CA1337115C - Vaccination against rabies-related diseases - Google Patents
Vaccination against rabies-related diseasesInfo
- Publication number
- CA1337115C CA1337115C CA000573293A CA573293A CA1337115C CA 1337115 C CA1337115 C CA 1337115C CA 000573293 A CA000573293 A CA 000573293A CA 573293 A CA573293 A CA 573293A CA 1337115 C CA1337115 C CA 1337115C
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- rabies
- virus
- protein
- cvs
- cfa
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Methods of vaccinating to induce protective immunity to rabies and rabies-related viruses are taught wherein certain synthetic, genetically engineered, or rabies-derived polypeptides are used. The sequence of the polypeptides is derived from the N
protein. Both B and T cells are stimulated by these antigenic polypeptides to provide immunity to rabies and other related infections.
protein. Both B and T cells are stimulated by these antigenic polypeptides to provide immunity to rabies and other related infections.
Description
VACCINATION AGAINST RAP~.~-RELATED VIRUSES
This invention was made under grants from the National Institutes of Health. The United States Government has certain rights in the invention.
TECHNICAL ~LD OF THE INVENTION
This invention relates to rabies virus and rabies-related viruses. More particularly it relates to methods of immllni~ine host anim~l.c to protect against infections with rabies and rabies-related viruses.
BACKGROUND OF THE INVENTION
Rabies virus continues to be endemic in most areas of the world. It causes an acute central nervous system ~lice~ce which is normally fatal to hllm~n.c and domestic and wild ~nim~lc.
The virus structure is bullet-sh~ped~ consisting of a nucleo-capsid core surrounded by a membrane envelope. The nucleocapsid is comprised of a single, non-segmented strand of RNA together with RNA transcriptase (L), phosphoprotein (NS), and nucleoprotein (N).
The N protein, which is the major portion of the nucleocapsid, is noncovalently bound to the RNA to form the helical ribonucleo-protein (RNP) complex. Two viral proteins are associated with the viral envelope, the major surface antigen (G) which is glyco~sylated and the matrix protein (M) which is thought to be located on the inner leaflet of the lipid bilayer, associating with both the C-terminal dom~in of the membrane-in~serted G protein and the RNP
structure.
Virus-neutralizing antibodies raised in ~nim~l.c again~st rabies virus, only recognize the G protein. The level of antibody production has been thought to correlate with the degree of protection afforded against live virus infection. See Crick, Post Graduate Medical Journal, Vol. 49, p. 551 (1973) and Sikes, et al., Journal of American Veterinary Medical Association, Vol. 150, p.
1491 (1971). However, there are indications that antibody alone is not sufficient to protect from viral infection. For instance, passive immunization with anti-rabies antibodies without a vaccine as po~st e~L,o~lre therapy,`does not decrease the probability of infec-tion. Nicholco~, et al., Journal of Infect. Diseases, Vol. 140, p. 176 (1979). In addition, there exist effective vaccine~s which do not induce high level antibody production. One such vaccine is produced by Norden Labs.
It i~s p~ccihle that resistance to infection by rabies virus may require both virus-neutralizing antibodies and effector T-cell responses. Both the G protein and the nucleoprotein (N) have been shown to stimulate proliferation of rabies antigen-specific T cell lines. Most such T cell lines respond strongly to N protein and less strongly to G protein. A minority of rabies reactive T cell lines respond to G protein, but not at all to N protein. Celis et al., Journal of Immunology, Vol. 136, pp. 692-697, (1986). It has not been shown previously that N protein provides any protection against rabies infection in immunocompetent ~nim~ls or hllm~n.c Current vaccines against rabies consist of an inactivated rabies virus. These vaccines do not provide any cross-protection against the rabies-related virus Mokola. Preparation of current vaccines involves growth of the virus on permissive animal cells or embryos. This method of production requires the h~n-llinE of the virus by workers which entails an undesirable risk of infection.
Further, some inactivated virus vaccines can cause adverse side effects, such as demyelinating allergic encephalitis and systemic reactions, in a proportion of the vaccine recipients.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for inducing protective immunity to rabies and rabies-related viruses.
It is another object of the present invention to provide a rabies-related virus vaccine which does not require growth of viruses.
It is yet another object of the present invention to provide a rabies-related virus vaccine which can be chemically synth~-ci7ed.
It is still another object of the present invention to provide a rabies-related virus vaccine which can be produced in a micro-organism.
It is an object of the present invention to provide a rabies-related virus vaccine which stimulates proliferation of both T
and B cells.
It is another object of the present invention to provide a method of inducing protective immunity to rabies-related virus which does not cause demyelinating allergic encephalitis.
In accordance with these objects, the present invention provides an improved method for inducing protective immunity to rabies-related viruses comprising administering a priming injection of an antigen and one or more booster injections, the improvement comprising employing a polypeptide having amino acid sequence homology to rabies-related virus nucleoprotein as said priming injection, said polypeptide being substantially free of rabies-related virus glycoprotein, said polypeptide having the ability to induce proliferation of rabies-related virus-specific human T cells.
In another embodiment of the present invention, a biologically pure sample of a polypeptide is provided capable of inducing a proliferative response in rabies-related virus-specific human T cells, said polypeptide having sequence homology with the nucleoprotein of rabies-related virus and having the ability to induce protective immunity to rabies-related viruses .
In yet another embodiment of the present invention a method is provided for inducing protective immunity to a rabies-related virus comprising administering an injection of a preparation comprising a polypeptide having sequence homology with the nucleoprotein of a rabies-related virus, said polypeptide being capable of inducing a proliferative response in rabies-related virus-specific human T cells, said polypeptide being substantially free of rabies-related virus glycoprotein.
According to an aspect of the invention, a method of inducing protective immunity to rabies-related virus comprises:
administering to a human or domestic animal as a priming injection a preparation which comprises the nucleoprotein of a rabies-related virus, the preparation 4a is substantially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one or more booster injections which comprises an inactivated rabies virus.
According to another aspect of the invention, a method of inducing protective immunity to rabies-related virus consisting essentially of the step of:
administering to a human or domestic animal one injection of a preparation which comprises the nucleoprotein of a rabies-related virus, the preparation is substantially free of rabies-related virus glycoprotein.
According to a further aspect of the invention, a method of inducing protective immunity to rabies-related virus consisting essentially of:
administering to a human or domestic animal a priming injection which comprises the nucleoprotein of a rabies-related virus, the nucleoprotein is substantially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one booster injection which comprises the nucleoprotein of a rabies-related virus, the nucleoprotein is substantially free of rabies-related virus glycoprotein.
The vaccines of this invention can be produced without growing the rabies virus and they do not cause the adverse side effects associated with inactivated virus vaccines. In addition, the vaccine provides protection against heterologous rabies-related virus strains.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that the nucleoprotein of rabies virus, a protein which is internal to the membranous envelope of the virus, can serve as an effective inducer of both humoral and ce~ r immllne respon~ses. Further, a particular region of the nucleoprotein (N protein), amino acid residues 313-337, has been found to contain an epitope identified by certain anti-rabies antibodies. In addition, this isolated region of the protein (as well as two other regions, cu-,esL,onding to amino acid rP-si-luP-s 369-383 and 394-408) is able to stimulate proliferation of rabies antigen-specific human T celLs in the presence of irradiated autologous mono~llclear cells. Other suitable peptides may be found which are able to stimulate proliferation of rabies antigen-specific human T cells and which can provide protective immunity to rabies infection. Stimulation of proliferation of T celLs can be tested according to the method of Celis et al, J. Immunol. 56:426-433 (1985), and in Example 6 below.
Protective immunity can be tested according to standard methods known in the art and described in Example 7 below.
