AU608354B2 - Vaccination against rabies-related viruses - Google Patents

Description

AU-AI -21364/8842 WORLD INTELLECTUAL PROPERTY ORGAI4ZAT16N.

International Bureau

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PCI'

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATiON TRtJ.ATY (PCT) 1) Interqational Patent Classification 4 (11) International Publication Number: WO 89/ 00861 A61K 39/205, C07K 7/10 Al (43) International Publication Date: February 1989 (09.02.89) I !E (European pavent), BG, BR, CH, CH (European (22) International Ffiling Date: 29 July 1988 (29.07.88) patent), DE, DE (European patent), DK, Fl, FR (European patent), GB, GB (European patent), HU, IT (31) Priority A4pplication Number: 079,639 (Epean paten), C, G, W, NL NLU, (Europpeptent), OCR, SD, E, E N (European pa-U (32) Priority Date: 30 July 1987 (30.07,87) tn) O O D E E(uoenptn)

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(33) Priority Country-, US Pubifshed With international search report.

Beflore the expiration of the time limit for amending the (71) Applicant: THE WISTAR INSTITUTE [US/US]; claims and to be republished in the event of the r(.ceipt Thirty-Sixth Street at Spruce, Phifladelphia, PA 19104 of amendments.

2 ru APR 1989 (72) Inventors-, DIETZSCHOLD, Bernhprd ,3034 Goshen Road, Newton Square, PA !9073 KOPROWS KI, Hilary ;334 Fairhill Road, Wyneewood, PA 19096 (US), (74) Agents: HOSCHEIT, Dale, H. et al., Banner, Birch, 1M~AR 1989 McKie Beckett, 0Oe Thomas Circle, Wash- ATeNT t itoDC 20005

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(54) Title; VACCINATION AGAINST RAB IES-RE1LATED VI RUS 117 1,71is do(,Ih~ ~1tills tie amendments Inac li ndf-T SectIoll 49 an1d is corvect [or' N-V lao N-V1 2b

NH

2 -tyr-glu-ala-ala-glu-Ieu-thr-1ys-thr~asp-valala -leu--ala-asp.

N H 2 -his-phe-val-gly-cys-tyr-met-gly-glu-val-argser-Ieu-asn-ala- thr-v ai-fle-ala-ala-cys-ala-prohls-glu.

N H 2 -tyr-phe-sfc-gly-glu-thr-arg-ser-pro-glu-alaval-tyr-t-r-arg.

(57) Abstract M~ethods of vaccinating to induce protective immunity to rabies anid rabies-related viruses are taught wherein certain synthetic, genetically engineered, or rabies-derived polypeptides are used. The sequence of the polypaptides is deriv~C4 from ttiz N ptotein. Both B and T cells are stimulated by these antigenic polypeptides to provide ifnmnuaity to rabies and other related infections.

WO 89/00861 PCT/LS88/02529 VACCINATION AGAINST RABIES-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 FIELD OF THE INVENTION This invention relates to rabies virus and rabies-related viruses. iMore particularly it relates to methods of immunizing host animals 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 disease which is normally fatal to humans and domestic and wild animals.

The virus structure is bullet-shaped, consisting of a nucleocapsid core surrounded by a membrane envelope. The nucleocapsid is comprised of a single, non-segmented strand of RNA together with RNA transcriptase phosphoprotein and nucleoprotein The N protein, which is the major portion of the nucleocapsid, is noncovalently bound to the RNA to form the helical ribonucleoprotein (RNP) complex. Two viral proteins are associated with the C IC i WO 89/00861 PCT/US88/02529 -2viral envelope, the major surface antigen which is glycosylated and the matrix protein which is thought to be located on the inner leaflet of the lipid bilayer, associating with both the C-terminal domain of the membrane-inserted G protein and the RNP structure.

Virus-neutralizing antibodies raised in animals against 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 post-exposure therapy, does not decrease the probability of infection, Nicholson, et al., Journal of Infect. Diseases, Vol. 140, p. 176 (1979). In addition, there exist effective vaccines which do not induce high level antibody prcduction. One such vaccine is produced by Norden Labs.

