Construction of Vero cell-adapted rabies vaccine strain by five amino acid substitutions in HEP-Flury strain.

Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.

One-dose intradermal rabies booster enhances rabies antibody production and avidity maturation.

The incidence of rabies in Thailand reached its peak in 2018 with 18 human deaths. Preexposure prophylaxis (PrEP) vaccination is thus recommended for high-risk populations. WHO has recently recommended that patients who are exposed to a suspected rabid animal and have already been immunized against rabies should receive a 1-site intradermal (ID) injection of 0.1 mL on days 0 and 3 as postexposure prophylaxis (PEP). In Thailand, village health and livestock volunteers tasked with annual dog vaccination typically receive only a single lifetime PrEP dose and subsequent boosters solely upon confirmed animal bites. However, the adequacy of a single PrEP dose for priming and maintaining immunity in this high-risk group has not been evaluated. Therefore, our study was designed to address two key questions: (1) sufficiency of single-dose PrEP-to determine whether a single ID PrEP dose provides adequate long-term immune protection for high-risk individuals exposed to numerous dogs during their vaccination duties. (2) Booster efficacy for immune maturation-to investigate whether one or two additional ID booster doses effectively stimulate a mature and sustained antibody response in this population. The level and persistence of the rabies antibody were determined by comparing the immunogenicity and booster efficacy among the vaccination groups. Our study demonstrated that rabies antibodies persisted for more than 180 days after cost-effective ID PrEP or the 1st or the 2nd single ID booster dose, and adequate antibody levels were detected in more than 95% of participants by CEE-cELISA and 100% by indirect ELISA. Moreover, the avidity maturation of rabies-specific antibodies occurred after the 1st single ID booster dose. This smaller ID booster regimen was sufficient for producing a sufficient immune response and enhancing the maturation of anti-rabies antibodies. This safe and effective PrEP regimen and a single visit involving a one-dose ID booster are recommended, and at least one one-dose ID booster regimen could be equitably implemented in at-risk people in Thailand and other developing countries. However, an adequate antibody level should be monitored before the booster is administered.

Protective Activity of Inactivated Rabies Vaccine Using Flagellin-Based Adjuvant.

Rabies is a zoonotic disease with high lethality. Most human deaths are associated with the bites received from dogs and cats. Vaccination is the most effective method of preventing rabies disease in both animals and humans. In this study, the ability of an adjuvant based on recombinant Salmonella typhimurium flagellin to increase protective activity of the inactivated rabies vaccine in mice was evaluated. A series of inactivated dry culture vaccine for dogs and cats "Rabikan" (strain Shchelkovo-51) with addition of an adjuvant at various dilutions were used. The control preparation was a similar series of inactivated dry culture vaccine without an adjuvant. Protective activity of the vaccine preparations was evaluated by the NIH potency test, which is the most widely used and internationally recommended method for testing effectiveness of the inactivated rabies vaccines. The value of specific activity of the tested rabies vaccine when co-administered with the adjuvant was significantly higher (48.69 IU/ml) than that of the vaccine without the adjuvant (3.75 IU/ml). Thus, recombinant flagellin could be considered as an effective adjuvant in the composition of future vaccine preparations against rabies virus.

Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia.

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Comparative analysis of rabies pathogenic and vaccine strains detection by RIG-I-like receptors.

Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year globally. RABV infection is characterized by the suppression of the interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) that initiates IFN signaling currently remain elusive. Here, we showed that RABV RNAs are primarily recognized by the RIG-I RLR, resulting in an IFN response in the infected cells, but this response varied according to the type of RABV used. Pathogenic RABV strain RNAs, Tha, were poorly detected in the cytosol by RIG-I and therefore caused a weak antiviral response. However, we revealed a strong IFN activity triggered by the attenuated RABV vaccine strain RNAs, SAD, mediated by RIG-I. We characterized two major 5' copy-back defective interfering (5'cb DI) genomes generated during SAD replication. Furthermore, we identified an interaction between 5'cb DI genomes, and RIG-I correlated with a high stimulation of the type I IFN signaling. This study indicates that wild-type RABV RNAs poorly activate the RIG-I pathway, while the presence of 5'cb DIs in the live-attenuated vaccine strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection thus strongly stimulates the IFN response.