As normally practiced in the art, rabies prophylaxis is performed by a series of injections, the first one being termed a priming injection and subsequent ones termed boosters. Methods for minictering such injections, including al,prop-iate formulations, dosages, anatomical localizations and timing are generally known in the medical and veterinary sciences. Generally, the dosage desired is such that will induce a protective response without causing side effects or immlJne tolerance. Standard inactivated rabies virus vaccine, for example, may be used in the practice of the present invention as a booster inoculation. The inactivation of virus i~s generally performed by treatment with a chemical, such as -6- ~ 337~ ~ 5 beta-propiolactone. Such a vaccine is av~ hle, for example, from Institut Merieux, Lyon, France.
For the purposes of the present invention the term ~rabies-related virus~ is meant to encompass both rabies as well as rabies-related viruses such as the Mokola and European bat virus (e.g. the Duvenhage 6 strain). This definition is used because of the finding that the vaccines of the present invention provide cross-protection to heterologous virus strains. This is a great benefit provided by the present invention over prior vaccines.
Subunits of the rabies-related virus, such as N protein or RNP
complex, may be prepared and purified according to known methods.
See, for example, Virology 124:330 (1983) and J. Virol. ~:241 (19~1).
Even purer preparations of N protein can be obtained by preparative gel electrophoresis, as described in J. Virol, 44:596 (1982). Other purification steps may be used, as are known in the art.
The useful polypeptides of the present invention can be most easily prepared by chemical synthesis, according to the solid phase method described by Merrifield, Adv. Enzymol., 32:221-296 (1969).
Alternatively, proteolysis of N protein can be accomplished using enzymes or chemicals, e.g., trypsin, 3-bromo-3-methyl-2 [(2-nitro-phenyl) thiol] -3 H-indole skatole, cyanogen bromide, or protease V8. Fragments af ter cleavage can be separated by gel electro-phoresis, for example. Although many fragments can be found which are reactive with monoclonal antibodies raised against rabies N protein, only some f ragments are also able to stimulate rabies-specific T cell proliferation. The inventors have found one polypeptide to be particularly useful among a set of proteolytic cleavage products of N protein. This fragment, termed N-V12b, was originally produced by protease V-8 treatment of N protein, and later chemically syn~h~-si7ed. Fragments N-V10c and D30 were also found to stimulate T cell proliferation, although to a lesser degree.
Fragment N-V12b corresponds to the amino acids 313-337 of N
protein; fragment N-V10c co~ onds to amino acids 369-383; and fragment D30 corr~ol ds to amino acids 394-405. The sequences are shown below in Table 4. The sequence of the N protein has been previously determined by Tordo et al., Nucleic Acids Research, Vol.
14, pp. 2671-2683 (1986).
When the synthetic peptides are coupled to a protein, e.g.
keyhole limpet hemocyanin, either via thiol groups (Biochemistry 18:690-697 (1979)) or via amino groups with glutar~ ldehyde, they are found to stimulate anti-N protein antibodies in rabbits.
However, the antibody titer was lower in anti-N-V12b serum than in anti-N-VlOc serum.
Although the sequences of the polypeptides are defined herein with particularity (Table 4) it will be apparent to one skilled in the art that some of the amino acids can be conservatively substituted without losing the beneficial characteristics. It is also apparent to one skilled in the art that changes in length can be made without altering the antigenicity. The minimllm nllmher of amino acids necessary to comprise the epitopes has not been determined.
The polypeptides of the present invention can also be prepared through genetic enEineering. That is to say that parts or all of the N gene can be cloned into suitable expression vectors, as are known in the art. Organisms transformed with the cloned gene or portion thereof can be grown to produce the polypeptide products. Recovery of the polypeptide products is within the ordinary skill of the art. Comhin~tions or concatemers of antigenic polypeptide fragments can also be used. These can be made by synthesis, çlo~ine, or post-synthetic covalent bo~line.
Liposomes, which can be employed in the practice of the present invention, are known in the art. They are used as a macromolecular carrier for the polypeptides to ensure antigenicity.
Other proteins or synthetic nanoparticles may also be used as carriers. In the case of RNP a~lminictration no carrier is required.
Liposomes may be formed, e.g., according to the method of Thiholeau, ~Genetic variation among influenza viruses," Acad. Press, N.Y. (1981) p. 587. Generally, a mixture of lipids in particular ratios, such as ph~-sphatidyl rholine~ cholesterol, and lysophos-phatidyl choline, are mixed under conditions to form closed lipid vesicles. Many such conditions and methods are known in the art.
In the practice of the present invention the polypeptides are coupled to a saturated fatty acid having from about 15 to about 21 carbon atoms, before mixing with the lipid composition to form liposomes. Suitable fatty acids include, palmitic, stearic and oleic acids. Conjugates may be formed by the method of Hopp, Molecular Immunology, 21:13-16 (1984), wherein peptide fragment spacers of gly-gly-lys-(NH2)2 are added to the N-terminus of the polypeptide, and the alpha and e~ilon amino groups of lysine are used to couple with the fatty acids.
The biologically pure s~mples of the polypeptides of the present invention are substantially f ree of glycoprotein of rabies virus. They are sufficiently pure that after injection into mice, rabies glycoprotein~pecific antibody or T cells are not induced. In addition, rabies glycoprotein specific T cells would not be stimulated in vitro to proliferate in the presence of the preparation.
The following examples are illustrative only and are not intended to limit the scope of the invention. The invention is defined by the claims appended below.
9 133ill5 This example describes the selection of a multiple variant virus of rabies virus.
Seven anti-glycoprotein monoclonal antibodies c~p~hle of neutralizing parent CVS-11 rabies virus were used to sequentially select antigenic variants. Serial dilutions of virus were mixed with monoclonal antibody diluted 1:100, overlayed with nutrient agar, and after 4 to 5 days of incubation at 35C in a 5% carbon lioxide atmosphere, plaques were selected. Viruses able to replicate in the presence of the monoclonal antibodies used for their selection were recovered. The seventh generation variant CVS-V7 was not neutralized by any of 40 diiferent rabies virus-specific neutralizing monoclonal antibody. The nucleoprotein antigen of the CVS-V7 virus rem~ined immllnologically indistinguishable from that of the CVS-11 parent virus.
This ex~mple shows the protection afforded by vaccination with the CVS-V7 virus, as well as the virus neutralizing antibodies it induced against parent strain CVS-11.
Vaccines prepared f rom the CVS-V7 variant viruses, were used to immunize mice. Groups of nine 4-week old female ICR mice were immllni7pd with 0.2 ml of 5 serial dilutions (2000-3 ng) of the CVS-V7 inactivated virus vaccine on days 0 and 7. The inactivated virus vaccine was prepared with beta-propiolactone and adjusted to a protein concentration of 100 ug/ml. The viruses had been grown on BHK 21 cell monolayers and purified as described in Journal of Virology, Vol. 21, pp. 626-635 (1977). On day 14, vaccinated and unvaccinated control mice were bled and infected intracerebrally with 0.03 ml (50 MIC LD50) of CVS-11. (CVS-11 parent virus was used in the ch~llenge because the CVS-V~ variant virus was not pathogenic for adult mice.) The ~nim~l.c were observed for 3 weeks and mortalities were recorded daily. The effective dose was calculated for each vaccine as described in Atansiu, P., ~Quanti-tative assay and potency test of antirabies serum," In: Laboratory Techniques In Rabies, 2nd Ed., Geneva: World Health Organization, 1966, pp. 16~-~2. The neutralizing activity of the mouse immune sera for CVS-ll was determined as described in Lumio et al., Lance, Vol. i, p. 3~8 (1986).
The geometric mean tighter (GMT) was calculated for each vaccination group. Multiple group comparisons for differences in GMT were tested by one way analysis of variance and two-group GMT were compared statistically by a one-tailed test.
