An Immunoinformatic-Based In Silico Identification on the Creation of a Multiepitope-Based Vaccination Against the Nipah Virus.

The zoonotic viruses pose significant threats to public health. Nipah virus (NiV) is an emerging virus transmitted from bats to humans. The NiV causes severe encephalitis and acute respiratory distress syndrome, leading to high mortality rates, with fatality rates ranging from 40% to 75%. The first emergence of the disease was found in Malaysia in 1998-1999 and later in Bangladesh, Cambodia, Timor-Leste, Indonesia, Singapore, Papua New Guinea, Vietnam, Thailand, India, and other South and Southeast Asian nations. Currently, no specific vaccines or antiviral drugs are available. The potential advantages of epitope-based vaccines include their ability to elicit specific immune responses while minimizing potential side effects. The epitopes have been identified from the conserved region of viral proteins obtained from the UniProt database. The selection of conserved epitopes involves analyzing the genetic sequences of various viral strains. The present study identified two B cell epitopes, seven cytotoxic T lymphocyte (CTL) epitopes, and seven helper T lymphocyte (HTL) epitope interactions from the NiV proteomic inventory. The antigenic and physiological properties of retrieved protein were analyzed using online servers ToxinPred, VaxiJen v2.0, and AllerTOP. The final vaccine candidate has a total combined coverage range of 80.53%. The tertiary structure of the constructed vaccine was optimized, and its stability was confirmed with the help of molecular simulation. Molecular docking was performed to check the binding affinity and binding energy of the constructed vaccine with TLR-3 and TLR-5. Codon optimization was performed in the constructed vaccine within the Escherichia coli K12 strain, to eliminate the danger of codon bias. However, these findings must require further validation to assess their effectiveness and safety. The development of vaccines and therapeutic approaches for virus infection is an ongoing area of research, and it may take time before effective interventions are available for clinical use.

Mapping the distribution of Nipah virus infections: a geospatial modelling analysis.

BACKGROUND: Nipah virus is a zoonotic paramyxovirus responsible for disease outbreaks with high fatality rates in south and southeast Asia. However, knowledge of the potential geographical extent and risk patterns of the virus is poor. We aimed to establish an integrated spatiotemporal and phylogenetic database of Nipah virus infections in humans and animals across south and southeast Asia. METHODS: In this geospatial modelling analysis, we developed an integrated database containing information on the distribution of Nipah virus infections in humans and animals from 1998 to 2021. We conducted phylodynamic analysis to examine the evolution and migration pathways of the virus and meta-analyses to estimate the adjusted case-fatality rate. We used two boosted regression tree models to identify the potential ecological drivers of Nipah virus occurrences in spillover events and endemic areas, and mapped potential risk areas for Nipah virus endemicity. FINDINGS: 749 people and eight bat species across nine countries were documented as being infected with Nipah virus. On the basis of 66 complete genomes of the virus, we identified two clades-the Bangladesh clade and the Malaysia clade-with the time of the most recent common ancestor estimated to be 1863. Adjusted case-fatality rates varied widely between countries and were higher for the Bangladesh clade than for the Malaysia clade. Multivariable meta-regression analysis revealed significant relationships between case-fatality rate estimates and viral clade (p=0·0021), source country (p=0·016), proportion of male patients (p=0·036), and travel time to health-care facilities (p=0·036). Temperature-related bioclimate variables and the probability of occurrence of Pteropus medius were important contributors to both the spillover and the endemic infection models. INTERPRETATION: The suitable niches for Nipah virus are more extensive than previously reported. Future surveillance efforts should focus on high-risk areas informed by updated projections. Specifically, intensifying zoonotic surveillance efforts, enhancing laboratory testing capacity, and implementing public health education in projected high-risk areas where no human cases have been reported to date will be crucial. Additionally, strengthening wildlife surveillance and investigating potential modes of transmission in regions with documented human cases is needed. FUNDING: The Key Research and Development Program of China.

Foals of mares vaccinated for Hendra virus have a suboptimal response to HeV vaccination.

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals.

Current status of diagnostic assays for emerging zoonotic viruses: Nipah and Hendra.