It is possible 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 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. Cells 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 animals or humans

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WVO 89/00861 PCT/US88/02529 -3- 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 handling 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.

CUMMARY OF THE INV&-TTICEN-- It is an object of the present invention to provide a methd for inducing protective immunity to rabies and rabies-r elted viruses, It is another object of the present inventionn to provide a rabies-related virus vaccine which does not ,rquire growth of viruses, It is yet another object of the p esent invention to provide a rabies-related virus vaccine which .a be chemically synthesized, It is still another objec of the present invention to provide a rabies-related virus vacc de which can be produced in a microorganism. 7 It is an object of the present invention to provide a rabies-related'virus vaccine which stimulates proliferation 'f both T and B celi It is another object of the present invention to provide a method of inducing protective immunity to rabies-related virus *whiee5aeef~^ifi-i(tgeee^t4g -4- The present invention provides a method of inducing protective immunity to rabies-related virus comprising: administering to a human or domestic animal as a priming injection a preparation comprising the nucleoprotein of a rabies-related virus, tsad prepiration being substantially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one or more booster injections comprising an inactivated rabies virus.

The present invention also provides a method of inducing protective immunity to rabies-related virus consisting essentially of the step of: administering to a human or domestic animal one I injection of a preparation comprising the nucleoprotein of a 15 rabies-related virus, said preparation being substantially Sfree of rabies-related virus glycoprotein, and a method of inducing protective immunity to rabies-related virus consisting essentially of: administering to a human or domestic animal a priming injection comprising the nucleoprotein of a rabiesrelated virus, said nucleoprotein being substantially free of rabies-related virus glycoprotein; and ,,tt administering to the human or domestic animal one booster injection comprising the nucleoprotein of a 25 rabies-related virus, said nucleoprotoin being substanti ally free of rabies-related virus glycoprotein.

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i, i ~r ernmrrrrr~r~ NVO 89/0061 PCT/L'S88/02529 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 cellular immune responses. 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, corresponding to amino acid residues 369-383 and 394-408) is able to stimulate proliferation of rabies antigen-specific human T cells in the presence of irradiated autologous mononuclear 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 Cells 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 ncrz ally 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 administering such injections, including appropriate 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 immune 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 is generally performed by treatment with a chemical, such as WO 89/00861 PCT/US88/02529 -6beta-propiolactone. Such a vaccine is available, for example, from Institut Merieux, Lyon, France.

For the purposes of the present invention the term "rabiesrelated virus" is meant to encompass both rabies as well as rabies-related viruses such as the Mokola and European bat virus the Duvenhage 6 strain). This definition is used because of the finding that the vaccines of the preste;t 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. 7:241 (1971).

Even purer preparations of N protein can be obtained by preparative gel electrophoresis, as described tn 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, trypsin, 3-bromo-3-methyl-2 [(2-nitrophenyl) thiol] -3 H-indole skatole, cyanogen bromide, or protease V8. Fragments after cleavage can be separated by gel electrophoresis, for example. Although many fragments can be found which are reactive with monoclonal antibodies raised against rabies N protein, only some fragments 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 synthesized. Fragments N-V10c and D30 were also WO 89/00861 PCT/'S88/02529 -7found 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-VlOc corresponds to amino acids 369-383; and fragment D30 corresponds 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 gt lups (Biochemistry 18:690-697 (1979)) or via amino groups with glutardialdehyde, they are found to stimulate anti-N protein antibodies in rabbits, However, the antibody titer was lower in anti-N-Vl2b serum than in 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 minimum number of amino acids necessary to comprise the epitopes has not been determined.

The polypeptides of the present invention can also be prepared through genetic engineering. 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. Combinations or concatemers of antigenic polypeptide fragments can also be used. These can be made by synthesis, cloning, or post-synthetic covalent bonding.

WO 89/00861 PCT/US88/02520 -8- 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 administration no carrier is required.

