CN101072879B - Improved adenoviral vectors and uses thereof - Google Patents

Abstract

The present invention relates to recombinant adenoviral vectors based on adenoviruses that encounter pre-existing immunity in a minority of the human population and which harbour a chimeric capsid. The chimeric capsid comprises fiber proteins that have at least the knob domain of a human adenovirus that binds to the Coxsackievirus and Adenovirus Receptor (CAR) and a hexon protein from an adenovirus serotype that encounters pre-existing immunity in a low percentage of the human population.

Description

The adenovirus carrier and the application thereof of improvement

Invention field

The present invention relates to field of medicaments, be particularly related to field by using the reorganization chimeric adenoviral vectors in vaccine composition, comprise therapeutic nucleic acids to treat and prevent.

Background of invention

Recombinant adenoviral vector is widely used in gene therapy and vaccine.Up to now, 51 kinds of different adenoviral serotypes have been differentiated.The most deep research has been carried out in application such as gene therapy to subgroup C adenovirus, and especially serotype 2 and 5 (Ad2 and Ad5) is in this area widespread use.Reorganization Ad5 is used for multiple various objectives, comprises immunization.Importantly, be illustrated in based on the vaccine of Ad5 carrier and caused strong protective immune response in the several animal models.In addition, carrying out the extensive clinical trial to the HIV immunization, (WO 01/02607 wherein to use recombinant vectors based on Ad5; WO 02/22080; Shiver et al.2002; Letvin et al.2002; Shiver and Emini.2004).Yet, obviously be subjected to the restriction of the high seroprevalence of Ad5 specificity neutralizing antibody (NAb) among the crowd probably based on the application of the vaccine of the HIV of reorganization Ad5 carrier and other pathogenic agent.Research in mouse and rhesus monkey has demonstrated the remarkable immunogenicity that suppresses based on the vaccine of Ad5 of existence of anti-Ad5 immunity.Early time data from 1 clinical trial phase shows that this problem also may occur in (Shiver 2004) in the human body.

A kind ofly be hopeful to get around the previous strategy that has infected the existence of the immunity that is pre-existing in the individuality of modal adenovirus hominis (as Ad5) and comprise the adenoviral serotype exploitation recombinant vectors that never meets with this immunity that is pre-existing in.Through differentiating that useful especially adenovirus hominis carrier is based on serotype 11,26,34,35,48,49 and 50, as (also see Vogels et al.2003 as described in) as described in WO 00/70071, WO 02/40665 and the WO 2004/037294.Other researchist has been found that adenovirus 24 (Ad24) also is to make us interested especially, is rare serotype (WO 2004/083418) because shown it.

A kind of similar strategy is based on using simian adenovirus, because these adenovirus typically do not infect human body.They present low seroprevalence in the human sample.Yet they also can be used for human body, because these viruses can (WO 03/000283 at the Infection in Vitro human body; WO2004/037189).

Illustrated vaccine based on adenoviral serotype 35 (Ad35) carrier can cause not by anti-Ad5 immunity strong cellullar immunologic response (the Barouch et al.2004 that obviously suppresses; Vogels et al.2003).Similarly, chimpanzee adenovirus being shown causes and is subjected to anti-Ad5 immunity to influence minimum immunne response (Farina et al.2001; Pinto et al.2003).Confirm neutralizing antibody (NAb) and CD8 in recent years ⁺The T lymphocyte responses all helps anti-Ad5 immunity, and Ad5 specificity NAb seems to bring into play main effect (Sumida et al.2004).Although this progress looks like very useful scheme, show that in the research of the Ad5 immunity that is not pre-existing in the immunogenicity of vaccine in mouse based on the Ad35 carrier is lower than the vaccine (Barouch et al.2004) based on the Ad5 carrier.

Obviously, this area needs other adenovirus carrier, and it does not meet with the immunity that is pre-existing in the host, but still is immunogenic and can induces at by the strong immune response that is inserted into the heterologous nucleic acids encoded protein matter in the entrained nucleic acid of carrier.

The accompanying drawing summary

Fig. 1 (A) illustrates the seroprevalence per-cent from Ad5, AdI1 and Ad35 in the sample in the U.S., Haiti, Botswana, Zambia and South Africa; (B) NAT from anti-Ad5, AdI1 and Ad35 in the U.S. and the sample from Africa is shown.Fig. 1 C illustrates the NAT of anti-Ad5, Ad5f35, Ad5f35p35, Ad35f5 and Ad35 in these samples.

Fig. 2 (A) illustrates nucleotide sequence (the SEQ ID NO:1 of coding Ad35k5 scleroproein (fiber protein); Underscore illustrates the part of coding Ad5 fiber knob); (B) the fibrinous aminoacid sequence of Ad35k5 (SEQ ID NO:2 is shown; The underscore place represents the part of Ad5 fiber knob); (C) the fibrinous nucleotide sequence of coding Ad35f5 (SEQ IDNO:4 is shown; The underscore place illustrates the segmental part of Ad35 fiber that coding keeps, and the BsiWI cloning site is represented with lowercase); (D) the fibrinous aminoacid sequence of Ad35f5 (SEQ ID NO:5 is shown; The Ad35 fiber fragment part that the representative of underscore place keeps); (E) coding Ad35k2 6 fibrinous nucleotide sequences (SEQ ID NO:80 is shown; The underscore place illustrates the part of coding Ad26 fiber knob); (F) the fibrinous aminoacid sequence of Ad35k26 (SEQ ID NO:81 is shown; The underscore place represents Ad26 fiber knob part); (G) the fibrinous nucleotide sequence of coding Ad35k49 (SEQ ID NO:82 is shown; The underscore place illustrates the part of coding Ad49 fiber knob); (H) the fibrinous aminoacid sequence of Ad35k49 (SEQ ID NO:83 is shown; The underscore place illustrates Ad49 fiber knob part).

Fig. 3 illustrates Ad5, the Ad35fib5.BSU, Ad35.BSU and the Ad35 carrier that carry SIV gag encoding gene to mouse (naive mice) injection of not carrying out experiment and compares the t cell response of institute's inductive at SIV gag with the mouse of injecting blank carrier (Ad5.empty and Ad35.empty).Y-axis is represented SFC/10 ⁶Splenocyte.

Fig. 4 illustrates Ad5, Ad35k5 and the immunogenicity of Ad35 carrier in the mouse that did not carry out experiment of expressing SIV Gag, with 10 ⁹Vp (A) and 10 ⁸Vp (B) immunity is perhaps with 10 ⁸Inject once (C) or twice (D) 10 before each carrier immunity of vp ¹⁰Vp Ad5-Empty carries out pre-immunity (pre-immunization).In all situations, a plurality of time points after immunity pass through D ^b/ AL11 tetramer binding analysis is estimated Gag-specific C D8 ⁺The T lymphocyte responses.

Fig. 5 illustrates the anti-carrier immunity that is caused by the Ad35k5 carrier in the mouse.(A) never carried out the experiment the C57/BL6 mouse the 0th the week with 10 ⁹Vp Ad35k5-Gag initial immunity is also all with 10 the 4th ⁹Vp Ad5-Gag or Ad35-Gag strengthen.(B) usefulness of estimating in the viral neutralization analysis based on luciferase at Ad5 and Ad35 10 ⁹The mice serum sample of vp Ad5-Gag, Ad35k5-Gag or Ad35-Gag injection.

Fig. 6 is illustrated in Ad5, the Ad35k5 of expression HIV-I Env in the mouse and the immunogenicity of Ad35 carrier.To never carry out the Balb/c mouse of experiment with 10 ⁸Vp Ad5-Env, Ad35k5-Env or Ad35-Env immunity.(A) the Env specificity cellular immunity response by mixed peptide (pooled peptide) and MIlO epitope peptide specificity IFN-γ ELISPOT analyzing evaluation.(B) the Env specific humoral immune response of evaluating by ELISA.

Fig. 7 was illustrated in for the 0th week with 10 ¹¹The immunogenicity of Ad5, Ad35k5 and Ad35 carrier in the rhesus monkey of vp Ad5-Gag (A, B), Ad35-Gag (C, D) and Ad35k5-Gag (E, F) initial immunity.In the 12nd week, all monkeys are all accepted the homology booster immunization.Env-and the Gag-specificity cellular immunity response a plurality of time points after immunity are by mixed peptide IFN-γ ELISPOT analyzing evaluation (A, C, E).Carrier specificity NAb tires by Ad5 and Ad35 virus neutralization analysis evaluation (B, D, F).

Fig. 8 is illustrated in the research that Fig. 7 describes CD4 in the monkey ⁺And CD8 ⁺The T lymphocyte responses.PBMC that the use CD8 that the 16th week obtained from monkey after immunity exhausts and that CD4 exhausts carries out mixed peptide IFN-γ ELISPOT and analyzes.Monkey accepts 10 in the 0th week and the 12nd week ¹¹Vp (A) Ad5-Gag, (B) Ad35-Gag or (C) Ad35k5-Gag.

Fig. 9 is illustrated in acceptance (A) 10 ⁹Vp, (B) 10 ⁸Vp or (C) 10 ⁷Not the carrying out of vp the immunogenicity of Ad5-Gag, Ad5HVR48 (1)-Gag and Ad5HVR48 (1-7) carrier in the mouse of experiment.Gag-specific C D8 ⁺The a plurality of time points of T lymphocyte responses after immunity pass through D ^bThe evaluation of/ALll tetramer binding analysis.

Figure 10 is illustrated in immunity 8 weeks and 4 all injections twice 10 before ¹⁰The pre-immunity of vp Ad5-Empty, accept (A) 10 ⁹Vp, (B) 10 ⁸Vp or (C) 10 ⁷The immunogenicity of Ad5-Gag, Ad5HVR48 (1)-Gag and Ad5HVR48 (1-7) carrier in the mouse of vp.

Figure 11 illustrates based on the aminoacid sequence of the hexon of Ad5 (SEQ ID NO:12), has wherein mixed 7 the corresponding HVR (underscore place) (Ad5HVR48 (1-7)) from Ad48 at 7 HVR places.

Figure 12 illustrates based on the aminoacid sequence of the hexon of Ad5 (SEQ ID NO:13), has wherein mixed 7 the corresponding HVR (underscore place) (Ad5HVR35 (1-7)) from Ad35 at 7 HVR places.

Figure 13 illustrates based on the aminoacid sequence of the hexon of Ad5 (SEQ ID NO:14), has wherein mixed 7 the corresponding HVR (underscore place) (Ad5HVR11 (1-7)) from AdI1 at 7 HVR places.

Figure 14 illustrates based on the aminoacid sequence of the hexon of Ad5 (SEQ ID NO:15), wherein mixes 7 the corresponding HVRs (underscore place) (Ad5HVR26 (1-7)) of l from Ad26 at 7 HVR places.

Figure 15 illustrates based on the aminoacid sequence of the hexon of Ad5 (SEQ ID NO:16), has wherein mixed 7 the corresponding HVR (underscore place) (Ad5HVRPan9 (1-7)) from the Pan9 adenovirus at 7 HVR places.

Figure 16 is illustrated under the background (not to be had and infects, 1 ° of graph; 2 ° of Ab) reach gag dyeing in Ad35k5 (two crowdes: #4, #5), Ad35k26 (two crowdes: twice of #3, #28) and the metainfective cell of Ad35k49 (three crowdes: #4, #5, #9).

Figure 17 illustrates Ad5HVR48 (1-7) ^*The aminoacid sequence (SEQID NO:84) of middle hexon wherein lacks HVR1 sequence as shown in Table IV, and the HVR2 sequence shown in the Table IV is replaced by the QG joint, and the HVR3 to HVR7 of Table IV definition is replaced between Ad5 and Ad48.

Figure 18: as Figure 17, this figure is at Ad5HVR35 (1-7) ^*(SEQ ID NO:85).

Figure 19: as Figure 17, this figure is at Ad5HVR26 (1-7) ^*(SEQ ID NO:86).

Figure 20; As Figure 17, this figure is at Ad5HVR49 (1-7) ^*(SEQ ID NO:87).

Figure 21 be with the pre-immunity of Ad5-Empty secondary (double pre-immunization), subsequently the 0th day with the Ad35-Gag initial immunity, the 28th day with shown in three kinds of different carriers strengthen after, CD8 in the mouse ⁺The synoptic diagram of T lymphocyte responses.

