cDNA.In addition, also can with as site-specific mutagenesis, L-Ala scan, other known these class technology are introduced sudden change in cDNA in the technology of PCR mutagenesis or this area.
The alternative approach that makes up encoding gene group or anti-genomic cDNA comprise use improved PCR condition reverse transcription-PCR (for example, as people such as Cheng, institute of NAS reports 91:5695-5699,1994 is described, is incorporated herein by reference) so that the quantity of subunit cDNA composition reduces to one or two fragment.In other embodiments, can use different promoters (for example T3, SP6) or different ribozyme (as hepatitis D virus).Can be with different dna vectors (for example clay) breeding, to adapt to bigger genome or anti-genome better.
By transfection, electroporation, machinery insertion, transduction etc., can be in proper host cell, in supporting the infectious cell of productivity PIV such as HEp-2, FRhL-DBS2, LLC-MK2, MRC-5 and Vero cell, insert encoding gene group or anti-genomic isolating polynucleotide (for example cDNA).For example, the transfection by calcium phosphate mediation (people such as Wigler, cell 14:725,1978; Corsaro and Pearson, somatic cell genetics (Somatic Cell Genetics) 7:603,1981; Graham and van der Eb, virusology 52:456,1973), electroporation (people such as Neumann, EMBO J.1:841-845,1982), the transfection of DEAE-dextran mediation (people such as Ausubel, compile, " modern molecular biology method ", John Wiley and Sons, Inc., NY, 1987), the transfection of positively charged ion lipid mediation (people such as Hawley-Nelson, focus (Focus) 15:73-79,1993) transfection reagent such as the LipofectACE (above-mentioned each reference all are incorporated herein by reference) such as (Life Technologies) that maybe can buy can introduce the transfection of isolating polynucleotide sequence in cultured cells.
As mentioned above, in certain embodiments of the invention, the PIV albumen of N, P, L and other hope is by one or more helper virus codings, and they are different from encoding gene group or anti-genomic on phenotype.The PIV albumen of N, P, L and other hope also can be by one or more expression vector codes, and they can be identical, or are different from encoding gene group or anti-genomic, and various combination.Can comprise other protein when wishing, by himself carrier, or by the proteic carrier of PIV of coding one or more N, P, L and other hope, or complete genome group or anti-genome encoding.
Provide infectivity PIV clone of the present invention to allow the multiple change of reorganization generation in PIV genome (or anti-genome), the sudden change that produces the phenotypic alternation of the caused hope of determining." infectious clone " is meant cDNA or its synthetic or other products, can directly mix the RNA in the infectious virion, and it can be transcribed into genome or the antigenomic RNA that can be used as the genomic templates that produces infective virus or subviral particle.As mentioned above, can be by multiple routine techniques (as site-directed mutagenesis) sudden change as described in the importing in genome or anti-genomic cDNA copy.The application that genome or anti-genome cDNA subfragment are assembled into complete genome group described herein or anti-genome cDNA has each zone can both separate operation (its medium and small cDNA is easier to operation than big cDNA), so can easily be assembled into the advantage of global cDNA.Therefore, the subfragment of complete anti-genome or genome or selection can be used as the template of the mutagenesis of oligonucleotide guidance.This can be by the intermediate of strand phasmid form, as use Bio-Rad laboratory (Richmond, CA) MUTA-gen test kit, or directly use the method for double-stranded plasmid as template, as Stratagene (La Jolla, CA) Chameleon mutagenesis kit, or by using Oligonucleolide primers or containing the polymerase chain reaction of the template of purpose sudden change.The subfragment of sudden change can be assembled into complete anti-genome or genome cDNA then.Known multiple other induced-mutation technique can routine be used for producing the purpose sudden change at anti-genome of PIV of the present invention or genome cDNA.
Therefore, in an illustrative embodiment, utilize available from the MUTA-gene phasmid vitro mutagenesis test kit of Bio-Rad and introduce sudden change.In brief, will encode PIV genome or anti-genomic cDNA are cloned among the plasmid pTZ18U, and are used for transforming CJ236 cell (LifeTechnologies).Recommend preparation phasmid goods by manufacturer.By introducing the Nucleotide that changes, be designed for the oligonucleotide of mutagenesis at genome or anti-genomic desired location.Amplification contains the genome of genetics change or the plasmid of anti-genome section then.
Sudden change may be different from single nucleotide alteration, introduces, lacks or replace the big cDNA fragment that contains one or more genes or genome section.The genome section can be corresponding to structure and/or functional domain, for example, proteinic kytoplasm, stride film or extracellular domain, avtive spot, combine or the chemically interactive site of other biological with different proteins as mediation, the epi-position site for example stimulates the site of antibodies and/or body fluid or cell-mediated immune responses, or the like.In this, for the genome section in little functional domain of coded protein such as epi-position site, useful genome section is about 15-35 Nucleotide, to about 50,75,100,200-500 and 500-1500 or more Nucleotide.
The ability of introducing described sudden change in infectivity PIV has many purposes, comprises that PIV causes a disease and the operation of immunogen mechanism.For example, by introducing the sudden change of eliminating or reducing protein expression level or produce mutein, can operate the function that PIV albumen comprises N, P, M, F, HN and L albumen and C, D and V ORF product.Several genes group RNA constitutional features, as promotor, intergenic region with transcribe signal, also can be with method and composition routine operation of the present invention.For example, in the replicate(determination) of using the PIV minimal genome, use complete anti-genome cDNA, can easily determine the effect (people such as Dimock of trans-acting albumen and cis acting RNA sequence, Journal of Virology 67:2772-8,1993, be incorporated herein by reference), its rescue dependent status can be used for characterizing the mutant that those may can't recover owing to inhibition in not relying on the infective virus that duplicates.
Some displacement, insertion, disappearance or the rearrangement that make gene among the reorganization of the present invention PIV or genome section are (for example, the encode displacement of genome section in a kind of protein of selection or protein zone, for example kytoplasm tail, membrane-spanning domain or extracellular domain, epi-position site or zone, binding site or zone, avtive spot or contain the zone of avtive spot, or the like) relevant on structure or function with existing " corresponding " gene or genome section from identical or different PIV or other sources.These modify the new recon that generations and wild-type or parent PIV or other virus strain are compared the phenotypic alternation with hope.For example, this class recon can be expressed a kind of chimeric protein, and this chimeric protein contains kytoplasm tail and/or the membrane-spanning domain of a kind of PIV that merges with the extracellular domain of another kind of PIV.The typical recon of other of this class is expressed multiple protein district, as multiple immunogenicity district.
When this used, " correspondence " gene, genome section, protein or protein district were generally from allos source (for example, from different PIV genes, or representing (being homology or equipotential) gene or genome section identical in different PIV types or the strain).The typical counterpart of selecting in this specification sheets has total constitutional features, and for example, a kind of corresponding protein of each counterpart codified or protein domain are as cytoplasm domain, membrane-spanning domain, extracellular domain, binding site or zone, epi-position site or zone etc.Corresponding domain and encoding gene group section thereof are included in the set of the species that have the sequence variations that a certain size and common biological activity determine in structural domain or the genome section variant.
Corresponding gene and genome section, and other polynucleotide that are used to produce reorganization PIV of the present invention disclosed herein, usually with the polynucleotide of selecting " canonical sequence ", for example, the corresponding sequence with another kind is selected has basic sequence identity.When this used, " canonical sequence " was a kind of definite sequence, as the correlated basis of sequence, for example, the fragment of full-length cDNA or gene, or global cDNA or gene order.Usually, canonical sequence length is at least 20 Nucleotide, usually at least 25 Nucleotide, at least 50 Nucleotide usually.Because two kinds of polynucleotide all can (1) contain between two kinds of polynucleotide similarly sequence (promptly, the part of complete polynucleotide sequence), (2) can further contain sequences different between two kinds of polynucleotide, general by go up the relatively sequence of two kinds of polynucleotide at " contrast window ", to identify the also regional area of comparative sequences similarity, carry out two kinds of sequences contrasts between (or multiple) polynucleotide.When this uses, " contrast window " is meant the conceptual segment of at least 20 continuous nucleotide positions, wherein a kind of polynucleotide sequence can be compared with the canonical sequence of 20 continuous nucleotides at least, and for the best parallelism of two kinds of sequences, this polynucleotide sequence is compared with canonical sequence (do not contain and add or disappearance) in the part of contrast in the window can contain 20% or still less interpolation or disappearance (being breach).The best parallelism of sequence that is used for parallelism contrast window can carry out with following method: local homology's algorithm of Smith and Waterman (applied mathematics progress (Adv.Appl.Math.) 2:482,1981), homology parallelism algorithm (the molecular biology magazine 48:443 of Needleman and Wunsch, 1970), (institute of NAS reports 85:2444 in the similarity method retrieval of Pearson and Lipman, 1988) (all quote as a reference), the computer of these algorithms is carried out the (GAP in the Wisconsin genetics software package 7.0 editions, BESTFIT, FASTA and TFASTA, the genetics computer set, 575 Science Dr., Madison, WI, be incorporated herein by reference), or by range estimation, select the best parallelism that different methods produces (that is, on the contrast window, producing the highest percentage ratio of sequence similarity).Term " sequence identity " is meant that two kinds of polynucleotide sequences are on the contrast window identical (that is, on Nucleotide-Nucleotide basis).The method of calculation of term " sequence identity percentage ratio " are: the sequence that compares two kinds of best parallelisms on the contrast window, determine to exist on two kinds of sequences the positional number of identical nucleic acid base (for example A, T, C, G, U or I), produce the positional number of coupling, the matched position number is divided by the total number of positions (being window size) in the contrast window, again the result be multiply by 100, obtain the percentage ratio of sequence identity.The feature of polynucleotide sequence represented in term " basic identity " when this uses, wherein these polynucleotide contain a kind of on the contrast window of at least 20 nucleotide positions, usually on the window of 25-50 Nucleotide at least, compare with canonical sequence, at least 85% sequence identity is arranged, 90%-95% sequence identity at least preferably, the sequence of at least 99% sequence identity more generally, wherein by on the contrast window relatively canonical sequence with may comprise canonical sequence altogether 20% or the lower disappearance or the polynucleotide sequence of interpolation, come sequence of calculation identity percentage ratio.Canonical sequence can be the subclass of big sequence.
Except these polynucleotide sequences relation, generally also select reorganization PIV encoded protein matter of the present invention and protein zone, conservatively concern promptly, have basic sequence identity or sequence similarity with having of selecting with reference to polypeptide.When being used for polypeptide, term " sequence identity " is meant to have identical or similar amino acid whose peptide (being conservative substitution) in the corresponding position.Term " basic sequence identity " is meant two kinds of peptide sequences, when best parallelism, as with program GAP or BESTFIT during with default breach amount parallelism, they have at least 80% identity, at least 90% sequence identity preferably, at least 95% sequence identity more preferably, or more (for example 99% sequence identity).Term " basic similarity " is meant two kinds of peptide sequences of total corresponding sequence similarity percentage ratio.Preferably, residue position inequality is because conservative amino acid replacement and difference.Conservative amino acid replacement is meant the interchangeability of the residue with similar side chain.For example, the one group of amino acid that contains aliphatic lateral chain is glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; The one group of amino acid that contains aliphatic series-hydroxyl side chain is Serine and Threonine; The one group of amino acid that contains the amide containing side chain is l-asparagine and glutamine; The one group of amino acid that contains aromatic side chains is propylhomoserin in the benzene, tyrosine and tryptophane; The one group of amino acid that contains basic side chain is Methionin, arginine and Histidine; The one group of amino acid that contains sulfur-containing side chain is halfcystine and methionine(Met).Preferred conservative amino acid replacement group is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.20 kinds of naturally occurring amino acid whose abbreviations of Shi Yonging are herein used (E.S.Golub and D.R.Gren compile for " immunology-synthetic ", second edition, Sinauer Associates, Sunderland, MA 1991, is incorporated herein by reference) according to routine.20 kinds of amino acid whose steric isomers of routine (for example D-amino acid), alpha-non-natural amino acid such as α, α-disubstituted amino acid, N-alkyl amino acid, lactic acid and other unconventional amino acid also can be the suitable components that is used for polypeptide of the present invention.Unconventional amino acid whose example comprises: 4-Hydroxyproline, Gla, ε-N, N, N-trimethyl lysine, ε-N-acetyllysine, O-Phosphoserine, N-acetylserine, N-formylmethionine, 3-Methyl histidine, 5-oxylysine, ω-N-methylarginine and other similar amino acid and imino-acid (for example 4-oxyproline).And, also can be by modified amino acids such as glycosylation, phosphorylations.
In order to select to determine viability, attenuation and immunogenic standard according to well-known method according to candidate vaccine virus of the present invention.The virus of wishing most in vaccine of the present invention must keep viability; has stable attenuation phenotype; displaying duplication (even if lower level) in by the host of immunity; and can in the vaccinate, cause immunne response effectively, be enough to provide the protection of the serious disease that causes at wild-type virus infection subsequently.Reorganization PIV of the present invention is not only survival and more suitable than former vaccine candidate strain attenuation; and more stable on the genetics in vivo---keep stimulating protective immune response; and enlarge the ability of the protection that causes by multiple modification in some cases; for example; induce protection at different virus strain or subgroup; or because the protection of different amynologic basis, for example secretor type and serum immune globulin, cellular immunization etc.
Can be with multiple well-known, the external and body inner model detection reorganization PIV of the present invention that generally acknowledges, with confirm suitable attenuation, to the regressive resistance of phenotype be used for the immunogenicity of vaccine.In external test, be aspect the ts phenotype for example in the temperature sensitivity of virus replication, aspect the phenotype of little plaque or other hope, detect the virus of modifying (for example, PIV multiple attenuation, biology deutero-or reorganization).Further in the PIV infected animal model, detect the virus of modifying.Described several animal models, and summed up in the many pieces of reference of quoting herein.Be used to estimate the attenuation and the immunogenic PIV model system of the strain of PIV vaccine candidate, comprise rodent and non-human primate, accept extensively in the art, thus obtained data are relevant with PIV infection, attenuation and immunogenicity in human body.
According to above description, the present invention also provides the isolating infectivity reorganization PIV composition that uses for vaccine.The attenuated virus that is vaccine composition is isolating and generally is the form of purifying.Isolating be meant not in the wild-type virus natural surroundings as the nasopharynx of infected individuality in PIV.More generally, the isolating attenuated virus that comprises as cell culture or other artificial medium components, it can be bred at this, is characterised in that the setting of control.For example, attenuation PIV of the present invention can separate and add stablizer with the cell cultures deposits yields that infects from cell culture.
For vaccine use, can directly in vaccine preparation, use according to the reorganization PIV of production of the present invention, or when wishing with the known freeze drying process freeze-drying of technician.Freeze dried virus generally is maintained at about 4 ℃.When prepare using, freeze-drying virus is rebuild with stabilizing solution, as salt solution or contain SPG, Mg
++And HEPES, it contains or does not contain adjuvant, further describes as following.
PIV vaccine of the present invention contains a kind of PIV of immunogen significant quantity of producing as described here as activeconstituents.The virus of modifying can import among the host with physiology acceptable carrier and/or adjuvant.Useful carrier is known in the art, comprises, for example: water, buffered water, 0.4% salt solution, 0.3% glycine, hyaluronic acid etc.The aqueous solution that produces can be packed or freeze-drying, and as mentioned above, freeze-dried preparation combines with sterile solution before using.Composition can contain the acceptable auxiliary substance of pharmacy at needs during near physiological condition, as pH regulator and buffer reagent, tension regulator, wetting agent etc., for example sodium acetate, Sodium.alpha.-hydroxypropionate, sodium-chlor, Repone K, calcium chloride, Arlacel-20, Emulphor FM etc.Acceptable adjuvant comprises incomplete Freund's adjuvant, MPL
TM(3-o-deacylated tRNA base monophogphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT) and IL-12 (Genetics Institute, Cambridge MA), and other known suitable adjuvants of this area.
After using the immunity of PIV composition as described here, by aerosol, droplet, per os, part or other approach, host's immunity system is by producing the special antibody of PIV albumen such as F and HN glycoprotein vaccine reaction.Because with the PIV inoculation of the immunogen significant quantity that produces as described here, the host is at least partly or entirely immunity of PIV infection, or the moderate of the particularly lower respiratory tract of tolerance development or severe PIV infection.
The host who uses vaccine can be any Mammals to PIV or closely related virus infection sensitivity, and this host can produce the antigenic protective immune response to the inoculation strain.Therefore, the invention provides the method for producing vaccine into the multiple mankind and veterinary purpose.
Susceptible PIV infected or have this dangerous host use the vaccine composition that contains PIV of the present invention, to improve host's immunne response ability itself.This amount is defined as " the effective dosage of immunogen ".In this purposes, the accurate amount of the PIV that uses is depended in the effective dose host's state of health and body weight, method of application, preparation nature etc., but be generally each host about 10
3-10
7Plaque forming unit (PFU) or more virus are more generally as each host about 10
4-10
6PFU virus.In any case vaccine preparation should be able to provide is enough to effectively protect host patient to avoid the modification PIV of the present invention of the amount of serious or life-threatening PIV infection.
The PIV that produces according to the present invention can combine with the virus of other PIV serotypes or strain, to realize the protection to multiple PIV serotype or strain.In addition, as described here, by also can realize protection in conjunction with the protective epitope who is building up to multiple serotype in a kind of virus or strain to multiple PIV serotype or strain.Generally using differently when viral, they will mix and use simultaneously, but but their also separate administration.Can cause protection with a strain immunity at the not homophyletic of identical or different serotype.
In some cases, with PIV vaccine of the present invention with can induce to other pathogenic agent particularly the vaccine of the protective response of other children's viruses combine and wish.In another aspect of this invention; as described here; by in PIV genome or anti-genome, mixing the sequence of these protective antigens of coding; be used for producing infectivity PIV, PIV can be with the carrier of the protective antigen that acts on other pathogenic agent such as respiratory syncytial virus (RSV) or Measles virus.
All experimenters, determine accurate amount and the time of using and the repetition of the reorganization PIV vaccine used according to patient's state of health and body weight, method of application, preparation nature etc.Dosage is generally every patient about 10
3-10
7Plaque forming unit (PFU) or more virus are more generally as every patient about 10
4-10
6PFU virus.In any case vaccine preparation should be able to provide the attenuation PIV that is enough to effective stimulus or induces the amount of anti-PIV immunne response, for example, can determine by complement combination, plaque neutralization and/or enzyme-linked immunosorbent assay etc.In this, also monitor the sign and the symptom of individual upper respiratory disease.As chimpanzee is used, the attenuated virus of vaccine in vaccinate's nasopharynx with than wild-type virus low about 10 times or more horizontal growth, or with level low about 10 times or more horizontal growth than the PIV of incomplete attenuation.
In newborn infant and baby, may need repeatedly to use and cause effective immune level.Using should be from first month, and carries out at certain intervals with whole the Childhood, and as 2 months, 6 months, 1 year and 2, this was to keep effective protection level that natural (wild-type) PIV is infected necessary.Similarly, individual to repeating or the responsive especially adult of serious PIV infection as the old-age group that kinsfolk, the cardio-pulmonary function for the treatment of personnel, children on health care personnel, daytime are impaired, can need repeatedly immunity to set up and/or protect protective immune response.In being tested and appraised and the amount of secretor type and serotype antibody, can monitoring institute's inductive immune level, and regulate dosage or repeated inoculation where necessary, with the protection level that keeps wishing.And then, can specify different vaccine viruses to using of isoacceptor group not.For example, the express cell factor or be rich in t cell epitope other proteinic engineering PIV strains may to adult rather than to baby's particularly advantageous.
The PIV vaccine that produces according to the present invention can with another subgroup of expressing PIV or antigenic viral combination of strain, to realize protection to multiple PIV subgroup or strain.In addition, as described here, vaccine virus also can mix the multiple PIV strain that is implemented among a kind of PIV clone or the protective epitope of subgroup.
PIV vaccine of the present invention can cause when individual during with postoperative infection wild-type PIV, the generation that serious lower respiratory illness such as pneumonia and bronchitis are had the immunne response of protectiveness.Though natural circulation virus still can cause infection, particularly upper respiratory tract infection,, has the possibility of the rhinitis of very big reduction owing to inoculation with because the possible resistance that wild-type virus infection subsequently causes is strengthened.After the inoculation, but the host with detection level produces serum and secretor type antibody, they can be in vitro and in vivo in homology (identical subgroup) wild-type virus.In many cases, host's antibody wild-type virus of different non-vaccine subgroups that also can neutralize.
Compare with the wild-type virus of natural circulation in human body, preferred PIV vaccine candidate strain of the present invention shows the virulence that greatly reduces.These viruses are attenuations fully, make infection symptoms not to take place most of in by immune body.In some cases, attenuated virus still can be to nonvaccinated individual the propagation.Yet its virulence is fully eliminated, make the inoculation or accidental host in serious lower respiratory infection does not take place.
For example, by quantitatively by the virus that exists in the respiratory tract of immune body, and with wild-type PIV or be evaluated as the amount that other attenuations PIV of candidate vaccine strain produces and compare, can determine the attenuation level of HIV vaccine candidate strain.For example, compare with the levels of replication of wild-type virus, attenuated virus of the present invention will have the copy limit of higher degree in the upper respiratory tract of height susceptible host, for example low 10 to 1000 times.In order further to reduce the development of the rhinorrhea relevant with virus replication in the upper respiratory tract, ideal vaccine candidate virus all should show flat the duplicating of restricting water supply in the upper respiratory tract and lower respiratory tract.Yet attenuated virus of the present invention must have sufficient infectivity and immunogenicity in human body, to provide protection in the individuality of inoculation.The method of measuring PIV level in the infected individuals nasopharynx is well-known in the literature.
Also can be by monitoring the induction of immunity level that vaccine of the present invention provides with the amount of secretor type and serum antibody in measuring.According to these mensuration, can regulate vaccine dose where necessary, or repeated inoculation, with the protection level that keeps wishing.Further, different vaccine viruses can help not isoacceptor group.For example, the expression of transformation other proteinic PIV strains of being rich in t cell epitope are particularly conducive to adult rather than baby.
In another aspect of this invention, use PIV as of short duration respiratory tract Vectors in Gene Therapy.According to this embodiment, the sequence that reorganization PIV genome or anti-genome have mixed a kind of goal gene product of encoding.The goal gene product is subjected to the control of identical or different promotor, thereby control PIV expresses.PIV albumen by coexpression reorganization PIV genome or anti-genome and N, P, L and other hope produces, and the sequence infectivity PIV that contains the goal gene product of encoding can use the patient.Use generally is by aerosol, atomizer or to other topical applications of treatment patient's respiratory tract.To be enough to cause to treat or the amount administered recombinant PIV of the hope gene product expression of prevention level.The representative gene product that available this method is used preferably is applicable to transient expression, comprise for example interleukin II, interleukin 4, gamma-interferon, GM-CSF, G-CSF, erythropoietin and other cytokines, film conduction-modifying agent (CFTR), xanthoglobulin-guanine phosphoribosyltransferase, cytotoxin, tumor suppressor gene, sense-rna and vaccine antigen are striden in glucocerebrosidase, Phenylalanine hydroxylase, cystic fibrosis.
The following example is in order to illustrate, rather than restriction.These embodiment have proved according to aforesaid method and have made up the representative chimeric PIV with one or more heterologous antigen determinants.In one embodiment, the HA gene of Measles virus inserts in one of the JS wild-type of HPIV3 or three kinds of genes connections of attenuated strain as alia gene, and promptly N/P, P/M or HN/L connect, and recover the reorganization embedded virus.Insert measles HA gene at genomic three the different positions places of HPIV3 the useful construct scope that is used for shifting to the PIV carrier antigenic determinant of foreign pathogens has been described.In addition, more near the genetic unit expection of the insertion of 3 ' leader will with than the homologous genes unit higher level that is positioned at far-end transcribe and express, this will cause the tightr adjusting (people such as Collins of allogeneic gene expression, the third edition, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, 1205-1243 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。
With by deutero-rHPIV3 compare, in wild-type rHPIV3 background, contain the chimeric rHPIV that Measles virus HA inserts and efficiently duplicate, but in hamster, duplicate limited external.Similarly, containing the chimeric HPIV3 of reorganization that Measles virus HA inserts in attenuation rHPIV3 background can duplicate in external and hamster, level a little less than by deutero-attenuation rHPIV3cp45L mutated viruses.The proteic amount of HA that contains the cell expressing that the attenuation rHPIV3-Measles virus HA recon of HA gene infected in N/P or P/M connect is high, even surpasses seen in the cell that infects wild Measles virus.In the upper respiratory tract of hamster, containing Measles virus HA during N/P or P/M connect, to insert the levels of replication of segmental rHPIV3cp45L lower 10 times than rHPIV3-cp45L parental virus, shows the attenuation of gene insertion can unexpectedly causing HPIV3 carrier.These results that determine unique host range phenotype are unexpected.
Importantly, infect hamster with the every kind of reorganization embedded virus that detects and to induce high-caliber HPIV3 antibody and Measles virus antibody.Highly tolerate duplicating of HPIV3 challenge virus with having the recombinate animal of chimeric HPIV3 immunity of attenuation that HA inserts.Although the wild-type Measles virus can not efficiently be duplicated in hamster, thereby can not use in Attack Research, the protectiveness of attenuation reorganization chimeric owing to inductive high level neutralizing antibody rapidly obviously.In human body, these levels relevant with high-caliber measles resistance (people such as Chen, transmissible disease magazine 162:1036-42,1990).
Further proof in an embodiment, with the attenuated chimeric reorganization HPIV carrier of one or more antigenic determinants combination of HPIV3 main chain and HPIV1 also can be as expressing the antigenic carrier of other external sources (for example HPIV2 or non-PIV virus).This aspect of the present invention utilizes effective growth of HPIV3 main chain and the antigenic determinant that outstanding attenuation characteristic is carried multiple allos pathogenic agent, example such as HPIV1 and HPIV2.By between the genetic unit of F that contains HPIV1 and HN ORF, inserting the genetic unit that contains HPIV2 HN gene ORF, modify the cDNA of coding cPIV3-1 (the not attenuation recon of the major antigen of a kind of HPIV1 of having) or rPIV3-1cp45 (a kind of attenuation recon that carries the HPIV1 major antigen).The reorganization embedded virus that is named as rPIV3-1.2HN and rPIV3-1cp45.2HN is easy to reply, and can efficiently duplicate in tissue culture.The every kind of virus type that shows the replication in vitro temperature sensitivity of certain level is similar to its rPIV3-1 or rPIV3-1cp45 parental virus.Being inserted in of PIV2 HN makes all attenuations of rPIV3-1 and rPIV3-cp45 in the hamster, this discovery is similar in rJS and rPIV3cp45 that to insert Measles virus HA viewed.Insert segmental these antigen rPIV3-1 recon and infect hamster and can induce serum antibody response with containing PIV2 HN gene at HPIV1 and HPIV2.
Therefore, can use a kind of attenuation rHPIV3 or the strain of rHPIV3-1 vaccine candidate, infect the respiratory tract of susceptible host, thereby induce external source protective antigen that external genetic unit expresses as carrier, and to the strong antibody response of HPIV carrier itself.The existence of three kinds of antigen serotypes of HPIV; this does not cause tangible cross protection; allow with the different HPIV variant of antigen that contains identical or different heterologous antigen determinant; as protective antigen, antigenic domain or the epi-position of Measles virus or one or more different virus or microbial pathogen, more effectively continuous immunity baby.Continuous immunity can develop the primary immune response to exogenous protein; subsequently with containing one or more heterologous antigen determinants, as being strengthened in the different HPIV course of infection of the secondary antigen of protective antigen, antigenic domain or the epi-position of Measles virus or one or more different virus or microbial pathogen.Like this, strengthen to avoid with the different HPIV carrier of antigen to the immunity of a kind of HPIV carrier inductive.In this manual, successful immunity to the animal of PIV3 immunity realizes with the PIV3-1 vaccine candidate strain of attenuation, confirm feasibility with the different PIV virus continuous immunity of serotype, even (people such as Tao still feasible when the total protein except that HN and F of these PIV, vaccine 17:1100-8,1999).In this research, under in advance to the PIV3 immunity or the situation of denying, in hamster, measure immunogenicity and the usefulness that rPIV3-1.cp45L attacks PIV1.RPIV3-1.cp45L can effectively infect the hamster that infects with wild-type or attenuation PIV3 in advance, but rPIV3-1.cp45L virus duplicating in the animal of PIV3-immunity reduced by 5 times approximately.Yet the rPIV3-1.cp45L immune induction of the animal of PIV3 immunity is to the intensive serum antibody response of PIV1, and makes being replicated in the lower respiratory tract of PIV1 challenge virus reduce by 1000 times, reduces by 200 times in the upper respiratory tract.These results prove, the chimeric rPIV3-1.cp45L candidate vaccine even also can induce immunity to PIV1 in to the animal of PIV3 immunity of recombinating.This has shown the feasibility of using the continuous immunity scheme, wherein the chimeric rPIV3-1.cp45L of administered recombinant or other PIV vaccine viruses after attenuated live PIV3 vaccine.Because rPIV3-1.cp45L is easy to induce the protective immunity to itself, so also induce effective immunne response that its any carrier that carries is delivered protective antigen.PIV and RSV also have the anomalous property that can infect respiratory tract again, though infect generally not relevant with serious disease again.Therefore, the vaccine constructs based on carrier of the present invention can be used for by for the second time, for the third time or use same HPIV carrier for the 4th time or reply by continuous use different carriers booster immunization.
In preferred vaccinization method of the present invention, wish with the different PIV carrier continuous immunity babies that express identical heterologous antigen determinant such as Measles virus HA.This continuous immunity can be induced the high-titer antibody of anti-heterologous protein, and it is peculiar that this is that secondary antibodies is replied.In one embodiment, use for example early stage baby of the proteic attenuated chimeric HPIV3 immunity of Measles virus HA (for example 2-4 month big baby) of expressing heterologous antigenic determinant, also be suitable for causing the immunne response of anti-HPIV3.Useful in this manual a kind of typical vaccine candidate strain is rcp45L (HAP-M) recon.Subsequently, for example when big, once more, but used and first kind of second kind of different PIV vector construction body immunity baby that antigenicity is different at 4 months.A kind of typical vaccine candidate strain in this specification sheets is to express Measles virus HA gene and the HPIV1 antigenic determinant rPIV3-1 cp45L virus as the functional essential glycoprotein of carrier.After inoculation for the first time, the vaccinate will cause to PIV3 HN and F albumen with to the proteic primary antibody of Measles virus HA and reply, but do not cause PIV1 HN and the proteic primary antibody of F not replied.After with the rPIV3-1 cp45L that expresses Measles virus HA immunity for the second time; the vaccinate can be easily by vaccine infection owing to not containing anti-PIV1 HN and the proteic antibody of F; and will develop the primary antibody of PIV1 HN and F protective antigen is replied and the proteic high titre secondary antibodies of allos Measles virus HA is replied.Can develop a kind of similar continuous immunity scheme; wherein be used in disclosed one or more chimerics virus in place and cause continuously HPIV3, stimulate initial and secondary, high titre protective response simultaneously measles or another kind of non-PIV pathogenic agent then to the immunity of HPIV2.This continuous immunity strategy, the different serotypes of preferably using PIV can effectively be avoided the immunity of first grade carrier inductive as the primary and secondary carrier, and this is the factor of the availability of the final restriction carrier that only contains a kind of serotype.
Further according to this aspect of the present invention, the TYPICAL COMBINED vaccination regimen can mix two kinds, three kinds, four kinds maybe can reach six kinds or more separate chimeric HPIV vaccine virus, they are for example 1,2 or used (for example, in how special vaccine mixture) in 4 months when big simultaneously in elementary inoculation step.For example, can use two or more whole vaccine viruses that maybe can reach based on HPIV, they express one or more antigenic determinants (being whole antigen, immunogenicity territory or epi-position) dividually, are selected from: the F albumen of the F albumen of the G albumen of RSV subgroup A, RSV subgroup A, the G albumen of RSV subgroup B, RSV subgroup B, the HA albumen of Measles virus and/or the F albumen of Measles virus.The uniting of these identical vaccine constructs based on PIV3 strengthen using and repeat when can be at 2 months big.Subsequently, for example when big, can in secondary inoculation step, use 2-6 kind or the more different attenuated live vaccine virus of antigenicity separately based on HPIV at 4 months.For example, secondary inoculation can comprise uses mixture or the several formulations that contains multiple HPIV3-1 vaccine constructs simultaneously, these construct co expression are from the RSV G of subgroup A, from the RSV F of subgroup A, from the RSV F of subgroup B, RSV G, Measles virus HA and/or Measles virus F from subgroup B, or from the antigenic determinant of these proteinic any combinations.Secondary immunity causes the immune strengthening to every kind of allos RSV and Measles virus protein or its antigenic determinant.At 6 months when big, inoculation step for the third time, comprise and use 1-6 or how isolating attenuated live vaccine recon based on the PIV3-2 carrier, can be co-administered, they separately or co expression from the RSV G of subgroup A, from the RSV F of subgroup A, from the RSV G of subgroup B, RSV F, Measles virus HA and/or Measles virus F from subgroup B, or its antigenic determinant.Randomly, in this step of vaccination regimen, rPIV3 and rPIV3-1 vaccine can use in strengthening preparation.Like this, in infantile preceding 6 months, can induce the peculiar strong immunity of the whole secondary antibodies of anti-PIV1, PIV2, PIV3, RSV A, RSV B and Measles virus.This associating/continuous immunity strategy, can induce the secondary antibodies of multiple viral respiratory disease substance is replied, a kind of highly effective and immunization protocol extremely flexibly is provided, and this is required with other pathogenic agent immunity at each of three kinds of PIV viruses in early days in infancy.
In other aspects of the present invention, separately use and in the strain of HPIV vaccine candidate, insert heterologous nucleotide sequence, for example regulate the attenuation level of candidate vaccine recon for the upper respiratory tract.Therefore, in rHPIV, insert the guiding exogenous protein and express and make the nucleotide sequence of virus attenuation in the animal host, or separately use Nucleotide and insert that to make candidate vaccine virus attenuation all be possible.In order to determine that the domination gene inserts some principle to the effect of attenuation, in the genetic unit insertion wild-type HPIV3 main chain with different lengths, and quarantine genetic unit length is to the effect of attenuation.Transform these novel gene unit and insert, make it not contain important ORF, this can estimate the effect of the genetic unit length that does not rely on the proteinic effect of genetic expression.These heterologous sequences are between the alia gene unit insertion HN and L gene of 168nt-3918nt as size.In addition, contrast cDNA structure and virus, wherein the insertion of similar size is placed in 3 '-non-coding region of HN gene, does not therefore comprise the interpolation of alia gene.These viruses can be used to estimate the influence of the increase of total genome length and gene dosage to attenuation.Estimate that the unitary insertion of alia gene can reduce the gene transcription of inserting the downstream, site, this will influence expressed proteinic overall abundance and ratio.So the place proves, length makes the upper respiratory tract and the lower respiratory tract attenuation of wild-type virus for hamster greater than gene insertion or the extension of about 3000nt.The gene of the about 2000nt of length inserts and further makes the strain of rHPIV3cp45L vaccine candidate for upper respiratory tract attenuation.In a word, gene inserts and may have the dual function that makes candidate vaccine virus attenuation and induce the protective effect of second kind of virus of antagonism.Gene in gene 3 '-non-coding region that can not expression of other proteins matter extends also attenuation voluntarily.In these methods of the present invention, it is the determinative of attenuation that gene inserts length.
GU in the reorganization PIV of the present invention and NCR insert and produce a kind of attenuation phenotype, it is characterized in that duplicating in the external body that efficiently duplicates and reduce, and be other paramyxovirus to be inserted a kind of phenotype of describing in the past.The mechanism that GU inserts the attenuation that causes may be because main following one or more factors of effect in vivo.The unitary interpolation of alia gene can reduce the transcriptional level of downstream gene because have a kind of gradient of transcribing, wherein more near the gene of promotor with than transcribing further from the higher speed of the gene of promotor.If gene product is limited, or the special ratio of efficiently duplicating required gene product changes, and reduces the reduction that the downstream gene product that causes expresses by the mRNA abundance and can cause attenuation.Can think and transcribe the result that gradient is the transcriptase mixture that comes off from template during transcribing and connect shifting by gene.In addition, the increase of the genome and the outer mRNA length overall of transcribing can improve the level of viral double-stranded RNA, but its higher levels of antiviral activity of inducing interferon system subsequently.At last, the aggregate level of genome duplication can reduce owing to the increase of genome or anti-genome length.This may be owing to replicative enzyme mixture between geneome RNA or antigenomic RNA replicative phase comes off from template.The minimizing that can be used for being packaged in the genome amount in the virosome can cause the reduction of viral yield, and this will cause attenuation.
The mechanism of being inserted the attenuation that causes by NCR may be because following one or more factors.The extra length that the HN mRNA 3 ' that is caused by NCR holds can cause the unstable of mRNA, and causes the reduction of HN protein expression.In addition, the increase of genomic length overall and HN mRNA extra length can improve the level of viral double-stranded RNA, the higher levels of antiviral activity of this energy inducing interferon system.In addition, the aggregate level of genome duplication can reduce owing to the increase of genome or anti-genome length.This may be owing to replicative enzyme mixture between geneome RNA or antigenomic RNA replicative phase comes off from template.The minimizing that can be used for being packaged in the genome amount in the virosome can cause the reduction of viral yield, and this will cause attenuation.At last, Nucleotide can reduce the transcriptional level of downstream gene outside HN gene 3 ' the terminal interpolation, because the transcriptase mixture can come off from template during the outer Nucleotide of HN gene 3 ' end is transcribed.
In the external and body that GU in PIV3 and NCR insert growth properties be different from cited above before with the discovery of other mononegavirale RNA viruses.Expressed protein is checked in insertion with Pretesting, thereby can not determine and insert the independent action of length to growing in the virosome.This discovery proof is inserted greater than the GU of 3kb and NCR and to be caused the attenuation phenotype that does not rely on expressed protein.Short insertion for example greater than the insertion of about 2kb, causes further attenuation in part attenuation acceptor.Equally unexpectedly, GU and NCR are inserted in and cause in the limited body under the situation that does not exist limited body to duplicate and duplicate.In addition, when being inserted as GU or NCR infix form, can see attenuation phenotype in the body---other insertions that proved are the GU form.Therefore, the GU of coded protein or NCR insert to duplicate in the body do not cause and weaken a kind of unique way that reduces negative strand virus member in the body of representative.Example I
The encode structure of the anti-genomic cDNA of a kind of chimeric PIV3/ Measles virus-HA and the acquisition of infective virus
Full length cDNA clone had been described in the past, p3/7 (131) 2G
+The anti-genome of complete 15462 Nucleotide of its coding JS PIV3 wt virus, and pFLCcp45L, it is coded in the anti-genome that contains the JS wt derivative of three special temperature sensitive mutations of cp45 among the L ORF of PIV3 (people such as Durbin, virusology 235:323-332,1997a; People such as Skiadopoulos, Journal of Virology 72:1762-8,1998, be incorporated herein by reference).These clones are used to insert the HA gene of Measles virus as carrier, to produce the proteic attenuation HPIV3 of the HA chimeric construct body of wild-type and expressing heterologous antigenic determinant such as Measles virus.Containing every kind of measles HA gene, to insert segmental size be 6 multiple, makes the embedded virus that is obtained by cDNA meet 6 principle (1997b is incorporated herein by reference for people such as Durbin, virusology 234:74-83).The structure of the chimeric HPIV3 cDNA of the coding proteic total length of Measles virus HA in N/P or P/M connection
With PmlI-BamHI fragment (the anti-genomic nt 1215-3903 of the PIV3) subclone of p3/7 (131) 2G+ in the modified plasmid pUC119 that in multiple clone site, contains a PmII site { pUC119 (PmlI-BamHI) }.With Kunkel method (people such as Kunkel, Enzymology method 154:367-382 1987, are incorporated herein by reference) pUC119 (PmlI-BamHI) is carried out independently strand mutagenesis reaction twice; First set reaction is CTTAAG (pAflII N-P) by the CTAAAT series jump with anti-genomic nts 1677-1682 place, in 3 ' (downstream)-non-coding region of N gene, introduce an AflII site, the reaction that separates for the second time is CTTAAG (pAflII P-M) by the TCAATC series jump with anti-genomic nts 3693-3698 place, introduces an AflII site in 3 '-non-coding region of P gene.
Utilize the HA ORF of reverse transcriptional PCR (RT-PCR) by Edmonston wild-type virus amplification Measles virus Edmonston strain.The nt sequence of Edmonston wild-type HA open reading frame (ORF) is GenBank preserving number #U03669, is incorporated herein by reference (noticing that this sequence does not just contain the ORF of upstream 3 nts or terminator codon).According to manufacturer's recommend method, with TRIzol-LS (Life Technologies, Gaithersburg, MD) purifying Measles virus RNA from clarifying substratum.(Clontech, Palo Alto CA) carry out RT-PCR to use Advantage RT-for-PCR and Advantage-HF PCR test kit according to recommend method.The complete ORF, the flank that produce across Measles virus HA gene with primer are the PCR fragment of PIV3 non-coding sequence and AflII restriction site.Forward primer 5 '-TTAATCTTAAGAATATACAAATAAGAAAAACTTAGGATTAAAGAGCGATGTCACCA CAACGAGACCGGATAAATGCCTTCTAC-3 ' (SEQ IDNO.13) coding derives from the upstream, AflII site (italic) that the N/P gene connects the PIV3 non-coding sequence of nts3699-3731 (underscore marks), contain the beginning part (runic) of GE, IG and GS sequence (Figure 1A) and measles HA ORF, the front is three kinds of non-HPIV3 that indicate in primer, non-Measles virus nts.Reverse primer 5 '-ATTATTGCTTAAGGTTTGTTCGGTGTCGTTTCTTTGTTGGATCCTATCTGCGATTG GTTCCATCTTC-3 ' (SEQ ID NO.14) coding derives from the downstream, AflII site (italic) (in just complementary strand) of PIV3 non-coding sequence nt3594-3623 (underscore marks) of P gene and the end (runic) of measles HA ORF.Then the PCR fragment that obtains is digested with AflII, be cloned into respectively among p (AflII N-P) and the p (AflII P-M), produce pUC119 (HA N-P) and pUC119 (HA P-M).(Foster city CA) checks order on complete AflII insertion fragment to pUC119 (HA N-P) and pUC119 (HA P-M), confirms that sequence is correct for ABI prism, PE Applied Biosystems to stop the cycle sequencing rapid reaction with rhodamine.
(people such as Durbin as previously mentioned, virusology 235:323-332,1997a, be incorporated herein by reference), the PmlI-BamHI fragment of pUC119 (HA N-P) and pUC119 (HA P-M) is cloned into respectively among the anti-genome cDNA plasmid of total length p3/7 (131) 2G+, produces pFLC (HAN-P) and pFLC (HA P-M) (Fig. 1).Then the XhoI-NgoMI fragment (nt 7437-15929) of pFLCcp45L is cloned in the XhoI-NgoMI window of pFLC (HA N-P) and pFLC (HA P-M), produces pFLCcp45L (HA N-P) and pFLCcp45L (HA P-M).Three seed amino acids in the L gene of pFLCcp45L coding PIV3 cp45 change (amino acid position 942,992 and 1558), this causes the most of temperature sensitivities and the attenuation (people such as Skiadopoulos of cp45 vaccine candidate virus, Journal of Virology 72:1762-8,1998, be incorporated herein by reference), these sudden changes are shifted in the segmental transfer of XhoI-NgoMI.The structure of the chimeric cDNA of the coding proteic total length HPIV3 of HA in HN/L connects
Make up the chimeric cDNA of HPIV3 by PCR, to comprise the heterologous polynucleotide sequence, as Measles virus HA gene, its coding Measles virus heterologous antigen determinant, flank is the non-coding region of transcribing signal and HPIV3 HN gene.The alia gene that this cDNA design is used as after the HN gene combines with the rPIV3 carrier.At first, with Kunkel mutagenesis (people such as Kunkel, Enzymology method 154:367-382,1987, be incorporated herein by reference), introduce a StuI site in 3 '-non-coding region of HN gene, method is that the AGACAA series jump with anti-genome nts 8598-8603 place is AGGCCT, generation plasmid p3/7 (131) 2G-Stu (Figure 1B).Make up with three fragments through PCR then and contain the cDNA (seeing Figure 1B) that flank is the measles HA ORF of HPIV3 sequence.The left hand upstream fragment of PCR synthetic gene for the first time.Forward primer 5 '-GACAATAGGCCTAAAAGGGAAATATAAAAAACTTAGGAGTAAAGTTACGCAATCC-3 ' (SEQ ID NO.15) contains a StuI site (italic), be HPIV3 sequence (underscore marks) afterwards, it comprises the downstream end (HPIV3 nts8602-8620), intergenic region, gene start signal of HN gene and from the sequence (HPIV3 nt 6733-6753) of HN gene downstream end.Reverse primer 5 '-GTAGAACGCGTTTATCCGGTCTCGTTGTGGTGACATCTCGAATTTGGATTTGTCTA TTGGGTCCTTCC-3 ' (SEQ ID NO.16) contains a MluI site (italic) in measles HA ORF starting point (runic) downstream, be the complementary sequence (underscore marks) of HPIV3 nts 6744-6805 afterwards, they are parts of upstream HN non-coding region.The MluI site that exists in the Measles virus ORF that introduces is by nt 27 is changed into C by T (in wild-type Edmonston HA gene), and nt30 is changed into G by C produces.These two kinds of changes all are non-coding in Measles virus ORF.Carry out PCR with p3/7 (131) 2G-Stu as template.The product that obtains is called PCR fragment 1, and flank is the StuI site of 5 ' end and the MluI site of 3 ' end, and at the preceding 36nt that contains measles HA ORF from the downstream of the non-coding sequence of HPIV3 HN gene.The right hand end of the synthetic HN gene of PCR reaction for the second time.Forward primer 5 '-GTAGAACGCGTTTATCCGGTCTCGTTGTGGTGACATCTCGAATTTGGATTTGTCTA TTGGGTCCTTCC-3 ' (SEQ ID NO.16) contains the end (runic) of XmaI (italic) and measles HA ORF, is the HPIV3 nts 8525-8566 (underscore marks) that represents HN gene downstream non-translational region part subsequently.Reverse primer 5 '-CCATGTAATTGAATCCCCCAACACTAGC-3 ' (SEQ ID NO.17) across HPIV3 nts 11448-11475, is arranged in the L gene.Pcr template is p3/7 (131) 2G-Stu.The PCR fragment 2 that is produced by this reaction contains about 2800nt of the L ORF of the last 35nt of measles HA ORF and PIV3, and flank is an XmaI site and a SphI site (the natural HPIV3 site 11317 that is present in).PCR reaction for the third time is by the centre portions of the maximum of template cDNA pTM-7 amplification measles HA ORF, and pTM-7 is a kind of plasmid of the HA ORF that contains Measles virus Edmonston strain that provides of ATCC.Sequential analysis to this plasmid shows, with be used for comparing with the pTM-7 of the Edmonston wild-type HA sequence of inserting during P-M is connected to N-P, the Measles virus HA ORF that is contained among the PTM-7 contains 2 amino acid differences, and they are positioned at amino acid sites 46 (F is to S) and site 481 (Y is to N).Forward primer 5 '-CGGATAAACGCGTTCTACAAAGATAACC-3 ' (SEQ ID NO.18) (the MluI site is an italic) and reverse primer 5 '-CGGATAAACGCGTTCTACAAAGATAACC-3 ' (SEQ ID NO.18) (the XmaI site is an italic) amplification contain the PCR fragment 3 of the nts 19-1838 of measles HA ORF.In order to assemble these fragments, PCR fragment 1 usefulness StuI and MluI digestion, and PCR fragment 3 usefulness MluI and XmaI digestion.Contained the StuI-XmaI window of the pUC118 in a StuI site by three fragment clonings that reconnect these two kinds of digestion in the polyclone district to modifying the back then.The plasmid that obtains, pUC118 (HA 1+3), with StuI and MluI digestion, and PCR fragment 2 usefulness XmaI and SphI digestion.Then these two kinds of digestion products are cloned into the StuI-SphI window of p3/7 (131) 2G-Stu, produce plasmid pFLC (HA HN-L).Then with rhodamine stop the cycle sequencing rapid reaction (ABI prism, PE Applied Biosystems, Foster city, CA) order-checking comprises the StuI-SphI fragment of complete measles HA ORF.Chimeric construct body sequence is confirmed.Like this, to be the HPIV3 Measles virus HA ORF that transcribes signal be inserted into during N/P, the P/M of the anti-genome cDNA carrier that contains wild-type HPIV3 or HN/L connect as alia gene flank, or contain during the N/P of anti-genome cDNA carrier of attenuation HPIV3 or P/M connect.The acquisition of chimeric rPIV3 wild-type and the proteic rcp45L of expression Measles virus HA
5 kinds of total length carrier cDNA that contain measles HA ORF as isolated genes, with support plasmid { pTM (N), pTM (P no C), pTM (L) } and LipofectACE (Life Technologies) is together, difference transfection 6 well culture plates (Costar, Cambridge, MA) the HEp-2 cell in, (people such as Durbin, virusology 235:323-332,1997 simultaneously as previously mentioned; People such as Durbin, virusology 234:74-83,1997, be incorporated herein by reference), using the MVA-T7 cells infected, MVA-T7 is a kind of replication defect type vaccinia virus recon of the phage t7 polymerase protein of encoding.PTM (P no C) is derivative people such as (, virusology 261:319-330,1999) Durbin of pTM (P), wherein because the sudden change of C initiator codon, and C ORF expression silencing.At 32 ℃ of following incubations after 3 days, the transfection cutting T25 that goes down to posterity is shaken on the fresh individual layer of Vero cell in the bottle, and in 5 days (be called and go down to posterity 1) of 32 ℃ of following incubations.(people such as Durbin as previously mentioned, virusology 235:323-332,1997, be incorporated herein by reference), determine by plaque titration to go down to posterity on the LLC-MK2 monolayer culture thing existence of HPIV3 in 1 cutting wherein shows plaque with the special immunoperoxidase staining with the special monoclonal antibody of measles HA of HPIV3 HN.
(people such as Hall, virus research 22:173-184 1992, are incorporated herein by reference) as previously mentioned, by plaque purification on the LLC-MK2 cell three times, biology is cloned in rPIV3 (HA HN-L) virus that exists in the suitable 1 cutting supernatant liquor that goes down to posterity.Utilization is continuous 2 times of dilution Vero cell monolayers on 96 orifice plates, by end dilution eventually, and biology clone rPIV3 (HA N-P), rcp45L (HA N-P), rPIV3 (HA P-M) and rcp45L (HA P-M) from 1 cutting that goes down to posterity separately.32 ℃ of amplifications are taken turns or third round end dilution biology clone's recombinant virus twice eventually from the third round plaque purification or from second in LLC-MK2 cell { rPIV3 (HA HN-L) } or Vero cell { rPIV3 (HA N-P), rcp45L (HA N-P), rPIV3 (HA P-M), rcp45L (HA P-M) } then, produce virus with further sign.As the first step that confirms and characterize the chimeric PIV3 of reorganization that expresses Measles virus HA glycoprotein, use 3 kinds of different primers to analyzing every kind of 1 cutting that goes down to posterity by RT-PCR; Each inserts segmental each position to being used for HA ORF.First primer comprises N/P and inserts the site the PIV3 fragment of amplification across total length HPIV3 genome nucleotide 1596-1968.This clip size is increased to 2298 Nucleotide owing to insert measles HA ORF between N and P gene.Second primer comprises P/M and inserts the site the PIV3 fragment of amplification across total length HPIV3 genome nucleotide 3438-3866.The measles HA ORF that inserts between P and M gene makes this clip size be increased to 2352 Nucleotide.The 3rd primer is to the PIV3 fragment of amplification across the anti-genome nucleotide 8466-8649 of total length.The measles HA ORF that inserts between HN and L gene makes this clip size be increased to 2211 Nucleotide, and it comprises HN/L and inserts the site.All 5 kinds of viruses that obtain all contain the insertion fragment of suitable size in place.The generation of every kind of PCR product depends on the existence of ThermoScript II, shows that every kind all derives from RNA, rather than derives from the cDNA of pollution.
