A single dose of recombinant adenoviral vector rabies vaccine expressing two copies of glycoprotein protects mice from lethal virus challenge.

INTRODUCTION: Rabies is a fatal infectious disease, that poses a major public health threat in developing countries. With an annual death toll of approximately 59,000, more than half of which are children, an urgent need exists for a safe, affordable, and effective preventive measure against rabies virus infection. METHODOLOGY: A recombinant rabies vaccine called Ad5-dRVG was constructed by introducing two copies of the rabies virus glycoprotein into a human adenoviral vector. Virus-neutralizing assays and virus challenge experiments were employed to evaluate the Ad5-dRVG vaccine. RESULTS: Our findings demonstrate that a single dose of Ad5-dRVG, administered either intramuscularly or orally, elicited significantly stronger immune responses than Ad5-RVG. Moreover, both vaccines provided complete protection in mice. Notably, the vaccine exhibited remarkable efficacy even at low doses, suggesting potential cost reduction in production. CONCLUSIONS: The development of the Ad5-dRVG recombinant rabies vaccine represents a significant advancement in rabies prevention. Its enhanced immunogenicity, demonstrated efficacy and potential cost savings make it a promising candidate for widespread use.

Rabies Virus Regulates Inflammatory Response in BV-2 Cells through Activation of Myd88 and NF-κB Signaling Pathways via TLR7.

Rabies is a fatal neurological infectious disease caused by rabies virus (RABV), which invades the central nervous system (CNS). RABV with varying virulence regulates chemokine expression, and the mechanisms of signaling pathway activation remains to be elucidated. The relationship between Toll-like receptors (TLRs) and immune response induced by RABV has not been fully clarified. Here, we investigated the role of TLR7 in the immune response induced by RABV, and one-way analysis of variance (ANOVA) was employed to evaluate the data. We found that different RABV strains (SC16, HN10, CVS-11) significantly increased CCL2, CXCL10 and IL-6 production. Blocking assays indicated that the TLR7 inhibitor reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). The activation of the Myd88 pathway in BV-2 cells stimulated by RABV was TLR7-dependent, whereas the inhibition of Myd88 activity reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). Meanwhile, the RABV stimulation of BV-2 cells resulted in TRL7-mediated activation of NF-κB and induced the nuclear translocation of NF-κB p65. CCL2, CXCL10 and IL-6 release was attenuated by the specific NF-κB inhibitor used (p < 0.01). The findings above demonstrate that RABV-induced expression of CCL2, CXCL10 and IL-6 involves Myd88 and NF-κB pathways via the TLR7 signal.

A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice.

Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Application prospects of the 2BS cell-adapted China fixed rabies virus vaccine strain 2aG4-B40.

BACKGROUND: Rabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine. METHODS AND RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml). CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.

Toward the Development of a Pan-Lyssavirus Vaccine.

In addition to the rabies virus (RABV), 16 more lyssavirus species have been identified worldwide, causing a disease similar to RABV. Non-rabies-related human deaths have been described, but the number of cases is unknown, and the potential of such lyssaviruses causing human disease is unpredictable. The current rabies vaccine does not protect against divergent lyssaviruses such as Mokola virus (MOKV) or Lagos bat virus (LBV). Thus, a more broad pan-lyssavirus vaccine is needed. Here, we evaluate a novel lyssavirus vaccine with an attenuated RABV vector harboring a chimeric RABV glycoprotein (G) in which the antigenic site I of MOKV replaces the authentic site of rabies virus (RABVG-cAS1). The recombinant vaccine was utilized to immunize mice and analyze the immune response compared to homologous vaccines. Our findings indicate that the vaccine RABVG-cAS1 was immunogenic and induced high antibody titers against both RABVG and MOKVG. Challenge studies with different lyssaviruses showed that replacing a single antigenic site of RABV G with the corresponding site of MOKV G provides a significant improvement over the homologous RABV vaccine and protects against RABV, Irkut virus (IRKV), and MOKV. This strategy of epitope chimerization paves the way towards a pan-lyssavirus vaccine to safely combat the diseases caused by these viruses.

A phase 2b, Randomized, double blinded comparison of the safety and efficacy of the monoclonal antibody mixture SYN023 and human rabies immune globulin in patients exposed to rabies.