Summary of the invention

The present invention has disclosed recombinant adenoviral vector newly developed, in order to the conveying of improvement gene, inoculation and gene therapy.In a preferred embodiment, described carrier is based on the recombinant adenovirus of adenoviral serotype 35 (Ad35), and wherein Ad35 fiber knob is replaced by fiber knob in conjunction with the serotype of Coxsackie virus and adenovirus receptor (CAR) at least.More preferably, this serotype is that (generation is called Ad5 fiber knob Ad35k5Carrier, wherein have only knob to be replaced, perhaps produce and be called Ad35f5Carrier, wherein the part of knob, axle (shaft) and afterbody is replaced).The axle and the afterbody of fiber can carry main chain serotype, i.e. Ad35 is the carrier of homologous serotype mutually and the invention still further relates to wherein axle construction territory and fiber knob serotype.In Ad35f5, guarantee partly that from the tail region of main chain serotype correct interaction with the capsid remainder is to produce stable carrier.Carrier of the present invention comprises interested therapeutic nucleic acids, preferably can be used for inoculating the nucleic acid of purpose.

The present invention relates to recombinant adenoviral vector, they meet with the lower immunity that is pre-existing in and still can induce at the antigenic strong immune response by the nucleic acid encoding that described carrier comprised in most of crowds.This recombinant vectors based on adenoviral serotype such as AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49, Ad50 or simian adenovirus that carries chimeric capsid by generation is realized.Described chimeric capsid comprises scleroproein, and described scleroproein part is based on the scleroproein from the adenovirus hominis serotype different with the main chain carrier, and in the most preferred embodiment, described scleroproein is in conjunction with (by its knob structural domain) CAR acceptor.Preferentially the many different adenovirus in conjunction with CAR can be used for providing fiber knob, as the adenovirus hominis of A, C, D, E and F subgroup with also in conjunction with the ovine adenovirus of CAR.Being used to the preferred adenoviral serotype of fiber knob structural domain is provided is the adenovirus hominis of subgroup C, more preferably Ad2 and Ad5.

In another embodiment, the virus that the present invention produces is based on the preferably recombinant adenovirus of Ad5 of subgroup C adenovirus, it makes replication defect type by functional deficiency E1 zone, and wherein the hexon in the viral capsid is a chimeric protein, and one or more hypervariable region (HVR) is by the HVR displacement derived from rare adenoviral serotype thus.This rare serotype is the NAb in most of individualities among the experience crowd not.Being used to the preferred serotype of HVR is provided is Ad35 and Ad4 8.Preferably, described recombinant virus comprises and waits to be transported to the host to prevent or the interested heterologous nucleic acids of therapeutic purpose.

Describe in detail

As mentioned above, be restricted based on the application of the recombinant vectors of Ad5 existence owing to the previous neutralizing antibody (NAb) that infects wild-type virus and in most of individual humans, existed.Although the different capsid proteins that exist in the virus coat are induced antibody in the host, the main target position that has confirmed Ad5 specificity NAb is Ad5 hexon (Surnida et al.2005).Produce such viewpoint thus, promptly it is no longer found by existing NAb thus by changing described hexon, can produce the adenovirus carrier of improvement, this for have anti-Ad5 NAb's or before used based on the previous vaccine inoculation of Ad5 or for the first time/use in the strengthened scheme based on the people of the carrier initial immunity of Ad5 helpful.Yet it is not easy to do changing hexon.Six adjacent structural reforms accommodations completely often just are only possible between the adenovirus of identical Ad subgroup, and produce very poor virus (the Youil et al.2002 of viability; Gall et al.1998; Roy et al.1998).If this is a limiting factor, then must contain hexon from another subgroup C adenovirus based on the carrier of Ad5.Yet,, rare because those serotypes are not considered to even be not also to be that most these carriers are useless all.Most of individualities all once met with the serotype of subgroup C among the crowd.The preferred six adjacent bodies that use from the serotype that should not meet with the NAb that has existed.Those rare serotypes are mainly found in subgroup B (AdI1, Ad34, Ad35, Ad50) and D (Ad24, Ad26, Ad48 and Ad49).

The inventor discloses first now by (again) and limits the special part of six adjacent bodies and pass through to use conservative approach and available structure and sequence data, can differentiate and exchange some zone, produce producible recombinant virus, described virus be the survival and can produce to sufficiently high and tire.Being used to the preferred serotype of hexon or its relevant portion is provided is AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50, because known these serotypes meet with the lower immunity that is pre-existing in (seeing WO 00/70071).More preferably AdI1, Ad26, Ad35 and Ad48, and Ad48 is most preferred serotype.Non-in this respect adenovirus hominis also is interesting.A preferred example is chimpanzee adenovirus Pan9.

The zone of being differentiated is 7 surface rings, is also referred to as six adjacent body hypervariable regions (HVR).Six adjacent body mutabilities in the adenoviral serotype concentrate in these 7 rings (Crawford-Miksza andSchnurr.1996).Should understand the HVR that the invention is not restricted to as described in embodiment, use Ad48.This further confirms by the discriminating of HVR among Ad5, AdI1, Ad26, Ad34, Ad35, Ad48, Ad49 and the Ad50 of relative broad sense (seeing Table II) and definition (seeing Table IV) relatively more that limit, that minimize (minimalistic) more.Ad48 is the example of all other serotypes, these other serotypes also meet with the lower immunity that is pre-existing in and have benefited from also can be used for producing chimeric adenovirus by the known advantages (strong immunogenicity is easy to produce etc.) and the benefit of rare serotype of the subgroup C adenovirus of Ad5 representative.Obviously,, wherein preferably use another serotype main chain,, then advantageously strengthen with the carrier that does not meet with the NAb that produces at causing carrier as Ad35 if first/booster shot scheme is used in expectation.Therefore, the combination as AdI1, Ad24, Ad26, Ad35, Ad48, Ad49, Ad50 etc. and the HVR of another rare serotype (as above-mentioned any rare serotype) also is a part of the present invention.The non-limitative example of this carrier is Ad5HVR48, Ad5HVR35, Ad35HVR11, Ad35HVR48, Ad11HVR35 and Ad11HVR48, has at least 1, more preferably 6, metathetical HVR between 7 serotype most preferably.Therefore, preferred at least one HVR that adopts from a rare serotype inserts in the six adjacent bodies of main chain serotype.The inventor has illustrated the corresponding HVR that all 7 HVR of Ad5 is replaced into Ad48, produces survival and producible carrier, and described carrier meets with any immunity that is pre-existing in hardly in the mouse of blank Ad5 virus immunity.Yet, if only replace the no effect of first HVR (in viral genome as seen) from left side ITR to right side ITR.This does not mean that a displacement may not be enough.The inventor thinks that a zone has more immunogenicity than other zone, and what wait to study is which area contribution maximum in the zone of these 7 discriminatings, and which HVR is not necessarily needing to be replaced in background or the application-specific.Some individuality is compared with other individuality, can produce different immunne responses at different HVR.In addition, the carrier that contains chimeric six adjacent bodies only exists in the host in the active background of medium NAb can provide benefit.According to the example that provides, owing to give 10 continuously 2 times ¹⁰The blank Ad5 carrier of vp and the immunity that is pre-existing in that produces is very high.Yet all HVR are all replaced in the most preferably described hexon, and the killer opportunity that produces carrier is provided like this, and the NAb that described carrier is not pre-existing among the host detects.

In order to produce the adenovirus carrier of improvement, at first to Ad5 specificity neutralizing antibody (NAb) epitope mapping.Mapping shows the Ad5 seroprevalence and tires very high in the crowd (in the U.S. is about 50%, is 90% in part developing country).In addition, as disclosed herein, the significant Ad5 specificity of discovery feature NAb is almost just at hexon (seeing that also Sumida et al.2005 is described).On the contrary, in corresponding physical environment, significantly do not suppress the Ad5 vaccine immunogenicity at fibrinous Ad5 specific antibody, described environment promptly with the relevant environment of finding that in human body anti-Ad5NAb tires.Opposite with Ad5, find Ad35 and AdI1 seroprevalence and the crowd of the many different zones in the world of tiring in very low.

The present invention relates to new adenovirus carrier, it carries rare (perhaps rare at least neutral) Ad serotype such as the six adjacent bodies of AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50, and preferably from the knob of the fiber at least structural domain of the serotype of CAR acceptor interaction, as most of adenovirus hominis serotypes and ovine adenovirus from subgroup A, C, D, E and F.Therefore as the main chain carrier and carry himself hexon and find seldom that in the crowd the preferred serotype of NAb is AdI1 and Ad35 relatively with it.The preferred serotype that reaches at its fiber as the main chain carrier is Ad5.This area is fully recognized that the cell that plays a major role is dendritic cell in immunne response, because these cells can effectively be presented immunogenic peptide in its surface, thereby cause the strong immune response at this peptide.Preferred carrier is those carriers from adenovirus subgroup B because it infects the ability of dendritic cell very effectively in this area, perhaps carry with effective means identification and infect the fibrinous carrier of the adenovirus of dendritic cell, see that for example WO 02/24730 and WO 00/70071 are described.These carriers probably use CD46 as acceptor.Astoundingly, as shown in the present, the inventor finds, for the carrier that carries the known knob of fiber at least by the interactional adenovirus carrier of CAR (being Ad5), when this fiber knob was cloned in the top of the carrier that meets with the immunity that lower level is pre-existing in, it made immunne response be enhanced.The immunne response of considering hope is by dendritic cell, and this is very mysterious.

Be understood that described knob structural domain is the main structural domain that participates in identification adenovirus carrier bonded specific cell acceptor, those skilled in the art can be easy to itself and fibrinous rest part are made a distinction (by sequence to when about the general knowledge of acceptor interaction).The CAR that had before illustrated on Ad5 fiber knob and the cell surface interacts, and the effective virus of mediation is adhered to before virus enters, and this is promotion (Bergelson et al.1997 by penton base portion (penton base) and cell integrin and further; Bewley et al.1999; Roelvink et al.1998 and 1999; Wickham et al.1993), and Type B virus as AdI1 and Ad35 by CD46 interaction (Gagger et al.2003).The present invention relates to adenovirus carrier, wherein receptors bind structural domain (being limited by the knob structural domain) is exchanged at least.Therefore the latter's carrier still comprises the fibrous texture territory, the axle of the autonomous chain carrier of Tathagata and/or the part of afterbody.In fact wish to use the main chain carrier to the small part tail region to guarantee and the moderately stable interaction of other capsid protein, produce stable recombinant vectors.

Chimeric adenoviral vectors of the present invention with only based on Ad5 or Ad35 and the carrier that does not have a chimeric fiber compare more effective aspect vaccine and the gene therapy.The chimeric adenoviral that carries chimeric Ad5-Ad35 fiber known in the art.Disclosed the Ad5 carrier that carries chimeric fiber, wherein at least the knob structural domain (WO 00/31285, and WO 00/52186 derived from Ad35; WO02/24730; Shayakhmetov et al.2003; Rea et al.2001).Smith et al. (2003) has disclosed a kind of chimeric fiber, and wherein afterbody and axle are from Ad35, and the knob structural domain is from Ad5.All carriers in these reference of quoting are all based on Ad5, point out described carrier because the existence of Ad5 six adjacent bodies and still meet with the immunity that is pre-existing in the virus vector capsid.The data acknowledgement that Ophorst et al. (2004) provides this phenomenon.Recombinant replication-defective type Ad35 carrier and the method that produces it also are that known in the art (WO 00/70071; WO02/40665), and Ganesh et al. (2003) has disclosed the application based on the carrier of Ad35 that comprises fiber, and wherein the fiber knob of Ad35 is by the displacement of Ad5.Yet, described carrier carry a marker gene (green fluorescent protein, GFP) and obviously present lower transduction and render a service.Therefore, Ganesh etc. do not disclose or propose the vaccine by the knob metathetical carrier of Ad5 based on the knob at least of Ad35 wherein, have said nothing of the advantage relevant with its application.Ganesh etc. have also disclosed a kind of carrier based on the Ad35 with complete Ad5 fiber.Must be noted that this carrier comprises complete Ad5 fiber rather than chimeric fiber, therefore different with the carrier of the present invention's announcement.Ganesh etc. remind the exchange of part fiber to find lower transduction effectiveness.Carrier of the present invention has good transduction to be renderd a service, and chimeric fiber guarantees that stabilize anchor fixes in the residue capsid.Those skilled in the art do not have motivation to expect carrying out such inoculation, its objective is the known fiber knob that very effectively infects the serotype (as those serotypes of subgroup B, for example AdI1, Ad34 and Ad35) of dendritic cell is replaced into the main fiber knob that passes through the serotype of CAR cells infected.

