Full-length G glycoprotein directly extracted from rabies virus with detergent and then stabilized by amphipols in liquid and freeze-dried forms.

Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV-G/A8-35 in a solution of up to 1 year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed long-term stability for 2-years at 5, 25, and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions.

Rabies virus glycoprotein enhances spatial memory via the PDZ binding motif.

Rabies is a life-threatening viral infection of the brain. Rabies virus (RABV) merely infects excitable cells including neurons provoking drastic behaviors including negative emotional memories. RABV glycoprotein (RVG) plays a critical role in RABV pathogenesis. RVG interacts with various cytoplasmic PDZ (PSD-95/Dlg/ZO-1) containing proteins through its PDZ binding motif (PBM). PTZ domains have crucial role in formation and function of signal transduction. Hippocampus is one of the cerebral regions that contain high load of viral antigens. We examined impact of RVG expression in the dorsal hippocampus on aversive as well as spatial learning and memory performance in rats. Two microliter of the lentiviral vector (~108 T.U./ml) encoding RVG or ∆RVG (deleted PBM) genomes was microinjected into the hippocampal CA1. After 1 week, rat's brain was cross-sectioned and RVG/∆RVG-expressing neuronal cells were confirmed by fluorescent microscopy. Passive avoidance and spatial learning and memory were assessed in rats by Shuttle box and Morris water maze (MWM). In the shuttle box, both RVG and ∆RVG decreased the time spent in the dark compartment compared to control (p < 0.05). In MWM, RVG and ∆RVG did not affect the acquisition of spatial task. In the probe test, RVG-expressing rats spent more time in the target quadrant, and also reached the platform position sooner than control group (p < 0.05). Rats expressing ∆RVG significantly swam farther from the hidden platform than RVG group (p < 0.05). Our data indicate RVG expression in the hippocampus strengthens aversive and spatial learning and memory performance. The boosting effect on spatial but not avoidance memory is mediated through PBM.

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus.

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431-1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.

Rabies virus matrix protein targets host actin cytoskeleton: a protein-protein interaction analysis.

Multifunctional matrix protein (M) of rabies virus (RABV) plays essential roles in the pathogenesis of rabies infection. Identification of M protein interacting partners in target hosts could help to elucidate the biological pathways and molecular mechanisms involved in the pathogenesis of this virus. In this study, two-dimensional Far-western blotting (2D-Far-WB) technique was applied to find possible matrix protein partners in the rat brainstem. Recombinant RABV M was expressed in Pichia pastoris and was partially purified. Subsequently, 2D-Far-WB-determined six rat brainstem proteins interacted with recombinant M proteins that were identified by mass spectrometry. Functional annotation by gene ontology analysis determined these proteins were involved in the regulation of synaptic transmission processes, metabolic process and cell morphogenesis-cytoskeleton organization. The interaction of viral M protein with selected host proteins in mouse Neuro-2a cells infected with RABV was verified by super-resolution confocal microscopy. Molecular docking simulations also demonstrated the formation of RABV M complexes. However, further confirmation with co-immunoprecipitation was only successful for M-actin cytoplasmic 1 interaction. Our study revealed actin cytoplasmic 1 as a binding partner of M protein, which might have important role(s) in rabies pathogenesis.

Rabies Virus-Inspired Metal-Organic Frameworks (MOFs) for Targeted Imaging and Chemotherapy of Glioma.

The blood-brain barrier (BBB) restricts access to the brain of more than 98 % of therapeutic agents and is largely responsible for treatment failure of glioblastoma multiforme (GBM). Therefore, it is of great importance to develop a safe and efficient strategy for more effective drug delivery across the BBB into the brain. Inspired by the extraordinary capability of rabies virus (RABV) to enter the central nervous system, we report the development and evaluation of the metal-organic framework-based nanocarrier MILB@LR, which closely mimicked both the bullet-shape structure and surface functions of natural RABV. MILB@LR benefited from a more comprehensive RABV-mimic strategy than mimicking individual features of RABV and exhibited significantly enhanced BBB penetration and brain tumor targeting. MILB@LR also displayed superior inhibition of tumor growth when loaded with oxaliplatin. The results demonstrated that MILB@LR may be valuable for GBM targeting and treatment.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method.

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Structure of a rabies virus polymerase complex from electron cryo-microscopy.

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

Proteomic Profiling of Purified Rabies Virus Particles.

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus (RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC-MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.