BACKGROUND: SYN023 is an anti-rabies monoclonal antibody mixture administered as part of post-exposure prophylaxis regimens. The rabies virus neutralizing antibody (RVNA) concentration generally accepted as an adequate immune response to vaccination is ≥ 0.5 IU/mL. METHODS: Within 54 h of potential rabies exposure, 448 patients in two risk substrata of WHO Category III exposure were randomized to receive either 0.3 mg/kg SYN023 or 0.133 mL/kg human rabies immunoglobulin (HRIG) injected in and around the wound site(s) plus a course of rabies vaccination. Patients were followed for safety and absence of rabies for ≥ 365 days. RESULTS: GMT RVNA was higher with SYN023 throughout the 2-week post-treatment period. In the primary analysis group (n = 368), 99.4 % of SYN023 recipients versus 4.5 % of HRIG recipients had protective RVNA levels on Day 4. On Day 8, 98.1 % SYN023 versus 12.2 % HRIG recipients were protected. The SYN023:HRIG ratio of geometric mean titer of RVNA (RVNA GMTs) on Day 8 (19.42) exceeded the 10 % superiority margin (P < 0.0001) indicating higher Day 8 RVNA with SYN023. On Day 99, the SYN023:HRIG RVNA GMT ratio (0.66) was below the non-inferiority margin of 20 % (P = 0.9485) suggesting some moderation of vaccine immune response by SYN023 relative to HRIG. The ratio of percent SYN023:HRIG recipients achieving RVNA ≥ 0.5 IU/mL on Day 99 (0.98) met the non-inferiority margin of 20 % (P = 0.013) indicating anti-rabies immune response with SYN023 was non-inferior to HRIG despite this effect. There were no probable/confirmed rabies cases in any patient. Study regimens were well tolerated. CONCLUSIONS: SYN023 provided higher RVNA than HRIG soon after rabies exposure. By Day 99 post-treatment, GM RVNA with SYN023 was lower than HRIG, however, the percent of SYN023 recipients with a protective response was not inferior at this time point. No rabies cases were reported in the study. The SYN023 safety profile was acceptable. CLINICALTRIALS: gov ID: NCT03961555.

Randomized, blind, controlled phase III clinical trial: Assessing the immunogenicity and safety of freeze-dried human rabies vaccine (vero cell) with a 4-dose regimen (2-1-1) in a 10-60 year-old demographic.

OBJECTIVE: The aim of this study is to demonstrate that the freeze-dried human rabies vaccine (Vero cell), administered in a four-dose schedule (2-1-1) to the 10-60 years old population, has immunogenicity that is not inferior to the approved five-dose schedule and similar vaccines with a four-dose schedule, and to evaluate its safety. METHOD: A total of 1800 individuals were enrolled and divided into three groups: four-dose test group, four-dose control group, and five-dose control group. The rabies virus neutralizing antibodies were measured using the Rapid Fluorescent Focus Inhibition Test to assess immunogenicity, and the incidence of adverse events and serious adverse events were statistically analyzed. RESULTS: The seroconversion rates 14 days after the first dose and 14 days after the complete course of vaccination were 100% in all three groups. The antibody GMC of the four-dose test group was higher than that of the five-dose control group, but slightly lower than the four-dose control group. Seven days after the first dose, both four-dose regimen groups showed higher seroconversion rates and antibody GMCs compared to the five-dose regimen group, proving that the immunogenic effect of the four-dose regimen seven days post-first vaccination is superior to the five-dose regimen. The overall incidence of adverse events showed no significant difference between the four-dose test group and the five-dose control group, but was significantly lower in the four-dose test group compared to the four-dose control group. CONCLUSION: The vaccine in the four-dose test group is equivalent in immunogenic effect to the four-dose control group vaccine and superior to the five-dose control group vaccine; the safety of the vaccine in the four-dose test group is equivalent to the five-dose control group vaccine and superior to the four-dose control group vaccine. CLINICALTRIALS: gov number: NCT05549908.

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.

One step closer to a transmissible vaccine for rabies virus.