Because the host mainly is owing to the NAb at hexon for the neutralization activity of adenovirus carrier, therefore adenovirus carrier also can be based on height neutral serotype, Ad5 for example, wherein six adjacent bodies are by the six adjacent bodies exchanges of rare neutral serotype such as AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.An example of this carrier is referred to as Ad5h35 (based on the carrier of Ad5, it comprises the six adjacent bodies that replace Ad5 from the hexon of Ad35).Those skilled in the art are based on information revealed of the present invention and can imagine and produce different may making up based on the available knowledge in this area.Obviously be difficult to exchange whole six adjacent bodies (Gall et al.1998; Youil et al.2002).Disclose a kind of mode new and practicality in the embodiment of the invention and made up chimeric hexon.The embodiment 8 of WO 00/03029 has disclosed the another kind of method that the hexon clone who uses other serotype comprises the carrier of chimeric capsid.

Recombinant adenoviral vector of the present invention, promptly based on those carriers (AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and the Ad50 that in the crowd, meet with the low active serotype that is pre-existing in usually, and some simian adenovirus), it carries the knob structural domain of the adenovirus that causes high immunne response usually, has represented in the adenovirus that causes high immunne response usually and owing to being carried in the world the detected hexon of immunity that is not pre-existing among the crowd at high proportion and has escaped improvement on the active adenovirus of the neutralization that the is pre-existing in basis.In other words reach the illustration in institute of the present invention, Ad35fib5 and Ad35k5 have all represented the improvement on Ad5 and Ad35 carrier basis.

Suppose that the Ad5 carrier has high-caliber anti-Ad5 immunity in the crowd, it is fully suppressed by anti-carrier immunity probably, is like this based on the experimental result of carrying out in mouse and monkey at least.The level of supposing anti-Ad35 immunity among the crowd is low, and then Ad35 and Ad35 related vector have substantial advantage than Ad5 carrier in this respect.In other words, Ad35, Ad35fib5 and Ad35k5 carrier are not suppressed by anti-carrier immunity basically.Yet, based on the vaccine of Ad35 carrier with compare based on the vaccine of Ad5 carrier in immunogenicity aspect the antibody of antigenicity inset lower.

Importantly, the inventor has disclosed first and has carried difference but the low neutralization carrier of (allos) at least knob structural domain of hyperimmunization originality carrier is more effective than the main chain carrier with its natural fiber aspect induce immune response.Therefore, this carrier representative surmounts the technology and the practice progress of present available adenovirus carrier.Those skilled in the art can utilize knowledge provided herein, clone other adenovirus carrier based on identical principle.For example, the invention still further relates to the reorganization simian adenovirus, it carries the knob structural domain of the fiber of immunogenicity adenovirus hominis.Except the reorganization simian adenovirus, other non-adenovirus hominis is also contained in the present invention as reorganization ox and hepatitis infectiosa canis virus.The invention still further relates to the adenovirus hominis of other low neutralizationization, it can be used as the main chain carrier, carries the structural domain of knob at least of other immunogenicity adenovirus.Many different combinations are arranged, but the hexon at least that is limited to adenovirus should not meet with the high-caliber immunity that is pre-existing in, knob structural domain identification CAR acceptor at least simultaneously, thus guarantee in using the host of described carrier, to produce high immunne response.The general knowledge of those skilled in the art by this area can be distinguished described feature, with the immunity of determining to be pre-existing in, determines the immunne response level that causes and obtains the strategy of reorganization of the present invention (chimeric) carrier.

The fibrinous function dependent interaction of powerful immunogenicity prompting Ad5 of Ad35k5 vaccine carrier.Ad5 fiber knob decision combines with high-affinity receptor CAR's, effectively is transported in cell at virion also to play an important role aspect the nucleus.Although accurately mechanism is also determined, the enhanced immunogenicity of Ad35k5 carrier reflects the known organism function of Ad5 fiber knob probably.As described herein, reorganization Ad35k5 carrier mainly presents the neutralization pattern of reorganization Ad35 carrier, and escapes the anti-Ad5 immunity of low/medium level effectively.These observed results are consistent with previous research, show that Ad5 specificity NAb is primarily aimed at the Ad5 hexon.Support the observations that also having of this model is such, promptly contain the fibrinous Ad5 carrier of Ad35 and can not avoid anti-Ad5 immunity (Ophorst et al.2004).Yet as described herein, high-caliber anti-Ad5 immunity in fact can reduce the immunogenicity of Ad35k5 carrier, has reflected probably than low liter but can know that detected NAb is at the Ad5 scleroproein.Notice that owing to anti-Ad5 immunity causes the immunogenic reduction of Ad35k5 be partly, tiring at extra high Ad5 specificity NAb just can detect down.

Another restriction of Ad35k5 carrier is its high relatively vp/pfu ratio, and it is than high about 10 times of the Ad35 carrier that observes.This observes the viral integrity of prompting and stability can jeopardize by comprising chimeric fiber albumen, although this still needs research and confirms.

As described herein, the immunogenic rhesus monkey of contrast Ad5, Ad35k5 and Ad35 carrier studies confirm that the result who obtains in mouse.This is correlated with, because mouse lacks the immunogenicity that therefore best Ad35 acceptor CD46 has also underestimated the Ad35 carrier.In rhesus monkey, the Ad5 carrier causes fast and the high carrier specificity NAb that tires that it prevents the homology booster immunization effectively just as expected.On the contrary, Ad35 compares with the Ad5 carrier with the Ad35k5 carrier and causes that all lower carrier specificity NAb tires, and it promotes the reinforcement that these are replied after using homologous vector once more.The strong immune response that supposition observes in the monkey of accepting the Ad35k5 carrier has reflected the fact of replying by force and can effectively be strengthened that these carriers are first probably.

The present invention confirms can make up the chimeric reorganization of capsid Ad carrier to make up the useful immunology and the serological property of different Ad serotypes.On behalf of a generation, produce chimeric Ad carrier be used to inoculate New Policy with two generation Ad carriers of the improvement of gene therapy.

According to a preferred embodiment of the invention, the present invention relates to a kind of duplicate deficit type recombinant adenovirus serotype 35 (Ad35) carrier, it comprises chimeric fiber albumen, this chimeric fiber albumen comprises from the structural domain of knob at least in conjunction with the adenoviral serotype of CAR, and wherein said recombinant vectors further comprises interested treatment nucleic acid.Treatment nucleic acid is meant that coding can be used for diagnosing, treating and/or preventing property treatment Mammals, preferred people's human cytokines metallic substance such as the nucleic acid of protein, peptide or polypeptide.The example of therapeutic protein is the protein that causes immunne response in tumor inoculation.Other example is the protein that can be used for treating genetic diseases, as is used for gene therapy those.Preferred therapeutic protein be derived from or based on or (directly) clone from the protein of bacterium, parasite or infection entity such as virus.Adenovirus carrier can highly be applicable to the inoculation purpose at for example following virus: human immunodeficiency virus (HIV), SIV, Ebola virus, rabies virus, hsv (HSV), hepatitis C virus (HCV) etc.The antigenic example of HIV deutero-that can be encoded in adenoviral nucleic acid is nef, gag, pol and env.The antigenic example of HSV is antigen 9D.In carrier of the present invention, also can use the parasitic therapeutic protein that causes malaria freely.The particularly preferred protein that can clone in these carriers is the protein from plasmodium falciparum (Plasmodium falciparum), as ring spore (CS) albumen and LSA-I.Other preferred protein can be from mycobacterium (Mycobacterium) bacterial strain, particularly cause phthisical those protein as mycobacterium tuberculosis (Mycobacterium tuberculosis).From the preferred antigens of this bacterium be 10.4,85A, 85B and 85C antigen, therefore it also can be used in the carrier of the present invention.Be not considered to therapeutic protein with the protein typical case who marks to carry out in vitro study, so GFP, luciferase or CAT are not regarded as curative.

Suitable promotor and poly-(A) sequence of can be used for antigen expressed are known in the art, include but not limited to CMV, Ad2 MLP, SV40 etc.Suitable transcription termination sequence can be for example derived from SV40 or BGH.Described antigenic encoding sequence can be the codon of optimizing for optimum expression in the preferred people of Mammals.Codon optimization method is known in the art.

The present invention relates to duplicate deficit type recombinant adenovirus, be selected from adenoviral serotype 11,24,26,34,35,48,49 and 50, described adenovirus comprises chimeric fiber albumen, this chimeric fiber albumen comprises the structural domain of knob at least in conjunction with the adenoviral serotype of CAR, and wherein said recombinant vectors further comprises the heterologous nucleic acids of interested therapeutic of coding or antigen protein.Preferred serotype is subgroup B serotype, and more preferably serotype 11,34 and 35, most preferably serotype 35 (Ad35).The present invention relates to these serotype, because they meet with the immunity that is pre-existing in of neutrality in the human serum sample who takes from whole world healthy individual of very low per-cent.As used herein, chimeric fiber albumen is meant the scleroproein that comprises from the part of at least two different adenoviral serotypes, and wherein the knob structural domain is from the adenovirus in conjunction with CAR.Preferred described fibrinous tail structure territory is the main chain carrier, like this by having increased the stability of carrier with the suitable interaction of carrier capsid.In a preferred embodiment, in conjunction with the knob structural domain of CAR from the adenoviral serotype of subgroup C, more preferably from the fiber of serotype A d5.

Interested therapeutic protein used herein relates to the protein that can be used in Mammals therapeutic treatment such as the gene therapy.Interested antigen protein used herein relates in host or host cell when expressing and causes the protein of interest matter of immunne response.This immunne response is essential for different inoculation backgrounds: an example is a tumor inoculation, wherein strengthened expressing removing of this proteinic tumour cell at the immunne response of interested antigen protein, and another preferably to use be in prophylactic treatment, as inoculate with prevention or significantly suppress pathogenic agent as the infection of virus, bacterium, yeast or parasite to the host.Therefore, preferred described interested antigen protein comprises from virus, bacterium, parasite or zymic protein.Recombinant adenovirus of the present invention also can be used for process in the infection that taken place of treatment and cause immunne response at interested antigen protein, therefore prevention duplicate, packing etc.In other words, described carrier also can be used for pre-anti-virus and is transmitted to next host from the host who has infected.

In one embodiment, recombinant adenovirus of the present invention is included in the heterologous nucleic acids under the allogeneic promoter control.

Of the present invention one preferred aspect in, described antigen protein comprises the protein from virus, wherein said virus is retrovirus, HSV or Ebola virus, if described antigen protein is a retrovirus, then preferred described virus is ape and monkey or human immune deficiency retrovirus, and wherein said heterologous nucleic acids preferably comprises and is selected from proteic one or more gene of following coding immunodeficiency virus: gag, pol, env and nef.

In another aspect of this invention, described interested antigen protein is from the parasite that causes malaria, wherein said protein preferably from plasmodium, more preferably the circumsporozoite protein of plasmodium falciparum or liver specific antigen (LSA-I, LSA-3) or its immunogenicity part.

The present invention relates to the new duplicate deficit type recombinant adenovirus as medicine, it comprises heterologous nucleic acids and chimeric fiber, and wherein said adenovirus is selected from adenoviral serotype 11,24,26,34,35,48,49 and 50.

The invention further relates to recombinant adenoviral vector of the present invention and be used for the application preventative or medicine that therapeutic treatment malaria infection, Ebola virus infection, HSV infection, HCV infection or HIV infect in production.

