RABV antigenic peptide loaded polymeric nanoparticle production, characterization, and preliminary investigation of its biological activity.

Nanoparticle-based antigen carrier systems have become a significant area of research with the advancement of nanotechnology. Biodegradable polymers have emerged as particularly promising carrier vehicles due to their ability to address the limitations of existing vaccine systems. In this study, we successfully encapsulated the G5-24 linear peptide, located between amino acids 253 and 275 in the primary sequence of the rabies virus G protein, into biodegradable and biocompatible PLGA copolymer using the double emulsion solvent evaporation method. The resulting nanoparticles had a size of approximately 230.9 ± 0.9074 nm, with a PDI value of 0.168 ± 0.017 and a zeta potential value of -9.86 ± 0.132 mV. SEM images confirmed that the synthesized nanoparticles were uniform in size and distribution. Additionally, FTIR spectra indicated successful peptide loading into the nanoparticles. The encapsulation efficiency of the peptide-loaded nanoparticles was 73.3%, with a peptide loading capacity of 48.2% and a reaction yield of 30.4%. Peptide release studies demonstrated that 65.55% of the peptide was released in a controlled manner over 28 d, following a 'biphasic burst release' profile consistent with the degradation profile of PLGA. This controlled release is particularly beneficial for vaccine studies. Cytotoxicity tests revealed that the R-NP formulation did not induce cytotoxicity in fibroblast cells and enhanced NO production in macrophages, indicating its potential for vaccine development.

Self-assembling nanoparticle engineered from the ferritinophagy complex as a rabies virus vaccine candidate.

Over the past decade, there has been a growing interest in ferritin-based vaccines due to their enhanced antigen immunogenicity and favorable safety profiles, with several vaccine candidates targeting various pathogens advancing to phase I clinical trials. Nevertheless, challenges associated with particle heterogeneity, improper assembly and unanticipated immunogenicity due to the bulky protein adaptor have impeded further advancement. To overcome these challenges, we devise a universal ferritin-adaptor delivery platform based on structural insights derived from the natural ferritinophagy complex of the human ferritin heavy chain (FTH1) and the nuclear receptor coactivator 4 (NCOA4). The engineered ferritinophagy (Fagy)-tag peptide demonstrate significantly enhanced binding affinity to the 24-mer ferritin nanoparticle, enabling efficient antigen presentation. Subsequently, we construct a self-assembling rabies virus (RABV) vaccine candidate by noncovalently conjugating the Fagy-tagged glycoprotein domain III (GDIII) of RABV to the ferritin nanoparticle, maintaining superior homogeneity, stability and immunogenicity. This vaccine candidate induces potent, rapid, and durable immune responses, and protects female mice against the authentic RABV challenge after single-dose administration. Furthermore, this universal, ferritin-based antigen conjugating strategy offers significant potential for developing vaccine against diverse pathogens and diseases.

Development of novel canine phage display-derived neutralizing monoclonal antibody fragments against rabies virus from immunized dogs.

Animal rabies is a potentially fatal infectious disease in mammals, especially dogs. Currently, the number of rabies cases in pet dogs is increasing in several regions of Thailand. However, no passive postexposure prophylaxis (PEP) has been developed to combat rabies infection in animals. As monoclonal antibodies (MAbs) are promising biological therapies for postinfection, we developed a canine-neutralizing MAb against rabies virus (RABV) via the single-chain variable fragment (scFv) platform. Immunized phage-displaying scFv libraries were constructed from PBMCs via the pComb3XSS system. Diverse canine VHVLκ and VHVLλ libraries containing 2.4 × 108 and 1.3 × 106 clones, respectively, were constructed. Five unique clones that show binding affinity with the RABV glycoprotein were then selected, of which K9RABVscFv1 and K9RABVscFv16 showed rapid fluorescent foci inhibition test (RFFIT) neutralizing titers above the human protective level of 0.5 IU/ml. Finally, in silico docking predictions revealed that the residues on the CDRs of these neutralizing clones interact mainly with similar antigenic sites II and III on the RABV glycoprotein. These candidates may be used to develop complete anti-RABV MAbs as a novel PEP protocol in pet dogs and other animals.