As can be seen in Table 1, virus neutralizing activity as well as protection from death were provided by the CVS-V7 vaccine. As the CVS-V~ virus, from which the vaccine was prepared, was not neutralized by any of 40 rabies glycoprotein-specific monoclonal antiho~liP-s and yet was able to confer protection against ch~llPnge with the parental CVS-11 strain, it is concluded that the glyco-protein is not the sole factor in determining the relative efficacy of rabies prophylaxis.
This example demonstrates the protection afforded and the virus neutralizing antibody induced by means of rabies virus subunit vaccines which are formulated with liposomes.
The glycoprotein and ribonucleoprotein (RNP) were purified from the CVS-V~ variant virus as described in Diet7chold et al., CVS-V7-G- CVS-V7-N- CVS-V7-(N+G)-Vaccine CVS-V7-Virus Liposome Liposome Liposome Vaccine VNA against Mortality VNA agalnst MortalityVNA a~ainstIViortallty VNA agalnstiviortallty Concen- GMT (range) Rate GiviT (range) Rate GMT (range) GMT (range) tration [ng]
10000 (27o-l62o) 0/7 (<10-810) 1/s 7/7 588 1/6 2000 (60 540) 2/7 (290 540) 2/7 0 7/7 (260 810) 1/7 400 (60-540 3/7 (<10 180) 4/~ 7/7 323 1/7 (<10-540) 3/7 (211o 60) 3/6 0 7/7 (<10-540) 1/7 1~ (<10-540) 3/7 (<10 30) 5/6 o 7/7 (<10-50) 5/7 Effective 33.3 ng 588 ng 45 ng (ED50) i Table 1 - Protective activities of an inactivated CV~ S-V7 virus vaccine and CVS-V7 subunit vaccines against an i.c. chalienge infection with CV~l 1 virus.
Isolation and Purification of a Polymeric Form of the Glycoprotein of Rabies Virus, J. Gen. Virol. 1978, 40 131-135 and Schneider et al., Rabies Group-Specific Ribonucleoprotein Antigen and a Test System for Grouping and Typing of Rhabdoviruses 1973, J. Viral. 11, 748-755.
The proteins were inserted into liposomes as described in Thibodeau, Genetic Variation Among Influenze Viruses, Acad. Press, N.Y. 1981, p. 587. The amount of rabies protein liposomes used per injection is expressed as weight of the total complex. Mice were injected intracerebrally on days O and 7 and ch~llenged at day 14 as described in Example 2. The resulting mortality rates and amount of virus neutralizing antibody produced are shown in Table 1.
The RNP incorporated into liposomes induced no detectable VNA against CVS-ll nor any detectable protection from death. The CVS-V7-derived G protein incorporated into liposomes induced low titers of VNA and poor protection from death. However, when the RNP was combined with G protein, the treatment was significantly better than either protein alone, and produced results roughly comparable to those obtained with the whole virus vaccine. This too, shows the importance of N protein in immunity to rabies.
This ex~mple demor~ctrates that rabies virus RNP can augment the function of B cells producing neutralizing antibody.
Groups of mice were primed with either 5 ug of N protein (from the ERA strain of rabies) plus complete Freund's adjuvant (CFA) or CFA alone. Ten days after priming both groups of An;m~l~
received serial dilutions of inactivated rabies virus vaccine or isolated rabies glycoprotein. Mice that were primed with RNP plus CFA and boosted with rabies virus vaccine developed significantly higher VNA titers than those mice primed with CFA alone and then boosted with a rabies virus vaccine. The results are shown in Table 2. In addition, the effective dose of rabies virus vaccine was determined and found to be about 10-fold lower in the mice which had been primed with RNP.
The mice which received booster vaccinations consisting of just rabies virus glycoprotein developed only low levels of VNA and were poorly protected against a lethal ch~lle~Ee infection with rabies virus, which can be seen in the right half of Table 2. This suggests that there must be common antigens between the priming and boosting immllni7ations in order to induce an effective immune response.
The mortality rates shown in Table 2 were determined by ch~llengin~ all mice with an intracerebral innoculation with 50 MIC
LD50 Of rabies virus and observing the survival up to 3 weeks post-infection.
This ~x~mplPs demonctrates that protective immunity can be induced using rabies virus RNP to an intramuscular (I.M.) ch~llpn~e infection with homologous or heterologous rabies virus strains.
Purified RNP of the ERA strain of rabies or of the rabies-related strain Mokola was used to immunize Balb/c mice against an (I.M.) ch~llenEe with the rabies strain CVS-24. The RNP
was isolated and purified according to the method of Schn~ider, et al., J. Virol., Vol. 11, pp. ~48-~55 (19~3). Groups of 10 to 20 mice were immunized intraperitoneally (I.P.) with ERA-or Mokola-RNP
plus CFA, or with CFA alone, or subcutaneollcly (S.C.) with ERA-RNP alone. Four weeks after immunization, the mice were ch~ nged (I.M.) with eight mouse I.M. LD50 f CVS-24 rabies virus Booster immunization with Booster immunization with Vaccine ERA-virus ERA~
tration Priming with Priming with Priming with Priming with (ng) ERA-N + CfA CFA ERA N + CFA CFA
VNA a~aalnst Mortallty Vna a~alr6t Mortdlty VNA a~al~t Mortallty VNA agalr~t Mortallty CVS-l r Rate CVS-ll I?ate CVS~ ate CVS-l 1 Rate GMT (tan~e) GM~ (rar~e) GMT (rar~e) GlvlT (rrar~e) 5000 (1~C~4~0) 3/7 (<10-180) 5/5 (6C~80) / 28 6/7 489 39 6/7 25 6/7 ~ 7/7 (6C~1620) 4/7 (10-180) (10-60) (c10~60) 138 10 13 1.3 200 (20-1620) 3/7 (<lO~S0) 6 n (10-20) 6/7 (<1C~20) 7/7 34 26 4 1.3 (<1C~270) 5/6 (20~60) 6/7(<1C~20) 7/7 (<10-20) 7/7 8 (<10-540) 6/7 (<1C~30) 7/7(<1C~20) 7/7 6/6 Effective ~,~
oDs5eO) ~25r~3 >5000ng ~5000 ng ,5000 n~
~able 2 - Effect of RNP - priming on VNA tite~ and mortality rates (see Table 3) or two mouse I.M. LD50 f rabies-related virus Duvenhage 6 (see Table 4).
As can be seen in Table 3, 80% of the mice that received ERA-RNP plus CFA intraperitoneally or ERA-RNP alone subcatane-ously survived the ch~llenee. In addition, 90% of the mice that were immunized intraperitoneally with Mokola-RNP plus CFA survived the ch~llen~e infection. In contrast only 10% of the mice that received only complete Freund Adjuvant (CFA) succumbed to rabies.
It is known that neutralizing antibody produced against the Mokola virus does not neutralize rabies virus; therefore, this experiment clearly demonstrates that the protection conferred by RNP is not due to neutralizing antibody which may have been induced by very small, undetectable amounts of glycoprotein. In addition, the results shown in Table 4 demonstrate that RNP from both the ERA strain and from the Mokola virus induce protective immunity against the rabies-related European bat virus strain Duvenhage 6. Taken together these results demonstrate that RNP
purified from rabies virus and rabies-related viruses can induce protective immunity against heterologous viruses.
The synthetic peptides shown below in Table 5 were synth~-ci7ed according to the method of Merrified, cited above. In a typical coupling reaction, the tboc group of the amino terminus was removed with 50% trifluoroacetic acid (TFA). After neutralization with N, N-diisopropylethylamine (DIEA), a 4- to 6-molar excess of preformed tboc-amino acid-pentafluorophenylester (Kisfaludy et al., Liebigs Ann. Chem. pp. 1421-1429 (19~3)) and a 1-molar equivalent of DIEA in dichloromethane (1:1) were added. After b~hbling with N2 gas for 1.5-2 h, the resin was analyzed for the presence of free amino groups as described by Kaiser et al. (Anal. Biochem., 34:595-598 (1970)). Couplings were repeated until less than 1% free NH2 groups were found.