With infection multiplicity (MOI) 5, shake the LLC-MK2 cell monolayer in the bottle with rcp45L (HA N-P), rcp45L (HA P-M), rJS infection T25, or false the infection.With MOI 5, infect T25 with the strain of Measles virus Edmonston wild-type and shake Vero cell monolayer in the bottle.For measles Edmonston virus infection is selected the Vero cell monolayer, because Measles virus can not well grow in the LLC-MK2 cell.In infection back 24 hours, wash this individual layer with the DMEM (LifeTechnologies) that lacks methionine(Met).Adding concentration in the substratum that lacks DMEM is 10 μ Ci/ml's
35S methionine(Met), and shake to every and to add 1ml in the bottle is then 32 ℃ of following incubations 6 hours.Harvested cell and with PBS washing 3 times.With cell precipitation be resuspended to 1ml RIPA damping fluid 1% (w/v) Sodium desoxycholate, 1% (v/v) Triton X-100 (Sigma), 0.2% (w/v) SDS, 150mM NaCl, 50mM Tris-HCl, among the pH7.4}, freeze thawing, centrifugal 5 minutes of 6500 * G clarification.Cell extract is transferred in the new eppendorf pipe, and in every kind of sample, added and to discern Measles virus HA glycoprotein (79-XV-V17,80-III-B2,81-1-366) (people such as Hummel, Journal of Virology 69:1913-6,1995; People such as Sheshberadaran, virus journal (Arch.Virol.) 83:251-68,1985, be incorporated herein by reference) maybe can discern HN albumen (101/1,403/7, the 166/11) (people such as van Wyke Coelingh of PIV3, virusology 160:465-72,1987, be incorporated herein by reference) the monoclonal antibody mixture, and under constant mixing 4 ℃ of incubations 2 hours.(MO) suspension spends the night the precipitation immunocomplex subsequently 4 ℃ of following constant mixing for Sigma, St.Louis by add 200 μ l, 10% albumin A Sepharose pearl in each sample.With every kind of sample of 90 μ l, 1 * sample loading buffer suspension, and add 10 μ l reductive agents.70 ℃ of heating were recommended according to manufacturer after 10 minutes, every kind of sample of 20 μ l is added on the 4-12% polyacrylamide gel (NuPAGE, Novex, San Diego, CA).Desiccant gel and radioautograph (Fig. 2).With anti-measles HA monoclonal antibody precipitation coding a kind of proteinic rcp45L (HA P-M) and rcp45L (HA N-P), it is identical with reliable measles HA albumen size.Rcp45L (HA P-M) and rcp45L (HA N-P) express Measles virus HA albumen, and degree is higher than the Edmonston wild-type strain of Measles virus, show that these constructs can be connected with P/M by the N/P of attenuated strain rcp45L to efficiently express Measles virus HA.According to the reactivity of the anti-HN monoclonal antibody of PIV3, confirm that rcp45L (HA N-P) and rcp45L (HA P-M) are based on HPIV3's.RPIV3 parent and rPIV3 (HA) embedded virus is in the temperature sensitivity of replication in vitro
The temperature sensitivity level of duplicating of the chimeric rPIV3 of Measles virus HA insertion is carried in assessment,, compares with parent vector virus under differing temps with estimation, and HA inserts the levels of replication (table 1) whether segmental acquisition changes embedded virus.On the LLC-MK2 of 96 orifice plates cell monolayer, in the L-15 that is supplemented with 5%FBS, 4mM glutamine and 50 μ g/ml gentamicins, carry out continuous 10 times of dilutions of rcp45L, rcp45L (N-P), rcp45L (HA P-M), rPIV3 (HA HN-L), rPIV3 (HAP-M) or rJS, and 32,36,37,38,39 or 40 ℃ of following incubations 6 days.Detect virus by hemocyte absorption, and be expressed as log
10TCID
50/ ml.What is interesting is, carry the chimeric derivant of two kinds of wild-type vector virus of Measles virus HA gene, rPIV3 (HAHN-L) and rPIV3 (HA P-M) duplicate under 40 ℃ by slightly restricted (table 1).Carry two kinds of attenuation rPIV3 of Measles virus HA gene, rcp45L (N-P) and rcp45L (HA P-M) have the temperature sensitivity level that is similar to rcp45L parent vector virus, and rcp45L (HA P-M) is slightly higher than its parent's temperature sensitivity.Therefore, contain the segmental virus of insertion duplicating in tissue culture and be similar to parent vector rPIV3, temperature sensitivity just improves slightly.These results show that rPIV3 can easily insert segmental carrier as holding HA at the different loci place, and not bigger change of replication in vitro, rPIV3 (HA) embedded virus can easily adapt to the further interpolation of one or more attenuation sudden changes.Table 1. is expressed the proteic reorganization of Measles virus HA HPIV3 at the virus titer (log of the replication-competent virus under permissive temperature and the elevated temperature under assigned temperature as alia gene in N-P, P-M or HN-L connection
10TCID
50/ ml) 32 ℃ of 1 36 ℃ of 37 ℃ of 38 ℃ of 39 ℃ of 40 ℃ of rcp45L
28.2 8.2 7.2 5.26 3.4 3.0rcp45L (HA P-M)
37.4 6.7 5.2 4.2 1.4 1.4rcp45L (HA N-P)
37.4 7.2 5.7 4.2 2.2≤1.2rPIV3 (HA HN-L)
47.7 8.2 7.0 7.7 6.7 5.2rPIV3 (HA P-M)
47.7 7.4 6.7 6.2 6.2 4.7PIV3-rJS
58.7 the reorganization ts derivative of the JS wild-type strain of 9.0 9.0 8.4 8.2 9.01. permissive temperature 2.HPIV3 contains 3 attenuation amino-acid substitutions that derive from cp45.3. express the recombinant attenuated ts derivative of the proteic JS wild-type of Measles virus HA HPIV3.4. express the proteic reorganization wild-type of Measles virus HA HPIV3.5. reorganization wild-type HPIV3, the JS strain.
6. the titre that marks of underscore is represented the lower limit temperature, reduces by 100 times or more when observe titre than 32 ℃ this moment, and is defined as the temperature of ending of virus.
Example II
The chimeric rPIV3 that contains the measles virus antigens determinant efficiently duplicates in hamster, and induces anti-
The high-titer antibody rPIV3 (HA) of HPIV3 and anti-measles virus is duplicated mensuration with the immunogenicity level in hamster
To contain the levels of replication of chimeric rPIV3 of measles virus antigens determinant and its parent rPIV3 relatively, with determine determinant such as HA insert segmental acquisition whether obviously change its duplicate with inductor in the ability of immunne response.In two kinds of different experiments, every group of 6 or 7 4-6 golden Syria hamster intranasal vaccination in age in week 0.1ml contained 10
6.0The EMEM (Life Tchnologies) (table 2 and table 3) of the rJS of PFU, rcp45L, rcp45L (HAP-M), rcp45L (HA N-P), rPIV3 (HA HN-L) or rPIV3 (HA P-M).At postvaccinal the 4th day, kill hamster, take out lung and concha.With concha and lung respectively 10% and 20%w/v L-15 (Quality Biologicals, Gaithersburg, MD) homogenate in the suspension, quick freezing sample.The virus that on 96 orifice plates of LLC-NK2 cell monolayer, exists in the titre sample, and 32 ℃ of following incubations 7 days.Detect virus by hemocyte absorption, and calculate the average log of every group of hamster
10TCID
50/ g.Insert HA gene (table 2) and be limited in and duplicate 4-20 doubly in the upper respiratory tract in wild-type rJS, can reach 5 times in lower respiratory tract, the acquisition that shows the HA gene has only been duplicated minimal effect to wild-type rJS virus in hamster.Chimeric each---the reorganization parental virus that in L albumen, contains three attenuation ts sudden changes---low 10 times that in the hamster upper respiratory tract, duplicate (table 3) of two kinds of rcp45 (HA) antigen than rcp45L, but identical with the rcp45L parent in lower respiratory tract.Therefore, for two kinds of rcp45 (HA) antigen chimeric each, duplicating in the hamster upper respiratory tract has slight but statistics reduces significantly, shows that rcp45L acquisition HA gene has strengthened the attenuation for the upper respiratory tract, but then strengthen for lower respiratory tract.Therefore, the HA gene inserts acting in the upper respiratory tract quite that wild-type or attenuation PIV3 duplicate.The replication-competent virus of table 2. wild-type rPIV3 (HA) embedded virus in the hamster upper respiratory tract and lower respiratory tract
1Number of animals virus titer (log
10TCID
50/ gm ± S.E.
2)
[Tukey-Kramer grouping]
3
[Tukey-Kramer grouping]
3
Studied the ability of chimeric rHPIV3 (HA) virus induction then to the immunne response of HPIV3 and Measles virus.As mentioned above at the 0th day with 10
6.0The rJS of PFU, rPIV3 (HA P-M), rcp45L, rcp45L (HA P-M) or rcp45L (HA N-P) (table 4) infect every group of only golden Syria hamster of 6-24.Measure (people such as van WykeCoelingh by foregoing hemagglutinin-inhibition (HAI), virusology 143:569-582,1985, be incorporated herein by reference) estimate serum antibody response to HPIV3, and reduce and to measure (people such as Coates with foregoing 60% plaque, U.S.'s epidemiology magazine (Am.J.Epidemiol.) 83:299-313 1966, is incorporated herein by reference) assessment is to the serum antibody response of Measles virus.With these results with use 10 at the 0th day intramuscular
5.0The result of another cotton mouse control group of attenuated live measles virus (Moraten strain) relatively.Using cotton mouse rather than hamster in this group, is because Measles virus has only faint infectivity to hamster.As seen in Table 4, every kind of PIV3 (HA) embedded virus can cause the strong serum neutralizing antibody of Measles virus is replied.The amount of the serum neutralizing antibody that attenuation derivative rcp45L (HA P-M) causes is compared with homologue rPIV3 (HA P-M) in the wild-type background, does not have significant difference.In addition, in rPIV3 (HA) the recon inductive Measles virus and the serum antibody level than 5 times of the mean heights that is reached with the immunity of attenuated live seat exanthema virus vaccine intramuscular.In addition, the serum antibody response to HPIV3 that all embedded viruses produce is also stronger, and infects produce suitable with wild-type rJS.Table 4.rPIV3 (HA) antigen embedded virus causes the outstanding serum antibody response virus to Measles virus and PIV3
1Number of animals is anti-to the serum of HPIV3 to the serum antibody of Measles virus
Tire (60% plaque reduce in body reply (HAI tires,
With tire, average reciprocal average reciprocal
log
2±S.E.
2)?????????log
2±S.E.)
The 0th day the 25th day the 0th day the 25th day rcp45L
318≤3.3 ± 0≤3.3 ± 0≤2.0 ± 0 10.7 ± 0.2rcp45L (HA P-M)
424≤3.3 ± 0 12.8 ± 0.1≤2.0 ± 0 9.2 ± 0.2rcp45L (HA N-P)
56≤3.3 ± 0 13.4 ± 0.4≤2.0 ± 0 10.8 ± 0.3rPIV3 (HA P-M)
66≤3.3 ± 0 13.3 ± 0.3≤2.0 ± 0 10.3 ± 0.2 Measles virus 4≤3.3 ± 0 10.8 ± 0.2≤2.0 ± 0≤2.0 ± 0rJS
86≤3.3 ± 0≤3.3 ± 0≤2.0 ± 0 10.7 ± 0.21. the 0th day to all animals with the 0.1ml inoculum in 10
6.0The dosage intranasal administration virus of PFU, exception are to accept the Measles virus group of virus by intramuscular injection.2. standard error.3. in L albumen, contain three recombinant attenuated HPIV3 that derive from temperature sensitive (ts) sudden change of cp45.4.cp45 the recombinant attenuated HPIV3 in the background contains Measles virus HA ORF in the P/M of rPIV3 non-coding region.5.cp45 the recombinant attenuated HPIV3 in the background contains Measles virus HA ORF in the N/P of rPIV3 non-coding region.6. the reorganization HPIV3 that in the P/M of wild-type rPIV3 non-coding region, contains Measles virus HA ORF.7. in dividing other research, by to 4 cotton mouse intramuscular injection with in the 0.1ml inoculum 10
5The dosage of pfu is used attenuated live Measles Vaccine virus, Moraten strain.Other all animals are hamster.8. wild-type HPIV3 recombinates.
Use similarly every group of rsv infection and each 6 hamster of control group the 25th day with the 0.1ml inoculum of intranasal administration in 10
6.0The biology deutero-HPIV3 wild-type virus virus attack of pfu.Took out lung and concha at the 4th day, and handle as mentioned above.The virus that on 96 orifice plates of LLC-MK2 cell monolayer, exists in the titre sample, and 32 ℃ of following incubations 7 days.Detect virus by hemocyte absorption, calculate the average log of every group of hamster
10TCID
50/ g.As shown in table 5, accepted the hamster of embedded virus, no matter be attenuation or the wild-type main chain in, protection antagonism wild-type HPIV3 duplicating in the upper respiratory tract and lower respiratory tract of attacking highly all.Therefore; although the acquisition of Measles virus HA gene is to the slight abated effect that duplicates of rcp45 (HA) embedded virus; but infect the protection that can induce high-caliber antagonism HPIV3 with rcp45L (HA P-M) or rcp45L (HA N-P), its duplicating in the hamster upper respiratory tract and lower respiratory tract is reduced about 1000 times and has been shown this point.Because the wild-type Measles virus can not efficiently be duplicated in hamster, so it can not be used for attacking this host.Yet the strain of expection attenuated chimeric rcp45L (HA) vaccine candidate will be very effective to Measles virus, because can induce high-caliber neutralizing antibody, that is, on average tiring is higher than 1: 5000.In the suitable level of Measles virus antibody and the human body to the strong resistance of Measles virus disease relevant (people such as Chen, transmissible disease magazine 162:1036-42 1990, are incorporated herein by reference).Table 5. attenuation and wild-type HPIV3 measles HA embedded virus have the height protective immunity animal of duplicating of antagonism attack wild-type PIV3 in the hamster upper respiratory tract and lower respiratory tract and use material
1Number of animals virus titer (log
10TCID
50/ g) reduction (the log of titre
10)
[Tukey-Kramer grouping]
3
Concha lung concha lung RSV 6 7.0 ± 0.3[A] 5.7 ± 0.4[A] NA
2NArcp45L (HA P-M) 6 3.4 ± 0.3[B] 2.9 ± 0.0[B] 3.6 2.8rcp45L (HA N-P), 6 2.6 ± 0.3[B] 3.4 ± 0.2[B] 4.4 2.3rPIV3 (HA P-M), 6 2.0 ± 0.3[B] 3.2 ± 0.1[B] 5.0 2.5rcp45L 6 1.9 ± 0.2[B, C] 3.6 ± 0.1[B] 5.1 2.1rJS 6＜1.4 ± 0.0[C] 2.9 ± 0.2[B]＞all groups of 5.7 2.81. all use in the 0.1ml inoculum of intranasal administration 106Pfu biology deutero-JS wild-type PIV3 attacks.2. inapplicable.3. by the Tukey-Kramer check average virus titer is distributed to the similar group of statistics (A-C).Therefore, the mean value of each row of different letters significantly different (α=0.05), the row of same letter do not have significant difference.
The EXAMPLE III coding contains HPIV2 HN gene as the structure of the anti-genome cDNA of the chimeric HPIV3-1 carrier that inserts the outer transcribing/translation unit between F and the HN gene and the acquisition of infective virus
RPIV3-1 is the chimeric HPIV3 of a kind of reorganization, wherein HN and F gene be replaced by the HN of HPIV1 and F gene (referring to, for example, people such as Skiadopoulos, vaccine 18:503-510,1999; People such as Tao, vaccine 17:1100-1108,1999; The U.S. Patent Application Serial 09/083,793 of application on May 22nd, 1998; The United States Patent (USP) series number 09/458,813 of application on December 10th, 1999; The United States Patent (USP) series number 09/459,062 of application on December 10th, 1999 all is incorporated herein by reference).In the present embodiment, the HN gene of HPIV2 inserts in the rPIV3-1 embedded virus, as the carrier that produces chimeric derived virus, contains the heterologous antigen determinant from HPIV2 of introducing, the protection of can create antagonism HPIV1 and HPIV2.HPIV2 HN gene also inserts in the attenuation derivative of rPIV3-1, and this derivative is called as rPIV3-1cp45, contains 12 in 15 cp45 sudden changes of inserting in the rPIV3 main chain, promptly, sudden change in the gene except that HN and F (people such as Skiadopoulos, vaccine 18:503-510,1999).The source of HPIV2 wild-type virus is wild-type strain V9412-6 (being called PIV2/V94) (people such as Tao, vaccine 17:1100-1108,1999), and it is isolating in the Vero cell of the nose washing lotion that obtained from the children of natural infection HPIV2 in 1994.Plaque purification PIV2/V94 is three times on the Vero cell, increases twice on the Vero cell with the OptiMEM tissue culture medium (TCM) that does not contain FBS then.By using the reverse transcription (RT) of hexabasic at random basic sequence and Superscript Preamplification system (LifeTechnologies), subsequently by using Advantage cDNA synthetic agent box (Clontech, Palo Alto, CA) and introduce the PCR (Fig. 3 A) of synthetic primer that flank is the NcoI-HindIII site of HN cDNA, produce the cDNA clone of the HN gene of PIV2/V94 from virosome RNA.The sequence of these primers is: (the HPIV distinguished sequence is capitalization, line out below the restriction site, non-HPIV or with nts that wt changes be small letter, initial and terminator codon are runic), upstream HPIV2 HN 5 '-gggccATGGAAGATTACAGCAAT-3 ' (SEQ ID NO.19); Downstream HPIV2 HN 5 '-caataagcTTAAAGCATTAGTTCCC-3 ' (SEQ ID NO.20).HN PCR fragment digests with NcoI-HindIII, is cloned among the pLit.PIV31HNhc, produces pLit.32HNhc (Fig. 3 B).With the ThermoSequenase test kit and
33(NJ) the HPIV2 HN heterologous gene that checks order fully inserts fragment to the terminator of P-mark, confirms that it contains the reliable sequence of PIV2/94 HN coding region for PharmaciaAmersham, Piscataway.
Utilize PCR and Deep Vent heat-stable DNA polymerase further to modify HPIV2 HN gene (New England Biolab among the pLit.32HNhc, Beverly, MA), be used for cloning to introduce the PpuMI site to unique PpuMI site of p38 ' Δ PIV31hc, Fig. 3 C (people such as Skiadopoulos, vaccine 18:503-510,1999).The sequence of these primers is that (the HPIV distinguished sequence is capitalization, the relevant limit site marks with underscore, and non-PIV nt or the nt that compares change with wt are small letter): upstream HPIV2 HN 5 '-gcgatgggcccGAGGAAGGACCCAATAGACA-3 ' (SEQ ID NO.21); Downstream HPIV2 HN 5 '-cccgggtcctgATTTCCCGAGCACGCTTTG-3 ' (SEQ IDNO.22).The modification cDNA that contains HPIV2 HN ORF by (from left to right) 5 ' end contain the HPIV3 HN in PpuMI site part 5 ' non-translational region (5 '-UTR), 3 '-UTR of HPIV2 HNORF, HPIV3 HN, sequence and the HPIV3 HN a complete set of HPIV3 that is connected coupling with the L gene transcribe signal (being gene termination, intergenic region and gene homing sequence), HPIV3L part 5 '-UTR, hold the PpuMI site of interpolation to form (Fig. 3 C) 3 '.This fragment digests with PpuMI, inserts among p38 ' the Δ PIV31hc with PpuMI digestion, produces p38 ' Δ PIV31hc.2HN (Fig. 3 D).The PpuMI box that inserts of order-checking fully, find it with design consistent.The insertion fragment that derives from p38 ' Δ PIV31hc.2HN is separated into 8.5kb BspEI-SphI fragment, and import respectively in the BspEI-SphI window of pFLC.2G+.hc or pFLCcp45, produce pFLC.31hc.2HN or pFLC.31hc.cp45.2HN (Fig. 3, E and F).As previously mentioned, pFLC.2G+.hc and pFLCcp45 are encode the respectively anti-genomic clone of total length (people such as Skiadopoulos, Journal of Virology 73:1374-81,1999 of wt rPIV3-1 and rPIV3cp45; People such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference).
As previously mentioned, in the presence of MVA-T7, add the HEp-2 cell that pTM (N), pTM (P no C) and pTM (L) support plasmid to infect to converge (people such as Durbin, virusology 235:323-332 with pFLC.31hc.2HN or pFLC.31hc.cp45.2HN, 1997, be incorporated herein by reference).By adding TPCK trypsin catalog number (Cat.No.) 3741, WorthingtonBiochemical Corp., Freehold, NJ), and go down to posterity and titration contains the virus of HPIV1HN and F glycoprotein the reorganization embedded virus that activation is obtained by transfection (people such as Tao, Journal of Virology 72:2955-2961 as previously mentioned, 1998, be incorporated herein by reference).As previously mentioned, go down to posterity chimeric recombinant virus rPIV3-1.2HN that purifying obtains and rPIV3-1cp45.2HN (people such as Tao, vaccine 17:1100-1108 by plaque-plaque-plaque on the LLC-MK2 individual layer in the agarose tectum, 1999, be incorporated herein by reference).
In order to determine whether rPIV3-1.2HN and rPIV3-1cp45.2HN reorganization contains allos HPIV2 HN gene, the viral RNA of every kind of reorganization embedded virus that amplification obtains on the LLC-MK2 cell, and concentrate (people such as Mbiguino by polyoxyethylene glycol (PEG) precipitation, virological method magazine (J.Virol.Methods) 31:161-170,1991, be incorporated herein by reference).Extract virosome RNA (vRNA) with Trizol (Life Technologies), and use (the Life Technologies of Superscript Preamplification system as mentioned above as template, Gaithersburg, MD) and the at random synthetic first chain cDNA of hexabasic basic primer.Use AdvantagecDNA synthetic agent box (Clontech, Palo Alto, CA) and the special primer of HPIV1 F and HPIV1 HN coding region (for HPIV1 F is 5 '-AGTGGCTAATTGCATTGCATCCACAT-3 ' (SEQ ID NO.23), for HPIV1 HN is 5 '-GCCGTCTGCATGGTGAATAGCAAT-3 ' (SEQ IDNO.24)), pcr amplification synthetic cDNA.The relative position of PIV1 F and HN primer marks with arrow in Fig. 3 and Fig. 4.The dna fragmentation of digest amplification, and analysis (Fig. 4) on sepharose.The data not shown of rPIV3-1cp45.2HN, but structurally quite and be confirmed.RPIV3-1.2HN and rPIV3-1cp45.2HN all contain the insertion fragment of expection size, and the digestion pattern of multiple restriction enzyme has confirmed purpose identity and verity.Also confirmed to exist among the rPIV3-1cp45.2HN cp45 sudden change.
In order to confirm that the rPIV3-1.2HN embedded virus expresses HPIV2 HN, in containing the tryptic serum-free OptiMEM of 0.25 μ g/mlTPCK, shake LLC-MK2 individual layer in the bottle with MOI 5 usefulness PIV2/V94, rIV3-1 or rPIV3-1.2HN infection T25.At 32 ℃ of following incubations after 18 hours, (MD) washing is shaken bottle 3 times for Bio Whittacker, Walkersville not have methionine(Met) and halfcystine DMEM with 5ml.(Pharmacia Amersham, Piscataway, 1ml NJ) do not have methionine(Met) and halfcystine DMEM feeder cell, and 32 ℃ of following incubations 18 hours with replenishing 120 μ Ci ProMix 35S-methionine(Met)s and 35S-halfcystine mixture then.Cell is scraped in the substratum, in micro tube through of short duration centrifugation, and with cold PBS washing 3 times.Every kind of cell precipitation is resuspended to 1ml RIPA damping fluid (1% Sodium desoxycholate that contains 250 units/ml Benzonase (Sigma), 1%Triton X-100,0.2%SDS, 150mM NaCl, 50mM Tris-HCl, pH7.4) in, freeze thawing was once clarified with 12000 * g in micro tube in centrifugal 5 minutes.Clarifying supernatant liquor is transferred in the micro tube of cleaning, is mixed (people such as Tsurudome, virusology 171:38-48 1989, are incorporated herein by reference) with 50 anti-HPIV2 HN monoclonal antibody (mAb) 150S1, and under mixing 4 ℃ of incubations 3 hours.By in every pipe, adding 0.2ml 10% albumin A sepharose suspension (in the RIPA damping fluid) precipitation monoclonal antibody, and under mixing 4 ℃ of incubations 18 hours.Wash pearl three times with the RIPA damping fluid, centrifugation in short-term in micro tube.Every kind of sample suspends with 90 μ l, 1 * sample loading buffer, and at 4-12%SDS polyacrylamide gel (PAGE; NOVEX, San Diego CA) goes up analysis 10 μ l.Gel drying and radioautograph (Fig. 5).MAb a kind of protein of precipitation from the LLC-MK2 cell of rPIV3-1.2HN and PIV2/V94 infection that PIV2 HN is special, but from the cell that rPIV3-1 infects, do not precipitate, the expection size is the 86Kd HN albumen (people such as Rydbeck of HPIV2, general virology magazine 69:931-5,1988, be incorporated herein by reference).
The rPIV3-1 virus that EXAMPLE IV is carried the HPIV2 antigenic determinant shows and temperature like the parent vector virus type
Responsive phenotype
Estimate the temperature sensitivity level that rPIV3-1.2HN and rPIV3-1.cp45.2HN duplicate in the LLC-MK2 cell, compare with parent vector virus determining, whether the HN ORF that obtains HPIV2 as the rPIV3-1 wild-type or the attenuated virus of carrier can change the temperature sensitivity level (Fig. 6) that the chimeric derivant that carries HPIV2 heterologous antigen determinant of generation duplicates.RPIV3-1.2HN and rPIV3-1.cp45.2HN with contrast virus, with having replenished the tryptic 1 * L15 of 0.5 μ g/ml TPCK with serial dilution in 1: 10, and are used for infection LLC-MK2 individual layer in 96 orifice plates in quadruplicate.The culture plate that infects placed differing temps following 7 days, used 0.2% guinea-pig red blood cell (in 1 * PBS) by hemocyte determining adsorption virus titer afterwards.Virus titer is expressed as log
10TCID
50± standard error (S.E.).As shown in table 6, rPIV3-1.2HN and rPIV3-1.cp45.2HN show and its parent vector virus, promptly lack HPIV2 HN and insert segmental rPIV3-1 and the similar temperature sensitivity level of rPIV3-1.cp45.This shows, introduces extra a transcribing/translation unit among rPIV3-1.2HN and the rPIV3-1.cp45.2HN and can significantly not change its temperature sensitivity level at replication in vitro.The rPIV3-1 virus that table 6. carries PIV2 HN insertion has the temperature sensitive phenotype that is similar to its parental virus.The titre of virus in the time of 32 ℃
aDiffering temps (℃) under titre reduce (log
10TCID
50)
(log
10TCID
50) 35 °
b36 ° of 37 ° of 38 ° of 39 ° of 40 ° of PIV2/V9412 7.8 0.3 (0.1)
c0.0 (0.4) (0.4) 0.0PIV1/Wash64 8.5 1.5 1.1 1.4 0.6 0.5 0.9rPIV3/JS 7.9 0.3 0.1 0.1 (0.3) (0.4) 0.4PIV3 cp45 7.8 0.5 0.3 1.3 3.4
d6.8 6.9rPIV3-1 8.0 0.8 0.5 0.6 0.9 1.1 2.6rPIV3-1.2HN 8.3 0.5 (0.3) 0.3 0.6 1.5 2.6rPIV3-1cp45 8.0 0.5 0.4 3.4 4.8 6.6 7.5rPIV3-1 8.0 0.3 1.4 2.9 5.3 7.6 7.6cp45.2HN
aData presented is the mean value of twice experiment.
bData in the time of 35 ℃ are from once experiment.
cDigitized representation titre in the bracket increases.
dThe value representation that underscore marks is by temperature, and the titre of the virus titer of this moment during with 32 ℃ compared and shown and reduce by 100 times or more.
EXAMPLE V
RPIV3-1.cp45.2HN embedded virus duplicating and immunogenicity in animal
For levels of replication in the body of determining embedded virus, 6 every group golden Syria hamster intranasal vaccinations contain 10
5.3TCID
50(or 10
6Pfu) 0.1ml 1 * L15 substratum (table 7) of virus infected after 4 days, killed hamster and took out lung and concha.Measure virus titer, be expressed as average log
10TCID
50/ gram tissue (table 7).Express the rPIV3-1 of PIV2 HN gene, be called as rPIV2-1.2HN, more restricted than its rPIV3-1 parent aspect duplicating, the virus titer in the hamster upper respiratory tract and lower respiratory tract reduces by 30 times and has shown this point.Therefore, inserting proteic the transcribing of expression PIV2 HN/translation unit in rPIV3-1 makes virus for the hamster attenuation.It is viewed a little more than insert the similar units that contains measles HA ORF in the reorganization JS of wild-type PIV3 strain to insert the attenuation contain the transcribing of PIV2 HN ORF/translation unit in rPIV3-1.Aspect duplicating, limited 1000 times in rPIV3-1.cp45.2HN virus than rPIV3-1cp45 parent, show that PIV2 HN inserts and cp45 suddenlys change attenuation adds and.By adding, should regulate the attenuation level than 12 sudden changes that exist among rPIV3-1.cp45.2HN cp45 sudden change still less.Table 7. is expressed chimeric rPIV3-1 (rPIV3-1.2HN) attenuation experiment numbers virus virus titer in specified tissue in the hamster respiratory tract of PIV2 HN glycoprotein
(log
10TCID
50/g±S.E.)
c
The NT lung
rPIV3-1??????????6.9±0.1[A]
d???????6.0±0.3[A]1
a
rPIV3-1.2HN??????5.4±0.2[B]?????????4.4±0.4[C]
rPIV3-1??????????6.7±0.1[A]?????????6.6±0.2[A]
rPIV3-1.2HN??????5.1±0.1[B，C]??????5.2±0.2[B]2
b
rPIV3-1cp45??????4.6±0.3[C]?????????1.8±0.4[D]
rPIV3-1cp45.2HN??1.5±0.1[D]?????????≤1.2[D]
rPIV3/JS?????????6.5±0.2[A]?????????6.7±0.1[A]
Rcp45 4.9 ± 0.2[B, C] 1.2 ± 0.04[D]
aEvery group of 6 animal in the 0th day intranasal vaccination 0.1ml substratum 10
6Pfu specifies virus.
bEvery group of 6 hamsters were in the 0th day as experiment 1 intranasal vaccination 10
5.3TCID
50Specify virus.
cTook out lung and concha in the 4th day.Measure the virus titer in the tissue, titre is expressed as log
10TCID
50/ gram ± standard error (S.E.).The NT=concha.
dAccording to the Duncan multiple range test, the mean value of each row of different letter representations is different (a=0.05) significantly, and the row with same letter do not have significant difference.
Because the protective antigen of a kind of rPIV3-1.2HN expressing viral PIV1 (F and HN glycoprotein) and PIV2 (having only HN glycoprotein), infect this virus and will induce resistance PIV1 or the attack of PIV2 wild-type virus.In order to confirm this point, use 10 as mentioned above
5.3TCID
50Immune every group of 12 golden Syria hamsters in the virus nose.Half hamster was attacked with PIV2 at the 29th day, all the other half attacked with PIV1 in the 32nd day.Take out hamster lung and concha tissue in back 4 days in attacking, and measure the titre (table 8) of challenge virus as mentioned above.Before immunity and after 28 days, obtain serum, and detect the NAT of its anti-PIV1 and PIV2.Table 8. is expressed the attack immunity virus of chimeric rPIV3-1 virus (rPIV3-1.2HN) the protection hamster antagonism PIV1 and the PIV2 of PIV2 HN glycoprotein
aThe serum NAT of anti-appointment virus (average log2 reciprocal ± SE)
bTitre (the log of challenge virus in the specified tissue
10TCID
50/ g ± S.E.)
c
PIV1???????????????????PIV2???????????????????????PIV1???????????????????????PIV2
Around NT NT after lung lung rPIV3/JS ≤ 4.0 ± 0.0 ≤ 4.0 ± 0.0 4.5 ± 0.1 4.6 ± 0.2 5.4 ± 0.2 5.1 ± 0.1 6.8 ± 0.2 6.0 ± 0.3PIV2 ≤ 4.0 ± 0.0 ≤ 4.0 ± 0.0 4.3 ± 0.2 9.6 ± 0.2 5.7 ± 0.2 5.7 ± 0.2 ≤ 1.2 ≤ 1.2rPIV3-1 4.2 ± 0.1 8.5 ± 0.3 4.0 ± 0.0 4.2 ± 0.1 ≤ 1.2 ≤ 1.2 6.3 ± 0.1 6.5 ± 0.2rPIV3-1.2HN ≤ 4.0 ± 0.0 6.2 ± 0.2 4.1 ± 0.1 8.3 ± 0.2 2.3 ± 0.5 ≤ 1.2 ≤ 1.2 ≤ 1.2rPIV3-1cp45 ≤ 4.0 ± 0.0 6.2 ± 0.4 ≤ 4.0 ± 0.0 4.0 ± 0.0 3.6 ± 0.3 2.7 ± 0.5 6.0 ± 0.1 5.7 ± 0.4rPIV3-1cp45.2HN 4.0 ± 0.9 4.1 ± 0.1 4.0 ± 0.0 4.2 ± 0.1 5.1 ± 0.2 4.8 ± 0.2 6.8 ± 0.1 6.6 ± 0.2aEvery group of 12 hamster in the 0th day with 10
5.3TCID
50Specify virus immunity.
bSerum dilutes with OptiMEM at 1: 10, and passes through in the hot deactivation in 30 minutes of 56 ℃ of following incubations.Measure the serum NAT on LLC-MK2, tiring is expressed as average log reciprocal
2± standard error (SE).
cHalf hamster of each immune group in the 29th day with 10
6TCID
50PIV2 attacks, all the other half in the 32nd day with 10
6TCID
50PIV1 attacks.At back 4 days collection organization's samples of attack, the challenge virus titre is expressed as log
10TCID
50/ gram tissue ± SE.The NT=concha.
As expection, PIV3 do not cause the resistance of PIV1 or PIV2 people such as (, 17:1100-1108,1999) Tao, induces the complete resistance that PIV2 and PIV1 challenge virus are duplicated respectively and infect PIV2 wild-type virus and rPIV3-1 in advance.These viruses at a kind of protection of virus are different with only providing; rPIV3-1.2HN can induce the antibody of anti-PIV1 and PIV2; and comprising strong resistance to PIV1 and PIV2, every kind of virus duplicating in the upper respiratory tract of the hamster of rPIV3-1.2HN immunity and lower respiratory tract reduced 1000-10000 this point doubly has been described.This shows that a kind of chimeric PIV that recombinates can induce the resistance at two kinds of Human virus's pathogenic agent.Yet the derivative that contains the rPIV3-1.2HN of cp45 sudden change can not be induced the obvious resistance that wild-type PIV1 or PIV2 challenge virus are duplicated, and shows this special reorganization embedded virus undue attenuation in hamster.Can in rPIV3-1.2HN, introduce the cp45 sudden change of one or several selection, rather than whole 12 sudden changes, and the attenuation horizontal adjustment that makes rPIV3-1.2HN is to proper level.
Example VI
The structure that contains the cDNA of the coding rHPIV3 virus that Nucleotide inserts
As mentioned above, between the N/P or the connection of P/M gene of attenuated carrier virus rPIV3cp45L, and between connecting, N/P, the P/M of wild-type PIV3 and HN/L insert measles HAORF, further limit its duplicating in the hamster upper respiratory tract, show in the arbitrary position of HPIV3 genome and insert the attenuation that another kind of gene can strengthen candidate vaccine virus.Of the present invention these typical aspect, gene inserts fragment relatively large (about 1900 nts).Provide other embodiment at this, shown the optional attenuation level that segmental size is determined the recombinant virus of generation of inserting.Its evaluation method be between HPIV3 HN and L ORF, introduce derive from a kind of allos virus as the sequence of the different lengths of RSV A2 strain as single-gene unit (GU).These insert fragment and lack any tangible ORF through special design, and may influencing of expressed protein do not make observed any effect complicated thus.Because the effect that the expression of the genome length that increases and other mRNA causes prepares the second series construct, wherein introduce the insertion fragment of similar size in order to distinguish to the downstream of HN gene non-coding region (NCR).Therefore, make the rPIV3 of two series contain the insertion that length increases: in GU series, insert fragment as the alia gene interpolation of the outer mRNA of coding, and in NCR series, fragment is inserted in preparation to make the gene number constant.The structure that contains the cDNA of the coding rHPIV3 virus that GU and 3 '-NCR inserts
Make up in based on the plasmid pUC118-Stu of pUC and insert sudden change, this plasmid contains the XhoI-SphI fragment (HPIV3 nts 7437-11317) of total length HPIV3 clone p3/7 (131) 2G-stu.Make up two kinds of different plasmids as receptor plasmid, be used to insert GU and HN gene 3 '-NCR and extend (Fig. 6).In each, in unique StuI site, insert the synthetic oligonucleotide duplex that contains multiple clone site.The sequence of inserting to GU insertion plasmid contains (IG) trinucleotide sequence and L gene-initial (GS) signal sequence between HN gene-termination (GE) signal sequence, conservative gene respectively, instructs the cis acting sequence (Fig. 6) of HN genetic transcription termination and insertion sequence transcription initiation.In multiple clone site, contain other unique restriction endonuclease sites and be beneficial to subsequently screening and subclone.Design and make up 3 '-NCR similarly and extend receptor plasmid, but it is in 5 '-terminal cis acting GE, IG and the GS sequence (Fig. 6 B, table 9) of lacking.RSV anti-geneome plasmid d53RSV site or subgene group plasmid pUC118FM2 (table 9) digest with suitable restriction enzyme, and separate to wish the fragment of size, and be connected to unique HpaI site (Fig. 6 that GU or HN gene 3 '-NCR extend receptor plasmid separately by agarose gel electrophoresis; Table 9).Screening and cloning identifies that promptly all read the direction (Fig. 7) that frame all contains a plurality of terminator codons to insert the clone of RSV restricted fragment in the other direction.The short synthetic oligonucleotide duplex that also with size is 13-17 Nucleotide in case of necessity inserts in GU or 3 '-NCR receptor plasmid, makes it to meet " 6 principles " (table 9) to change genome length.The short synthetic oligonucleotide size of special RSV sequence and interpolation is summarized in the table 9.By being used for all restriction sites order-checking plasmid clones of subclone, and will meeting XhoI-SphI fragment that containing of 6 principle insert sudden change and extend as GU or HN gene NCR and be cloned among total length PIV3 cDNA plasmid p3/7 (131) 2G+.A kind of insertion fragment that comprises 1908 GU insertion also is placed in the anti-genome cDNA of three kinds of L sudden changes of carrying cp45.Table 9. is used for producing GU multiple clone site in the anti-genome of source restricted fragment size (nts) RSV that genetic unit (GU) and HN gene 3 ' non-coding region (NCR) extend the Nucleotide that inserts, and (58 GU insert the NCR multiple clone site, and (32 NCR insert
Restriction site and nt position nt)+6 oligonucleotide principles
e(total nts of insertion) nt)+6 oligonucleotide principles
e(total nts of insertion) 97
aSspl-Sspl; 7272-7369+58,+13 168 nd nd212
bHpal-Hpal; 12243-12455 nd nd+32,+14 258603
bSspl-Sspl; 309-912+58,+17 678 nd nd925
bHpal-Hpal; 12455-13380+58,+13 996+32,+15 9721356
B, cHincII-HincII; 5060-6417+58,+14 1428+32,+16 14041850
B, dHpal-Hpal; 12455-13380+5,8+0 1908 nd nd3079
bEcoRV-Ec/13611; 1403-4482 nd nd+32,+15 31263845
bScal-Scal; The source of 344-4189+58,+15 3918+32+17 3894a.RSV sequence is pUC118FM2, a kind of segmental plasmid (Juhasz of subgene group cDNA that contains RSV subgroup A as previously mentioned, K. wait the people, Journal of Virology 71:5814-5819,1997) source of .b.RSV sequence is D53sites, a kind of plasmid that contains complete RSV subgroup A cDNA sequence as previously mentioned, wherein containing the point mutation of several introducings. aforementioned D53sites plasmid is used for producing Whitehead, S. wait the people, Journal of Virology 72:4467-4471,1998 described rAsites virus .c. cut product with 1357 nt restriction endonuclease enzymes of prediction and compare, and the 1356nt fragment of gel-purified contains 1 nt disappearance.D.1850 the nt fragment is in the following oligonucleotide insertion of the product .e. MluI restriction site of two 3 ' the 925nt restricted fragments that are connected with 3 ', makes the exogenous array of all insertions meet 6 principle: 13mer:CGCGGCAGGCCTG (SEQ ID NO.25); 14mer:CGCGGCGAGGCCTG (SEQ ID NO.26); 15mer:CGCGAGGCCTCCGCG (SEQ ID NO.27); 16mer:CGCGCCGCGGAGGCCT (SEQ ID NO.28); 17mer:CGCGCCCGCGGAGGCCT (SEQ ID NO.29) .nd does not carry out.Contain the acquisition of the reorganization PIV3 that inserts sudden change
(Life Technologies MD), supports plasmid pTM (N), pTM (P no C) and pTM (L) (people such as Durbin, virusology 235:323-332,1997 with containing the anti-genome cDNA derivative of total length that inserts sudden change and three kinds to use LipofectACE; People such as Durbin, virusology 261:319-330,1999, be incorporated herein by reference) transfection 6 orifice plates (Costar, MA) the HEp-2 individual layer in, and infect individual layer (people such as Durbin, virusology 235:323-332,1997 with MVA-T7 as previously mentioned; People such as Skiadopoulos, Journal of Virology 72:1762-8,1998, be incorporated herein by reference).At 32 ℃ of following incubations after 4 days, the transfection cutting T-25 that goes down to posterity is shaken on the LLC-MK2 cell in the bottle, at 32 ℃ of following incubation 4-8 days.The clarifying medium supernatant of plaque purification (people such as Durbin, virusology 235:323-332,1997 on the LLC-MK2 cell as previously mentioned; People such as Hall, virus research 22:173-184,1992; People such as Skiadopoulos, Journal of Virology 72:1762-8,1998, be incorporated herein by reference).Every kind of biology clone's recombinant virus is increasing twice under 32 ℃ in the LLC-MK2 cell, produce virus in order to further sign.Utilize polyethylene glycol precipitation concentrating virus (people such as Mbiguino from clarifying substratum, virological method magazine 31:161-170,1991, be incorporated herein by reference), and extract virosome RNA (vRNA) with Trizol reagent (Life Technologies).The Superscript II Preamplification system (Life Technologies) of hexabasic basic primer carries out reverse transcription to vDNA with containing at random.(Clontech CA) and justice (PIV nt 7108-7137) and antisense primer (PIV3 nt 10605-10576) amplified fragments are arranged, is used for restriction endonuclease and digests or sequential analysis with Advantage cDNA PCR test kit.Utilize agarose gel electrophoresis (Fig. 8) and sequencing analysis PCR fragment.The every kind of rPIV3 that obtains inserts mutant and contains the insertion of specifying size, with their biological property of postevaluation.
Example VII A contains GU or NCR and inserts segmental rHPIV3 virus duplicate the multistep growth curve in animal and tissue culture
RPIV3 GU is compared with rcp45L with rPIV3 wt with the growth in vitro character that NCR inserts mutant.As shown in Figure 9, contain replication rate and peak virus titer and the rPIV3 wt undistinguishable of every kind of rPIV3 of GU or NCR insertion, show that the sequence of inserting length at least 3918 nts does not influence viral replication in vitro.Contain rPIV the duplicating in hamster that GU inserts
Hamster intranasal vaccination 10
6.0TCID
50RPIV3wt, rcp45
LOr specified contain one of sudden change rPIV3 that GU inserts (table 10).After infection, take out lung and concha on the 4th day, and measure the levels of replication of every kind of virus.The big or small 168nt of being does not significantly reduce virus duplicating in the hamster respiratory tract to the insertion of the Gu of 1908nt.Yet, between the HN of wild-type PIV3 and L ORF, insert 3918 nt genetic units and cause virus duplicating in concha and lung to reduce by 5 and 25 times respectively.This shows that this big or small genetic unit inserts for the wild-type virus attenuation, and short size below the 2000nt, does not influence wild-type virus duplicating in the hamster respiratory tract according to appointment.Therefore, can change the attenuation level that GU length is determined the hope of PIV vaccine virus.Table 10.rPIV3 GU inserts the replication-competent virus of mutant in the hamster respiratory tract
aAverage virus titer (log
10TCID
50/ g ± S.E.
b)
Concha lung rPIV3 wt 5.9 ± 0.2 6.0 ± 0.2r168 nt GU ins 5.9 ± 0.1 6.4 ± 0.1r678 nt GU ins 6.1 ± 0.1 6.2 ± 0.1r996 nt GU ins 5.5 ± 0.2 5.4 ± 0.2r1428 nt GU ins 5.9 ± 0.1 5.3 ± 0.6r1908 nt GU ins 5.6 ± 0.1 5.7 ± 0.2r3918 nt GU ins 5.2 ± 0.2 4.6 ± 0.3rcp45L3.1 ± 0.0 1.7 ± 0.2r1908 nt GU ins/cp45
L1.8 ± 0.2 1.5 ± 0a. hamster, 8 every group, intranasal administration in the 0.1ml inoculum 10
6.0TCID
50Virus.After 4 days, take out lung and concha, and measure virus titer down at 32 ℃.B.S.E.: standard error.
As mentioned above, the HA gene of insertion Measles virus further makes every kind of virus for the hamster attenuation in rJS wild-type and attenuation cp45L virus.Because the HA mrna length of Measles virus is 1936 nt, we have checked that the gene of similar size inserts the influence that (1908 nt) duplicates rcp45L.1908 genes insert and are different from the insertion of Measles virus HA gene, because it can not synthesize big polypeptide.(the r1908 nt GU ins/cp45 in the table 10 when 1908nt GU insertion combines with cp45L polysaccharase amino-acid substitution
L), the attenuation in the upper respiratory tract strengthens about 20 times.Combine consideration, these discoveries show that the GU of the about 3918nt of length inserts and can make wild-type PIV3 virus for the hamster attenuation, and the GU of only about half of this size inserts the PIV3 vaccine candidate strain attenuation that can further make attenuation.Therefore, GU inserts and may have dual function in the design of recombiant vaccine.First effect is a kind of protective antigen of coding pathogenic agent, and second effect provides the attenuation phenotype.Contain rPIV the duplicating in hamster that HN gene 3 '-NCR inserts
The hamster intranasal vaccination contrasts virus or contains the rPIV3 virus (table 11) of the insertion sudden change of expansion HN gene 3 '-NCR length.Take out lung and concha after 4 days in inoculation, measure the levels of replication of virus in every kind of tissue as mentioned above.Size does not significantly reduce virus duplicating in the hamster respiratory tract (table 3) for 258nt inserts to the HN gene NCR of 1404nt.Yet, the insertion of 3126nt makes the upper respiratory tract of infected hamster and the virus titer in the lower respiratory tract reduce by 16 times, and 3894nt HN gene NCR insertion makes virus duplicating in the upper respiratory tract and lower respiratory tract reduce by 12 times, and prompting increases genome length also has attenuation to virus replication.Table 11.rPIV3 NCR inserts the replication-competent virus of mutant in the hamster respiratory tract
aAverage virus titer (log
10TCID
50/ g ± S.E.
b)
Concha lung rPIV3 wt 6.2 ± 0.1 6.4 ± 0.1r258 nt NCR ins 5.9 ± 0.1 6.5 ± 0.1r972 nt NCR ins 5.9 ± 0.1 6.6 ± 0.1r1404 nt NCR ins 5.9 ± 0.2 6.6 ± 0.1r3126 nt NCR ins 5.0 ± 0.1 5.2 ± 0.1r3894 nt NCR ins 5.1 ± 0.1 5.3 ± 0.1rcp45L3.4 ± 0.1 1.9 ± 0.2a. hamster, 8 every group, intranasal administration in the 0.1ml inoculum 10
6.0TCID
50Virus.After 4 days, take out lung and concha, and measure virus titer down at 32 ℃.The b.S.E.=standard error.The evaluation of the temperature sensitivity level that GU and NCR insert
Measuring the efficiency of plating (EOP) (table 12) of rPIV under permission and non-permissive temperature on the LLC-MK2 individual layer as mentioned above.In the time of 32 ℃, contain size and insert and big or smallly have the plaque morphology that is similar to rPIV3wt to the virus of the NCR insertion of 3894nt for 258nt to the GU of 3918nt for 168nt.Yet, when 39 ℃ and higher temperature, contain all viruses of inserting sudden change and all have little plaque phenotype (table 12).Size is not temperature sensitive virus for 996nt when the GU of 3918nt insertion is created in 40 ℃.Yet, slight temperature sensitive virus when containing virus that 1404nt or bigger HN gene NCR insert and being created in 40 ℃, its temperature sensitivity gradient and insertion are in proportion.To cp45
LAdding 1908nt GU in the main chain inserts when being created in 38 ℃ and rcp45
LCompare the almost high 100 times virus of temperature sensitivity, prove that 1908nt GU inserts that the ts phenotype that sudden change causes with L gene ts cause adds and.Table 12.rPIV3 GU and NCR insert the plaque of mutant under permission and non-permissive temperature and form the virus titer (log of efficient virus under assigned temperature
10PFU/ml)
32 ℃ of 37 ℃ of 38 ℃ of 39 ℃ of 40 ℃ of rPIV3 wt, 7.8 ND ND, 7.4 7.5r168 nt GU ins, 7.8 ND ND 7.5
a6.7
aR678 nt GU ins 7.9 ND ND 7.3
a7.0
aR996 nt GU ins 7.7 ND ND 7.0
a6.3
aR1428 nt GU ins 7.8 ND ND 7.4
a6.4
aR1908 nt GU ins 7.6 ND ND 6.5
a6.0
aR3918 nt GU ins 6.3 ND ND 5.7
a5.0
aR258 nt NCR ins 8.1 ND ND 7.4
a7.5
aR972 nt NCR ins 8.2 ND ND 7.8
a7.8
aR1404 nt NCR ins 6.7 ND ND 5.2
a＜3.7r3126 nt NCR ins 7.4 ND ND 6.4
b4.5
aR3894 nt NCR ins 7.4 ND ND 5.3
a5.0
aRcp45
L7.8 7.3 6.0＜0.7 NDr1908 nt GU ins/cp45
L6.7 5.0
a3.0
a＜0.7 NDrcp45 8.1 6.7 5.7
a2.0
aNDa. incubation was counted plaque by immunoperoxidase staining after 6 days under assigned temperature.Numeric representation lower limit that mark with underscore and runic degree warm in nature, the titre during at this moment with 32 ℃ is compared, and the efficiency of plating of this moment reduces by 100 times at least, and this is defined as the temperature of ending of plaque formation.
