Rabies Glycoprotein Research Articles

Use of recombinant vaccinia-rabies glycoprotein virus for oral vaccination of wildlife against rabies: innocuity to several non-target bait consuming species.

The pathogenicity of a vaccinia recombinant virus expressing the rabies glycoprotein (VVTGgRAB) was tested in several wild animal species which could compete with the natural rabies host, the red fox (Vulpes vulpes) in consuming vaccine baits in Europe. The following species were included in this study: wild boar (Sus scrofa), Eurasian badger (Meles meles), wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), bank vole (Clethrionomys glareolus), common vole (Microtus arvalis), field vole (Microtus agrestis), water vole (Arvicola terrestris), common buzzard (Buteo buteo), kestrel (Falco tinnunculus), carrion crow (Corvus corone), magpie (Pica pica) and jay (Garrulus glandarius). During the observation period, the 107 animals given the VVTGgRAB vaccine orally did not show any clinical signs. Daily monitoring for 28 days and postmortem examination did not result in the detection of pox lesions in the oral mucosa or the skin in mammals or the unfeathered portions of birds. VVTGgRAB seems to multiply in the mammalian species tested, since rabies seroconversion was observed in all of them. Birds failed to develop demonstrable rabies virus-neutralizing antibody. A seroconversion against vaccinia virus was observed in two of four wild boars. Serological results obtained in badgers and wild boars also demonstrates the absence of direct or indirect horizontal transmission of the recombinant virus. The potential of the recombinant virus for the immunization of badgers against rabies also was investigated. Only 50% of the badgers orally administered with 1 x 10(8.3) TCID50 of this vaccine were protected against rabies.

The use of immunosome technology for vaccines against rabies and other viral diseases

Subunit viral vaccines present several advantages. They are free of nucleic acids (of viral and/or cellular origin) and proteins of cellular and/or serum origin; they contain only the relevant antigen. For rabies virus, the antigen which induces the virus-neutralizing antibody (VNAb) is the glycoprotein (GP), which is anchored, in form of spikes, to the viral membrane. The GP may be extracted from the virion but, after solubilization and purification, it appears to be poorly immunogenic. In order to restore its immunogenicity, GP molecules are anchored to preformed liposomes (unilamellar phospholipid vesicles) to mimic their native structure and environment. The subunit vaccine obtained by this technique is called an "immunosome" (IMS). Rabies immunosomes exhibit structural and immunological properties very similar to those of the viral particle. The rabies glycoprotein molecules, anchored to the lipid bilayer of the liposome, correctly expose the immunodominant epitope involved in VNAb induction and induce a strong specific humoral immune response. They also induce a specific cellular immune response. As a result IMS have a highly protective activity when tested with either pre- or post-exposure potency tests. Immunosome technology may be applied to other purified membrane proteins or amphiphilic peptides to restore their immunogenicity.

Rabies virus-specific T cell hybridomas: identification of class II MHC-restricted T-cell epitopes using synthetic peptides.

Rabies virus-specific T-cell hybridomas were produced from immune mice by somatic cell fusion. Cloned T-cell hybridomas were studied for antigen specificity using purified virus, a recombinant vaccinia virus expressing rabies glycoprotein and synthetic peptides containing amino acid sequences of rabies viral antigens. Two closely situated T-cell epitopes of rabies glycoprotein, and one of rabies nucleoprotein were identified using synthetic peptides corresponding to amino acid sequences of these proteins. The major histocompatibility gene complex elements that determine the recognition of antigen by these T-cell hybrids were determined using mouse fibroblasts (L cells) transfected with and expressing the I-Ad and I-Ed genes. Some of the T cell hybridomas exhibited significant cytotoxic activity against target cells expressing surface rabies antigens. This T cell mediated cytotoxicity requires cell-to-cell contact between target and effector cells since no by-stander cytotoxicity was observed. The results are discussed in the context of their significance for the design of newer subunit vaccines to prevent rabies infection.

Rabies oral immunization.

My interest in oral immunization dates to 1950 when I fed a child the live, attenuated polio virus vaccine. During the ensuing several years, many more children were immunized with this vaccine, and these efforts culminated in mass oral immunization of 250,000 infants in the Belgian Congo, during the epidemic of true infantile paralysis. This immunization required six weeks to perform. Our current interest in oral immunization stems from the fact that our real problem, as far as rabies is concerned, is not domestic animals, and not even vaccines for humans, but for effective vaccines for wildlife. The experience, particularly of Europeans with foxes, has indicated that the eradication of rabies in wildlife cannot be achieved by reducing the reservoir species by killing, gassing, or any other rather barbaric method. Instead, immunization of the wild reservoir animals offers an alternative approach (Steck et al. 1982). The questions are, how and what type of vaccine could be used, and how effective it would be — and this is the subject of my contribution. I will be discussing only one type of vaccine — the vaccinia-rabies glycoprotein recombinant vaccine (V-RG). I do not need to present details about the vaccine, other than that it is obtained by the cDNA derived from the mRNA for rabies glycoprotein incorporated into a construct plasmid from which it is in turn incorporated with vaccinia virus TK gene plasmid (Kieny et al. 1984). Transfection of vaccinia-infected cells with the plasmid allows double reciprocal recombination in vivo and final construction of the V-RG recombinant virus vaccine with the rabies glycoprotein gene interrupting the vaccinia TK gene sequence.KeywordsRabies VirusOral ImmunizationOral VaccinationSubmandibular Lymph NodeRabies Virus GlycoproteinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.