Peptides were cleaved and deblocked with HF/thio~nicol (10:1) at 0C for 30 min, and the peptide was extracted with 0.1 M
NH4HCO3 and lyophili7ed~ The crude peptide was then dissolved in 0.1 M NH4HCO3 and purified on a BioGelT~ P4 column calibrated with 0.1 M NH4HCO3. The eluted peptide was then applied to a Vydac~ 2P C18 reverse-phase column and eluted with methanol-water (8:2). The elution of the peptide was monitored with a UV
detector at 214 nm. To verify the amino acid sequence, aliquots of the peptide were subjected to amino acid analysis and amino acid sequencing.
The peptides were tested for the ability to stimulate T-cell proliferation in the presence of antigen-presenting populations of cells of the same HLA-DR type. At concentrations of 0.4 ug/ml to 25 ug/ml of protein, fragments N-V12b and N-VlOc, and N protein isolated from ERA strain virus, stimulated proliferation of the T-cells. Hepatitis B antigens, used as controls, caused no stimulation. At low concentrations, N-V12b was more stimulatory than N protein, whereas at higher concentrations the reverse was true.
The rabies specific T-cell lines were isolated and tested as described in Celis et al., J. Immunol. 56:426-433 (1985).
This e~mrle demonstrates the protective c~p~hilities of the synthetic peptides N-V10 and N-V12b against the rabies strain CVS-24.
Immunization of Balb/C mice with Rabies-RNP and Mokola-RNP again~t an l.M. challenge with CVS-24 Antigen Route of Mortality (%) Immunization + CFA 2/10 (20) 10 ug ERA-RNP S.C. 2/10 (20) 10 ug MOK-RNP l.P. 1/10 (10) + CFA
CFA l.P. 18/20 (90) 1 3371 1 5 -i~- T~BLE 4 Immunization of Balb/C mice with ERA-RNP and Mokola-RNP against an l.M. challence with DUV 6 virus Antigen Immunization Mortality (%) 10 ug ERA-RNP + CFA l.P. 0/10 (0) 10 ug Mok-RNP + CFA l.P. 1/10 (10) CFA l.P. 6/10 (60) N-VlOc NH2-tyr-glu-ala-ala-glu-leu-thr-lys-thr-asp-val-ala-leu-ala-asp.
N-V12b NH2-his-phe-val-gly-cys-tyr-met-gly-glu-val-arg-ser-leu-asn-ala-thr-val-ile-ala-ala-cys-ala-pro-his-glu.
D-30 NH2-tyr-phe-ser-gly-glu-thr-arg-ser-pro-glu-ala-val-tyr-thr-arg.
-Groups of 5 to 10 mice were immunized intraperitoneally with N-V12b-liposomes plus CFA, N-VlOc-liposomes plus CFA, or lipo-somes plus CFA. The mice were then ch~llenged with various amounts of CVS-24 virus.
The lirllsomes were formed by the method of Thibodeau, Genetic Variations Among Influenza Viruses, Acad. Press, N.Y., 58~
(1981). The peptides were incorporated into liposomes as described above in ~x~mple 3. To facilitate the incorporation of the peptides into liposomes, palmitic acid was linked to the amino terminal end of the peptide as described by Hopp, Mol. Immunol., Vol. 21, pp.
13-16, 1984. In experiment No. 1, two times the mouse I.M. LD50 was used as ch~llen~e. As shown in Table 6, 88% of the mice that received the N-V12b-liposome vaccine survived while none of the mice immunized by N-VlOc-liposome survived three weeks after the ch~llenge.
In experiment No. 2, four times the mo~Lse I.M. LD50 wa,s used as a ch~llenge. Sixty-two percent of the mice immunized with peptide N-V12b-lip~some vaccine survived while only 12% of the mice which received the control vaccine of liposomes plus CFA
survived the ch~llenge.
In experiment No. 3, 8 times the mouse I.M. LD50 was used as a ch~ nge. Sixty percent of the mice immunized with the N-V12b-lip~some vaccine survived the ch~llenEe, while only 10 and 20 percent respectively of the mice immunized with N-V10 liposomes plus CFA and liposome control vaccine plus CFA survived.
Thus, peptide N-V12b provides good protection from rabies virus ~h~llen~e when incor~orated into liposomes and administered with CFA. Peptide N-VlOc however does not provide good -2l- l 3371 l 5 TABLE 6 Protective activitie~ of N-Y1 2b peptide liposomes in mice - to i.m. challen~e with CVS-24 Experiment #1, i.m. challenge with 2 MIM LD 50 Concentratlon (u~) Vacclne R te 15 N-V10-Liposomes +-CFA 5J5 15 N-V1 2b-Liposomes + CFA 1 /8 Experiment #2, i.m. challenge with 4 MIM LD
so Vacclne Vacclne Mortallty Concentratlon (u~) ~ a e N-V12b-Liposomes + CFA 3/8 --- Liposome ~ CFA 7/8 Experiment #3, I.m. challenge with 8 MIM LD
Concentratlon (u~) Vacclne R te 15 N-V12b-Liposomes + CFA 4/10 15 N-V10 Liposomes + CFA 9/10 --- Liposomes +CFA 8/10 -Z2- l 337 1 1 5 protection against rabies virus ch~llenge~ even though it is able to stimulate rabies specific T cell proliferation.
Peptides consisting of 15 amino acids were synth~-si~ed which together correspond to the entire amino acid sequence of the ERA-N
protein. These were synth~si~ed a,s described above in Example 6.
Each peptide was screened for T cell proliferative activity in vitro and for protective activity in vivo as described above for peptides N-V12b and N-V10c.
The results are displayed in Table ~. Peptide nllmher 30D
demon~strated both significant T cell stimulatory activity as well a~s partial but significant protection against rabies viru~s ch~llenge.
Peptide D30 protected 60% of the immunized mouse population from a ch~llenge of 8 times the mouse I.M. LD50 of CVS-24 viru~s.
TI~BLE 7 T-Cell Stimulatory and Protective Activities of synthetic N-peptides Peptide No.T-cell Stimulatory Activity Mor~lit~ b~ Averace Su~.val Tima Days Liposo- e ctr. - / ~ b ' .-/- ~ . .
+ / "b ' .-- /' ~- O
- / ~b .9 + t` / b1 ) .1 O~lu - 7/l - / b + /' 'o - /' ~1 ~.1 _/ ~, . 7 + / "b . 3 - / b .5 - / ,~ 0.2 /- , 9.
+ -/- b 9 + / % 1 .
++ , /'0~ 1 ~ 2~ +++ 4/9 ( %) V1 2b- - 711~ . ~%) V1 b- I - 1 / 1 ( %) V12"- 1 - 1 ( V1 2~- /
V1 C +++ 9/1 (9 ~) .'
This invention was made under grants from the National Institutes of Health. The United States Government has certain rights in the invention.
TECHNICAL ~LD OF THE INVENTION
This invention relates to rabies virus and rabies-related viruses. More particularly it relates to methods of immllni~ine host anim~l.c to protect against infections with rabies and rabies-related viruses.
BACKGROUND OF THE INVENTION
Rabies virus continues to be endemic in most areas of the world. It causes an acute central nervous system ~lice~ce which is normally fatal to hllm~n.c and domestic and wild ~nim~lc.
The virus structure is bullet-sh~ped~ consisting of a nucleo-capsid core surrounded by a membrane envelope. The nucleocapsid is comprised of a single, non-segmented strand of RNA together with RNA transcriptase (L), phosphoprotein (NS), and nucleoprotein (N).