In an especially preferred embodiment, the present invention relates to a kind of chimeric duplicate deficit type recombinant adenovirus, it comprises from the knob structural domain of the fiber of Ad5 and from the hexon of the serotype different with Ad5, described knob structural domain has as aminoacid sequence shown in Fig. 2 B underscore, wherein further preferred described hexon from the adenoviral serotype of low neutralizationization.The serotype of low neutralizationization is to meet with active those serotypes of neutralization that are pre-existing in the NAb form in minority healthy individual sample.In a special embodiment, the present invention relates to a kind of chimeric replication defect type reorganization Ad35 carrier, it comprises Ad35 six adjacent bodies and chimeric fiber albumen, and described chimeric fiber albumen has aminoacid sequence shown in the SEQ ID NO:2.

Six adjacent body HVR exchanges

Because the Ad5 specificity NAb of advantage is primarily aimed at the Ad5 hexon, have reason to think therefore that containing suddenly change the surely new reorganization Ad5 carrier of exchange of target in six adjacent bodies can escape the Ad5 six adjacent body specificity NAb of advantage.This is not a new idea, has carried out some in the art and has attempted this to produce " stealth " adenovirus carrier.Yet, as described below, none success.

As if the amino acid mutability in the hexon of different serotypes more than 99% concentrates in 7 short relatively hypervariable regions (HVR), and described hypervariable region is differentiated on the surface that is positioned at six adjacent bodies exposures by Crawford-Miksza and Schnurr (1996).These authors have contrasted the hexon of 15 kinds of different adenovirus (AdI, Ad2, Ad5, Ad β, Ad8, Ad9, AdI2, AdI5, AdI6, Ad31, Ad40, Ad41, Ad48, BAV3 and MAV1).Described 7 HVR are in 250 variable residues of ring 1 and 2 (being also referred to as 11 and 12) by discriminating, and wherein HVR1 to HVR6 is in ring 1, and HVR7 is in ring 2.Crystal and colleague (see WO 98/40509; US 6,127, and 525; US 6,153, and 435) based on the discovery of Crawford-Miksza et al. (1996), whole rings 1 of main chain carrier (Ad5) and the ring 1 and 2 of 2 usefulness Ad2 are replaced (Gall et al.1998).Also produced and wherein only encircled 2 by the metathetical carrier.Produced the virus of survival.Ad2 and Ad5 all are subgroup C adenovirus, even after two rings are all replaced, still have cross neutralization, show at least that when described exchange is in identical subgroup this exchange does not produce by the ability at the identification of the neutralizing antibody of wild-type adenovirus hexon and reduces the carrier that maybe can not discern.Yet in order to attempt to obtain this carrier, they also attempt replacing the ring of Ad5, with its ring displacement with Ad7.These trials have been failed, and produce the virus of survival, and the exchange that shows a kind of subgroup and another kind of subgroup is impossible generation, are impossible based on Crawford-Miksza to the discriminating of HVR at least.Certainly,, can realize essential genetic modification, generation all plasmid/clays of reorganization (chimeric) virus of should encoding by common Protocols in Molecular Biology by the understanding of future to the viral genome structure.Yet, owing to unknown cause but probably owing to the key factor of six complicated adjacent body structures and the effect in capsid forms thereof, the virus (Rux et al.2003) that when six adjacent body coding regions are modified, does not obtain to survive.Gall et al. (1998) suggestion only exchanges outside ring and does not exchange six whole adjacent bodies.Mentioned that also other capsid protein such as penton can have significant neutralizing epitope to explain the failure of Ad5-Ad2 exchange.

The inventor also attempts to produce based on chimeric adenoviral Ad5, that carry chimeric hexon, its HVR that comprises Ad35 or Ad48 replaces Ad5HVR, such as Crawford-Miksza and Schnurr (1996) and Rux and Burnett (2000) guidance.These trials have been failed.This discovery with Crystal etc. is consistent, and Crystal can not produce any recombinant virus (seeing above-mentioned) that six adjacent body portions of different subgroups are wherein exchanged.

In order to address this problem, Rux et al. (2003) has disclosed new high resolving power crystallographic refinement, and it forms different with in the Ad2 that resolves previous discovery and the Ad5 six adjacent body structures by Ad2 and Ad5 six adjacent body structures.Produced the new qualification to the zone in the hexon like this, showing has 9 HVR rather than 7.Rux et al. (2003) also differentiated when the new carrier based on adenovirus of design should ruined hexon part.

The qualification that the inventor also attempts to provide based on Rux et al. (2003) produces chimeric adenoviral, and it comprises the chimeric hexon of the HVR with exchange.Yet, fail to produce the virus of survival once more.Obviously, based on the qualification of this area about six adjacent body HVR, can not produce have one or more between main chain carrier and another serotype, at least between different subgroup adenoviral serotypes by the recombinant adenovirus of metathetical HVR.

The inventor has differentiated 7 different HVR of 9 HVR that 7 HVR differentiating with Crawford-Miksza andSchnurr (1996) and Rux et al. (2003) differentiate now in adenovirus hominis.Broad sense of the present invention is limited to shown in the Table II, and more minimized being limited to shown in the Table IV.The independent displacement of these HVR produces the virus of survival.The inventor's maximum contribution is that this is anyly first can produce the chimeric adenoviral that comprises chimeric hexon per capita, wherein is not that whole rings is all exchanged, but the wherein unique HVR of exchange.This also is the recombinant adenovirus that has successfully obtained to comprise chimeric hexon first, and wherein six adjacent bodies comprise the part from the adenoviral serotype of two different subgroups.Again the theoretical basis that limits HVR is to use conservative amino acid as binding site.Yet do not get rid of by change N and/or C-terminal one or two or perhaps in addition three amino acid can produce the carrier of survival.Yet the qualification that this area before provided is not enough to provide the carrier of the survival with chimeric six adjacent bodies.Yet qualification provided herein needn't be followed in strictness, because change slightly also can provide good result's (seeing Table the contrast of II and Table IV).Preferably, the HVR sequence of discriminating (sequence shown in SEQ ID NO:17-79 and the 88-150) is the zone that is exchanged between serotype.

As described in embodiment, make up carrier based on Ad5, it contains one or more HVR by Ad35 (subgroup B) or Ad48 (subgroup D) exchange.Obviously, can exchange one or more HVR.NAb is at any HVR probably, and preferred great majority (if not all) replace the wild-type HVR of main chain carrier from the HVR of rare serotype.On the other hand, this extensive exchange can cause more being difficult to produce the carrier of survival.The present invention has disclosed all 7 HVR that differentiate in the main chain carrier (for example Ad5) all can the corresponding HVR displacement by 7 of rare serotype (for example Ad48).This also points out the HVR based on Table IV ^*The exchange that limits also produces the virus of survival.

Interval region between the HVR preferably keeps the zone of main chain serotype, and is proteinic correct folding to guarantee.Although also can use the subgroup C serotype such as the Ad2 of other common use and widespread use, preferred main chain is Ad5.When exchange during HVR, preferably at least 1, more preferably 2 in addition more preferably 3,4,5,6 and most preferably 7 HVR exchanged.

As disclosed, the Ad5HVR35 that produces compares under the condition that has anti-Ad5 immunity with reorganization Ad5 carrier with the Ad5HVR48 carrier and has more immunogenicity, because the HVR of the six adjacent bodies that the NAb that is primarily aimed at the hexon of Ad5 in the individuality that Ad5 infects no longer can be by Ad35 and Ad48 neutralizes.These characteristics have supported that to all HVR that take from all known rare serotypes all be correct, and described serotype can be selected from AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.

Shown in hereinafter, all 7 HVR that contain Ad48 replace Ad5HVR48 (1-7) virus of 7 HVR of Ad5 to produce not by the immunity interferential virus that is pre-existing in by previous injection Ad5 virus induction.This makes the technician can use the carrier based on Ad5 in numerous backgrounds, for example for the first time-strengthened scheme in, wherein need be identical with the acceptor identification maintenance of strengthening between the carrier at first carrier.The another kind of application is to use based on the carrier of Ad5 to inoculate or the therapeutic application in the individuality that meets with wild-type Ad5 infection before treatment.

Based on the discriminating of HVR as described herein, the numerous combinations between main chain carrier and other adenoviral serotype are feasible now.These knowledge can be extrapolated to six adjacent bodies of all known person and non-adenovirus hominis.No matter when need first-strengthened scheme (be not only wherein need based on the background of Ad5 carrier), knowledge provided herein all can be used for making up the carrier at the immunity that is pre-existing in of six adjacent body HVR that has that the stealth ability escapes promptly that the virus formerly used exists.Should understand the chimeric HVR carrier of the present invention's announcement can further modify by means known in the art.For example, can change fiber with produce selectively targeted ability (for example from the fiber knob of subgroup B virus place based on can the target smooth muscle cell on the carrier of subgroup C, former generation inoblast, dendritic cell etc.).The example of this carrier is based on the carrier of Ad5, and it comprises from the HVR of for example Ad48 and from the knob of fiber at least of for example Ad35 (major decision of acceptor identification bunch).This carrier all in the present invention about comprising the scope that its HVR is the adenovirus carrier of chimeric six adjacent bodies.

Those skilled in the art can make up and produce the recombinant replication-defective adenoviral of survival now by means of knowledge provided herein, its one or more HVR is by the HVR displacement of other adenovirus.In addition, HVR sequence provided by the invention makes the technician can remove those sequences now and uses these positions in the six adjacent bodies to import other material, as the target part with the adenovirus target in interested cell (reaching in vivo), radioactivity binding site external with at external tracking adenovirus and some B cell epitope (as described in Worgall et al.2005).

The present invention relates to based on the recombinant replication-defective adenoviral of the serotype of first kind of subgroup batch, described adenovirus comprises chimeric hexon, wherein said hexon comprises from the sequence of the serotype of described first kind of subgroup and from least one hypervariable region (HVR) sequence of second kind of subgroup serotype, and wherein said first kind and second kind of subgroup are inequality.Preferred described first kind of subgroup is subgroup A, C, D, E or F.The also preferably wherein said second kind of subgroup of the present invention be subgroup B or D batch.The serotype that more preferably wherein said first kind of subgroup is subgroup C and described subgroup C be the serotype of Ad5 and wherein said subgroup B or D be selected from AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50 batch.

In a preferred embodiment of the invention, described chimeric hexon comprises preceding 6 HVR sequences (HVR1-HVR6) or all 7 HVR sequences (HVR1-HVR7) of described second kind of subgroup serotype.

In another preferred embodiment, described hexon keeps the aminoacid sequence of main chain virus between the HVR sequence.Therefore, one or more HVR sequence of this paper elaboration can be lacked, is suddenlyd change, replaced by joint displacement or by the HVR sequence from another serotype, and the sequence that particularly preferably is reservation connection HVR does not add change.This makes the technician that the stable virus owing to the six adjacent body-backbone structures that provide by non-HVR sequence can be provided.Sequence between the therefore preferred HVR sequence is from the serotype of described first kind of subgroup.