Rabies in Cats-An Emerging Public Health Issue.

Human rabies cases today are predominantly associated with infection from rabid domestic dogs. Unlike dogs, a common global reservoir species that perpetuates rabies viruses (RABV) within their populations, domestic cats are much less frequently reported or vaccinated. Epidemiologically, cats are important vectors of lyssaviruses but are not viral reservoirs. Typically, cats are incidental hosts only, infected with the predominant lyssavirus in their geographic locale. Human cases associated with rabid cats have occurred in Africa, Asia, Europe and throughout the Americas. As adept, solitary hunters, wild and domestic felids are at risk of lyssavirus infection based upon interactions with infected prey, such as bats, or from transmission by other mesocarnivores, such as rabid dogs, foxes, jackals, raccoons, and skunks. Current veterinary vaccines provide safe and effective immunity in cats against phylogroup I lyssaviruses, such as RABV, but not against divergent lyssaviruses in phylogroups II-IV. With the focus upon the global elimination of canine rabies, the emergence of rabies in cats represents a concerning trend. Clearly, education about the occurrence of rabies in cats needs to be improved, as well as the routine vaccination of cats to reduce the associated risks to public health, agriculture, and conservation biology from a One Health perspective.

Notes from the Field: Enhanced Surveillance for Raccoon Rabies Virus Variant and Vaccination of Wildlife for Management - Omaha, Nebraska, October 2023-July 2024.

On September 28, 2023, a kitten aged approximately 6 weeks found in Omaha, Nebraska, had test results positive for rabies at the Nebraska Veterinary Diagnostic Center (NVDC) after dying with neurologic signs and having bitten and scratched its caretakers. Preliminary investigation identified 10 exposed persons for whom postexposure prophylaxis (PEP)† was recommended. Subsequent variant-typing by NVDC yielded a presumptive positive result for the Eastern raccoon rabies virus variant (RRVV), which CDC confirmed on October 6.

Molecular characterisation of human rabies in Tanzania and Kenya: a case series report and phylogenetic investigation.

BACKGROUND: Rabies remains a major public health problem in low- and middle-income countries. However, human rabies deaths are rarely laboratory-confirmed or sequenced, especially in Africa. Five human rabies deaths from Tanzania and Kenya were investigated and the causative rabies viruses sequenced, with the aim of identifying implications for rabies control at individual, healthcare and societal levels. CASE PRESENTATION: The epidemiological context and care of these cases was contrasting. Four had a clear history of being bitten by dogs, while one had an unclear biting history. Two individuals sought medical attention within a day of being bitten, whereas three sought care only after developing rabies symptoms. Despite seeking medical care, none of the cases received complete post-exposure prophylaxis: one patient received only tetanus vaccination, one did not complete the post-exposure vaccination regimen, one followed an off-label vaccination schedule, and two did not receive any post-exposure vaccinations before the onset of symptoms. These cases highlight serious gaps in health-seeking behaviour, and in health systems providing appropriate care following risky exposures, including in the accessibility and effectiveness of post-exposure prophylaxis as it is administered in the region. CONCLUSIONS: The viral genomic and epidemiological data confirms dog-mediated rabies as the cause of each of these deaths. The phylogenetic investigation highlights the transboundary circulation of rabies within domestic dog populations, revealing distinct rabies virus clades with evidence of regional spread. These findings underscore the importance of coordinated cross-border control efforts between the two countries. Urgent action is needed to improve awareness around the need for emergency post-exposure vaccines that should be accessible in local communities and administered appropriately, as well as investment in coordinated dog vaccination to control dog-mediated rabies, the underlying cause of these deaths.

Adapted Milwaukee protocol for rabies treatment in a Brazilian indigenous child: case report.