Liposomes may be formed, according to the method of Thibodeau, "Genetic variation among influenza viruses," Acad. Press, N.Y. (1981) p. 587. Generally, a mixture of liOids in particular ratios, such as phosphatidyl choline, cholesterol, and lysophosphatidyl 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 epsilon amino groups of lysine are used to couple with the fatty acids.

The bio'ogically pure samples of the polypeptides of the present invention are substantially free of glycoprotein of rabies virus. They are sufficiently pure that after injection into mice, rabies glycoprotein-specific 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, ~g i~l W O 89/00861 PCT/L'S88/02529 -9- EXAMPLE 1 This example describes the selection of a multiple variant virus of rabies virus.

Seven anti-glycoprotein monoclonal antibodies capable 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 35 0 C in a 5% carbon dioxide 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 different rabies virus-specific neutralizing monoclonal antibody. The nueleoprotein antigen of the CVS-V7 virus remained immunologically indistinguishable from that of the CVS-11 parent virus.

EXAMPLE 2 This example 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 from the CVS-V7 varifint viruses, were used to immunize mice, Groups of nine 4-week old female ICR mice were immunized 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 wih 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

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1 VWO 89/00861 PCT/US88/02529 with 0.03 ml (50 MIC LD 5 0 of CVS-11, (CVS-11 parent virus was used in the challenge because the CVS-V7 variant virus was not pathogenic for adult mice.) The animals were observed for 3 weeks and mortalities were recorded daily. The effective dose was calculated for each vaccine as described in Atansiu, "Quanti, tative assay and potency test of antirabies serum," In: Laboratory Techniques In Rabies, 2nd Ed., Geneva: World Health Organization, 1966, pp. 167-72, The neutralizing activity of the mouse immune sera for CVS-11 was determined as described in Lumio et al., Lance, Vol i, p. 378 (1986).

The geometric mean tighter (GMT) was calculated for eac, vaccination group, Multiple group comparisons for differences iti 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 wel.

as protection from death wero provided by the CVS-V7 vaccine, As the CVS-V7 virus, from which the vaccine was prepared, was not neutralized by any of 40 rabies glycoprotein-speclfic monoclonal antibodies and yet was able to confer protection against challenge with the parental CVS-11 strain, it is concluded that the glycoprotein is not the sole factor in determining the relative efficacy o: rabies prophylaxis, EXAMPLE 3 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-V7 variant virus as described in Dietzchold et al.

WO 89/00861 WO 8900861PCT/CS88/02529 Id) z 0

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t i o WO 89/00861 PCT/LS88/02529 -12- 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 descried 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 0 and 7 and challenged 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-11 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 viris vaccine, This too, shows the importance of N protein in immunity to rabies, EXAMPLE 4 This example demonstrates that rabies virus RNP can augment the function of B cells producing neutralizing antibody, Groups of mice were primed with eith7r 5 ug of N protein (from the ERA strain of rabies) plus completa Freund's adjuvant (CFA) or CFA alone. Ten days after priming both groups of animals 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 (iters than those mite primed with CFA alone and then WO 89/00861 PCT/US88/02529 -13boosted 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 challenge 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 immunizations in order to induce an effective immune response.

The mortality rates shoo'n in Table 2 were determined by challenging all mice with an intracerebral innoculation with 50 MIC

LD

5 0 of rabies virus and observing the survival up to 3 weeks post-infection, EXAMPLE This examples denonstrates that protective immunity can be induced using rabies virus RNP to an intramuscular challenge infection with homologous or heterologous rabies virus strains, Purified RNP of the ERA strain of rables or of the rabies-related strain Mokola was used to immunize Balb/c mice against an challenge with the rabies strain CVS-24, The RNP was isolated and purified according to the method of Schneider, et al., J. Virol., Vol. 11, pp. 748,,755 (1973). (Goups of 10 to 20 mice were immunized intraperitoneally with ERA-or Mokola-RNP plus CFA, or with CFA alone, or subcutaneously with ERA-RNP alone. Four weeks after immunization, the mice were challenged with eight mouse I.M. LDg 0 of CVS-24 rabies virus vow Voccine Concentroion ing) Booster immuni_-lion with ERA-viri.s Booster irmmnunizotion with

ERA-G

Prminwg with Priming with PYImIng wh I Priming with ERA-N +CFA CFA ERAN CFA CFA L W"ZoIa ?airW

MOMOMY

crVuo auto GMT PC"* GM! o1o.