A recent study in PLOS Biology shows that a betaherpesvirus circulating with the vampire bat, Desmodus rotundus, could serve as an effective vector for a transmissible vaccine capable of reducing the risk of rabies virus spillover in Peru.

Different but Not Unique: Deciphering the Immunity of the Jamaican Fruit Bat by Studying Its Viriome.

A specialized and fine-tuned immune response of bats upon infection with viruses is believed to provide the basis for a "friendly" coexistence with these pathogens, which are often lethal for humans and other mammals. First insights into the immunity of bats suggest that bats have evolved to possess their own strategies to cope with viral infections. Yet, the molecular details for this innocuous coexistence remain poorly described and bat infection models are the key to unveiling these secrets. In Jamaican fruit bats (Artibeus jamaicensis), a New World bat species, infection experiments with its (putative) natural viral pathogens Tacaribe virus (TCRV), rabies virus (RABV), and the bat influenza A virus (IAV) H18N11, have contributed to an accurate, though still incomplete, representation of the bat-imposed immunity. Surprisingly, though many aspects of their innate and adaptive immune responses differ from that of the human immune response, such as a contraction of the IFN locus and reduction in the number of immunoglobulin subclasses, variations could also be observed between Jamaican fruit bats and other bat species.

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.

Lyssaviruses and the Fatal Encephalitic Disease Rabies.

Lyssaviruses cause the disease rabies, which is a fatal encephalitic disease resulting in approximately 59,000 human deaths annually. The prototype species, rabies lyssavirus, is the most prevalent of all lyssaviruses and poses the greatest public health threat. In Africa, six confirmed and one putative species of lyssavirus have been identified. Rabies lyssavirus remains endemic throughout mainland Africa, where the domestic dog is the primary reservoir - resulting in the highest per capita death rate from rabies globally. Rabies is typically transmitted through the injection of virus-laden saliva through a bite or scratch from an infected animal. Due to the inhibition of specific immune responses by multifunctional viral proteins, the virus usually replicates at low levels in the muscle tissue and subsequently enters the peripheral nervous system at the neuromuscular junction. Pathogenic rabies lyssavirus strains inhibit innate immune signaling and induce cellular apoptosis as the virus progresses to the central nervous system and brain using viral protein facilitated retrograde axonal transport. Rabies manifests in two different forms - the encephalitic and the paralytic form - with differing clinical manifestations and survival times. Disease symptoms are thought to be due mitochondrial dysfunction, rather than neuronal apoptosis. While much is known about rabies, there remain many gaps in knowledge about the neuropathology of the disease. It should be emphasized however, that rabies is vaccine preventable and dog-mediated human rabies has been eliminated in various countries. The global elimination of dog-mediated human rabies in the foreseeable future is therefore an entirely feasible goal.

Innate Immune Signaling and Role of Glial Cells in Herpes Simplex Virus- and Rabies Virus-Induced Encephalitis.

The environment of the central nervous system (CNS) represents a double-edged sword in the context of viral infections. On the one hand, the infectious route for viral pathogens is restricted via neuroprotective barriers; on the other hand, viruses benefit from the immunologically quiescent neural environment after CNS entry. Both the herpes simplex virus (HSV) and the rabies virus (RABV) bypass the neuroprotective blood-brain barrier (BBB) and successfully enter the CNS parenchyma via nerve endings. Despite the differences in the molecular nature of both viruses, each virus uses retrograde transport along peripheral nerves to reach the human CNS. Once inside the CNS parenchyma, HSV infection results in severe acute inflammation, necrosis, and hemorrhaging, while RABV preserves the intact neuronal network by inhibiting apoptosis and limiting inflammation. During RABV neuroinvasion, surveilling glial cells fail to generate a sufficient type I interferon (IFN) response, enabling RABV to replicate undetected, ultimately leading to its fatal outcome. To date, we do not fully understand the molecular mechanisms underlying the activation or suppression of the host inflammatory responses of surveilling glial cells, which present important pathways shaping viral pathogenesis and clinical outcome in viral encephalitis. Here, we compare the innate immune responses of glial cells in RABV- and HSV-infected CNS, highlighting different viral strategies of neuroprotection or Neuroinflamm. in the context of viral encephalitis.

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.

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.

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.

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.