Antigenic variations of recent street rabies virus.

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.

Migration of gastric cancer is suppressed by recombinant Newcastle disease virus (rL-RVG) via regulating α7-nicotinic acetylcholine receptors/ERK- EMT.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) have been reported to be overexpressed in malignancies in humans and is associated with tumorigenesis and cell migration. In previous studies of gastric cancer, alpha7 nicotinic acetylcholine receptor (α7-nAChR) overexpression leads to epithelial-mesenchymal transition (EMT) and promotes the migration of gastric cancer cells. Recombinant avirulent LaSota strain of Newcastle disease virus (NDV) expressing the rabies virus glycoprotein (rL-RVG) may promote apoptosis of gastric cancer cells and reduces the migration of lung cancer metastasis. However, whether rL-RVG inhibits migration of gastric cancer cells and what the underlying functional mechanism is remains unknown. METHODS: The gastric cancer cell lines BGC and SGC were randomly divided into 3 groups: rL-RVG, NDV and Phosphate Buffered Solution (PBS) control groups. Furthermore,we adopted ACB and MLA,α7nAChR-siRNA for the overexpression and silencing of α7-nAChR.Corynoxenine was used for inhibiting the MEK-ERK pathway. Western blot, Immunofluoresce,cell proliferation assays,cell migration analyses through wound-healing assays and Transwell assays were used to explore the underlying mechanisms. A mouse xenograft model was used to investigate the effects of rL-RVG,NDV on tumor growth. RESULTS: In this study, our findings demonstrate that rL-RVG suppressed the migration of gastric cancer cells and reduced EMT via α7-nAChR in vitro. Furthermore rL-RVG decreased the phosphorylation levels of the MEK/ERK signaling pathway such as down-regulating the expression of P-MEK and P-ERK. Additionally, rL-RVG also reduced the expression level of mesenchymal markers N-cadherin and Vimentin and enhanced the expression of the epithelial marker E-cadherin. Lastly, rL-RVG inhibited nicotinic acetylcholine receptors (nAChRs) to suppress cell migration and epithelial to mesenchymal transition (EMT) in gastric cell. We also found that rL-RVG suppresses the growth of gastric cancer subcutaneous tumor cells in vivo. CONCLUSION: rL-RVG inhibits α7-nAChR-MEK/ERK-EMT to suppress migration of gastric cancer cells.

Transcriptional Control and mRNA Capping by the GDP Polyribonucleotidyltransferase Domain of the Rabies Virus Large Protein.

Rabies virus (RABV) is a causative agent of a fatal neurological disease in humans and animals. The large (L) protein of RABV is a multifunctional RNA-dependent RNA polymerase, which is one of the most attractive targets for developing antiviral agents. A remarkable homology of the RABV L protein to a counterpart in vesicular stomatitis virus, a well-characterized rhabdovirus, suggests that it catalyzes mRNA processing reactions, such as 5'-capping, cap methylation, and 3'-polyadenylation, in addition to RNA synthesis. Recent breakthroughs in developing in vitro RNA synthesis and capping systems with a recombinant form of the RABV L protein have led to significant progress in our understanding of the molecular mechanisms of RABV RNA biogenesis. This review summarizes functions of RABV replication proteins in transcription and replication, and highlights new insights into roles of an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase, domain of the RABV L protein in mRNA capping and transcription initiation.

Multiplex Neural Circuit Tracing With G-Deleted Rabies Viral Vectors.