GWOOVO

GM! at I(Mar" cvS-1Y am*n GM4T (2"gW) f

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la 4x 5/5 87 7/7 286/7 3/7 (<10-180 (W80) 10-1w 89 39 25 6677 1 6/7 671 1000 (60_162M 47 (10-180) (10- (1o 138 10 13 J13 200 20-1620) 3/7 (<10-6m (10-20) 6/7 17/7 34 26 4 1.3 4) (<10-270) 5/6 o6M 6 (10-20) 7/7 (0120, 7/7 26 10 1.5 8 (<1030= 7/7 6/6 Effective 625ng 50SOOO ng 500 ng Tobe 2 J,?ect Of RNP priming on VNA Ifters ond mortoity rates

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WO 89/00861 PCT/US88/02529 (see Table 3) or two mouse I.M. LD 5 0 of 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 subcataneously survived the challenge. In addition, 90% of the mice that were immunized intraperitoneally with Mokola-RNP plus CFA survived the challenge 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 demonstraaes 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 demorntrate that RNP purified from rabies virus and rabies-related viruses can induce protective immunity against heterologous viruses.

EXAMPLE 6 The synthetic peptides shown below in Table 5 were synthesized 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-penltafluorophenylester (Kisfaludy et al., Liebigs Ann, Chem. pp. 1421-1429 (1973)) and a 1-molar equivalent of DIEA in dichloromethane were added. After bubbling with

N

2 gas for 1.5-2 h, the resin was analyzed for the presence of free I I II WO 89/00861 PCT/LS88/02529 -16amino groups as described by Kaiser et al. (Anal. Biochem., 34:595-598 (1970)). Couplings were repeated until less than 1% free

NH

2 groups were found.

Peptides were cleaved and deblocked with HF/thioanisol (10:1) at 0°C for 30 min, and the peptide was extracted with 0.1 M

NH

4

HCO

3 and lyophilized. The crude peptide was then dissolved in 0.1 M NH 4

HCO

3 and purified on a BioGel M P4 column calibrated with 0.1 M NH4HCO 3 The eluted peptide was then applied to a Vydac RP C18 reverse-phase column and eluted with methanolwater 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 "I-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 ug/ml of protein, fragments N-V12b and N-V10c, 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 Cells et al., J. Immunol, 56:426-433 (1985).

EXAMPLE 7 This example demonstrates the protective capabilities of the synthetic peptides N-V10 and N-V12b against the rabies strain CVS-24.

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Immunization of Baib/C mice with Rabies-RNP and Mokola-RNP against an I.M. challenge with CVS-24 Antigen Route of otlt Antigen Immunization Motly(% ug ERA-RNP I.P. 2/10 CFA ug ERA-RNP S.C. 2110 ug MOK-RNP 1/10 CFA E CFA LID. 18/20

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Immunization of Baib/C mice with ERA-RNP and Mokola-RNP against an t.M. chailence with DUV 6 virus Antigen Route ofMotly Antigen Immunization Motly(% ug ERA-RNP CFA I.P. 0/10 (0) ug Mok-RNP +CFA 1/10 CFA I.P. 6/10 00)

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WVO 89/0086 1 PCT/US88/02529 TABLE N-VlOc N -Vi2b N H 2 -tyr-glu-ala-ala-glu-Ieu-thr-lys-thr-asp-valala-leu-ala-asp.

N H 2 -his-phe-val-gly-cys- tyr-me t-gly-glu--val-argser-leu-asn- ala- thr-v al-ile-al a-al a-c ys-ala-prohis-glu.

N H 2 tyr- phe-ser-gly-gl u- t hr-a rg-2-r- pro-glu- alaval-tyr-thr-arg.