The N protein, which is the major portion of the nucleocapsid, is noncovalently bound to the RNA to form the helical ribonucleo-protein (RNP) complex. Two viral proteins are associated with the viral envelope, the major surface antigen (G) which is glyco~sylated and the matrix protein (M) which is thought to be located on the inner leaflet of the lipid bilayer, associating with both the C-terminal dom~in of the membrane-in~serted G protein and the RNP
structure.
Virus-neutralizing antibodies raised in ~nim~l.c again~st rabies virus, only recognize the G protein. The level of antibody production has been thought to correlate with the degree of protection afforded against live virus infection. See Crick, Post Graduate Medical Journal, Vol. 49, p. 551 (1973) and Sikes, et al., Journal of American Veterinary Medical Association, Vol. 150, p.
1491 (1971). However, there are indications that antibody alone is not sufficient to protect from viral infection. For instance, passive immunization with anti-rabies antibodies without a vaccine as po~st e~L,o~lre therapy,`does not decrease the probability of infec-tion. Nicholco~, et al., Journal of Infect. Diseases, Vol. 140, p. 176 (1979). In addition, there exist effective vaccine~s which do not induce high level antibody production. One such vaccine is produced by Norden Labs.
It i~s p~ccihle that resistance to infection by rabies virus may require both virus-neutralizing antibodies and effector T-cell responses. Both the G protein and the nucleoprotein (N) have been shown to stimulate proliferation of rabies antigen-specific T cell lines. Most such T cell lines respond strongly to N protein and less strongly to G protein. A minority of rabies reactive T cell lines respond to G protein, but not at all to N protein. Celis et al., Journal of Immunology, Vol. 136, pp. 692-697, (1986). It has not been shown previously that N protein provides any protection against rabies infection in immunocompetent ~nim~ls or hllm~n.c Current vaccines against rabies consist of an inactivated rabies virus. These vaccines do not provide any cross-protection against the rabies-related virus Mokola. Preparation of current vaccines involves growth of the virus on permissive animal cells or embryos. This method of production requires the h~n-llinE of the virus by workers which entails an undesirable risk of infection.
Further, some inactivated virus vaccines can cause adverse side effects, such as demyelinating allergic encephalitis and systemic reactions, in a proportion of the vaccine recipients.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for inducing protective immunity to rabies and rabies-related viruses.
It is another object of the present invention to provide a rabies-related virus vaccine which does not require growth of viruses.
It is yet another object of the present invention to provide a rabies-related virus vaccine which can be chemically synth~-ci7ed.
It is still another object of the present invention to provide a rabies-related virus vaccine which can be produced in a micro-organism.
It is an object of the present invention to provide a rabies-related virus vaccine which stimulates proliferation of both T
and B cells.
It is another object of the present invention to provide a method of inducing protective immunity to rabies-related virus which does not cause demyelinating allergic encephalitis.
In accordance with these objects, the present invention provides an improved method for inducing protective immunity to rabies-related viruses comprising administering a priming injection of an antigen and one or more booster injections, the improvement comprising employing a polypeptide having amino acid sequence homology to rabies-related virus nucleoprotein as said priming injection, said polypeptide being substantially free of rabies-related virus glycoprotein, said polypeptide having the ability to induce proliferation of rabies-related virus-specific human T cells.
In another embodiment of the present invention, a biologically pure sample of a polypeptide is provided capable of inducing a proliferative response in rabies-related virus-specific human T cells, said polypeptide having sequence homology with the nucleoprotein of rabies-related virus and having the ability to induce protective immunity to rabies-related viruses .
In yet another embodiment of the present invention a method is provided for inducing protective immunity to a rabies-related virus comprising administering an injection of a preparation comprising a polypeptide having sequence homology with the nucleoprotein of a rabies-related virus, said polypeptide being capable of inducing a proliferative response in rabies-related virus-specific human T cells, said polypeptide being substantially free of rabies-related virus glycoprotein.
According to an aspect of the invention, a method of inducing protective immunity to rabies-related virus comprises:
administering to a human or domestic animal as a priming injection a preparation which comprises the nucleoprotein of a rabies-related virus, the preparation 4a is substantially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one or more booster injections which comprises an inactivated rabies virus.
According to another aspect of the invention, a method of inducing protective immunity to rabies-related virus consisting essentially of the step of:
administering to a human or domestic animal one injection of a preparation which comprises the nucleoprotein of a rabies-related virus, the preparation is substantially free of rabies-related virus glycoprotein.
According to a further aspect of the invention, a method of inducing protective immunity to rabies-related virus consisting essentially of:
administering to a human or domestic animal a priming injection which comprises the nucleoprotein of a rabies-related virus, the nucleoprotein is substantially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one booster injection which comprises the nucleoprotein of a rabies-related virus, the nucleoprotein is substantially free of rabies-related virus glycoprotein.
The vaccines of this invention can be produced without growing the rabies virus and they do not cause the adverse side effects associated with inactivated virus vaccines. In addition, the vaccine provides protection against heterologous rabies-related virus strains.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that the nucleoprotein of rabies virus, a protein which is internal to the membranous envelope of the virus, can serve as an effective inducer of both humoral and ce~ r immllne respon~ses. Further, a particular region of the nucleoprotein (N protein), amino acid residues 313-337, has been found to contain an epitope identified by certain anti-rabies antibodies. In addition, this isolated region of the protein (as well as two other regions, cu-,esL,onding to amino acid rP-si-luP-s 369-383 and 394-408) is able to stimulate proliferation of rabies antigen-specific human T celLs in the presence of irradiated autologous mono~llclear cells. Other suitable peptides may be found which are able to stimulate proliferation of rabies antigen-specific human T cells and which can provide protective immunity to rabies infection. Stimulation of proliferation of T celLs can be tested according to the method of Celis et al, J. Immunol. 56:426-433 (1985), and in Example 6 below.
Protective immunity can be tested according to standard methods known in the art and described in Example 7 below.
As normally practiced in the art, rabies prophylaxis is performed by a series of injections, the first one being termed a priming injection and subsequent ones termed boosters. Methods for minictering such injections, including al,prop-iate formulations, dosages, anatomical localizations and timing are generally known in the medical and veterinary sciences. Generally, the dosage desired is such that will induce a protective response without causing side effects or immlJne tolerance. Standard inactivated rabies virus vaccine, for example, may be used in the practice of the present invention as a booster inoculation. The inactivation of virus i~s generally performed by treatment with a chemical, such as -6- ~ 337~ ~ 5 beta-propiolactone. Such a vaccine is av~ hle, for example, from Institut Merieux, Lyon, France.
For the purposes of the present invention the term ~rabies-related virus~ is meant to encompass both rabies as well as rabies-related viruses such as the Mokola and European bat virus (e.g. the Duvenhage 6 strain). This definition is used because of the finding that the vaccines of the present invention provide cross-protection to heterologous virus strains. This is a great benefit provided by the present invention over prior vaccines.
Subunits of the rabies-related virus, such as N protein or RNP
complex, may be prepared and purified according to known methods.
See, for example, Virology 124:330 (1983) and J. Virol. ~:241 (19~1).
Even purer preparations of N protein can be obtained by preparative gel electrophoresis, as described in J. Virol, 44:596 (1982). Other purification steps may be used, as are known in the art.
The useful polypeptides of the present invention can be most easily prepared by chemical synthesis, according to the solid phase method described by Merrifield, Adv. Enzymol., 32:221-296 (1969).
Alternatively, proteolysis of N protein can be accomplished using enzymes or chemicals, e.g., trypsin, 3-bromo-3-methyl-2 [(2-nitro-phenyl) thiol] -3 H-indole skatole, cyanogen bromide, or protease V8. Fragments af ter cleavage can be separated by gel electro-phoresis, for example. Although many fragments can be found which are reactive with monoclonal antibodies raised against rabies N protein, only some f ragments are also able to stimulate rabies-specific T cell proliferation. The inventors have found one polypeptide to be particularly useful among a set of proteolytic cleavage products of N protein. This fragment, termed N-V12b, was originally produced by protease V-8 treatment of N protein, and later chemically syn~h~-si7ed. Fragments N-V10c and D30 were also found to stimulate T cell proliferation, although to a lesser degree.