Because r3918nt GU inserts mutant and r3126nt and r3894nt NCR insertion mutant and efficiently duplicates external, but duplicating in the hamster respiratory tract is restricted, these recons show new host range attenuation phenotype.
According to top embodiment, proved that reorganization HPIV3 (rHPIV3) provides a kind of effective carrier, is used to be expressed as the external source protective antigen of other extra gene, as Measles virus hemagglutinin (HA) glycoprotein gene.In another embodiment, rHPIV3-1 antigen embedded virus, a kind of PIV3 F and HN gene are replaced by the reorganization HPIV3 of its HPIV1 counterpart, and a kind of effective carrier is provided, and are used for HPIV2 hemagglutinin-neuraminidase (HN) glycoprotein.In all situations, design also makes up outer source coding sequence, makes it to be in a cover HPIV3 gene initial sum gene termination and transcribes under the signal control, in other extra gene insertion vector genome, and is expressed as isolating mRNA by the HPIV3 polysaccharase.
RHPIV3 or rHPIV3-1 insert fragment expression Measles virus HA by a kind of extra gene or HPIV2 HN glycoprotein is determined after duplicating through the number wheel stable.With the rHPIV3 carrier of expressing Measles virus HA or express hamster that the rHPIV3-1 carrier of HPIV2 HN glycoprotein infects and induce to HPIV3 and Measles virus or respectively to the protective immune response of HPIV1 and HPIV2.Therefore; a kind of rHPIV3 carrier of expression Measles virus protective antigen is induced the protective immune response to two kinds of human pathogens; that is, HPIV3 is the immunne response at the glycoprotein that exists in the carrier main chain, and Measles virus is served as reasons and inserts the HA protective antigen that the alia gene among the rHPIV3 is expressed.Therefore Measles virus glycoprotein is not incorporated in the infectivity HPIV3 vector virus, estimates that it expresses the tropism who does not change carrier, does not make it in the Measles virus specific antibody and responsive yet.Similarly; the antigenic a kind of rHPIV3-1 carrier of expression HPIV2 protectiveness HN is induced the protective immune response at two kinds of human pathogens; promptly; HPIV1 is the immunne response at the glycoprotein that exists in the carrier main chain, and HPIV2 serves as reasons and inserts the HA protective antigen that the alia gene among the rHPIV3-1 is expressed.
Example VII A I expresses a kind of rHPIV3 can reach three kinds of extra exogenous virus glycoprotein and induces and anti-ly can reach three kinds of viruses
Protection antibody
Modify a kind of recombinant vaccine virus and have several advantages to induce immunity to multiple pathogenic agent.Development is expressed at antigenic a kind of attenuation main chain of multiple pathogenic agent more feasible and quicker at other attenuated vaccine of branch of every kind of pathogenic agent than development.Every kind of pathogenic agent provides different attacks for the proof of operation, attenuation and security and efficient, and each of the range of pathogen of development attenuation form is a fearful job.Preparation, handle with to use a kind of vaccine virus also simpler, easier than bearing these activity with several different attenuated virus.The quantity that reduces virus vaccines also will help to simplify the complicated procedures of paediatrics immunity.Several attenuated virus can be used as a kind of mixture, but this makes vaccine development complicated, because every kind of composition must be respectively safe, so also be safety and effectively as mixture.A specific question using virus mixture is the common phenomenon of viral interferential, and wherein the virus of one or more in the mixture is disturbed duplicating of one or more other compositions.This can cause duplicating and immunogenic reduction of one or more compositions.Utilize a kind of carrier main chain can avoid this common problem.Equally, because some virus has special safety issue as Measles virus, compare with the mixture of the virus of attenuation respectively, wherein each must separate development and confirmation, and using a kind of more benign virus will be safer as PIV as having extra antigenic carrier.
In the present embodiment, make up reorganization HPIV, and as carrier, be used for for the vaccine virus development, having satisfied more than one the extra gene that duplicates with the immunogenicity feature.Particularly, present embodiment has been described design, structure, answer and the sign of the rHPIV3 that expresses a kind of, the two or three extra gene that derives from following tabulation: (i) hemagglutinin-neuraminidase (HN) of HPIV1 (Washington/20993/1964 strain); The (ii) HN of HPIV2 (V9412 strain); The (iii) hemagglutinin (HA) of Measles virus wild-type Edmonston strain; The 3918-nt that (iv) is called genetic unit (GU) translates reticent synthetic gene (the same).The gene that adds is inserted between the nucleoprotein (N) and phosphorprotein (P) gene of rHPIV3, between P and membranin (M) gene, or between HN and big polysaccharase (L) gene.Therefore, present disclosure proves, successfully utilizes the HPIV3 carrier that is modified to divalence, trivalent or tetravalent vaccine recon can induce multivalent immunogen, for example, and at the immunity of carrier itself and one or both other pathogenic agent.
Between N/P and P/M gene, insert HPIV1 HN and HPIV2 HN gene is following carries out: modify plasmid pUC119 (AflII N-P) by site-directed mutagenesis, a kind of subclone (Durbin of the anti-genome cDNA of HPIV3, Journal of Virology 74:6821-31,2000, be incorporated herein by reference), (CTAAAT becomes CTTAAG with the downstream non-coding region to (i) HPIV3 N gene, HPIV3 nts 1677-1682) or (ii) insert unique AflII site in the downstream non-coding region of HPIV3 P gene (TCAATC becomes CTTAAG, HPIV3 nts 3693-3698).Modify each AflII site by inserting an oligonucleotide duplex then, interstitial granules pUC (GE/GS-N-H) in producing respectively
N-PAnd pUC (GE/GS-N-H)
P-MThe duplex that inserts contains the HPIV3 gene and stops (IG) trinucleotide sequence and HPIV3 gene initial (GS) sequence between (GE) sequence, conservative gene, and they are to instruct Transcription Termination and initial cis acting signal (Figure 10) respectively.In multiple clone site, contain other unique restriction endonuclease sites, be beneficial to subsequently subclone and screening, comprise the NcoI and the HindIII site that are used to add HPIV1 and HPIV2 HN ORF.Therefore, the external source ORF that is inserted in the multiple clone site will be subjected to one group of HPIV3 to transcribe signal control, and be expressed as mRNA separately by the HPIV3 polysaccharase.Multiple clone site also contains a MluI site, is used for inserting where necessary the oligonucleotide of different lengths, so that complete insertion sequence meets 6 principle (people such as Calain, Journal of Virology 67:4822-30,1993; People such as Durbin, virusology 234:74-83,1997; 1999a; People such as Skiadopoulos, virusology 272:225-34,2000).
HPIV1 HN ORF can be used as NcoI-HindIII restricted fragment people such as (, Journal of Virology 72:2955-2961,1998) Tao of p38 ' Δ 31hc# 6, inserts pUC (GE/GS-N-H)
N-PAnd pUC (GE/GS-N-H)
P-MThe NcoI-HindIII site in, produce pUC 1HN respectively
N-PWith pUC 1HN
P-MIn unique MluI restriction site, insert the short oligonucleotide duplex, meet 6 principle to regulate sequence.Then these chimeric subgene group cDNA are cloned among the anti-genome cDNA p3/7 of the total length HPIV3 that is called as pFLCHPIV3 wt (131) 2G+, produce pFLC HPIV3 1HN respectively
N-PWith pFLC HPIV3 1HN
P-M(Figure 11 therefrom separates the plasmid from last second kind and the third recombinant virus).
HPIV2 HN ORF can be used in the NcoI-HindIII restricted fragment of p32Hnhc#31hc people such as (, Journal of Virology 72:2955-2961,1998) Tao, inserts pUC (GE/GS-N-H)
N-PAnd pUC (GE/GS-N-H)
P-MThe NcoI-HindIII site in, produce pUC2HN respectively
N-PWith pUC 2HN
P-MIn unique MluI restriction site, insert the short oligonucleotide duplex, meet 6 principle to regulate sequence.By mistake, the oligonucleotide of insertion is a kind of short Nucleotide, need make the genome of the virus of acquisition meet 6 principle.Therefore, contain all cDNA that HIV2 HN gene inserts and do not meet 6 principle.But, from every kind of cDNA, can both obtain virus.These chimeric subgene group cDNA are cloned among the anti-genome cDNA pFLC of the total length PIV3 HPIV3 wt, produce pFLC PIV3 2HN respectively
(N-P)With pFLC PIV3 2HN
(P-M)(Figure 11 therefrom separates the plasmid from last the 4th kind and the 5th kind recombinant virus).
Assemble other reorganization HPIV3 anti-genome cDNAs, what they can contain various combination and position in the HPIV3 main chain reaches three kinds of extra foreign genes (Figure 11).Assemble these anti-genome cDNAs by above-mentioned subgene group cDNA, wherein the HN of HPIV1 or HPIV2 is inserted between N and P gene or P and the M gene.Other subclones that are used to assemble contain Measles virus HA gene as mentioned above between P/M gene or HN/L gene.As mentioned above, the another kind of subclone that uses in assembling contains 3918nt GU between HN and L gene.
It is as follows to contain the segmental recon of two or more extra insertions: rHPIV3 1HN
N-P2HN
P-M(Figure 11, from last the 6th recon) contains the HPIV1 HN and the HPIV2 HN gene that insert respectively between N/P and the P/M gene; RHPIV3 1HN
N-P2HN
P-MHA
HN-L(Figure 11, the 7th recon) contains HPIV1HN, the HPIV2 HN and the Measles virus HA that insert respectively between N/P, P/M and the HN/L gene; RHPIV3 1HN
N-P2HN
P-M3918GU
HN-L(Figure 11, the most following) contains HPIV1 HN and the HPIV2HN gene that inserts respectively between N/P and the P/M gene, contains 3918-nt GU in addition and insert fragment between HN and L gene.
It should be noted that the penult of these constructs, rHPIV3 1HN
N-P2HN
P-MHA
HN-L(Figure 11, from last the 7th construct) contains the protective antigen of following four kinds of pathogenic agent: HPIV3 (HN and F), HPIV1 (HN), HPIV2 (HN) and Measles virus (HA).The length overall of inserting the exogenous array in this recon is about 5.5 kb, is 36% of HPIV3 genome total length 15462 nt.Last recon, rHPIV3-1HN
N-P2HN
P-MGU
HN-L(Figure 11, the most following), length is about 23kb.It is longer by 50% than wild-type HPIV3, and is all longer than all the biology deutero-described in the past or reorganization paramyxovirus.Contain recovery and replication in vitro that one, two or three extra genes insert segmental reorganization rHPIV3
The anti-genome cDNA of total length HPIV3 and support plasmid pTM (N), pTM (P no C) and pTM (L) and LipofectACE (Life Technologies that contain one or more extra genes of allos paramyxovirus protective antigen, Gaithersburg, MD) distinguish transfection 6 orifice plate (Costar together, Vambridge, MA) the HEp-2 monolayer culture thing in, utilize above-mentioned technology (people such as Durbin, virusology 235:323-332,1997 simultaneously; People such as Skiadopoulos, virusology 272:225-34,2000, be incorporated herein by reference), using the MVA-T7 cells infected, MVA-T7 is a kind of replication defect type vaccinia virus recon of the phage t7 polymerase protein of encoding.After 32 ℃ of following incubations can reach 4 days, with the transfection cutting 25cm that goes down to posterity
2T25 shakes on the LLC-MK2 monolayer culture thing in the bottle, and in 32 ℃ of following incubation cells 5 days.Further on 32 ℃ of viruses that will reclaim from cell conditioned medium liquid down go down to posterity the LLC-MK2 cell, amplicon virus.(1999a is incorporated herein by reference for people such as Skiadopoulos, Journal of Virology 73:1374-81) as previously mentioned, by plaque purification on the LLC-MK2 cell, the biology clone contains one or more foreign genes and inserts segmental rHPIV3.Amplification derives from the viral suspension of biology clone's virus on the LLC-MK2 cell, produces 10
7-10
9TCID
50The whole titre of/ml, this is similar to the general titre scope that obtains of rHPIV3.Measure recombinant virus 39 ℃ of abilities of growing down.It is shocking, contain several rHPIV3 (rHPIV3-1HN that one or more foreign genes insert
N-P, rHPIV3-1HN
N-P2HN
P-MHA
HN-L, rHPIV3-1HN
N-P2HN
P-M3918GU
HN-L) duplicating in the time of 39 ℃ compare limited 100-1000 doubly with duplicating under permissive temperature.
Isolated viral RNA (vRNA) (referring to, people such as Skiadopoulos, Journal of Virology 73:1374-81,1999a is incorporated herein by reference) from biology clone's reorganization embedded virus as mentioned above.It is used to use and the reverse transcription and the polymerase chain reaction that insert the special primer that the site links to each other as template.By restriction endonuclease digestion and the product analysing amplified, insert segmental the existence and identity to confirm each external source to the part dna sequencing of joining region.In all situations, all confirmed the correct sequence of expection.Express rHPIV3 duplicating in the hamster respiratory tract a kind of, two or three extra external source protective antigen
Show in the past, express a kind of rHPIV3 or rHPIV3-1 virus of extra viral protective antigen gene and can efficiently duplicate in vitro and in vivo, and induce at the protective immune response of two kinds of vector virus with the virus of extra antigen gene representative.Yet, do not know whether rHPIV can hold two or more extra genes, and keep and efficiently to duplicate and to induce ability in vitro and in vivo at two kinds of carriers and expressed extra antigenic protective immune response.Present embodiment shows that this is possible really.
Every group of 8 hamster intranasal vaccinations 10
6TCID
50Contain one or more extra foreign genes and insert segmental every kind of rHPIV3 or contrast virus (table 13).Infect and take out concha and lung after 4 days, as mentioned above by 32 ℃ of serial dilutions on LLC-MK2 monolayer culture thing, the virus that exists in the quantitative tissue homogenate (referring to, people such as Skiadopoulos, Journal of Virology 73:1374-81,1999a).Utilize guinea-pig red blood cell to detect virus by hemocyte absorption, the average virus titer of each group is expressed as log
10TCID
50(50% TCID/gram tissue ± SE).Table 13. contains that one or more extra genes insert fragments, the reorganization HPIV3 that expresses HPIV1, HPIV2 or Measles virus glycoprotein gene duplicates group number in the hamster upper respiratory tract and lower respiratory tract
aVirus
bAverage virus titer (log
10TCID
50/ g ± S.E.)
The concha titre reduces (log
10)
dThe lung titre reduces (log
10)
d1 rHPIV3 1HN
(N-P)4.5 ± 0.2 1.8 3.9 ± 0.2 3.02 rHPIV3 1HN
(P-M)3.5 ± 0.2 2.8 4.3 ± 0.2 2.33 rHPIV3 2HN
(N-P)5.4 ± 0.2 0.9 5.3 ± 0.3 1.64 rHPIV3 2HN
(P-M)6.3 ± 0.1 0.0 6.3 ± 0.5 0.65 rHPIV3 HA
(N-P)5.3 ± 0.2 1.0 5.8 ± 0.4 1.16 rHPIV3 HA
(P-M)6.0 ± 0.2 0.3 7.3 ± 0.2-0.47 rHPIV3 HA
(HN-L)6.0 ± 0.1 0.3 6.6 ± 0.2 0.38 rHPIV3 1HN
(N-P)2HN
(P-M)5.2 ± 0.1 1.1 5.0 ± 0.3 1.99 rHPIV3 1HN
(N-P)2HN
(N-P)HA
(HN-L)1.6 ± 0.1 4.7 2.5 ± 0.1 4.410 rHPIV3 1HN
(N-P)2HN
(N-P)3918GU
(HN-L)2.0 ± 0.3 4.3 1.8 ± 0.2 5.111 rHPIV3 cp45 4.6 ± 0.1 1.7 2.1 ± every group of 8 hamsters of 0.2 4.812 rHPIV3 wt, 6.3 ± 0.1-6.9 ± 0.1-a..B. every hamster inoculates 10 in the 0.1ml inoculum
6TCID
50Virus.C. by under 32 ℃ on LLC-MK2 monolayer culture thing serial dilution titre virus.D. compare the reduction of virus replication with rHPIV3 wt (group 12).
More than show, by between HPIV3 HN and the L gene, between N and the P gene or the rHPIV3 of the extra gene insertion fragment expression Measles virus HA between P and the M gene duplicating in the hamster upper respiratory tract and lower respiratory tract appropriate limited (about 10-20 times).This is confirmed in the present embodiment, wherein contains Measles virus HA and weakens as the rHPIV3 of the single extra gene between N/P, P/M or the HN/L and can reach 10 times (table 13, groups 5,6,7).Similarly, the appropriateness of duplicating that insertion HPIV2 HN gene is also shown in the hamster respiratory tract between HPIV3N and P gene or between P and the M gene reduces (about 10-20 doubly) (table 13, group 3,4).On the contrary, inserting HPIV1 HN gene between P and M gene or N and P gene causes duplicating in the hamster upper respiratory tract and lower respiratory tract to be reduced above about 100 times (table 13, groups 3 and 4).Because HPIV1HN, HPIV2 HN and Measles virus HA gene insert all roughly the same size (be respectively 1794nt, 1781nt, 1926nt), this can not be because the length of inserting.Therefore, the higher attenuation level of introducing the HPIV1HN gene and being provided may be because HPIV1 HN protein expression is special, other attenuations that the HPIV3 carrier is duplicated.Therefore, in some cases, as HPIV1HN, owing to insert another kind of gene, extra antigen can make rHPIV3 surpass the attenuation of appropriateness for the attenuation of hamster.
The inspection of the data of his-and-hers watches 13 shows, inserts the site and also works in the limit levels that chimeric rHPIV3 duplicates in the hamster respiratory tract.Between rHPIV3 N and P gene, insert Measles virus HA gene or HPIV2 HN gene and can make duplicating more in the hamster upper respiratory tract and lower respiratory tract reduce (table 13, comparative group 3 and group 4, group 5 and group 6) than between P and M gene, inserting.For the insertion of HPIV1 HN gene, not obvious to this site-specific attenuation that the HPIV3 carrier duplicates, this may be because covered by the special stronger attenuation of HPIV1 HN.
The chimeric recombinant virus of rHPIV3 shows a kind of attenuation gradient, and it is the function that extra gene inserts number of fragments.The virus that contains the gene of three kinds of interpolations shows the strongest effect, and duplicating in the upper respiratory tract of infected hamster and lower respiratory tract reduces about 10000-108000 doubly (table 13, group 9,10).Contain two kinds of genes and insert the moderate attenuation level of the chimeric recombinant virus demonstration of segmental rHPIV3, reduce about 12-80 doubly (table 3, group 8).Contain a kind of extra gene the chimeric recombinant virus of rHPIV3 (except contain HPIV1 HN gene) only reduce about 10-25 times (the group 3-7 in the table 13).Importantly, containing a kind of, two or three extra gene inserts the chimeric recombinant virus of segmental rHPIV3 and all duplicates in all animals.The maximum attenuation of these viruses promptly contains the virus of three kinds of extra genes, and duplicating in the upper respiratory tract and lower respiratory tract more weakened than rcp45 (group 11).Express the immunogenicity of rHPIV3 in hamster a kind of, two or three extra external source protective antigen
A kind of with containing as mentioned above, two or three extra gene inserts segmental HPIV1 wt, HPIV2 wt, rHPIV3 wt and rHPIV3 and infects hamster.Collect serum sample in immunity after 3 days and 28 days, and by in the special virus of HPIV1 or Measles virus and assay method, or the blood clotting that is used for HPIV3 or HPIV2 HN specific antibody suppresses (HJAI) assay method, mensuration HPIV1, HPIV2, HPIV3 or the special antibody (table 14) of Measles virus.Except the virus that contains three kinds of extra genes insertions, all rHPIV3 viruses all cause the strong immune response to the HPIV3 main chain.At rHPIV3 1HN
N-P2HN
N-PHA
HN-LOr rHPIV3 1HN
N-P2HN
N-P3918GU
HN-LTo reduce or do not take place may be because these viruses duplicating too in hamster weakened to immunne response in the hamster that infects.Equally, the antigenic immunne response to load in the virus that contains three kinds of foreign genes also maybe can not detect than low.On the contrary, the virus that contains one or both foreign genes insertions can be induced each additional antigenic immunne response, proves that the foreign gene of load has immunogenicity in hamster, and as at rHPIV3 1HN
N-P2HN
N-P(table 14; Group 11) the same among the embodiment, can be used for inducing to three kinds of different virus---the strong immune response of HPIV1, HPIV2 and HPIV3.Table 14 in hamster to the immunne response of the antigenic rHPIV3 carrier of one or more Additional Protections immunity of expressing HPIV1, HPIV2 or Measles virus
aThe anti-serum of specifying virus of group number virus
bAntibody titer (average log
2± SE)
HPIV3
cHPIV1
dHPIV2
cMeasles virus
f1 rHPIV3wt 10.00 ± 0---2 HPIV2wt＜2.0 ± 0-8.0 ± 0.0-3 HPIV1wt＜2.0 ± 0 5.4 ± 0.3--4 rHPIV3 HA
(N-P)9.5 ± 0.2--12.4 ± 0.45 rHPIV3 HA
(P-M)8.7 ± 1.4--11.8 ± 0.26 rHPIV3 HA
(HN-L)9.0 ± 0--8.1 ± 0.67 rHPIV3 1HN
(N-P)9.5 ± 0.2 3.4 ± 0.6--8 rHPIV3 1HN
(P-M)7.2 ± 0.8 2.7 ± 0.3--9 rHPIV3 2HN
(N-P)9.8 ± 0.5-9.3 ± 0.8-10 rHPIV3 2HN
(P-M)10.0 ± 0.5-8.3 ± 1.1-11 rHPIV3 1HN
(N-P)2HN
(N-P)9.6 ± 0.7 5.5 ± 0.4 8.3 ± 0.8-12 rHPIV3 1HN
(N-P)2HN
(N-P)HA
(HN-L)＜2.0 ± 0 1.0 ± 0.3＜2.0 ± 0.0＜313 rHPIV3 1HN
(N-P)2HN
(N-P)3918GU
(HN-L)4.3 ± 0.7 2.3 ± 0.6＜2.0 ± 0.0-14 rHPIV3 cp45 7.7 ± 0.2---a. intranasal vaccination 10
6TCID
50The average antibody of the hamster of rHPIV3 group (n=6) is replied, hemagglutinin-neuraminic acid albumen or the Measles virus hemagglutinin (HA) of these expressing virals HPIV1 (1HN), HPIV2 (2HN) of insertion between the N of rHPIV3 and P gene (N-P), P and M (P-M) or HN and L gene (HN-L).B. collected serum in preceding 3 days and back 28 days in immunity.C. the average hemagglutination inhibition antibody (HAI) of HPIV3 is tired.D. to the average NAT of HPIV1.E. to the average HAI antibody titer of HPIV2.F. (60% plaque reduces neutralization, PRN) to the average NAT of Measles virus.
Example I X
RHPIV3-NB is used for the proteic application of Measles virus HA as attenuated carrier
The conduct of the animal virus of attenuation is the basis in order to the Jennerian method of development vaccine at the application of the vaccine of antigen relevant people homologue in human body because host range limits.Find that 3 type bovine parainfluenza virus (BPIV3) Kansas (Ka) strains are compared with 3 type human parainfluenza viruses (HPIV3) and in rhesus monkey, duplicate 100-1000 restriction doubly, in human body, also show attenuation (people such as Coelingh, transmissible disease magazine 157:655-62,1988; People such as Karron, transmissible disease magazine 171:1107-14,1995b all is incorporated herein by reference).Once produced chimeric recombinant human haemadsorption virus 1 (HPIV3) virus of living in the past, nucleoprotein (N) open reading frame (ORF) that it contains BPIV3 Ka replaces HPIV3 N ORF.This chimeric recon was called as cKa-N (2000a is incorporated herein by reference for people such as Bailly, Journal of Virology 74:3188-3195) in the past, was called as rHPIV3-N herein
BShould study ORF in advance from exchange N, because in PIV3 albumen, BPIV3 and HPIV3 N albumen have amino acid sequence identity (the 85%) (people such as Bailly of moderate level, virogene (Virus Genes) 20:173-82,2000b is incorporated herein by reference), and show that this BPIV3/HPIV3 N recon is the (people such as Bailly who lives, Journal of Virology 74:3188-3195,2000a is incorporated herein by reference).This representative a kind of " improved Jennerian " method wherein has only a kind of gene subgroup in the vaccine virus to derive from the animal counterpart.RHPIV3-N
BAt LLC-MK2 monkey kidney and hunt for a short time grow in the bovine kidney cells titre suitable with the BPIV3 parental virus with rHPIV3 (people such as Bailly, Journal of Virology 74:3188-3195,2000a).Therefore, the proteic allos character of N can not stop rHPIV3-N
BExternal duplicating.Yet, rHPIV3-N
BDuplicating in rhesus monkey be restricted to the degree that is similar to the BPIV3 parental virus (people such as Bailly, Journal of Virology 74:3188-3195,2000a).This determines that BPIV3 N albumen is the restrictive determinative of host range that BPIV3 duplicates in primate.
So rHPIV3-N
BEmbedded virus combines the antigenic determinant of HPIV3 and host range restriction and the attenuation phenotype of BPIV3.Have in 5115 amino acid altogether between the N of HPIV3 and BPIV3 albumen 79 differences (people such as Bailly, virogene 20:173-82,2000b).Many in these 79 amino acid differences may cause rHPIV3-N
BHost range attenuation phenotype.Because the restriction of expection host range is based on a large amount of amino acid differences, so expection rHPIV3-N
BThe attenuation phenotype in addition in the body that prolongs, duplicate after to be still genetics stable.Although its duplicating in rhesus monkey is limited, rHPIV3-N
BCan induce the high-level resistance that monkey wild-type (wt) HPIV3 is attacked, the resistance of this level can't be distinguished mutually with wt rHPIV3 immunity is caused.RHPIV3-N
BInfectivity, attenuation and immunogenicity show, this novel embedded virus be as outstanding candidate's strain of HPIV3 vaccine (people such as Bailly, Journal of Virology 74:3188-3195,2000a).In addition, as described below, show that at this these embedded viruses are the outstanding candidate's strains that are used to express the attenuated carrier of Additional Protection antigen such as Measles virus HA.The carrier components of the embedded virus that produces can be induced the immunne response at HPIV3, and the extra gene of adding can be induced at its immunne response of allos pathogenic agent separately.In this specific embodiments, preparation can be induced the two valency attenuated vaccine viruses to the immunne response of HPIV3 and Measles virus simultaneously.
Show that more than rHPIV3 can be as expressing the proteic carrier of Measles virus hemagglutinin (HA).In two embodiment, express the rcp45 of Measles virus HA gene
LHA (N-P) and rcp45HA (HN-L) attenuated carrier contain the point mutation of three attenuation amino acid in the carrier main chain.RHPIV3-N of the present invention
BCarrier may be more stable than the carrier that has the attenuation phenotype owing to three amino acid point mutation.The same demonstration, the HA that inserts as extra gene in rHPIV3 can make wt HPIV3 and attenuation HPIV3cp45
LDuplicating in hamster weakens.In addition, in HPIV3, insert 1908nt gene insertion fragment and do not make wild-type main chain attenuation, contain p45 but can improve
LWhat the main chain of sudden change duplicated in hamster weakens level.Therefore, plan is to the rHPIV3-N of host range restriction
BInsert Measles virus HA gene in the virus, further to weaken it in external and/or intravital growth.Influence the insertion fragment of duplicating in external or the body for special vaccine of development such as rHPIV3-N
BHas problem.Particularly, the height-limited candidate's virus of replication in vitro will be difficult to produce, and may can not be used as vaccine by undue attenuation and duplicate height-limited candidate's virus in the body.Also do not know to express the rHPIV3-N of Measles virus HA glycoprotein
BEmbedded virus is called as rHPIV3-N
BHA
(P-M), in primate, whether have satisfied immunogenicity, because used all researchs of the HPIV3 that expresses HA all in rodent model, to carry out in the past to HPIV3 and Measles virus.
Present embodiment has described use reverse genetic technology in detail and has produced rHPIV3-N
BHA
(P-M), show surprisingly, to rHPIV3-N
BThe middle HA gene that inserts can not further limit its duplicating in rhesus monkey.The attenuation of inserting may be caused the N of the host range restriction of duplicating in primate
BThe genetic elements that exists in the gene is covered.On the contrary, rHPIV3-N
BHA
(P-M)Attenuation satisfactorily in rhesus monkey.Use rHPIV3-N
BHA
(P-M)The immunity rhesus monkey can be induced the resistance that the wtHPIV3 challenge virus is duplicated; and the high-level neutralizing antibody that stimulates anti-Measles virus, this level is known to have protectiveness (people such as Chen, transmissible disease magazine 162:1036-42 in human body; 1990, be incorporated herein by reference).PFLC HPIV3-N
BHA
(P-M)Structure, it is a kind of chimeric ox/anti-genome cDNA of people PIV3, coding replaces the BPIV3 N gene ORF of rHPIV3 N gene ORF, and inserts Measles virus HA gene between rHPIV3 P and the M gene as extra gene
With the anti-genome cDNA plasmid of two step structure total lengths pFLC HPIV3-N
BHA
(P-M)(Figure 12).The pLeft-N that makes up at first, in the past
BPlasmid contains 3 ' half section (HPIV3 nts 1-7437 of the anti-genome cDNA of HPIV3, one position, back is the intragenic XhoI of HN site), its HPIV3N ORF is replaced by ORF (people such as Bailly, the Journal of Virology 74:3188-3195 of BPIV3,2000a is incorporated herein by reference).Downcut the PshAI-NgoMIV fragment from this plasmid.Notice that the PshAI site is arranged in the site 2147 (seeing Figure 12) of the anti-genome sequence of HPIV3, and the NgoMIV site is present in the carrier sequence, so this all HPIV3 sequences in downstream, PshAI site have been removed.This fragment is replaced with the plasmid pLeft HA that made up in the past
(P-M)The PshAI-NgoMIV fragment, it contain HPIV3 transcribe under the signal control and insert HPIV3 N and the P gene between Measles virus HA ORF (Durbin, Journal of Virology 74:6821-31 2000, are incorporated herein by reference).This produces pLeft-N
BHA
P-MSubsequently, with pLeft-N
BHA
P-M11899 nt NgoMIV-XhoI fragments, it contains 3 ' half section of the anti-genome cDNA of HPIV3, comprise BPIV3 N gene ORF and Measles virus HA gene and insert fragment, be cloned in the NgoMIV-XhoI window of rRight, rRight is the plasmid (people such as Durbin of 5 ' half section (PIV3 nts 7462-15462) of the anti-genome cDNA of a kind of HPIV3 of coding, virusology 235:323-332,1997a).This produces pFLC HPIV3-N
BHA
P-M, a kind of plasmid that contains the anti-genome cDNA of total length of HPIV3, it contains BPIV3 N ORF and replaces HPIV3 N ORF, and contains between the P that inserts HPIV3 and the M gene the additionally Measles virus HA gene of gene of conduct.Express the recovery of the chimeric rHPIV3 of ox N gene and Measles virus HA gene
From using pFLC HPIV3-N
BHA
P-MReclaim rHPIV3-N in the HEp-2 cell of transfection
BHA
P-MFor realizing this point, use pFLC HPIV3-N
BHA
P-MWith support plasmid pTM (N), pTM (P no C), pTM (L) and LipofectACE (Life Technologies, Gaithersburg, MD) transfection 6 well culture plate (Costar together, Cambridge, MA) the HEp-2 cell in, simultaneously as mentioned above, use the MVA-T7 cells infected, MVA-T7 is a kind of replication defect type vaccinia virus recon of the phage t7 polymerase protein of encoding.At 32 ℃ of following incubations after 4 days, with the transfection cutting 25cm that goes down to posterity
2Shake on the LLC-MK2 in the bottle, and 32 ℃ of following incubations 5 days.By further on the LLC-MK2 cell, going down to posterity the virus that amplification is reclaimed under 32 ℃ from cell conditioned medium liquid.As mentioned above, by plaque purification on the LLC-MK2 cell, biology clone rHPIV3-N
BHA
P-MUnder 32 ℃ on LLC-MK2 monolayer culture thing amplification derive from the viral suspension of biology clone's virus.Isolated viral RNA (vRNA) from biology clone's reorganization embedded virus as mentioned above.Use across the special Oligonucleolide primers of BPIV3 N ORF or Measles virus HA gene carrying out RT-PCR, and as mentioned above by restriction endonuclease digestion and the analysing amplified cDNA of part dna sequencing.This has confirmed that BPIV3 N ORF displacement and the extra gene of Measles virus HA insert segmental existence.
As previously mentioned (Durbin, Journal of Virology 74:6821-31,2000, be incorporated herein by reference), by using special mouse monoclonal antibody and the mountain sheep anti mouse peroxidase link coupled antibody of Measles virus HA albumen, immunostaining is at rHPIV3-N
BHA
P-MThe plaque that forms on the LLC-MK2 monolayer culture thing that infects has confirmed the proteic expression of Measles virus HA at first.RHPIV3-N
BHA
P-MIn the rhesus monkey respiratory tract with rHPIV3-N
BIdentical level is duplicated
Determine that then Measles virus HA inserts segmental acquisition and whether significantly reduces rHPIV3-N
BDuplicating in the upper respiratory tract and lower respiratory tract is as viewed when inserting extra gene in the attenuation HPIV3 main chain that lacks the chimeric composition of ox.Also determine rHPIV3-N
BHA
P-MWhether efficiently duplicate, to induce immunne response in vivo HPIV3 and Measles virus.Compare rHPIV3-N
BHA
P-MWith rHPIV3-N
BParent and wt HPIV3 and wt BPIV3 duplicating in the rhesus monkey upper respiratory tract and lower respiratory tract (table 15).As previously mentioned (people such as Bailly, Journal of Virology 74:3188-3195,2000a), the seronegative rhesus monkey of HPIV3 and Measles virus simultaneously in the intranasal (IN) and interior (IT) approach of tracheae inoculate each position and contain 10 for one milliliter
5TCID
50The L15 substratum of viral suspension.Collect nasopharynx (NP) swab sample at metainfective the 1st day to the 10th day, and after infection, collected lavage of trachea (TL) sample on the the 2nd, 4,6,8,10 day.By 32 ℃ of serial dilutions on the LLC-MK2 cell monolayer, the virus that exists in quantitative NP and the TL sample, the titre of acquisition is expressed as log
10TCID
50/ ml (table 15).
This contrast shows, rHPIV3-N
BHA
(P-M)Embedded virus is levels of replication and its rHPIV3-N in the rhesus monkey upper respiratory tract and lower respiratory tract
BParental virus is identical.This levels of replication is also suitable with BPIV3 virus parent, proves rHPIV3-N
BHA
(P-M)Kept rHPIV3-N
BWith the attenuation phenotype of BPIV, unexpectedly, to rHPIV3-N
BInserting Measles virus HA gene in the genome can not make this virus duplicating further in the rhesus monkey respiratory tract weaken.Chimeric people/ox PIV3 duplicating satisfactorily in the rhesus monkey upper respiratory tract and lower respiratory tract that table 15 contains the Measles virus hemagglutinin gene weakened, and can induce the antibody of anti-HPIV3 and Measles virus, and protection antagonism HPIV3 wild-type virus is attacked
A. this research comprises in every group and accepts rHPIV3-N
BHA
(P-M)4 monkeys and accept rHPIV3wt, rHPIV3-N
BOr 2 monkeys of BPIV3 Ka.Remove and accept rHPIV3-N
BHA
(P-M)Group outside, data presented comprises from people such as Bailey, Journal of Virology 74:3188-3195,2000 and people such as Schmidt, Journal of Virology 74:8922-8929, the historical data of 2000 researchs of being reported.People such as b.Schmidt, Journal of Virology 74:8922-8929,2000 historical data.C. inoculate 10 in each position 1ml inoculum in the monkey nose and in the tracheae
5TCID
50Virus.Collected nasopharynx (NP) swab sample at metainfective the 1st to 10 day.The the 2nd, 4,6,8,10 day collection lavage of trachea (TL) sample after infection.Datedrawn no matter, the peak virus titer mean value .S.E.=standard error of every animal in a group. on the LLC-MK2 cell, carrying out titration of virus under 32 ℃.The detection limit of virus titer is 10 TCID
50/ ml.D. in this research, from monkey, to collect serum, and attacked animal with HPIV3 then in the 31st day after immunity. in the twice former research, the sampling in the 28th day after immunity is also attacked monkey.E. measure simultaneously this research that collect with serum former twice research.Serum HIA titre is expressed as average log reciprocal
2± standard error, SE.F. the 59th day with parenteral (IM) use 10
6Pfu Measles virus Moraten vaccine strain immune animal is (that is, immunity for the first time is after 87 days) collection serum after .28 days.Data presented is available from the sample of only collecting from animal in this research.The average NAT of anti-wild-type Measles virus is expressed as average log reciprocal
2Standard error.PRN, plaque reduces neutralization.G. the immunity back is 28 or 31 days, 10 in the monkey nose and in each position 1ml inoculum of the interior inoculation of tracheae
6TCID
50Wt HPIV3.After attack, collected NP and TL sample on the the 0th, 2,4,6,8 day.The NP that obtained at the 0th day and the titre＜2.0log of TL sample
10TCID
50/ ml.H. except that the 5th group, data presented is studied from this.Use rHPIV3-N
BHA
(P-M)The high-titer antibody that the immunity rhesus monkey can be induced anti-HPIV3 and Measles virus, and protection monkey antagonism HPIV3 attacks
| Group number immunity virus aAnimal number a | To replying of immunity | The the 28th or 31 day to replying that HPIV3wt attacks | The 59th day to using replying of measles virus vaccines (Moraten) | ||||
| Virus replication | Serum antibody response | Virus replication | Serum antibody response | Serum antibody response | |||
| The average peak virus titer c(log 2±TCID 50/ml±SE) | Serum HAI antibody titer (the average log reciprocal of the 28/31st day anti-HPIV3 2± ????SE) d，e | The average antibody of anti-Measles virus tire (60% PRFN, average log reciprocal 2± SE) (back 31 days of immunity) f | Average HPIV3 virus peak titre g (log 10±TCID 50/ml±SE) | The HAI antibody titer of the 56/59th day anti-HPIV3 (average log reciprocal 2±SE) h | The average antibody of anti-Measles virus tire (60% PRFN, average log reciprocal 2± SE) (back 87 days of immunity for the first time) f | ||
| NP swab lavage of trachea | The NP swab | Lavage of trachea | |||||
| 1????rHPIV3?wt???????6 2????rHPIV3-N B???????8 3????rHPIV3-N B??????4 ?????HA (P-M)4????BPIV3?Ka????????8 5????none b??????????4 | ??4.9±0.4????3.2±0.6 ??2.6±0.6????2.0±0.4 ??2.2±0.6????2.8±0.6 ??2.3±0.2????1.9±0.2 ?????ND??????????ND | ???9.3±0.6 ???7.3±0.3 ???6.8±0.3 ???5.0±0.4 ?????＜2 | ??＜5.5±0.0 ??＜5.5±0.0 ??9.6±0.5 ????ND ????ND | ????0.5±0.0 ????1.4±0.9 ????1.2±0.7 ????2.9±0.3 ????4.5±0.3 | ??0.5±0.0 ??0.5±0.0 ??2.3±0.2 ??2.0±0.5 ??4.5±0.2 | ????12.0±0.0 ????9±1.0 ????11.5±0.3 ????11.5±0.3 ????12.0±0.6 | ????8.2±0.8 ????10.1±0.4 ????10.2±0.4 ????ND ????ND |
Use rHPIV3-N
BHA
(P-M)The rhesus monkey of immunity can produce the serum antibody (table 15) of high-caliber anti-HPIV3 and Measles virus.Use guinea-pig red blood cell suppress to measure (HAI) quantitatively serum HPIV3 antibody (Durbin, Journal of Virology 74:6821-31 2000, are incorporated herein by reference) by hemagglutinin as previously mentioned, tiring is expressed as average inverse (reciprocal) log
2± SE.RHPIV3-N
BHA
P-MAnd rHPIV3-N
BCan induce high-caliber anti-HPIV3 serum HAI antibody, prove that these attenuation recons can be induced the antigenic strong immunization of HPIV3 carrier main chain is replied.Also find to use rHPIV3-N
BHA
P-MThe rhesus monkey of immunity can produce high-caliber serum Measles virus neutralizing antibody in immunity after 31 days; the required level of horizontal exceeding protection human body antagonism viral infection of measles (people such as Chen; transmissible disease magazine 162:6821-31,2000), tiring is expressed as average log reciprocal
2± SE (table 15).
In order relatively to use attenuated live rHPIV3-N
BHA
P-MWith rHPIV3-N
BThe ability of the protection that antagonism wt HPIV3 is provided is infected in virus vaccines candidate strain, primary infection after 31 days with 10
6TCID
50Wt HPIV3 IN and IT attack monkey (table 15).After attack the 2nd, 4,6,8 day collect Nasopharyngeal swabs and lavage of trachea sample.Virus by existing in the quantitative sample of serial dilution on LLC-MK2 monolayer culture thing as mentioned above.RHPIV3-N
BHA
P-MAnd rHPIV3-N
BProtection suitable, that high-caliber antagonism wt HPIV3 attacks is provided, and wt HPIV3 is doubly being shown this point by the duplicating reduction 100-1000 in the respiratory tract of the monkey of immunity.This proof is inserted Measles virus HA gene and can not be reduced in the HPIV3 glycoprotein inductive protection level that exists in the attenuated virus carrier main chain in chimeric ox/people PIV3.
Compare rHPIV3-N then
BHA
P-MWith the immunogenicity of Moraten strain in rhesus monkey of the live,attenuated measles virus vaccine of permitting, the Moraten strain is the kind that PIV3 and Measles virus are all efficiently duplicated.At the 59th day, with 10
6RHPIV3 or rHPIV3-N are infected in pfu live,attenuated measles virus vaccine Moraten strain parenteral (IM) immunity in advance
BHA
P-MRhesus monkey, gathered serum sample at the 87th day, and analyze the neutralizing antibody (table 15) of anti-Measles virus.Before accepting the Moraten vaccine, do not contact (table 15, group 1 and group 2) in the animal of Measles virus, Moraten vaccine institute inductive measles specific antibody tire roughly with at rHPIV3-N
BHA
P-M(table 15, the group 2) of observing in the animal of immunity is identical.Therefore, express the rHPIV3-N of HA glycoprotein Measles virus
BHA
P-MCarrier induces the ability of virucidin suitable with the Moraten vaccine in primate.
RHPIV3-N
BHA
P-MA significant advantage that is better than the Moraten vaccine as measles virus vaccines is, the PIV carrier can intranasal in approach use, and live,attenuated measles virus vaccine can not infect by this approach, this may be the attenuation cultivated of its pair cell and the result of adaptation.This makes can use rHPIV3-N in early days in infancy
BHA
P-MImmunity, this stage is because the neutralization of maternal antibody and immunosuppressive action and can not be with the age group of current live,attenuated measles virus vaccine such as Moraten strain immunity (Durbin, Journal of Virology 74:6821-31 2000, are incorporated herein by reference).Also narrated other advantages above, comprised PIV carrier well-grown in cell culture, lack mixing of Measles virus HA in virosome, this will stop the tropism who changes the PIV carrier, and stop the immunosuppression of Measles virus inductive.
Can not inoculate viral infection of measles effectively causes the whole world to have above 2700 death of child every day.RHPIV3-N
BHA
(P-M)Candidate vaccine provides the main diseases of serious pediatric disease because of, the i.e. unique opportunity of HPIV3 and Measles virus immunity.The measles virus vaccines that are different from current permission, we expect to use chimeric rHPIV3-N
BHA
(P-M)And other people-Niu chimeric vector construct induces in baby and the children below 6 months the strong immunization of for example Measles virus replied, they express the major antigen determinant (Durbin of Measles virus or other allos pathogenic agent, Journal of Virology 74:6821-31,2000).Need a kind of effective immunization strategy that is used for baby and children, to satisfy the purpose of the World Health Organization's to 2010 year elimination measles.Particularly, in order to eradicate, it is favourable using the measles virus vaccines that do not comprise the infectivity Measles virus.
The application of body
For using in the present invention, make up reorganization chimeric people-Niu PIV, wherein BPIV3 F and HN gene are replaced by the gene of HPIV3.This chimeric ox-Human virus rB/HPIV3 that recombinates is presented at and has the ability fully in the cell culture to duplicate, and in rhesus monkey, show the limited attenuation phenotypic characteristic of host range of BPIV3, and (people such as Schmidt is disclosed in the U.S. Patent Application Serial 09/586,479 of application on June 1st, 2000 to have hyperimmunization originality and protectiveness; People such as Schmidt, Journal of Virology 74:8922-9,2000, be incorporated herein by reference).This is another example of " improved Jennerian " method, and is useful in the compositions and methods of the invention, but in this case, and a complete set of virus " inside " gene source is in BPIV3, and has only antigenic determinant to derive from HPIV3.
As mentioned above, be different from each and must separate attenuation, confirmation and can be with the complex mixture of the interactional different virus of uncertain mode, preferred vaccine has many reality and security consideration based on a kind of PIV3 main chain.In addition, the restriction of the host range of BPIV3 provides a kind of attenuation phenotype that should be extremely stable.In the present embodiment, design, rescue also characterize reorganization chimeric people-Niu PIV3 (rB/HPIV3), its coding respiratory syncytial virus (RSV) G or F glycoprotein, and they are main RSV neutralization and protective antigen.This embodiment shows that rB/HPIV3 easily accepts external source RSV gene, and does not significantly reduce it at external or intravital duplicating efficiency, thereby is valuable candidate vaccine and carrier.It is the bad and problem of unstable of growth in vitro of RSV feature that this carrier will not exist.The chimeric rB/HPIV3 virus of coding reorganization, contain RSV subgroup A G or F ORF structure as the anti-genome cDNA of extra gene
Make up the full-length cDNA of BPIV3 Kansas strain, wherein the F of bovine viral and HN glycoprotein gene have been replaced by the corresponding gene (U.S. Patent Application Serial 09/586,479 of people such as Schmidt and application on June 1st, 2000 of HPIV3 JS strain (rB/HPIV3); People such as Schmidt, Journal of Virology 74:8922-9,2000, be incorporated herein by reference).For using in the present invention, modify this cDNA, make it contain other three unique restriction enzyme recognition site.Especially, introduce a BlpI site before, before the N gene termination sequence, introduce an AscI site, before P gene end sequence, introduce a NotI site at N ORF (Nucleotide (nt) 103-109).Introduce these restriction enzyme recognition site and be beneficial to the insertion of the extra gene of external source in chimeric B/HPIV3 viral genome.Design these sites, make them not destroy any BPIV3 and duplicate and transcribe cis-acting elements.This specific embodiments will be narrated to the insertion in BlpI site (Figure 13).
In order to insert in the promotor near-end BlpI site of B/HPIV3, RSV subgroup A glycoprotein gene G that describes before modifying and F (GenBank preserving number M74568) are (Figure 13).This strategy is that to express every kind of allos ORF be the other mRNA that separates, therefore be imported into make before it to BPIV3 gene start signal in the rB/HPIV3 genome, be important for BPIV3 gene termination signal afterwards.BlpI inserts the gene start signal (Figure 13) that the N gene is followed in the site.Therefore, in order to insert, need to insert a BlpI site and add BPIV3 gene termination signal, intergenic region, gene start signal and BlpI site at upstream termination, to modify RSV ORF in its downstream in this site.For RSV A G ORF, the forward PCR primer that uses is (5 ' to 3 ') AATTCGCTTAGCGATGTCCAAAAACAAGGACCAACGCACCGC (SEQID NO.30), reverse primer is (5 ' to 3 ') AAAAAGCTAAGCGCTAGCCTTTAATCCTAAGTTTTTCTTACTTTTTTTACTACTGG CGTGGTGTGTTGGGTGGAGATGAAGGTTGTGATGGG (SEQ ID NO.31) (BlpI lines out below the site, and ORF translation initiation and termination triplet are runic).For RSV A F ORF, the forward PCR primer that uses is (5 ' to 3 ') AAAGGCCTGCTTAGCAAAAAGCTAGCACAATGGAGTTGCTAATCCTCAAAGCAAAT GCAATTACC (SEQ ID NO.32), reverse primer is (5 ' to 3 ') AAAAGCTAAGCGCTAGCTTCTTTAATCCTAAGTTTTTCTTACTTTTATTAGTTACT AAATGCAATATTATTTATACCACTCAGTTGATC (SEQ IDNO.33) (BlpI lines out below the site, and ORF translation initiation and termination triplet are runic).