.i i yri~l WO 89/00861 PCT/US88/02529 Groups of 5 to 10 mice were immunized intraperitoneally with N-V12b-liposomes plus CFA, N-VlOc-liposomes plus CFA, or liposomes plus CFA. The mice were then challenged with various amounts of CVS-24 virus, The liposomes were formed by the method of Thibodeau, Genetic Variations Among Influenza Viruses, Acad. Press, 587 (1981). The peptides were incorporated into liposomes as described above in Example 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, LD 5 0 was used as challenge. As shown in Table 6, 88% of the mice that received the N-Vl2b-liposome vaccine survived while none of the mice immunized by N-VlOc-liposome survived three weeks after the challenge.

In experiment No. 2, four times the mouse I.M. LD 5 0 was used as a challenge, Sixty-two percent of the mice immunized with peptide N-V12b-liposome vaccine survived while only 12% of the mice which received the Control vaccine of liposomes plus CFA survived the challenge, In experiment No. 3, 8 times the mouse IM, LD 5 0 was used as a challenge. Sixty percent of the mice immunized with the N-Vl2b-liposome vaccine survived the challenge, while only 10 and percent respectively of the mice immunized with N liposomes plus CFA and liposome control vaccine plus CFA survived.

Thus, peptide N-V12b provides good protection from rabies virus challenge when incorporated into liposomes and administered with CFA. Peptide N-VOc however does not provide good

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WO 89/00861 W 0 8900861PCT/US88/02529 iADLL b -21- Protective activities of N*V12b peptide ilposomes In mice to l.m. challenge with CVS124 Experiment 91, Im. challenge with 2 MIM LD o Vaccine Mortality Concentratlion u)VcieRt IsN'VIOLUosornos *-CFA I NI.Vtbosom~s CFA Its Experiment 92, 1^m challenge with 4 MIMA LID Vaccine Vaccine mortality Concentration Nog) note Is N-YM2-I.Ocecmo CFA 3/S Upoorm*CFA 7/8 Experiment 03, l.m. challenge with I MIM LO Vaccine Mortality Concentration (us) Veceine note is N-VtbLiposome.+CFA 4110 LUPooine CFA 9/10 Uposines+CPA 11110 WO 89/00861 PCT/US88/025 2 9 -22protection against rabies virus challenge, even though it is able to stimulate rabies specific T cell proliferation.

EXAMPLE 8 Peptides consisting of 15 amino acids were synthesized which together correspond to the entire amino acid sequence of the ERA-N protein. These were synthesized as 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-VlOc.

The results are displayed in Table 7, Peptide number demonstrated both significant T cell stimulatory activity as well as partial but significant protection against rabies virus challenge, Peptide D30 protected 60% of the immunized mouse population from a challenge of 8 times the mouse I.M. LD 5 0 of CVS-24 virus, 1.

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T-Ceii Stimuiatory and Protective Activities of synthetic N-peptides Pood& No- T-cell Stimulatory Activity Mortmlhv Averse* Sufvival Tis Oz- Upoem~e ct. ailo too%) 10.1 110 (90%l 10.1 7110 10.1 W10 9D 7/10 0-0 110 0/10 1'J.1 130 si10 9.1 140D 10/10 (100) 0.3 150 7/10 170) 0.0 ISD 01 0.3 170 *7/10 9.6 150 'silo (o0n) 10.1 2001 .7110 0.7 220 9 /10 9.3 23D si10 240 11 10.1 25D 9/10 9.71__ 260 +7/10 (70j%) 0.6 260D 9/10 10.3 300 411 10.5 V12b 41 Vl2b-I 7/10 9.4 V12b.I 10111 9.4 V12b-tIl 101 91%) VI2b-IV 010100%) VIOC 9/10 (90M%) 9.1

Claims (5)

1. A method of inducing protective immunity to rabies-related virus comprising: administering to a human or domestic animal as a priming injection a preparation comprising the nucleoprotein of a rabies-related virus, said preparation being substant- ially free of rabies-related virus glycoprotein; and administering to the human or domestic animal one or more booster injections comprising an inactivated rabies virus.