In highly preferred embodiment, the invention provides adenovirus batch, wherein first kind of subgroup is subgroup C, and at least one HVR sequence of wherein said second kind of subgroup serotype is selected from SEQ ID NO:24-79 and SEQ ID NO:95-150.In preferred embodiment kind, described HVR1 sequence is selected from SEQ ID NO:17,24,31,38,45,52,59,66 and 73, wherein the HVR2 sequence is selected from SEQ ID NO:18,25,32,39,46,53,60,67 and 74, wherein the HVR3 sequence is selected from SEQ ID N0:19,26,33,40,47,54,61,68 and 75, wherein the HVR4 sequence is selected from SEQ ID NO:20,27,34,41,48,55,62,69 and 76, wherein the HVR5 sequence is selected from SEQ ID NO:21,28,35,42,49,56,63,70 and 77, wherein the HVR6 sequence is selected from SEQ ID NO:22,29,36,43,50,57,64,71 and 78, and wherein the HVR7 sequence is selected from SEQ IDNO:23,30,37,44,51,58,65,72 and 79.In other embodiment preferred, HVR1 sequence (HVR1 ^*) be selected from SEQ ID NO:88,95,102,109,116,123,130,137 and 144, wherein HVR2 sequence (HVR2 ^*) be selected from SEQ ID NO:89,96,103,110,117,124,131,138 and 145, wherein HVR3 sequence (HVR3 ^*) be selected from SEQ ID NO:90,97,104,111,118,125,132,139 and 146, wherein HVR4 sequence (HVR4 ^*) be selected from SEQ ID NO:91,98,105,112,119,126,133,140 and 147, wherein HVR5 sequence (HVR5 ^*) be selected from SEQ ID NO:92,99,106,113,120,127,134,141 and 148, wherein HVR6 sequence (HVR6 ^*) be selected from SEQ ID NO:93,100,107,114,121,128,135,142 and 149, reach wherein HVR7 sequence (HVR7 ^*) be selected from SEQ ID NO:94,101,108,115,122,129,136,143 and 150.More preferably, the HVR1 sequence in the disappearance Table II or those HVR1 sequences shown in the Table IV in the main chain carrier.The reason in disappearance HVR1 zone is the crystalline structure according to the adenovirus hexon, and the flanking sequence of HVR1 is very close, represents that these flanking sequences can directly connect and do not lose structure, except disappearance.In another preferred embodiment, those HVR2 sequences shown in HVR2 sequence shown in the Table II or the Table IV can be replaced by short circuit head, and more preferably the joint displacement of being made up of two amino acid is most preferably replaced by the QG joint.Equally, the HVR2 flanking sequence is also very close in crystalline structure.Yet, be far apart owing to seeming that it is compared slightly with HVR1, therefore preferably include little bridge joint (bridging linker), preferably 1,2,3 or 4 amino acid whose joint is more preferably 2 amino acid whose joints.Removing HVR from six adjacent bodies can be the ideal solution that prevents at the NAb attack of the immunogenicity determinant of being presented by HVR.Yet, consider crystalline structure, disappearance HVR3-7 from hexon and do not destroy its one-piece construction and therefore its function of performance and act on seemingly infeasible in capsid forms.

Although the six adjacent bodies of Ad24 also are preferred for its HVR, the sequence of inventor's hexon of also unknown this special " rare " serotype when filing an application.Yet according to the teachings provided herein, the technician can determine the HVR in any adenovirus hexon now, comprises preferred serotype A d24.The application of arbitrary HVR of Ad24 also is a part of the present invention.

The invention still further relates to the application of isolating nucleic acid in making up chimeric hexon of at least one HVR sequence of six adjacent bodies of the adenovirus hominis serotype of coding subgroup B, described six adjacent bodies further comprise the sequence of the adenovirus of subgroup A, C, D, E or F.In addition, the application of the isolating nucleic acid of at least one HVR sequence of six adjacent bodies of the adenovirus hominis serotype of the subgroup D that the present invention relates to encode in making up chimeric hexon, described six adjacent bodies further comprise the sequence of the adenovirus of subgroup A, B, C, E or F.Preferred described at least one HVR sequence is selected from SEQ ID NO:24-79 and 95-150.

Table III has been summarized different embodiments of the present invention.This has expressed the chimeric replication-defective adenoviral that the present invention relates to based on the main chain adenovirus, this main chain adenovirus comprises penton and the hexon of serotype X, wherein the HVR of the n in the hexon is the HVR of serotype Y, further comprise fiber, it comprises the afterbody of serotype F and the knob of axle and serotype K, wherein:

-X is the adenoviral serotype that is selected from adenovirus hominis 1 to 51, ape and monkey serotype, dog serum type, sheep serum type, porcine blood serum type and bovine serum type;

-Y is the adenoviral serotype that is selected from adenovirus hominis 11,24,26,34,35,48,49 and 50, and wherein X and Y can be identical or different, and wherein n is the HVR of 0 to 7 any number, and condition is if X Ф is Y, then n=1 to 7;

-F is the adenoviral serotype that is selected from adenovirus hominis 1 to 51, ape and monkey serotype, dog serum type, sheep serum type, porcine blood serum type and bovine serum type, and wherein F, X and Y can be identical or different; And

-K mainly uses the adenoviral serotype of CAR as cell receptor, and wherein K, F, X and Y can be identical or different.Preferably with the fibrinous afterbody of penton protein-interacting be not to be serotype X in the situation of X at least partially in F.

Should understand the main chain adenovirus carrier can be any adenovirus (reorganization produces, and it does not duplicate in normal non-packing cell thus) that can be applicable to human body.Six interior any known person adenovirus (serotype 1 to 51) and known simian adenovirus, hepatitis infectiosa canis virus, cow adenovirus, ovine adenovirus and porcine adenoviruses of subgroup A, B, C, D, E and F all can be used as the main chain carrier, as long as it is reaching as long as it can be applicable to human body therapy of replication defective.Described main chain carrier is by the disappearance funtion part at least one E1 district but replication defective preferably lacks whole functional E1 zones.Although described main chain carrier typical case comprises zone in other early stage and late period, but the technician recognizes the possibility of supplying some essential adenovirus protein by alternate manner, for example by advancing nucleic acid essential in its genome and supply with helper virus or by packing cell being transformed make thus it to comprise stable integration.Typically, virus of the present invention comprises the adenoviral gene group with E1 disappearance, and preferred E3 also lacks, to provide the space interested heterologous nucleic acids is cloned in E1 district or E3 district or the two.Remaining areas such as E2, E4, ITR ' s and the existence usually of zone in late period are although these zones can be supplied separately at production period in packing cell.Described carrier preferably comprises E4orf6 zone (perhaps from another serotype), and the E1B-55K albumen that exists in itself and the package cell line is compatible, discloses as WO03/104467.Working as described recombinant vectors like this has and known package system such as cell PER.

Or during the inconsistent original E4orf6 of the E1B-55K of 293 Ad5 that exist in cell zone, can in described cell, produce.

Described main chain serotype is above being represented with X.In addition, described main chain virus can through engineering approaches, and it contains the HVR (Y represents with serotype) of one or more adenoviral serotype that is selected from people AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50 thus.Obviously, during one of Y serotype, HVR can not carry out through engineering approaches when X, because these serotypes are considered to " rare " serotype, and only meets with the lower immunity that is pre-existing in the most of individualities of crowd.Therefore, when X was one of Y serotype, the number of the HVR of exchange (number of being represented by n) can be 0, but also can be 1 to 7; And when X was not one of Y serotype, then at least one HVR was from Y serotype, and wherein preferred gradually a plurality of HVR are from Y serotype, and most preferably all seven HVR all are exchanged for the respective regions of Y serotype.

Main chain virus of the present invention comprises and the viral penton albumen of homologous serotype mutually of main chain.Described main chain virus is chimeric, be meant six adjacent bodies be chimeric (one or more HVR and exchange are arranged or replace) and/or scleroproein be chimeric and/or scleroproein from the different serotype of main chain virus.The possibility that has X=Y=F=K, the Ad48 carrier of for example recombinating is because Ad48 is in conjunction with CAR.Yet this carrier is not chimeric in above-mentioned implication when n=0.Non-chimeric reorganization AdI1, Ad34, Ad35 and Ad50 do not meet above-mentioned qualification, because fiber knob should be always from preferential serotype in conjunction with CAR, these B subgroup viruses then are not.

Fiber comprises the afterbody from serotype F from the serotype of representing with F, comprises from the axle of serotype F and comprises knob from the serotype of representing with K.Obviously, when K was the serotype identical with F, described scleroproein was not chimeric.K is preferential and the serotype of CAR acceptor interaction, and K can be the serotype identical with X or different with it.Obviously, some human serum type interacts as and CAR preferential from those serotypes of subgroup B, but preferably interacts with another kind of cell receptor such as CD46.Therefore, when K was identical with X, then X was not one of subgroup B adenovirus.Equally, when K is the serotype different with F (and therefore described fiber is chimeric), with at least a portion of the interactional afterbody of penton in the capsid be serotype X, cause correct and stable capsid formation like this.Because most of capsid protein encoding genes of known adenovirus can obtain in the art, so those skilled in the art can differentiate each afterbody, axle and knob zone known and adenovirus that will be revealed.

The non-limitative example of chimeric replication-defective adenoviral of the present invention is preferably: Ad5HVR11 (1-7), Ad5HVR24 (1-7), Ad5HVR26 (1-7), Ad5HVR34 (1-7), Ad5HVR35 (1-7), Ad5HVR48 (1-7), Ad5HVR49 (1-7), Ad5HVR50 (1-7), Ad5HVRPan9 (1-7), Ad5HVR11 (1-6), Ad5HVR24 (1-6), Ad5HVR26 (1-6), Ad5HVR34 (1-6), Ad5HVR35 (1-6), Ad5HVR48 (1-6), Ad5HVR49 (1-6), Ad5HVR50 (1-6), Ad5HVRPan9 (1-6), Ad5HVR11 (1), Ad5HVR24 (1), Ad5HVR26 (1), Ad5HVR34 (1), Ad5HVR35 (1), Ad5HVR48 (1), Ad5HVR49 (1), Ad5HVR50 (1), Ad5HVRPan9 (1), Ad11k5, Ad24k5, Ad26k5, Ad34k5, Ad35k5, Ad48k5, Ad49k5, Ad50k5, Pan9k5, Ad11f5, Ad24f5, Ad26f5, Ad34f5, Ad35f5, Ad48f5, Ad49f5, Ad50f5, Pan9f5, Ad11k26, Ad34k26, Ad35k26, Ad50k26, Pan9k26, Ad11f26, Ad34f26, Ad35f26, Ad50f26, Pan9f26, Ad11k49, Ad34k49, Ad35k49, Ad50k49, Pan9k49, Ad11f49, Ad34f49, Ad35f49, Ad50f49, Pan9f49, Ad2HVR11 (1-7), Ad2HVR24 (1-7), Ad2HVR26 (1-7), Ad2HVR34 (1-7), Ad2HVR35 (1-7), Ad2HVR48 (1-7), Ad2HVR49 (1-7), Ad2HVR50 (1-7), Ad2HVRPan9 (1-7), Ad2HVR11 (1-6), Ad2HVR24 (1-6), Ad2HVR26 (1-6), Ad2HVR34 (1-6), Ad2HVR35 (1-6), Ad2HVR48 (1-6), Ad2HVR49 (1-6), Ad2HVR50 (1-6), Ad2HVRPan9 (1-6), Ad2HVR11 (1), Ad2HVR24 (1), Ad2HVR26 (1), Ad2HVR34 (1), Ad2HVR35 (1), Ad2HVR48 (1), Ad2HVR49 (1), Ad2HVR50 (1), Ad2HVRPan9 (1), Ad11k2, Ad24k2, Ad26k2, Ad34k2, Ad35k2, Ad48k2, Ad49k2, Ad50k2, Pan9k2, Ad11f2, Ad24f2, Ad26f2, Ad34f2, Ad35f2, Ad48f2, Ad49f2, Ad50f2 and Pan9f2.

Embodiment

International seroprevalence and the NAb of embodiment 1:Ad5, Ad35 and AdI1 tire

Ad specificity neutralizing antibody (NAb) is replied by evaluating as the described viral neutralization analysis based on luciferase of Sprangers et al. (2003).In brief, with the A549 human lung carcinoma cell with 1 * 10 ⁴The density of individual cells/well is plated in the 96 hole flat boards, and the Ad-luciferase reporter gene construct of the not reproducible that lacks with E1 in the serum of 2 times of serial dilutions of 200 μ l reaction volumes infects, and infection multiplicity (moi) is 500.After insulation 24 hours, uciferase activity uses Steady-Glo Luciferase Reagent system (Promega) to measure in the cell.During 90% neutralization is tired and is meant and the maximum serum dilution of 90% uciferase activity.

Except the research that WO 00/70071 discloses, experimentize and in developing world, the seroprevalence and the NAb of Ad5 and other Ad serotype are tired with evaluation.Use the described viral neutralization analysis of Sprangers et al. (2003), use the serum sample of the health adult that derives from the U.S. (N=19), Haiti (N=67), Botswana (N=57), Zambia (N=29) and South Africa (N=59) based on luciferase.Shown in Figure 1A, be 50% at U.S. Ad5 seroprevalence, have low relatively median and tire.On the contrary, be 82% at Haiti Ad5 seroprevalence, be 93% in Botswana, be 93% in Zambia, be 88% in South Africa.In addition, median Ad5-specificity NAb potency ratio is tired high more than 10 times (Figure 1B) at the median that the U.S. finds in these samples.These data have been expanded previous discovery (Kostense et al.2004; Vogels et al.2003) and confirm that Ad5-specificity NAb almost is that general and high tiring exists at developing world.On the contrary, AdI1 and Ad35 seroprevalence and tire very low in these colonies.