BACKGROUND: This case report describes the treatment of a 12-year-old indigenous Brazilian girl from the Maxakali group with rabies using the adapted Milwaukee Protocol. CASE PRESENTATION: The patient suffered a superficial bat bite on her right elbow, reported on April 5, 2022. Despite receiving immunoglobulin, a vaccine, and antiviral medications such as amantadine and sapropterin, the patient succumbed to the disease 25 days after hospital admission. The report highlights the inherent challenges in treating rabies due to the virus's neurotropic nature and the difficulties in delivering antiviral drugs to the central nervous system. The case underscores the need for early antiviral intervention and calls for more studies to validate and improve treatment protocols for rabies in vulnerable populations, particularly those with genetic and immunological susceptibilities like the Maxakali indigenous group. CONCLUSION: The findings suggest that while the Milwaukee Protocol offers some hope, significant obstacles remain in achieving successful outcomes in rabies cases.

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.

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 centre (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.

Role of Neurotropic Viruses in Brain Metastasis of Breast Cancer: Mechanisms and Therapeutic Implications.

Neurotropic viruses have been implicated in altering the central nervous system microenvironment and promoting brain metastasis of breast cancer through complex interactions involving viral entry mechanisms, modulation of the blood-brain barrier, immune evasion, and alteration of the tumour microenvironment. This narrative review explores the molecular mechanisms by which neurotropic viruses such as Herpes Simplex Virus, Human Immunodeficiency Virus, Japanese Encephalitis Virus, and Rabies Virus facilitate brain metastasis, focusing on their ability to disrupt blood-brain barrier integrity, modulate immune responses, and create a permissive environment for metastatic cell survival and growth within the central nervous system. Current therapeutic implications and challenges in targeting neurotropic viruses to prevent or treat brain metastasis are discussed, highlighting the need for innovative strategies and multidisciplinary approaches in virology, oncology, and immunology.

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.

The roles of rabies virus structural proteins in immune evasion and implications for vaccine development.

Rabies is a zoonotic infectious disease that targets the nervous system of human and animals and has about 100% fatality rate without treatment. Rabies virus is a bullet-like viral particle composed of five structural proteins, including nucleoprotein (N), phosphorylated protein (P), matrix protein (M), glycoprotein (G), and large subunit (L) of RNA-dependent RNA polymerase. These multifunctional viral proteins also play critical roles in the immune escape by inhibiting specific immune responses in the host, resulting in massive replication of the virus in the nervous system and abnormal behaviors of patients such as brain dysfunction and hydrophobia, which ultimately lead to the death of patients. Herein, the role of five structural proteins of rabies virus in the viral replication and immune escape and its implication for the development of vaccines were systemically reviewed, so as to shed light on the understanding of pathogenic mechanism of rabies virus.

Factors affecting the outcome of primary rabies vaccination in young cats.

Limited data exist on the factors affecting feline rabies vaccination outcomes during primary immunization. This study aimed to assess if specific factors (signalment, vaccination count, vaccine brand, and time since last vaccination) correlated with meeting global antibody titer standards and absolute titers in young cats given monovalent inactivated rabies vaccines. Analyzing a dataset from cats tested before their first annual booster using the FAVN test, logistic and linear regression models were applied. Among 379 cats, 94.2 % achieved titers meeting or exceeding the standard threshold (≥0.5 IU/ml). Time since last vaccination proved to be the primary predictor of vaccination success. Cats receiving two vaccinations tended toward higher titers. Age, sex, breed, and vaccine type showed no impact on outcomes. The present study indicates that vaccination failure in young cats is uncommon, and that the time interval from the latest vaccination is the single most important predictor of successful rabies vaccination.

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.

Investigation of Rabies virus in wild mammals of the atlantic forest in Rio de Janeiro, Brazil.