2. A etahod 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 comprising the nucleoprotein of a t« rabies-related virus, said preparation being substantially free of rabies-related virus glycoprotein.

3. A method of inducing protective immunity to rabies-related virus consisting essentially of: administering to a human or domestic animal a priming injection comprising the nucleoprotein of a rabies- related virus, said nucleoprotein being substantially free of rabies-related virus glycoprotein; and acdiinistering to the human or domestic animal one booster injection comprising the nucleoprotein of a rabies-related virus, said nucleoprotein being substant- ially free of rabies-related virus glycoprotein.

4. A method of inducing protective immunity to rabies-related virus substantially as hereinbefore describ- U4 1U_ Vr 00 ed with reference to the Examples. DATED this 6th day of zecemnber, 1990. THE WISTAR INSTITUT1E WATERMARK PATENT TRADEMARK ATTOV,'NEYS 290 BURWOOD ROAD3, HAWTHORN, VICTORIA, AUSTRALIA. TAS:13B(10 ITRNATIONAL SEARCH REPORT Intarnational Application No PCT '-USEB'02525 1. CLASSIFICATfoh OF SUBJECT MATTERIS e eil CIoss1ii3tIOn symoci accly, indicate ll) According to international Patent ctasiricallon (IPC) of to Dotn Nationat Classirication and )PC IPC A 61 K 39/205. C 07 K 7/10 11 FIELDS SEARtCHED Minimum Documentation Soarchioo ClassificaionSysatem Classification Symbols ,PC A 61 K; C£12 N Documentation SoarirnI'd other tnen Minimum Documentation to theg Extent that such Documents a t nctuovo In the Fields SearchoO Ill. DOCUMENTS CONSIDERED TO SE1 Category Citation of Douymeni, It with inication, whisi appropriate, at the relevant passages ~i Relevant to Claim No E\ FiX roc. Natl. Acad, Sci. ik3A, Vol. 84, p. 9165-9169, 114'12 December 1987 k(8 DIETZSCHOLD et al.r; iInduct,on of protective imminity against rabies by, immu- nization uith rabies virus ribonucleoprotein" see the vncle document E,X Virus Research. 8 (1987) p. 103-125 11 12 8 DIETZSCHOLD et al.i 'Localiation and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins" t see pages 103-105, table 2, page 116 and pages