The immunodominance target position of embodiment 2:Ad5 specificity neutralizing antibody

Utilize sample in the previous embodiment further to determine the dominant antigen target position of Ad5-specificity NAb among the U.S. and Sub-Saharan Africa (the sub-Saharan Africa).Supposing does not have detectable NAb cross reactivity between Ad5 and the Ad35, use the chimeric Ad5/Ad35 virus of capsid be expressed in the viral neutralization analysis as the luciferase of target position.In having the complete virion situation of wild-type growth kinetics, these carriers by Ad5 and Ad35 six adjacent bodies, penton, and fibrinous various combination form (Havenga et al.2002; Reaet al.2001).The chimeric vector that uses in this research comprises Ad5f35 (Ad5 that contains Ad35 fiber knob, axle and the chimeric afterbody of Ad35-Ad5), Ad5f35p35 (Ad5 that contains Ad35 fiber and axle, the chimeric afterbody of Ad35-Ad5 and penton) and Ad35f5 (Ad35 that contains Ad5 fiber knob, axle and the chimeric afterbody of Ad5-Ad35).

Shown in Fig. 1 C, observe similar NAb at Ad5, Ad5f35 and Ad5f35p35 and tire, all based on Ad5.Because the capsid of Ad5f35p35 carrier contains Ad5 six adjacent bodies rather than Ad35 deutero-fiber and Ad35 deutero-penton, so the most Ad5-specificity of these Notes of Key Datas NAb activity is at Ad5 six adjacent bodies.Also be measured to lower but obviously can detectedly tire at reorganization Ad35f5 (being also referred to as Ad35fib5), show that Ad5 fiber-specific NAb is than tire low 5 to 10 times of Ad5 six adjacent body specificity NAb in these samples at this.Use these viruses not detect Ad5 penton specificity NAb, but at the similar NAb of Ad5f35 and Ad5f35p35 tire prompting penton specificity NAb in this neutralization to a lot of less relatively effects.

The generation of embodiment 3:Ad35f5 and Ad35k5

The vaccine based on Ad35 of reorganization is immunogenic in the situation that has anti-Ad5 immunity.Yet, reorganization Ad35 vaccine in the situation of not anti-Ad5 immunity inherently than the immunogenicity low (Barouch et al.2004) of reorganization Ad5 vaccine.This problem overcomes by making up the chimeric reorganization of capsid Ad35 carrier now, in the described carrier at least Ad35 fiber knob by Ad5 fiber knob displacement (Ad35k5 is meant that knob replaces, and Ad35f5 is meant most of fiber replacements).

Reorganization Ad35k5 carrier is by producing with chimeric fiber protein coding gene displacement Ad35 scleroproein encoding gene, and described chimeric fiber protein coding gene is made up of Ad35 fiber afterbody that is connected with Ad5 fiber knob (the 133rd to 314 amino acids) and axle (the 1st to 132 amino acids).Ad5 fiber-specific antibody does not weaken reorganization Ad5 vaccine immunogenicity.Ad35 fiber knob and CAR do not interact, because it is subgroup B adenovirus (Roelvink et al.1998), still generation utilizes CD46 as its acceptor (Gagger et al.2003).Different scleroproeins guides these viruses to enter transport pathway (intracellular trafficking pathway) in the different cells.Previous research has illustrated Ad5 fiber knob and has promoted virus to escape from from early stage endosome fast to enter kytoplasm, cause viral genome effectively transposition to nucleus, and make virion remain in the endosome in late period from the fiber knob of the subfamily B adenovirus that comprises AdI1 and Ad35, and cell surface (Shayakhmetov et al.2003) is got back in most of fraction recirculation.

The recombinant nucleic acid of following clones coding Ad35k5 carrier.The zone of coding Ad5 fiber knob is synthesized by PCR and is advanced pBR.Ad35.PacI-rITR.dE3 plasmid (the pBr.Ad35.PR Δ E3 the described in=WO 2004/001032) to replace the respective regions of coding Ad35 fiber knob as the BsiWI-Nhel fragment cloning.Then mutant pBR.Ad35k5.PacI-rITR.dE3 plasmid and pWE.Ad35.pIX-EcoRV (WO2004/001032) clay and the joint plasmid pAdApt35-SIVGag cotransfection that comprises SIVmac239 Gag gene are advanced in the PER.C6/55K cell (to see WO 00/70071 and WO 02/40665) that dual homologous recombination produces the Ad35k5-SIVGag carrier of reorganization.With this carrier through plaque purification, order-checking, amplification and by the CsCl gradient centrifugation with after general purification process known to the skilled is learned purifying.The nucleotides sequence of chimeric fiber is shown in Fig. 2 A (SEQ ID NO:1), and its aminoacid sequence is shown in Fig. 2 B (SEQ ID NO:2).

Ad35f5 carrier typical case as WO 00/70071 at Ad35fibl6 and WO 00/03029 at generation as described in the Ad5fibXX; The PCR product of part afterbody of the Ad5 fiber that is connected with Ad5 fiber knob with the Ad5 fibre axis of wherein will encoding places the remainder of Ad35 fiber afterbody.Use the main chain carrier of E3 disappearance and use BsiWI and NheI to carry out clone's program as restriction/cloning site.For carrier, see for details in WO 03/104467 and WO2004/001032 described.

Specifically, carry out following program to make up pBr/Ad35.pacI-rITRfib5: plasmid pBr/Ad35.PacI-rITRNotI (pBr/Ad35.PRn the described in=WO 2004/001032) is carried out PCR, use the primer DF35-1:5 '-CAC TCACCA CCT CCA ATT CC-3 ' (SEQ ID NO:6) and the DF35-2:5 '-CGG GATCCC GTA CGG GTA GAC AGG GTT GAA GG-3 ' (SEQ ID NO:7) that contain BamHI and BsiWI site.This PCR produces the dna fragmentation of about 650bp, from fiber 35 tail region, imports BsiWI site and BamHI site by it.Then, plasmid pBr/Ad35.PRn is carried out PCR, use the primer DF35-3:5 '-CGG GATCCG CTA GCT GAA ATA AAG TTT AAG TGT TTT TAT TTA AAATCA C-3 ' (SEQ ID NO:8) and the primer DF35-4:5 '-CCA GTT GCA TTGCTT GGT TGG-3 ' (SEQ ID NO:9) that contain BamHI and NheI site.This PCR produces the dna fragmentation of about 1400bp, begins to the E4 zone after the terminator codon of fiber 35.PBr/Ad35.PRn is digested with MIuI and NdeI, remove the dna fragmentation (therefore removing most of fibers 35 zones) of 2850bp from the Ad35 main chain.With the PCR fragment of DF35-1+DF35-2 with MIuI and BamHI digestion and with the PCR fragment of DF35-3+DF35-4 with BamHI and NdeI digestion.These two PCR fragments are all connected the pBr/Ad35PRn plasmid of into cutting, produce the pBr/Ad35PRndFIB construct.Then, use new BsiWI that imports and NheI site Ad5 fiber sequence to be imported in the Ad35 main chain, use primer 35F5-5-F:5 '-CGG GAA CGT ACG ACA CGGAAA CCG GTC CTC C-3 ' (SEQ ID NO:10) and 35F5-R:5 '-CGG CTAGCT AGC TTA TTC TTG GGC AAT GTA TGA AA-3 ' (SEQ ID NO:11) to produce described PCR product by producing the PCR product as template with Ad5DNA.Afterwards, as described at the pBr/Ad35PRNdE3 construct, lack the E3 zone.

Those skilled in the art can produce these clones, insert fragment, produce the PCR product and lack some fragment and produce at last virus by using conventional molecular cloning and adenovirus generation knowledge in package cell line.In addition, acquisition can be in vivo with the production and the purification process of the adenovirus of external application batch also be known in the art.

All carriers that the present invention discusses all are by using general CsCl purification process to obtain, finding its in the PER.C6/55K package cell line stable go down to posterity (data not shown goes out) more than at least 5 generations.The speed of growth of Ad35k5 carrier and output and parental generation Ad35 carrier similar (data not shown goes out).Yet Ad35k5 carrier (100-1000) is compared with Ad35 carrier (10-100), high about 10 times of the ratio of virion (vp) and plaque forming unit (pfu).

Also study carrier and whether still can discern its acceptor (the Ad5 fiber is in conjunction with CAR, and the Ad35 fiber is in conjunction with CD46) separately by fiber knob natural by it or that replace.These specificitys interactions (data not shown goes out) have been determined with chimeric vector and primary based on the carrier of Ad5 and Ad35.

According to identical strategy, produce virus based on chimeric replication defect type Ad35, it comprises the knob of Ad26 or Ad49.These viruses are known as Ad35k26 and Ad35k49, and used rare serotype A d26 and Ad49 fiber knob the CAR binding ability and also be the hexon that exists in the main chain carrier capsid of rare serotype, at this by the Ad35 illustration.Represent the nucleotide sequence and the aminoacid sequence of Ad35k26 and Ad35k49 chimeric fiber to be shown in Fig. 2 E to 2H respectively.

Contrast Ad35k5, Ad35k26 and Ad35k49 virus are in the external ability that transgene expression is provided.All carriers all carry the SIVGag transgenosis.With 7.5 * 10 ⁴Individual A549 cell infected 2 hours with the main seed culture of viruses virus (master virus seed stock viruses) (virus of unknown concentration) in the 2ml volume.Washed cell and cultivating 48 hours then.Use the anti-p27 monoclonal antibody of 2F12 to pass through fluidic cell surveying assay Gag dyeing subsequently by dyeing in the born of the same parents.Therefore the restriction of this experiment is the given number that does not use virion, can not infer clearly that Ad35k49 compares with other carrier whether to be grown to higher tire or whether it has higher specific activity.In any case, this carrier duplicating/growing or expression level aspect as if having some advantage than other carrier.The Three Represents that Figure 16 illustrates Ad35k49 batch is compared with the representative batch of Ad35k5 and Ad35k26 expression level in the significantly higher born of the same parents is shown.The adenovirus carrier Ad35k49 of reorganization is the preferred embodiment of the invention.

In order to produce adenovirus carrier well in packing cell, this area is recognized usually should be with compatible by the regional E4orf6 albumen that produces of the E4 of virus vector by its stable gene being integrated into the E1B-55K albumen that obtains in the genome of packing cell.Previous find the E4orf6 albumen of Ad35 with usually at the known package cell as 293 cells and PER.

Obtainable E1B-55K albumen is incompatible in the cell (sees WO 03/104467; WO 2004/001032; WO 2004/018627; WO 95/34671).Use the new clone of having integrated in the genome from the E1B-55K of Ad35 in order to avoid, and therefore can be so that those production platforms of determining that provide as the PER.C6 cell can be provided the technician, produce the E4orf6 zone of Ad35 main chain wherein and use other construct from the E4orf6 regional replacement of Ad5, consistent with WO03/104467 content described in detail.The displacement in E4orf6 zone is applied in all carriers based on Ad35, but is not further used in immunogenicity research as herein described and the target research.

Embodiment 4: the immunogenicity contrast of reorganization Ad35f5 and Ad35k5 and Ad5 and Ad35

The immunogenicity of carrier is carried the carrier of chimeric fiber and Ad5-SIVGag and Ad35-SIVGag by contrast and is evaluated.

With the mouse (N=5 only/group) that never carried out experiment through intramuscular with 10 ¹⁰VpAd5-SIVGag, Ad35-SIVGag, Ad35.BSU.SIVGag and Ad35fib5.BSU.SIVGag immunity.Term BSU is meant in adenoviral gene group nucleic acid and uses endogenous pIX promotor, and it guarantees in the correct expression based on the recombinant virus production period pIX gene of Ad35.See that about the detailed description of BSU construct WO 2004/001032 is described.With control mice (N=3 only/group) with blank Ad5 and the immunity of Ad35 carrier.After 28 days, blood sampling and use SIV gag ELIspot as mentioned below analyze and determine immunne response.

Fig. 3 illustrates these result of experiment, shows that all carriers are compared with blank carrier all can induce the proteinic correct t cell response about SIV gag, shows that described insertion body provides immunogenic protein behind the injection mouse.