What would be the impact on the rabies risk of reducing the waiting period before dogs are imported? A modelling study based on the European Union legislation.

AIMS: Lyssavirus rabies (RABV) is responsible for a major zoonotic infection that is almost always lethal once clinical signs appear. Rabies can be (re)introduced into rabies-free areas through transboundary dog movements, thus compromising animal and human health. A number of measures have been implemented to prevent this happening, one of which is the waiting period (WP) after anti-rabies vaccination and serological testing. This WP ensures that antibodies assessed through the serological test are due to the vaccine, not to infection. Indeed, if antibodies are due to RABV infection, the dog should display clinical signs within this WP and would not therefore be imported. METHODS AND RESULTS: Within a framework of quantitative risk assessment, we used modelling approaches to evaluate the impact of this WP and its duration on the risk of introducing rabies via the importation of dogs into the European Union. Two types of models were used, a classical stochastic scenario tree model and an individual-based model, both parameterised using scientific literature or data specifically applicable to the EU. Results showed that, assuming perfect compliance, the current 3-month waiting period was associated with a median annual number of 0.04 infected dogs imported into the EU. When the WP was reduced, the risk increased. For example, for a 1-month WP, the median annual number of infected dogs imported was 0.17 or 0.15 depending on the model, which corresponds to a four-fold increase. CONCLUSION: This in silico study, particularly suitable for evaluating rare events such as rabies infections in rabies-free areas, provided results that can directly inform policymakers in order to adapt regulations linked to rabies and animal movements.

Prevenção é a melhor forma de evitar infecção por raiva em humanos

Problema de saúde pública que pode afetar seres humanos, rebanhos e animais domésticos, a raiva é uma doença infecciosa viral aguda e grave, com letalidade próxima a 100%.

Safety and immunogenicity of a serum-free purified Vero rabies vaccine in healthy adults: A randomised phase II pre-exposure prophylaxis study.

A serum-free, highly purified Vero cell rabies vaccine (PVRV-NG) is under development. We previously demonstrated that pre-exposure prophylaxis (PrEP) with PVRV-NG had a satisfactory safety profile and was immunogenically non-inferior to the licensed purified Vero cell rabies vaccine in adults. Here, we evaluated the safety and immunogenic non-inferiority of PrEP with PVRV-NG compared to the licensed human diploid cell vaccine (HDCV) in healthy adults (NCT01784874). Participants received three vaccinations (days 0, 7, and 28) as PrEP with or without a booster injection after 12 months. Rabies virus neutralising antibodies (RVNA) were evaluated on days 0, 28 (subgroup only), and 42, and Months 6, 12, and 12 + 14 days (booster group only). Non-inferiority (first primary objective) was based on the proportion of participants with RVNA titres ≥ 0.5 IU/mL (World Health Organization criteria for seroconversion) on day 42, expected to be ≥ 99% (second primary objective). Safety was evaluated after each dose and monitored throughout the study. At day 42, PVRV-NG was non-inferior to HDCV and the first primary objective was met; seroconversion was observed for 98.3% of PVRV-NG recipients and 99.1% of HDCV recipients. As < 99% of participants in the PVRV-NG group had RVNA titres ≥ 0.5 IU/mL, the second primary objective was not met. Booster vaccination produced a strong increase in RVNA titres for all groups, primed with PVRV-NG or HDCV. RVNA geometric mean titres tended to be higher for HDCV than PVRV-NG primary vaccine recipients. In a complementary evaluation using alternative criteria for seroconversion (complete virus neutralization at 1:5 serum dilution), 99.6% and 100% of participants in the PVRV-NG and HDCV groups, respectively, achieved seroconversion across the vaccine groups. No major safety concerns were observed during the study. PVRV-NG was well tolerated, with a similar safety profile to HDCV in terms of incidence, duration, and severity of adverse events after primary and booster vaccinations. ClinicalTrials.gov number: NCT01784874.