With BlpI digestion PCR product, and be cloned into the standard molecule clone technology in the full length cDNA clone of modification.The full-length cDNA that contains RSV A G ORF that obtains is named as pB/HPIV3-G
A1, the plasmid that contains F ORF is named as pB/HPIV3-F
A1.Utilize restriction enzyme digestion and automatic sequencing to confirm the nucleotide sequence of every kind of gene of insertion.All constructs are designed to all make that whole genome nucleotide length is 6 multiple, and this is the needs (people such as Calain, Journal of Virology 67:4822-30 1993, are incorporated herein by reference) of effective rna replicon.RB/HPIV3-G1 and rB/HPIV3-F1 are from the answer of cDNA
Respectively from cDNA pB/HPIV3-G
A1 and pB/HPIV3-F
A1 replys rB/HPIV3-G1 and rB/HPIV3-F1 virus.This realizes by method of describing in the past, wherein supports plasmid transfection HEp-2 cell together with separately anti-genome cDNA and BPIV3 N, P and L.Simultaneously with the vaccinia virus recombinant MVA strain cells infected of expressing the T7 rna polymerase gene.Clone regressive recombinant virus by last eventually continuously dilution biology in the Vero cell.By the viral RNA that separates the self-infection cell is carried out RT-PCR, restriction enzyme digestion subsequently and dna sequencing confirm to exist RSV G or the F gene that inserts in the main chain of regressive every kind of recombinant virus.The insertion gene in the regressive recombinant virus and the sequence of flanking region are identical with initial anti-genome cDNA.RB/HPIV3-G1 and rB/HPIV3-F1 virus are efficiently duplicated in cell culture
(people such as Bailly as previously mentioned, Journal of Virology 74:3188-3195,2000a, be incorporated herein by reference), infect the LLC-MK2 cell monolayer in triplicate by infection multiplicity (MOI) with 0.01, and sample is collected at the interval with 24 hours in 7 day time, determines rB/HPIV3-G1 and the rB/HPIV3-F1 many cycling depositions kinetics in the LLC-MK2 cell.These two kinds of viruses and BPIV3 Ka, HPIV3 JS, rBIV3 Ka and rB/HPIV3 are compared (Figure 14).Two kinds of parental virus that contain HPIV3 glycoprotein, as if promptly HPIV3 and rB/HPIV3 slightly duplicate soon than other.Yet these 6 kinds of whole titres that virus reached are similar, have only the replication of exception a: rB/HPIV3-F1 to compare reduction about 8 times (Figure 14) with other viruses.This may be the result who contains this big gene at the promotor proximal location, maybe may be the result of second kind of expressing fusion protein, or is these two kinds of reasons simultaneously.The proposition of a kind of possibility in back is because rB/HPIV3-F1 can induce big plasmodial discovery, and is viewed quite with the wild-type RSV infection, and greater than viewed with rB/HPIV3 or other parental virus.Comparatively speaking, rB/HPIV3-G1 brings out lower cytopathic effect and less synplasm in LLC-MK2, be similar to rB/HPIV3.But rB/HPIV3-F1 and rB/HPIV3-G1 can grow at least 10 in LLC-MK2 cell and Vero cell
7TCID
50The whole titre of/ml.This shows all permissions fully of growth of every kind of virus, and this will allow to save the production of vaccine of cost.RB/HPIV3-G1 and rB/HPIV3-F1 virus are efficiently duplicated in the hamster respiratory tract
Estimate the ability that rB/HPIV3-G1 and rB/HPIV3-F1 duplicate in the hamster upper respiratory tract and lower respiratory tract.RB/HPIV3 parental virus, and BPIV3 and the biologically-derived virus of HPIV3, parallel in contrast comparison (table 16).Every kind of virus is all with 10
6TCID
50The dosage intranasal administration, a winding is subjected to rB/HPIV3-G1 and rB/HPIV3-F1.After infection, killed every group animal in the 4th day and the 5th day, by the virus titer in serial dilution mensuration concha and the lung.The levels of replication of rB/HPIV3-G1 in respiratory tract extremely is similar to HPIV3 JS and BPIV3Ka, undistinguishable statistically.As if rB/HPIV3-F1 be replicated in the 4th day and the 5th day lower slightly than other, but compare with biological BPIV3 virus, this species diversity is not remarkable statistically, fully duplicate in primate that BPIV3 virus is former and the clinical study and can induce protective immune response (people such as Coelingh, virusology 162:137-143 1988; People such as Karron, paediatrics transmissible disease magazine 15:650-654,1996, all be incorporated herein by reference).Equally, at the 4th day, as if the virus titer of rB/HPIV3-G1 and rB/HPIV3-F1 polyinfection reduced in lower respiratory tract slightly, but not remarkable statistically.At the 5th day, one of contrast virus BPIV3 Ka duplicating slightly in lower respiratory tract reduced: this was not remarkable statistically yet, shows that these little differences may be unimportant.Therefore, as if rB/HPIV3-G1 and rB/HPIV3-F1 virus can be duplicated fully in vivo, have 0.9 kb G or the extra gene of 1.8 kb F although be right after promotor.Table 16 can efficiently duplicate immunity virus at the rB/HPIV3 that the promotor proximal location contains RSV G or the extra gene of F ORF conduct in the hamster respiratory tract
aThe 4th day average virus titer of number of animals
bThe 5th day average virus titer
b
(log
10?TCID
50/g±S.E.)
c??????(log
10?TCID
50/g±S.E.)
c
Concha lung concha lung rB/HPIV3-G1 6 5.9 ± 0.1 (AB) 5.1 ± 0.6 (A) 5.5 ± 0.2 (A) 5.6 ± 0.4 (AC) rB/HPIV3-F1 6 5.1 ± 0.3 (B) 4.6 ± 0.2 (A) 5.7 ± 0.2 (AB) 3.6 ± 0.2 (BD) rB/HPIV3-G1 ﹠amp; rB/HPIV3-F1 6 5.7±0.3 (BC) 4.3±0.8 (A) 5.6±0.2 (A) 5.9±0.2 (A)rB/H PIV3 6 6.2±0.2 (AC) 5.2±0.6 (A) 6.5±0.1 (B) 5.7±0.6 (AC)HPIV3 JS wild type 6 6.6±0.1 (A) 6.5±0.1 (A) 6.0±0.2 (AB) 6.0±0.4 (A)BPIV3 Ka wild type 6 5.8±0.1 (AB) 6.1±0.2 (A) 5.3±0.2 (A) 4.2±0.5 (CD)
aIn the hamster intranasal vaccination 0.1ml inoculum 10
6TCID
50Virus.
bAs point out that the 4th day or the 5th day kill animals after inoculation by in titration on LLC-MK2 (PIV3) or HEp-2 (RSV) cell under 32 ℃, are measured the virus titer in concha and the lung.The detection limit of virus is 10
2.45TCID
50/ gram tissue.The S.E.=standard error.
cBy the Tukey-Kramer check average virus titer is distributed to similar group (A, B, C, D).In each row, the average titers variant statistically (p＜0.05) of different letters, the titre that marks with biliteral is compared with the titre that marks with arbitrary letter does not have significant difference.The serum antibody of rB/HPIV3-G1 and anti-HPIV3 of rB/HPIV3-F1 virus induction and RSV
Infect hamster with rB/HPIV3-G1, rB/HPIV3-F1 or rB/HPIV3 as mentioned above.Another winding is subjected to rB/HPIV3-G1 and rB/HPIV3-F1, another group RSV intranasal infection.After infecting 5 days, collect serum sample, and, suppress (HAI) antibody test by hemagglutinin and measure HPIV3 HN specific antibody (table 18) by RSV F albumen or the special ELISA test determination RSV specific antibody (table 17) of RSV G albumen.Respectively by the F specificity of rB/HPIV3-F1 or rB/HPIV3-G1 virus induction or G specific antibody tire respectively 2 times to 4 times to the wild-type RSV inductive.The animal of inoculation rB/HPIV3-F1 and rB/HPIV3-G1 also has F specificity and the G specific antibody that height is tired.Except the high ELISA of anti-RSV G and F tires, among rB/HPIV3-G1 and the rB/HPIV3-F1 RSV that also induction ratio wt RSV institute inductive is higher and serum antibody titer (table 18).Every kind of virus all can be induced the PIV3 specific antibody of necessarily tiring, and it and parental virus rB/HPIV3 can't distinguish (table 18).Therefore, the rB/HPIV3 carrier that contains the F of RSV or G gene induces the strong immunization that RSV is inserted fragment and PIV carrier to reply.Table 17 can be induced anti-RSV G or the proteic antibody response immunity of F virus with expressing RSV G or F ORF as the rB/HPIV3 immunity hamster of extra gene
aThe proteic serum IgG of the anti-RSV F of the proteic serum of the anti-RSV G of every group animal
IgG ELISA titre
bThe ELISA titre
b
(average log reciprocal
2± S.E.) (average log reciprocal
2± S.E.)
The 26th day before log
2The 26th day log before increasing
2Increase rB/HPIV3-G1 12 6.0 ± 0.4
c12.5 ± 0.5 6.5 6.7 ± 0.5
c7.5 ± 0.5 0.8rB/HPIV3-F1 12 6.3 ± 0.3 7.2 ± 0.3 0.9 6.8 ± 0.3 16.2 ± 0.5 9.4rB/HPIV3-G1﹠amp; RB/HPIV3-F1 12 6.5 ± 0.6 12.0 ± 0.9 5.5 7.3 ± 0.5 14.7 ± 0.4 7.4rB/HPIV3 12 6.5 ± 0.4 8.0 ± 0.4 1.5 7.3 ± 0.7 8.3 ± 0.8 1.0RSV 12 6.8 ± 0.3 10.8 ± 0.4 4.0 7.3 ± 0.5 15.7 ± 0.4 8.2
aIn the hamster intranasal vaccination 0.1ml inoculum 10
6TCID
50Virus.
bAfter inoculation, gathered serum sample on the 26th day, as state by anti-FSV G of the special elisa assay of glycoprotein or the proteic antibody of F.
cThe non-special background level of titre representative antibody in this susceptibility ELISA of serum specimen before.The neutralizing antibody that table 18 can be induced anti-RSV with the rB/HPIV3 immunity hamster of expressing RSV G or F ORF, and the hemagglutinin of anti-HPIV3 suppresses (HAI) antibody mediated immunity virus
aEvery group animal is replied the serum neutralizing antibody of RSV
bSerum HAI antibody response to HPIV3
c
(average log reciprocal
2± S.E.)
d(average log reciprocal
2± S.E.)
d
The 26th day rB/HPIV3-G1 12≤3.3 10.0 ± 0.3 (A)≤2 10.0 ± 0.5 (A) rB/HPIV3-F1 12≤3.3 9.3 ± 0.5 (A)≤2 8.8 ± 0.1 (A) rB/HPIV3-G1﹠amp before the 26th day before; RB/HPIV3-F1 12≤3.3 10.8 ± 0.4 (A)≤2 8.8 ± 0.3 (A) rB/HPIV3 12≤3.3 0.8 ± 0.8 (B)≤2 9.5 ± 0.8 (A) RSV 12≤3.3 8.1 ± 1.2 (A)≤2≤2 (B)
aIn the hamster intranasal vaccination 0.1ml inoculum 10
6TCID
50Specify PIV3 or 10
6PFU RSV.
bAfter inoculation, gathered serum sample on the 26th day, reduce neutralization test by 60% plaque and measure antibody titer.
cAfter inoculation, gathered serum sample on the 26th day, measure antibody titer by the hemagglutinin inhibition test.
dBy the Tukey-Kramer check average virus titer is distributed to similar group (A, B).In each row, the average titer of different letters has significant difference (p＜0.05).RB/HPIV3-G1 and rB/HPIV3-F1 virus can be induced the resistance that HPIV3 and RSV challenge virus are duplicated
With rB/HPIV3, rB/HPIV3-G1, rB/HPIV3-F1 immunity hamster, or after 28 days by intranasal vaccination 10
6TCID
50HPIV3 or 10
6PFU RSV adds the strain of rB/HPIV3-F1 vaccine candidate with rB/HPIV3-G1 and attacks.Kill animals after 5 days is taken out concha and lung, measures virus titer (table 19).The animal of having accepted parent rB/HPIV3 virus or G1 and F1 derivative shows the high-level resistance that the HPIV3 challenge virus is duplicated, and does not have significant difference between experimental group.Accept the high-level resistance of the animal demonstration of rB/HPIV3-G1 or rB/HPIV3-F1 or these two kinds of viruses to the RSV challenge virus.As if the protection level of efficiency that rB/HPIV3-F1 virus is attacked RSV be lower than rB/HPIV3-G1 virus or RSV contrast more or less.Yet this species diversity does not have the significance difference.Therefore, the rB/HPIV3 carrier that contains RSV F or G gene can be induced level and the complete suitable protection usefulness of infectivity RSV.Table 19 can be induced the resistance immunity of HPIV3 and RSV attack viral after infecting 28 days with rB/HPIV3-G1 and/or rB/HPIV3-F1 immunity hamster
aThe average HPIV3 titre of number of animals
bAverage RSV titre
c
(log
10TCID
50/g±S.E.)
d????(log
10PFU/g±S.E.)
d
Concha lung concha lung rB/HPIV3-G1 6 2.3 ± 0.1 (A) 3.1 ± 0.2 (A) 1.9 ± 0.2 (AB)≤1.7 (A) rB/HPIV3-F1 6 2.6 ± 0.2 (A) 3.1 ± 0.1 (A) 2.9 ± 0.4 (BC) 2.1 ± 0.2 (A) rB/HPIV3-G1﹠amp; RB/HPIV3-F1 6 2.8 ± 0.2 (A) 2.8 ± 0.3 (A) 1.8 ± 0.1 (A) 1.9 ± 0.4 (A) rB/HPIV3 6 2.3 ± 0.5 (A) 3.6 ± 0.4 (A) 4.1 ± 0.5 (C) 3.5 ± 0.4 (B) RSV 6 5.6 ± 0.2 (B) 5.2 ± 0.2 (B) 1.9 ± 0.3 (AB)≤1.7 (A)
aIn every group 6 hamster intranasal vaccination 0.1ml inoculums 10
6TCID
50Specify PIV3 or 10
6PFU RSV.
bOn the LLC-MK2 cell, carry out the HPIV3 titration.The detection limit of virus is 10
1.7TCID
50/ gram tissue.
cMeasure the amount of RSV by plaque counting on the HEp-2 cell.The detection limit of virus is 10
1.7PFU/ restrains tissue.
dBy the Tukey-Kramer check average virus titer is distributed to similar group (A, B, C).In each row, the average titer of different letters has significant difference (p＜0.05).The titre that marks with biliteral is compared with the titre that marks with arbitrary letter does not have significant difference.
Embodiment XI
RB/HPIV3.1 is as the application that is used for PIV2 hemagglutinin HN and the proteic carrier of F
Chimeric rHPIV3-1 virus, it contains the HPIV3 main chain, and wherein HPIV3 HN and F gene have been replaced by its HPIV1 counterpart, as the proteic useful carrier of HPIV2 HN as extra gene.This chimeric vector, rHPIV3-1.2HN can induce the resistance that HPIV1 and HPIV2 are duplicated in this proof in hamster.These discoveries have illustrated the surprising handiness of PIV expression system.For example; the rHPIV3-1.2HN recombinant virus contains each the element that derives from three kinds of serotypes of HPIV that can cause important diseases: with the HN of serotype 1 and the internal gene of F glycoprotein gene bonded serotype 3, as the HN protective antigen of the serotype 2 of extra gene.
Present embodiment provides the another kind of method that produces based on the carrier bacterin of PIV, is used to provide protection antagonism PIV1 and PIV2.In the present embodiment, modify rB/HPIV3 by the corresponding protein that people PIV3 HN and F albumen is replaced into HPIV1.This virus is named as rHPIV3-1, contains PIV1 HN and the F glycoprotein part as the carrier main chain, is used for inducing neutralizing antibody and to the immunity of HPIV1.This virus is in the present embodiment as carrier, to express HN and the F albumen of HPIV2 separately or together as extra gene.Reclaim three kinds of viruses, show survival: rB/HPIV3.1-2F fully; RB/HPIV3.1-2HN; Or rB/HPIV3.1-2F, 2HN is all as extra genetic expression PIV2 F and/or HN gene.RB/HPIV3.1-2F, 2HN, by two kinds of extra genetic expression PIV2 F and/or HN albumen, and by carrier main chain expression PIV1 F and HN gene, thereby express two kinds of main protection antigen, that is, derive from a kind of PIV1 of virus and F and the HN glycoprotein of PIV2.This method makes protection usefulness the best of vaccine, and makes the production cost minimum, because it has only promptly realized immunogenic raising with a kind of virus.Comparing with the mixture of several viruses, also may be simpler, safer and more effective with a kind of multivalent virus immunity baby and children.The chimeric rB/HPIV3.1 virus of coding reorganization, contain HPIV2 F and HN gene structure as the anti-genome cDNA of extra gene
Make up full-length cDNA as previously mentioned, wherein the F of bovine viral and HN glycoprotein gene have been replaced by the corresponding gene (people such as Schmidt, Journal of Virology 74:8922-9 2000, are incorporated herein by reference) of HPIV3 JS strain (rB/HPIV3).Modify this cDNA, make it to contain other three unique restriction enzyme recognition site (Figure 15).Especially, introduce a BlpI site before, before N gene end sequence, introduce an AscI site (nt 1676-83), before P gene end sequence, introduce a NotI site (nt3674-3681) at N ORF (Nucleotide (nt) 103-109).Subsequently, the corresponding gene that F and the HN glycoprotein gene of rB/HPIV3 is replaced into HPIV1.For realizing this point, the subclone 3.1hcR6 of the rHPIV3-1 full-length cDNA that PCR mutagenesis was described in the past (people such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference), it contains at HPIV3 transcribes the HPIV1 F under the signal control and the ORF of HN glycoprotein gene, before the F gene, produce a SgrAI restriction enzyme recognition site through PCR mutagenesis, before HN gene end sequence, produce a BsiWI site, this is similar to the position in SgrAI that introduces among the forward direction rB/HPIV3 and BsiWI site (people such as Schmidt, Journal of Virology 74:8922-9,2000).The mutagenesis forward primer that is used for producing the SgrAI site is (5 ' to 3 ') CGGCCGTGACGCGTCTCCGCACCGGTGTATTAAGCCGAAGCAAA (SEQ ID NO.34) (SgrAI lines out below the site), and the mutagenesis reverse primer is (5 ' to 3 ') CCCGAGCACGCTTTGCTCCTAAGTTTTTTATATTTCCCGTACGTCTATTGTCTGAT TGC (SEQ ID NO.35) (BsiWI lines out below the site).With SgrAI and BsiWI site the HPIV3 F among the rB/HPIV3 and HN gene are replaced with HPIV1 F and HN gene from the 3.1hcR6 plasmid of modifying as the unique DNA fragment.This produces the anti-genome cDNA pB/HPIV3.1 of total length, and it contains in the background from BPIV3 at HPIV3 and transcribes HPIV1 F and HN open reading frame under the signal control.
Next step, in order to insert pB/HPIV3.1 respectively in NotI and AscI site, HPIV2 F that describes before modifying and G open reading frame (GenBank preserving number AF213351 and AF213352) are (Figure 15).This strategy is used for expressing PIV2 F and HN ORF is the other mRNA that separates, therefore be imported into make before it to PIV3 gene start signal in the rB/HPIV3 genome, be important for PIV3 gene termination signal afterwards.NotI inserts the site before the gene of P gene stops letter (Figure 15).Therefore,, need add a NotI site at its downstream end, modify HPIV2 F ORF by inserting a NotI site and add BPIV3 gene termination signal, intergenic region and gene start signal at upstream termination in order to insert in this site.For HPIV2 F ORF, the forward PCR primer that uses is (5 ' to 3 ') AAAATATAGCGGCCGCAAGTAAGAAAAACTTAGGATTAAAGGCGGATGGATCACCT GCATCCAATGATAGTATGCATTTTTGTTATGTACACTGG (SEQ ID NO.36), reverse primer is (5 ' to 3 ') AAAATATAGCGGCCGCTTTTACTAAGATATCCCATATATGTTTCCATGATTGTTC TTGGAAAAGACGGCAGG (SEQ ID NO.37) (NotI lines out below the site, and ORF translation initiation and termination triplet are runic).For HPIV2 HNORF, add with as above for the described identical cis-acting elements of HPIV2 F, but replace NotI, add an AscI site inserting segmental each side, be beneficial in the N-P gene connects, clone.The forward PCR primer that uses is (5 ' to 3 ') GGAAAGGCGCGCCAAAGTAAGAAAAACTTAGGATTAAAGGCGGATGGAAGATTACA GCAATCTATCTCTTAAATCAATTCC (SEQ ID NO.38), and reverse primer is (5 ' to 3 ') GGAAAGGCGCGCCAAAATTAAAGCATTAGTTCCCTTAAAAATGGTATTATTTGG (SEQ ID NO.39).
With NotI (HPIV2 F inserts fragment) or AscI (HPIV2 HN inserts fragment) digestion PCR product, and be cloned into the standard molecule clone technology in the full length cDNA clone of modification.The full-length cDNA that contains HPIV2 F ORF that obtains is named as pB/HPIV3.1-2F, and the full-length cDNA that contains HPIV2 HN ORF is named as pB/HPIV3.1-2HN, contains F and HN and inserts segmental plasmid and be named as pB/HPIV3.1-2F, 2HN.Utilize restriction enzyme digestion and automatic sequencing to confirm the nucleotide sequence of every kind of gene of insertion.All constructs are designed to all make that whole genome nucleotide length is 6 multiple, and this is the needs (people such as Calain, Journal of Virology 67:4822-30 1993, are incorporated herein by reference) of effective rna replicon.The genome nucleotide length that obtains is as follows: pB/HPIV3.1:15492; PB/HPIV3.1-2HN:17250; PB/HPIV3.1-2F:17190; PB/HPIV3.1-2F, 2HN:18948.RB/HPIV3.1, rB/HPIV3.1-2F, rB/HPIV3.1-2HN and rB/HPIV3.1-2F, the 2HN embedded virus is by the acquisition of cDNA
From cDNA pB/HPIV3.1, pB/HPIV3.1-2F, pB/HPIV3.1-2HN and pB/HPIV3.1-2F, 2HN obtains rB/HPIV3.1, rB/HPIV3.1-2F, rB/HPIV3.1-2HN and rB/HPIV3.1-2F, 2HN embedded virus respectively.This realizes by method of describing in the past, wherein supports plasmid transfection HEp-2 cell together with separately anti-genome cDNA and BPIV3 N, P and L.Simultaneously with the vaccinia virus recombinant MVA strain cells infected of expressing the T7 rna polymerase gene.People such as (, Journal of Virology 72:2955-2961,1998) Tao as previously mentioned adds pig trypsinase to activate HPIV1 F albumen in cell culture medium.Clone the recombinant virus that obtains by last eventually continuously dilution biology in the Vero cell.All recombinant viruses all can efficiently duplicate, and induce CPE in 5 days inherent Vero cells, and to make cell monolayer be that hemocyte absorption is positive.By the viral RNA that separates the self-infection cell is carried out RT-PCR, restriction enzyme digestion subsequently and dna sequencing confirm to exist HPIV2 F and the HN gene that inserts in the main chain of the every kind of recombinant virus that obtains.Insertion gene in the recombinant virus that obtains is identical with initial anti-genome cDNA with the sequence of flanking region.
Embodiment XIIrHPIV3-1 cp45
LPurposes as Measles virus hemagglutinin (HA) protein carrier: continuous immunity
The development of strategy
More than shown chimeric rHPIV3-1 virus, it contains the HPIV3 main chain that HPIV3 HN and F gene have been replaced by the HPIV1 counterpart, can be used as the proteic useful carrier of HPIV2 HN as extra gene.This chimeric vector, rHPIV3-1.2HN can induce the resistance that HPIV1 and HPIV2 are duplicated in hamster.This discovery has illustrated the surprising handiness of PIV expression system.For example, this special virus, rHPIV3-1.2HN contains each the element that derives from three kinds of serotypes of HPIV: with the HN of serotype 1 and the internal gene of F glycoprotein gene bonded serotype 3, as the HN protective antigen of the serotype 2 of extra gene.Also prepare another kind of derivative, rHPIV3-1.2HNcp45
L, it contains the attenuation sudden change from the strain of cp45 HPIV3 vaccine candidate.
Therefore, the PIV carrier can be described to contain three kinds of compositions: the interior carrier backbone groups because of, can contain attenuation sudden change as desired; The carrier glycoprotein gene, it can be identical or allos serotype; The extra gene of one or more of the protective antigen of other pathogenic agent of encoding.In most applications, these extra antigens do not mix in the virosome, therefore can not change neutralization or tropism's feature of virus.Therefore, every kind of PIV carrier is two valencys or polyvalent vaccine, and wherein carrier itself can induce counterweight to want the immunity of human pathogen, and every kind of extra antigen can be induced the immunity to another kind of pathogenic agent.
In the present embodiment, utilize rHPIV3-1 virus and attenuation rHPIV3-1cp45 thereof
LDerivative has further proved the handiness of PIV carrier system as the Measles virus HA of vector expression as extra gene.This provides the new bivalent vaccine candidate strain of a kind of HPIV1 of being used for and Measles virus.Therefore, Measles virus HA can be with rHPIV3 that contains serotype 3 antigenic determinants and the load of attenuation derivative thereof, or with rHPIV3-1 that contains serotype 1 antigenic determinant and the load of attenuation derivative thereof.
Three kinds of serotypes (1,2,3) of it should be noted that HPIV do not cause tangible cross protection, and each represents a kind of important human pathogen that needs vaccine.This has increased available three kinds of serotypes at PIV and at allos pathogenic agent load protection antigen continuous immunity baby's possibility.Particularly, with the carrier preimmunization of the antigenic determinant that contains allos serotype, should influence or the not influence PIV carrier immunity that contains a kind of serotype antigen determinant minimumly.This provides at extra antigen and three kinds of chances that HPIV serotype is carried out continuous immunity and reinforcement (preferentially with 4-6 week or longer interval), and these genes can be expressed in the carrier main chain or the extra genetic expression of conduct.
Present embodiment has described reverse genetic technical development attenuated live HPIV1 candidate vaccine rHPIV3-1HA in detail
P-MCp45
LApplication, its is as the extra main Measles virus protective antigen of genetic expression, HA glycoprotein (Durbin; Journal of Virology 74:6821-31; 2000, be incorporated herein by reference), in baby and children, to be used for inducing immunne response to Measles virus and HPIV1.Also develop a kind of continuous immunity program, wherein use rHPIV3 HA
P-MCp45
LCandidate vaccine (containing serotype 3 antigenic determinants) is used rHPIV3-1 HA subsequently
P-MCp45
LCandidate vaccine (containing serotype 1 antigenic determinant) immunity.Hamster with these virus immunities produces HPIV3 and the antigenic antibody of HPIV1 that exists in the anti-carrier main chain, also keeps the antigenic high-titer antibody of anti-load, and this antigen is the Measles virus HA by HPIV3 and the extra antigen presentation of HPIV1 candidate vaccine virus conduct.RHPIV3-1 HA as extra genetic expression Measles virus HA
(P-M)With rHPIV3-1 HA
(P-M)Cp45
L, wild-type and attenuation form the structure of rHPIV3-1
Make up two kinds of total length plasmids as mentioned above, pFLC HPIV3-1 HA
(P-M)With pFLCHPIV3-1 HA
(P-M)Cp45
L(Figure 16) (referring to, Durbin, Journal of Virology 74:6821-31,2000; People such as Skiadopoulos, Journal of Virology 72:1762-8,1998; People such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference).With above-mentioned pFLC HPIV3HA
(P-M)Make up pFLC HPIV3-1 HA
(P-M), wherein wild-type Measles virus Edmonston strain HA gene ORF inserts between the P and M gene of rHPIV3 as extra gene.With BspEI and SphI digestion pFLC HPIV3 HA
(P-M), separate the cDNA fragment that lacks 6487 bp BspEI-SphI sequences.Then, contain the anti-genome cDNA plasmid of the total length pFLC 2G+.hc that PIV1 F and HN ORF replace the corresponding ORF of HPIV3 (people such as Tao with BspEI and SphI digestion, Journal of Virology 72:2955-2961,1998), the 6541 bp fragments (plasmid nts 4830-11371) that will contain the HPIV1 glycoprotein gene in the HPIV3 main chain are inserted pFLC HPIV3HA
P-MThe BspEI-SphI window in, produce pFLC HPIV3-1 HA
P-M(Figure 15).The cp45L that exists among L gene ORF sudden change (coded amino acid displacement Ser-942 is that His, Leu-992 are that Phe and Thr-1558 are the point mutation of Ile) is the main ts of HPIV3 cp45 candidate vaccine and att determinative (people such as Skiadopoulos, Journal of Virology 72:1762-8,1998), demonstration causes rHPIV3-1 cp45 in the past
LDuplicating in the hamster respiratory tract weakens (people such as Tao, vaccine 17:1100-8,1999).Modify pFLC HPIV3-1 HA then
P-M, these three kinds of ts sudden changes that make it to encode produce pFLC HPIV3-1 HA
P-MCp45
L(Figure 16).This passes through to pFLCHPIV3-1 HA
P-MThe SphI-NgoMIV window in insert pFLC HPIV3-1 cp45
LSphI-NgoMIV restriction endonuclease fragment (plasmid nts 11317-15929) (people such as Skiadopoulos, Journal of Virology 72:1762-8,1998) and realize.RHPIV3-1 HA
(P-M)With rHPIV3-1 HA
(P-M)Cp45
LAcquisition
Use pFLC HPIV3-1 HA respectively
(P-M)Or pFLC HPIV3-1 HA
(P-M)Cp45
LWith support plasmid pTM (N), pTM (P no C), pTM (L) and LipofectACE (LifeTechnologies, Gaithersburg, MD) together, transfection 6 well culture plate (Costar, Cambridge, MA) the HEp-2 cell in, (people such as Skiadopoulos, vaccine 18:503-10 simultaneously as previously mentioned, 1999b, be incorporated herein by reference), use the MVA-T7 cells infected, MVA-T7 is a kind of replication defect type vaccinia virus recon of the phage t7 polymerase protein of encoding.Containing in the tryptic substratum incubation under 32 ℃ after 4 days, with the transfection cutting 25cm that goes down to posterity
2Shake on the LLC-MK2 in the bottle, and 32 ℃ of following incubations 5 days.Further containing the virus that goes down to posterity and from cell conditioned medium liquid, reclaim on the tryptic LLC-MK2 monolayer culture thing under 32 ℃, with amplicon virus.As previously mentioned (people such as Skiadopoulos, vaccine 18:503-10,1999b), and by in end dilution eventually on LLC-MK2 monolayer culture thing under 32 ℃, biology clone rPIV3-1 HA
P-MWith rPIV3-1 HA
P-MCp45
LAmplification derives from the viral suspension of biology clone's virus on LLC-MK2 monolayer culture thing.
Isolated viral RNA (vRNA) from biology clone's reorganization embedded virus as mentioned above.Use rHPIV3-1 HA
P-MOr rHPIV3-1 HA
P-MCp45
LVRNA inserts the special Oligonucleolide primers of fragment or cp45 sudden change as template with across HA gene in the L gene, carries out RT-PCR.Analyze the RT-PCR product by restriction endonuclease digestion and part dna sequencing as mentioned above to the PCR product.There is the cp45L transgenation in the Measles virus HA gene that existence is inserted between P that this has confirmed at rHPIV3-1 and the M gene and its attenuation derivative.RHPIV3-1 HA
(P-M)Cp45
LThe proof of attenuation phenotype in hamster
Every group of 6 golden Syria hamster intranasal vaccinations 10
6TCID
50RHPIV3-1, rHPIV3-1 HA
P-M, rHPIV3-1 cp45
LOr rHPIV3-1 HA
P-MCp45
LInoculate after 4 days and to take out concha and lung, and measure as previously mentioned virus titer (people such as Skiadopoulos, vaccine 18:503-10,1999b).Titre is expressed as average log
10TCID
50/ gram tissue (table 20).Reorganization rHPIV3-1 HA
P-MDuplicate with suitable level with its parent rHPIV3-1 wt, show that the another kind of transcriptional units that inserts coding HA gene ORF can not further make virus for the hamster attenuation.RHPIV3-1 HA
P-MCp45
LAnd rHPIV3-1 cp45
LThe parent duplicates with similar level in the upper respiratory tract and lower respiratory tract, shows rHPIV3-1 HA
P-MCp45
LDuplicating satisfactorily in hamster weakens, and can not further make chimeric rHPIV3-1 cp45 and insert measles HA ORF
LThe parental virus attenuation.The rPIV3-1 of table 20 wild-type and attenuation form and the replication-competent virus of rPIV3-1 HA virus in the hamster respiratory tract
aAverage virus titer
b(log
10TCID
50/ g ± S.E.)
Concha lung rPIV3-1 wt 6.3 ± 0.1 6.6 ± 0.2rPIV3-1 HA
P-M6.0 ± 0.1 5.7 ± 0.7rPIV3-1 cp45
L4.1 ± 0.2 1.8 ± 0.2rPIV3-1 HA
P-MCp45
L4.4 every group of 6 hamster intranasal vaccinations 10 of ± 0.2 1.9 ± 0.2a.
6TCID
50Specify virus.B.4 a day back gathers in the crops lung and concha.By in serial dilution on LLC-MK2 monolayer culture thing under 32 ℃, the virus that exists in the titration tissue homogenate.Guinea-pig red blood cell is used for hemocyte absorption.
A kind of with attenuation rHPIV3 HA
P-MCp45
LAttenuation rHPIV3-1 HA is used in chimeric candidate strain subsequently
P-MCp45
LThe continuous immunity program of vaccine candidate strain immunity can be induced the HPIV3 and the antigenic antibody of HPIV1 of anti-carrier main chain, and induces and keep the high-titer antibody of anti-total load antigen Measles virus HA.
Use rHPIV3-1 HA
P-MCp45
LImmunity hamster group can be induced the strong immunization of HPIV1 and Measles virus is replied (table 21, group 6), shows that rHPIV3-1 may be the effective carrier of Measles virus HA as rHPIV3.
Checked then and used rHPIV3 HA
P-MCp45
LWith rHPIV3-1 HA
P-MCp45
LThe feasibility of continuous immunity hamster.With 10
6TCID
50RHPIV3 HA
P-MCp45
L(table 21, group 1,2,3), rHPIV3 cp45
L(group 4) or L15 substratum contrast (group 5) immune hamster group (table 21).Immunity is after 5 days, with 10 for the first time
6TCID
50RHPIV3-1 HA
P-MCp45
L(group 1,4), rHPIV3-1 cp45
L(group 2,5) or the immune hamster group of L15 substratum contrast (group 3).Before immunity for the first time, immunity for the first time collects serum sample after 58 days and after immune 35 days for the second time.Use rHPIV3-1 cp45
LThe animal of immunity (table 21, group 4) produces the strong antibody response to HPIV3, uses rHPIV3-1 HA
P-MCp45
LThe animal of immunity (group 1,2,3) produces the strong antibody response to HPIV3 and Measles virus.Animal in the group 4 is used rHPIV3-1 cp45 in advance
LRHPIV3-1 HA was used in immunity subsequently at the 59th day
P-MCp45
LImmunity.When the 94th day measures, these animals contain anti-HPIV3 that height tires and Measles virus antibody and the anti-HPIV1 antibody of level from low to high.This shows, HPIV3-1 chimeric virus even under the situation that has the HPIV3 immunity, also can induce to the HPIV1 antigen of carrier with to the proteic immunne response of the HA of load, but animal to the immunity of HPIV3 in its immunogenicity have to a certain degree minimizing.As organize as indicated in the replying of hamster in 4 rHPIV3-1 HA
P-MCp45
LVaccine obviously has immunogenicity in advance to the animal of HPIV3 immunity.Used rHPIV3-1 cp45 at the 0th day
LThese animals of immunity are used rHPIV3-1 HA the 59th
P-MCp45
LAfter the immunity 35 days, produced the neutralizing antibody that the appropriate height of anti-Measles virus is tired at the 94th day.It should be noted that and at first use rHPIV3 HA
P-MCp45
LRHPIV3-1 HA is used in immunity then
P-MCp45
LThe hamster of immunity (group 1, table 21) reached than the independent rHPIV3 of using HA at the 94th day
P-MCp45
LThe higher Measles virus serum NAT of hamster group (group 3) of immunity shows rHPIV3-1 HA
P-MCp45
LCan be used at rHPIV3 HA
P-MCp45
LThe immunity back keeps high antimeasles serum neutralizing antibody of tiring.Because the hamster in the group 1 is being used rHPIV3 HA for the first time
P-MCp45
LThe antibody of the anti-Measles virus HA that generation this height in immunity back is tired therefore can not be at rHPIV3-1 HA
P-MCp45
LDetect these tire 4 times or more risings after the immunity.
For the mankind, can in preceding 4 months, use HPIV1 vaccine such as rHPIV3-1HA at life
P-MCp45
LAfter two months, use HPIV3 vaccine such as rHPIV3 HA
P-MCp45
L(1999b is incorporated herein by reference for people such as Skiadopoulos, vaccine 18:503-510).Different with rodent, the baby produces antibiosis especially and orders the antigenic low liter antibody of the viral glycoprotein of using in preceding 6 months, this is (people such as Karron, paediatrics transmissible disease magazine 14:10-6,1995a because the immunosuppression of immature, the maternal antibody of immunology and other factors; People such as Karron, transmissible disease magazine 172:1445-1450,1995b; People such as Murphy, clinical microbiology magazine (J.Clin.Microbiol.) 24:894-8,1986, all be incorporated herein by reference).Therefore need rHPIV3-1 HA most probably
P-MCp45
LTo the booster action of Measles virus HA antibody titer, use rHPIV3 HA in preceding 6 months at life
P-MCp45
LBe easy to observe among the baby of immunity.Present embodiment shows, with two kinds of different attenuated live PIV vaccine continuous immunity animals of serology of all expressing Measles virus HA is possible, with HPIV3 and the antigenic antibody of HPIV1 that produces anti-carrier main chain, and keep anti-load antigen---the high-titer antibody of Measles virus HA.Table 21 rHPIV3 HA
(P-M)Cp45
LUse rHPIV3-1 HA subsequently
(P-M)Cp45
LThe continuous immunity hamster can be induced the immunity to following three kinds of viruses, that is, and and HPIV1, HPIV3 and Measles virus, and high-level the Measles virus antibody titer that keeps
A. collected serum in preceding 5 days and back 58 days in immunity for the first time.Implement immunity for the second time after 59 days in immunity for the first time, and (the 94th day) collects serum once more after 35 days.B. average serum PIV3 HAI antibody titer is expressed as average log reciprocal
2± standard error SE.C. the average serum neutralizing antibody of anti-HPIV1 is expressed as average log reciprocal
2± SE.D. the average serum neutralizing antibody of anti-wild-type Measles virus is expressed as average log reciprocal
2± standard error, PRN, plaque reduces neutralization.
| Group number | Pool-size | The virus that use (the 0th day) during immunity for the first time | To immune for the first time immunne response | The virus that use (the 59th day) during immunity for the second time | To immune for the second time immunne response a | ||||
| The serum HAI antibody titer (log of anti-HPIV3 2±SE) b(the 58th day) | The serum antibody titer of anti-HPIV1 c(log 2± SE) (the 58th day) | The serum antibody titer of anti-Measles virus d(60％PRN，log 2± SE) (the 58th day) | The serum HAI antibody of anti-HPIV3 is imitated rank b(log 2± SE) (the 94th day) | The serum NAT of anti-HPIV1 c(log 2± SE) (the 94th day) | The serum antibody titer of anti-Measles virus d(60％PRN，log 2± SE) (the 94th day) | ||||
| ????1 | ????8 | ?rPIV3?HA (P-M)cp45 L | ????10.8±0.4 | ????≤0.5±0.0 | ????12.5±0.4 | ?rPIV3-1?HA (P-M)????cp45 L | ??11.5±0.5 | ??0.9±0.2 | ????13.1±0.3 |
| ????2 | ????8 | ?rPIV3?HA (P-M)cp45 L | ????10.9±0.4 | ????≤0.5±0.0 | ????13.2±0.4 | ??rPIV3-1?cp45 L | ??10.5±0.5 | ??1.2±0.3 | ????12.8±0.4 |
| ????3 | ????6 | ?rPIV3?HA (P-M)cp45 L | ????9.3±0.3 | ????≤0.5±0.0 | ????12.7±0.4 | ????none | ??9.6±0.9 | ??1.1±0.4 | ????12.3±0.2 |
| ????4 | ????8 | ?rPIV3?cp45 L | ????9.6±0.6 | ????≤0.5±0.0 | ????＜3.3 | ??rPIV3-1?HA (P-M)????cp45 L | ??9.0±0.7 | ??0.9±0.3 | ????7.3±0.3 |
| ????5 | ????6 | ????none | ????＜2±0.0 | ????≤0.5±0.0 | ????＜3.3 | ??rPIV3-1?cp45 L | ??＜2±0.0 | ??4.8±0.6 | ????＜3.3 |
| ????6 | ????8 | ?rPIV3-1?HA (P-M)cp45 L | ?????3.0±0.4 | ????10.5±0.4 | |||||
Embodiment XIII
Express the structure and the sign of the proteic chimeric HPIV3-2 vaccine recon of chimeric glycoprotein
Present embodiment has described in detail with the reverse genetic technology and has been created in the attenuated live PIV2 candidate vaccine virus of using among baby and the children.As described in for HPIV3-1 chimeric construct body, acquisition contains the chimeric PIV3-PIV2 virus of reorganization of total length PIV2 glycoprotein in wild-type PIV3 main chain preliminary trial does not produce infective virus.Yet, when with chimeric HN and F ORF rather than total length PIV2 ORF structure full-length cDNA, obtain the PIV2-PIV3 embedded virus of living.The virus that obtains, be called as rPIV3-2CT, wherein PIV2 extracellular domain and membrane-spanning domain and PIV3 cytoplasm domain merge, and rPIV3-2TM, wherein PIV2 extracellular domain and PIV3 stride film and cytoplasmic tail fusion, although have similarly---being different---external and interior phenotype of body.Therefore, successfully obtain the PIV2-PIV3 embedded virus and as if only need the HN of PIV3 or the kytoplasm tail of F glycoprotein.
RPIV3-2 reorganization embedded virus shows strong host range phenotype, that is, they efficiently duplicate external, but duplicate very limited in the body.Attenuation in this body takes place when suddenling change less than any adding from cp45.Although rPIV3-2CT and rPIV3-2TM efficiently duplicate external, they show HN and the proteic chimeric interior attenuation phenotype of body of determining of F itself of PIV2 and PIV3 at the upper respiratory tract and the lower respiratory tract camber attenuation of hamster and cercopithecus aethiops (AGM).Comprise the phenotype of growing in the external body that efficiently duplicates with limitation in height be the vaccine candidate strain very wish.Although this attenuation is arranged, they have hyperimmunization originality and the PIV2 wild-type virus is attacked in two kinds of species protectiveness.Be positioned at HN and outer further rPIV3-2CT of modification of PIV3 cp45 sudden change and the rPIV3-2TM of F encoding sequence by introducing 12, produce rPIV3-2CTcp45 and rPIV3-2TMcp45.These derivatives efficiently duplicate external, but in hamster and AGM further attenuation, show that glycoprotein attenuation chimeric and that the cp45 sudden change is determined adds and.These find to determine that PIV3-2CT and rPIV3-2TM recon are the preferred candidate strains of using in attenuated live PIV2 vaccine.Virus and cell
The wild-type PIV1 strain of using in this research, PIV1/Washington/20993/1964 (PIV1/Wash64) (people such as Murphy, infect and immune 12:62-68,1975, be incorporated herein by reference), breeding (people such as Tao, Journal of Virology 72:2955-2961 in LLC-MK2 cell (ATCC CCL 7.1) as previously mentioned, 1998, be incorporated herein by reference).The PIV wild-type virus, the V9412-6 strain is called as PIV2/V94, is isolating in the Vero cell from ill children's nose washing in 1994.PIV2/V94 plaque purification three times on the Vero cell increases twice on the Vero cell with the OptiMJEM that does not contain FBS afterwards.(people such as Durbin, virusology 235:323-332 1997, are incorporated herein by reference) are bred in wild-type cDNA deutero-recon PIV3/JS strain (rPIV3/JS) as previously mentioned.Express bovine vaccine Ankara virus (MVA) recon of the modification of phage t7 RNA polymerase and be so kind as to give (people such as Wyatt, virusology 210:202-205 1995, are incorporated herein by reference) by L.Wyatt and doctor B.Moss.
HEp-2 cell (ATCC CCL 23) remain in the MEM that contains 10% foetal calf serum, 50 μ g/ml gentamicin sulphates and 2mM glutamine (Life Technologies, Gaithersburg, MD) in.The Vero cell below 150 times of going down to posterity remain in the serum free medium VP-SFM that contains 50 μ g/ml gentamicin sulphates and 2mM glutamine (preparation 96-0353SA, LifeTechnologies) in.Virosome RNA separates, the reverse transcription of virogene and pcr amplification, and automatic sequencing
Gene genetic mark for crohn virus gene or confirmation reorganization embedded virus, amplicon virus on cultured cells as previously mentioned, and concentrate (people such as Mbiguino, virological method magazine 31:161-170 by polyethylene glycol precipitation, 1991, be incorporated herein by reference).From the virus precipitation, extract virosome RNA with Trizol reagent (Life Technologies), and be used as the template of the reverse transcription (RT) of using Superscript Preamplification system (Life Technologies).Further use Advantage cDNA test kit (Clontech, Palo Alto, CA) pcr amplification cDNA.In order to clone or to check order, recommend (Schleicher﹠amp according to manufacturer; Schuell, Keene is NH) with the NA45 DEAE film DNA that purifying RT-PCR increases from sepharose.(CA) (Perkin Elmer, Forster City CA) checks order with ABI 310 genetic analysis instrument for Perkin Elmer, Forster City to stop the cycle sequencing test kit with rhodamine.Encode complete PIV2 F and HN albumen or chimeric F and HN albumen, contain the structure of the anti-genome cDNA of chimeric PIV3-PIV2 in PIV2 deutero-extracellular domain and PIV3 deutero-kytoplasm tail territory
Make up the DNA of coding total length PIV3 antigenomic RNA, for the described strategy of PIV3-PIV1 people such as (, Journal of Virology 72:2955-2961,1998) Tao PIV3 F and HN ORF are replaced with its PIV2 homologue before wherein using.Figure 17 has shown the details of this structure.Be concentrated in the PIV2/V94 that breeds in the Vero cell, and from the virus precipitation, extract virosome RNA (vRNA) with Trizol reagent.With hexabasic basic primer and SuperScriptPreamplification system are by F and the HN ORF of vRNA reverse transcription PIV2/V94 at random, the primer of using cDNA Advantage test kit and PIV2 F and HN gene specific afterwards respectively is to pcr amplification (1,2 and 3,4; Table 22).With the cDNA fragment of the PIV2 F ORF of NcoI and BamHI digest amplification, be connected to the NcoI-BamHI window (people such as Tao, Journal of Virology 72:2955-2961 1998, are incorporated herein by reference) of pLit.PIV31.Fhc, produce pLit.PIV32Fhc.BspEI site in the PIV3 full-length cDNA is unique, and we plan to exchange fragment (seeing Figure 17-19) with it between cDNA.Therefore, remove the BspEI site of in PIV2 F ORF, finding and do not influence aminoacid sequence by site-directed mutagenesis.With the cDNA fragment of NcoI and HindIII digestion PIV2 HN ORF, be connected to NcoI-HindIII window people such as (, Journal of Virology 72:2955-2961,1998) Tao of pLit.PIV31.HNhc, produce pLit.PIV32HNhc.PIV2ORF among order-checking pLit.PIV32Fhc and the pLit.PIV32HNhc finds that sequence is as design.The nucleotide sequence of PIV2 F and HN ORF is submitted to GenBank.PLit.PIV32Fhc and pLit.PIV32HNhc use PpuMI and SpeI digestion, and the assembling back produces pLit.PIV32hc.The 4 kb BspEI-SpeI fragments of pLit.PIV32hc are imported the BspEI-SpeI window (people such as Skiadopoulos, vaccine 18:503-510 1999, are incorporated herein by reference) of p38 ' Δ PIV31hc, produce p38 ' Δ PIV32hc.6.5kb fragment with BspEI and SphI digestion p38 ' Δ PIV32hc generation, it contains PIV2 total length F and HN ORF, imports in the BspEI-SphI window of pFLC.2G+.hc (people such as Tao, Journal of Virology 72:2955-2961,1998), produce pFLC.PIV32hc (Figure 17; Table 23=SEQ IDNO.60).The primer primer gene direction position that table 22. uses in the structure of the chimeric anti-genome cDNA of PIV3-2 total length is used for its structure or characterizes sequence
Begin to stop 1 PIV2 F adopted PIV2 F initiator codon 20 downstream pFLC.PIV32hc gtaccATGgATCACCTGCATCCAAT are arranged
5070
b5091 (SEQ ID NO.40) 2 PIV2 F antisense PIV2 F terminator codons, 20 upstream pFLC.PIV32hc tgtggatccTAAGATATCCCATATATGTTTC
6732
b6705
b(SEQ ID NO.41) 3 PIV2 HN have adopted PIV2 HN initiator codon 18 downstream pFLC.PIV32hc gggccATGGAAGATTACAGCAAT
6837
b6856
b(SEQ ID NO.19) 4 PIV2 HN antisense PIV2 HN terminator codons 17 upstream pFLC.PIV32hc caataagcTTAAAGCATTAGTTCCC
8558
b8538
b(SEQ ID NO.20) 5 PIV2 F have justice 5069
c5088
cPFLC.PIV32TM ATGCATCACCTGCATCCAAT
(SEQ ID NO.42) 6 PIV2 F antisenses 6538
c6517
cPFLC.PIV32TM TAGTGAATAAAGTGTCTTGGCT
(SEQ ID NO.43) 7 PIV2 HN have justice 6962
c6985
cPFLC.PIV32TM CATGAGATAATTCATCTTGATGTT
(SEQ ID NO.44) 8 PIV2 HN antisenses 8560
c8537
cPFLC.PIV32TM agcTTAAAGCATTAGTTCCCTTAA
(SEQ ID NO.45) 9 PIV3 F have justice 6539
c6566
cPFLC.PIV32TM ATCATAATTATTTTGATAATGATCATTA
(SEQ ID NO.46) 10 PIV3 F antisenses 5068
c5050
cPFLC.PIV32TM GTTCAGTGCTTGTTGTGTT
(SEQ ID NO.47) 11 PIV3 HN have justice 8561
c8587
cPFLC.PIV32TM TCATAATTAACCATAATATGCATCAAT
(SEQ ID NO.48) 12 PIV3 HN antisenses 6961
c6938
cPFLC.PIV32TM GATGGAATTAATTAGCACTATGAT
(SEQ ID NO.49) 13 PIV2 F have justice 5069
d5088
dPFLC.PIV32CT ATGCATCACCTGCATCCAAT
(SEQ ID NO.50) 14 PIV2 F antisenses 6607
d6589
dPFLC.PIV32CT GATGATGTAGGCAATCAGC
(SEQ ID NO.51) 15 PIV2 HN have justice 6887
d6904
dPFLC.PIV32CT ACTGCCACAATTCTTGGC
(SEQ ID NO.52) 16 PIV2 HN antisenses 8536
d8511
dPFLC.PIV32CT TTAAAGCATTAGTTCCCTTAAAAATG
(SEQ ID NO.53) 17 PIV3 F have justice 6620
d6642
dPFLC.PIV32CT AAGTATTACAGAATTCAAAAGAG
(SEQ ID NO.54) 18 PIV3 F antisenses 5068
d5050
dPFLC.PIV32CT GTTCAGTGCTTGTTGTGTT
(SEQ ID NO.47) 19 PIV3 HN have justice 8525
d8551
dPFLC.PIV32CT TCATAATTAACCATAATATGCATCAAT
(SEQ ID NO.48) 20 PIV3 HN antisenses 6898
d6879
dPFLC.PIV32CT CTTATTAGTGAGCTTGTTGC
(SEQ ID NO.55) 21 PIV2 F have justice 6608
C, d6630
C, dChimera confirmation ACCGCAGCTGTAGCAATAGT
(SEQ ID NO.56) 22 PIV2 HN antisenses 7522
c7502
cChimera confirmation GATTCCATCACTTAGGTAAAT
7501
d7481
d(SEQ ID NO.57) 23 PIV3 M have justice 4759
C, d4780
C, dChimera confirmation GATACTATCCTAATATTATTGC
(SEQ ID NO.58) 24 PIV3 L antisenses 9100
c9081
cChimera confirmation GCTAATTTTGATAGCACATT
9076
d9057
dAll primer anotated of (SEQ ID NO.59) a., because the PIV distinguished sequence is capitalization, non-PIV sequence is a small letter, the initial sum terminator codon is a runic, lines out below the restriction site.B. numeral is the nt position among the anti-genome cDNA construct of the total length pFLC.PIV32hc.C. numeral is the nt position among total length anti-genome cDNA construct pFLC.PIV32TM and the pFLC.PIV32TMcp45.D. numeral is the nt position among total length anti-genome cDNA construct pFLC.PIV32CT and the pFLC.PIV32CTcp45.