120-23 A 3. gen, Viro'. (1985), Vol. 66, p. 2125-2133, 11. 12 (M LAFON et "Antigenic Sites on the ERA Rabies Virts NuQleoprotein and Non-structural Protein" see the v/hole O.ncument *special categories of cited documents; Iii later document Published shot the Internatioal filing date -A do Cument defining the generel state of the ai which Is no of priority date and not in conflict with theo spolict lion but cited to understbnd the principle or theory undciiring the coneide(so to be, of particutar relevance invention earlier document but Pkibtiahed on or einot the international dou ntopaiclreevce teceidinnin wfii itedoetblhtepblctodaef nhe "Xy" document of particular reloeance; the claimed Invention riling datecannot be consalereit nove[ ot c40ifl1 be considered to *il' document which may throw do~ubts on Priority claim(s) or involve an inventive step citation or or speciel reason (as specified) cannot be considered to Involve an Inventive slap when the documerst referring to an oral disclosure, use, exhibition or document to combined with one or more other such doCu" other miliahs menta. auch combination being obvious to a parson skiiied documnt publiahed prior to the internaetional filino date but tn tioe orn, later thant theo priority date claimed document meomber of the same patent ftnly IV. CEIRTIFICATION________ Date of the Actual Completion of the International Search Date of Mailing of this International Search, RepolI 27th October 1988 28 NOV 1W8 International Searching Authority sigiso-TAuthori mo EUROPEAN PATENT OFFICE Form PCT/ISAI210 leecond shoatt (January 19811 international A150lication No. PCT/US88., 0252F Ili. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THlE &ECOND NIHEET) Category Citation, of Document with incicatton. *we ameo~ nate. Of tre revant oaes Ralevart X Nucleic Acids Research. Vol. 14. No. 6. 1986, 11 Y p. 2671-83 TORDO et al I: "Primary 12 structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VS\J" see page 2680 Y The Journal of Immunology, Vol. 136, No. 2, 11, I January 15, 1986, p. 692-96 (E CELlS et al.): "'Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-y" see page 602, right column. last paragraph and page 696. right dolumn page 697 x VEP, Al, 0 237 686 (I\STITUT PASTEUR, CENTRE NATIONAL 11 DE LA RECHERCHE SCIENTIFIQUE) 12 23 September 1987 see claim I and page 6, lines 40-50 Y EP, AZ, 0 203 676 (THE WISTAR INSTITUTE OF ANATOMY 11, 12 AND BIOLOGY) 3 Decsoiber 1966 see page 1, line 10, page 2, lines 17-26, page 3, lines 25-32 and claim 1 A J. gen, \Jiiol. (1983) 64, p. 1649-1656 11, 12 (W H WUNNER et "Rabies Subunit Vaccines" see page 1649, first paragraph and page 1655, lines 2-3 Form PCT ISA110 ($W$1tshl 0111- I1 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLI CATION INO. -CT/US88/02>L2- SA 27164-' Tis 2nncx li.kt the patent family mcmbers relating to (he psientdi ri Inic cjurd in lb 78inhnecnrcrIinncd inirrnationnl search report. The members are 2S contained in the Eurropeani Patent Oflice 11)1' Mle on 17/10 8 The Ptirnpean Patent Ofrice is in no way liable (or lhcse pnrhictfirs h~ch nre nmerely given for the jrrrrpnqe of infoemation. Patent document Plihlctinn fPotent fnmilv Pulricntron cited in xcarch report I atI mcmhe-r(e) 'I da (C EP-A- 0237686 EP-A- 0203676 23-09-8 7 03-12-86 None None r(11 More 40t2i% A10111t lhk% aInnx See ortical imirmnai or it, riuropran Pitent ()Miee. No, 128 -lnlernitOMa A:DI~ce cr ri,. TASU88/ 02 529 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V,S) OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This inteornatlons asafr rpart fias not been ealaolishect in resect of certain claims undo[ AtCls 17(2) for the followint; reasons: IQ~ Claim numbers because they telate to suoiect matter not required to be aeartnea by ito Ai.thorlty, namely: See PCT Rule 39.l(iv) Methods for treatment of the human or animal body bv means of surgery or therapy, as veil as diagnostic methods. 2 claim numbert;.......becoat they (elate to Daris of !h-3 International application that co not comply with the pruscribed require- m~nta to auch an extant that no meaningful inlterntionafl5 searcyi can DO CAriOO Oul, &lPiC11.1y numbers bescause they are deaetroerit clisn and are n-ot drafted in accordaince wrt ed isfond and Vurd seteoncs of PCT Buis 6.4(a), VlE ONSERVATIONS WHERE UNITY OF INVENTION IS LACK~ING This Internastional 5earchlng Authority found muliple Inventions In tisl International application as follows; IE] all required addllcnral search fees were timely paid by the applicant, I!de Intarnatlonal 3saich report covers all searchable claims kt the International application. 2.E As only some of the requited additional search fees were Illrnly paid by the applicant, this lernatuonal oearch report covers only thos.. claima of the International application tor which toe were Paid, speicltically claimat 3M No required additional searchr tees were timely paid by the applicant, Consequently. this International search report is restricted to the l4rventlon first mentioned In the clalms; it is covered by claim numbers: A .flA all searchable claims could be aseched wlthout eflort just1ifig an additional too, the Intrn.tonal Seatching Authority dId not Invlte payment of any addtitional lee. Roerit an Protest 0] The additional sarch Iee wereii atcompan~ed by apollcent's proledt. L3 No protest accompaniedt the payment oftadditilonal sarch toem Form PCT/ISAI21O (supplemental shoet (January t085)