Interferon-Inducible GTPase 1 Impedes the Dimerization of Rabies Virus Phosphoprotein and Restricts Viral Replication.

Rabies, caused by rabies virus (RABV), is an ancient zoonosis and still a major public health problem for humans, especially in developing countries. RABV can be recognized by specific innate recognition receptors, resulting in the production of hundreds of interferon-stimulated genes (ISGs), which can inhibit viral replication at different stages. Interferon-inducible GTPase 1 (IIGP1) is a mouse-specific ISG and belongs to the immunity-related GTPases (IRGs) family. IIGP is reported to constrain intracellular parasite infection by disrupting the parasitophorous vacuole membrane. However, the role of IIGP1 in restricting viral replication has not been reported. In this present study, we found that IIGP1 was upregulated in cells and mouse brains upon RABV infection. Overexpression of IIGP1 limited RABV replication in cell lines and reduced viral pathogenicity in a mouse model. Consistently, deficiency of IIGP1 enhanced RABV replication in different parts of mouse brains. Furthermore, we found that IIGP1 could interact with RABV phosphoprotein (P protein). Mutation and immunoprecipitation analyses revealed that the Y128 site of P protein is critical for its interaction with IIGP1. Further study demonstrated that this interaction impeded the dimerization of P protein and thus suppressed RABV replication. Collectively, our findings for the first reveal a novel role of IIGP1 in restricting a typical neurotropic virus, RABV, which will provide fresh insight into the function of this mouse-specific ISG.IMPORTANCE Interferon and its downstream products, ISGs, are essential in defending against pathogen invasion. One of the ISGs, IIGP1, has been found to constrain intracellular parasite infection by disrupting their vacuole membranes. However, the role of IIGP1 in limiting viral infection is unclear. In this study, we show that infection with a typical neurotropic virus, RABV, can induce upregulation of IIGP1, which, in turn, suppresses RABV by interacting with its phosphoprotein (P protein) and thus blocking the dimerization of P protein. Our study provides the first evidence that IIGP1 functions in limiting viral infection and provides a basis for comprehensive understanding of this important ISG.

Single amino acid change at position 255 in rabies virus glycoprotein decreases viral pathogenicity.

Previous studies have indicated that the amino acid at position 333 in the glycoprotein (G) is closely related to rabies virus (RABV) pathogenicity. However, whether there are other amino acid residues in G that relate to pathogenicity remain unclear. The aim of this study is to find new amino acid residues in G that could strongly reduce RABV pathogenicity. The present study found that the pathogenicity of a virulent strain was strongly attenuated when the amino acid glycine (Gly) replaced the aspartic acid (Asp) at position 255 in G (D255G) as intracranial (i.c.) infection with this D255G mutant virus did not cause death in adult mice. The indexes of neurotropism of the D255G mutant strain and the parent GD-SH-01 are 0.72 and 10.0, respectively, which indicate that the D255G mutation decreased the neurotropism of RABV. In addition, the D255G mutation significantly decreased RABV replication in the mouse brain. Furthermore, the D255G mutation enhanced the immune response in mice, which contributed to the clearance of RABV after infection. The Asp255 â†’ Gly255 mutation was genetically stable in vitro and in vivo. In this study, we describe a new referenced amino acid site in G that relates to the pathogenicity of RABV.

Inactivated rabies vaccines: Standardization of an in vitro assay for residual viable virus detection.

Human rabies, a neglected viral zoonosis, is preventable through domestic animals vaccination and post-exposure prophylaxis using inactivated rabies vaccines. During vaccine production, several mandatory in vivo quality control trials, such as potency, live virus, and safety, are responsible for the use of large numbers of laboratory animals. Over the years, global organizations encouraged the development of alternative methods to reduce, replace and refine the use of animals in the pharmaceutical industry. In this study we standardized an in vitro assay for determination of residual live virus combining viral isolation techniques with direct immunofluorescence detection and viral quantification by a molecular method. Standardization of viral recovery steps and quantification by RT-qPCR were performed and the combined method was shown to be 3 fold more sensitive than the in vivo assay. It was possible to identify viral suspensions cultures, which still had residual viable rabies virus particles, evidencing the importance to implement this method in quality control schemes of rabies vaccine production. In addition, this developed assay is more practical, inexpensive and less time consuming, producing results in just 4 days, which may allow greater agility in the internal quality control of the vaccine. The in vitro method may reduce 2/3rd of laboratory animals numbers used for this purpose, since it can be applied in the intermediate quality control of inactivated rabies vaccine production.