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.

Review of Oral Rabies Vaccination of Dogs and Its Application in India.

Oral rabies vaccines (ORVs) have been in use to successfully control rabies in wildlife since 1978 across Europe and the USA. This review focuses on the potential and need for the use of ORVs in free-roaming dogs to control dog-transmitted rabies in India. Iterative work to improve ORVs over the past four decades has resulted in vaccines that have high safety profiles whilst generating a consistent protective immune response to the rabies virus. The available evidence for safety and efficacy of modern ORVs in dogs and the broad and outspoken support from prominent global public health institutions for their use provides confidence to national authorities considering their use in rabies-endemic regions. India is estimated to have the largest rabies burden of any country and, whilst considerable progress has been made to increase access to human rabies prophylaxis, examples of high-output mass dog vaccination campaigns to eliminate the virus at the source remain limited. Efficiently accessing a large proportion of the dog population through parenteral methods is a considerable challenge due to the large, evasive stray dog population in many settings. Existing parenteral approaches require large skilled dog-catching teams to reach these dogs, which present financial, operational and logistical limitations to achieve 70% dog vaccination coverage in urban settings in a short duration. ORV presents the potential to accelerate the development of approaches to eliminate rabies across large areas of the South Asia region. Here we review the use of ORVs in wildlife and dogs, with specific consideration of the India setting. We also present the results of a risk analysis for a hypothetical campaign using ORV for the vaccination of dogs in an Indian state.

Household survey on owned dog population and rabies knowledge in selected municipalities in Bulacan, Philippines: A cross-sectional study.

BACKGROUND: Despite the effort to eradicate rabies in the Philippines, human rabies cases have not decreased in the past decade. Rabid dogs pose the most significant hazard in the countries with the highest burden of rabies, and 70% rabies vaccine coverage is recommended for dogs in high-risk areas. Ascertaining the owned dog population and community knowledge on rabies can help improve vaccine coverage and information campaigns. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional survey in six randomly selected communities (five urban, one rural) in Central Luzon, Philippines. We first conducted the complete mapping of 9,173 households and then randomly selected 727 households. More than half (54.1%) of the households owned dogs (1.21 dogs/household). In the 727 households, we identified 878 owned dogs and 3256 humans. According to these results, the dog-to-human ratio was approximately 1:3.7. Only 8.8% of households reported a history of dog bite in 2019. Among dog-owning households, 31% reported that they allow their dogs to roam freely. Of the recorded dogs, 35.9% have never been vaccinated, and only 3.5% were spayed or castrated. Factors associated with lower rabies knowledge include (1) no education aOR: 0.30 (0.16-0.59), and (2) only primary school education aOR: 0.33 (0.22-0.49). In contrast, factors associated with higher knowledge include (1) owning a dog and not allowing them to roam freely aOR: 2.01 (1.41-2.87) and (2) owning a dog and allowing them to roam freely aOR: 1.84 (1.17-2.92), when compared to those with no dogs. CONCLUSIONS/SIGNIFICANCE: We identified a larger dog population in the community than the usual estimates (1:10), suggesting that annual vaccine needs in the Philippines must be reassessed. Our survey shows a relatively good understanding of rabies; however, awareness of the concept of rabies as a disease, and how animals and humans can acquire it, is lacking.

Current characteristics of animal rabies cases in Thailand and relevant risk factors identified by a spatial modeling approach.

The situation of human rabies in Thailand has gradually declined over the past four decades. However, the number of animal rabies cases has slightly increased in the last ten years. This study thus aimed to describe the characteristics of animal rabies between 2017 and 2018 in Thailand in which the prevalence was fairly high and to quantify the association between monthly rabies occurrences and explainable variables using the generalized additive models (GAMs) to predict the spatial risk areas for rabies spread. Our results indicate that the majority of animals affected by rabies in Thailand are dogs. Most of the affected dogs were owned, free or semi-free roaming, and unvaccinated. Clusters of rabies were highly distributed in the northeast, followed by the central and the south of the country. Temporally, the number of cases gradually increased after June and reached a peak in January. Based on our spatial models, human and cattle population density as well as the spatio-temporal history of rabies occurrences, and the distances from the cases to the secondary roads and country borders are identified as the risk factors. Our predictive maps are applicable for strengthening the surveillance system in high-risk areas. Nevertheless, the identified risk factors should be rigorously considered and integrated into the strategic plans for the prevention and control of animal rabies in Thailand.