Fragment N-V12b corresponds to the amino acids 313-337 of N
protein; fragment N-V10c co~ onds to amino acids 369-383; and fragment D30 corr~ol ds to amino acids 394-405. The sequences are shown below in Table 4. The sequence of the N protein has been previously determined by Tordo et al., Nucleic Acids Research, Vol.
14, pp. 2671-2683 (1986).
When the synthetic peptides are coupled to a protein, e.g.
keyhole limpet hemocyanin, either via thiol groups (Biochemistry 18:690-697 (1979)) or via amino groups with glutar~ ldehyde, they are found to stimulate anti-N protein antibodies in rabbits.
However, the antibody titer was lower in anti-N-V12b serum than in anti-N-VlOc serum.
Although the sequences of the polypeptides are defined herein with particularity (Table 4) it will be apparent to one skilled in the art that some of the amino acids can be conservatively substituted without losing the beneficial characteristics. It is also apparent to one skilled in the art that changes in length can be made without altering the antigenicity. The minimllm nllmher of amino acids necessary to comprise the epitopes has not been determined.
The polypeptides of the present invention can also be prepared through genetic enEineering. That is to say that parts or all of the N gene can be cloned into suitable expression vectors, as are known in the art. Organisms transformed with the cloned gene or portion thereof can be grown to produce the polypeptide products. Recovery of the polypeptide products is within the ordinary skill of the art. Comhin~tions or concatemers of antigenic polypeptide fragments can also be used. These can be made by synthesis, çlo~ine, or post-synthetic covalent bo~line.
Liposomes, which can be employed in the practice of the present invention, are known in the art. They are used as a macromolecular carrier for the polypeptides to ensure antigenicity.
Other proteins or synthetic nanoparticles may also be used as carriers. In the case of RNP a~lminictration no carrier is required.
Liposomes may be formed, e.g., according to the method of Thiholeau, ~Genetic variation among influenza viruses," Acad. Press, N.Y. (1981) p. 587. Generally, a mixture of lipids in particular ratios, such as ph~-sphatidyl rholine~ cholesterol, and lysophos-phatidyl choline, are mixed under conditions to form closed lipid vesicles. Many such conditions and methods are known in the art.
In the practice of the present invention the polypeptides are coupled to a saturated fatty acid having from about 15 to about 21 carbon atoms, before mixing with the lipid composition to form liposomes. Suitable fatty acids include, palmitic, stearic and oleic acids. Conjugates may be formed by the method of Hopp, Molecular Immunology, 21:13-16 (1984), wherein peptide fragment spacers of gly-gly-lys-(NH2)2 are added to the N-terminus of the polypeptide, and the alpha and e~ilon amino groups of lysine are used to couple with the fatty acids.
The biologically pure s~mples of the polypeptides of the present invention are substantially f ree of glycoprotein of rabies virus. They are sufficiently pure that after injection into mice, rabies glycoprotein~pecific antibody or T cells are not induced. In addition, rabies glycoprotein specific T cells would not be stimulated in vitro to proliferate in the presence of the preparation.
The following examples are illustrative only and are not intended to limit the scope of the invention. The invention is defined by the claims appended below.
9 133ill5 This example describes the selection of a multiple variant virus of rabies virus.
Seven anti-glycoprotein monoclonal antibodies c~p~hle of neutralizing parent CVS-11 rabies virus were used to sequentially select antigenic variants. Serial dilutions of virus were mixed with monoclonal antibody diluted 1:100, overlayed with nutrient agar, and after 4 to 5 days of incubation at 35C in a 5% carbon lioxide atmosphere, plaques were selected. Viruses able to replicate in the presence of the monoclonal antibodies used for their selection were recovered. The seventh generation variant CVS-V7 was not neutralized by any of 40 diiferent rabies virus-specific neutralizing monoclonal antibody. The nucleoprotein antigen of the CVS-V7 virus rem~ined immllnologically indistinguishable from that of the CVS-11 parent virus.
This ex~mple shows the protection afforded by vaccination with the CVS-V7 virus, as well as the virus neutralizing antibodies it induced against parent strain CVS-11.
Vaccines prepared f rom the CVS-V7 variant viruses, were used to immunize mice. Groups of nine 4-week old female ICR mice were immllni7pd with 0.2 ml of 5 serial dilutions (2000-3 ng) of the CVS-V7 inactivated virus vaccine on days 0 and 7. The inactivated virus vaccine was prepared with beta-propiolactone and adjusted to a protein concentration of 100 ug/ml. The viruses had been grown on BHK 21 cell monolayers and purified as described in Journal of Virology, Vol. 21, pp. 626-635 (1977). On day 14, vaccinated and unvaccinated control mice were bled and infected intracerebrally with 0.03 ml (50 MIC LD50) of CVS-11. (CVS-11 parent virus was used in the ch~llenge because the CVS-V~ variant virus was not pathogenic for adult mice.) The ~nim~l.c were observed for 3 weeks and mortalities were recorded daily. The effective dose was calculated for each vaccine as described in Atansiu, P., ~Quanti-tative assay and potency test of antirabies serum," In: Laboratory Techniques In Rabies, 2nd Ed., Geneva: World Health Organization, 1966, pp. 16~-~2. The neutralizing activity of the mouse immune sera for CVS-ll was determined as described in Lumio et al., Lance, Vol. i, p. 3~8 (1986).
The geometric mean tighter (GMT) was calculated for each vaccination group. Multiple group comparisons for differences in GMT were tested by one way analysis of variance and two-group GMT were compared statistically by a one-tailed test.
As can be seen in Table 1, virus neutralizing activity as well as protection from death were provided by the CVS-V7 vaccine. As the CVS-V~ virus, from which the vaccine was prepared, was not neutralized by any of 40 rabies glycoprotein-specific monoclonal antiho~liP-s and yet was able to confer protection against ch~llPnge with the parental CVS-11 strain, it is concluded that the glyco-protein is not the sole factor in determining the relative efficacy of rabies prophylaxis.
This example demonstrates the protection afforded and the virus neutralizing antibody induced by means of rabies virus subunit vaccines which are formulated with liposomes.
The glycoprotein and ribonucleoprotein (RNP) were purified from the CVS-V~ variant virus as described in Diet7chold et al., CVS-V7-G- CVS-V7-N- CVS-V7-(N+G)-Vaccine CVS-V7-Virus Liposome Liposome Liposome Vaccine VNA against Mortality VNA agalnst MortalityVNA a~ainstIViortallty VNA agalnstiviortallty Concen- GMT (range) Rate GiviT (range) Rate GMT (range) GMT (range) tration [ng]
10000 (27o-l62o) 0/7 (<10-810) 1/s 7/7 588 1/6 2000 (60 540) 2/7 (290 540) 2/7 0 7/7 (260 810) 1/7 400 (60-540 3/7 (<10 180) 4/~ 7/7 323 1/7 (<10-540) 3/7 (211o 60) 3/6 0 7/7 (<10-540) 1/7 1~ (<10-540) 3/7 (<10 30) 5/6 o 7/7 (<10-50) 5/7 Effective 33.3 ng 588 ng 45 ng (ED50) i Table 1 - Protective activities of an inactivated CV~ S-V7 virus vaccine and CVS-V7 subunit vaccines against an i.c. chalienge infection with CV~l 1 virus.