The sequence of table 23 (SEQ ID NO.60) pFLC.PIV32,15492bp, sense orientation
( Show only insert )
1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACTTAGGAT
61 TAAAGACATT GACTAGAAGG TCAAGAAAAG GGAACTCTAT AATTTCAAAA ATGTTGAGCC
121 TATTTGATAC ATTTAATGCA CGTAGGCAAG AAAACATAAC AAAATCAGCC GGTGGAGCTA
181 TCATTCCTGG ACAGAAAAAT ACTGTCTCTA TATTCGCCCT TGGACCGACA ATAACTGATG
241 ATAATGAGAA AATGACATTA GCTCTTCTAT TTCTATCTCA TTCACTAGAT AATGAGAAAC
301 AACATGCACA AAGGGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG
361 AGCTCTACCT AACAACAAAT GGAAGTAATG CAGATGTCAA GTATGTCATA TACATGATTG
421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTGTGGT TAAGACGAGA GAGATGATAT
481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT
541 TGCAGAACGG CAGGAACAAT TCAACAATTG AAGACCTTGT CCACACATTT GGGTATCCAT
601 CATGTTTAGG AGCTCTTATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTAGTA
661 TCTCAGGGTT AAGAAAAGGC TTTTTCACCC GATTGGAAGC TTTCAGACAA GATGGAACAG
721 TGCAGGCAGG GCTGGTATTG AGCGGTGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT
781 CTCAACAGAG CTTGGTAACT CTTATGGTTG AAACATTAAT AACAATGAAT ACCAGCAGAA
841 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACTACATA AGAGATGCAG
901 GTCTCGCTTC ATTCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAATG GCAGCTTTGA
961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT
1021 CAAAGGGACC ACGCGCTCCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG
1081 CACCAGGCAA CTATCCTGCC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTACAAAATA
1141 GAGCCATGCA ACAGTATGTG ACGGGAAGAT CATATCTAGA CATTGATATG TTCCAGCTAG
1201 GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AACACTGGAA GATGAACTTG
1261 GAGTGACACA CGAATCTAAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG
1321 AGACATCTTT CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC
1381 CAGAACAATT CGAACATAGA GCAGATCAAG AACAAAATGG AGAACCTCAA TCATCCATAA
1441 TTCAATATGC CTGGGCAGAA GGAAATAGAA GCGATGATCA GACTGAGCAA GCTACAGAAT
1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACTAAACAAG AGACTCAACG
1561 ACAAGAAGAA ACAAAGCAGT CAACCACCCA CTAATCCCAC AAACAGAACA AACCAGGACG
1621 AAATAGATGA TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA
1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCCT ATCATACCGG AATATAGGGT
1741 GGTAAATTTA GAGTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGCTAA
1801 AAACTATCAA ATCATGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA CTAATATCTC
1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA
1921 AAACGACACA ATCAACACAA GAACCCAGCA ACTCAGTGCC ACCATCTGTC AACCAGAAAT
1981 CAAACCAACA GAAACAAGTG AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG
2041 GTCATCACAC GAATGTACAA CAGAAGCAAA AGATAGAAAT ATTGATCAGG AAACTGTACA
2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG
2161 AATCCCCAGA AGCATCACAG ATTCTAAAAA TGGAACCCAA AACACGGAGG ATATTGATCT
2221 CAATGAAATT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTGCAAA
2281 TGTTCCAAGC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG
2341 TGATCATGGA AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT
2401 TACTGCTGCA ACACCAGATG ATGAAGAAGA AATACTAATG AAAAATAGTA GGACAAAGAA
2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA
2521 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC
2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA
2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCATGGAATC TCATCATCGA
2701 CAACAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC
2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC
2821 AAATGGAAAG GAAAGGAAGG ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC
2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAAGACAA
2941 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAATGTAGAT ACTGCATCAA AGATAGATTT
3001 CCTGGCAGGA TTAGTCATAG GGGTTTCAAT GGACAACGAC ACAAAATTAA CACAGATACA
3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT
3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT
3181 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT
3241 CAAAACAAAA TTGAAAGAAG AAAAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGC
3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA
3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT
3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA
3481 TCTCTCACAA AGCACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA
3541 AGAAGTATCT GAATTAATGG ACATGTTCAA TGAAGATGTC AACAATTGCC AATGATCCAA
3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTGTCAACAC
3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA AGAAAAACTT
3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA
3781 CATTCCCAGA ATCATCATTC TCTGAAAATG GTCATATAGA ACCATTACCA CTCAAAGTCA
3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAGTTGCCAA GATCGGAAAT CCACCAAAAC
3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG
3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG
4021 GATCATTACC AATCGGATTG GCTAAGTACA CTGGGAATGA CCAGGAATTG TTACAAGCCG
4081 CAACCAAACT GGATATAGAA GTGAGAAGAA CAGTCAAAGC GAAAGAGATG GTTGTTTACA
4141 CGGTACAAAA TATAAAACCA GAACTGTACC CATGGTCCAA TAGACTAAGA AAAGGAATGC
4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA
4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC
4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA
4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG
4441 AAAAATCACT GAATTTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT
4501 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCTTTAGGAC
4561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA
4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA
4681 ATCTAGTTAT CTGGGCTTCA TCAGTAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT
4741 CTTTACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGTT GGGAAAATCA
4801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA
4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC
4921 TCGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAAATAG GAGAAATCAA AACAAAAGGT
4981 ACAGAACACC AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAAA
5041 GAGACCGGCA ACACAACAAG CACTGAACAC CATGGATCAC CTGCATCCAA TGATAGTATG
5101 CATTTTTGTT ATGTACACTG GAATTGTAGG TTCAGATGCC ATTGCTGGAG ATCAACTCCT
5161 CAATGTAGGG GTCATTCAAT CAAAGATAAG ATCACTCATG TACTACACTG ATGGTGGCGC
5221 TAGCTTTATT GTTGTAAAAT TACTACCCAA TCTTCCCCCA AGCAATGGAA CATGCAACAT
5281 CACCAGTCTA GATGCATATA ATGTTACCCT ATTTAAGTTG CTAACACCCC TGATTGAGAA
5341 CCTGAGCAAA ATTTCTGCTG TTACAGATAC CAAACCCCGC CGAGAACGAT TTGCAGGAGT
5401 CGTTATTGGG CTTGCTGCAC TAGGAGTAGC TACAGCTGCA CAAATAACCG CAGCTGTAGC
5461 AATAGTAAAA GCCAATGCAA ATGCTGCTGC GATAAACAAT CTTGCATCTT CAATTCAATC
5521 CACCAACAAG GCAGTATCCG ATGTGATAAC TGCATCAAGA ACAATTGCAA CCGCAGTTCA
5581 AGCGATTCAG GATCACATCA ATGGAGCCAT TGTCAACGGG ATAACATCTG CATCATGCCG
5641 TGCCCATGAT GCACTAATTG GGTCAATATT AAATTTGTAT CTCACTGAGC TTACTACAAT
5701 ATTTCATAAT CAAATAACAA ACCCTGCGCT GACACCACTT TCCATCCAAG CTTTAAGAAT
5761 CCTCCTCGGT AGCACCTTGC CAATTGTCAT TGAATCCAAA CTCAACACAA AACTCAACAC
5821 AGCAGAGCTG CTCAGTAGCG GACTGTTAAC TGGTCAAATA ATTTCCATTT CCCCAATGTA
5881 CATGCAAATG CTAATTCAAA TCAATGTTCC GACATTTATA ATGCAACCCG GTGCGAAGGT
5941 AATTGATCTA ATTGCTATCT CTGCAAACCA TAAATTACAA GAAGTAGTTG TACAAGTTCC
6001 TAATAGAATT CTAGAATATG CAAATGAACT ACAAAACTAC CCAGCCAATG ATTGTTTCGT
6061 GACACCAAAC TCTGTATTTT GTAGATACAA TGAGGGTTCC CCGATCCCTG AATCACAATA
6121 TCAATGCTTA AGGGGGAATC TTAATTCTTG CACTTTTACC CCTATTATCG GGAACTTTCT
6181 CAAGCGATTC GCATTTGCCA ATGGTGTGCT CTATGCCAAC TGCAAATCTT TGCTATGTAA
6241 GTGTGCCGAC CCTCCCCATG TTGTGTCTCA AGATGACAAC CAAGGCATCA GCATAATTGA
6301 TATTAAGAGG TGCTCTGAGA TGATGCTTGA CACTTTTTCA TTTAGGATCA CATCTACATT
6361 CAATGCTACA TACGTGACAG ACTTCTCAAT GATTAATGCA AATATTGTAC ATCTAAGTCC
6421 TCTAGACTTG TCAAATCAAA TCAATTCAAT AAACAAATCT CTTAAAAGTG CTGAGGATTG
6481 GATTGCAGAT AGCAACTTCT TCGCTAATCA AGCCAGAACA GCCAAGACAC TTTATTCACT
6541 AAGTGCAATC GCATTAATAC TATCAGTGAT TACTTTGGTT GTTGTGGGAT TGCTGATTGC
6601 CTACATCATC AAGCTGGTTT CTCAAATCCA TCAATTCAGA GCACTAGCTG CTACAACAAT
6661 GTTCCACAGG GAGAATCCTG CCGTCTTTTC CAAGAACAAT CATGGAAACA TATATGGGAT
6721 ATCTTAGGAT CCCTACAGAT CATTAGATAT TAAAATTATA AAAAACTTAG GAGTAAAGTT
6781 ACGCAATCCA ACTCTACTCA TATAATTGAG GAAGGACCCA ATAGACAAAT CCAAATCCAT
6841 GGAAGATTAC AGCAATCTAT CTCTTAAATC AATTCCTAAA AGGACATGTA GAATCATTTT
6901 CCGAACTGCC ACAATTCTTG GCATATGCAC ATTAATTGTG CTATGTTCAA GTATTCTTCA
6961 TGAGATAATT CATCTTGATG TTTCCTCTGG TCTTATGAAT TCTGATGAGT CACAGCAAGG
7021 CATTATTCAG CCTATCATAG AATCATTAAA ATCATTGATT GCTTTGGCCA ACCAGATTCT
7081 ATATAATGTT GCAATAGTAA TTCCTCTTAA AATTGACAGT ATCGAAACTG TAATACTCTC
7141 TGCTTTAAAA GATATGCACA CCGGGAGTAT GTCCAATGCC AACTGCACGC CAGGAAATCT
7201 GCTTCTGCAT GATGCAGCAT ACATCAATGG AATAAACAAA TTCCTTGTAC TTGAATCATA
7261 CAATGGGACG CCTAAATATG GACCTCTCCT AAATATACCC AGCTTTATCC CCTCAGCAAC
7321 ATCTCCCCAT GGGTGTACTA GAATACCATC ATTTTCACTC ATCAAGACCC ATTGGTGTTA
7381 CACTCACAAT GTAATGCTTG GAGATTGTCT TGATTTCACG GCATCTAACC AGTATTTATC
7441 AATGGGGATA ATACAACAAT CTGCTGCAGG GTTTCCAATT TTCAGGACTA TGAAAACCAT
7501 TTACCTAAGT GATGGAATCA ATCGCAAAAG CTGTTCAGTC ACTGCTATAC CAGGAGGTTG
7561 TGTCTTGTAT TGCTATGTAG CTACAAGGTC TGAAAAAGAA GATTATGCCA CGACTGATCT
7621 AGCTGAACTG AGACTTGCTT TCTATTATTA TAATGATACC TTTATTGAAA GAGTCATATC
7681 TCTTCCAAAT ACAACAGGGC AGTGGGCCAC AATCAACCCT GCAGTCGGAA GCGGGATCTA
7741 TCATCTAGGC TTTATCTTAT TTCCTGTATA TGGTGGTCTC ATAAATGGGA CTACTTCTTA
7801 CAATGAGCAG TCCTCACGCT ATTTTATCCC AAAACATCCC AACATAACTT GTGCCGGTAA
7861 CTCCAGCAAA CAGGCTGCAA TAGCACGGAG TTCCTATGTC ATCCGTTATC ACTCAAACAG
7921 GTTAATTCAG AGTGCTGTTC TTATTTGTCC ATTGTCTGAC ATGCATACAG AAGAGTGTAA
7981 TCTAGTTATG TTTAACAATT CCCAAGTCAT GATGGGTGCA GAAGGTAGGC TCTATGTTAT
8041 TGGTAATAAT TTGTATTATT ATCAACGCAG TTCCTCTTGG TGGTCTGCAT CGCTCTTTTA
8101 CAGGATCAAT ACAGATTTTT CTAAAGGAAT TCCTCCGATC ATTGAGGCTC AATGGGTACC
8161 GTCCTATCAA GTTCCTCGTC CTGGAGTCAT GCCATGCAAT GCAACAAGTT TTTGCCCTGC
8221 TAATTGCATC ACAGGGGTGT ACGCAGATGT GTGGCCGCTT AATGATCCAG AACTCATGTC
8281 ACGTAATGCT CTGAACCCCA ACTATCGATT TGCTGGAGCC TTTCTCAAAA ATGAGTCCAA
8341 CCGAACTAAT CCCACATTCT ACACTGCATC GGCTAACTCC CTCTTAAATA CTACCGGATT
8401 CAACAACACC AATCACAAAG CAGCATATAC ATCTTCAACC TGCTTTAAAA ACACTGGAAC
8461 CCAAAAAATT TATTGTTTAA TAATAATTGA AATGGGCTCA TCTCTTTTAG GGGAGTTCCA
8521 AATAATACCA TTTTTAAGGG AACTAATGCT TTAAGCTTAA TTAACCATAA TATGCATCAA
8581 TCTATCTATA ATACAAGTAT ATGATAAGTA ATCTGCAATC AGACAATAGA CAAAAGGGAA
8641 ATATAAAAAA CTTAGGAGCA AAGCGTGCTC GGGAAATGGA CACTGAATCT AACAATGGCA
8701 CTGTATCTGA CATACTCTAT CCTGAGTGTC ACCTTAACTC TCCTATCGTT AAAGGTAAAA
8761 TAGCACAATT ACACACTATT ATGAGTCTAC CTCAGCCTTA TGATATGGAT GACGACTCAA
8821 TACTAGTTAT CACTAGACAG AAAATAAAAC TTAATAAATT GGATAAAAGA CAACGATCTA
8881 TTAGAAGATT AAAATTAATA TTAACTGAAA AAGTGAATGA CTTAGGAAAA TACACATTTA
8941 TCAGATATCC AGAAATGTCA AAAGAAATGT TCAAATTATA TATACCTGGT ATTAACAGTA
9001 AAGTGACTGA ATTATTACTT AAAGCAGATA GAACATATAG TCAAATGACT GATGGATTAA
9061 GAGATCTATG GATTAATGTG CTATCAAAAT TAGCCTCAAA AAATGATGGA AGCAATTATG
9121 ATCTTAATGA AGAAATTAAT AATATATCGA AAGTTCACAC AACCTATAAA TCAGATAAAT
9181 GGTATAATCC ATTCAAAACA TGGTTTACTA TCAAGTATGA TATGAGAAGA TTACAAAAAG
9241 CTCGAAATGA GATCACTTTT AATGTTGGGA AGGATTATAA CTTGTTAGAA GACCAGAAGA
9301 ATTTCTTATT GATACATCCA GAATTGGTTT TGATATTAGA TAAACAAAAC TATAATGGTT
9361 ATCTAATTAC TCCTGAATTA GTATTGATGT ATTGTGACGT AGTCGAAGGC CGATGGAATA
9421 TAAGTGCATG TGCTAAGTTA GATCCAAAAT TACAATCTAT GTATCAGAAA GGTAATAACC
9481 TGTGGGAAGT GATAGATAAA TTGTTTCCAA TTATGGGAGA AAAGACATTT GATGTGATAT
9541 CGTTATTAGA ACCACTTGCA TTATCCTTAA TTCAAACTCA TGATCCTGTT AAACAACTAA
9601 GAGGAGCTTT TTTAAATCAT GTGTTATCCG AGATGGAATT AATATTTGAA TCTAGAGAAT
9661 CGATTAAGGA ATTTCTGAGT GTAGATTACA TTGATAAAAT TTTAGATATA TTTAATAAGT
9721 CTACAATAGA TGAAATAGCA GAGATTTTCT CTTTTTTTAG AACATTTGGG CATCCTCCAT
9781 TAGAAGCTAG TATTGCAGCA GAAAAGGTTA GAAAATATAT GTATATTGGA AAACAATTAA
9841 AATTTGACAC TATTAATAAA TGTCATGCTA TCTTCTGTAC AATAATAATT AACGGATATA
9901 GAGAGAGGCA TGGTGGACAG TGGCCTCCTG TGACATTACC TGATCATGCA CACGAATTCA
9961 TCATAAATGC TTACGGTTCA AACTCTGCGA TATCATATGA AAATGCTGTT GATTATTACC
10021 AGAGCTTTAT AGGAATAAAA TTCAATAAAT TCATAGAGCC TCAGTTAGAT GAGGATTTGA
10081 CAATTTATAT GAAAGATAAA GCATTATCTC CAAAAAAATC AAATTGGGAC ACAGTTTATC
10141 CTGCATCTAA TTTACTGTAC CGTACTAACG CATCCAACGA ATCACGAAGA TTAGTTGAAG
10201 TATTTATAGC AGATAGTAAA TTTGATCCTC ATCAGATATT GGATTATGTA GAATCTGGGG
10261 ACTGGTTAGA TGATCCAGAA TTTAATATTT CTTATAGTCT TAAAGAAAAA GAGATCAAAC
10321 AGGAAGGTAG ACTCTTTGCA AAAATGACAT ACAAAATGAG AGCTACACAA GTTTTATCAG
10381 AGACCCTACT TGCAAATAAC ATAGGAAAAT TCTTTCAAGA AAATGGGATG GTGAAGGGAG
10441 AGATTGAATT ACTTAAGAGA TTAACAACCA TATCAATATC AGGAGTTCCA CGGTATAATG
10501 AAGTGTACAA TAATTCTAAA AGCCATACAG ATGACCTTAA AACCTACAAT AAAATAAGTA
10561 ATCTTAATTT GTCTTCTAAT CAGAAATCAA AGAAATTTGA ATTCAAGTCA ACGGATATCT
10621 ACAATGATGG ATACGAGACT GTGAGCTGTT TCCTAACAAC AGATCTCAAA AAATACTGTC
10681 TTAATTGGAG ATATGAATCA ACAGCTCTAT TTGGAGAAAC TTGCAACCAA ATATTTGGAT
10741 TAAATAAATT GTTTAATTGG TTACACCCTC GTCTTGAAGG AAGTACAATC TATGTAGGTG
10801 ATCCTTACTG TCCTCCATCA GATAAAGAAC ATATATCATT AGAGGATCAC CCTGATTCTG
10861 GTTTTTACGT TCATAACCCA AGAGGGGGTA TAGAAGGATT TTGTCAAAAA TTATGGACAC
10921 TCATATCTAT AAGTGCAATA CATCTAGCAG CTGTTAGAAT AGGCGTGAGG GTGACTGCAA
10981 TGGTTCAAGG AGACAATCAA GCTATAGCTG TAACCACAAG AGTACCCAAC AATTATGACT
11041 ACAGAGTTAA GAAGGAGATA GTTTATAAAG ATGTAGTGAG ATTTTTTGAT TCATTAAGAG
11101 AAGTGATGGA TGATCTAGGT CATGAACTTA AATTAAATGA AACGATTATA AGTAGCAAGA
11161 TGTTCATATA TAGCAAAAGA ATCTATTATG ATGGGAGAAT TCTTCCTCAA GCTCTAAAAG
11221 CATTATCTAG ATGTGTCTTC TGGTCAGAGA CAGTAATAGA CGAAACAAGA TCAGCATCTT
11281 CAAATTTGGC AACATCATTT GCAAAAGCAA TTGAGAATGG TTATTCACCT GTTCTAGGAT
11341 ATGCATGCTC AATTTTTAAG AATATTCAAC AACTATATAT TGCCCTTGGG ATGAATATCA
11401 ATCCAACTAT AACACAGAAT ATCAGAGATC AGTATTTTAG GAATCCAAAT TGGATGCAAT
11461 ATGCCTCTTT AATACCTGCT AGTGTTGGGG GATTCAATTA CATGGCCATG TCAAGATGTT
11521 TTGTAAGGAA TATTGGTGAT CCATCAGTTG CCGCATTGGC TGATATTAAA AGATTTATTA
11581 AGGCGAATCT ATTAGACCGA AGTGTTCTTT ATAGGATTAT GAATCAAGAA CCAGGTGAGT
11641 CATCTTTTTT GGACTGGGCT TCAGATCCAT ATTCATGCAA TTTACCACAA TCTCAAAATA
11701 TAACCACCAT GATAAAAAAT ATAACAGCAA GGAATGTATT ACAAGATTCA CCAAATCCAT
11761 TATTATCTGG ATTATTCACA AATACAATGA TAGAAGAAGA TGAAGAATTA GCTGAGTTCC
11821 TGATGGACAG GAAGGTAATT CTCCCTAGAG TTGCACATGA TATTCTAGAT AATTCTCTCA
11881 CAGGAATTAG AAATGCCATA GCTGGAATGT TAGATACGAC AAAATCACTA ATTCGGGTTG
11941 GCATAAATAG AGGAGGACTG ACATATAGTT TGTTGAGGAA AATCAGTAAT TACGATCTAG
12001 TACAATATGA AACACTAAGT AGGACTTTGC GACTAATTGT AAGTGATAAA ATCAAGTATG
12061 AAGATATGTG TTCGGTAGAC CTTGCCATAG CATTGCGACA AAAGATGTGG ATTCATTTAT
12121 CAGGAGGAAG GATGATAAGT GGACTTGAAA CGCCTGACCC ATTAGAATTA CTATCTGGGG
12181 TAGTAATAAC AGGATCAGAA CATTGTAAAA TATGTTATTC TTCAGATGGC ACAAACCCAT
12241 ATACTTGGAT GTATTTACCC GGTAATATCA AAATAGGATC AGCAGAAACA GGTATATCGT
12301 CATTAAGAGT TCCTTATTTT GGATCAGTCA CTGATGAAAG ATCTGAAGCA CAATTAGGAT
12361 ATATCAAGAA TCTTAGTAAA CCTGCAAAAG CCGCAATAAG AATAGCAATG ATATATACAT
12421 GGGCATTTGG TAATGATGAG ATATCTTGGA TGGAAGCCTC ACAGATAGCA CAAACACGTG
12481 CAAATTTTAC ACTAGATAGT CTCAAAATTT TAACACCGGT AGCTACATCA ACAAATTTAT
12541 CACACAGATT AAAGGATACT GCAACTCAGA TGAAATTCTC CAGTACATCA TTGATCAGAG
12601 TCAGCAGATT CATAACAATG TCCAATGATA ACATGTCTAT CAAAGAAGCT AATGAAACCA
12661 AAGATACTAA TCTTATTTAT CAACAAATAA TGTTAACAGG ATTAAGTGTT TTCGAATATT
12721 TATTTAGATT AAAAGAAACC ACAGGACACA ACCCTATAGT TATGCATCTG CACATAGAAG
12781 ATGAGTGTTG TATTAAAGAA AGTTTTAATG ATGAACATAT TAATCCAGAG TCTACATTAG
12841 AATTAATTCG ATATCCTGAA AGTAATGAAT TTATTTATGA TAAAGACCCA CTCAAAGATG
12901 TGGACTTATC AAAACTTATG GTTATTAAAG ACCATTCTTA CACAATTGAT ATGAATTATT
12961 GGGATGATAC TGACATCATA CATGCAATTT CAATATGTAC TGCAATTACA ATAGCAGATA
13021 CTATGTCACA ATTAGATCGA GATAATTTAA AAGAGATAAT AGTTATTGCA AATGATGATG
13081 ATATTAATAG CTTAATCACT GAATTTTTGA CTCTTGACAT ACTTGTATTT CTCAAGACAT
13141 TTGGTGGATT ATTAGTAAAT CAATTTGCAT ACACTCTTTA TAGTCTAAAA ATAGAAGGTA
13201 GGGATCTCAT TTGGGATTAT ATAATGAGAA CACTGAGAGA TACTTCCCAT TCAATATTAA
13261 AAGTATTATC TAATGCATTA TCTCATCCTA AAGTATTCAA GAGGTTCTGG GATTGTGGAG
13321 TTTTAAACCC TATTTATGGT CCTAATACTG CTAGTCAAGA CCAGATAAAA CTTGCCCTAT
13381 CTATATGTGA ATATTCACTA GATCTATTTA TGAGAGAATG GTTGAATGGT GTATCACTTG
13441 AAATATACAT TTGTGACAGC GATATGGAAG TTGCAAATGA TAGGAAACAA GCCTTTATTT
13501 CTAGACACCT TTCATTTGTT TGTTGTTTAG CAGAAATTGC ATCTTTCGGA CCTAACCTGT
13561 TAAACTTAAC ATACTTGGAG AGACTTGATC TATTGAAACA ATATCTTGAA TTAAATATTA
13621 AAGAAGACCC TACTCTTAAA TATGTACAAA TATCTGGATT ATTAATTAAA TCGTTCCCAT
13681 CAACTGTAAC ATACGTAAGA AAGACTGCAA TCAAATATCT AAGGATTCGC GGTATTAGTC
13741 CACCTGAGGT AATTGATGAT TGGGATCCGG TAGAAGATGA AAATATGCTG GATAACATTG
13801 TCAAAACTAT AAATGATAAC TGTAATAAAG ATAATAAAGG GAATAAAATT AACAATTTCT
13861 GGGGACTAGC ACTTAAGAAC TATCAAGTCC TTAAAATCAG ATCTATAACA AGTGATTCTG
13921 ATGATAATGA TAGACTAGAT GCTAATACAA GTGGTTTGAC ACTTCCTCAA GGAGGGAATT
13981 ATCTATCGCA TCAATTGAGA TTATTCGGAA TCAACAGCAC TAGTTGTCTG AAAGCTCTTG
14041 AGTTATCACA AATTTTAATG AAGGAAGTCA ATAAAGACAA GGACAGGCTC TTCCTGGGAG
14101 AAGGAGCAGG AGCTATGCTA GCATGTTATG ATGCCACATT AGGACCTGCA GTTAATTATT
14161 ATAATTCAGG TTTGAATATA ACAGATGTAA TTGGTCAACG AGAATTGAAA ATATTTCCTT
14221 CAGAGGTATC ATTAGTAGGT AAAAAATTAG GAAATGTGAC ACAGATTCTT AACAGGGTAA
14281 AAGTACTGTT CAATGGGAAT CCTAATTCAA CATGGATAGG AAATATGGAA TGTGAGAGCT
14341 TAATATGGAG TGAATTAAAT GATAAGTCCA TTGGATTAGT ACATTGTGAT ATGGAAGGAG
14401 CTATCGGTAA ATCAGAAGAA ACTGTTCTAC ATGAACATTA TAGTGTTATA AGAATTACAT
14461 ACTTGATTGG GGATGATGAT GTTGTTTTAG TTTCCAAAAT TATACCTACA ATCACTCCGA
14521 ATTGGTCTAG AATACTTTAT CTATATAAAT TATATTGGAA AGATGTAAGT ATAATATCAC
14581 TCAAAACTTC TAATCCTGCA TCAACAGAAT TATATCTAAT TTCGAAAGAT GCATATTGTA
14641 CTATAATGGA ACCTAGTGAA ATTGTTTTAT CAAAACTTAA AAGATTGTCA CTCTTGGAAG
14701 AAAATAATCT ATTAAAATGG ATCATTTTAT CAAAGAAGAG GAATAATGAA TGGTTACATC
14761 ATGAAATCAA AGAAGGAGAA AGAGATTATG GAATCATGAG ACCATATCAT ATGGCACTAC
14821 AAATCTTTGG ATTTCAAATC AATTTAAATC ATCTGGCGAA AGAATTTTTA TCAACCCCAG
14881 ATCTGACTAA TATCAACAAT ATAATCCAAA GTTTTCAGCG AACAATAAAG GATGTTTTAT
14941 TTGAATGGAT TAATATAACT CATGATGATA AGAGACATAA ATTAGGCGGA AGATATAACA
15001 TATTCCCACT GAAAAATAAG GGAAAGTTAA GACTGCTATC GAGAAGACTA GTATTAAGTT
15061 GGATTTCATT ATCATTATCG ACTCGATTAC TTACAGGTCG CTTTCCTGAT GAAAAATTTG
15121 AACATAGAGC ACAGACTGGA TATGTATCAT TAGCTGATAC TGATTTAGAA TCATTAAAGT
15181 TATTGTCGAA AAACATCATT AAGAATTACA GAGAGTGTAT AGGATCAATA TCATATTGGT
15241 TTCTAACCAA AGAAGTTAAA ATACTTATGA AATTGATCGG TGGTGCTAAA TTATTAGGAA
15301 TTCCCAGACA ATATAAAGAA CCCGAAGACC AGTTATTAGA AAACTACAAT CAACATGATG
15361 AATTTGATAT CGATTAAAAC ATAAATACAA TGAAGATATA TCCTAACCTT TATCTTTAAG
15421 CCTAGGAATA GACAAAAAGT AAGAAAAACA TGTAATATAT ATATACCAAA CAGAGTTCTT
15481 CTCTTGTTTG GT
In second kind of strategy (Figure 18), make up chimeric PIV3-PIV2 F and HN ORF rather than complete ORF exchange, wherein use PCR, Vent archaeal dna polymerase (NEB, Beverly, MA) and the special primer of PIV2 F (5 in the table 22,6) and HN (7 in the table 22,8) to respectively by the PIV2 F and the HNORF district of pLit.PIV32Fhc and pLit.PIV32HNhc amplification coding extracellular domain.Simultaneously, with the special primer of PCR, Vent archaeal dna polymerase and PIV3 F (9 in the table 22,10) and HN (11 in the table 22,12) to respectively from its cDNA subclone pLit.PIV3.F3a and pLit.PIV3.HN4 (people such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference) middle PIV3 F and HN ORF district of deleting the coding extracellular domain.The PIV2 of purifying amplification and F and the HN cDNA fragment of PIV3 from sepharose, connection produces pLit.PIV32FTM and pLit.PIV32HNTM respectively.Digest chimeric F and HN construct with PpuMI and SpeI, being assembled together produces pLit.PIV32TM, stops sequencing kit to the complete order-checking in the special district of its PIV with rhodamine subsequently, finds as design.Then the 4 kb BspEI-SpeI fragments of pLit.PIV32TM are inserted in the BspEI-SpeI window of p38 ' Δ PIV31hc and produced p38 ' Δ PIV32TM.The 6.5kb BspEI-SphI fragment that will contain p38 ' the Δ PIV32TM of chimeric F of PIV3-PIV2 and HN gene is introduced pFLC.2G+.hc and pFLCcp45 (people such as Skiadopoulos respectively, Journal of Virology 73:1374-81,1999, be incorporated herein by reference) the BspEI-SphI window in, produce pFLC.PIV32TM (table 24; SEQ ID NO.61) and pFLC.PIV32TMcp45.The segmental nucleotide sequence of BspEI-SpeI that contains chimeric PIV3-PIV2F and HN gene is submitted to GenBank.
The sequence of table 24 (SEQ ID NO.61) pFLC.PIV32TM, 15498bp, sense orientation
( Show only anti- genome )
1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACTTAGGAT
61 TAAAGACATT GACTAGAAGG TCAAGAAAAG GGAACTCTAT AATTTCAAAA ATGTTGAGCC
121 TATTTGATAC ATTTAATGCA CGTAGGCAAG AAAACATAAC AAAATCAGCC GGTGGAGCTA
181 TCATTCCTGG ACAGAAAAAT ACTGTCTCTA TATTCGCCCT TGGACCGACA ATAACTGATG
241 ATAATGAGAA AATGACATTA GCTCTTCTAT TTCTATCTCA TTCACTAGAT AATGAGAAAC
301 AACATGCACA AAGGGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG
361 AGCTCTACCT AACAACAAAT GGAAGTAATG CAGATGTCAA GTATGTCATA TACATGATTG
421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTGTGGT TAAGACGAGA GAGATGATAT
481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT
541 TGCAGAACGG CAGGAACAAT TCAACAATTG AAGACCTTGT CCACACATTT GGGTATCCAT
601 CATGTTTAGG AGCTCTTATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTAGTA
661 TCTCAGGGTT AAGAAAAGGC TTTTTCACCC GATTGGAAGC TTTCAGACAA GATGGAACAG
721 TGCAGGCAGG GCTGGTATTG AGCGGTGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT
781 CTCAACAGAG CTTGGTAACT CTTATGGTTG AAACATTAAT AACAATGAAT ACCAGCAGAA
841 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACTACATA AGAGATGCAG
901 GTCTCGCTTC ATTCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAATG GCAGCTTTGA
961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT
1021 CAAAGGGACC ACGCGCTCCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG
1081 CACCAGGCAA CTATCCTGCC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTACAAAATA
1141 GAGCCATGCA ACAGTATGTG ACGGGAAGAT CATATCTAGA CATTGATATG TTCCAGCTAG
1201 GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AACACTGGAA GATGAACTTG
1261 GAGTGACACA CGAATCTAAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG
1321 AGACATCTTT CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC
1381 CAGAACAATT CGAACATAGA GCAGATCAAG AACAAAATGG AGAACCTCAA TCATCCATAA
1441 TTCAATATGC CTGGGCAGAA GGAAATAGAA GCGATGATCA GACTGAGCAA GCTACAGAAT
1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACTAAACAAG AGACTCAACG
1561 ACAAGAAGAA ACAAAGCAGT CAACCACCCA CTAATCCCAC AAACAGAACA AACCAGGACG
1621 AAATAGATGA TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA
1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCCT ATCATACCGG AATATAGGGT
1741 GGTAAATTTA GAGTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGCTAA
1801 AAACTATCAA ATCATGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA CTAATATCTC
1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA
1921 AAACGACACA ATCAACACAA GAACCCAGCA ACTCAGTGCC ACCATCTGTC AACCAGAAAT
1981 CAAACCAACA GAAACAAGTG AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG
2041 GTCATCACAC GAATGTACAA CAGAAGCAAA AGATAGAAAT ATTGATCAGG AAACTGTACA
2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG
2161 AATCCCCAGA AGCATCACAG ATTCTAAAAA TGGAACCCAA AACACGGAGG ATATTGATCT
2221 CAATGAAATT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTGCAAA
2281 TGTTCCAAGC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG
2341 TGATCATGGA AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT
2401 TACTGCTGCA ACACCAGATG ATGAAGAAGA AATACTAATG AAAAATAGTA GGACAAAGAA
2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA
2521 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC
2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA
2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCATGGAATC TCATCATCGA
2701 CAACAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC
2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC
2821 AAATGGAAAG GAAAGGAAGG ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC
2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAAGACAA
2941 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAATGTAGAT ACTGCATCAA AGATAGATTT
3001 CCTGGCAGGA TTAGTCATAG GGGTTTCAAT GGACAACGAC ACAAAATTAA CACAGATACA
3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT
3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT
3181 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT
3241 CAAAACAAAA TTGAAAGAAG AAAAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGC
3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA
3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT
3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA
3481 TCTCTCACAA AGCACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA
3541 AGAAGTATCT GAATTAATGG ACATGTTCAA TGAAGATGTC AACAATTGCC AATGATCCAA
3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTGTCAACAC
3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA AGAAAAACTT
3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA
3781 CATTCCCAGA ATCATCATTC TCTGAAAATG GTCATATAGA ACCATTACCA CTCAAAGTCA
3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAGTTGCCAA GATCGGAAAT CCACCAAAAC
3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG
3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG
4021 GATCATTACC AATCGGATTG GCTAAGTACA CTGGGAATGA CCAGGAATTG TTACAAGCCG
4081 CAACCAAACT GGATATAGAA GTGAGAAGAA CAGTCAAAGC GAAAGAGATG GTTGTTTACA
4141 CGGTACAAAA TATAAAACCA GAACTGTACC CATGGTCCAA TAGACTAAGA AAAGGAATGC
4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA
4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC
4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA
4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG
4441 AAAAATCACT GAATTTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT
4501 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCTTTAGGAC
4561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA
4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA
4681 ATCTAGTTAT CTGGGCTTCA TCAGTAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT
4741 CTTTACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGTT GGGAAAATCA
4801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA
4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC
4921 TCGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAAATAG GAGAAATCAA AACAAAAGGT
4981 ACAGAACACC AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAAA
5041 GAGACCGGCA ACACAACAAG CACTGAACAT GCATCACCTG CATCCAATGA TAGTATGCAT
5101 TTTTGTTATG TACACTGGAA TTGTAGGTTC AGATGCCATT GCTGGAGATC AACTCCTCAA
5161 TGTAGGGGTC ATTCAATCAA AGATAAGATC ACTCATGTAC TACACTGATG GTGGCGCTAG
5221 CTTTATTGTT GTAAAATTAC TACCCAATCT TCCCCCAAGC AATGGAACAT GCAACATCAC
5281 CAGTCTAGAT GCATATAATG TTACCCTATT TAAGTTGCTA ACACCCCTGA TTGAGAACCT
5341 GAGCAAAATT TCTGCTGTTA CAGATACCAA ACCCCGCCGA GAACGATTTG CAGGAGTCGT
5401 TATTGGGCTT GCTGCACTAG GAGTAGCTAC AGCTGCACAA ATAACCGCAG CTGTAGCAAT
5461 AGTAAAAGCC AATGCAAATG CTGCTGCGAT AAACAATCTT GCATCTTCAA TTCAATCCAC
5521 CAACAAGGCA GTATCCGATG TGATAACTGC ATCAAGAACA ATTGCAACCG CAGTTCAAGC
5581 GATTCAGGAT CACATCAATG GAGCCATTGT CAACGGGATA ACATCTGCAT CATGCCGTGC
5641 CCATGATGCA CTAATTGGGT CAATATTAAA TTTGTATCTC ACTGAGCTTA CTACAATATT
5701 TCATAATCAA ATAACAAACC CTGCGCTGAC ACCACTTTCC ATCCAAGCTT TAAGAATCCT
5761 CCTCGGTAGC ACCTTGCCAA TTGTCATTGA ATCCAAACTC AACACAAAAC TCAACACAGC
5821 AGAGCTGCTC AGTAGCGGAC TGTTAACTGG TCAAATAATT TCCATTTCCC CAATGTACAT
5881 GCAAATGCTA ATTCAAATCA ATGTTCCGAC ATTTATAATG CAACCCGGTG CGAAGGTAAT
5941 TGATCTAATT GCTATCTCTG CAAACCATAA ATTACAAGAA GTAGTTGTAC AAGTTCCTAA
6001 TAGAATTCTA GAATATGCAA ATGAACTACA AAACTACCCA GCCAATGATT GTTTCGTGAC
6061 ACCAAACTCT GTATTTTGTA GATACAATGA GGGTTCCCCG ATCCCTGAAT CACAATATCA
6121 ATGCTTAAGG GGGAATCTTA ATTCTTGCAC TTTTACCCCT ATTATCGGGA ACTTTCTCAA
6181 GCGATTCGCA TTTGCCAATG GTGTGCTCTA TGCCAACTGC AAATCTTTGC TATGTAAGTG
6241 TGCCGACCCT CCCCATGTTG TGTCTCAAGA TGACAACCAA GGCATCAGCA TAATTGATAT
6301 TAAGAGGTGC TCTGAGATGA TGCTTGACAC TTTTTCATTT AGGATCACAT CTACATTCAA
6361 TGCTACATAC GTGACAGACT TCTCAATGAT TAATGCAAAT ATTGTACATC TAAGTCCTCT
6421 AGACTTGTCA AATCAAATCA ATTCAATAAA CAAATCTCTT AAAAGTGCTG AGGATTGGAT
6481 TGCAGATAGC AACTTCTTCG CTAATCAAGC CAGAACAGCC AAGACACTTT ATTCACTAAT
6541 CATAATTATT TTGATAATGA TCATTATATT GTTTATAATT AATATAACGA TAATTACAAT
6601 TGCAATTAAG TATTACAGAA TTCAAAAGAG AAATCGAGTG GATCAAAATG ACAAGCCATA
6661 TGTACTAACA AACAAATAAC ATATCTACAG ATCATTAGAT ATTAAAATTA TAAAAAACTT
6721 AGGAGTAAAG TTACGCAATC CAACTCTACT CATATAATTG AGGAAGGACC CAATAGACAA
6781 ATCCAAATTC GAGATGGAAT ACTGGAAGCA TACCAATCAC GGAAAGGATG CTGGTAATGA
6841 GCTGGAGACG TCTATGGCTA CTCATGGCAA CAAGCTCACT AATAAGATAA TATACATATT
6901 ATGGACAATA ATCCTGGTGT TATTATCAAT AGTCTTCATC ATAGTGCTAA TTAATTCCAT
6961 CCATGAGATA ATTCATCTTG ATGTTTCCTC TGGTCTTATG AATTCTGATG AGTCACAGCA
7021 AGGCATTATT CAGCCTATCA TAGAATCATT AAAATCATTG ATTGCTTTGG CCAACCAGAT
7081 TCTATATAAT GTTGCAATAG TAATTCCTCT TAAAATTGAC AGTATCGAAA CTGTAATACT
7141 CTCTGCTTTA AAAGATATGC ACACCGGGAG TATGTCCAAT GCCAACTGCA CGCCAGGAAA
7201 TCTGCTTCTG CATGATGCAG CATACATCAA TGGAATAAAC AAATTCCTTG TACTTGAATC
7261 ATACAATGGG ACGCCTAAAT ATGGACCTCT CCTAAATATA CCCAGCTTTA TCCCCTCAGC
7321 AACATCTCCC CATGGGTGTA CTAGAATACC ATCATTTTCA CTCATCAAGA CCCATTGGTG
7381 TTACACTCAC AATGTAATGC TTGGAGATTG TCTTGATTTC ACGGCATCTA ACCAGTATTT
7441 ATCAATGGGG ATAATACAAC AATCTGCTGC AGGGTTTCCA ATTTTCAGGA CTATGAAAAC
7501 CATTTACCTA AGTGATGGAA TCAATCGCAA AAGCTGTTCA GTCACTGCTA TACCAGGAGG
7561 TTGTGTCTTG TATTGCTATG TAGCTACAAG GTCTGAAAAA GAAGATTATG CCACGACTGA
7621 TCTAGCTGAA CTGAGACTTG CTTTCTATTA TTATAATGAT ACCTTTATTG AAAGAGTCAT
7681 ATCTCTTCCA AATACAACAG GGCAGTGGGC CACAATCAAC CCTGCAGTCG GAAGCGGGAT
7741 CTATCATCTA GGCTTTATCT TATTTCCTGT ATATGGTGGT CTCATAAATG GGACTACTTC
7801 TTACAATGAG CAGTCCTCAC GCTATTTTAT CCCAAAACAT CCCAACATAA CTTGTGCCGG
7861 TAACTCCAGC AAACAGGCTG CAATAGCACG GAGTTCCTAT GTCATCCGTT ATCACTCAAA
7921 CAGGTTAATT CAGAGTGCTG TTCTTATTTG TCCATTGTCT GACATGCATA CAGAAGAGTG
7981 TAATCTAGTT ATGTTTAACA ATTCCCAAGT CATGATGGGT GCAGAAGGTA GGCTCTATGT
8041 TATTGGTAAT AATTTGTATT ATTATCAACG CAGTTCCTCT TGGTGGTCTG CATCGCTCTT
8101 TTACAGGATC AATACAGATT TTTCTAAAGG AATTCCTCCG ATCATTGAGG CTCAATGGGT
8161 ACCGTCCTAT CAAGTTCCTC GTCCTGGAGT CATGCCATGC AATGCAACAA GTTTTTGCCC
8221 TGCTAATTGC ATCACAGGGG TGTACGCAGA TGTGTGGCCG CTTAATGATC CAGAACTCAT
8281 GTCACGTAAT GCTCTGAACC CCAACTATCG ATTTGCTGGA GCCTTTCTCA AAAATGAGTC
8341 CAACCGAACT AATCCCACAT TCTACACTGC ATCGGCTAAC TCCCTCTTAA ATACTACCGG
8401 ATTCAACAAC ACCAATCACA AAGCAGCATA TACATCTTCA ACCTGCTTTA AAAACACTGG
8461 AACCCAAAAA ATTTATTGTT TAATAATAAT TGAAATGGGC TCATCTCTTT TAGGGGAGTT
8521 CCAAATAATA CCATTTTTAA GGGAACTAAT GCTTTAAGCT TCATAATTAA CCATAATATG
8581 CATCAATCTA TCTATAATAC AAGTATATGA TAAGTAATCA GCAATCAGAC AATAGACAAA
8641 AGGGAAATAT AAAAAACTTA GGAGCAAAGC GTGCTCGGGA AATGGACACT GAATCTAACA
8701 ATGGCACTGT ATCTGACATA CTCTATCCTG AGTGTCACCT TAACTCTCCT ATCGTTAAAG
8761 GTAAAATAGC ACAATTACAC ACTATTATGA GTCTACCTCA GCCTTATGAT ATGGATGACG
8821 ACTCAATACT AGTTATCACT AGACAGAAAA TAAAACTTAA TAAATTGGAT AAAAGACAAC
8881 GATCTATTAG AAGATTAAAA TTAATATTAA CTGAAAAAGT GAATGACTTA GGAAAATACA
8941 CATTTATCAG ATATCCAGAA ATGTCAAAAG AAATGTTCAA ATTATATATA CCTGGTATTA
9001 ACAGTAAAGT GACTGAATTA TTACTTAAAG CAGATAGAAC ATATAGTCAA ATGACTGATG
9061 GATTAAGAGA TCTATGGATT AATGTGCTAT CAAAATTAGC CTCAAAAAAT GATGGAAGCA
9121 ATTATGATCT TAATGAAGAA ATTAATAATA TATCGAAAGT TCACACAACC TATAAATCAG
9181 ATAAATGGTA TAATCCATTC AAAACATGGT TTACTATCAA GTATGATATG AGAAGATTAC
9241 AAAAAGCTCG AAATGAGATC ACTTTTAATG TTGGGAAGGA TTATAACTTG TTAGAAGACC
9301 AGAAGAATTT CTTATTGATA CATCCAGAAT TGGTTTTGAT ATTAGATAAA CAAAACTATA
9361 ATGGTTATCT AATTACTCCT GAATTAGTAT TGATGTATTG TGACGTAGTC GAAGGCCGAT
9421 GGAATATAAG TGCATGTGCT AAGTTAGATC CAAAATTACA ATCTATGTAT CAGAAAGGTA
9481 ATAACCTGTG GGAAGTGATA GATAAATTGT TTCCAATTAT GGGAGAAAAG ACATTTGATG
9541 TGATATCGTT ATTAGAACCA CTTGCATTAT CCTTAATTCA AACTCATGAT CCTGTTAAAC
9601 AACTAAGAGG AGCTTTTTTA AATCATGTGT TATCCGAGAT GGAATTAATA TTTGAATCTA
9661 GAGAATCGAT TAAGGAATTT CTGAGTGTAG ATTACATTGA TAAAATTTTA GATATATTTA
9721 ATAAGTCTAC AATAGATGAA ATAGCAGAGA TTTTCTCTTT TTTTAGAACA TTTGGGCATC
9781 CTCCATTAGA AGCTAGTATT GCAGCAGAAA AGGTTAGAAA ATATATGTAT ATTGGAAAAC
9841 AATTAAAATT TGACACTATT AATAAATGTC ATGCTATCTT CTGTACAATA ATAATTAACG
9901 GATATAGAGA GAGGCATGGT GGACAGTGGC CTCCTGTGAC ATTACCTGAT CATGCACACG
9961 AATTCATCAT AAATGCTTAC GGTTCAAACT CTGCGATATC ATATGAAAAT GCTGTTGATT
10021 ATTACCAGAG CTTTATAGGA ATAAAATTCA ATAAATTCAT AGAGCCTCAG TTAGATGAGG
10081 ATTTGACAAT TTATATGAAA GATAAAGCAT TATCTCCAAA AAAATCAAAT TGGGACACAG
10141 TTTATCCTGC ATCTAATTTA CTGTACCGTA CTAACGCATC CAACGAATCA CGAAGATTAG
10201 TTGAAGTATT TATAGCAGAT AGTAAATTTG ATCCTCATCA GATATTGGAT TATGTAGAAT
10261 CTGGGGACTG GTTAGATGAT CCAGAATTTA ATATTTCTTA TAGTCTTAAA GAAAAAGAGA
10321 TCAAACAGGA AGGTAGACTC TTTGCAAAAA TGACATACAA AATGAGAGCT ACACAAGTTT
10381 TATCAGAGAC CCTACTTGCA AATAACATAG GAAAATTCTT TCAAGAAAAT GGGATGGTGA
10441 AGGGAGAGAT TGAATTACTT AAGAGATTAA CAACCATATC AATATCAGGA GTTCCACGGT
10501 ATAATGAAGT GTACAATAAT TCTAAAAGCC ATACAGATGA CCTTAAAACC TACAATAAAA
10561 TAAGTAATCT TAATTTGTCT TCTAATCAGA AATCAAAGAA ATTTGAATTC AAGTCAACGG
10621 ATATCTACAA TGATGGATAC GAGACTGTGA GCTGTTTCCT AACAACAGAT CTCAAAAAAT
10681 ACTGTCTTAA TTGGAGATAT GAATCAACAG CTCTATTTGG AGAAACTTGC AACCAAATAT
10741 TTGGATTAAA TAAATTGTTT AATTGGTTAC ACCCTCGTCT TGAAGGAAGT ACAATCTATG
10801 TAGGTGATCC TTACTGTCCT CCATCAGATA AAGAACATAT ATCATTAGAG GATCACCCTG
10861 ATTCTGGTTT TTACGTTCAT AACCCAAGAG GGGGTATAGA AGGATTTTGT CAAAAATTAT
10921 GGACACTCAT ATCTATAAGT GCAATACATC TAGCAGCTGT TAGAATAGGC GTGAGGGTGA
10981 CTGCAATGGT TCAAGGAGAC AATCAAGCTA TAGCTGTAAC CACAAGAGTA CCCAACAATT
11041 ATGACTACAG AGTTAAGAAG GAGATAGTTT ATAAAGATGT AGTGAGATTT TTTGATTCAT
11101 TAAGAGAAGT GATGGATGAT CTAGGTCATG AACTTAAATT AAATGAAACG ATTATAAGTA
11161 GCAAGATGTT CATATATAGC AAAAGAATCT ATTATGATGG GAGAATTCTT CCTCAAGCTC
11221 TAAAAGCATT ATCTAGATGT GTCTTCTGGT CAGAGACAGT AATAGACGAA ACAAGATCAG
11281 CATCTTCAAA TTTGGCAACA TCATTTGCAA AAGCAATTGA GAATGGTTAT TCACCTGTTC
11341 TAGGATATGC ATGCTCAATT TTTAAGAATA TTCAACAACT ATATATTGCC CTTGGGATGA
11401 ATATCAATCC AACTATAACA CAGAATATCA GAGATCAGTA TTTTAGGAAT CCAAATTGGA
11461 TGCAATATGC CTCTTTAATA CCTGCTAGTG TTGGGGGATT CAATTACATG GCCATGTCAA
11521 GATGTTTTGT AAGGAATATT GGTGATCCAT CAGTTGCCGC ATTGGCTGAT ATTAAAAGAT
11581 TTATTAAGGC GAATCTATTA GACCGAAGTG TTCTTTATAG GATTATGAAT CAAGAACCAG
11641 GTGAGTCATC TTTTTTGGAC TGGGCTTCAG ATCCATATTC ATGCAATTTA CCACAATCTC
11701 AAAATATAAC CACCATGATA AAAAATATAA CAGCAAGGAA TGTATTACAA GATTCACCAA
11761 ATCCATTATT ATCTGGATTA TTCACAAATA CAATGATAGA AGAAGATGAA GAATTAGCTG
11821 AGTTCCTGAT GGACAGGAAG GTAATTCTCC CTAGAGTTGC ACATGATATT CTAGATAATT
11881 CTCTCACAGG AATTAGAAAT GCCATAGCTG GAATGTTAGA TACGACAAAA TCACTAATTC
11941 GGGTTGGCAT AAATAGAGGA GGACTGACAT ATAGTTTGTT GAGGAAAATC AGTAATTACG
12001 ATCTAGTACA ATATGAAACA CTAAGTAGGA CTTTGCGACT AATTGTAAGT GATAAAATCA
12061 AGTATGAAGA TATGTGTTCG GTAGACCTTG CCATAGCATT GCGACAAAAG ATGTGGATTC
12121 ATTTATCAGG AGGAAGGATG ATAAGTGGAC TTGAAACGCC TGACCCATTA GAATTACTAT
12181 CTGGGGTAGT AATAACAGGA TCAGAACATT GTAAAATATG TTATTCTTCA GATGGCACAA
12241 ACCCATATAC TTGGATGTAT TTACCCGGTA ATATCAAAAT AGGATCAGCA GAAACAGGTA
12301 TATCGTCATT AAGAGTTCCT TATTTTGGAT CAGTCACTGA TGAAAGATCT GAAGCACAAT
12361 TAGGATATAT CAAGAATCTT AGTAAACCTG CAAAAGCCGC AATAAGAATA GCAATGATAT
12421 ATACATGGGC ATTTGGTAAT GATGAGATAT CTTGGATGGA AGCCTCACAG ATAGCACAAA
12481 CACGTGCAAA TTTTACACTA GATAGTCTCA AAATTTTAAC ACCGGTAGCT ACATCAACAA
12541 ATTTATCACA CAGATTAAAG GATACTGCAA CTCAGATGAA ATTCTCCAGT ACATCATTGA
12601 TCAGAGTCAG CAGATTCATA ACAATGTCCA ATGATAACAT GTCTATCAAA GAAGCTAATG
12661 AAACCAAAGA TACTAATCTT ATTTATCAAC AAATAATGTT AACAGGATTA AGTGTTTTCG
12721 AATATTTATT TAGATTAAAA GAAACCACAG GACACAACCC TATAGTTATG CATCTGCACA
12781 TAGAAGATGA GTGTTGTATT AAAGAAAGTT TTAATGATGA ACATATTAAT CCAGAGTCTA
12841 CATTAGAATT AATTCGATAT CCTGAAAGTA ATGAATTTAT TTATGATAAA GACCCACTCA
12901 AAGATGTGGA CTTATCAAAA CTTATGGTTA TTAAAGACCA TTCTTACACA ATTGATATGA
12961 ATTATTGGGA TGATACTGAC ATCATACATG CAATTTCAAT ATGTACTGCA ATTACAATAG
13021 CAGATACTAT GTCACAATTA GATCGAGATA ATTTAAAAGA GATAATAGTT ATTGCAAATG
13081 ATGATGATAT TAATAGCTTA ATCACTGAAT TTTTGACTCT TGACATACTT GTATTTCTCA
13141 AGACATTTGG TGGATTATTA GTAAATCAAT TTGCATACAC TCTTTATAGT CTAAAAATAG
13201 AAGGTAGGGA TCTCATTTGG GATTATATAA TGAGAACACT GAGAGATACT TCCCATTCAA
13261 TATTAAAAGT ATTATCTAAT GCATTATCTC ATCCTAAAGT ATTCAAGAGG TTCTGGGATT
13321 GTGGAGTTTT AAACCCTATT TATGGTCCTA ATACTGCTAG TCAAGACCAG ATAAAACTTG
13381 CCCTATCTAT ATGTGAATAT TCACTAGATC TATTTATGAG AGAATGGTTG AATGGTGTAT
13441 CACTTGAAAT ATACATTTGT GACAGCGATA TGGAAGTTGC AAATGATAGG AAACAAGCCT
13501 TTATTTCTAG ACACCTTTCA TTTGTTTGTT GTTTAGCAGA AATTGCATCT TTCGGACCTA
13561 ACCTGTTAAA CTTAACATAC TTGGAGAGAC TTGATCTATT GAAACAATAT CTTGAATTAA
13621 ATATTAAAGA AGACCCTACT CTTAAATATG TACAAATATC TGGATTATTA ATTAAATCGT
13681 TCCCATCAAC TGTAACATAC GTAAGAAAGA CTGCAATCAA ATATCTAAGG ATTCGCGGTA
13741 TTAGTCCACC TGAGGTAATT GATGATTGGG ATCCGGTAGA AGATGAAAAT ATGCTGGATA
13801 ACATTGTCAA AACTATAAAT GATAACTGTA ATAAAGATAA TAAAGGGAAT AAAATTAACA
13861 ATTTCTGGGG ACTAGCACTT AAGAACTATC AAGTCCTTAA AATCAGATCT ATAACAAGTG
13921 ATTCTGATGA TAATGATAGA CTAGATGCTA ATACAAGTGG TTTGACACTT CCTCAAGGAG
13981 GGAATTATCT ATCGCATCAA TTGAGATTAT TCGGAATCAA CAGCACTAGT TGTCTGAAAG
14041 CTCTTGAGTT ATCACAAATT TTAATGAAGG AAGTCAATAA AGACAAGGAC AGGCTCTTCC
14101 TGGGAGAAGG AGCAGGAGCT ATGCTAGCAT GTTATGATGC CACATTAGGA CCTGCAGTTA
14161 ATTATTATAA TTCAGGTTTG AATATAACAG ATGTAATTGG TCAACGAGAA TTGAAAATAT
14221 TTCCTTCAGA GGTATCATTA GTAGGTAAAA AATTAGGAAA TGTGACACAG ATTCTTAACA
14281 GGGTAAAAGT ACTGTTCAAT GGGAATCCTA ATTCAACATG GATAGGAAAT ATGGAATGTG
14341 AGAGCTTAAT ATGGAGTGAA TTAAATGATA AGTCCATTGG ATTAGTACAT TGTGATATGG
14401 AAGGAGCTAT CGGTAAATCA GAAGAAACTG TTCTACATGA ACATTATAGT GTTATAAGAA
14461 TTACATACTT GATTGGGGAT GATGATGTTG TTTTAGTTTC CAAAATTATA CCTACAATCA
14521 CTCCGAATTG GTCTAGAATA CTTTATCTAT ATAAATTATA TTGGAAAGAT GTAAGTATAA
14581 TATCACTCAA AACTTCTAAT CCTGCATCAA CAGAATTATA TCTAATTTCG AAAGATGCAT
14641 ATTGTACTAT AATGGAACCT AGTGAAATTG TTTTATCAAA ACTTAAAAGA TTGTCACTCT
14701 TGGAAGAAAA TAATCTATTA AAATGGATCA TTTTATCAAA GAAGAGGAAT AATGAATGGT
14761 TACATCATGA AATCAAAGAA GGAGAAAGAG ATTATGGAAT CATGAGACCA TATCATATGG
14821 CACTACAAAT CTTTGGATTT CAAATCAATT TAAATCATCT GGCGAAAGAA TTTTTATCAA
14881 CCCCAGATCT GACTAATATC AACAATATAA TCCAAAGTTT TCAGCGAACA ATAAAGGATG
14941 TTTTATTTGA ATGGATTAAT ATAACTCATG ATGATAAGAG ACATAAATTA GGCGGAAGAT
15001 ATAACATATT CCCACTGAAA AATAAGGGAA AGTTAAGACT GCTATCGAGA AGACTAGTAT
15061 TAAGTTGGAT TTCATTATCA TTATCGACTC GATTACTTAC AGGTCGCTTT CCTGATGAAA
15121 AATTTGAACA TAGAGCACAG ACTGGATATG TATCATTAGC TGATACTGAT TTAGAATCAT
15181 TAAAGTTATT GTCGAAAAAC ATCATTAAGA ATTACAGAGA GTGTATAGGA TCAATATCAT
15241 ATTGGTTTCT AACCAAAGAA GTTAAAATAC TTATGAAATT GATCGGTGGT GCTAAATTAT
15301 TAGGAATTCC CAGACAATAT AAAGAACCCG AAGACCAGTT ATTAGAAAAC TACAATCAAC
15361 ATGATGAATT TGATATCGAT TAAAACATAA ATACAATGAA GATATATCCT AACCTTTATC
15421 TTTAAGCCTA GGAATAGACA AAAAGTAAGA AAAACATGTA ATATATATAT ACCAAACAGA
15481 GTTCTTCTCT TGTTTGGT
In the third strategy (Figure 19), make up chimeric PIV3-PIV2 F and HN gene, wherein use the special primer of PCR, Vent archaeal dna polymerase and PIV2 F (13 in the table 22,14) and PIV2 HN (15 in the table 22,16) to respectively by the PIV2 F and the HN ORF district of pLit.PIV32Fhc and pLit.PIV32HNhc amplification coding extracellular domain and membrane-spanning domain.Simultaneously, with the special primer of PCR, Vent archaeal dna polymerase and PIV3 F (17 in the table 22,18) and PIV3 HN (19 in the table 22,20) to respectively from its cDNA subclone pLit.PIV3.F3a and pLit.PIV3.HN4 (people such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference) middle deletion coding extracellular domain and the PIV3 F of membrane-spanning domain and the part of O RF of HN gene.The F of gel-purified PIV2 and PIV3 and HN cDNA fragment, connection produces pLit.PIV32FCT and pLit.PIV32HNCT respectively.Digest chimeric F and HN construct with PpuMI and SpeI, being assembled together produces pLit.PIV32CT, to the complete order-checking in the special district of PIV, finds as design.Then the 4 kb BspEI-SpeI fragments of pLit.PIV32CT are introduced in the BspEI-SpeI window of p38 ' Δ PIV31hc and produced p38 ' Δ PIV32CT.The 6.5kb BspEI-SphI fragment that will contain p38 ' the Δ PIV32CT of PIV3-PIV2 F and HN mosaic gene is introduced respectively in the BspEI-SphI window of pFLC.2G+.hc and pFLCcp45, produces pFLC.PIV32CT (table 25; SEQ ID NO.62) and pFLC.PIV32CTcp45.The segmental nucleotide sequence of this BspEI-SpeI is submitted to GenBank.