With the successful control of rabies transmitted by dogs in Brazil, wild animals have played a relevant epidemiological role in the transmission of rabies virus (RABV). Bats, non-human primates and wild canines are the main wild animals that transmit RABV in the country. It is worth highlighting the possibility of synanthropic action of these species, when they become adapted to urban areas, causing infections in domestic animals and eventually in humans. This work aimed to evaluate the circulation of RABV in the Pedra Branca Forest, an Atlantic Forest area, located in the state of Rio de Janeiro, Southeast Brazil. Saliva and blood samples were obtained from 60 individuals of eight species of bats, captured with mist nets, and 13 individuals of callitrichid primates, captured with tomahawk traps. Saliva samples were subjected to Reverse Transcription Polymerase Chain Reaction (RT-PCR), targeting the RABV N gene, with all samples being negative. Blood samples of all animals were submitted to the Rapid Fluorescent Focus Inhibition Test (RFFIT) to detect neutralizing antibodies (Ab) for RABV. Six bat samples (8%) were seropositive for RABV with antibody titers greater than or equal to 0.1 IU/mL. The detection of Ab but not viral RNA indicates exposure rather than current RABV transmission in the analyzed populations. The results presented here reinforce the importance of serological studies in wildlife to access RABV circulation in a region.

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.

Geographic Distribution of Rabies Virus and Genomic Sequence Alignment of Wild and Vaccine Strains, Kenya.

Rabies, a viral disease that causes lethal encephalitis, kills ≈59,000 persons worldwide annually, despite availability of effective countermeasures. Rabies is endemic in Kenya and is mainly transmitted to humans through bites from rabid domestic dogs. We analyzed 164 brain stems collected from rabid animals in western and eastern Kenya and evaluated the phylogenetic relationships of rabies virus (RABV) from the 2 regions. We also analyzed RABV genomes for potential amino acid changes in the vaccine antigenic sites of nucleoprotein and glycoprotein compared with RABV vaccine strains commonly used in Kenya. We found that RABV genomes from eastern Kenya overwhelmingly clustered with the Africa-1b subclade and RABV from western Kenya clustered with Africa-1a. We noted minimal amino acid variances between the wild and vaccine virus strains. These data confirm minimal viral migration between the 2 regions and that rabies endemicity is the result of limited vaccine coverage rather than limited efficacy.

Rabies virus circulation in a highly diverse bat assemblage from a high-risk area for zoonoses outbreaks in the Brazilian Amazon.

Bats are the second most diverse order of mammals and play a central role in ecosystem dynamics. They are also important reservoirs of potentially zoonotic microorganisms, of which rabies virus is the most lethal among the bat-transmitted zoonotic pathogens. Importantly, recent outbreaks of human rabies have been reported from the Brazilian Amazon. Here we present a survey of bat species and rabies virus (RABV) circulation in a bat assemblage in the Marajó region, northern Brazil. Using data from mist-net captures and bioacoustic sampling, 56 bat species were recorded along the Jacundá River basin over a 10-day expedition in November 2022. For the investigation of RABV, we used the direct fluorescent antibody test (DFAT) and the rapid fluorescent focus inhibition test (RFFIT). In total, 159 bat individuals from 22 species were investigated for RABV. Five adults of the common vampire bat, Desmodus rotundus, showed RABV-specific antibodies in serum samples. Additionally, we report on local residents with injuries caused by D. rotundus bites and the occurrence of colonies of non-hematophagous bats from different species roosting inside human residences. This scenario raises concerns about the risks of new cases of human rabies and other zoonotic diseases associated with bats in the region and highlights the need for epidemiological surveillance and mitigation measures to prevent outbreaks of emerging infectious diseases.

Development of mRNA rabies vaccines.

Rabies, primarily transmitted to humans by dogs (accounting for 99% of cases). Once rabies occurs, its mortality rate is approximately 100%. Post-exposure prophylaxis (PEP) is critical for preventing the onset of rabies after exposure to rabid animals, and vaccination is a pivotal element of PEP. However, high costs and complex immunization protocols have led to poor adherence to rabies vaccinations. Consequently, there is an urgent need to develop new rabies vaccines that are safe, highly immunogenic, and cost-effective to improve compliance and effectively prevent rabies. In recent years, mRNA vaccines have made significant progress in the structural modification and optimization of delivery systems. Various mRNA vaccines are currently undergoing clinical trials, positioning them as viable alternatives to the traditional rabies vaccines. In this article, we discuss a novel mRNA rabies vaccine currently undergoing clinical and preclinical testing, and evaluate its potential to replace existing vaccines.