Rabies Vaccine Candidate Research Articles

A novel inactivated oral rabies vaccine with the incorporation of U-OMP19 enhances the immunogenicity by reducing viral proteins degradation and activating dendritic cells in a mouse model

Currently, dogs, especially stray dogs, and/or wild animals are the main sources of rabies transmission, and oral vaccination is the most practical way to control rabies in these animals. Safety and efficacy are two key criteria for developing oral vaccines. Concerning the efficacy of oral vaccines, degradation of immunogens by gastrointestinal fluid is a major challenge, resulting in suboptimal immune responses after vaccination. For safety reasons, inactivated vaccines are the most optimal choice. In the present study, a recombinant rabies virus (RABV) with un-lipidated outer membrane protein 19 (U-OMP19) of Brucella spp incorporated into RABV virions, designated as LBNSE-OMP19-G, was constructed and rescued. We found that U-OMP19 was incorporated into LBNSE-OMP19-G virion, which could protect RABV G protein from digestion by gastrointestinal fluids in vitro. Moreover, the immunogenicity of LBNSE-OMP19-G as an inactivated oral vaccine was evaluated, and the inactivated LBNSE-OMP19-G could activate more dendritic cells (DCs) and promote the generation of follicular helper T (TFH) cells, germinal center (GC) B cells, and plasma cells in immunized mice compared with those in mice immunized with parent virus LNBSE, which consequently induced a higher level of virus neutralizing antibody and provided better protection after a lethal challenge of rabies. These data indicate that LBNSE-OMP19-G, which has good safety and immunogenicity, could be a potential inactivated oral rabies vaccine candidate.

A single dose of an ALVAC vector-based RABV virus-like particle candidate vaccine induces a potent immune response in mice, cats and dogs

Rabies, caused by the Rabies virus (RABV), is a highly fatal zoonotic disease. Existing rabies vaccines have demonstrated good immune efficacy, but the complexity of immunization procedures and high cost has impeded the elimination of RABV, particularly in the post-COVID-19 era. There is a pressing need for safer and more effective rabies vaccines that streamline vaccination protocols and reduce expense. To meet this need, we have developed a potential rabies vaccine candidate called ALVAC-RABV-VLP, utilizing CRISPR/Cas9 gene editing technology. This vaccine employs a canarypox virus vector (ALVAC) to generate RABV virus-like particles (VLPs). In mice, a single dose of ALVAC-RABV-VLP effectively activated dendritic cells (DCs), follicular helper T cells (Tfh), and the germinal center (GC)/plasma cell axis, resulting in durable and effective humoral immune responses. The survival rate of mice challenged with lethal RABV was 100%. Similarly, in dogs and cats, a single immunization with ALVAC-RABV-VLP elicited a stronger and longer-lasting antibody response. ALVAC-RABV-VLP induced superior cellular and humoral immunity in both mice and beagles compared to the commercial inactivated rabies vaccine. In conclusion, ALVAC-RABV-VLP induced robust protective immune responses in mice, dogs and cats, offering a novel, cost-effective, efficient, and promising approach for herd prevention of rabies.

Rabies virus-based oral and inactivated vaccines protect minks against SARS-CoV-2 infection and transmission1

Minks are highly susceptible to SARS-CoV-2, and have transmitted SARS-CoV-2 to humans. Oral immunization is one of the most promising strategies to prevent SARS-CoV-2 infection and transmission in minks. Here, we generated three recombinant rabies viruses (RABV), rERAG333E/S6P, rERAG333E/DS6P and rERAG333E/BA2S6P, expressing the prefusion-stabilized SARS-CoV-2 spike protein of wild-type (S6P), δ (DS6P) or BA.2 (BA2S6P) strain based on an oral rabies vaccine candidate (rERAG333E). Oral or inactivated immunization of the three RABVs monovalent or trivalent were safe, and induced robust RABV neutralizing antibody and cross-antibody responses against the three SARS-CoV-2 in mice and minks. The challenge tests showed that two doses of rERAG333E-S6P as an oral or inactivated vaccine completely protected mice against mouse-adapted SARS-CoV-2 infection in the upper and lower respiratory tracts, and largely prevented viral replication and lung damage caused by wild-type SARS-CoV-2 infection in minks. Notably, we also confirmed that two doses of rERAG333E-S6P as an oral or inactivated vaccine can largely protect minks against wild-type SARS-CoV-2 transmission via respiratory droplets. Our findings suggest that rERAG333E-based COVID-19 vaccines appear to be suitable oral candidates to protect minks from SARS-CoV-2 infection and transmission, and may serve as inactivated vaccines for further investigation in humans.