Isolation and Purification of a Polymeric Form of the Glycoprotein of Rabies Virus, J. Gen. Virol. 1978, 40 131-135 and Schneider et al., Rabies Group-Specific Ribonucleoprotein Antigen and a Test System for Grouping and Typing of Rhabdoviruses 1973, J. Viral. 11, 748-755.
The proteins were inserted into liposomes as described in Thibodeau, Genetic Variation Among Influenze Viruses, Acad. Press, N.Y. 1981, p. 587. The amount of rabies protein liposomes used per injection is expressed as weight of the total complex. Mice were injected intracerebrally on days O and 7 and ch~llenged at day 14 as described in Example 2. The resulting mortality rates and amount of virus neutralizing antibody produced are shown in Table 1.
The RNP incorporated into liposomes induced no detectable VNA against CVS-ll nor any detectable protection from death. The CVS-V7-derived G protein incorporated into liposomes induced low titers of VNA and poor protection from death. However, when the RNP was combined with G protein, the treatment was significantly better than either protein alone, and produced results roughly comparable to those obtained with the whole virus vaccine. This too, shows the importance of N protein in immunity to rabies.
This ex~mple demor~ctrates that rabies virus RNP can augment the function of B cells producing neutralizing antibody.
Groups of mice were primed with either 5 ug of N protein (from the ERA strain of rabies) plus complete Freund's adjuvant (CFA) or CFA alone. Ten days after priming both groups of An;m~l~
received serial dilutions of inactivated rabies virus vaccine or isolated rabies glycoprotein. Mice that were primed with RNP plus CFA and boosted with rabies virus vaccine developed significantly higher VNA titers than those mice primed with CFA alone and then boosted with a rabies virus vaccine. The results are shown in Table 2. In addition, the effective dose of rabies virus vaccine was determined and found to be about 10-fold lower in the mice which had been primed with RNP.
The mice which received booster vaccinations consisting of just rabies virus glycoprotein developed only low levels of VNA and were poorly protected against a lethal ch~lle~Ee infection with rabies virus, which can be seen in the right half of Table 2. This suggests that there must be common antigens between the priming and boosting immllni7ations in order to induce an effective immune response.
The mortality rates shown in Table 2 were determined by ch~llengin~ all mice with an intracerebral innoculation with 50 MIC
LD50 Of rabies virus and observing the survival up to 3 weeks post-infection.
This ~x~mplPs demonctrates that protective immunity can be induced using rabies virus RNP to an intramuscular (I.M.) ch~llpn~e infection with homologous or heterologous rabies virus strains.
Purified RNP of the ERA strain of rabies or of the rabies-related strain Mokola was used to immunize Balb/c mice against an (I.M.) ch~llenEe with the rabies strain CVS-24. The RNP
was isolated and purified according to the method of Schn~ider, et al., J. Virol., Vol. 11, pp. ~48-~55 (19~3). Groups of 10 to 20 mice were immunized intraperitoneally (I.P.) with ERA-or Mokola-RNP
plus CFA, or with CFA alone, or subcutaneollcly (S.C.) with ERA-RNP alone. Four weeks after immunization, the mice were ch~ nged (I.M.) with eight mouse I.M. LD50 f CVS-24 rabies virus Booster immunization with Booster immunization with Vaccine ERA-virus ERA~
tration Priming with Priming with Priming with Priming with (ng) ERA-N + CfA CFA ERA N + CFA CFA
VNA a~aalnst Mortallty Vna a~alr6t Mortdlty VNA a~al~t Mortallty VNA agalr~t Mortallty CVS-l r Rate CVS-ll I?ate CVS~ ate CVS-l 1 Rate GMT (tan~e) GM~ (rar~e) GMT (rar~e) GlvlT (rrar~e) 5000 (1~C~4~0) 3/7 (<10-180) 5/5 (6C~80) / 28 6/7 489 39 6/7 25 6/7 ~ 7/7 (6C~1620) 4/7 (10-180) (10-60) (c10~60) 138 10 13 1.3 200 (20-1620) 3/7 (<lO~S0) 6 n (10-20) 6/7 (<1C~20) 7/7 34 26 4 1.3 (<1C~270) 5/6 (20~60) 6/7(<1C~20) 7/7 (<10-20) 7/7 8 (<10-540) 6/7 (<1C~30) 7/7(<1C~20) 7/7 6/6 Effective ~,~
oDs5eO) ~25r~3 >5000ng ~5000 ng ,5000 n~
~able 2 - Effect of RNP - priming on VNA tite~ and mortality rates (see Table 3) or two mouse I.M. LD50 f rabies-related virus Duvenhage 6 (see Table 4).
As can be seen in Table 3, 80% of the mice that received ERA-RNP plus CFA intraperitoneally or ERA-RNP alone subcatane-ously survived the ch~llenee. In addition, 90% of the mice that were immunized intraperitoneally with Mokola-RNP plus CFA survived the ch~llen~e infection. In contrast only 10% of the mice that received only complete Freund Adjuvant (CFA) succumbed to rabies.
It is known that neutralizing antibody produced against the Mokola virus does not neutralize rabies virus; therefore, this experiment clearly demonstrates that the protection conferred by RNP is not due to neutralizing antibody which may have been induced by very small, undetectable amounts of glycoprotein. In addition, the results shown in Table 4 demonstrate that RNP from both the ERA strain and from the Mokola virus induce protective immunity against the rabies-related European bat virus strain Duvenhage 6. Taken together these results demonstrate that RNP
purified from rabies virus and rabies-related viruses can induce protective immunity against heterologous viruses.
The synthetic peptides shown below in Table 5 were synth~-ci7ed according to the method of Merrified, cited above. In a typical coupling reaction, the tboc group of the amino terminus was removed with 50% trifluoroacetic acid (TFA). After neutralization with N, N-diisopropylethylamine (DIEA), a 4- to 6-molar excess of preformed tboc-amino acid-pentafluorophenylester (Kisfaludy et al., Liebigs Ann. Chem. pp. 1421-1429 (19~3)) and a 1-molar equivalent of DIEA in dichloromethane (1:1) were added. After b~hbling with N2 gas for 1.5-2 h, the resin was analyzed for the presence of free amino groups as described by Kaiser et al. (Anal. Biochem., 34:595-598 (1970)). Couplings were repeated until less than 1% free NH2 groups were found.
Peptides were cleaved and deblocked with HF/thio~nicol (10:1) at 0C for 30 min, and the peptide was extracted with 0.1 M
NH4HCO3 and lyophili7ed~ The crude peptide was then dissolved in 0.1 M NH4HCO3 and purified on a BioGelT~ P4 column calibrated with 0.1 M NH4HCO3. The eluted peptide was then applied to a Vydac~ 2P C18 reverse-phase column and eluted with methanol-water (8:2). The elution of the peptide was monitored with a UV
detector at 214 nm. To verify the amino acid sequence, aliquots of the peptide were subjected to amino acid analysis and amino acid sequencing.
The peptides were tested for the ability to stimulate T-cell proliferation in the presence of antigen-presenting populations of cells of the same HLA-DR type. At concentrations of 0.4 ug/ml to 25 ug/ml of protein, fragments N-V12b and N-VlOc, and N protein isolated from ERA strain virus, stimulated proliferation of the T-cells. Hepatitis B antigens, used as controls, caused no stimulation. At low concentrations, N-V12b was more stimulatory than N protein, whereas at higher concentrations the reverse was true.
The rabies specific T-cell lines were isolated and tested as described in Celis et al., J. Immunol. 56:426-433 (1985).
This e~mrle demonstrates the protective c~p~hilities of the synthetic peptides N-V10 and N-V12b against the rabies strain CVS-24.