Neural circuits interconnect to organize large-scale networks that generate perception, cognition, memory, and behavior. Information in the nervous system is processed both through parallel, independent circuits and through intermixing circuits. Analyzing the interaction between circuits is particularly indispensable for elucidating how the brain functions. Monosynaptic circuit tracing with glycoprotein (G) gene-deleted rabies viral vectors (RVΔG) comprises a powerful approach for studying the structure and function of neural circuits. Pseudotyping of RVΔG with the foreign envelope EnvA permits expression of transgenes such as fluorescent proteins, genetically-encoded sensors, or optogenetic tools in cells expressing TVA, a cognate receptor for EnvA. Trans-complementation with rabies virus glycoproteins (RV-G) enables trans-synaptic labeling of input neurons directly connected to the starter neurons expressing both TVA and RV-G. However, it remains challenging to simultaneously map neuronal connections from multiple cell populations and their interactions between intermixing circuits solely with the EnvA/TVA-mediated RV tracing system in a single animal. To overcome this limitation, here, we multiplexed RVΔG circuit tracing by optimizing distinct viral envelopes (oEnvX) and their corresponding receptors (oTVX). Based on the EnvB/TVB and EnvE/DR46-TVB systems derived from the avian sarcoma leukosis virus (ASLV), we developed optimized TVB receptors with lower or higher affinity (oTVB-L or oTVB-H) and the chimeric envelope oEnvB, as well as an optimized TVE receptor with higher affinity (oTVE-H) and its chimeric envelope oEnvE. We demonstrated independence of RVΔG infection between the oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems and in vivo proof-of-concept for multiplex circuit tracing from two distinct classes of layer 5 neurons targeting either other cortical or subcortical areas. We also successfully labeled common input of the lateral geniculate nucleus to both cortico-cortical layer 5 neurons and inhibitory neurons of the mouse V1 with multiplex RVΔG tracing. These oEnvA/oTVA, oEnvB/oTVB, and oEnvE/oTVE systems allow for differential labeling of distinct circuits to uncover the mechanisms underlying parallel processing through independent circuits and integrated processing through interaction between circuits in the brain.

1H, 15N and 13C resonance assignments of the C-terminal domain of the P protein of the Nishigahara strain of rabies virus.

The C-terminal domain of the P protein of rabies virus is a multifunctional domain that interacts with both viral and host cell proteins. Here we report the 1H, 13C and 15N chemical shift assignments of this domain from P protein of the Nishigahara strain of rabies virus, a pathogenic laboratory strain well established for studies of virulence functions of rabies virus proteins, including P protein. The data and secondary structure analysis are in good agreement with the reported predominantly helical structure of the same domain from the CVS strain of rabies solved by crystallography. These assignments will enable future solution studies of the interactions of the P protein with viral and host proteins, and the effects of post-translational modifications.

Novel mass spectrometry based detection and identification of variants of rabies virus nucleoprotein in infected brain tissues.

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms.

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro.

Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families.IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens-Ebola virus, influenza virus, SARS CoV, and rabies virus-self-associate. This observation across various viral families suggests that transmembrane domain interactions may be broadly relevant and serve as a new target for therapeutic development.

Targeted transport of nanocarriers into brain for theranosis with rabies virus glycoprotein-derived peptide.

For successful theranosis of brain diseases, limited access of therapeutic molecules across blood-brain barrier (BBB) needs be overcome in brain delivery. Currently, peptide derivatives of rabies virus glycoprotein (RVG) have been exploited as delivery ligands to transport nanocarriers across BBB and specifically into the brain. The targeting peptides usually conjugate to the nanocarrier surface, and the cargoes, including siRNA, miRNA, DNA, proteins and small molecular chemicals, are complexed or encapsulated in the nanocarriers. The peptide ligand of the RVG-modified nanocarriers introduces the conjugated targeted-delivery into the brain, and the cargoes are involved in disease theranosis. The peptide-modified nanocarriers have been applied to diagnose and treat various brain diseases, such as glioma, Alzheimer's disease, ischemic injury, protein misfolding diseases etc. Since the targeting delivery system has displayed good biocompatibility and desirable therapeutic effect, it will raise a potential application in treating brain diseases.

A novel approach to a rabies vaccine based on a recombinant single-cycle flavivirus vector.

The RepliVax® vaccine (RV) platform is based on flavivirus genomes that are rationally attenuated by deletion. These single-cycle RV vaccine candidates targeting flavivirus pathogens have been demonstrated to be safe, highly immunogenic, and efficacious in animal models, including non-human primates. Here we show utility of the technology for delivery of a non-flavivirus immunogen by engineering several West Nile-based RV vectors to express full-length rabies virus G protein. The rabies virus G protein gene was incorporated in place of different West Nile structural protein gene deletions. The resulting RV-RabG constructs were demonstrated to replicate to high titers (8 log10 infectious particles/ml) in complementing helper cells. Following infection of normal cells, they provided efficient rabies virus G protein expression, but did not spread to surrounding cells. Expression of rabies virus G protein was stable and maintained through multiple rounds of in vitro passaging. A sensitive neurovirulence test in 2-3 day old neonatal mice demonstrated that RV-RabG candidates were completely avirulent indicative of high safety. We evaluated the RV-RabG variants in several animal models (mice, dogs, and pigs) and demonstrated that a single dose elicited high titers of rabies virus-neutralizing antibodies and protected animals from live rabies virus challenge (mice and dogs). Importantly, dogs were protected at both one and two years post-immunization, demonstrating durable protective immunity. The data demonstrates the potential of the RepliVax® technology as a potent vector delivery platform for developing vaccine candidates against non-flavivirus targets.