The sequence of table 25 (SEQ ID NO.62) pFLC.PIV32CT, 15474bp, sense orientation
( Show only insert )
1 ACCAAACAAG AGAAGAAACT TGTCTGGGAA TATAAATTTA ACTTTAAATT AACTTAGGAT
61 TAAAGACATT GACTAGAAGG TCAAGAAAAG GGAACTCTAT AATTTCAAAA ATGTTGAGCC
121 TATTTGATAC ATTTAATGCA CGTAGGCAAG AAAACATAAC AAAATCAGCC GGTGGAGCTA
181 TCATTCCTGG ACAGAAAAAT ACTGTCTCTA TATTCGCCCT TGGACCGACA ATAACTGATG
241 ATAATGAGAA AATGACATTA GCTCTTCTAT TTCTATCTCA TTCACTAGAT AATGAGAAAC
301 AACATGCACA AAGGGCAGGG TTCTTGGTGT CTTTATTGTC AATGGCTTAT GCCAATCCAG
361 AGCTCTACCT AACAACAAAT GGAAGTAATG CAGATGTCAA GTATGTCATA TACATGATTG
421 AGAAAGATCT AAAACGGCAA AAGTATGGAG GATTTGTGGT TAAGACGAGA GAGATGATAT
481 ATGAAAAGAC AACTGATTGG ATATTTGGAA GTGACCTGGA TTATGATCAG GAAACTATGT
541 TGCAGAACGG CAGGAACAAT TCAACAATTG AAGACCTTGT CCACACATTT GGGTATCCAT
601 CATGTTTAGG AGCTCTTATA ATACAGATCT GGATAGTTCT GGTCAAAGCT ATCACTAGTA
661 TCTCAGGGTT AAGAAAAGGC TTTTTCACCC GATTGGAAGC TTTCAGACAA GATGGAACAG
721 TGCAGGCAGG GCTGGTATTG AGCGGTGACA CAGTGGATCA GATTGGGTCA ATCATGCGGT
781 CTCAACAGAG CTTGGTAACT CTTATGGTTG AAACATTAAT AACAATGAAT ACCAGCAGAA
841 ATGACCTCAC AACCATAGAA AAGAATATAC AAATTGTTGG CAACTACATA AGAGATGCAG
901 GTCTCGCTTC ATTCTTCAAT ACAATCAGAT ATGGAATTGA GACCAGAATG GCAGCTTTGA
961 CTCTATCCAC TCTCAGACCA GATATCAATA GATTAAAAGC TTTGATGGAA CTGTATTTAT
1021 CAAAGGGACC ACGCGCTCCT TTCATCTGTA TCCTCAGAGA TCCTATACAT GGTGAGTTCG
1081 CACCAGGCAA CTATCCTGCC ATATGGAGCT ATGCAATGGG GGTGGCAGTT GTACAAAATA
1141 GAGCCATGCA ACAGTATGTG ACGGGAAGAT CATATCTAGA CATTGATATG TTCCAGCTAG
1201 GACAAGCAGT AGCACGTGAT GCCGAAGCTC AAATGAGCTC AACACTGGAA GATGAACTTG
1261 GAGTGACACA CGAATCTAAA GAAAGCTTGA AGAGACATAT AAGGAACATA AACAGTTCAG
1321 AGACATCTTT CCACAAACCG ACAGGTGGAT CAGCCATAGA GATGGCAATA GATGAAGAGC
1381 CAGAACAATT CGAACATAGA GCAGATCAAG AACAAAATGG AGAACCTCAA TCATCCATAA
1441 TTCAATATGC CTGGGCAGAA GGAAATAGAA GCGATGATCA GACTGAGCAA GCTACAGAAT
1501 CTGACAATAT CAAGACCGAA CAACAAAACA TCAGAGACAG ACTAAACAAG AGACTCAACG
1561 ACAAGAAGAA ACAAAGCAGT CAACCACCCA CTAATCCCAC AAACAGAACA AACCAGGACG
1621 AAATAGATGA TCTGTTTAAC GCATTTGGAA GCAACTAATC GAATCAACAT TTTAATCTAA
1681 ATCAATAATA AATAAGAAAA ACTTAGGATT AAAGAATCCT ATCATACCGG AATATAGGGT
1741 GGTAAATTTA GAGTCTGCTT GAAACTCAAT CAATAGAGAG TTGATGGAAA GCGATGCTAA
1801 AAACTATCAA ATCATGGATT CTTGGGAAGA GGAATCAAGA GATAAATCAA CTAATATCTC
1861 CTCGGCCCTC AACATCATTG AATTCATACT CAGCACCGAC CCCCAAGAAG ACTTATCGGA
1921 AAACGACACA ATCAACACAA GAACCCAGCA ACTCAGTGCC ACCATCTGTC AACCAGAAAT
1981 CAAACCAACA GAAACAAGTG AGAAAGATAG TGGATCAACT GACAAAAATA GACAGTCCGG
2041 GTCATCACAC GAATGTACAA CAGAAGCAAA AGATAGAAAT ATTGATCAGG AAACTGTACA
2101 GAGAGGACCT GGGAGAAGAA GCAGCTCAGA TAGTAGAGCT GAGACTGTGG TCTCTGGAGG
2161 AATCCCCAGA AGCATCACAG ATTCTAAAAA TGGAACCCAA AACACGGAGG ATATTGATCT
2221 CAATGAAATT AGAAAGATGG ATAAGGACTC TATTGAGGGG AAAATGCGAC AATCTGCAAA
2281 TGTTCCAAGC GAGATATCAG GAAGTGATGA CATATTTACA ACAGAACAAA GTAGAAACAG
2341 TGATCATGGA AGAAGCCTGG AATCTATCAG TACACCTGAT ACAAGATCAA TAAGTGTTGT
2401 TACTGCTGCA ACACCAGATG ATGAAGAAGA AATACTAATG AAAAATAGTA GGACAAAGAA
2461 AAGTTCTTCA ACACATCAAG AAGATGACAA AAGAATTAAA AAAGGGGGAA AAGGGAAAGA
2521 CTGGTTTAAG AAATCAAAAG ATACCGACAA CCAGATACCA ACATCAGACT ACAGATCCAC
2581 ATCAAAAGGG CAGAAGAAAA TCTCAAAGAC AACAACCACC AACACCGACA CAAAGGGGCA
2641 AACAGAAATA CAGACAGAAT CATCAGAAAC ACAATCCTCA TCATGGAATC TCATCATCGA
2701 CAACAACACC GACCGGAACG AACAGACAAG CACAACTCCT CCAACAACAA CTTCCAGATC
2761 AACTTATACA AAAGAATCGA TCCGAACAAA CTCTGAATCC AAACCCAAGA CACAAAAGAC
2821 AAATGGAAAG GAAAGGAAGG ATACAGAAGA GAGCAATCGA TTTACAGAGA GGGCAATTAC
2881 TCTATTGCAG AATCTTGGTG TAATTCAATC CACATCAAAA CTAGATTTAT ATCAAGACAA
2941 ACGAGTTGTA TGTGTAGCAA ATGTACTAAA CAATGTAGAT ACTGCATCAA AGATAGATTT
3001 CCTGGCAGGA TTAGTCATAG GGGTTTCAAT GGACAACGAC ACAAAATTAA CACAGATACA
3061 AAATGAAATG CTAAACCTCA AAGCAGATCT AAAGAAAATG GACGAATCAC ATAGAAGATT
3121 GATAGAAAAT CAAAGAGAAC AACTGTCATT GATCACGTCA CTAATTTCAA ATCTCAAAAT
3181 TATGACTGAG AGAGGAGGAA AGAAAGACCA AAATGAATCC AATGAGAGAG TATCCATGAT
3241 CAAAACAAAA TTGAAAGAAG AAAAGATCAA GAAGACCAGG TTTGACCCAC TTATGGAGGC
3301 ACAAGGCATT GACAAGAATA TACCCGATCT ATATCGACAT GCAGGAGATA CACTAGAGAA
3361 CGATGTACAA GTTAAATCAG AGATATTAAG TTCATACAAT GAGTCAAATG CAACAAGACT
3421 AATACCCAAA AAAGTGAGCA GTACAATGAG ATCACTAGTT GCAGTCATCA ACAACAGCAA
3481 TCTCTCACAA AGCACAAAAC AATCATACAT AAACGAACTC AAACGTTGCA AAAATGATGA
3541 AGAAGTATCT GAATTAATGG ACATGTTCAA TGAAGATGTC AACAATTGCC AATGATCCAA
3601 CAAAGAAACG ACACCGAACA AACAGACAAG AAACAACAGT AGATCAAAAC CTGTCAACAC
3661 ACACAAAATC AAGCAGAATG AAACAACAGA TATCAATCAA TATACAAATA AGAAAAACTT
3721 AGGATTAAAG AATAAATTAA TCCTTGTCCA AAATGAGTAT AACTAACTCT GCAATATACA
3781 CATTCCCAGA ATCATCATTC TCTGAAAATG GTCATATAGA ACCATTACCA CTCAAAGTCA
3841 ATGAACAGAG GAAAGCAGTA CCCCACATTA GAGTTGCCAA GATCGGAAAT CCACCAAAAC
3901 ACGGATCCCG GTATTTAGAT GTCTTCTTAC TCGGCTTCTT CGAGATGGAA CGAATCAAAG
3961 ACAAATACGG GAGTGTGAAT GATCTCGACA GTGACCCGAG TTACAAAGTT TGTGGCTCTG
4021 GATCATTACC AATCGGATTG GCTAAGTACA CTGGGAATGA CCAGGAATTG TTACAAGCCG
4081 CAACCAAACT GGATATAGAA GTGAGAAGAA CAGTCAAAGC GAAAGAGATG GTTGTTTACA
4141 CGGTACAAAA TATAAAACCA GAACTGTACC CATGGTCCAA TAGACTAAGA AAAGGAATGC
4201 TGTTCGATGC CAACAAAGTT GCTCTTGCTC CTCAATGTCT TCCACTAGAT AGGAGCATAA
4261 AATTTAGAGT AATCTTCGTG AATTGTACGG CAATTGGATC AATAACCTTG TTCAAAATTC
4321 CTAAGTCAAT GGCATCACTA TCTCTACCCA ACACAATATC AATCAATCTG CAGGTACACA
4381 TAAAAACAGG GGTTCAGACT GATTCTAAAG GGATAGTTCA AATTTTGGAT GAGAAAGGCG
4441 AAAAATCACT GAATTTCATG GTCCATCTCG GATTGATCAA AAGAAAAGTA GGCAGAATGT
4501 ACTCTGTTGA ATACTGTAAA CAGAAAATCG AGAAAATGAG ATTGATATTT TCTTTAGGAC
4561 TAGTTGGAGG AATCAGTCTT CATGTCAATG CAACTGGGTC CATATCAAAA ACACTAGCAA
4621 GTCAGCTGGT ATTCAAAAGA GAGATTTGTT ATCCTTTAAT GGATCTAAAT CCGCATCTCA
4681 ATCTAGTTAT CTGGGCTTCA TCAGTAGAGA TTACAAGAGT GGATGCAATT TTCCAACCTT
4741 CTTTACCTGG CGAGTTCAGA TACTATCCTA ATATTATTGC AAAAGGAGTT GGGAAAATCA
4801 AACAATGGAA CTAGTAATCT CTATTTTAGT CCGGACGTAT CTATTAAGCC GAAGCAAATA
4861 AAGGATAATC AAAAACTTAG GACAAAAGAG GTCAATACCA ACAACTATTA GCAGTCACAC
4921 TCGCAAGAAT AAGAGAGAAG GGACCAAAAA AGTCAAATAG GAGAAATCAA AACAAAAGGT
4981 ACAGAACACC AGAACAACAA AATCAAAACA TCCAACTCAC TCAAAACAAA AATTCCAAAA
5041 GAGACCGGCA ACACAACAAG CACTGAACAT GCATCACCTG CATCCAATGA TAGTATGCAT
5101 TTTTGTTATG TACACTGGAA TTGTAGGTTC AGATGCCATT GCTGGAGATC AACTCCTCAA
5161 TGTAGGGGTC ATTCAATCAA AGATAAGATC ACTCATGTAC TACACTGATG GTGGCGCTAG
5221 CTTTATTGTT GTAAAATTAC TACCCAATCT TCCCCCAAGC AATGGAACAT GCAACATCAC
5281 CAGTCTAGAT GCATATAATG TTACCCTATT TAAGTTGCTA ACACCCCTGA TTGAGAACCT
5341 GAGCAAAATT TCTGCTGTTA CAGATACCAA ACCCCGCCGA GAACGATTTG CAGGAGTCGT
5401 TATTGGGCTT GCTGCACTAG GAGTAGCTAC AGCTGCACAA ATAACCGCAG CTGTAGCAAT
5461 AGTAAAAGCC AATGCAAATG CTGCTGCGAT AAACAATCTT GCATCTTCAA TTCAATCCAC
5521 CAACAAGGCA GTATCCGATG TGATAACTGC ATCAAGAACA ATTGCAACCG CAGTTCAAGC
5581 GATTCAGGAT CACATCAATG GAGCCATTGT CAACGGGATA ACATCTGCAT CATGCCGTGC
5641 CCATGATGCA CTAATTGGGT CAATATTAAA TTTGTATCTC ACTGAGCTTA CTACAATATT
5701 TCATAATCAA ATAACAAACC CTGCGCTGAC ACCACTTTCC ATCCAAGCTT TAAGAATCCT
5761 CCTCGGTAGC ACCTTGCCAA TTGTCATTGA ATCCAAACTC AACACAAAAC TCAACACAGC
5821 AGAGCTGCTC AGTAGCGGAC TGTTAACTGG TCAAATAATT TCCATTTCCC CAATGTACAT
5881 GCAAATGCTA ATTCAAATCA ATGTTCCGAC ATTTATAATG CAACCCGGTG CGAAGGTAAT
5941 TGATCTAATT GCTATCTCTG CAAACCATAA ATTACAAGAA GTAGTTGTAC AAGTTCCTAA
6001 TAGAATTCTA GAATATGCAA ATGAACTACA AAACTACCCA GCCAATGATT GTTTCGTGAC
6061 ACCAAACTCT GTATTTTGTA GATACAATGA GGGTTCCCCG ATCCCTGAAT CACAATATCA
6121 ATGCTTAAGG GGGAATCTTA ATTCTTGCAC TTTTACCCCT ATTATCGGGA ACTTTCTCAA
6181 GCGATTCGCA TTTGCCAATG GTGTGCTCTA TGCCAACTGC AAATCTTTGC TATGTAAGTG
6241 TGCCGACCCT CCCCATGTTG TGTCTCAAGA TGACAACCAA GGCATCAGCA TAATTGATAT
6301 TAAGAGGTGC TCTGAGATGA TGCTTGACAC TTTTTCATTT AGGATCACAT CTACATTCAA
6361 TGCTACATAC GTGACAGACT TCTCAATGAT TAATGCAAAT ATTGTACATC TAAGTCCTCT
6421 AGACTTGTCA AATCAAATCA ATTCAATAAA CAAATCTCTT AAAAGTGCTG AGGATTGGAT
6481 TGCAGATAGC AACTTCTTCG CTAATCAAGC CAGAACAGCC AAGACACTTT ATTCACTAAG
6541 TGCAATCGCA TTAATACTAT CAGTGATTAC TTTGGTTGTT GTGGGATTGC TGATTGCCTA
6601 CATCATCAAG TATTACAGAA TTCAAAAGAG AAATCGAGTG GATCAAAATG ACAAGCCATA
6661 TGTACTAACA AACAAATAAC ATATCTACAG ATCATTAGAT ATTAAAATTA TAAAAAACTT
6721 AGGAGTAAAG TTACGCAATC CAACTCTACT CATATAATTG AGGAAGGACC CAATAGACAA
6781 ATCCAAATTC GAGATGGAAT ACTGGAAGCA TACCAATCAC GGAAAGGATG CTGGTAATGA
6841 GCTGGAGACG TCTATGGCTA CTCATGGCAA CAAGCTCACT AATAAGACTG CCACAATTCT
6901 TGGCATATGC ACATTAATTG TGCTATGTTC AAGTATTCTT CATGAGATAA TTCATCTTGA
6961 TGTTTCCTCT GGTCTTATGA ATTCTGATGA GTCACAGCAA GGCATTATTC AGCCTATCAT
7021 AGAATCATTA AAATCATTGA TTGCTTTGGC CAACCAGATT CTATATAATG TTGCAATAGT
7081 AATTCCTCTT AAAATTGACA GTATCGAAAC TGTAATACTC TCTGCTTTAA AAGATATGCA
7141 CACCGGGAGT ATGTCCAATG CCAACTGCAC GCCAGGAAAT CTGCTTCTGC ATGATGCAGC
7201 ATACATCAAT GGAATAAACA AATTCCTTGT ACTTGAATCA TACAATGGGA CGCCTAAATA
7261 TGGACCTCTC CTAAATATAC CCAGCTTTAT CCCCTCAGCA ACATCTCCCC ATGGGTGTAC
7321 TAGAATACCA TCATTTTCAC TCATCAAGAC CCATTGGTGT TACACTCACA ATGTAATGCT
7381 TGGAGATTGT CTTGATTTCA CGGCATCTAA CCAGTATTTA TCAATGGGGA TAATACAACA
7441 ATCTGCTGCA GGGTTTCCAA TTTTCAGGAC TATGAAAACC ATTTACCTAA GTGATGGAAT
7501 CAATCGCAAA AGCTGTTCAG TCACTGCTAT ACCAGGAGGT TGTGTCTTGT ATTGCTATGT
7561 AGCTACAAGG TCTGAAAAAG AAGATTATGC CACGACTGAT CTAGCTGAAC TGAGACTTGC
7621 TTTCTATTAT TATAATGATA CCTTTATTGA AAGAGTCATA TCTCTTCCAA ATACAACAGG
7681 GCAGTGGGCC ACAATCAACC CTGCAGTCGG AAGCGGGATC TATCATCTAG GCTTTATCTT
7741 ATTTCCTGTA TATGGTGGTC TCATAAATGG GACTACTTCT TACAATGAGC AGTCCTCACG
7801 CTATTTTATC CCAAAACATC CCAACATAAC TTGTGCCGGT AACTCCAGCA AACAGGCTGC
7861 AATAGCACGG AGTTCCTATG TCATCCGTTA TCACTCAAAC AGGTTAATTC AGAGTGCTGT
7921 TCTTATTTGT CCATTGTCTG ACATGCATAC AGAAGAGTGT AATCTAGTTA TGTTTAACAA
7981 TTCCCAAGTC ATGATGGGTG CAGAAGGTAG GCTCTATGTT ATTGGTAATA ATTTGTATTA
8041 TTATCAACGC AGTTCCTCTT GGTGGTCTGC ATCGCTCTTT TACAGGATCA ATACAGATTT
8101 TTCTAAAGGA ATTCCTCCGA TCATTGAGGC TCAATGGGTA CCGTCCTATC AAGTTCCTCG
8161 TCCTGGAGTC ATGCCATGCA ATGCAACAAG TTTTTGCCCT GCTAATTGCA TCACAGGGGT
8221 GTACGCAGAT GTGTGGCCGC TTAATGATCC AGAACTCATG TCACGTAATG CTCTGAACCC
8281 CAACTATCGA TTTGCTGGAG CCTTTCTCAA AAATGAGTCC AACCGAACTA ATCCCACATT
8341 CTACACTGCA TCGGCTAACT CCCTCTTAAA TACTACCGGA TTCAACAACA CCAATCACAA
8401 AGCAGCATAT ACATCTTCAA CCTGCTTTAA AAACACTGGA ACCCAAAAAA TTTATTGTTT
8461 AATAATAATT GAAATGGGCT CATCTCTTTT AGGGGAGTTC CAAATAATAC CATTTTTAAG
8521 GGAACTAATG CTTTAATCAT AATTAACCAT AATATGCATC AATCTATCTA TAATACAAGT
8581 ATATGATAAG TAATCAGCAA TCAGACAATA GACAAAAGGG AAATATAAAA AACTTAGGAG
8641 CAAAGCGTGC TCGGGAAATG GACACTGAAT CTAACAATGG CACTGTATCT GACATACTCT
8701 ATCCTGAGTG TCACCTTAAC TCTCCTATCG TTAAAGGTAA AATAGCACAA TTACACACTA
8761 TTATGAGTCT ACCTCAGCCT TATGATATGG ATGACGACTC AATACTAGTT ATCACTAGAC
8821 AGAAAATAAA ACTTAATAAA TTGGATAAAA GACAACGATC TATTAGAAGA TTAAAATTAA
8881 TATTAACTGA AAAAGTGAAT GACTTAGGAA AATACACATT TATCAGATAT CCAGAAATGT
8941 CAAAAGAAAT GTTCAAATTA TATATACCTG GTATTAACAG TAAAGTGACT GAATTATTAC
9001 TTAAAGCAGA TAGAACATAT AGTCAAATGA CTGATGGATT AAGAGATCTA TGGATTAATG
9061 TGCTATCAAA ATTAGCCTCA AAAAATGATG GAAGCAATTA TGATCTTAAT GAAGAAATTA
9121 ATAATATATC GAAAGTTCAC ACAACCTATA AATCAGATAA ATGGTATAAT CCATTCAAAA
9181 CATGGTTTAC TATCAAGTAT GATATGAGAA GATTACAAAA AGCTCGAAAT GAGATCACTT
9241 TTAATGTTGG GAAGGATTAT AACTTGTTAG AAGACCAGAA GAATTTCTTA TTGATACATC
9301 CAGAATTGGT TTTGATATTA GATAAACAAA ACTATAATGG TTATCTAATT ACTCCTGAAT
9361 TAGTATTGAT GTATTGTGAC GTAGTCGAAG GCCGATGGAA TATAAGTGCA TGTGCTAAGT
9421 TAGATCCAAA ATTACAATCT ATGTATCAGA AAGGTAATAA CCTGTGGGAA GTGATAGATA
9481 AATTGTTTCC AATTATGGGA GAAAAGACAT TTGATGTGAT ATCGTTATTA GAACCACTTG
9541 CATTATCCTT AATTCAAACT CATGATCCTG TTAAACAACT AAGAGGAGCT TTTTTAAATC
9601 ATGTGTTATC CGAGATGGAA TTAATATTTG AATCTAGAGA ATCGATTAAG GAATTTCTGA
9661 GTGTAGATTA CATTGATAAA ATTTTAGATA TATTTAATAA GTCTACAATA GATGAAATAG
9721 CAGAGATTTT CTCTTTTTTT AGAACATTTG GGCATCCTCC ATTAGAAGCT AGTATTGCAG
9781 CAGAAAAGGT TAGAAAATAT ATGTATATTG GAAAACAATT AAAATTTGAC ACTATTAATA
9841 AATGTCATGC TATCTTCTGT ACAATAATAA TTAACGGATA TAGAGAGAGG CATGGTGGAC
9901 AGTGGCCTCC TGTGACATTA CCTGATCATG CACACGAATT CATCATAAAT GCTTACGGTT
9961 CAAACTCTGC GATATCATAT GAAAATGCTG TTGATTATTA CCAGAGCTTT ATAGGAATAA
10021 AATTCAATAA ATTCATAGAG CCTCAGTTAG ATGAGGATTT GACAATTTAT ATGAAAGATA
10081 AAGCATTATC TCCAAAAAAA TCAAATTGGG ACACAGTTTA TCCTGCATCT AATTTACTGT
10141 ACCGTACTAA CGCATCCAAC GAATCACGAA GATTAGTTGA AGTATTTATA GCAGATAGTA
10201 AATTTGATCC TCATCAGATA TTGGATTATG TAGAATCTGG GGACTGGTTA GATGATCCAG
10261 AATTTAATAT TTCTTATAGT CTTAAAGAAA AAGAGATCAA ACAGGAAGGT AGACTCTTTG
10321 CAAAAATGAC ATACAAAATG AGAGCTACAC AAGTTTTATC AGAGACACTA CTTGCAAATA
10381 ACATAGGAAA ATTCTTTCAA GAAAATGGGA TGGTGAAGGG AGAGATTGAA TTACTTAAGA
10441 GATTAACAAC CATATCAATA TCAGGAGTTC CACGGTATAA TGAAGTGTAC AATAATTCTA
10501 AAAGCCATAC AGATGACCTT AAAACCTACA ATAAAATAAG TAATCTTAAT TTGTCTTCTA
10561 ATCAGAAATC AAAGAAATTT GAATTCAAGT CAACGGATAT CTACAATGAT GGATACGAGA
10621 CTGTGAGCTG TTTCCTAACA ACAGATCTCA AAAAATACTG TCTTAATTGG AGATATGAAT
10681 CAACAGCTCT ATTTGGAGAA ACTTGCAACC AAATATTTGG ATTAAATAAA TTGTTTAATT
10741 GGTTACACCC TCGTCTTGAA GGAAGTACAA TCTATGTAGG TGATCCTTAC TGTCCTCCAT
10801 CAGATAAAGA ACATATATCA TTAGAGGATC ACCCTGATTC TGGTTTTTAC GTTCATAACC
10861 CAAGAGGGGG TATAGAAGGA TTTTGTCAAA AATTATGGAC ACTCATATCT ATAAGTGCAA
10921 TACATCTAGC AGCTGTTAGA ATAGGCGTGA GGGTGACTGC AATGGTTCAA GGAGACAATC
10981 AAGCTATAGC TGTAACCACA AGAGTACCCA ACAATTATGA CTACAGAGTT AAGAAGGAGA
11041 TAGTTTATAA AGATGTAGTG AGATTTTTTG ATTCATTAAG AGAAGTGATG GATGATCTAG
11101 GTCATGAACT TAAATTAAAT GAAACGATTA TAAGTAGCAA GATGTTCATA TATAGCAAAA
11161 GAATCTATTA TGATGGGAGA ATTCTTCCTC AAGCTCTAAA AGCATTATCT AGATGTGTCT
11221 TCTGGTCAGA GACAGTAATA GACGAAACAA GATCAGCATC TTCAAATTTG GCAACATCAT
11281 TTGCAAAAGC AATTGAGAAT GGTTATTCAC CTGTTCTAGG ATATGCATGC TCAATTTTTA
11341 AGAATATTCA ACAACTATAT ATTGCCCTTG GGATGAATAT CAATCCAACT ATAACACAGA
11401 ATATCAGAGA TCAGTATTTT AGGAATCCAA ATTGGATGCA ATATGCCTCT TTAATACCTG
11461 CTAGTGTTGG GGGATTCAAT TACATGGCCA TGTCAAGATG TTTTGTAAGG AATATTGGTG
11521 ATCCATCAGT TGCCGCATTG GCTGATATTA AAAGATTTAT TAAGGCGAAT CTATTAGACC
11581 GAAGTGTTCT TTATAGGATT ATGAATCAAG AACCAGGTGA GTCATCTTTT TTGGACTGGG
11641 CTTCAGATCC ATATTCATGC AATTTACCAC AATCTCAAAA TATAACCACC ATGATAAAAA
11701 ATATAACAGC AAGGAATGTA TTACAAGATT CACCAAATCC ATTATTATCT GGATTATTCA
11761 CAAATACAAT GATAGAAGAA GATGAAGAAT TAGCTGAGTT CCTGATGGAC AGGAAGGTAA
11821 TTCTCCCTAG AGTTGCACAT GATATTCTAG ATAATTCTCT CACAGGAATT AGAAATGCCA
11881 TAGCTGGAAT GTTAGATACG ACAAAATCAC TAATTCGGGT TGGCATAAAT AGAGGAGGAC
11941 TGACATATAG TTTGTTGAGG AAAATCAGTA ATTACGATCT AGTACAATAT GAAACACTAA
12001 GTAGGACTTT GCGACTAATT GTAAGTGATA AAATCAAGTA TGAAGATATG TGTTCGGTAG
12061 ACCTTGCCAT AGCATTGCGA CAAAAGATGT GGATTCATTT ATCAGGAGGA AGGATGATAA
12121 GTGGACTTGA AACGCCTGAC CCATTAGAAT TACTATCTGG GGTAGTAATA ACAGGATCAG
12181 AACATTGTAA AATATGTTAT TCTTCAGATG GCACAAACCC ATATACTTGG ATGTATTTAC
12241 CCGGTAATAT CAAAATAGGA TCAGCAGAAA CAGGTATATC GTCATTAAGA GTTCCTTATT
12301 TTGGATCAGT CACTGATGAA AGATCTGAAG CACAATTAGG ATATATCAAG AATCTTAGTA
12361 AACCTGCAAA AGCCGCAATA AGAATAGCAA TGATATATAC ATGGGCATTT GGTAATGATG
12421 AGATATCTTG GATGGAAGCC TCACAGATAG CACAAACACG TGCAAATTTT ACACTAGATA
12481 GTCTCAAAAT TTTAACACCG GTAGCTACAT CAACAAATTT ATCACACAGA TTAAAGGATA
12541 CTGCAACTCA GATGAAATTC TCCAGTACAT CATTGATCAG AGTCAGCAGA TTCATAACAA
12601 TGTCCAATGA TAACATGTCT ATCAAAGAAG CTAATGAAAC CAAAGATACT AATCTTATTT
12661 ATCAACAAAT AATGTTAACA GGATTAAGTG TTTTCGAATA TTTATTTAGA TTAAAAGAAA
12721 CCACAGGACA CAACCCTATA GTTATGCATC TGCACATAGA AGATGAGTGT TGTATTAAAG
12781 AAAGTTTTAA TGATGAACAT ATTAATCCAG AGTCTACATT AGAATTAATT CGATATCCTG
12841 AAAGTAATGA ATTTATTTAT GATAAAGACC CACTCAAAGA TGTGGACTTA TCAAAACTTA
12901 TGGTTATTAA AGACCATTCT TACACAATTG ATATGAATTA TTGGGATGAT ACTGACATCA
12961 TACATGCAAT TTCAATATGT ACTGCAATTA CAATAGCAGA TACTATGTCA CAATTAGATC
13021 GAGATAATTT AAAAGAGATA ATAGTTATTG CAAATGATGA TGATATTAAT AGCTTAATCA
13081 CTGAATTTTT GACTCTTGAC ATACTTGTAT TTCTCAAGAC ATTTGGTGGA TTATTAGTAA
13141 ATCAATTTGC ATACACTCTT TATAGTCTAA AAATAGAAGG TAGGGATCTC ATTTGGGATT
13201 ATATAATGAG AACACTGAGA GATACTTCCC ATTCAATATT AAAAGTATTA TCTAATGCAT
13261 TATCTCATCC TAAAGTATTC AAGAGGTTCT GGGATTGTGG AGTTTTAAAC CCTATTTATG
13321 GTCCTAATAC TGCTAGTCAA GACCAGATAA AACTTGCCCT ATCTATATGT GAATATTCAC
13381 TAGATCTATT TATGAGAGAA TGGTTGAATG GTGTATCACT TGAAATATAC ATTTGTGACA
13441 GCGATATGGA AGTTGCAAAT GATAGGAAAC AAGCCTTTAT TTCTAGACAC CTTTCATTTG
13501 TTTGTTGTTT AGCAGAAATT GCATCTTTCG GACCTAACCT GTTAAACTTA ACATACTTGG
13561 AGAGACTTGA TCTATTGAAA CAATATCTTG AATTAAATAT TAAAGAAGAC CCTACTCTTA
13621 AATATGTACA AATATCTGGA TTATTAATTA AATCGTTCCC ATCAACTGTA ACATACGTAA
13681 GAAAGACTGC AATCAAATAT CTAAGGATTC GCGGTATTAG TCCACCTGAG GTAATTGATG
13741 ATTGGGATCC GGTAGAAGAT GAAAATATGC TGGATAACAT TTCAAAACT ATAAATGATA
13801 ACTGTAATAA AGATAATAAA GGGAATAAAA TTAACAATTT CTGGGGACTA GCACTTAAGA
13861 ACTATCAAGT CCTTAAAATC AGATCTATAA CAAGTGATTC TGATGATAAT GATAGACTAG
13921 ATGCTAATAC AAGTGGTTTG ACACTTCCTC AAGGAGGGAA TTATCTATCG CATCAATTGA
13981 GATTATTCGG AATCAACAGC ACTAGTTGTC TGAAAGCTCT TGAGTTATCA CAAATTTTAA
14041 TGAAGGAAGT CAATAAAGAC AAGGACAGGC TCTTCCTGGG AGAAGGAGCA GGAGCTATGC
14101 TAGCATGTTA TGATGCCACA TTAGGACCTG CAGTTAATTA TTATAATTCA GGTTTGAATA
14161 TAACAGATGT AATTGGTCAA CGAGAATTGA AAATATTTCC TTCAGAGGTA TCATTAGTAG
14221 GTAAAAAATT AGGAAATGTG ACACAGATTC TTAACAGGGT AAAAGTACTG TTCAATGGGA
14281 ATCCTAATTC AACATGGATA GGAAATATGG AATGTGAGAG CTTAATATGG AGTGAATTAA
14341 ATGATAAGTC CATTGGATTA GTACATTGTG ATATGGAAGG AGCTATCGGT AAATCAGAAG
14401 AAACTGTTCT ACATGAACAT TATAGTGTTA TAAGAATTAC ATACTTGATT GGGGATGATG
14461 ATGTTGTTTT AGTTTCCAAA ATTATACCTA CAATCACTCC GAATTGGTCT AGAATACTTT
14521 ATCTATATAA ATTATATTGG AAAGATGTAA GTATAATATC ACTCAAAACT TCTAATCCTG
14581 CATCAACAGA ATTATATCTA ATTTCGAAAG ATGCATATTG TACTATAATG GAACCTAGTG
14641 AAATTGTTTT ATCAAAACTT AAAAGATTGT CACTCTTGGA AGAAAATAAT CTATTAAAAT
14701 GGATCATTTT ATCAAAGAAG AGGAATAATG AATGGTTACA TCATGAAATC AAAGAAGGAG
14761 AAAGAGATTA TGGAATCATG AGACCATATC ATATGGCACT ACAAATCTTT GGATTTCAAA
14821 TCAATTTAAA TCATCTGGCG AAAGAATTTT TATCAACCCC AGATCTGACT AATATCAACA
14881 ATATAATCCA AAGTTTTCAG CGAACAATAA AGGATGTTTT ATTTGAATGG ATTAATATAA
14941 CTCATGATGA TAAGAGACAT AAATTAGGCG GAAGATATAA CATATTCCCA CTGAAAAATA
15001 AGGGAAAGTT AAGACTGCTA TCGAGAAGAC TAGTATTAAG TTGGATTTCA TTATCATTAT
15061 CGACTCGATT ACTTACAGGT CGCTTTCCTG ATGAAAAATT TGAACATAGA GCACAGACTG
15121 GATATGTATC ATTAGCTGAT ACTGATTTAG AATCATTAAA GTTATTGTCG AAAAACATCA
15181 TTAAGAATTA CAGAGAGTGT ATAGGATCAA TATCATATTG GTTTCTAACC AAAGAAGTTA
15241 AAATACTTAT GAAATTGATC GGTGGTGCTA AATTATTAGG AATTCCCAGA CAATATAAAG
15301 AACCCGAAGA CCAGTTATTA GAAAACTACA ATCAACATGA TGAATTTGAT ATCGATTAAA
15361 ACATAAATAC AATGAAGATA TATCCTAACC TTTATCTTTA AGCCTAGGAA TAGACAAAAA
15421 GTAAGAAAAA CATGTAATAT ATATATACCA AACAGAGTTC TTCTCTTGTT TGGT
The cDNA that makes up is designed to the anti-genome of whole PIV3-2 and meets 6 principle (people such as Calain, Journal of Virology 67:4822-30,1993; People such as Durbin, virusology 234:74-83,1997, be incorporated herein by reference).It is 15498nt that PIV3-2 among the pFLC.PIV32TM inserts fragment length, and the length among the pFLC.PIV32CT is 15474 nt.These length overalls do not comprise two 5 ' terminal G residues of T7 promotor contribution, are removed in Recovery Process because suppose them.The transfection and the answer of the chimeric PIV3-PIV2 virus of recombinating
The HEp-2 cell monolayer grows in 6 orifice plates and converges, and is basic as state and carry out transfection (people such as Tao, Journal of Virology 72:2955-2961 1998, are incorporated herein by reference).Contain among the MEM of 12 μ l LipofectACE (Life Technologies) with the 5 anti-genome cDNAs of μ g PIV3-PIV2 and three kinds at 0.2ml and to support plasmids, 0.2 μ g pTM (N), 0.2 μ g pTM (P no C), 0.1 μ g pTM (L), the HEp-2 individual layer in hole of transfection.Infection multiplicity (MOI) with 3 is used the MVA-T7 cells infected in containing the 0.8ml serum-free MEM of 50 μ g/ml gentamicins and 2mM glutamine simultaneously.Simultaneously the chimeric anti-genome cDNA pFLC.2G+.hc of transfection people such as (, Journal of Virology 72:2955-2961,1998) Tao is as positive control.After 12 hours, transfection media is replaced with the fresh serum-free MEM that 1.5ml is supplemented with 50 μ g/ml gentamicins and 2mM glutamine at 32 ℃ of following incubations.Cells transfected incubation 2 days again under 32 ℃.The pig trypsin p-trypsinase of the 3rd day adding gamma-irradiation after transfection; T1311, Sigma, St Louis, MO) to final concentration be 0.5 μ g/ml.The harvested cell culture supernatant, and on go down to posterity (be called and go down to posterity 1) shake fresh Vero cell monolayer in the bottle to T25.After absorption is spent the night, the transfection cutting is replaced with the fresh tryptic VP-SFM of 0.5 μ g/ml p-that is supplemented with.Go down to posterity 1 training at foster thing 32 ℃ of following incubations 4 days, the virus of results amplification, and in the presence of 0.5 μ g/ml p-trypsinase, further on the Vero cell 32 ℃ go down to posterity (be called and go down to posterity 2) 4 days.Adsorb viral existence in 2 cultures of determining to go down to posterity by the hemocyte of using 0.2% guinea-pig red blood cell (RBC).Be supplemented with three end dilutions eventually continuously that the Vero cell that keeps among 2mM glutamine, 50 μ g/ml gentamicins and the tryptic VP-SFM of 0.5 μ g/ml p-carries out by being used in, be further purified virus.For the third time eventually after the dilution of end, to the further amplicon virus of Vero cell three times, this viral suspension is used for characterizing in further external and the body.Utilize the order-checking of PCR product and restriction analysis determining to the chimeric character of vRNA
In order to analyze the genetic construction of vRNA, to LLC-MK2 cell amplification reorganization PIV and concentrated.From the virus precipitation, extract vRNA, and record with Superscript Preamplification system inversion.RT-PCR carries out (21 in the table 22,22 and 23,24) with Advantage cDNA synthetic agent box and PIV2 or the special primer of PIV3.By limiting digestion or gel-purified analysis and passing through sequencing analysis RT-PCR product.PIV's duplicates among the LLC-MK2
By with the MOI 0.01 triplicate LLC-MK2 cell monolayer that converges on 6 orifice plates that infects, assess the growth of PIV virus in tissue culture.Remove inoculum in absorption under 32 ℃ after 1 hour.Cell is added the 2ml/ hole and is contained 50 μ g/ml gentamicins and the tryptic OptiMEM I of 0.5 μ g/ml p-with serum-free OptiMEM I washing 3 times, and at 32 ℃ of following incubations.With 24 hours intervals, from every hole, take out 0.5ml equal portions substratum, freeze suddenly, and adding 0.5ml contains the tryptic fresh culture of p-in culture.(people such as Tao, Journal of Virology 72:2955-2961 1998, are incorporated herein by reference) cover with fluid as previously mentioned, under 32 ℃ on the LLC-MK2 cell monolayer virus in the titration equal portions, determine titration end point by hemocyte absorption, titre is expressed as log
10TCID
50/ ml.The chimeric PIV3-PIV2 virus of recombinating duplicating under differing temps
Virus is with having replenished 2mM glutamine and the tryptic 1 * L15 serial dilution of 0.5 μ g/ml p-.With the LLC-MK2 individual layer in virus infection 96 orifice plates of dilution.(people such as Skiadopoulos, vaccine 18:503-510,1999 as previously mentioned.Be incorporated herein by reference) with the culture plate that infects incubation 7 days under differing temps.As above measure virus titer.The chimeric PIV3-PIV2 virus of recombinating duplicating in the hamster respiratory tract, immunogenicity and protection efficient
Every group of 6 golden Syria hamster intranasal vaccinations 10
5.3TCID
50Reorganization or biology deutero-virus.Inoculate and kill hamster after 4 days, take out lung and concha, prepare to be used for quantitatively (people such as Skiadopoulos, vaccine 18:503-510,1999 of virus.Be incorporated herein by reference).The titre of every group of 6 hamsters is expressed as average log
10TCID
50/ gram tissue.