Immunogenicity of rabies virus G mRNA formulated with lipid nanoparticles and nucleic acid immunostimulators in mice

Rabies is a preventable zoonotic disease caused by rabies virus (RABV) with high mortality. Messenger RNA (mRNA) vaccines have opened up new avenues for vaccine development and pandemic preparedness with potent scalability, which may overcome the only licensed rabies inactived vaccine’ shortcoming of time and cost wasting. Here, we designed an RABV mRNA vaccines expressed RABV G protein and capsulated with lipid nanoparticle (LNP) and different nucleic acid immunostimulator (CPG 1018, CPG 2395 and Poly I:C) and then assessed the immunogenicity and protective capacity in mice. While RABV mRNA capsulated with LNP and CPG 1018 could induce more potent humoral response with highest and durable RABV-G specific IgG titers and virus neutralizing titers, but also induced stronger RABV G-specific cell-mediated immunity (CMI) responses, including the highest proportions of interferon-γ (IFN-γ) and tumor necrosis factor alpha (TNFα)- producing CD4+/CD8 + T cells according to a flow cytometry assay in mice. In addition, in the pre- and post-exposure challenge assays, LNP + CPG 1018 capsulated RABV G mRNA induced 100 % protection against 25 LD50 of RABV infection with highest inhibition efficacy of viral replication with the decreased virus genome detected by qRT-PCR. These results showed that RABV G mRNA capsulated with LNP immune-stimulating nucleic acids CPG 1018 showed promise as a safe and economical rabies vaccine candidate.

Incorporation of Escherichia coli heat-labile enterotoxin B subunit into rabies virus particles enhances its immunogenicity in mice and dogs

Although inactivated vaccines against rabies have the advantage of high safety, effective protection against rabies virus (RABV) infection often requires multiple, high-dose immunization. Incorporating a molecular adjuvant into the viral particles has been found to be a useful strategy to promote the immune effectiveness of inactivated vaccines. In this study, we constructed a recombinant virus, rCVS11-LTB, which chimerically expresses a molecular adjuvant heat-labile enterotoxin B subunit (LTB) protein on the surface of the RABV particles. Immunogenicity in vivo was found to be promoted by rCVS11-LTB through the activation of dendritic cells (DCs). Our results demonstrated that inactivated rCVS11-LTB was able to induce higher levels of virus-neutralizing antibodies (VNAs) in both mice and dogs than the parent virus rCVS11, to enhance the cellular immune response and T cell immune memory in mice, and was also able to provide 100% protection in mice from lethal doses of rabies virus, indicating its potential as a safe and effective inactivated rabies vaccine candidate.

A recombinant rabies virus chimera expressing the DC-targeting molecular MAB2560 shows enhanced vaccine immunogenicity through activation of dendritic cells.

Rabies, caused by the rabies virus (RABV), is an ancient and neglected zoonotic disease posing a large public health threat to humans and animals in developing countries. Immunization of animals with a rabies vaccine is the most effective way to control the epidemic and the occurrence of the disease in humans. Therefore, the development of cost-effective and efficient rabies vaccines is urgently needed. The activation of dendritic cells (DCs) is known to play an important role in improving the host immune response induced by rabies vaccines. In this study, we constructed a recombinant virus, rCVS11-MAB2560, based on the reverse genetic system of the RABV CVS11 strain. The MAB2560 protein (a DC-targeting molecular) was chimeric expressed on the surface of the viral particles to help target and activate the DCs when this virus was used as inactivated vaccine. Our results demonstrated that inactivated rCVS11-MAB2560 was able to promote the recruitment and/or proliferation of DC cells, T cells and B cells in mice, and induce good immune memory after two immunizations. Moreover, the inactivated recombinant virus rCVS11-MAB2560 could produce higher levels of virus-neutralizing antibodies (VNAs) in both mice and dogs more quickly than rCVS11 post immunization. In summary, the recombinant virus rCVS11-MAB2560 chimeric-expressing the molecular adjuvant MAB2560 can stimulate high levels of humoral and cellular immune responses in vivo and can be used as an effective inactivated rabies vaccine candidate.

A recombinant rabies vaccine that prevents viral shedding in rabid common vampire bats (Desmodus rotundus).