Immunization of Balb/C mice with Rabies-RNP and Mokola-RNP again~t an l.M. challenge with CVS-24 Antigen Route of Mortality (%) Immunization + CFA 2/10 (20) 10 ug ERA-RNP S.C. 2/10 (20) 10 ug MOK-RNP l.P. 1/10 (10) + CFA
CFA l.P. 18/20 (90) 1 3371 1 5 -i~- T~BLE 4 Immunization of Balb/C mice with ERA-RNP and Mokola-RNP against an l.M. challence with DUV 6 virus Antigen Immunization Mortality (%) 10 ug ERA-RNP + CFA l.P. 0/10 (0) 10 ug Mok-RNP + CFA l.P. 1/10 (10) CFA l.P. 6/10 (60) N-VlOc NH2-tyr-glu-ala-ala-glu-leu-thr-lys-thr-asp-val-ala-leu-ala-asp.
N-V12b NH2-his-phe-val-gly-cys-tyr-met-gly-glu-val-arg-ser-leu-asn-ala-thr-val-ile-ala-ala-cys-ala-pro-his-glu.
D-30 NH2-tyr-phe-ser-gly-glu-thr-arg-ser-pro-glu-ala-val-tyr-thr-arg.
-Groups of 5 to 10 mice were immunized intraperitoneally with N-V12b-liposomes plus CFA, N-VlOc-liposomes plus CFA, or lipo-somes plus CFA. The mice were then ch~llenged with various amounts of CVS-24 virus.
The lirllsomes were formed by the method of Thibodeau, Genetic Variations Among Influenza Viruses, Acad. Press, N.Y., 58~
(1981). The peptides were incorporated into liposomes as described above in ~x~mple 3. To facilitate the incorporation of the peptides into liposomes, palmitic acid was linked to the amino terminal end of the peptide as described by Hopp, Mol. Immunol., Vol. 21, pp.
13-16, 1984. In experiment No. 1, two times the mouse I.M. LD50 was used as ch~llen~e. As shown in Table 6, 88% of the mice that received the N-V12b-liposome vaccine survived while none of the mice immunized by N-VlOc-liposome survived three weeks after the ch~llenge.
In experiment No. 2, four times the mo~Lse I.M. LD50 wa,s used as a ch~llenge. Sixty-two percent of the mice immunized with peptide N-V12b-lip~some vaccine survived while only 12% of the mice which received the control vaccine of liposomes plus CFA
survived the ch~llenge.
In experiment No. 3, 8 times the mouse I.M. LD50 was used as a ch~ nge. Sixty percent of the mice immunized with the N-V12b-lip~some vaccine survived the ch~llenEe, while only 10 and 20 percent respectively of the mice immunized with N-V10 liposomes plus CFA and liposome control vaccine plus CFA survived.
Thus, peptide N-V12b provides good protection from rabies virus ~h~llen~e when incor~orated into liposomes and administered with CFA. Peptide N-VlOc however does not provide good -2l- l 3371 l 5 TABLE 6 Protective activitie~ of N-Y1 2b peptide liposomes in mice - to i.m. challen~e with CVS-24 Experiment #1, i.m. challenge with 2 MIM LD 50 Concentratlon (u~) Vacclne R te 15 N-V10-Liposomes +-CFA 5J5 15 N-V1 2b-Liposomes + CFA 1 /8 Experiment #2, i.m. challenge with 4 MIM LD
so Vacclne Vacclne Mortallty Concentratlon (u~) ~ a e N-V12b-Liposomes + CFA 3/8 --- Liposome ~ CFA 7/8 Experiment #3, I.m. challenge with 8 MIM LD
Concentratlon (u~) Vacclne R te 15 N-V12b-Liposomes + CFA 4/10 15 N-V10 Liposomes + CFA 9/10 --- Liposomes +CFA 8/10 -Z2- l 337 1 1 5 protection against rabies virus ch~llenge~ even though it is able to stimulate rabies specific T cell proliferation.
Peptides consisting of 15 amino acids were synth~-si~ed which together correspond to the entire amino acid sequence of the ERA-N
protein. These were synth~si~ed a,s described above in Example 6.
Each peptide was screened for T cell proliferative activity in vitro and for protective activity in vivo as described above for peptides N-V12b and N-V10c.
The results are displayed in Table ~. Peptide nllmher 30D
demon~strated both significant T cell stimulatory activity as well a~s partial but significant protection against rabies viru~s ch~llenge.
Peptide D30 protected 60% of the immunized mouse population from a ch~llenge of 8 times the mouse I.M. LD50 of CVS-24 viru~s.
TI~BLE 7 T-Cell Stimulatory and Protective Activities of synthetic N-peptides Peptide No.T-cell Stimulatory Activity Mor~lit~ b~ Averace Su~.val Tima Days Liposo- e ctr. - / ~ b ' .-/- ~ . .
+ / "b ' .-- /' ~- O
- / ~b .9 + t` / b1 ) .1 O~lu - 7/l - / b + /' 'o - /' ~1 ~.1 _/ ~, . 7 + / "b . 3 - / b .5 - / ,~ 0.2 /- , 9.
+ -/- b 9 + / % 1 .
++ , /'0~ 1 ~ 2~ +++ 4/9 ( %) V1 2b- - 711~ . ~%) V1 b- I - 1 / 1 ( %) V12"- 1 - 1 ( V1 2~- /
V1 C +++ 9/1 (9 ~) .'
Claims (3)
1. The use of an injectable preparation for inducing protective immunity to rabies-related virus, said preparation comprising:
the nucleoprotein of a rabies-related virus, said preparation being substantially free of rabies-related virus glycoprotein.
the nucleoprotein of a rabies-related virus, said preparation being substantially free of rabies-related virus glycoprotein.
2. The use of an injectable preparation as a priming injection for inducing protective immunity to rabies-related virus, said preparation comprising:
the nucleoprotein of a rabies-related virus, said preparation being substantially free of rabies-related virus glycoprotein.
the nucleoprotein of a rabies-related virus, said preparation being substantially free of rabies-related virus glycoprotein.
3. The use of an injectable preparation as a booster injection for inducing protective immunity to rabies-related virus, said preparation comprising:
the nucleoprotein of a rabies-related virus, said nucleoprotein being substantially free of rabies-related virus glycoprotein.
the nucleoprotein of a rabies-related virus, said nucleoprotein being substantially free of rabies-related virus glycoprotein.
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| Application Number | Priority Date | Filing Date | Title |
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| US7963987A | 1987-07-30 | 1987-07-30 | |
| US079,639 | 1987-07-30 |
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| CA000573293A Expired - Fee Related CA1337115C (en) | 1987-07-30 | 1988-07-28 | Vaccination against rabies-related diseases |
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| EP (1) | EP0326598A1 (en) |
| CA (1) | CA1337115C (en) |
| WO (1) | WO1989000861A1 (en) |
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| FR2645173B1 (en) * | 1989-03-29 | 1994-06-17 | Pasteur Institut | CLONING AND EXPRESSION OF GENES ENCODING PEPTIDES AND / OR FRAGMENTS OF MOKOLA VIRUS PEPTIDES, APPLICATION TO THE PREPARATION OF A V |
| AU2359195A (en) * | 1994-04-19 | 1995-11-10 | Thomas Jefferson University | Viral ribonucleocapsid as an immunological enhancer |
| CA2364150A1 (en) * | 2001-12-07 | 2003-06-07 | Mireille Lafage | Polypeptides inducing apoptosis, polynucleotides that code for them and their therapeutic applications |
| WO2011163446A2 (en) | 2010-06-24 | 2011-12-29 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Pan-lyssavirus vaccines against rabies |
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| DE3683688D1 (en) * | 1985-04-19 | 1992-03-12 | Wistar Inst | VACCINE FOR THE GENERATION OF AN IMMUNOGENIC T-CELL REPLY PROTECTING A VIRUS. |
| EP0237686A1 (en) * | 1986-03-18 | 1987-09-23 | Institut Pasteur | DNA sequences derived from the rabies virus genome |
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1988
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