Research Advances on the Interactions between Rabies Virus Structural Proteins and Host Target Cells: Accrued Knowledge from the Application of Reverse Genetics Systems.

Rabies is a lethal zoonotic disease caused by lyssaviruses, such as rabies virus (RABV), that results in nearly 100% mortality once clinical symptoms appear. There are no curable drugs available yet. RABV contains five structural proteins that play an important role in viral replication, transcription, infection, and immune escape mechanisms. In the past decade, progress has been made in research on the pathogenicity of RABV, which plays an important role in the creation of new recombinant RABV vaccines by reverse genetic manipulation. Here, we review the latest advances on the interaction between RABV proteins in the infected host and the applied development of rabies vaccines by using a fully operational RABV reverse genetics system. This article provides a background for more in-depth research on the pathogenic mechanism of RABV and the development of therapeutic drugs and new biologics.

Toll-Like Receptor 4 Regulates Rabies Virus-Induced Humoral Immunity through Recruitment of Conventional Type 2 Dendritic Cells to Lymph Organs.

Rabies, caused by rabies virus (RABV), is fatal to both humans and animals around the world. Effective clinical therapy for rabies has not been achieved, and vaccination is the most effective means of preventing and controlling rabies. Although different vaccines, such as live attenuated and inactivated vaccines, can induce different immune responses, different expressions of pattern recognition receptors (PRRs) also cause diverse immune responses. Toll-like receptor 4 (TLR4) is a pivotal PRR that induces cytokine production and bridges innate and adaptive immunity. Importantly, TLR4 recognizes various virus-derived pathogen-associated molecular patterns (PAMPs) and virus-induced damage-associated molecular patterns (DAMPs), usually leading to the activation of immune cells. However, the role of TLR4 in the humoral immune response induced by RABV has not yet been revealed. Based on TLR4-deficient (TLR4-/-) and wild-type (WT) mouse models, we report that TLR4-dependent recruitment of the conventional type 2 dendritic cells (CD8α- CD11b+ cDC2) into secondary lymph organs (SLOs) is critical for antigen presentation. cDC2-initiated differentiation of follicular helper T (Tfh) cells promotes the proliferation of germinal center (GC) B cells, the formation of GCs, and the production of plasma cells (PCs), all of which contribute to the production of RABV-specific IgG and virus-neutralizing antibodies (VNAs). Collectively, our work demonstrates that TLR4 is necessary for the recruitment of cDC2 and for the induction of RABV-induced humoral immunity, which is regulated by the cDC2-Tfh-GC B axis. IMPORTANCE Vaccination is the most efficient method to prevent rabies. TLR4, a well-known immune sensor, plays a critical role in initiating innate immune response. Here, we found that TLR4-deficient (TLR4-/-) mice suppressed the induction of humoral immune response after immunization with rabies virus (RABV), including reduced production of VNAs and RABV-specific IgG compared to that occurred in wild-type (WT) mice. As a consequence, TLR4-/- mice exhibited higher mortality than that of WT mice after challenge with virulent RABV. Importantly, further investigation found that TLR4 signaling promoted the recruitment of cDC2 (CD8α+ CD11b-), a subset of cDCs known to induce CD4+ T-cell immunity through their MHC-II presentation machinery. Our results imply that TLR4 is indispensable for an efficient humoral response to rabies vaccine, which provides new insight into the development of novel rabies vaccines.

The role of interferon regulatory factor 7 in the pathogenicity and immunogenicity of rabies virus in a mouse model.