Vampire bat transmitted rabies (VBR) is a continuing burden to public health and agricultural sectors in Latin America, despite decades-long efforts to control the disease by culling bat populations. Culling has been shown to disperse bats, leading to an increased spread of rabies. Thus, non-lethal strategies to control VBR, such as vaccination, are desired. Here, we evaluated the safety and efficacy of a viral-vectored recombinant mosaic glycoprotein rabies vaccine candidate (RCN-MoG) in vampire bats (Desmodus rotundus) of unknown history of rabies exposure captured in México and transported to the United States. Vaccination with RCN-MoG was demonstrated to be safe, even in pregnant females, as no evidence of lesions or adverse effects were observed. We detected rabies neutralizing antibodies in 28% (8/29) of seronegative bats post-vaccination. Survival proportions of adult bats after rabies virus (RABV) challenge ranged from 55–100% and were not significantly different among treatments, pre- or post-vaccination serostatus, and route of vaccination, while eight pups (1–2.5 months of age) used as naïve controls all succumbed to challenge (P<0.0001). Importantly, we found that vaccination with RCN-MoG appeared to block viral shedding, even when infection proved lethal. Using real-time PCR, we did not detect RABV nucleic acid in the saliva samples of 9/10 vaccinated bats that succumbed to rabies after challenge (one was inconclusive). In contrast, RABV nucleic acid was detected in saliva samples from 71% of unvaccinated bats (10/14 sampled, plus one inconclusive) that died of the disease, including pups. Low seroconversion rates post-vaccination and high survival of non-vaccinated bats, perhaps due to earlier natural exposure, limited our conclusions regarding vaccine efficacy. However, our findings suggest a potential transmission-blocking effect of vaccination with RCN-MoG that could provide a promising strategy for controlling VBR in Latin America beyond longstanding culling programs.

Safety and immunogenicity of a simian-adenovirus-vectored rabies vaccine: an open-label, non-randomised, dose-escalation, first-in-human, single-centre, phase 1 clinical trial

Rabies kills around 60 000 people each year. ChAdOx2 RabG, a simian adenovirus-vectored rabies vaccine candidate, might have potential to provide low-cost single-dose pre-exposure rabies prophylaxis. This first-in-human study aimed to evaluate its safety and immunogenicity in healthy adults. We did a single-centre phase 1 study of ChAdOx2 RabG, administered as a single intramuscular dose, with non-randomised open-label dose escalation at the Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK. Healthy adults were sequentially allocated to groups receiving low (5 × 109 viral particles), middle (2·5 × 1010 viral particles), and high doses (5 x 1010 viral particles) of ChAdOx2 RabG and were followed up to day 56 after vaccination. The primary objective was to assess safety. The secondary objective was to assess immunogenicity with the internationally standardised rabies virus neutralising antibody assay. In an optional follow-up phase 1 year after enrolment, we measured antibody maintenance then administered a licensed rabies vaccine (to simulate post-exposure prophylaxis) and measured recall responses. The trial is registered with ClinicalTrials.gov, NCT04162600, and is now closed to new participants. Between Jan 2 and Oct 28, 2020, 12 adults received low (n=3), middle (n=3), and high doses (n=6) of ChAdOx2 RabG. Participants reported predominantly mild-to-moderate reactogenicity. There were no serious adverse events. Virus neutralising antibody concentrations exceeded the recognised correlate of protection (0·5 IU/mL) in three middle-dose recipients and six high-dose recipients within 56 days of vaccination (median 18·0 IU/mL). The median peak virus neutralising antibody concentrations within 56 days were 0·7 IU/mL (range 0·0-54·0 IU/mL) for the low-dose group, 18·0 IU/mL (0·7-18·0 IU/mL) for the middle-dose group, and 18·0 IU/mL (6·0-486·0 IU/mL) for the high-dose group. Nine participants returned for the additional follow-up after 1 year. Of these nine participants, virus neutralising antibody titres of more than 0·5 IU/mL were maintained in six of seven who had received middle-dose or high-dose ChAdOx2 RabG. Within 7 days of administration of the first dose of a licensed rabies vaccine, nine participants had virus neutralising antibody titres of more than 0·5 IU/mL. In this study, ChAdOx2 RabG showed an acceptable safety and tolerability profile and encouraging immunogenicity, supporting further clinical evaluation. UK Medical Research Council and Engineering and Physical Sciences Research Council.