Recombinant fowlpox virus inducing protective immunity in non-avian species.

The natural host of fowlpox virus is limited to avian species. When inoculated into non-avian tissue culture cells, however, fowlpox virus can initiate an abortive infection. A fowlpox virus was engineered to express rabies virus glycoprotein. On inoculation of the recombinant virus into either avian (permissive) or non-avian (non-permissive) cells, the rabies glycoprotein was expressed as a membrane-associated antigen. Inoculation of the fowlpox virus recombinant into six different species of mammal resulted in specific immune responses to both fowlpox antigens and to rabies glycoprotein. In mice, cats and dogs the immune response was sufficient to protect against a live rabies virus challenge. The results demonstrate the utility of a fowlpox virus vector in immunizing non-avian species against rabies in the absence of productive viral replication of the fowlpox vector.

Development of animal recombinant DNA vaccine and its efficacy in foxes.

Rabies is prevalent in most parts of the world. An extensive reservoir of the disease is present in the population of wild animals. The fox in particular is a major vector of the disease in North America and Europe. Although attempts to control rabies by vaccination of wild carnivores with attenuated rabies virus have met with some success, this approach remains controversial. The potential of a recombinant vaccinia virus expressing the rabies glycoprotein for the protection of foxes against rabies was examined. Both the parental (wild-type) and recombinant viruses were found to be innocuous to foxes. Inoculation of live recombinant virus via the subcutaneous, intradermal, or oral routes uniformly elicited high titers of neutralizing antibodies, and animals that received 10(8) plaque-forming units of the recombinant virus in bait resisted severe challenge with live rabies virus.

Genetic restriction and fine specificity of human T cell clones reactive with rabies virus.

Rabies virus-specific T cell clones isolated from a human vaccine recipient were studied for their fine specificity and genetic restriction using synthetic peptides of the viral Ag and mouse fibroblasts transfected with human MHC genes. Two clones were found to react with an epitope present in the rabies glycoprotein, which was presented by the HLA-DR7 molecule. Other T cell clones recognized synthetic epitopes corresponding to the rabies nucleoprotein in association with the HLA-DR7 or HLA-DQw3 molecule, and one clone responded to the viral nucleocapsid Ag in the presence of HLA-DPw4. T cell clones that exhibited different cross-reactivity patterns among several virus strains were found to recognize closely situated epitopes (within 15 amino acid residues), which were presented in the context of the same MHC molecule. The lack of recognition of a particular virus strain by a T cell clone was attributable in some cases to amino acid variations of the Ag that appear to affect the T cell's receptor for Ag specificity and not the ability of that epitope to associate with the corresponding MHC molecule. Comparisons of the T cell cross-reactivity patterns with various rabies and rabies-related viruses, the fine antigenic specificity, and MHC restriction may aid in understanding the role of individual amino acid variations among virus strains in the induction of cross-protective immunity.

Immunization of foxes against rabies with a vaccinia recombinant virus expressing the rabies glycoprotein.

Foxes were vaccinated orally (by bait), gastrically (by stomach tube) and by scarification with a vaccinia recombinant virus expressing the rabies glycoprotein. Neutralizing antibodies against rabies virus were detected at two weeks postvaccination in 8/8 foxes in the bait-fed group, in 3/6 foxes inoculated by stomach tube and in 2/2 of the scarified foxes. After challenge at three months postvaccination with street rabies virus, all foxes that had developed antibodies were protected. The high rate of seroconversion, high levels of antibodies, and resistance to challenge suggest that this recombinant virus might be a suitable vaccine for oral immunization of foxes against rabies.

Interleukin-2 production in vitro: a new approach to the study of rabies vaccine immunogenicity as appraised by testing different glycoprotein presentations

When injected as an immunosome presentation (molecules anchored to preformed liposome), rabies glycoprotein (GP) is capable of protecting animals against rabies either before or after viral infection. The presentation of the GP molecules in the correct form seems to be essential for the induction of antirabies protection. This condition must be taken in account in the making-up of a rabies subunit vaccine. In order to study the relationship between the immune responses induced by the rabies GP and its protective activity, different presentations of the GP were prepared. Purified glycoprotein molecules were associated under different physical forms: liposome-anchored, self-aggregated (rosettes) and associated with the viral lipids (virosomes). These presentations appeared different on electron microscopy. They also exhibited differences in the expression of an immunodominant epitope and in their protective activity. The non-specific immune response, as appraised by interferon production and natural cytotoxicity, was induced at a high level only by the purified viral particles. Specific immune responses (namely virus neutralizing antibody and interleukin-2 production) was induced at high levels only by the viral particle and by the liposome-anchored glycoprotein. A parallelism has previously been established between protection by glycoprotein preparations and interleukin-2 production in primed mice splenocytes. This suggests that the measure of interleukin-2 production in vitro could be used to evaluate the capability of a rabies antigen to induce a T-cell response and to confer protection.