Rabies is a zoonotic disease caused by the rabies virus (RABV). RABV can lead to fatal encephalitis and is still a serious threat in most parts of the world. Interferon regulatory factor 7 (IRF7) is the main transcriptional regulator of type I IFN, and it is crucial for the induction of IFNα/ß and the type I IFN-dependent immune response. In this study, we focused on the role of IRF7 in the pathogenicity and immunogenicity of RABV using an IRF7-/- mouse model. The results showed that the absence of IRF7 made mice more susceptible to RABV, because IRF7 restricted the replication of RABV in the early stage of infection. IRF7 deficiency affected the recruitment of plasmacytoid dendritic cells to the draining lymph nodes (dLNs), reduced the production of type I IFN and expression of IFN-stimulated genes. Furthermore, we found that the ability to produce specific RABV-neutralizing antibody was impaired in IRF7-/- mice. Consistently, IRF7 deficiency affected the recruitment of germinal-centre B cells to dLNs, and the generation of plasma cells and RABV-specific antibody secreting cells. Moreover, the absence of IRF7 downregulated the induction of IFN-γ and reduced type 1 T helper cell (Th1)-dependent antibody production. Collectively, our findings demonstrate that IRF7 promotes humoral immune responses and compromises the pathogenicity of RABV in a mouse model.

Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island, Indonesia.

Effective parenteral vaccines are available to control rabies in dogs. While such vaccines are successfully used worldwide, the period between vaccine boosters required to guarantee protection of the population against rabies varies between vaccines and populations. In Flores Island, Indonesia, internationally and locally produced rabies vaccines are used during annual vaccination campaigns of predominantly free-roaming owned domestic dogs. The study objective was to identify the duration of the presence and factors associated with the loss of adequate level of binding antibodies (≥0.5 EU/ml) following rabies vaccination in a domestic dog population on Flores Island. A total of 171 dogs that developed an antibody titre higher or equal to 0.5 EU/ml 30 days after vaccination (D30), were repeatedly sampled at day 90, 180, 270, and 360 after vaccination. On the day of vaccination (D0), an interview was performed with dog owners to collect information on dog characteristics (age, sex, body condition score (BCS)), history of rabies vaccination, kind of daily food, frequency of feeding, and origin of the dog. Serum samples were collected and the level of antibodies was quantitatively assessed using ELISA tests. Dogs were categorized as having an adequate level of binding antibodies (≥0.5 EU/ml) or inadequate level of binding antibodies (<0.5 EU/ml) at each time points examined. A total of 115, 72, 23, and 31 dogs were sampled at D90, D180, D270, and D360, respectively, with the highest proportion of antibodies ≥ 0.5 EU/ml (58%, 95% CI, 49-67%) at D90, which reduced gradually until D360 (35%, 95% CI, 19-52%). Multivariable logistic regression models showed that loss of adequate level of binding antibodies is significantly associated with dogs having no history of vaccination or vaccination applied more than 12 months before D0, being less than 12 months of age, and having a poor BCS. These results highlight the importance of BCS regarding the immune response duration and provide insights into frequency of vaccination campaigns required for the internationally available vaccine used on Flores Island. For dogs without vaccination history or vaccination being applied more than 12 months before D0, a booster is recommended within 3 months (a largest drop of antibodies was detected within the first 90 days) after the first vaccination to guarantee measurable protection of the population that lasts at least for one year.

U.S. dog importations during the COVID-19 pandemic: Do we have an erupting problem?

Dog importation data from 2018-2020 were evaluated to ascertain whether the dog importation patterns in the United States changed during the COVID-19 pandemic, specifically with regard to denial of entry. Dog denial of entry reports from January 1, 2018, to December 31, 2020, stored within the Centers for Disease Control and Prevention (CDC) Quarantine Activity Reporting System (QARS), were reviewed. Basic descriptive statistics were used to analyze the data. Reason for denial, country of origin, and month of importation were all examined to determine which countries of origin resulted in the largest number of denials, and whether there was a seasonal change in importations during the COVID-19 pandemic (2020), compared to previous years (2018 and 2019). During 2020, CDC denied entry to 458 dogs. This represents a 52% increase in dogs denied entry compared to the averages in 2018 and 2019. Dogs were primarily denied entry for falsified rabies vaccination certificates (56%). Three countries exported 74% of all dogs denied entry into the United States, suggesting that targeted interventions may be needed for certain countries. Increased attempts to import inadequately vaccinated dogs from countries with canine rabies in 2020 may have been due to the increased demand for domestic pets during the COVID-19 pandemic. Educational messaging should highlight the risk of rabies and the importance of making informed pet purchases from foreign entities to protect pet owners, their families, and the public.