INTRODUCTION: Nipah and Hendra viruses belong to the Paramyxoviridae family, which pose a significant threat to human health, with sporadic outbreaks causing severe morbidity and mortality. Early symptoms include fever, cough, sore throat, and headache, which offer little in terms of differential diagnosis. There are no specific therapeutics and vaccines for these viruses. AREAS COVERED: This review comprehensively covers a spectrum of diagnostic techniques for Nipah and Hendra virus infections, discussed in conjunction with appropriate type of samples during the progression of infection. Serological assays, reverse transcriptase Real-Time PCR assays, and isothermal amplification assays are discussed in detail, along with a listing of few commercially available detection kits. Patents protecting inventions in Nipah and Hendra virus detection are also covered. EXPERT OPINION: Despite several outbreaks of Nipah and Hendra infections in the past decade, in-depth research into their pathogenesis, Point-of-Care diagnostics, specific therapies, and human vaccines is lacking. A prompt and accurate diagnosis is pivotal for efficient outbreak management, patient treatment, and the adoption of preventative measures. The emergence of rapid point-of-care tests holds promise in enhancing diagnostic capabilities in real-world settings. The patent landscape emphasizes the importance of innovation and collaboration within the legal and business realms.

Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle.

Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

Enzyme-Linked Immunosorbent Assay Using Henipavirus-Receptor EphrinB2 and Monoclonal Antibodies for Detecting Nipah and Hendra Viruses.

The Nipah virus (NiV) and the Hendra virus (HeV) are highly pathogenic zoonotic diseases that can cause fatal infections in humans and animals. Early detection is critical for the control of NiV and HeV infections. We present the development of two antigen-detection ELISAs (AgELISAs) using the henipavirus-receptor EphrinB2 and monoclonal antibodies (mAbs) to detect NiV and HeV. The NiV AgELISA detected only NiV, whereas the NiV/HeV AgELISA detected both NiV and HeV. The diagnostic specificities of the NiV AgELISA and the NiV/HeV AgELISA were 100% and 97.8%, respectively. Both assays were specific for henipaviruses and showed no cross-reactivity with other viruses. The AgELISAs detected NiV antigen in experimental pig nasal wash samples taken at 4 days post-infection. With the combination of both AgELISAs, NiV can be differentiated from HeV. Complementing other henipavirus detection methods, these two newly developed AgELISAs can rapidly detect NiV and HeV in a large number of samples and are suitable for use in remote areas where other tests are not available.

Nipah Virus Neurotropism: Insights into Blood-Brain Barrier Disruption.

The genome of the Nipah virus (NiV) encodes a variety of structural proteins linked to a diverse array of symptoms, including fevers, headaches, somnolence, and respiratory impairment. In instances of heightened severity, it can also invade the central nervous system (CNS), resulting in more pronounced problems. This work investigates the effects of NiV on the blood-brain barrier (BBB), the vital physiological layer responsible for safeguarding the CNS by regulating the passage of chemicals into the brain selectively. To achieve this, the researchers (MMJAO, AM and MNMD) searched a variety of databases for relevant articles on NiV and BBB disruption, looking for evidence of work on inflammation, immune response (cytokines and chemokines), tight junctions (TJs), and basement membranes related to NiV and BBB. Based on these works, it appears that the affinity of NiV for various receptors, including Ephrin-B2 and Ephrin-B3, has seen many NiV infections begin in the respiratory epithelium, resulting in the development of acute respiratory distress syndrome. The virus then gains entry into the circulatory system, offering it the potential to invade brain endothelial cells (ECs). NiV also has the ability to infect the leukocytes and the olfactory pathway, offering it a "Trojan horse" strategy. When NiV causes encephalitis, the CNS generates a strong inflammatory response, which makes the blood vessels more permeable. Chemokines and cytokines all have a substantial influence on BBB disruption, and NiV also has the ability to affect TJs, leading to disturbances in the structural integrity of the BBB. The pathogen's versatility is also shown by its capacity to impact multiple organ systems, despite particular emphasis on the CNS. It is of the utmost importance to comprehend the mechanisms by which NiV impacts the integrity of the BBB, as such comprehension has the potential to inform treatment approaches for NiV and other developing viral diseases. Nevertheless, the complicated pathophysiology and molecular pathways implicated in this phenomenon have offered several difficult challenges to researchers to date, underscoring the need for sustained scientific investigation and collaboration in the ongoing battle against this powerful virus.

Research on emerging infectious diseases: pros and cons of centralised government controls.

As the world grapples with the constant threat of new pathogens, the role of government oversight in research and response efforts has become a topic of considerable debate in the academic community. In the recently released "SOP [standard operating procedure] for Nipah virus research in Kerala for studies involving human participants / human samples" by the Government of Kerala, the SOP, apart from administrative permission, requires the proposal to be cleared by the Institutional Research Committee at a Government Medical College, and the inclusion of an investigator from a government institution [1]. In these challenging times, it is crucial to weigh the pros and cons of stringent administrative controls to ensure an effective and ethical approach to tackling emerging infectious diseases.

Evaluation of the immunogenicity of an mRNA vectored Nipah virus vaccine candidate in pigs.

Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 µg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.

A potent Henipavirus cross-neutralizing antibody reveals a dynamic fusion-triggering pattern of the G-tetramer.

The Hendra and Nipah viruses (HNVs) are highly pathogenic pathogens without approved interventions for human use. In addition, the interaction pattern between the attachment (G) and fusion (F) glycoproteins required for virus entry remains unclear. Here, we isolate a panel of Macaca-derived G-specific antibodies that cross-neutralize HNVs via multiple mechanisms. The most potent antibody, 1E5, confers adequate protection against the Nipah virus challenge in female hamsters. Crystallography demonstrates that 1E5 has a highly similar binding pattern to the receptor. In cryo-electron microscopy studies, the tendency of 1E5 to bind to the upper or lower heads results in two distinct quaternary structures of G. Furthermore, we identify the extended outer loop ß1S2-ß1S3 of G and two pockets on the apical region of fusion (F) glycoprotein as the essential sites for G-F interactions. This work highlights promising drug candidates against HNVs and contributes deeper insights into the viruses.

Multi-epitope vaccine design using in silico analysis of glycoprotein and nucleocapsid of NIPAH virus.

According to the 2018 WHO R&D Blueprint, Nipah virus (NiV) is a priority disease, and the development of a vaccine against NiV is strongly encouraged. According to criteria used to categorize zoonotic diseases, NiV is a stage III disease that can spread to people and cause unpredictable outbreaks. Since 2001, the NiV virus has caused annual outbreaks in Bangladesh, while in India it has caused occasional outbreaks. According to estimates, the mortality rate for infected individuals ranges from 70 to 91%. Using immunoinformatic approaches to anticipate the epitopes of the MHC-I, MHC-II, and B-cells, they were predicted using the NiV glycoprotein and nucleocapsid protein. The selected epitopes were used to develop a multi-epitope vaccine construct connected with linkers and adjuvants in order to improve immune responses to the vaccine construct. The 3D structure of the engineered vaccine was anticipated, optimized, and confirmed using a variety of computer simulation techniques so that its stability could be assessed. According to the immunological simulation tests, it was found that the vaccination elicits a targeted immune response against the NiV. Docking with TLR-3, 7, and 8 revealed that vaccine candidates had high binding affinities and low binding energies. Finally, molecular dynamic analysis confirms the stability of the new vaccine. Codon optimization and in silico cloning showed that the proposed vaccine was expressed to a high degree in Escherichia coli. The study will help in identifying a potential epitope for a vaccine candidate against NiV. The developed multi-epitope vaccine construct has a lot of potential, but they still need to be verified by in vitro & in vivo studies.

Indiscriminate activities of different henipavirus polymerase complex proteins allow for efficient minigenome replication in hybrid systems.

The henipaviruses, including Nipah virus (NiV) and Hendra virus (HeV), are biosafety level 4 (BSL-4) zoonotic pathogens that cause severe neurological and respiratory disease in humans. To study the replication machinery of these viruses, we developed robust minigenome systems that can be safely used in BSL-2 conditions. The nucleocapsid (N), phosphoprotein (P), and large protein (L) of henipaviruses are critical elements of their replication machinery and thus essential support components of the minigenome systems. Here, we tested the effects of diverse combinations of the replication support proteins on the replication capacity of the NiV and HeV minigenomes by exchanging the helper plasmids coding for these proteins among the two viruses. We demonstrate that all combinations including one or more heterologous proteins were capable of replicating both the NiV and HeV minigenomes. Sequence alignment showed identities of 92% for the N protein, 67% for P, and 87% for L. Notably, variations in amino acid residues were not concentrated in the N-P and P-L interacting regions implying that dissimilarities in amino acid composition among NiV and HeV polymerase complex proteins may not impact their interactions. The observed indiscriminate activity of NiV and HeV polymerase complex proteins is different from related viruses, which can support the replication of heterologous genomes only when the whole polymerase complex belongs to the same virus. This newly observed promiscuous property of the henipavirus polymerase complex proteins likely attributed to their conserved interaction regions could potentially be harnessed to develop universal anti-henipavirus antivirals.IMPORTANCEGiven the severity of disease induced by Hendra and Nipah viruses in humans and the continuous emergence of new henipaviruses as well as henipa-like viruses, it is necessary to conduct a more comprehensive investigation of the biology of henipaviruses and their interaction with the host. The replication of henipaviruses and the development of antiviral agents can be studied in systems that allow experiments to be performed under biosafety level 2 conditions. Here, we developed robust minigenome systems for the Nipah virus (NiV) and Hendra virus (HeV) that provide a convenient alternative for studying NiV and HeV replication. Using these systems, we demonstrate that any combination of the three polymerase complex proteins of NiV and HeV could effectively initiate the replication of both viral minigenomes, which suggests that the interaction regions of the polymerase complex proteins could be effective targets for universal and effective anti-henipavirus interventions.

Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains.

Nipah virus (NiV) is a World Health Organization priority pathogen and there are currently no approved drugs for clinical immunotherapy. Through the use of a naïve human phage-displayed Fab library, two neutralizing antibodies (NiV41 and NiV42) targeting the NiV receptor binding protein (RBP) were identified. Following affinity maturation, antibodies derived from NiV41 display cross-reactivity against both NiV and Hendra virus (HeV), whereas the antibody based on NiV42 is only specific to NiV. Results of immunogenetic analysis reveal a correlation between the maturation of antibodies and their antiviral activity. In vivo testing of NiV41 and its mature form (41-6) show protective efficacy against a lethal NiV challenge in hamsters. Furthermore, a 2.88 Å Cryo-EM structure of the tetrameric RBP and antibody complex demonstrates that 41-6 blocks the receptor binding interface. These findings can be beneficial for the development of antiviral drugs and the design of vaccines with broad spectrum against henipaviruses.

Structure and design of Langya virus glycoprotein antigens.

Langya virus (LayV) is a recently discovered henipavirus (HNV), isolated from febrile patients in China. HNV entry into host cells is mediated by the attachment (G) and fusion (F) glycoproteins which are the main targets of neutralizing antibodies. We show here that the LayV F and G glycoproteins promote membrane fusion with human, mouse, and hamster target cells using a different, yet unknown, receptor than Nipah virus (NiV) and Hendra virus (HeV) and that NiV- and HeV-elicited monoclonal and polyclonal antibodies do not cross-react with LayV F and G. We determined cryoelectron microscopy structures of LayV F, in the prefusion and postfusion states, and of LayV G, revealing their conformational landscape and distinct antigenicity relative to NiV and HeV. We computationally designed stabilized LayV G constructs and demonstrate the generalizability of an HNV F prefusion-stabilization strategy. Our data will support the development of vaccines and therapeutics against LayV and closely related HNVs.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Infection and transmission of henipavirus in animals.

Henipavirus (HNV) is well known for two zoonotic viruses in the genus, Hendra virus (HeV) and Nipah virus (NiV), which pose serious threat to human and animal health. In August 2022, a third zoonotic virus in the genus Henipavirus, Langya virus (LayV), was discovered in China. The emergence of HeV, NiV, and LayV highlights the persistent threat of HNV to human and animal health. In addition to the above three HNVs, new species within this genus are still being discovered. Although they have not yet caused a pandemic in humans or livestock, they still have the risk of spillover as a potential threat to the health of humans and animals. It's important to understand the infection and transmission of different HNV in animals for the prevention and control of current or future HNV epidemics. Therefore, this review mainly summarizes the animal origin, animal infection and transmission of HNV that have been found worldwide, and further analyzes and summarizes the rules of infection and transmission, so as to provide a reference for relevant scientific researchers. Furthermore, it can provide a direction for epidemic prevention and control, and animal surveillance to reduce the risk of the global pandemic of HNV.

Horse populations are severely underestimated in a region at risk of Hendra virus spillover.

OBJECTIVE: To identify the size and distribution of the horse population in the Northern Rivers Region of NSW, including changes from 2007 to 2021, to better understand populations at risk of Hendra virus transmission. METHODS: Census data from the 2007 Equine Influenza (EI) outbreak were compared with data collected annually by New South Wales Local Land Services (LLS) (2011-2021), and with field observations via road line transects (2021). RESULTS: The horse populations reported to LLS in 2011 (3000 horses; 0.77 horses/km2) was 145% larger than that reported during the EI outbreak in 2007 (1225 horses; 0.32 horses/km2). This was inconsistent with the 6% increase in horses recorded from 2011 to 2020 within the longitudinal LLS dataset. Linear modelling suggested the true horse population of this region in 2007 was at least double that reported at the time. Distance sampling in 2021 estimated the region's population at 10,185 horses (3.89 per km2; 95% CI = 4854-21,372). Field sampling and modelling identified higher horse densities in rural cropland, with the percentage of conservation land, modified grazing, and rural residential land identified as the best predictors of horse densities. CONCLUSIONS: Data from the 2007 EI outbreak no longer correlates to the current horse population in size or distribution and was likely not a true representation at the time. Current LLS data also likely underestimates horse populations. Ongoing efforts to further quantify and map horse populations in Australia are important for estimating and managing the risk of equine zoonoses.