Interleukin 2 Increases Protection against Experimental Rabies

Vaccination with either whole inactivated rabies virus or immunosome (rabies glycoprotein anchored on liposomes) induces a high level of interleukin 2 (IL 2) production after in vitro specific stimulation of splenocytes from primed mice (9). On the contrary, infection with a live rabies virus does not specifically induce the production of IL 2: splenocytes from ill mice previously infected with wild rabies virus cannot be specifically stimulated by rabies antigens, whereas they can be non-specifically stimulated by a mitogen (Concanavalin A (Con A)). When injected in mice, exogenous IL 2 (purified rat IL 2 or human recombinant IL 2) exhibits an adjuvant effect on rabies virus vaccine or subunit vaccine tested in a pre-exposure potency test (NIH test). When injected in hamsters, according to a post-exposure potency test (infection with a wild rabies virus followed by vaccination), IL 2 has no adjuvant effect on the rabies vaccine. Nevertheless, when injected alone, IL 2 protects thirty to fifty percent of the infected animals treated (1 hour, 3 and 7 days post-infection) with 10 international units of human recombinant IL 2.

The association of the rabies glycoprotein with liposome (immunosome) induces an in vitro specific release of interleukin 2

BALB/c mice were primed by receiving a unique intraperitoneal injection of rabies virus antigens presented as complete inactivated virus (P.V. strain) or as purified glycoproteins either in the aggregated form or in physical combination with liposomes (i.e., in the form of “immunosomes”). The splenocytes of these mice were restimulated, 6–15 days after priming, in culture with rabies virus antigens, and antigen-specific IL-2 production was measured. It was found that rabies antigens presented as immunosomes were as active as the inactivated virus, whereas equivalent amounts of purified glycoproteins were inactive. The optimal amounts of rabies immunosomes used for priming was found to be 0.5 to 0.05 μg per mouse.

Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine.

Animal rabies control has been frustrated by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines. In this investigation, raccoons fed a vaccinia-rabies glycoprotein recombinant virus in a sponge bait developed rabies virus-neutralizing antibody (0.6-54.0 units) and resisted street rabies virus infection 28 and 205 days after feeding. Additional raccoons immunized by oral infusion with attenuated antigenic variants of rabies virus strains CVS-11 and ERA failed to develop rabies virus-neutralizing antibody. This work demonstrates the feasibility of a recombinant virus vaccine containing the rabies glycoprotein gene for immunization of raccoons, and possibly other wildlife, to obtain long-term protection against rabies.

How reliable is amino acid sequence homology in predicting similarity of structure and function of c-myc and Ad12 E1A oncogenic proteins?

Adenovirus E1A and c-myc genes are known to be capable of transforming primary rat cells when they occur in combination with either polyoma middle-T or T24 Harvey-ras1 genes. There was a low level of amino acid sequence homology between the nuclear adenovirus-12 (Ad12)E1A protein product (289 amino acids) and the c-myc protein based on optimal alignment and percentage identity. In contrast to others [Ralston R, Bishop JM (1983) Nature 306:803-806], we concluded that this low level of amino acid sequence homology was not significant, since rabies glycoprotein (RGP), which has no transforming function and localizes to the cell surface, had a similar low level of amino acid sequence homology to the c-myc protein. Furthermore, dot-matrix analysis, when used to test the overall level of amino acid sequence homology, showed no significant homology between c-myc and Ad12E1A, E1B, or RGP. Thus, low levels of amino acid sequence homology between two proteins may not be sufficient to predict structural and functional similarities between them reliably, even if the two proteins appear to share a common function.

Vaccinia virus containing DNA coding for rabies glycoprotein useful as vaccine and for the production of the glycoprotein and antisera

Vaccinia virus containing DNA coding for rabies glycoprotein useful as vaccine and for the production of the glycoprotein and antisera

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma.

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.

Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigen-induced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus.

Immunogenic properties of vaccinia recombinant virus expressing the rabies glycoprotein

Summary The ability to construct recombinant viruses expressing foreign genes clearlyhas great potential for vaccine development. In the case of rabies, the V-RG virus has proved to be highly immunogenic, and has outperformed conventional rabies vaccines in every comparative protection test. The question remains, however, whether recombinant vaccines based on vaccinia viruses will ever be accepted for human or veterinary use. In this regard, it should be noted that the lessons learned in the construction and evaluation of V-RG virus can be rapidly applied for the development of other recombinant viruses (bases on adenoviruses, for example).

Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins.

Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.

Expression of rabies virus glycoprotein from a recombinant vaccinia virus.

Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.