Study on an attenuated rabies virus strain CTN181-3

We have obtained an attenuated rabies virus CTN181-3. In this paper, we make a comprehensive studies on CTN181-3. CTN181-3 showed no pathogenicity by i. c. or o. i. inoculation in 3-week-old mice, lower pathogenic in 2-week-old mice, and no virulence by o. i. inoculation in 8-week-old golden hamsters. CTN181-3 showed high immunogenicity, which produced high level neutralizing antibodies, 100% sero-conversation and >5.0 IU/ml GMT by one dose i. m. or o. i. vaccination in mice and golden hamsters. Cellular immune response by one dose i. m. or o. i. inoculation was detected. Especially in PEP, reduced dose of vaccination resulted in 50% (one dose) and 100% (2 doses) protections in golden hamsters. Molecular basis of the attenuation indicated that eight substitutions compared to its parental virus strain CTN-1, among them the two substitutions at the G276 (Leu→Val) and L1496 (Met→Trp) were the critical attenuated site. The phenotypic and genotypic characteristics of CTN181-3 were highly stable, no reversion was occurred when the virus was multiple passaged in suckling mice brains, guinea pig submandibular glands or BSR/Vero cell cultures. The gene homology compared to the Chinese rabies isolates showed much higher than rabies vaccine strains used in China, suggesting CTN181-3 is a promising and suitable oral rabies vaccine candidate strain.

Recombinant adeno-associated virus serotype 9 AAV-RABVG expressing a Rabies Virus G protein confers long-lasting immune responses in mice and non-human primates

ABSTRACT Three or four intramuscular doses of the inactivated human rabies virus vaccines are needed for pre- or post-exposure prophylaxis in humans. This procedure has made a great contribution to prevent human rabies deaths, which bring huge economic burdens in developing countries. Herein, a recombinant adeno-associated virus serotype 9, AAV9-RABVG, harbouring a RABV G gene, was generated to serve as a single dose rabies vaccine candidate. The RABV G protein was stably expressed in the 293T cells infected with AAV9-RABVG. A single dose of 2 × 1011 v.p. of AAV9-RABVG induced robust and long-term positive seroconversions in BALB/c mice with a 100% survival from a lethal RABV challenge. In Cynomolgus Macaques vaccinated with a single dose of 1 × 1013 v.p. of AAV9-RABVG, the titres of rabies VNAs increased remarkably from 2 weeks after immunity, and maintained over 31.525 IU/ml at 52 weeks. More DCs were activated significantly for efficient antigen presentations of RABV G protein, and more B cells were activated to be responsible for antibody responses. Significantly more RABV G specific IFN-γ-secreting CD4+ and CD8+ T cells, and IL-4-secreting CD4+ T cells were activated, and significantly higher levels of IL-2, IFN-γ, IL-4, and IL-10 were secreted to aid immune responses. Overall, the AAV9-RABVG was a single dose rabies vaccine candidate with great promising by inducing robust, long-term humoral responses and both Th1 and Th2 cell-mediated immune responses in mice and non-human primates.

Inhibitory effect of concomitantly administered rabies immunoglobulins on the immunogenicity of commercial and candidate human rabies vaccines in hamsters

The World Health Organization protocol for rabies post-exposure prophylaxis (PEP) recommends extensive wound washing, immediate vaccination, and administration of rabies immunoglobulin (RIG) in severe category III exposures. Some studies have shown that RIG can interfere with rabies vaccine immunogenicity to some extent. We investigated the interference of RIG on a next generation highly purified Vero cell rabies vaccine candidate (PVRV-NG) versus standard-of-care vaccines in a previously described hamster model. The interference of either human (h) or equine (e) RIG on the immune response elicited by PVRV-NG, Verorab® (purified Vero cell rabies vaccine, PVRV), and Imovax® Rabies (human diploid cell rabies vaccine; HDCV) was evaluated using the 4-dose Essen PEP regimen. The anti-rabies seroneutralizing titers and specific serum IgM titers were measured by fluorescent antibody virus neutralization test and enzyme-linked immunosorbent assay, respectively, for the vaccines administered with or without RIG. The RIG interference on PVRV-NG, observed transiently at Day 7, was similar to that on PVRV and tended to be lower than that on HDCV using both read-outs. In summary, the results generated in the hamster model showed that RIG induced similar or less interference on PVRV-NG than the standard-of-care vaccines.

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

Advances in RNA Vaccines for Preventive Indications: A Case Study of A Vaccine Against Rabies.

There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies.

A simplified roller bottle platform for the production of a new generation VLPs rabies vaccine for veterinary applications

Rabies is a neglected disease with an estimated annual mortality of 55,000 human deaths, affecting mainly low-income countries. Over 95% of these cases result from virus transmission through the bite of infected dogs and for this reason there is a real need for a cheap and effective rabies veterinary vaccine to be used in mass vaccination campaigns. In this work, we describe the establishment of a simple platform for the production of a virus-like particles based rabies vaccine using mammalian cells and roller bottles as culture system. Adherent cells were cultured during more than 15 days and VLPs were continuously produced and secreted to the culture supernatant. Immunogenicity and protective efficacy of VLPs were tested through rabies virus neutralizing antibody test and NIH potency test. These viral particles induced high titer of long lasting neutralizing antibodies and protected mice against active virus challenge. Therefore, this development represents a promising platform for the production of a new generation and virus-free rabies vaccine candidate for veterinary applications.