Colloidal Manganese Salt Improves the Efficacy of Rabies Vaccines in Mice, Cats, and Dogs.

Rabies, caused by rabies virus (RABV), remains a serious threat to public health in most countries worldwide. At present, the administration of rabies vaccines has been the most effective strategy to control rabies. Herein, we evaluate the effect of colloidal manganese salt (Mn jelly [MnJ]) as an adjuvant of rabies vaccine in mice, cats, and dogs. The results showed that MnJ promoted type I interferon (IFN-I) and cytokine production in vitro and the maturation of dendritic cells (DCs) in vitro and in vivo. Besides, MnJ serving as an adjuvant for rabies vaccines could significantly facilitate the generation of T follicular helper (Tfh) cells, germinal center (GC) B cells, plasma cells (PCs), and RABV-specific antibody-secreting cells (ASCs), consequently improve the immunogenicity of rabies vaccines, and provide better protection against virulent RABV challenge. Similarly, MnJ enhanced the humoral immune response in cats and dogs as well. Collectively, our results suggest that MnJ can facilitate the maturation of DCs during rabies vaccination, which can be a promising adjuvant candidate for rabies vaccines. IMPORTANCE Extending the humoral immune response by using adjuvants is an important strategy for vaccine development. In this study, a novel adjuvant, MnJ, supplemented in rabies vaccines was evaluated in mice, cats, and dogs. Our results in the mouse model revealed that MnJ increased the numbers of mature DCs, Tfh cells, GC B cells, PCs, and RABV-specific ASCs, resulting in enhanced immunogenicity and protection rate of rabies vaccines. We further found that MnJ had the same stimulative effect in cats and dogs. Our study provides the first evidence that MnJ serving as a novel adjuvant of rabies vaccines can boost the immune response in both a mouse and pet model.

Virus-Like Vesicles Based on Semliki Forest Virus-Containing Rabies Virus Glycoprotein Make a Safe and Efficacious Rabies Vaccine Candidate in a Mouse Model.

Rabies is a fatal zoonosis that causes encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 108 focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (GC) B cells, plasma cells (PCs), and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe, and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest virus replicase (SFV nsP1 to nsP4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, but the VLVs that evolved grew to higher titers, reaching 108 FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity, and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved, demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.

The Route of Administration of Rabies Vaccines: Comparing the Data.

Cell culture rabies vaccines were initially licensed in the 1980s and are essential in the prevention of human rabies. The first post-exposure prophylaxis (PEP) vaccination regimen recommended by the World Health Organization (WHO) was administered intramuscularly over a lengthy three-month period. In efforts to reduce the cost of PEP without impinging on safety, additional research on two strategies was encouraged by the WHO including the development of less expensive production methods for CCVs and the administration of reduced volumes of CCVs via the intradermal (ID) route. Numerous clinical trials have provided sufficient data to support a reduction in the number of doses, a shorter timeline required for PEP, and the approval of the intradermal route of administration for PEP and pre-exposure prophylaxis (PreP). However, the plethora of data that have been published since the development of CCVs can be overwhelming for public health officials wishing to review and make a decision as to the most appropriate PEP and PreP regimen for their region. In this review, we examine three critical benchmarks that can serve as guidance for health officials when reviewing data to implement new PEP and PreP regimens for their region including: evidence of immunogenicity after vaccination; proof of efficacy against development of disease; and confirmation that the regimen being considered elicits a rapid anamnestic response after booster vaccination.