Another group was never carried out the mouse (N=8/group) of experiment with 10 ⁹Vp or 10 ⁸The Ad5-Gag of vp, Ad35-Gag and Ad35k5-Gag carrier (the SIV gag albumen of all encoding) immunity.The CD8 that vaccine causes ⁺The T lymphocyte responses passes through D ^b/ AL11 tetramer binding analysis makes evaluation with the following method: preparation is at dominance SIVmac239 Gag AL11 epitope peptide (AAVKNWMTQTL; SEQ ID NO:3; Barouch et al.2004) the tetrameric H-2D of folded around ^dMixture, and the P18-specific C D8 of the mouse that is used to dye ⁺The T lymphocyte uses means known in the art to carry out.Mouse blood is collected among the RPMI 1640 that contains the 40U/ml heparin.After splitting erythrocyte, utilize the D of the PE-mark of 0.1 μ g ^dAnti--CD8alpha rnAb (the Ly-2 of/P18 tetramer associating APC-mark; Caltag, Burlingame, CA, USA) dyeing P18-specific C D8 ⁺The T lymphocyte.This cell washed in containing the PBS of 2%FBS and containing among the PBS of 0.5ml of 1.5% Paraformaldehyde 96 fixing.Go up by double-colored fluidic cell surveying analytic sample at FACS Calibur (Becton Dickinson).Detect (Gated) CD8 of gate ⁺The lymphocytic D of T ^dThe tetrameric dyeing of/P18.Never carried out the CD8 of experiment ⁺The T lymphocyte is as negative control, presents＜0.1% tetramer dyeing.

As shown in Figure 4, all 3 kinds of vaccines are 10 ⁹The vp high dosage all is similar immunogenic (A).Importantly, 10 ⁸Vp is than low dosage (B), those immunne responses that the immunne response ratio reorganization Ad35-Gag that is caused by reorganization Ad35k5-Gag causes are obviously stronger, and (the average tetramer is replied p＜0.01 in the 14th day mouse organized, use the gauged variance analysis of band Bonferroni to carry out multiple comparisons), the order of magnitude and Ad5-Gag cause those reply almost similar (p=ns).This and previous observed result (Barouch et al.2004; Lemckert et al.2005) unanimity.These data show through engineering approaches Ad35 carrier, and it carries the knob structural domain of subgroup C adenovirus (for example Ad5) thus, cause it about inserting the immunogenicity of antigens improvement in the carrier.

These experiments are carried out under the situation of not anti-Ad5 immunity, Ad5 is shown produces similar immunne response with Ad35k5, and these are significantly higher than the Ad35 carrier.Be important to note that the Ad35k5 carrier carries Ad35 six adjacent bodies, it produces in having at the active individuality of neutralization of the hexon of Ad5 increases higher effectiveness aspect the immunne response.Therefore, Ad35k5 carrier ratio in the situation that has anti-Ad5 immunity is obviously more effective based on the carrier of Ad5-and Ad35.Ad5 presents lower immunogenicity owing to its natural hexon under these conditions, and Ad35 presents lower immunogenicity usually owing to its natural fiber knob structural domain.

Can not get rid of fibrinous neutralizing antibody can produce based on infection.Yet, believe that this under field conditions (factors) neutralization is active minimum, most of immunne response of host is at hexon.Be important to note that wherein contrasting recombinant vectors is to carry out under the condition of simulating nature (people) situation with the experiment with the immunity of parental generation carrier in advance, wherein wild-type virus infects natural host one or twice, the neutralizing antibody of inducing natural occurred level.In the mouse in the background of laboratory, use the immunne response that can produce extreme by multiple injection and high tiring.The experiment of simulating nature situation shows that the adenovirus carrier whether fibrinous neutralizing antibody is used for neutralization really is important.They have also disclosed the importance in the axle zone that has fiber, and this part is useful for the active also typical case of neutralization.

We infer the knob at least comprise the adenovirus (for example Ad2, Ad5 etc.) by CAR identification and host cells infected and further comprise from the carrier of the main immunogenic determinant (being hexon) of the capsid of neutral serotype (for example AdI1, Ad24, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50) at least is splendid inoculation carrier, inserts body immunne response, reproducible production, effective unknown up to now advantage such as transduction and good stability because they have made up the immunity that low carrier is pre-existing in the crowd, high antigenicity.

Embodiment 5:Ad5, Ad35k5 and the Ad35 immunogenicity in mouse with the immunity that is pre-existing in

Next, estimate of the immunogenic influence of anti-Ad5 immunity to these carriers.With C57/BL6 mouse group (N=4 only/group) before inoculation 4 weeks with 10 ¹⁰The pre-immunity of Vp Ad5-Empty is the anti-Ad5 immunity of/medium level low to produce once.To tire be 64 to 128 (Barouch et al.2004 to Ad5 specificity neutralizing antibody (NAb) in these mouse; Lemckert et al.2005; Sprangers et al.2003).Shown in Fig. 4 C, by 10 ⁸The tetramer that Vp Ad5-Gag causes ⁺CD8 ⁺The T lymphocyte responses in these mouse by basically eliminate.On the contrary, 10 ⁸Vp Ad35-Gag and Ad35k5-Gag reply the influence of the anti-Ad5 immunity that is not subjected to this level substantially.Importantly, confirm after immunity the 14th day, the immunogenicity of Ad35k5-Gag is better than Ad5-Gag (p＜0.001) and Ad35-Gag (p＜0.05) in these mouse.

Ad35k5-Gag escapes the ability of the anti-Ad5 immunity of low/medium level and found before that Ad5 specificity NAb was primarily aimed at the Ad5 hexon and (sees above-mentioned; Sumida et al.2005) discovery unanimity.Yet, in this previous research, also detect fibrinous low-level NAb at Ad5.Therefore in mouse, repeat this experiment with high-level anti-Ad5 immunity.With mouse 8 weeks and 4 all before inoculation with 10 ¹⁰Twice of the pre-immunity of vp Ad5-Empty.To tire be 8,192 to 16,384 (Barouch et al.2004 to Ad5 specificity NAb in these mouse; Lemckert et al.2005; Sprangers et al.2003), to the highest similar (the Kostense et al.2004 that tires that in the individuality of Sub-Saharan Africa, finds; Sumida et al.2005).Shown in Fig. 4 D, the tetramer that causes by Ad35k5-Gag ⁺CD8 ⁺The T lymphocyte responses is partly reduced in these mouse, to those reply similar (p=ns) by the Ad35-Gag inductive.Therefore, high-caliber anti-Ad5 immunity has partly suppressed the immunogenicity of Ad35k5 carrier.

For further detailed assessment by the carrier specificity immunity that chimeric Ad35k5-Gag carrier causes, carry out allos first-strengthen research and viral neutralization analysis.The C57/BL6 mouse group (N=4/group) of never carrying out experiment is all with 10 the 0th ⁹Vp Ad35k5-Gag initial immunity, all the 4th then with 10 ⁹Vp Ad5-Gag or Ad35-Gag strengthen.Shown in Fig. 5 A, the tetramer that causes by Ad35k5-Gag ⁺CD8 ⁺The T lymphocyte responses by Ad5-Gag but not Ad35-Gag effectively strengthen.These Notes of Key Datas Ad35 and Ad35k5 induce the anti-carrier immunity of the cross reactivity of essence, and Ad5 and Ad35k5 are the immunology uniqueness to a great extent.Consistent with these observed results, produce and those similar Ad35 specificity NAb of Ad35-Gag inductive with Ad35k5-Gag immunity mouse once, but be starkly lower than Ad5 specificity NAb (Fig. 5 B).Therefore, the Ad35k5 carrier presents to the Ad35 carrier but not the more similar serology collection of illustrative plates (serologic profile) of Ad5 carrier.

But in order to estimate these results' generalization, the immunogenicity of the Ad35k5 carrier of the another kind of antigen of evaluation expression in different strain mouse (Env:HIV-I 89.6P Env gpl20 sees that for clone's detailed description this paper and Vogels et al.2003 are described).With the Balb/c mouse group (N=4 only/group) of never carrying out experiment through intramuscular with 10 ⁹Vp Ad5-Env, Ad35k5-Env or Ad35-Env immunity are once.Cell that is caused by the Ad35k5-Env carrier and humoral immunoresponse(HI) are analyzed (Fig. 6 A) and Env specific ELISA (Fig. 6 B) is measured by IFN-γ ELISPOT between those are replied by Ad5-Env carrier and Ad35-Env carrier inductive.

The following ELISA that carries out.Measure serum antibody titer (the Barouch et al.2003 that mice immunized is specific to HIV-IEnv or SIV Gag by direct ELISA; Sumida et al.2005).It is dull and stereotyped with the PBS sealing that contains 2%BSA and 0.05%Tween-20 2 hours to be used for the 1 μ g/ml Recombinant HIV-I MN Env gpl20 in 100 μ l/ holes of PBS or 96 holes that SIVmac239 Gag p27 polypeptide (ImmunoDiagnostics) bag is spent the night.Then serum is added in the serial dilutions, be incubated 1 hour.This flat board with the PBS washing that contains 0.05%Tween-20 3 times, is used the anti-mouse secondary antibody of rabbit (Jackson Laboratories, the diluent insulation in 1: 2000 USA) 1 hour of the affinity purifying of peroxidase conjugated.Should wash 3 times by flat board,, stop, analyze at 450nm with Dynatech MR5000 ELISA plate reader with 1%HCl with the tetramethyl benzidine colour developing.

Embodiment 6:Ad5, Ad35k5 and the immunogenicity of Ad35 carrier in rhesus monkey

In order to confirm the immunogenicity research in mouse, carry out preliminary study to estimate the immunogenicity of these carriers in rhesus monkey.With 9 Mamu-A ^*The negative rhesus monkey of 01-(N=3/group) is used through intramuscular and expresses 10 of SIV Gag and HIV-I Env ¹¹Vp Ad5, Ad35 or the immunity of Ad35k5 carrier.In the 0th week rhesus monkey is carried out initial immunity and accepts the homology booster immunization in the 12nd week.Fig. 7 shows antigen and the carrier specificity immunne response in these animals.Gag-and Env-specificity cellular immunity response are analyzed by mixed peptide IFN-γ ELISPOT and are quantized, and carrier specificity NAb tires and determines based on the viral neutralization analysis of luciferase by a plurality of time points after immunity.As the above-mentioned neutralization analysis that carries out.

Just as expected, the Ad5 carrier causes behind initial immunity that high-frequency ELISPOT replys and (replys at average Gag+Env of the 12nd week and be 538SFC/10 ⁶PBMC), but these reply behind the homology booster immunization and do not increase substantially (at the 16th all average out to 608 SFC/10 ⁶PBMC; Fig. 7 A), probably reflected the quick generation (Fig. 7 B) of the high Ad5-specificity NAb that tires in these animals.On the contrary, initial immunity back Ad35 carrier cause antigen-specific ELISPOT reply than Ad5 carrier inductive those reply approximately low 2 times (at the 12nd all average out to 248SFC/10 ⁶PBMC).Interesting ground, these are replied after the homology booster immunization fully increases (at the 16th all average out to 876SFC/10 ⁶PBMC; Fig. 7 C), with the initial carrier specificity NAb than low liter consistent (Fig. 7 D) that produces in these animals.These Notes of Key Datas Ad35 carrier is compared with Ad5 and both caused lower antigen-specific immune response in rhesus monkey, also causes lower carrier specificity immunne response.

Behind initial immunity, the Ad35k5 carrier causes to Ad5 carrier inductive that those reply similar antigen-specific ELISPOT and replys (at the 12nd all average out to 578SFC/10 ⁶PBMC; Fig. 7 E).Importantly, these are replied after the homology booster immunization and fully strengthen (at the 16th all average out to 1736SFC/10 ⁶PBMC), probably reflected the initial low relatively carrier specificity NAb (Fig. 7 F) that produces in these monkeys.In fact, after booster immunization, the Ad35k5 carrier causes than Ad5 and all high 2 times Gag-and the Env-specificity ELISPOT of Ad35 carrier replys.In addition, Ad35k5 carrier the 16th week after immunity causes strong classification (fractionated) CD4 ⁺And CD8 ⁺The T lymphocyte responses is analyzed the PBMC (Fig. 8 A, B, C: be respectively Ad5, Ad35, Ad35k5) with CD4 exhaustion that uses CD8 to exhaust by ELISPOT and is determined.