Rational design of rabies vaccine formulation for enhanced stability.

BACKGROUND/AIM: Vaccines are often lyophilized in order to retain their stability and efficacy for a longer period of time. However, the same lyophilization process may also cause a major degradation of the vaccine, especially during early phases of manufacturing, leading to a loss of potency of the product. Many viral diseases, such as rabies, are acute and fatal unless the vaccine is administered prior to exposure or the onset of symptoms in the case of postexposure treatment. MATERIALS AND METHODS: We investigated the effect of lyophilization on the stability of the virus structure during rabies vaccine manufacturing using dynamic light scattering and transmission electron microscopy. RESULTS: Our results indicate that some viruses lose their stability and efficacy in the course of lyophilization if the pH of the cell culture medium is controlled by solvated CO2 because the structure of the rabies virus is very sensitive to the solution pH: the virus either aggregates or its shape is deformed at low solution pH, whereas at high pH empty capsid shells are formed. CONCLUSION: Based on our findings, we developed a new formulation for the rabies vaccine that is stable in different buffers owing to the prevention of pH upshift upon lyophilization.

The ectodomain of rabies virus glycoprotein determines dendritic cell activation.

The immune evasion of wild-type (wt) rabies virus (RABV) has been attributed to its glycoprotein (G), particularly to their inefficiency to bind/enter into dendritic cells (DCs). However, the domain responsible for G-mediated DC activation is not clear. In the present study, attempts were made to map the domain(s) on the G involved in differential DC activation using laboratory-adapted and wt viruses. Recombinant RABVs with exchange in each of the structural domains such as signal peptide (sp), ectodomain (et), transmembrane domain (tm), cytoplasmic tail (ct) of the G between wt and laboratory-adapted strains were constructed. Characterizations of these recombinant RABVs show that the viruses containing the sp, tm and ct from the wt G are capable of growing in high titer by efficient cell-to-cell spread, similar to laboratory-adapted virus. On the other hand, recombinant virus containing the et domain from wt G was inefficient in cell-to-cell spread and grew in lower levels, similar to the wt RABV. Analysis of DC activation shows that viruses containing sp and tm from wt G are efficient in binding to and activating DCs. However, viruses containing the et domain from wt G are incompetent in binding to and activating DCs. Analysis of the G expression in the infected cells suggests that the level of G expression is regulated solely by the ct domain, indicating the level of G expression and DC activation are governed by different domains. Together, our results demonstrate that G-mediated DC activation is regulated by the et domain while the level of G expression by the ct domain.

An accelerated rabies vaccine schedule based on toll-like receptor 3 (TLR3) agonist PIKA adjuvant augments rabies virus specific antibody and T cell response in healthy adult volunteers.

BACKGROUND: Rabies is a fatal disease where post-exposure prophylaxis (PEP) is crucial in preventing infection. However, deaths even after appropriate PEP, have been reported. The PIKA Rabies vaccine adjuvant is a TLR3 agonist that activates B and T cells leading to a robust immune response. METHODS: We conducted a phase I, open label, randomized study in healthy adults to assess the safety and immunogenicity of the PIKA Rabies vaccine and an accelerated vaccine regimen. Thirty-seven subjects were randomized into 3 groups: control vaccine classic regimen, PIKA vaccine classic regimen and PIKA vaccine accelerated regimen. Subjects were followed up for safety, rabies virus neutralizing antibodies (RVNA) and T cell responses. RESULTS: Both the control and PIKA Rabies vaccine were well tolerated. All adverse events (AEs) were mild and self-limiting. Seventy-five percent of subjects in the PIKA accelerated regimen achieved a RVNA titer ⩾0.5IU/mL on day 7, compared to 53.9% in the PIKA classic regimen (p=0.411) and 16.7% in control vaccine classic regimen (p=0.012). The PIKA rabies vaccine elicited multi-specific rabies CD4 mediated T cell response already detectable ex vivo at day 7 after vaccination and that was maintained at day 42. CONCLUSION: The investigational PIKA rabies vaccine was well tolerated and more immunogenic than the commercially available vaccine in healthy adults. Clinical trial registry: The study was registered with clinicaltrials.gov NCT02657161.