Every group of 12 golden Syria hamsters were at the 0th day intranasal infection 10
5.3TCID
50Virus, every group of 6 hamsters are back with 10 in 4 weeks
6TCID
50PIV1 or 10
6TCID
50PIV2 attacks.Attack and kill hamster after 4 days, take out lung and concha.(people such as Tao, Journal of Virology 72:2955-2961 1998, are incorporated herein by reference) measure the challenge virus titre in the tissue of results as previously mentioned.The titre of every group of 6 hamsters is expressed as average log
10TCID
50/ gram tissue.Inoculated before 3 days and gathered in the 28th day serum sample, (people such as van Wyke Coelingh, virusology 143:569-582 1985, are incorporated herein by reference) measure hemagglutinin-inhibitions mensuration (HAI) titre of anti-PIV1, PIV2 and PIV3 as previously mentioned.Titre is expressed as average log reciprocal
2The chimeric PIV3-PIV2 virus of recombinating duplicating in cercopithecus aethiops (AGM), immunogenicity and protection efficient
Every group of 4 AGM are in the 0th day nose and infect each position 10 in the tracheae
5TCID
50Virus.(people such as van Wyke Coelingh, virusology 143:569-582,1985) collect nose/larynx (NT) swab specimen and lavage of trachea thing 12 and 5 days respectively as previously mentioned.At the 29th day, at each position with 10
5TCID
50Hit the AGM of immunity in the PIV2/V94 nose with the tracheae inside fire attack.Collected NT swab specimen and lavage of trachea thing respectively 10 days and 5 days.Serum sample before the-3,28 and 60 days collect immunity, after immunity back and the attack respectively.People such as (, Journal of Virology 72:2955-2961,1998) Tao measures the virus titer in NT swab specimen and the lavage of trachea thing as previously mentioned.Titre is expressed as log
10TCID
50/ ml.People such as (, virusology 143:569-582,1985) van Wyke Coelingh measures the serum NAT of anti-PIV1 and PIV2 as previously mentioned.Titre is expressed as average log reciprocal
2The chimeric PIV3-PIV2 virus of recombinating duplicating and immunogenicity in chimpanzee
(people such as Whitehead, Journal of Virology 72:4467-4471 1998, are incorporated herein by reference) as previously mentioned, every group of 4 chimpanzee are in the 0th day nose and infect 10 in the tracheae
5TCID
50PIV2/V94 or rPIV3-2TM.Collected the NT swab specimen every day totally 12 days, and obtained the lavage of trachea thing at the 2nd, 4,6,8 and 10 day.People such as (, Journal of Virology 72:2955-2961,1998) Tao measures the virus titer in the sample as previously mentioned.The peak virus titer is expressed as log
10TCID
50/ ml.Respectively before the-3 and 28 days collect immunity with immunity after serum sample.(people such as van Wyke Coelingh, virusology 143:569-582 1985, are incorporated herein by reference) measure the serum NAT of anti-PIV1 and PIV2 as previously mentioned, and titre is expressed as average log reciprocal
2Embedded virus is lived in reorganization can not be from the chimeric cDNA acquisition of encode complete PIV2 F and the proteic PIV3-PIV2 of HN
More than describe and summarized in Figure 17 the structure of the chimeric cDNA of PIV3-PIV2, wherein the F of JS wild-type PIV3 and HN ORF are replaced by the ORF of PIV2/V94.Whole plasmid construction body, pFLC.PIV32hc (Figure 17), the chimeric antigenomic RNA of PIV3-PIV2 of a kind of 15392 nt that encode, it meets 6 principle.
Utilize LipofectACE to support plasmid pTM (N), pTM (P no C) and pTM (L) transfection HEp-2 cell monolayer with three kinds with pFLC.PIV32hc, use simultaneously MVA-T7 cells infected (people such as Tao as previously mentioned, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference).Can't from pFLC.PIV32hc obtain infective virus the transfection several times at first, and can from infect, obtain embedded virus with all of control plasmid pFLC.2G+.hc.
What meet these results is to take place to stop the possibility that reclaims the sudden change of rPIV3-2 virus from cDNA clone cells transfected outside the 4 kb BspEI-SpeI fragments of pFLC.PIV32hc.In order to check this possibility, the BspEI-SpeI fragment of p38 ' Δ PIV31hc and p38 ' Δ PIV32hc is exchanged.Except the SpeI-SphI fragment that contains PIV3 L gene order is exchanged, identical among regenerated p38 ' Δ PIV31hc and p38 ' Δ PIV32hc and Figure 17.The BspEI-SphI fragment of these two kinds of regenerated cDNA is introduced in the BspEI-SphI window of PIV3 full-length clone p3/7-(131) 2G+, by 5 other separate connection of branch, produce 10 pFLC.2G+.hc and pFLC.PIV32hc clone (be connected at every turn and produce 2 clones) respectively.(the PIV3 sequence outside the BspEI-SphI window of attention p3/7-(131) 2G+, pFLC.2G+.hc and pFLC.PIV32hc is identical).Therefore, this will replace any PIV3 main chain sequence, can obtain to have the puppet sudden change of the sequence of known function.In addition, assessed the functional of main chain simultaneously.10 pFLC.PIV32hc cDNA clones can not produce live virus, but 10 pFLC.2G+.hccDNA clones can both produce live virus.Although reclaim mode that height defective type PIV3 C-the knocks out recon transfection cutting that goes down to posterity, can not from pFLC.PIV32hc, reclaim viral (people such as Durbin, virusology 261:319-30 1999, are incorporated herein by reference) to be similar to successfully.Because except that the kytoplasm tail territory of F and HN, the every kind of unique composition that is used for producing pFLC.PIV32hc all can be used to successfully produce other recombinant viruses, so the failure that the mistake among the cDNA causes producing recombinant virus is very impossible.Certainly, better explain it is that one or more required PIV3 albumen of total length PIV2 F and HN glycoprotein and viral growth are incompatible.Embedded virus is obtained by chimeric IV3-PIV2 F of coding and the chimeric cDNA of the proteic PIV3-PIV2 of HN
Use other two kinds of strategies, make up the new anti-genome cDNA of chimeric PIV3-PIV2, wherein the extracellular domain of PIV3 F and HN glycoprotein or extracellular domain and membrane-spanning domain are replaced by its PIV2 counterpart.More than describe and in Figure 18,19 and 20, summarized the structure of four kinds of full-length cDNAs, that is, and pFLC.PIV32TM, pFLC.PIV32TMcp45, pFLC.PIV32CT and pFLC.PIV32CTcp45.PIV3-2 among whole plasmid pFLC.PIV32TM and the pFLC.PIV32CT inserts fragment length and is respectively 15498 nt and 15474 nt, and meet 6 principle, F and HN genes encoding chimeric glycoprotein albumen (people such as Calain, Journal of Virology 67:4822-30,1993 wherein; People such as Durbin, virusology 234:74-83,1997, be incorporated herein by reference).The verity of these four kinds of constructs is by being confirmed to order-checking of BspEI-SphI district and restriction analysis.
Obtain reorganization embedded virus, called after rPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45 and rPIV3-2CTcp45 respectively by full-length clone pFLC.PIV32TM, pFLC.PIV32CT, pFLC.PIV32TMcp45 or pFLC.PIV32CTcp45.Clone these virus by 3 last eventually continuously dilution biology on the Vero cell, in the Vero cell, increase three times then.The genetics of the chimeric PIV3-PIV2 virus of recombinating characterizes
Biology clone's chimeric PIV3-PIV2 virus rPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45 and rPIV3-2CTcp45 breed on the LLC-MK2 cell, concentrate then.In the RT-PCR that the specific gene fragment is carried out being increased, use the viral RNA (21 in the table 22,22 or 23) that from sedimentary virus, extracts with PIV2 or the special primer of PIV3.With the PIV2 special primer restriction enzyme digestion pattern from the RT-PCR product of rPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45 and rPIV3-2CTcp45 amplification all is different from PIV2/V94, uses the pattern of EcoRI, MfeI, NcoI or PpuMI desired as the cDNA of design.8 kinds of nucleotide sequences that different PIV3-PIV2 are connected in the F that Figure 20 has listed rPIV3-2TM and rPIV3-2CT and the HN gene.(people such as Skiadopoulos as previously mentioned, Journal of Virology 73:1374-81,1999, be incorporated herein by reference) also with RT-PCR and restriction enzyme digestion confirmed to exist among rPIV3-2TMcp45 and the rPIV3-2CTcp45 in 3 ' of NP-leader and gene initiator the cp45 mark.These results have confirmed the existence of the cp45 sudden change of the chimeric character of the PIV3-PIV2 virus that obtains and introducing.PIV3-PIV2 reorganization embedded virus can externally in the LLC-MK2 cell efficiently duplicate
The kinetics and the degree (Figure 21) of PIV3-PIV2 reorganization embedded virus replication in vitro have been estimated by many recursive copyings in the LLC-MK2 cell.At p-trypsin 0.5 μ g/ml) in the presence of in triplicate infect LLC-MK2 cell monolayer culture in 6 orifice plates with MOI 0.01 usefulness rPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45 or rPIV3-2CTcp45.In 6 days, from culture supernatant, take out sample with 24 hours intervals.Except that rPIV3-2CTcp45 (clone 2A1), every kind of reorganization embedded virus all duplicates with speed identical with the PIV2/V94 parental virus and similar level, show the chimeric speed of growth that can not change the reorganization embedded virus of the proteic PIV3-PIV2 of F and HN, and can both reach 10
7TCID
50/ ml or higher titre.In twice experiment, have only the rPIV3-2CTcp45 growth slightly fast, earlier reach its peak titre than PIV2/V94.The growth pattern of this acceleration may be a unidentified results of mutation among this clone, because the sister clone can not show this growth pattern.In the research of the following stated, use rPIV3-2CTcp45 clone 2A1.The temperature sensitivity level of rPIV3-2 embedded virus and cp45 derivative thereof
Detected the temperature sensitivity level that PIV3-PIV2 reorganization embedded virus duplicates, to determine whether rPIV3-2TM and rPIV3-2CT virus show the ts phenotype, and determine that these viruses obtain 12 cp45 sudden changes and whether make the cp45 derivative that carries these 12 PIV3 cp45 sudden changes that the temperature sensitivity feature (people such as Skiadopoulos of certain level is arranged, Journal of Virology 73:1374-81,1999, be incorporated herein by reference).(people such as Skiadopoulos, vaccine 18:503-510 1999, are incorporated herein by reference) measure this viral temperature sensitivity level (table 26) in the LLC-MK2 cell monolayer as previously mentioned.The titre of rPIV3-2TM and rPIV3-2CT is all quite constant under the different restrictive temperatures of permissive temperature (32 ℃) and test, shows that these recons are ts+.On the contrary, its cp45 derivative, rPIV3-2TMcp45 and rPIV3-2CTcp45 are ts, the temperature sensitivity level is similar to rPIV3-1cp45---carry the chimeric PIV3-PIV1 virus of complete PIV1 F and HN glycoprotein and identical one group of 12 cp45 sudden change.Therefore, the body outer property of rPIV3-2TM and rPIV3-2CT virus and cp45 derivative thereof is similar to rPIV3-1 and rPIV3-1cp45 respectively, character also will be similarly in the body of prompting rPIV3-2 and rPIV3-1 virus, but it is shocking that the fact is not like this.Table 26.rPIV3-2TM and rPIV3-2CT duplicating in the LLC-MK2 cell are not temperature sensitive, and the titre when containing the cp45 sudden change and causing 32 ℃ in cp45 temperature sensitive phenotype virus
aCompare during with 32 ℃, the titre under differing temps changes (log
10)
A, b
(log
10TCID
50)
35 ℃ of 36 ℃ of 37 ℃ of 38 ℃ of 39 ℃ of 40 ℃ of rPIV3/JS 7.9 0.3
D0.1 0.1 (0.3)
D(0.4) 0.4PIV3cp45
e7.8 0.5 0.3 1.3 3.4
d6.8 6.9PIV1/Wash64
e8.5 1.5 1.1 1.4 0.6 0.5 0.9rPIV3-1,8.0 0.8 0.5 0.6 0.9 1.1 2.6rPIV3-1cp45 8.0 0.5 0.4 3.4
d4.8 6.6 7.5PIV2/V9412
e7.8 0.3 (0.1) 0.0 (0.4) (0.4) 0.0rPIV3-2CT, 6.9 0.3 0.3 0.6 (0.1) 0.6 0.4rPIV3-2TM 8.3 0.3 (0.1) 0.3 0.6 1.0 2.1
dRPIV3-2CTcp45 8.0 0.8 (0.4) 2.0
d4.3 7.5 〉=7.6rPIV3-2TMcp45 8.0 0.3 0.6 2.4
d5.4 7.5 〉=7.6
aThe data of listing are the mean value of twice experiment.
bThe numeral titre in bracket does not reduce; Numeral titre in the bracket increases.
c35 ℃ data are only from once experiment.
dThe value representation virus titer that underscore marks reduces by 100 times minimum temperature than the titre under permissive temperature (32 ℃).This restrictive temperature is called as by temperature.
eBiology deutero-virus.RPIV3-2TM and rPIV3-2CT are attenuation, immunogenic in hamster and the height protectiveness, and the introducing of cp45 sudden change produces highly attenuated and virus low protectiveness
Every group of 6 hamster intranasal vaccinations 10
5.3TCID
50RPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45, rPIV3-2CTcp45 or contrast virus.Find that in the past rPIV3-1 virus is duplicated (people such as Skiadopoulos, vaccine 18:503-510,1999 as PIV3 and PIV1 parent in the hamster upper respiratory tract and lower respiratory tract; People such as Tao, Journal of Virology 72:2955-2961,1998, be incorporated herein by reference).PIV2 virus is efficiently duplicated in hamster, but rPIV3-2TM and rPIV3-2CT virus all copy as in the upper respiratory tract than PIV2 and the low 50-100 of PIV3 parent titre doubly, low 320-2000 times (table 27) in lower respiratory tract.This shows that chimeric PIV3-PIV2 F and HN glycoprotein cause a kind of unexpected attenuation phenotype in hamster.The derivative rPIV3-2TMcp45 and the rPIV3-2CTcp45 that contain cp45 sudden change have 50-100 attenuation doubly than its rPIV3-2 parent separately, only in concha, have just and can detectedly duplicate, and in lung detection less than.These rPIV3-2cp45 viruses are than the obvious attenuation more of rPIV3-1cp45, and displaying duplication reduces by 50 times in concha.Therefore, the attenuation that causes of the chimeric and cp45 of F and HN glycoprotein sudden change add and.Table 27.rPIV3-2TM and rPIV3-2CT virus are different from rPIV3-1, attenuation in the hamster respiratory tract, and the introducing of cp45 sudden change causes further attenuation.Group
eVirus titer (log in specified tissue
10TCID
50/ g ± S.E.)
b[Duncan] virus
aNT Log
10Drip lung Log
10Drip
Degree reduction degree reduces rPIV3/JS 5.9 ± 0.1[AB] 0 6.5 ± 0.1[A] 0,rPI,V3c,p45 4.5 ± 0.2[C] 1.4
c1.8 ± 0.2[E] 4.7
cPIV1/Wash64
d5.7 ± 0.1[B]-5.5 ± 0.1[B]-rPIV3-1 6.4 ± 0.2[A] 0 6.6 ± 0.2[A] 0rPIV3-1cp5 3.1 ± 0.1[D] 3.3
c1.2 ± 0.0[F] 5.4
cPIV2/V94
d6.2 ± 0.2[A] 0 6.4 ± 0.2[A] 0rPIV3-2CT 4.5 ± 0.4[C] 1.7
c3.1 ± 0.1[D] 3.3
cRPIV3-2TM 3.9 ± 0.3[C] 2.3
c3.9 ± 0.4[C] 2.5
cRPIV3-2CTcp45 1.4 ± 0.1[E] 4.8
c1.5 ± 0.2[E] 4.9
cRPIV3-2TMcp45 1.6 ± 0.2[E] 4.6
c1.4 ± 0.1[E] 5.0
c aEvery group of 6 hamsters were in intranasal vaccination 10 in the 0th day
5.3TCID
50Appointment virus.
bKilled hamster and taked its tissue sample at the 4th day.Measure the virus titer in the hamster tissue, the result is expressed as log
10TCID
50/ g ± standard error (SE).The NT=concha.
cBy relatively rPIV3cp45 and rPIV3/JS, rPIV3-1cp45 and rPIV3-1 and each PIV3-PIV2 mosaic and PIV2/V94 draw log
10Titre reduction value.
dBiology deutero-virus.
eThe classification of being analyzed by the Duncan multiple range test.
For the immunogenicity of measuring the PIV3-PIV2 embedded virus and protection efficient, at the 0th day with 10
5.3TCID
50RPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45, rPIV3-2CTcp45 or every group of 12 hamsters of contrast virus immunity.Every group of 6 hamster at the 29th day with 10
6TCID
50PIV1 attacks, all the other half attacked with PIV2 at the 32nd day.After attacking 4 days, kill hamster, take out lung and concha.Gather serum sample at the-3 days and the 28th day, and measure the HAI antibody titer of anti-PIV1, PIV2 and PIV3.Shown in table 28, although the growth in hamster weakens, all can produce the serum HAI antibody of anti-PIV2 with rPIV3-2TM or rPIV3-2CT immunity, it is on close level and infects institute's inductive in wild-type PIV2/V94.The restriction fully that causes the PIV2 challenge virus to duplicate with rPIV3-2TM and rPIV3-2CT immunity hamster.RPIV3-2TMcp45 and rPIV3-2CTcp45 can not cause detectable serum antibody response, only cause PIV2 challenge virus duplicating in lower respiratory tract to reduce 10-100 doubly (table 28) with any immune hamsters of this two kinds of viruses.Attack has the height protectiveness to wild-type PIV2 in hamster for table 28.rPIV3-2CT and rPIV3-2TM virus, but attack does not then have to PIV1
aEvery group of 12 hamster in the 0th day with 10
5.3TCID
50Appointment virus nose in immunity.
bA few days ago gather serum sample after 28 days in immunity with immunity.Detect the HAI antibody titer of anti-three kinds of PIV, antibody titer is expressed as log
2± standard error (SE).
cEvery group of 6 hamsters are with 10
6TCID
50PIV1 (the 29th day) or PIV2 (the 32nd day) nose in attack.Gather the hamster tissue after attacking 4 days, the virus titer in the specified tissue is expressed as log
10TCID
50/ g ± SE.RPIV3-2TM and rPIV3-2CT are attenuation, immunogenic in AGM and the height protectiveness, and the introducing of cp45 sudden change produces the highly attenuated and insufficient virus of protectiveness
| Immunity virus a | Anti-HAI antibody titer of specifying virus b(average log reciprocal 2±SE) | Challenge virus titre in the specified tissue c(log 10?TCID 50/g±SE) | |||||
| PIV1 | ?PIV2 | ||||||
| ????PIV1 | ????PIV2 | ????PIV3 | ????NT | Lung | ????NT | Lung | |
| ??rPIV3/JS???????????????≤1???????????????????≤1?????????????????10.2±0.1????????????6.2±0.2??????????????5.8±0.1?????????????5.9±0.2?????????????5.7±0.2 ??rPIV3cp45??????????????≤1???????????????????≤1?????????????????8.6±0.2?????????????5.9±0.3??????????????5.1±0.3?????????????5.6±0.2?????????????4.5±0.7 ??PIV1?????????????????6.7±0.2????????????????≤1???????????????????≤1????????????????1.3±0.1??????????????≤1.2±0.0???????????6.1±0.2?????????????6.2±0.3 ??rPIV3-1??????????????6.4±0.2????????????????≤1???????????????????≤1????????????????≤1.2±0.?????????????≤1.2±0.0???????????6.5±0.2?????????????5.0±0.6 ??rPIV3-1cp45??????????1.8±0.6????????????????≤1???????????????????≤1????????????????3.9±0.4??????????????1.6±0.3?????????????6.2±0.2?????????????4.5±0.6 ??PIV2???????????????????≤1????????????????4.0±0.0?????????????????≤1????????????????5.9±0.2??????????????5.5±0.1?????????????≤1.2±0.0???????????≤1.2±0.0 ??rPIV3-2CT??????????????≤1????????????????3.6±0.8?????????????????≤1????????????????5.3±0.1??????????????5.2±0.3?????????????≤1.2±0.0???????????≤1.2±0.0 ??rPIV3-2TM??????????????≤1????????????????4.5±0.2?????????????????≤1????????????????5.9±0.2??????????????4.4±0.3?????????????≤1.2±0.0???????????≤1.2±0.0 ??rPIV3-2CT.cp45?????????≤1???????????????????≤1???????????????????≤1????????????????6.2±0.2??????????????5.7±0.1?????????????5.3±0.2?????????????3.3±0.8 ??rPIV3-2TM.cp45?????????≤1???????????????????≤1???????????????????≤1????????????????5.8±0.3??????????????4.4±0.3?????????????5.5±0.2?????????????3.7±0.7 | |||||||
Some reorganization PIV3 and RSV virus can show attenuation (people such as Skiadopoulos, vaccine 18:503-510,1999 of different levels in rodent and primate; People such as Skiadopoulos, Journal of Virology 73:1374-81,1999; People such as Skiadopoulos, virusology 272:225-34,2000; People such as Whitehead, Journal of Virology 73:9773-9780,1999, all be incorporated herein by reference), show that attenuation has some species specificities.Therefore, estimated rPIV3-2 virus duplicating and the immunogenicity level in AGM.Every group of 4 AGM are in the 0th day nose and use each position 10 in the tracheae
5TCID
50RPIV3-2TM, rPIV3-2CT, rPIV3-2TMcp45, rPIV3-2CTcp45, PIV2/V94 or rPIV3-1.Virus titration as previously mentioned in NT swab specimen (gathering at 1-12 days) and the lavage of trachea thing (collection in the 2nd, 4,5,8 and 10 day) (people such as van Wyke Coelingh, virusology 143:569-582,1985, be incorporated herein by reference).Shown in table 29, rPIV3-2TM and rPIV3-2CT be attenuation in the AGM respiratory tract obviously, and the peak titre of the virus that discharges in the upper respiratory tract and lower respiratory tract is lower than its PIV2/V94 parental virus and shown this point.
The derivative rPIV3-2TMcp45 and the rPIV3-2CTcp45 that contain cp45 sudden change detect extremely low-level in NT swab and lavage of trachea thing---if any, show that F and HN glycoprotein attenuation chimeric and that the cp45 sudden change causes adds for AGM and hamster and.
In order to determine whether attack has protectiveness to PIV2 with PIV23-PIV2 embedded virus immunity AGM, at the 28th day with 10
5TCID
50PIV2 attacks and has infected the AGM (table 29) of rPIV3-2 virus.The virus titration as previously mentioned of existence in NT swab specimen (gathering at 29-38 days) and the lavage of trachea thing (gathering at the 30th, 32,34,36 and 38 day) (people such as Durbin, virusology 261:319-30,1999, be incorporated herein by reference).Shown in table 29, the high level that can induce the PIV2/V94 challenge virus to duplicate with rPIV3-2TM and rPIV3-2CT immunity limits.On the contrary, can not limit duplicating of PIV2/V94 challenge virus with rPIV3-2TMcp45 and rPIV3-2CTcp45 immunity AGM, and these animals produce the preceding anti-PIV2 serum neutralizing antibody of extremely low-level attack.The PIV2/V94 challenge virus duplicates in the AGM of rPIV3-2CT immunity limit fully with attack before anti-PIV2 serum antibody level than the AGM of rPIV3-2TM immunity high 2.5 times relevant, this will cause incomplete protection.Table 29.rPIV3-2CT or rPIV3-2TM duplicating in the cercopithecus aethiops respiratory tract are weakened, but still can induce the resistance that wild-type PIV2 is attacked
aEvery group of 4 cercopithecus aethiops are in the 0th day nose and inoculate 10 in the tracheae
5TCID
50Appointment virus.
bGathering nose at 1-12 days washes and throat swab (NT) combined sample.Gathered lavage of trachea (TL) sample at the 2nd, 4,6,8,10 day.On the LLC-MK2 individual layer, measure virus titer, be expressed as log
10TCID
50/ ml ± standard error (SE).
cBe determined at anti-PIV1 and PIV2 NAT in the serum sample of gathering in the 28th day.Tire and be expressed as average log reciprocal
2± SE.
dGathered the NT sample at 29-38 days.Gathered the TL sample at the 30th, 32,24,36,38 day.RPIV3-2TM duplicating in the chimpanzee respiratory tract weakened
| Immunity virus a | The average peak titre of the immunity virus of appointed part b????(log 10?TCID 50/g±SE) | Anti-serum NAT of specifying virus c(average log reciprocal 2±SE) | The average peak titre of PIV2/V94 challenge virus in the appointed part d(log 10?TCID 50/g±SE) | |||
| ????NT | ????TL | ????PIV1 | ????PIV2 | ????NT | ????TL | |
| ??rPIV3-1 ??PIV2/V94 ??rPIV3-2CT ??rPIV3-2TM ??rPIV3-2CTcp45 ??rPIV3-2TMcp45 | ????2.6±0.5 ????2.8±0.7 ????1.5±0.4 ????1.4±0.1 ????1.0±0.2 ????0.6±0.3 | ????3.2±0.1 ????5.0±0.3 ????0.5±0.2 ????1.6±0.7 ????≤0.2 ????≤0.2 | ????6.3±0.4 ????3.8±0.0 ????2.9±0.1 ????4.1±0.1 ????4.1±0.1 ????3.4±0.2 | ????3.1±0.3 ????7.1±0.7 ????7.2±0.1 ????5.9±0.2 ????5.3±0.0 ????4.6±0.6 | ????3.6±0.2 ????≤0.2 ????≤0.2 ????1.6±0.6 ????3.3±0.4 ????3.0±0.5 | ????3.3±0.7 ????≤0.2 ????≤0.2 ????1.3±0.9 ????3.5±0.3 ????4.1±0.2 |
Every group of 4 chimpanzee are in the 0th day nose and inoculate 10 in the tracheae
5TCID
50RPIV3-2TM or PIV2/V94.Gather NT swab sample (1-12 days) and lavage of trachea (the 2nd, 4,6,8,10 day) sample.Virus titer is measured (people such as Durbin, virusology 261:319-30 1999, are incorporated herein by reference) as previously mentioned, and the result is expressed as log
10TCID
50/ ml.Shown in table 30, rPIV3-2TM has the peak titre lower than wild-type parent PIV2/V94, and to be significantly shorter than the time length release of PIV2/V94, shows rPIV3-2TM attenuation in chimpanzee.With in hamster and AGM, compare PIV2/V94 wt virus low-level duplicating in chimpanzee, and rPIV3-2TM virus attenuation all in all these model hosts.Table 30.rPIV3-2TM attenuation in the chimpanzee respiratory tract, but still can cause the strong seroimmunity of PIV2 is replied
aInoculate 10 in every group of 4 chimpanzee noses and in the tracheae
5TCID
50Appointment virus.
bGather nose/larynx (NT) swab specimen and lavage of trachea thing (TL) respectively 12 days and 10 days, and measured virus titer.The peak titre is expressed as log
10TCID
50/ ml ± standard error (SE).
cBe determined at anti-NAT of specifying virus in the serum sample that virus inoculation gathers after preceding 3 days and 28 days.Tire and be expressed as average log reciprocal
2± SE.
dRelease duration has significant difference, p≤0.005, Student T check.
| The virus of inoculation a | The average peak titre of the virus that discharges in appointed part bVirus is released (log in the upper respiratory tract 10?TCID 50The average fate of/ml ± SE) put (my god | Anti-serum NAT of specifying virus c(average log reciprocal 2±SE) |
| ????NT??????????????????????TL????????????????????±SE) | Front and back | |
| ??PIV2/V94 ??rPIV3-2TM | ??2.9±0.6???????????????1.2±0.5???????????????8.8±1.1 d??2.0±0.3???????????????≤0.5±0.0?????????????2.5±0.7 d | ??≤2.8±0.0?????????????6.2±0.5 ??3.3±0.2???????????????4.3±0.4 |
As mentioned above, the main protection antigen of PIV is their HN and F glycoprotein.Therefore, in typical embodiments of the present invention, the attenuated live PIV candidate vaccine that is used for baby and children is included in chimeric HPIV3-1 and the HPIV3-2 virus that PIV3 background genome and anti-genome carry total length PIV1 and part PIV2 glycoprotein respectively.In the later case, with HN and F ORF rather than total length PIV2 ORF structure full-length cDNA.The virus that obtains is named as rPIV3-2CT, has phenotype in similarly external and the body, and wherein PIV2 extracellular domain and membrane-spanning domain and PIV3 stride film and cytoplasmic tail fusion.Especially, rPIV3-2 reorganization embedded virus shows the host range phenotype, that is, they efficiently duplicate external, and duplicate limited in the body.Attenuation takes place when the sudden change that does not contain from any interpolation of cp45 in this body.This is a kind of unexpected host range effect, is very to wish for vaccine use, in part because phenotype is not to be determined by the point mutation that is called as wt.Simultaneously, very favourable in external unrestricted growth to effective production of vaccine.
Though rPIV3-2CT and rPIV3-2TM efficiently duplicate external, they are at the upper respiratory tract and the lower respiratory tract camber attenuation of hamster and cercopithecus aethiops (AGM), illustrate that the HN of PIV2 and PIV3 itself and F are proteic to be entrenched in external a kind of attenuation phenotype that causes.Although attenuation, they still have the immunogenicity of height and the protectiveness of the wild virus attack of antagonism PIV2 in two species.Further modify rPIV3-2CT and rPIV3-2TM, method is to introduce 12 PIV3 cp45 sudden changes that are positioned at outside HN and the F encoding sequence, generation can external efficiently duplicate but in hamster and AGM the further rPIV3-2CTcp45 and the rPIV3-2TMcp45 of attenuation, show that glycoprotein attenuation chimeric and that the cp45 sudden change causes adds and.
Other people had once reported and had been used for influenza virus (people such as Belshe, New England Journal of Medicine 338:1405-1,1998; People such as Murphy; transmissible disease clinical practice (Infectious Diseases inClinical Practice) 2:174-181; 1993) and be used for rotavirus (people such as Perez-Schael; the production of the antigen embedded virus of the attenuation sudden change of protective antigen that contains a kind of virus New England Journal of Medicine 337:1181-7,1997) and another kind of virus.Contain the genomic virus of sections for these, more be easy to generate attenuation antigen chimeric, because high frequency ground producer group section is classified during coinfection again.The gene that attenuated live influenza virus vaccine candidate strain every year replaces with new popularity or infective virus by HA and NA gene with the attenuation donor virus and antigen upgrades.Also use recombinant DNA technology widely and make up the attenuated live antigen embedded virus vaccine that is used for flavivirus and paramyxovirus.For flavivirus, prepared the attenuated live vaccine candidate strain that is used for JEV (people such as Guirakhoo by the homologue that the cephacoria (prM) of attenuation yellow fever virus (YFV) and coating (E) district is replaced with japanese encephalitis virus (JEV) attenuated strain, virusology 257:363-72,1999).JEV-YFV antigen chimeric recombinant vaccine candidate strain is attenuation and immunogenicity (people such as Guirakhoo, virusology 257:363-72,1999) is arranged in vivo.The structure of this embedded virus and Nonstructural Protein all derive from attenuated live vaccine virus.Also passing flavivirus (Langat virus) and wild-type mosquito the lice of natural attenuation passes to step between leather 4 viruses and has prepared the antigen chimeric, the recon that find to produce passes the remarkable attenuation of parental virus (people such as Pletnev for mouse than lice, institute of NAS reports 95:1746-51,1998), but duplicate height-limited in the body of this embedded virus in the Vero cell.This is an example of the attenuation that the part uncompatibility causes between the Nonstructural Protein of being determined by Langat virus that carries out difference structure albumen and dengue virus.The third strategy is the production of exploring the tetravalence dengue virus vaccine, wherein be used in leather 4 main chains of stepping on that contain the attenuation deletion mutantion in 3 ' the non-structural area and make up the chimeric virus that contains the protective antigen of stepping on leather 1,2 or 3 C-type virus Cs of antigen (people such as Bray, institute of NAS reports 88:10342-6, and 1991; Journal of Virology 70:3930-7,1996).
Also produced the antigen embedded virus that is used for the mononegavirale RNA viruses.For example; can be according to method disclosed herein; by replace the corresponding protein of PIV3 in the strain of attenuation PIV3 vaccine candidate with the total length HN of 1 type parainfluenza virus and F albumen, make up the strain of the chimeric PIV1 vaccine candidate of antigen, this recon attenuation and PIV1 attacked protectiveness is arranged in laboratory animal.Similarly, can prepare the strain of typical antigen chimeric respiratory syncytial precursor virus (RSV) vaccine candidate, wherein use one or more RSV F and the G protective antigen of subgroup B virus, or its antigenic determinant is replaced the antigen of attenuation RSV subgroup A virus, the strain of generation attenuation RSV subgroup B vaccine candidate (referring to, international application no WO 97/06270; People such as Collins, institute of NAS report 92:11563-11567 (1995); The U. S. application of on July 5th, 1997 application number 08/892,403 (the international application no WO 98/02530 that are equivalent to announce, with the preferential U.S. Provisional Application of on May 23rd, 1997 application number 60/047,634,60/046 of application on May 9th, 1997,141,60/021 of application on July 15th, 1996,773), people such as Collins are in the United States Patent (USP) series number 09/291,894 of application on April 13rd, 1999; The U.S. Provisional Patent Application series number 60/129,006 of application on April 13rd, 1999; People such as Bucholz are in the U.S. Provisional Patent Application series number 60/143,132 of application on July 9th, 1999; With people such as Whitehead, Journal of Virology 73:9773-9780,1999, all be incorporated herein by reference).When in the wild-type virus background, carry out between PIV1 and the PIV3 virus and RSV subgroup A and RSV subgroup B virus between glycoprotein when exchange, the antigen embedded virus copies to the wild-type virus level in vitro and in vivo.These discoveries show, have high-caliber consistency between acceptor and donor virus, because chimeric processing has only seldom---attenuation if any---.These of PIV1 and PIV3 and RSV A and B glycoprotein exchange are found aspect several and the exchange between disclosed herein PIV2 and the PIV3 forms distinct contrast.
In this disclosure, total length PIV 2 HN or F albumen are replaced the proteic live-weight papova of PIV3 and can not be replied in this case, and the accidental sudden change that this obviously is attributable to introducing in cDNA makes up can successfully realize and exchange this for PIV1-PIV3 glycoprotein.This shows that one or more PIV3 albumen consistencies of encoding among PIV2 HN or F glycoprotein and the cDNA are relatively poor.When making up full-length cDNA, obtain two kinds of PIV2-PIV3 embedded viruses alive with chimeric HN and F ORF rather than with total length PIV2 ORF.Wherein a kind of embedded virus contains chimeric HN and F glycoprotein, and wherein PIV2 extracellular domain and PIV3 stride film district and cytoplasmic tail and merge, and another kind contains chimeric HN and F glycoprotein, wherein PIV2 extracellular domain and stride film district and the fusion of PIV3 cytoplasmic tail.External and the interior phenotype of body that two kinds of rPIV3-2 recons all have similar---although being different---.Therefore, as if the acquisition of PIV2-PIV3 embedded virus only needs the HN of PIV3 or the cytoplasmic tail of F glycoprotein.
In the former research to protein structure-functional analysis, made up and at vivoexpression chimeric HN or F albumen, and be used for to proteinic difference in functionality territory mapping (people such as Bousse, virusology 204:506-14,1994; People such as Deng, virusology journal supplementary issue (Arch.Virol.Suppl.) 13:115-30,1997; People such as Deng, virusology 253; 43-54,1999; People such as Deng, virusology 209; 457-69,1995; People such as Mebatsion, Journal of Virology 69:1444-1451,1995; People such as Takimoto, Journal of Virology 72:9747-54,1998; People such as Tanabayashi, Journal of Virology 70:6112-6118,1996; People such as Tsurudome, general virology magazine 79:279-89,1998; People such as Tsurudome, virusology 213:190-203,1995; People such as Yao, Journal of Virology 69:7045-53,1995).In reporting first, with a kind of by striding in the chimeric glycoprotein albumen insertion Measles virus infectious clone that Measles virus F cytoplasmic tail that film and extracellular domain merge forms with vesicular stomatitis virus G is proteic, replace Measles virus F and HN viral glycoprotein (people such as Spielhofer, Journal of Virology 72:2150-9,1998).Obtain reproducible but the very limited embedded virus of replication in vitro, as shown in delayed growth and low viral yield, show the external attenuation of height.This discovery obviously is different from the phenotype of expressing proteic reorganization PIV of the present invention of chimeric glycoprotein such as the demonstration of PIV2-PIV3 mosaic, and they efficiently duplicate external.
The external attenuated live vaccine candidate strain of efficiently duplicating for the extensive production of vaccine of needs of RPIV3-2 and other chimeric PIV viruses of the present invention is a kind of critical natures.Different with rPIV3-2CT and rPIV3-2TM, rPIV3-1 is attenuation not in vivo.Therefore, the HN of PIV2 and PIV3 and F proteic chimeric cause duplicating in the body weaken, this is the new discovery for the mononegavirale RNA viruses.It is machine-processed not clear that the host range that duplicates in this body limits.Importantly, infect the high-level resistance that to induce the PIV2 attack, show that the proteic antigenic structure of chimeric glycoprotein is most of or complete fully with these attenuations rPIV3-2CT and the strain of rPIV3-2TM vaccine candidate.Therefore rPIV3-2CT and rPIV3-2TM work as attenuated live PIV2 candidate vaccine virus, show the balance of wishing between attenuation in AGM and hamster and the immunogenicity.
The attenuation that the PIV3-PIV2 of F and HN glycoprotein is chimeric and cp45 sudden change is determined add and.The rPIV3-2 recon that contains the cp45 sudden change is highly attenuated in vivo, and the incomplete protection that PIV2 is attacked is provided in hamster, and few protection is provided in AGM.This is different from the discovery for rPIV3-1cp45, and the latter is attenuation satisfactorily in vivo, and the attack of the antagonism PIV1 that watches for animals.Chimeric and 12 cp45 sudden change of PIV3-PIV2 glycoprotein independently, add and the as if undue in vivo attenuation of attenuation combination.Obviously, if find rPIV3-2CT and rPIV3-2TM vaccine candidate strain insufficient attenuation in human body, cp45 attenuation sudden change should be one by one rather than as one group of 12 interpolation, to reach the balance of the required hope of the attenuated live PIV2 vaccine that is used for human body between attenuation and immunogenicity.Therefore, a kind of novel method that makes the paramyxovirus attenuation has been determined in these discoveries of herein listing, and the basis of estimating the strain of the chimeric attenuated live PIV2 vaccine candidate of these PIV3-PIV2 in human body is provided.Importantly, rPIV3-2CT or rPIV3-2TM virus also can be as the carriers of other PIV antigens or other viral protective antigens such as Measles virus HA albumen or its immunogenicity part.
Short summary above explanation and embodiment, used based on the viral answering system preparation of cDNA and characterized the chimeric PIV of reorganization that is configured to the carrier that carries allos virogene or genome section.Can be independently duplicated by the recombinant virus that cDNA obtains, and can breed in the mode identical with biology deutero-virus.In preferred embodiments, recombinate the strain of chimeric people PIV (HPIV) vaccine candidate preferably at one or more major antigen determinants that carry HPIV owing to one or more nucleotide modifications in the background of attenuation.Preferably, chimeric PIV of the present invention also expresses one or more protective antigens of another kind of pathogenic agent such as microbial pathogen.In these situations, HPIV is as the attenuated virus carrier, and is used for dual purpose: induce to one or more HPIV and to the protective immune response of the pathogenic agent of the external source protective antigen of deriving.As mentioned above, the main protection antigen of PIV is its HN and F glycoprotein.Other enveloped viruses; but the main protection antigen of the virus of infected person respiratory tract for example; it is their attachment protein; they can be expressed by one or more outer transcriptional units by the HPIV carrier; be also referred to as genetic unit, for example, HN albumen or other fusion roteins of the G albumen of RSV, the HA albumen of Measles virus, mumps virus; for example, the F albumen of RSV, Measles virus or mumps virus.It also is possible expressing the protective antigen of non-enveloped virus such as the L1 albumen of human papillomavirus, and they form virus-like particle (people such as Roden, Journal of Virology 70:5875-83,1996) in infected host.According to described, a large amount of protective antigens and the composition antigenic determinant thereof that derive from different pathogens can be integrated in chimeric PIV of the present invention, cause new efficient immune.
The present invention has overcome existing based on inherent difficulty in the vaccine development method of carrier, and for providing unique opportunity to multiple human pathogen immunity is one-year-old with interior baby.The research of development attenuated live PIV vaccine in the past unexpectedly shows, rPIV and attenuation thereof and chimeric derivant have following character, make their unique being suitable in non-segmented negative-strand RNA viruses, with vaccine as the multiple human pathogen of antagonism as the vector expression exogenous protein.Whether the technician has the feature that very is suitable as carrier with unpredictable people PIV, and these PIV trend towards poorer than model non-segmented negative-strand viral growth, generally can not fully show in molecular studies.To one's surprise equally, the intranasal administration approach of these vaccines has been proved to be to stimulate to the strong part of the heterologous antigen of carrier and expression and the effective way that general immunity is replied.In addition, this approach is for providing other advantages at the allos pathogenic agent immunity of infecting respiratory tract etc.
These character of PIV carrier have more than been described with the embodiment of rPIV3 carrier, it contains in (i) be expressed as isolated genes in wild-type and attenuation background Measles virus main and antigen, or (ii) in HPIV1 main and antigen, replace among the PIV3 and antigen, express in HPIV2 main in addition and antigen.These rPIV carriers wild-type and attenuation background constructing.In addition, description has herein proved the ability that is easy to modify PIV carrier main chain attenuation level.According to one of these methods, change that genome inserts segmental adjustable in length attenuation phenotype among the chimeric PIV of the present invention, extremely long to insert in the segmental wild-type derivative be tangible using for this.
To Measles virus or other microbial pathogenes immunity baby's trial, the present invention has 6 major advantages before comparing.First, to the PIV of the protective antigen that wherein inserts Measles virus or other microbial pathogenes recombinant vectors is a kind of attenuation rPIV that contains one or more attenuation genetic elements, known these elements can make virus for children's baby's respiratory tract attenuation (people such as Karron extremely, paediatrics transmissible disease magazine 15:650-654,1996; People such as Karron, transmissible disease magazine 171:1107-1114,1995a; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).The clinical evaluation in the past and the history of practice have greatly promoted in extremely young baby the evaluation to the derivative of these recons that contain the external source protectiveness.
Second advantage is; the rPIV main chain that contains other protective antigens of measles HA or another kind of human pathogen can be induced the duplicate protection immunne response; at (1) PIV; the necessary independent demand of aforesaid vaccine; (2) allos virus or other microbial pathogenes, its protective antigen is by vector expression.This is different from above-mentioned VSV-Measles virus HA recon, and the latter can be the Measles virus induction of immunity to a kind of human pathogen only, and is uncorrelated at most to the immunne response of carrier itself, or may be unfavorable.After a plurality of round-robin of process duplicate in tissue culture cells, still complete in the genome of encoding sequence at most of recombinant viruses of the foreign gene that in viral Mononegavirales purpose different members, inserts, the member who shows this viroid is as outstanding candidate's strain of carrier (people such as Bukreyev, Journal of Virology 70:6634-41,1996; People such as Schnell, institute of NAS reports 93:11359-65,1996a; People such as Singh, general virology magazine 80:101-6; People such as Yu, gene and cell (Genes Cells) 2:457-66,1997).
Another advantage of the present invention is that the application of the human pathogen main chain that vaccine needs can promote to add this attenuated live virus vector in intensive early stage immunization programs for children schedule.In addition, have a plurality of advantages by respiratory mucosa surface immunity.Attenuated live PIV3 can duplicate in the rhesus monkey respiratory tract, and can induce the protective immune response to itself in the presence of the PIV3 antibody that a large amount of parents obtain.Two kinds of candidate PIV3 vaccines infect in the extremely young baby's upper respiratory tract that contains the antibody that parent obtains and the ability of efficiently duplicating has also obtained proof (people such as Karron, paediatrics transmissible disease magazine 15:650-654,1996; People such as Karron, transmissible disease magazine 171:1107-1114,1995a; People such as Karron, transmissible disease magazine 172:1445-1450,1995b).This is different from the measles virus vaccines of current permission, and latter's infectivity when people's the upper respiratory tract is used is relatively poor, extremely sensitive to neutralizing to children's parenteral administration time the (people such as Black, New England Journal of Medicine 263:165-169,1960; People such as Kok, Trands.R.Soc.Trop.Med.Hyg.77:171-6,1983; People such as Simasathien, vaccine 15:329-34,1997).HPIV carrier duplicating in respiratory tract with the generation of stimulating mucosal IgA with to the general immunity of the exogenous antigen of HPIV carrier and expression.After contacting wild-type virus such as Measles virus naturally subsequently, it is that respiratory tract and whole body duplicate duplicating of position at inlet that the vaccine-induced part and the existence of general immunity should be able to limit it.
Another advantage of the present invention is that the chimeric HPIV that contains heterologous sequence can efficiently duplicate external, shows that the scale operation vaccine is feasible.This is different from duplicating of some mononegavirale RNA viruses, and inserting foreign gene can be in that vitro inhibition be this duplicates people such as (, Journal of Virology 70:6634-41,1996) Bukreyev.Therefore three kinds of antigen serotypes of HPIV---they can cause human important diseases, can be used as carrier and vaccine simultaneously---the different HPIV variant continuous immunity baby's of the existence antigenicity that also provides usefulness to carry identical exogenous protein chance.Like this; continuous immunity will cause the primary immune response to exogenous protein, be strengthened in the different HPIV variant process of can also carry identical or different exogenous protein with the postoperative infection antigenicity of---being the protective antigen of Measles virus or another kind of microbial pathogen---.Using homology HPIV carrier once more and also can strengthen the replying of HPIV and exogenous antigen, is the abnormal of HPIV but distinctive character (people such as Collins, the third edition because cause the multiple ability that infects again in human body, " wild virusology ", B.N.Fields, D.M.Knipe, P.M.Howley, R.M.Chanock, J.L.Melnick, T.P.Monath, B.Roizman and S.E.Straus compile, the first roll, the 1205-1243 page or leaf.Lippincott-Raven?Publishers，Philadelphia，1996)。
Another advantage is to introduce genetic unit and have several effects unexpected, that still very wish for producing attenuated virus in the PIV carrier.At first, insert to express as the genetic unit of Measles virus HA or PIV2HN and can give the PIV carrier host range phenotype that can't discern in the past, that is, the PIV carrier that obtains can efficiently duplicate external, but duplicates limited in the body in the upper respiratory tract and lower respiratory tract.These find to determine that the insertion of expressing the genetic unit of viral protective antigen is an attenuation factor for the PIV carrier, is the character of the hope of attenuated live virus vaccines of the present invention.
Compare with other all members of the mononegavirale virus of mononegavirale virales, the PIV carrier system has distinctive advantage.At first, once not to derive from human pathogen (for example, mouse HPIV1 (Sendai virus) (people such as Sakai, FEBSLett.456L221-6 as other mononegavirale viruses of great majority of carrier, 1999), vesicular stomatitis virus (RSV), it is a kind of cattle disease substance (people such as Roberts, Journal of Virology 72:4704-11,1998), with dog PIV2 (SV5) (people such as He, virusology 237:249-60,1997).For these non-human virus, the antigen that exists in the carrier main chain does not cause or only causes the extremely weak immunity to anyone viroid.Therefore, express for the human pathogen non-human virus vector of extra gene and only will induce resistance at a kind of human pathogen.In addition, virus can make the vaccinate contact the virus that it(?) may not can in the life runs into as VSV, SV5, rabies or Sendai virus as the application of carrier.It is useful how many infection of these non-human viruses and immunne responses that causes will be, and will cause the worry to safety, because there are not these viral experiences of human infection.
An important and special advantage of PIV carrier system is that the preferred intranasal administration approach that simulating nature infects can be induced mucous membrane and general immunity, and reduces the neutralization and the immunosuppressive action of the serum IgG of the maternal source that exists among the baby.When using RSV as carrier, these same advantages are possible in theory, and for example, we find that the insertion fragment strongly inhibited RSV greater than about 500 bp duplicates (people such as Bukreyev, institute of NAS reports 96:2367-72,1999).On the contrary, as described here, HPIV3 can hold several big genes easily and insert fragment.Recombinant RSV incorporate is unsuitable for carrying big insertion fragment and the PIV that recombinates is this very suitable discovery has obtained unexpected result.
Can propose, some other virus vector energy intranasal administrations, with the similar benefit of acquisition shown in the PIV carrier, but success as yet so far.For example, the vaccinia virus MVA strain of expression HPIV3 protective antigen is be evaluated as the attenuated live nose intradermal vaccine of antagonism HPIV3.Although this carrier is seemingly a kind of utmost point effectively expressing system in cell culture; but its (people such as Durbin inexplainably invalid aspect the induction of resistance in the primate upper respiratory tract; vaccine 16:1324-30; 1998); and in the presence of passive serum antibody, induce inexplainably invalid (people such as Durbin aspect the protective response; transmissible disease magazine 179:1345-51,1999).On the contrary, have been found that PIV3 and the strain of RSV vaccine candidate have protectiveness in the long class animal upper respiratory tract of non-human record and lower respiratory tract, even (people such as Crowe, 13:847-855,1995 also like this in the presence of passive serum antibody; People such as Durbin, transmissible disease magazine 179:1345-51,1999).
Particularly PIV3 has other advantages as the application of carrier.For example, set up condition, with high 10-1000 times the high titre PIV3 that in microcarrier is cultivated, obtains to reach than virus as RSV and Measles virus.The RSV infectivity also is unsettled, and this makes breeding, transportation, storage and processing become complicated.The RSV vaccine of development PIV load will be eliminated these problems.
Importantly, the PIV of two kinds of forms has carried out clinical evaluation widely as the candidate vaccine of intranasal administration, i.e. BPIV3 and attenuation HPIV3 cp45 strain.Find that each all is safe, immunogenic and phenotype is stable in children and baby.Also in children and baby, do not estimate other candidate's engineered vectors, particularly in this age group, intranasal administration is not estimated other available carriers.
Another advantage of PIV carrier system is that HPIV3 is as model in use, has identified the attenuations sudden change of a large amount of attenuation degree that can be introduced separately in the carrier main chain and obtain simultaneously to wish.For example, directly identified the special sudden change that causes HPIV3 cp45 attenuation mutant phenotype, and introduced in the wild-type recombinant virus by sequential analysis.Other attenuation sudden changes develop by " introducing " attenuation point mutation from Sendai virus and RSV.In some cases, can change with two kinds rather than a kind of Nucleotide and introduce some point mutation in recombinant virus, this can stablize sudden change and avoid replying and be wild-type.Lack C, D and C ORF expression and can make viral attenuation.In addition, also developed the embedded virus of HPIV3 and ox (B) PIV3, limited as the attenuation instrument with natural reservoir (of bird flu viruses) scope in primate with BPIV3.But also find some combined sequence attenuation, be replaced into the counterpart of HPIV2 as HPIV3 HN and F extracellular domain.Therefore, have a large amount of PIV attenuation sudden changes, they can be used for optionally making carrier main chain attenuation.