Phenotypic Consequence of Rearranging the N Gene of RABV HEP-Flury.

Nucleoprotein (N) is a key element in rabies virus (RABV) replication. To further investigate the effect of N on RABV, we manipulated an infectious cDNA clone of the RABV HEP-Flury to rearrange the N gene from its wild-type position of 1 (N-P-M-G-L) to 2 (P-N-M-G-L), 3 (P-M-N-G-L), or 4 (P-M-G-N-L), using an approach that left the viral nucleotide sequence unaltered. Subsequently, viable viruses were recovered from each of the rearranged cDNA and examined for their gene expression levels, growth kinetics in cell culture, pathogenicity in suckling mice and protection in mice. The results showed that gene rearrangement decreased N mRNA transcription and vRNA replication. As a result, all viruses with rearranged genomes showed worse replication than that of rHEP-Flury in NA cells at a MOI of 0.01, but equivalent or slightly better replication levels at a MOI of 3. Consequently, the lethality in suckling mice infected with N4 was clearly attenuated compared with rHEP-Flury. However, the protection to mice was not enhanced. This study not only gives us insight into the understanding of the phenotype of RABV N gene rearrangement, but also helps with rabies vaccine candidate construction.

Non-neutralizing antibodies elicited by recombinant Lassa\u2013Rabies vaccine are critical for protection against Lassa fever

Lassa fever (LF), caused by Lassa virus (LASV), is a viral hemorrhagic fever for which no approved vaccine or potent antiviral treatment is available. LF is a WHO priority disease and, together with rabies, a major health burden in West Africa. Here we present the development and characterization of an inactivated recombinant LASV and rabies vaccine candidate (LASSARAB) that expresses a codon-optimized LASV glycoprotein (coGPC) and is adjuvanted by a TLR-4 agonist (GLA-SE). LASSARAB elicits lasting humoral response against LASV and RABV in both mouse and guinea pig models, and it protects both guinea pigs and mice against LF. We also demonstrate a previously unexplored role for non-neutralizing LASV GPC-specific antibodies as a major mechanism of protection by LASSARAB against LF through antibody-dependent cellular functions. Overall, these findings demonstrate an effective inactivated LF vaccine and elucidate a novel humoral correlate of protection for LF.

Recombinant rabies virus with the glycoprotein fused with a DC-binding peptide is an efficacious rabies vaccine.

Our previous studies demonstrated that recruiting and/or activating dendritic cells (DCs) enhanced the immunogenicity of recombinant rabies viruses (rRABV). In this study, rRABV LBNSE with a small DC-binding peptide (designated as rLBNSE-DCBp) or a negative control peptide (designated as rLBNSE-DCCp) fused to the glycoprotein (G) was constructed and rescued. As expected, significantly more activated DCs were detected in rLBNSE-DCBp-immunized mice than those immunized with rLBNSE or rLBNSE-DCCp. Subsequently, significantly more generation of TFH and GC B cells were observed in rLBNSE-DCBp immunized mice than those in rLBNSE or rLBNSE-DCCp-immunized mice. In addition, significantly higher levels of virus neutralizing antibodies (VNAs) were observed in mice immunized with rLBNSE-DCBp than those immunized with rLBNSE or rLBNSE-DCCp, resulting in a better protection of rLBNSE-DCBp immunized mice against the lethal challenge. Taken together, our results suggest that rRABV with G fused with DCBp is a promising rabies vaccine candidate.

Phenotypic Consequences In vivo and In vitro of Rearranging the P Gene of RABV HEP-Flury.

Phosphoprotein (P) of the Rabies virus (RABV) is critically required for viral replication and pathogenicity. Here we manipulated infectious cDNA clones of the RABV HEP-Flury to translocate the P gene from its wild-type position 2 to 1, 3, or 4 in gene order, using an approach which left the viral nucleotide sequence unaltered. The recovered viruses were evaluated for the levels of gene expression, growth kinetics in cell culture, lethality in suckling mice and protection of mice. The results showed that viral replication was affected by the absolute value of N protein which was regulated by P protein. Viral lethality in suckling mice was consistent with the ratio of P mRNA in one complete transcription. The protection of mice induced by viruses was related to the antibody titer 5 weeks post-infection which might be regulated by G protein. However, the ability to induce cell apoptosis and viral spread were not only related to the viral replication but also to the ratio of related gene which affected by the gene position. These findings might not only improve the understanding of phenotype of RABV and P gene rearrangement, but also help rabies vaccine candidate construction.

Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate

Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.