Embodiment 7: contain the generation of the reorganization Ad5 carrier of chimeric hexon

The described Ad2 of Crawford-Miksza and Schnurr (1996), Rux and the described Ad5 of Burnett (2000), the described Ad5's of Rux et al. (2003) and Ad5's of the present invention HVR is shown in Table I.To the qualification of HVR, Table II provides 7 HVR sequences of adenovirus hominis Ad5, AdI1, Ad26, Ad35 and Ad48 and chimpanzee adenovirus 68 (Pan9) according to the present invention.Also show the specific position of six adjacent body sequences separately.

Structure is based on the carrier of Ad5, and it contains one or more HVR that exchanges from Ad35 (subgroup B) or Ad48 (subgroup D).

More in the embodiment of " minimized ", the HVR of above-mentioned adenoviral serotype is slightly short form in the present invention.These HVR are expressed as HVR (1-7) with asterisk in Table IV ^*In recombinant vectors, minimize to limit and be: HVR1 ^*From main chain, lack HVR2 ^*Replace with short two amino acid spacer regions (QG), and HVR3 ^*, HVR4 ^*, HVR5 ^*, HVR6 ^*And HVR7 ^*Replace from (*) counterpart of lacking of other serotype separately by it.More significantly, to the definition again of HVR7 narrower (contrast Table II and Table IV).

The part six adjacent body genes that contain required sequence are synthetic by GeneArt (Germany), and advance to contain as the ApaI-HpaI fragment cloning in the shuttle plasmid of complete Ad5 six adjacent body genes.From this shuttle plasmid, downcut bigger AscI-AscI fragment then, be used to replace corresponding Asc1-Asc1 fragment among the Ad5 clay pWE.Ad5.Af111-rITR.dE3 (, seeing that WO 02/40665 is described) based on the carrier in clay pWE.Ad5.Af111-rITR, disappearance E3 zone.Ad5 clay with sudden change advances the PER.C6/55K cell (at PER. with joint plasmid pAdApt-Gag (the gag albumen of coding simian immunodeficiency virus (SIV)) cotransfection then Has 55K E1B gene in the cell from Ad35, see WO 02/40665) in, homologous recombination produces Ad5HVR35 (l)-Gag, Ad5HVR48 (the 1)-Gag of reorganization of reorganization and Ad5HVR48 (the 1-7)-Gag virus of reorganization, wherein " 1 " is meant the only replacement of HVR1, and " 1-7 " is meant the replacement of all 7 independent HVR.With these carriers by general procedure known in the art through plaque purification, order-checking, amplification and by the CsCl gradient centrifugation purification.The sequence of six adjacent bodies is shown in Figure 11 among Ad5HVR48 (1-7)-Gag.

Except above-mentioned three kinds of viruses, also produced following recombinant vectors: Ad5HVR35 (1-6), Ad5HVR35 (1-7) (Figure 12), Ad5HVR11 (1-6), Ad5HVR11 (1-7) (Figure 13), Ad5HVR26 (1-6), Ad5HVR26 (1-7) (Figure 14), Ad5HVRPan9 (1-6) and Ad5HVRPan9 (1-7) (Figure 15)." 1-6 " is meant the replacement of HVR1-HVR6, the HVR7 of residue parental generation carrier.Obviously, based on some available parental generation carriers (as Ad2 and Ad5) and some known rare serotype, can produce more kinds of combinations according to instruction of the present invention.Can be used for providing HVR is Ad24, Ad34, Ad49 and Ad50 with other the rare adenovirus hominis serotype that produces " stealth " sample carrier.

Embodiment 8:Ad5, Ad5HVR48 (1) and Ad5HVR48 (1-7) virus is the mouse that never carried out experiment and have immunogenicity in the mouse of anti-Ad5 immunity

As mentioned above, reorganization Ad5-Gag, Ad5HVR48 (the 1)-Gag and Ad5HVR48 (the 1-7)-Gag virus of survival in packing cell, have been produced.The output of Ad5HVR48 (1)-Gag is similar to reorganization Ad5-Gag virus, and the speed of growth of Ad5HVR48 (1-7)-Gag virus, output and vp/pfu ratio hang down about 2 times than Ad5-Gag is viral.Gag expresses at first with 10 ⁹Or 10 ¹⁰Detect on the A549 cell that every kind of carrier of vp infects.The HPLC data show that the Gag expression is enough in the born of the same parents, and similar between different carriers (data not shown goes out) is although express a little less than Ad5-Gag from Ad5HVR48 (1-7)-Gag.This may be relevant with the lower slightly speed of growth.

The immunogenicity of virus is at first passed through with 10 ⁹, 10 ⁸And 10 ⁷Every kind of carrier immunity of vp was never carried out the C57/BL6 mouse (4 mouse/groups) of experiment and was studied.The CD8 that vaccine causes ⁺T lymphocyte responses such as the above-mentioned D that passes through ^b/ AL11 tetramer binding analysis evaluated for 2 weeks.The results are shown in Fig. 9.Obviously, all these three kinds of carriers all produce similar immunne response in these mouse that never carried out testing, although a little difference is arranged in the speed of growth and transgene expression.

Subsequently, the C57/BL6 mouse is injected twice 10 in 8 weeks and 4 weeks before with interested virus immunity respectively ¹⁰Vp Ad5-Empty carries out pre-immunity (Ad5Nab tires 8192 to 16384), to produce at the immunity that is pre-existing in based on the Ad5 carrier.(pre-first immunity 8 weeks of back) then are with mouse such as above-mentioned usefulness 10 ⁹, 10% and 10 ⁷Reorganization Ad5-Gag, Ad5HVR48 (1)-Gag of vp and Ad5HVR48 (1-7)-Gag immunity.Again, the CD8 that causes of vaccine ⁺T lymphocyte responses such as the above-mentioned D that passes through ^b/ AL11 tetramer binding analysis evaluated for 2 weeks.The results are shown in Figure 10.ELISPOT result has been reflected described tetramer analytical results, and analog result (data not shown goes out) is provided.

Just as expected, the Ad5-Gag carrier meets with the immunity that is pre-existing in these pre-mice immunized, cause being difficult to detected immunne response.Ad5HVR48 (1)-Gag virus can not be avoided high-caliber anti-Ad5 immunity, points out the described immunity that is pre-existing in the non-at least HVR1 of only limiting to.Importantly, the immunogenicity of Ad5HVR48 (1-7)-Gag virus is not subjected to the influence of the immunity that the Ad5 inductive is pre-existing in, show 7 six adjacent body HVR (as differentiating) and replace it, produce the carrier that is not subjected at the immunity that the is pre-existing in obstruction of wild-type protein with the corresponding HVR of rare serotype (for example Ad48) in the present invention by sudden change Ad5.To obtain analog result in the experiment of mouse with twice of blank Ad5 carrier initial immunity therein, representative has the situation (Figure 21) of the immunity that high level is pre-existing in.This experiment is carried out with every group of 4 C57/BL6 mouse.Be twice 10 of mouse group injections ¹⁰VpAd5-Empty carries out pre-immunity, to induce anti-Ad5 immunity.Tire with the Ad5NAb of the pre-mice immunized of Ad5-Empty and to be 8192-16384.Then with mouse the 0th day through intramuscular with 10 ⁹Vp Ad35-Gag initial immunity, then at the 28th day with 10 ⁹Vp Ad5-Gag, 10 ⁹Vp Ad35-Gag or 10 ⁹Vp Ad5HVR48 (1-7)-Gag strengthens.All volume injected all are 50 μ l.The X-axis upward arrow is represented immunity.Carry out D at the 0th, 7,14,21,28,35,42,49,56 day blood sampling ^bThe staining analysis of/AL11 the tetramer is to quantize the CD8 that vaccine causes ⁺The T lymphocyte responses.At the 56th day, also carry out IFN-γ ELISPOT and analyze, similar result (data not shown goes out) is shown.Ad5-Gag can not booster response, and supposition is because due to the anti-Ad5 immunity that is pre-existing in.Ad35-Gag can not booster response, and supposition is because due to the anti-Ad35 immunity of initial immunity inductive.Ad5HVR48 (1-7) effectively strengthens this and replys, and confirms that this carrier plays some new serotypes.So the background of inferring Ad5HVR48 (1-7) Ad5 failure therein reaches therein, and allos carrier (as Ad35) can be used as effective reinforcement carrier as in the background of initial immunity carrier.Make up previous research, we infer in the background that is pre-existing in anti-Ad5 immunity by the carrier Ad5 failure therein of Ad5HVR48 (1-7) representative be effective initial immunity carrier be again effectively to strengthen carrier.

Now these results make that people can be for the first time-strengthen using in the background carrier based on Ad5, and acceptor identification (mainly being undertaken by fiber and penton albumen) remains unchanged.

Table I: adenovirus hominis Ad2 (Crawford-Miksza and Schnurr, 1996), Ad5 (Rux and Burnett, 2000; Rux et al.2003) qualification of hypervariable region and in the hexon of Ad5 (the present invention).Based on the Ad2 sequence, Rux and Burnett (2000) meets qualification and the Crawford-Miksza qualification of the HVR of Ad5 very much.Attention these HVR in this table limit the change that 1 position is all arranged owing to lack the initial methionine residues in Rux and Burnett (2000) qualification (HVR1:137-181 etc.).

Table II

Table II (continuing)

Table III: chimeric replication-defective adenoviral vector of the present invention comprises the discriminating of different elements in the described carrier

The part of chimeric adenoviral/carrier	Characteristic features
		Skeleton serotype X (the not packaged cell of the element of carrier is supplied and got rid of fiber and hexon, comprises penton albumen)	Wherein X is selected from the adenovirus (comprising adenovirus hominis serotype 1 to 51, chimpanzee serotype, bovine serum type and dog serum type) that can be used for the human treatment
Six adjacent body HVR (n) Y	Wherein Y is selected from adenovirus hominis serotype 11,24,26,34,35,48,49 and 50, and n=0 to 7 wherein, and condition is if X ≠ Y, n=1 to 7
		Fiber delta knob F comprises afterbody T and axle S	Wherein F is selected from serotype 1 to 51, chimp ..., condition is if at least a portion of the T of X ≠ Y and the interactional F of penton is X

?Knob?K

Wherein K is selected from the adenoviral serotype that the CAR acceptor is used to infect on the main use cell (the adenovirus hominis serotype of subgroup A, C, D, E and F) and K can be X or different

Table IV

Table IV (continuing)

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Claims (3)

1. the recombinant replication-defective adenoviral based on Ad5 is characterized in that described recombinant replication-defective adenoviral comprises chimeric hexon, wherein

The HVR1 of Ad5 shown in the SEQ ID NO:17 is replaced by the HVR1 of the Ad48 shown in the SEQ ID NO:24;

The HVR2 of Ad5 shown in the SEQ ID NO:18 is replaced by the HVR2 of the Ad48 shown in the SEQ ID NO:25;

The HVR3 of Ad5 shown in the SEQ ID NO:19 is replaced by the HVR3 of the Ad48 shown in the SEQ ID NO:26;

The HVR4 of Ad5 shown in the SEQ ID NO:20 is replaced by the HVR4 of the Ad48 shown in the SEQ ID NO:27;

The HVR5 of Ad5 shown in the SEQ ID NO:21 is replaced by the HVR5 of the Ad48 shown in the SEQ ID NO:28;

The HVR6 of Ad5 shown in the SEQ ID NO:22 is replaced by the HVR6 of the Ad48 shown in the SEQ ID NO:29; And

The HVR7 of Ad5 shown in the SEQ ID NO:23 is replaced by the HVR7 of the Ad48 shown in the SEQ ID NO:30;

And wherein the hexon sequence between the HVR sequence is from Ad5.

2. the recombinant replication-defective adenoviral of claim 1, it comprises the chimeric six adjacent body sequences of SEQ ID NO:12.

3. each recombinant replication-defective adenoviral of aforementioned claim, it comprises waits to be transported to the host to prevent or the interested heterologous nucleic acids of therapeutic purpose.

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