Therefore, one aspect of the present invention disclosed herein relates to a kind of with the method for the reorganization PIV that selects as one or more protective antigens of the allos pathogenic agent of the extra gene of vector expression conduct.Allos pathogenic agent described herein comprises allos PIV, Measles virus and RSV.In the above-described embodiments, make up rHPIV3 and can reach 3 kinds of isolating extra genes insertion fragments as vector expression, wherein each all expresses different viral protective antigens.In addition, rHPIV3 can hold the total collection insertion length that is at least wild type gene group 50% easily.Prepare construct with following several different PIV carrier main chains, that is: wild-type HPIV3; The HPIV3 of attenuation form, wherein N ORF is replaced by the N ORF of BPIV3; The HPIV3-1 embedded virus, wherein the HN of HPIV3 and F ORF are replaced by the counterpart of HPIV1; A kind of HPIV3-1 of form, it is owing to exist three kinds of independently attenuation cp45 point mutation and attenuations in the L gene; A kind of BPIV3 of form, wherein HN and F gene are replaced by the counterpart of HPIV3.These carriers that contain one or more extra genes can efficiently duplicate external, prove that its business development is feasible, and they also can duplicate and induce the strong immune response to carrier and insertion fragment in vivo.Like this, can make up a kind of virus based on reorganization PIV, it can be induced at least 4 kinds of human pathogens is the immunne response of the pathogenic agent of PIV carrier itself and extra gene representative.
Second aspect of the present invention be, utilizes PIV as the excellent characteristic preparation of vaccine and the carrier vaccine at RSV.RSV is a kind of growth not as the pathogenic agent of PIV, and instability is because do not understand fully as yet former thereby induce the relatively poor immunne response of protectiveness.The development of attenuated live RSV vaccine has been carried out showing for this human pathogen 35 years more, has been reached suitable balance and have difficulty between immunogenicity and attenuation.Therefore, the attenuated live RSV vaccine that has a large amount of reasons to develop not to be based on infectivity RSV.Main protectiveness F of RSV and G antigen are the extra genetic expression of BPIV3 by the PIV carrier in this case, and do not need to produce the attenuated live vaccine based on infectivity RSV.
The 3rd aspect of the present invention described herein is the carrier based on PIV that the antigenic determinant of different PIV serotypes is carried in development.Owing between serotype, there is not cross protection substantially, therefore can develop a kind of method of the common PIV carrier continuous immunity that has changed with the protective antigen determinant.Therefore, as derive from rHPIV3, carry a kind of attenuation PIV carrier main chain of extra gene as desired, can be used for initial immunity.Immunity subsequently preferably after the first time 4-6 week or longer time, can be carried out with a kind of identical PIV carrier of form, and wherein the carrier glycoprotein gene has been replaced by the gene of allos PIV serotype such as rHPIV3-1.This carrier can contain identical extra gene, and they can provide additionally antigenic " reinforcement ", perhaps can contain different one group.Because immunity for the second time is to use the carrier format of the glycoprotein that contains allos PIV serotype to carry out, the special immunity of initial immunity inductive carrier has certain interference.In addition, can all be that the PIV carrier of different serotypes such as HPIV1 or HPIV2 carries out the immunity second time with all vector genes.Yet, use common one group of internal gene as being based on the rPIV3 of HPIV3 and the advantage of rPIV3-1 carrier, in every kind of construct, can use one group of attenuation sudden change, do not need to develop respectively attenuated strain for every kind of PIV serotype.Importantly, continuous immunity is followed the multivalence strategy: in each immunity, carrier itself induces counterweight to want the immunity of human pathogen, and every kind of extra insertion fragment is induced the immunity to another kind of pathogenic agent.
Although in order to be expressly understood, by embodiment in detail the present invention has been described in detail, the technician should be understood that in the scope of accessory claim book can implement some change and modification, and this shows by unconfined explanation.In this manual, for convenience of description, open text and other reference in aforementioned content, have been quoted.These reference all are incorporated herein by reference.
The preservation of biomaterial
Following material is preserved in US mode culture collection center according to the clause of budapest treaty, 10801 University Boulevard, Manassas, VA 20110-2209.Virus preserving number preservation date p3/7 (131) 2G (ATCC 97989) p3/7 on April 18th, 1997 (131) (ATCC 97990) P218 on April 18th, 1997 (131) (ATCC 97991) HPIV3 JS on the 18th cp45 April in 1997 (ATCC PTA-2419) on August 24th, 2000
Claims (179)
1. an isolating infectivity chimeric parainfluenza virus (PIV), it contains a kind of main nucleocapsid (N) albumen, a kind of nucleocapsid phosphorprotein (P), a kind of big polymerase protein (L) and partial or complete PIV vector gene group or anti-genome, described genome combines with one or more heterologous genes or the genome section of one or more antigenic determinants of one or more allos pathogenic agent of coding, forms a kind of chimeric PIV genome or anti-genome.
2. the chimeric PIV of claim 1, wherein among partial or complete PIV vector gene group or the anti-genome non-coding region or near one or more heterologous genes or the genome section of adding coding for antigens determinant, as extra gene or genome section.
3. the chimeric PIV of claim 1 wherein uses one or more heterologous genes of coding for antigens determinant or one or more corresponding gene or the genome section in genome section replacing section PIV vector gene group or the anti-genome.
4. the chimeric PIV of claim 1, wherein said one or more allos pathogenic agent are a kind of allos PIV, one or more PIV N, P, C, D, V, M, F, HN and/or L albumen or its fragment and described heterologous gene or genome section are encoded.
5. the chimeric PIV of claim 1, wherein vector gene group or anti-genome are partial or complete people PIV (HPIV) genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are from one or more allos PIV.
6. the chimeric PIV of claim 5, wherein said one or more allos PIV is selected from HPIV1, HPIV2 or HPIV3.
7. the chimeric PIV of claim 5, wherein vector gene group or anti-genome are partial or complete HPIV genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are from one or more allos HPIV.
8. the chimeric PIV of claim 7, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are from one or more allos HPIV.
9. the chimeric PIV of claim 8 wherein adds in partial or complete HPIV3 genome or anti-genome or permutation encoding is selected from one or more genes or the genome section of one or more HPIV1 antigenic determinants of HPIV1 HN and F glycoprotein and antigenic domain, fragment and epi-position.
10. the chimeric PIV of claim 8, wherein vector gene group or anti-genome are partial or complete HPIV3 JS genome or anti-genomes, and the heterologous gene of coding for antigens determinant or genome section belong to one or more allos HPIV.
11. the chimeric PIV of claim 10 wherein adds in partial or complete HPIV3 JS genome or anti-genome or permutation encoding is selected from one or more genes or the genome section of one or more antigenic determinants of HPIV1 HN and F glycoprotein and antigenic domain, fragment and epi-position.
12. the chimeric PIV of claim 9 is wherein with corresponding HPIV3 HN and F protein part in the HPIV1 gene substitution section H PIV3 vector gene group of the HPIV1 gene of coding HN and coding F glycoprotein or the anti-genome.
13. the chimeric PIV of claim 9, wherein mosaic gene group or anti-genome mix exist among the PIV3 JScp45 at least a and even can reach whole attenuation sudden changes, these sudden changes are selected from: in L albumen corresponding to Tyr942, the Leu992 of JS cp45 or the position of Thr1558, in N albumen corresponding to the residue Val96 of JS cp45 or the position of Ser389, with in C albumen corresponding to the position of the Ile96 of JS cp45, determine a kind of sudden change of amino-acid substitution; In the embedded virus 3 ' leader sequence corresponding to the nucleotide subsitution of the position of the Nucleotide 23,24,28 of JS cp45 or 45; And/or in N gene homing sequence corresponding to the sudden change of the position of the Nucleotide 62 of JS cp45.
14. the chimeric PIV of claim 8 wherein adds or mixes one or more genes or the genome section of one or more antigenic determinants of coding HPIV2 in partial or complete HPIV3 genome or anti-genome.
15. the chimeric PIV of claim 14 wherein adds or mixes one or more HPIV2 genes or the genome section of one or more HN of coding and/or F glycoprotein or its antigenic domain, fragment or epi-position in partial or complete HPIV3 vector gene group or anti-genome.
16. the chimeric PIV of claim 6 wherein adds or mixes multiple heterologous gene or the genome section of the antigenic determinant of the multiple allos PIV of coding in partial or complete HPIV vector gene group or anti-genome.
17. the chimeric PIV of claim 16 wherein adds or mixes described multiple heterologous gene or the genome section of the antigenic determinant of coding HPIV1 and HPIV2 in partial or complete HPIV3 vector gene group or anti-genome.
18. the chimeric PIV of claim 17, wherein in partial or complete HPIV3 vector gene group or anti-genome, add or mix one or more HN of coding and/or one or more HPIV1 genes or the genome section of F glycoprotein or its antigenic domain, fragment or epi-position and one or more HPIV2 genes or the genome section of encode one or more HN and/or F glycoprotein or its antigenic domain, fragment or epi-position.
19. the chimeric PIV of claim 18, wherein with the HPIV1 gene of coding HN and the corresponding HPIV3 HN of HPIV1 gene substitution and the F gene of coding F glycoprotein, form chimeric HPIV3-1 vector gene group or anti-genome, it is further modified by one or more genes or the constant gene segment C that add or mix one or more antigenic determinants of coding HPIV2.
20. the chimeric PIV of claim 19 wherein adds in chimeric HPIV3-1 vector gene group or anti-genome or mixes the transcriptional units of the open reading frame (ORF) that contains HPIV2 HN gene.
21. the chimeric PIV of claim 20, it is selected from rPIV3-1.2HN or rPIV3-1cp45.2HN.
22. the chimeric PIV of claim 1, wherein vector gene group or anti-genome are partial or complete people PIV (HPIV) genome or anti-genomes, and the allos pathogenic agent is selected from Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
23. the chimeric PIV of claim 22, wherein one or more heterologous antigen determinants are selected from Measles virus HN and F albumen, subgroup A or subgroup B respiratory syncytial virus F, G, SH and M2 albumen, mumps virus HN and F albumen, human mammilla tumor virus L 1 albumen, 1 type or 2 type human immunodeficiency virus gp160 albumen, the gB of hsv and cytomegalovirus, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM albumen, rabies virus G albumen, Epstein-Barr virus gp350 albumen; Filovirus G albumen, bunyavirus G albumen, flavivirus pre M, E and NS1 albumen and Alphavirus E albumen, and antigenic domain, fragment and epi-position.
24. the chimeric PIV of claim 22, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genomes, or contain partial or complete HPIV3 genome or anti-genomic chimeric HPIV genome or anti-genome, wherein add or mixed one or more genes or the genome section of one or more antigenic determinants of coding allos HPIV.
25. the chimeric PIV of claim 24, wherein the allos pathogenic agent is a Measles virus, and the heterologous antigen determinant is selected from Measles virus HA and F albumen and antigenic domain, fragment and epi-position.
26. the chimeric PIV of claim 25 wherein adds in HPIV3 vector gene group or anti-genome or mixes the transcriptional units of the open reading frame (ORF) that contains Measles virus HA gene.
27. the chimeric PIV of claim 26, it is selected from rPIV3 (HA HN-L), rPIV3 (HAN-P), rcp45L (HA N-P), rPIV3 (HA P-M) or rcp45L (HA P-M).
28. the chimeric PIV of claim 24, wherein vector gene group or anti-genome are a kind of chimeric HPIV genome or anti-genome, and it contains and adds or mixed one or more genes of one or more antigenic determinants of coding allos HPIV1 or the partial or complete HPIV3 genome or the anti-genome of genome section.
29. the chimeric PIV of claim 25, wherein the allos pathogenic agent is a Measles virus, and the heterologous antigen determinant is selected from Measles virus HA and F albumen and antigenic domain, fragment and epi-position.
30. the chimeric PIV of claim 29 wherein is replaced at the ORF of HPIV3 HN and F and adds in the HPIV3-1 vector gene group of ORF of HPIV1 HN and F or the anti-genome or mix the transcriptional units of the open reading frame (ORF) that contains Measles virus HA gene.
31. the chimeric PIV of claim 30, it is selected from rPIV3-1 HA
P-MOr rPIV3-1 HA
P-MCp45L.
32. the chimeric PIV of claim 1, wherein partial or complete PIV vector gene group or anti-genome combine with one or more extra heterologous genes or genome section, form chimeric PIV genome or anti-genome.
33. the chimeric PIV of claim 32, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genomes, and one or more extra heterologous genes or genome section are selected from HPIV1 HN, HPIV1 F, HPIV2 HN, HPIV2 F, measles HA and/or translate reticent synthetic gene unit.
34. the chimeric PIV of claim 33 wherein inserts one of ORF of HPIV1 HN and/or HPIV2 HN or both in HPIV3 vector gene group or anti-genome.
35. the chimeric PIV of claim 33 wherein inserts the ORF of HPIV1 HN, HPIV2 HN and Measles virus HA respectively between N/P, P/M and HN/L gene.
36. the chimeric PIV of claim 33 wherein inserts HPIV1 HN and HPIV2 HN gene respectively, and adds 3918-nt GU and insert fragment between HN and L gene between N/P and P/M gene.
37. the chimeric PIV of claim 33, it is selected from: rHPIV3 1HN
N-P, rHPIV31HN
P-M, rHPIV3 2HN
N-P, rHPIV3 2HN
P-N, rHPIV3 1HN
N-P2HN
P-M, rHPIV3 1HN
N-P2HN
P-MHAH
N-LWith rHPIV3 1HN
N-P2HN
P-M3918GUH
N-L
38. the chimeric PIV of claim 32, it contains and derives from protective antigens a kind of, two kinds, three kinds or four kinds pathogenic agent.
39. the chimeric PIV of claim 32, it contains the protective antigen that derives from the 1-4 kind pathogenic agent that is selected from HPIV3, HPIV1, HPIV2 and Measles virus.
40. the chimeric PIV of claim 32, wherein one or more extra heterologous genes or genome section add in recombination group or anti-genome that compare length overall with the wild-type HPIV3 genome length of 15462nt be 30%-50% or more exogenous array.
41. the chimeric PIV of claim 32 wherein adds a kind of attenuation phenotype that one or more extra heterologous genes or genome section are determined chimeric PIV, it is presented at duplicating in the upper respiratory tract and/or the lower respiratory tract and reduces 10-100 at least doubly.
42. the chimeric PIV of claim 1, wherein vector gene group or anti-genome are a kind of human-bovine chimeric PIV genome or anti-genome.
43. the chimeric PIV of claim 42, wherein human-bovine chimeric vector gene group or anti-genome combine with one or more heterologous genes or the genome section of one or more antigenic determinants of coding allos pathogenic agent, and this pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
44. the chimeric PIV of claim 42, wherein vector gene group or anti-genome contain one or more heterologous genes or partial or complete HPIV genome of genome section bonded or the anti-genome with BPIV.
45. the chimeric PIV of claim 44 is wherein with the ORF of the corresponding N of the transcriptional units replacement vector genome of open reading frame (ORF) of the ORF that contains BPIV3 N or the HPIV3 vector gene group in the anti-genome.
46. the chimeric PIV of claim 45, wherein vector gene group or anti-genome with insert segmental Measles virus HA gene as extra gene and combine.
47. the chimeric PIV of claim 48, it is rHPIV3-N
BHA
P-M
48. the chimeric PIV of claim 42, wherein vector gene group or anti-genome contain one or more heterologous genes or partial or complete BPIV genome of genome section bonded or the anti-genome with HPIV.
49. the chimeric PIV of claim 48; wherein in partial or complete cow genome group or anti-genome, add or mix coding HN and/or F glycoprotein or its one or more HPIV genes or genome section a kind of or panimmunity originality territory, fragment or epi-position, form vector gene group or anti-genome.
50. the chimeric PIV of claim 49 wherein with the corresponding BPIV3 HN of HPIV3 gene substitution and the F gene of coding HN and F glycoprotein, forms vector gene group or anti-genome.
51. the chimeric PIV of claim 50, wherein vector gene group or anti-genome with insert segmental respiratory syncytial virus (RSV) F as extra gene or the G gene combines.
52. the chimeric PIV of claim 51, it is selected from rBHPIV3-G1 or rB/HPIV3-F1.
53. the chimeric PIV of claim 49; wherein in partial or complete cow genome group or anti-genome, mix one or more HPIV1HN and/or the F gene or the genome section of coding one or more immunogenicity territories, fragment or epi-position; form vector gene group or anti-genome; it is further modified by mixing coding one or more HPIV2HN of one or more immunogenicity territories, fragment or epi-position and/or F gene or genome section, forms the mosaic gene group or the anti-genome of the protective antigen of expressing HPIV1 and HPIV2.
54. the chimeric PIV of claim 53, it is selected from rB/HPIV3.1-2F, rB/HPIV3.1-2HN or rB/HPIV3.1-2F, 2HN.
55. the chimeric PIV of claim 1, wherein modify vector gene group or anti-genome, a kind of chimeric glycoprotein albumen that makes it to encode has wherein mixed one or more heterologous antigen territories, fragment or the epi-position of allos PIV or non-PIV pathogenic agent, forms mosaic gene group or anti-genome.
56. the chimeric PIV of claim 55, wherein modify vector gene group or anti-genome, a kind of chimeric glycoprotein albumen that makes it to encode has wherein mixed one or more antigenic domains, fragment or epi-position from the different PIV of second kind of antigenicity, forms mosaic gene group or anti-genome.
57. the chimeric PIV of claim 55, wherein a kind of chimeric glycoprotein albumen that contains antigenic domain, fragment or epi-position of mosaic gene group or anti-genome encoding from two or more HPIV.
58. the chimeric PIV of claim 55, heterologous gene group section a kind of glycoprotein extracellular domain of encoding wherein comes corresponding glycoprotein extracellular domain in replacement vector genome or the anti-genome with it.
59. the chimeric PIV of claim 55, one or more heterologous gene group sections of the HPIV that second kind of antigenicity of one or more antigenic domains of permutation encoding, fragment or epi-position is different in HPIV vector gene group or anti-genome wherein, this chimeric glycoprotein albumen makes it to encode.
60. the chimeric PIV of claim 55 is wherein with coding glycoprotein extracellular domain with stride the heterologous gene group section replacement vector genome or the corresponding glycoprotein extracellular domain in the anti-genome in film district and stride the film district.
61. the chimeric PIV of claim 55, wherein chimeric glycoprotein albumen is selected from HPIV HN and F glycoprotein.
62. the chimeric PIV of claim 56, wherein PIV vector gene group or anti-genome are section H PIV3 genome or anti-genome, and second kind of different PIV of antigenicity is selected from HPIV1 or HPIV2.
63. the chimeric PIV of claim 62, wherein HPIV vector gene group or anti-genome are section H PIV3 genome or anti-genome, and second kind of different HPIV of antigenicity is HPIV2.
64. the chimeric PIV of claim 63 is wherein with one or more glycoprotein extracellular domain displacement HPIV3 vector gene groups of HPIV2 or one or more corresponding HN and the F glycoprotein extracellular domain in the anti-genome.
65. the chimeric PIV of claim 64 is wherein with the glycoprotein extracellular domain displacement HPIV3 vector gene group of HPIV2 HN and F glycoprotein or corresponding HN and the F glycoprotein extracellular domain in the anti-genome.
66. the chimeric PIV of claim 65, it is rPIV3-2TM.
67. the chimeric PIV of claim 55, it is further modified, and also can reach whole attenuation sudden changes to be incorporated in one or more that identify among the HPIV3 JS cp45.
68. the chimeric PIV of claim 55, it is rPIV3-2TMcp45.
69. the chimeric PIV of claim 55, wherein one of HN and/or F glycoprotein or both PIV2 extracellular domains and stride film district and one or more corresponding PIV3 cytoplasmic tails fusions.
70. the chimeric PIV of claim 69, the wherein extracellular domain of PIV2 HN and F glycoprotein and stride the film district and merge with corresponding PIV3 HN and F cytoplasmic tail.
71. the chimeric PIV of claim 70, it is rPIV3-2CT.
72. the chimeric PIV of claim 71, it is further modified, and also can reach whole attenuation sudden changes to be incorporated in one or more that identify among the HPIV3 JScp45.
73. the chimeric PIV of claim 72, it is rPIV3-2CTcp45.
74. the chimeric PIV of claim 55, it is further modified, also can reach whole attenuation sudden changes to be incorporated in one or more that identify among the HPIV3 JScp45, these sudden changes are selected from and cause in L albumen corresponding to Tyr942, the Leu992 of JS cp45 or the position of Thr1558, in N albumen corresponding to the residue Val96 of JS cp45 or the position of Ser389, in C albumen corresponding to the amino-acid substitution of the position of the Ile96 of JS cp45; In the embedded virus 3 ' leader sequence corresponding to the nucleotide subsitution of the position of the Nucleotide 23,24,28 of JS cp45 or 45; And/or in N gene homing sequence corresponding to the sudden change of the position of the Nucleotide 62 of JS cp45.
75. the chimeric PIV of claim 55 wherein adds or mixes multiple heterologous gene or the genome section of the antigenic determinant of the multiple allos PIV of coding in partial or complete HPIV vector gene group or anti-genome.
76. the chimeric PIV of claim 75, wherein said multiple heterologous gene or genome section coding be from the antigenic determinant of HPIV1 and HPIV2, and add or mix in partial or complete HPIV3 vector gene group or the anti-genome.
77. the chimeric PIV of claim 55 wherein makes this mosaic gene group or anti-genome attenuation by adding or mixing one or more gene or genome sections from ox PIV3 (BPIV3).
78. the chimeric PIV of claim 55, this mosaic gene group or anti-genome are modified in the wherein attenuation of the amino-acid substitution of the phenylalanine by introducing 456 places, a kind of HPIV3 of relating to L albumen position sudden change.
79. the chimeric PIV of claim 78, wherein the phenylalanine at 456 places, the proteic position of HPIV3 L is replaced into leucine.
80. the chimeric PIV of claim 55, wherein this mosaic gene group or anti-genome have mixed coding one or more one or more heterologous genes or genome sections from the antigenic determinant of respiratory syncytial virus (RSV) or Measles virus.
81. the chimeric PIV of claim 1, wherein, modify this mosaic gene group or anti-genome by adding or replacing one or more genetic stabilities that cause embedded virus raisings or change attenuation, body internal reaction originality or heterologous gene of in culture, growing or genome section.
82. the chimeric PIV of claim 1 wherein by being introduced in one or more attenuation sudden changes of identifying in biologically-derived sudden change PIV or other sudden change non-segmented negative-strand RNA viruses, modifies this mosaic gene group or anti-genome.
83. the chimeric PIV of claim 82 wherein contains exist among the PIV3 JS cp45 a kind of at least and even can reach whole attenuation sudden changes in this mosaic gene group or the anti-genome.
84. the chimeric PIV of claim 82, wherein this mosaic gene group or anti-genome contain at least a and even can reach the sudden change of whole attenuation, this sudden change causes in L albumen the Tyr corresponding to JS cp45
942, Leu
992Or Thr
1558The position, in N albumen corresponding to the residue Val of JS cp45
96Or Ser
389The position, in C albumen corresponding to the Ile of JS cp45
96The position, in F albumen corresponding to the Ile of JS cp45
420Or Ala
450, in HN albumen corresponding to the Val of JS cp45
384A kind of amino-acid substitution; In the embedded virus 3 ' leader sequence corresponding to the nucleotide subsitution of the position of the Nucleotide 23,24,28 of JS cp45 or 45; And/or in N gene homing sequence corresponding to the sudden change of the position of the Nucleotide 62 of JS cp45.
85. the chimeric PIV of claim 82, wherein this mosaic gene group or anti-genome contain the attenuation sudden change from the biologically-derived sudden change PIV of difference or other sudden change non-segmented negative-strand RNA viruses.
86. the chimeric PIV of claim 82, wherein this mosaic gene group or anti-genome mix a kind of attenuation sudden change, and its amino acid position place is corresponding to the amino acid position of the attenuation sudden change of identifying in allos sudden change minus-stranded rna virus.
87. the chimeric PIV of claim 86, wherein this attenuation sudden change is included in the amino-acid substitution of the proteic position of HPIV3 L 456 place's phenylalanines.
88. the chimeric PIV of claim 87, wherein the proteic position of HPIV3 L 456 place's phenylalanines are replaced into leucine.
89. the chimeric PIV of claim 82, wherein this mosaic gene group or anti-genome comprise at least a owing to multiple Nucleotide in the codon of mutagenesis changes and stable attenuation sudden change.
90. the chimeric PIV of claim 1, wherein mosaic gene group or anti-genome contain other nucleotide modifications, and it can cause the phenotypic alternation that is selected from growth characteristics change, attenuation, temperature sensitivity, acclimatization to cold, plaque size, host range restriction or immunogenicity change.
91. the chimeric PIV of claim 90, wherein said other nucleotide modifications change in vector gene group or the anti-genome or one or more PIV N, P, C, D, V, M, F, HN and/or L gene in heterologous gene or the genome section and/or 3 ' leader, 5 ' tail region and/or intergenic region.
92. the chimeric PIV of claim 91, wherein one or more PIV genes completely or partially lack, or owing to rna editing site mutation, phase shift mutation, definite amino acid whose sudden change or the one or more terminator codons of introducing, reduction or the elimination genetic expression in gene open reading frame (ORF) of change initiator codon.
93. the chimeric PIV of claim 92, wherein said other nucleotide modifications comprise the partially or completely disappearance of one or more C, D or V ORF, and one or more Nucleotide that maybe can reduce or eliminate this one or more C, D, V ORF expression change.
94. the chimeric PIV of claim 1, wherein further modified chimeric genome or anti-genome, a kind of cytokine makes it to encode.
95. the chimeric PIV of claim 1, it contains a kind of heterologous gene or genome section from respiratory syncytial virus (RSV).
96. the chimeric PIV of claim 95, wherein heterologous gene or genome section coding RSVF and/or G glycoprotein or its immunogenicity territory, fragment or epi-position.
97. the chimeric PIV of claim 1, it is a kind of virus.
98. the chimeric PIV of claim 1, it is a kind of subviral particle.
99. one kind stimulates the individual immunity system to induce the method for protection of antagonism PIV, comprises individuality is used a kind of immune q.s and chimeric PIV physiology acceptable carrier bonded claim 1.
100. the method for claim 99 is wherein with 10
3-10
7The dosage of PFU is used chimeric PIV.
101. the method for claim 99 is wherein used chimeric PIV to the upper respiratory tract.
102. the method for claim 99 is wherein used chimeric PIV by spraying, droplet or aerosol.
103. the method for claim 99, wherein vector gene group or anti-genome are people PIV3 (HPIV3), and chimeric PIV causes the immunne response at HPIV1 and/or HPIV2.
104. the method for claim 99, wherein chimeric PIV can cause at various human PIV and/or at the polyspecific immunne response of a kind of people PIV and a kind of non-PIV pathogenic agent.
105. the method for claim 99, wherein vector gene group or anti-genome are partial or complete people PIV (HPIV) genome or anti-genome, and the allos pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
106. the method for claim 99, wherein chimeric PIV can cause the polyspecific immunne response at people PIV (HPIV) and Measles virus.
107. the method for claim 106, wherein chimeric PIV can cause the polyspecific immunne response at HPIV3 and Measles virus.
108. the method for claim 99 is wherein used first kind of chimeric PIV and second kind of PIV according to claim 1, continuously or simultaneously to cause the polyspecific immunne response.
109. the method for claim 108, wherein second kind of PIV is second kind of chimeric PIV according to claim 1.
110. the method for claim 108 is wherein used first kind of chimeric PIV and second kind of PIV simultaneously in mixture.
111. the method for claim 108, wherein first kind of chimeric PIV and the second kind of PIV different variant of antigenicity that is HPIV.
112. the method for claim 111, wherein first kind of chimeric PIV contains one or more heterologous genes or partial or complete HPIV3 genome of genome section bonded or the anti-genome with one or more antigenic determinants of the different PIV of coding.
113. the method for claim 111, wherein first kind of chimeric PIV and second kind of PIV have all mixed one or more heterologous genes or the genome section of one or more antigenic determinants of the non-PIV pathogenic agent of encoding.
114. the method for claim 113, wherein first kind and second kind of chimeric PIV all are mixed with one or more heterologous genes or the genome section of one or more antigenic determinants of the same non-PIV pathogenic agent of coding.
115. a continuous immunity is to stimulate the individual immunity system, to bring out the method at the protection of multiple pathogenic agent; it comprises the first kind of attenuated chimeric HPIV that newborn infant to 4 a month big baby is used immune q.s; it expresses the antigenic determinant of non-PIV pathogenic agent and one or more antigenic determinants of HPIV3; use second kind of attenuated chimeric HPIV of immune q.s subsequently, it expresses the antigenic determinant of non-PIV pathogenic agent and one or more antigenic determinants of HPIV1 or HPIV2.
116. the continuous immunity method of claim 115, wherein first kind of attenuated chimeric HPIV is a kind of HPIV3 that expresses the measles virus antigens determinant, and second kind of attenuated chimeric HPIV is a kind of PIV3-1 embedded virus of expressing the measles virus antigens determinant and being mixed with one or more attenuation sudden changes of HPIV3 JS cp45.
117. the continuous immunity method of claim 115, wherein after inoculation for the first time, the vaccinate produces at PIV3 and non-PIV pathogenic agent and does not reply at the primary antibody of HPIV1 or HPIV2, after the immunity second time, the vaccinate infects second kind of attenuation HPIV easily, and the primary antibody of HPIV1 or HPIV2 and the secondary antibodies of tiring at the height of non-PIV pathogenic agent are replied in generation.
118. the continuous immunity method of claim 115, wherein first kind of chimeric PIV can cause the immunne response at HPIV3, and second kind of chimeric PIV causes the immunne response at HPIV1 or HPIV2, and wherein first kind and second kind of chimeric PIV can cause the immunne response at measles or RSV.
119. the continuous immunity method of claim 115, wherein non-PIV pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus (RSV), mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
120. the continuous immunity method of claim 115, wherein second kind of chimeric PIV contains partial or complete HPIV3 vector gene group or anti-genome, and it combines with one or more genes or the genome section of one or more HPIV1 of coding and/or HPIV2 HN and/or F glycoprotein or its antigenic domain, fragment or epi-position.
121. the continuous immunity method of claim 115, wherein the partial or complete HPIV3 vector gene group of first kind of chimeric PIV or anti-genome mix exist among the HPIV3 JS cp45 at least a and even can reach whole attenuation sudden changes, it is selected from: determine in L albumen corresponding to Tyr942, the Leu992 of JS cp45 or the position of Thr1558, in N albumen corresponding to the residue Val96 of JS cp45 or the position of Ser389, in C albumen corresponding to the sudden change of the position of the Ile96 of JS cp45; In embedded virus 3 ' leader sequence corresponding to the nucleotide subsitution of the position of the Nucleotide 23,24,28 of JS cp45 or 45; And/or in N gene homing sequence corresponding to the sudden change of the position of the Nucleotide 62 of JS cp45.
122. an immunogenic composition that is used for causing at the immunne response of PIV, it contains the chimeric PIV of the claim 1 of immunogenicity q.s in the physiology acceptable carrier.
123. the immunogenic composition of claim 122, it is with 10
3-10
7The dosage preparation of PFU.
124. the immunogenic composition of claim 122, preparation is in order to use the upper respiratory tract by spraying, droplet or aerosol.
125. the immunogenic composition of claim 122, wherein chimeric PIV can cause at one or more viral immunne responses that are selected from HPIV1, HPIV2 and HPIV3.
126. the immunogenic composition of claim 122, wherein chimeric PIV can cause at HPIV3 and be selected from HPIV1 and the immunne response of the another kind of virus of HPIV2.
127. the immunogenic composition of claim 122, wherein chimeric PIV can cause that this pathogenic agent is selected from the polyspecific immunne response of one or more HPIV and a kind of allos pathogenic agent: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
128. the immunogenic composition of claim 127, wherein chimeric PIV can cause the polyspecific immunne response at HPIV3 and Measles virus or respiratory syncytial virus.
129. the immunogenic composition of claim 122, it further contains second kind of chimeric PIV of with good grounds 1.
130. the immunogenic composition of claim 129, first kind of different variant of antigenicity that is HPIV with second kind of chimeric PIV wherein, and carry identical or different heterologous antigen determinant.
131. the immunogenic composition of claim 129, wherein first kind of chimeric PIV contains one or more heterologous genes or partial or complete HPIV3 genome of genome section bonded or the anti-genome with one or more antigenic determinants of the non-PIV allos pathogenic agent of coding.
132. the immunogenic composition of claim 129, wherein second kind of chimeric PIV is mixed with one or more heterologous genes or the genome section of one or more antigenic determinants of the same non-PIV allos pathogenic agent of coding.
133. the immunogenic composition of claim 129, wherein first kind of chimeric PIV can cause the immunne response at HPIV3, second kind of chimeric PIV can cause the immunne response at HPIV1 or HPIV2, and wherein first kind and second kind of chimeric PIV can cause immunne response at non-PIV pathogenic agent.
134. the immunogenic composition of claim 129, wherein the allos pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
135. the immunogenic composition of claim 129, wherein the allos pathogenic agent is selected from Measles virus or RSV.
136. the immunogenic composition of claim 129, wherein second kind of chimeric PIV contains HPIV1 gene or genome section bonded section H PIV3 vector gene group or the anti-genome with one or more antigenic determinants of coding HPIV1 HN and/or F glycoprotein.
137. the immunogenic composition of claim 129, wherein second kind of chimeric PIV contains one or more genes or genome section bonded partial or complete HPIV3 vector gene group or the anti-genome with one or more HPIV2 HN of coding and/or F glycoprotein or its antigenic domain, fragment or epi-position.
138. isolating polynucleotide, it contains a kind of chimeric PIV genome or anti-genome, partial or complete PIV vector gene group that wherein comprises or anti-genome combine with one or more genes or the genome section of one or more antigenic determinants of one or more allos pathogenic agent of coding, form chimeric PIV genome or anti-genome.
139. the isolating polynucleotide of claim 138, wherein within partial or complete PIV vector gene group or the anti-genomic non-coding region or near one or more heterologous genes or the genome section of adding coding for antigens determinant.
140. the isolating polynucleotide of claim 138 are wherein used one or more heterologous genes of coding for antigens determinant or one or more corresponding genes or the genome section in genome section replacing section or complete PIV vector gene group or the anti-genome.
141. the isolating polynucleotide of claim 138, wherein one or more allos pathogenic agent are a kind of allos PIV, one or more PIV N, P, C, D, V, M, F, HN and/or L albumen or its immunogen fragment, structural domain or epi-position and heterologous gene or genome section are encoded.
142. the isolating polynucleotide of claim 138, wherein vector gene group or anti-genome are partial or complete people PIV (HPIV) genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are one or more allos PIV.
143. the isolating polynucleotide of claim 142, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome, and the heterologous gene of coding for antigens determinant or genome section are HPIV1 and/or HPIV2.
144. the isolating polynucleotide of claim 138, wherein vector gene group or anti-genome are partial or complete people PIV (HPIV) genome or anti-genome, and the allos pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
145. the isolating polynucleotide of claim 144, wherein one or more heterologous antigen determinants are selected from: F, the G of Measles virus HA and F albumen, subgroup A and subgroup B respiratory syncytial virus, SH and M2 albumen, mumps virus HN and F albumen, human mammilla tumor virus L 1 albumen, 1 type or 2 type human immunodeficiency virus gp160 albumen, the gB of hsv and cytomegalovirus, gC, gD, E, gG, gH, gI, gJ, gK, gL, gM albumen, rabies virus G albumen, Epstein-Barr virus gp350 albumen; Filovirus G albumen, bunyavirus G albumen, flavivirus E and NS1 albumen and Alphavirus E albumen, and antigenic domain, fragment and epi-position.
146. the isolating polynucleotide of claim 138, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome or chimeric HPIV genome or anti-genome, are included in wherein to add or mixed one or more genes of one or more antigenic determinants of coding allos HPIV or the partial or complete HPIV3 genome or the anti-genome of genome section.
147. the isolating polynucleotide of claim 146, wherein the allos pathogenic agent is a Measles virus, and the heterologous antigen determinant is selected from Measles virus HA and F albumen and antigenic domain, fragment and epi-position.
148. the isolating polynucleotide of claim 147 wherein add in HPIV3 vector gene group or anti-genome or mix a kind of transcriptional units that contains the open reading frame (ORF) of Measles virus HA gene.
149. the isolating polynucleotide of claim 147 wherein are replaced at HPIV3 HN and FORF and add in the HPIV3-1 vector gene group of HPIV1 HN and F ORF or the anti-genome or mix a kind of transcriptional units that contains the open reading frame (ORF) of Measles virus HA gene.
150. the isolating polynucleotide of claim 138, wherein partial or complete PIV vector gene group or anti-genome combine with one or more extra heterologous genes or genome section, form chimeric PIV genome or anti-genome.
151. the isolating polynucleotide of claim 150, wherein vector gene group or anti-genome are partial or complete HPIV3 genome or anti-genome, and one or more extra heterologous genes or genome section are selected from HPIV1 HN, HPIV1 F, HPIV2 HN, HPIV2 F, measles HA and/or translate reticent synthetic gene unit.
152. the isolating polynucleotide of claim 138 wherein add a kind of, two kinds or whole HPIV1 HN, HPIV2 HN and Measles virus HAORF in vector gene group or anti-genome.
153. the isolating polynucleotide of claim 138 wherein add one or more HPIV1 HN and HPIV2 HN gene and 3918-nt GU and insert fragment in vector gene group or anti-genome.
154. the isolating polynucleotide of claim 150, wherein one or more extra heterologous genes or genome section add 30%-50% that length overall is wild-type HPIV3 genome length 15462 nt or longer exogenous array in recombination group or anti-genome.
155. the isolating polynucleotide of claim 138, wherein vector gene group or anti-genome are a kind of human-bovine chimeric genome or anti-genome.
156. the isolating polynucleotide of claim 155, wherein human-bovine chimeric vector gene group or anti-genome combine with one or more heterologous genes or the genome section of one or more antigenic determinants of a kind of allos pathogenic agent of coding, and this pathogenic agent is selected from: Measles virus, subgroup A and subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, 1 type and 2 type human immunodeficiency viruses, hsv, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, Alphavirus and influenza virus.
157. the isolating polynucleotide of claim 156, wherein vector gene group or anti-genome contain with from one or more heterologous genes or partial or complete HPIV genome of genome section bonded or the anti-genome of BPIV.
158. the isolating polynucleotide of claim 157, wherein in vector gene group or anti-genome with the corresponding N ORF of the transcriptional units displacement HPIV3 vector gene group of the open reading frame (ORF) that contains BPIV3 N ORF.
159. the isolating polynucleotide of claim 158, wherein vector gene group or anti-genome with insert segmental Measles virus HA gene as extra gene and combine.
160. the isolating polynucleotide of claim 138, wherein vector gene group or anti-genome contain with from one or more heterologous genes or partial or complete BPIV genome of genome section bonded or the anti-genome of HPIV.
161. the isolating polynucleotide of claim 160; wherein in partial or complete cow genome group or anti-genome, add or mix coding HN and/or F glycoprotein or its one or more HPIV genes or genome section a kind of or panimmunity prodomain, fragment or epi-position, form vector gene group or anti-genome.
162. the isolating polynucleotide of claim 161 wherein with the corresponding BPIV3 HN of HPIV3 gene substitution and the F gene of coding HN and F glycoprotein, form vector gene group or anti-genome.
163. the isolating polynucleotide of claim 162, wherein vector gene group or anti-genome with insert segmental respiratory syncytial virus (RSV) F as extra gene or the G gene combines.
164. the isolating polynucleotide of claim 138, wherein a kind of chimeric glycoprotein albumen that contains from antigenic domain, fragment or the epi-position of people PIV (HPIV) and allos pathogenic agent of mosaic gene group or anti-genome encoding.
165. the isolating polynucleotide of claim 164, wherein a kind of chimeric glycoprotein albumen that contains antigenic domain, fragment or epi-position of mosaic gene group or anti-genome encoding from two or more different PIV.
166. the isolating polynucleotide of claim 138 are wherein by being introduced in one or more attenuation sudden changes of identifying in biologically-derived sudden change PIV or other sudden change non-segmented negative-strand RNA viruses, modified chimeric genome or anti-genome.
167. the isolating polynucleotide of claim 138, wherein mosaic gene group or anti-genome are mixed with at least a and even can reach the attenuation sudden change that exists among whole PIV3 JS cp45.
168. the isolating polynucleotide of claim 138, wherein mosaic gene group or anti-genome are mixed with a kind of attenuation sudden change from the allos non-segmented negative-strand RNA viruses.
169. the isolating polynucleotide of claim 138, wherein mosaic gene group or anti-genome contain another kind of nucleotide modification, and it can cause a kind of phenotypic alternation that growth characteristics change, attenuation, temperature sensitivity, acclimatization to cold, plaque size, host range restriction or immunogenicity change that is selected from.
170. the isolating polynucleotide of claim 138, wherein said another kind of nucleotide modification changes in vector gene group or the anti-genome or one or more PIV N, P, C, D, V, M, F, HN and/or L gene in heterologous gene or the genome section and/or 3 ' leader, 5 ' tail region and/or intergenic region.
171. the isolating polynucleotide of claim 138, wherein one or more PIV genes completely or partially lack, or owing to rna editing site mutation, phase shift mutation, definite amino acid whose sudden change or the one or more terminator codons of introducing, reduction or the elimination genetic expression in gene open reading frame (ORF) of change initiator codon.
172. one or more isolating polynucleotide molecule production infectivity attenuated chimeric PIV particulate methods by coding PIV, it comprises:
In cell or acellular lysate, express a kind of expression vector that contains a kind of isolating polynucleotide and PIV N, P, L albumen, these polynucleotide contain and one or more heterologous genes of one or more antigenic determinants of one or more allos pathogenic agent of coding or partial or complete PIV vector gene group or the anti-genome of genome section bonded people or ox PIV, form a kind of chimeric PIV genome or anti-genome.
173. the method for claim 172, wherein chimeric PIV genome or anti-genome are expressed by two or more different expression vectors with N, P, L albumen.
174. expression vector, it contains a kind of promotor of effective connection, a kind of polynucleotide sequence and a kind of transcription terminator, this polynucleotide sequence comprises partial or complete PIV vector gene group or the anti-genome of people or ox PIV, they combine with heterologous gene or the genome section of one or more antigenic determinants of one or more allos pathogenic agent of coding, form a kind of mosaic gene group or anti-genome.
A 175. isolating infectivity recombinant parainfluenza virus (PIV), it contains a kind of main nucleocapsid (N) albumen, a kind of nucleocapsid phosphorprotein (P), a kind of big polymerase protein (L) and a kind of PIV genome or anti-genome, inserting length in this genome or anti-genomic non-coding region (NCR) is the polynucleotide of 150 Nucleotide (nt) to 4000 Nucleotide, or as isolating genetic unit (GU), these polynucleotide insert fragment and lack complete open reading frame (ORF), and give this reorganization PIV an attenuation phenotype.
176. the reorganization PIV of claim 175 wherein inserts these polynucleotide fragment and imports in PIV genome or the anti-genome, thereby make not coded protein of this insertion fragment with opposite direction, non-sense orientation.
177. the reorganization PIV of claim 175, wherein said polynucleotide insert fragment length and are about 2000nt or longer.
178. the reorganization PIV of claim 175, wherein said polynucleotide insert fragment length and are about 3000nt or longer.
179. the reorganization PIV of claim 175, wherein said reorganization PIV can efficiently duplicate external, and attenuation phenotype in the display body.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US17019599P | 1999-12-10 | 1999-12-10 | |
| US09/458,813 US7314631B1 (en) | 1997-05-23 | 1999-12-10 | Use of recombinant live-attenuated parainfluenza virus (PIV) as a vector to protect against disease caused by PIV and respiratory syncytial virus (RSV) |
| US09/459,062 | 1999-12-10 | ||
| US09/458,813 | 1999-12-10 | ||
| US09/459,062 US7250171B1 (en) | 1997-05-23 | 1999-12-10 | Construction and use of recombinant parainfluenza viruses expressing a chimeric glycoprotein |
| US60/170,195 | 1999-12-10 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010556188XA Division CN102766605A (en) | 1999-12-10 | 2000-12-08 | Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1347453A true CN1347453A (en) | 2002-05-01 |
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Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00805939A Pending CN1347453A (en) | 1999-12-10 | 2000-12-08 | Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens |
| CN201010556188XA Pending CN102766605A (en) | 1999-12-10 | 2000-12-08 | Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010556188XA Pending CN102766605A (en) | 1999-12-10 | 2000-12-08 | Use of recombinant parainfluenza viruses (PIVs) as vectors to protect against infection and disease caused by PIV and other human pathogens |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1179054A2 (en) |
| JP (1) | JP4795597B2 (en) |
| CN (2) | CN1347453A (en) |
| AU (1) | AU785148B2 (en) |
| CA (1) | CA2362685A1 (en) |
| IL (1) | IL144831A0 (en) |
| MX (1) | MXPA01008108A (en) |
| WO (1) | WO2001042445A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103773803A (en) * | 2014-01-23 | 2014-05-07 | 中国农业科学院特产研究所 | Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus |
| CN104093422A (en) * | 2012-01-24 | 2014-10-08 | 乔治亚大学研究基金公司 | Parainfluenza virus 5 based vaccines |
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| US6764685B1 (en) | 2000-03-21 | 2004-07-20 | Medimmune Vaccines, Inc. | Recombinant parainfluenza virus expression systems and vaccines |
| US8715922B2 (en) | 2001-01-19 | 2014-05-06 | ViroNovative | Virus causing respiratory tract illness in susceptible mammals |
| CN101921732A (en) | 2001-01-19 | 2010-12-22 | 维洛诺瓦蒂夫公司 | A virus causing respiratory tract illness in susceptible mammals |
| US7704509B2 (en) | 2001-11-21 | 2010-04-27 | United States Of America | Recovery of recombinant human parainfluenza virus type 1 (HPIV1) from cDNA and use of recombinant HPIV1 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens |
| WO2003072720A2 (en) | 2002-02-21 | 2003-09-04 | Medimmune Vaccines, Inc. | Recombinant parainfluenza virus expression systems and vaccines comprising heterologous antigens derived from metapneumovirus |
| US7081341B2 (en) | 2002-07-01 | 2006-07-25 | Wayne State University | Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance |
| AU2003270824B2 (en) | 2002-09-18 | 2009-02-19 | The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | Recovery of recombinant human parainfluenza virus type 2 (HPIV2) from cDNA and use of recombinant HPIV2 in immunogenic compositions and as vectors to elicit immune responses against PIV and other human pathogens |
| WO2005014626A2 (en) | 2003-02-28 | 2005-02-17 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Recombinant human metapneumovirus and its use |
| JP2007525178A (en) | 2003-04-25 | 2007-09-06 | メッドイミューン バクシーンズ，インコーポレイティド | Metapneumovirus strain, its use in vaccine formulations, use as a vector for expression of antigenic sequences, and virus propagation method |
| KR100999316B1 (en) | 2003-06-09 | 2010-12-10 | 와이어쓰 엘엘씨 | Improved method for the recovery of non-segmented negative-stranded RNA viruses from cDNA |
| DE102005006388A1 (en) | 2005-02-11 | 2006-08-24 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Replication-deficient RNA viruses as vaccines |
| WO2006122224A1 (en) | 2005-05-11 | 2006-11-16 | Wayne State University | Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance in pseudomonas aeruginosa |
| US8178659B2 (en) | 2005-05-11 | 2012-05-15 | Wayne State University | Targets for the identification of antibiotics that are not susceptible to antibiotic resistance |
| JP4905649B2 (en) * | 2006-03-08 | 2012-03-28 | 国立大学法人 長崎大学 | Fish vaccine, method for producing the same, and method for preventing fish infection |
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| US8362203B2 (en) | 2009-02-10 | 2013-01-29 | Wayne State University | Non-natural peptides as models for the development of antibiotics |
| US11273214B2 (en) | 2017-05-29 | 2022-03-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Recombinant chimeric bovine/human parainfluenza virus 3 expressing RSV G and its use |
| SG11202012398UA (en) * | 2018-06-27 | 2021-01-28 | Medicago Inc | Influenza virus hemagglutinin mutants |
| CN112771619A (en) * | 2018-08-28 | 2021-05-07 | 皇家飞利浦有限公司 | Method for evaluating the basis of genomic alignments |
| CN113462656B (en) * | 2021-03-24 | 2022-09-30 | 兰州生物制品研究所有限责任公司 | Human three-type parainfluenza virus cold-adaptation temperature-sensitive strain and application thereof |
| CN113999292B (en) * | 2021-10-27 | 2023-05-26 | 中国农业大学 | Polypeptide inhibitors targeting respiratory viruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100702523B1 (en) * | 1997-05-23 | 2007-04-04 | 더 가번먼트 오브 더 유나이티드 스테이츠 오브 아메리카, 에즈 레프리젠티드 바이 더 디파트먼트 오브 헬쓰 앤드 휴먼 서비시즈 | Production of attenuated parainfluenza virus vaccines from cloned nucleotide sequences |
| CA2378661A1 (en) * | 1999-07-09 | 2001-01-18 | Alexander C. Schmidt | Attenuated human-bovine chimeric parainfluenza virus (piv) vaccines |
-
2000
- 2000-12-08 CN CN00805939A patent/CN1347453A/en active Pending
- 2000-12-08 JP JP2001544321A patent/JP4795597B2/en not_active Expired - Fee Related
- 2000-12-08 CN CN201010556188XA patent/CN102766605A/en active Pending
- 2000-12-08 CA CA002362685A patent/CA2362685A1/en not_active Abandoned
- 2000-12-08 WO PCT/US2000/033293 patent/WO2001042445A2/en active Application Filing
- 2000-12-08 IL IL14483100A patent/IL144831A0/en unknown
- 2000-12-08 MX MXPA01008108A patent/MXPA01008108A/en not_active Application Discontinuation
- 2000-12-08 EP EP00984052A patent/EP1179054A2/en not_active Withdrawn
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104093422A (en) * | 2012-01-24 | 2014-10-08 | 乔治亚大学研究基金公司 | Parainfluenza virus 5 based vaccines |
| CN104093422B (en) * | 2012-01-24 | 2018-02-23 | 乔治亚大学研究基金公司 | Vaccine based on parainfluenza virus 5 |
| CN103773803A (en) * | 2014-01-23 | 2014-05-07 | 中国农业科学院特产研究所 | Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001042445A3 (en) | 2001-11-29 |
| CN102766605A (en) | 2012-11-07 |
| MXPA01008108A (en) | 2004-11-12 |
| AU2073101A (en) | 2001-06-18 |
| EP1179054A2 (en) | 2002-02-13 |
| JP4795597B2 (en) | 2011-10-19 |
| WO2001042445A2 (en) | 2001-06-14 |
| AU785148B2 (en) | 2006-10-05 |
| IL144831A0 (en) | 2002-06-30 |
| JP2003516148A (en) | 2003-05-13 |
| CA2362685A1 (en) | 2001-06-14 |
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