Immune responses in mice and pigs after oral vaccination with rabies virus vectored Nipah disease vaccines.

Nipah virus (NiV) is a re-emerging zoonotic pathogen that causes high mortality in humans and pigs. Oral immunization in free-roaming animals is one of the most practical approaches to prevent NiV pandemics. We previously generated a recombinant rabies viruses (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, rERAG333E, which contains a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) and serves as an oral vaccine for dog rabies. In this study, we generated two recombinant RABVs, rERAG333E/NiVG and rERAG333E/NiVF, expressing the NiV Malaysian strain attachment glycoprotein (NiV-G) or fusion glycoprotein (NiV-F) gene based on the rERAG333E vector platform. Both rERAG333E/NiVG and rERAG333E/NiVF displayed growth properties similar to those of rERAG333E and caused marked syncytia formation after co-infection in BSR cell culture. Adult and suckling mice intracerebrally inoculated with the recombinant RABVs showed NiV-G and NiV-F expression did not increase the virulence of rERAG333E. Oral vaccination with rERAG333E/NiVG either singularly or combined with rERAG333E/NiVF induced significant NiV neutralizing antibody against NiV and RABV, and IgG to NiV-G or NiV-F in mice and pigs. rERAG333E/NiVG and rERAG333E/NiVF thus appeared to be suitable candidates for further oral vaccines for potential animal targets in endemic areas of NiV disease and rabies.

Incorporating B cell activating factor (BAFF) into the membrane of rabies virus (RABV) particles improves the speed and magnitude of vaccine-induced antibody responses.

B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) superfamily of cytokines that links innate with adaptive immunity. BAFF signals through receptors on B cells, making it an attractive molecule to potentiate vaccine-induced B cell responses. We hypothesized that a rabies virus (RABV)-based vaccine displaying both antigen and BAFF on the surface of the same virus particle would target antigen-specific B cells for activation and improve RABV-specific antibody responses. To test this hypothesis, we constructed a recombinant RABV-based vector expressing virus membrane-anchored murine BAFF (RABV-ED51-mBAFF). BAFF was incorporated into the RABV particle and determined to be biologically functional, as demonstrated by increased B cell survival of primary murine B cells treated ex-vivo with RABV-ED51-mBAFF. B cell survival was inhibited by pre-treating RABV-ED51-mBAFF with an antibody that blocks BAFF functions. RABV-ED51-mBAFF also activated primary murine B cells ex-vivo more effectively than RABV as shown by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. In-vivo, RABV-ED51-mBAFF induced significantly faster and higher virus neutralizing antibody (VNA) titers than RABV while not adversely affecting the longevity of the vaccine-induced antibody response. Since BAFF was incorporated into the virus particle and genome replication was not required for BAFF expression in-vivo, we hypothesized that RABV-ED51-mBAFF would be effective as an inactivated vaccine. Mice immunized with 250 ng/mouse of ß-propriolactone-inactivated RABV-ED51-mBAFF showed faster and higher anti-RABV VNA titers compared to mice immunized with inactivated RABV. Together, this model stands as a potential foundation for exploring other virus membrane-anchored molecular adjuvants to make safer, more effective inactivated RABV-based vaccines.

Purification of rabies virus produced in Vero cells grown in serum free medium.

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease. Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield. Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media.

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ±â€¯0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ±â€¯0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines.

Rabies is an infectious viral disease, characterized as a neglected zoonosis, responsible for nearly 60,000 deaths annually. The virus is transmitted mainly by dogs in Africa and Asia, and wildlife in Europe and the Americas, to all mammals' species, causing severe encephalitis almost always fatal after the onset of neurological symptoms. Human rabies can be prevented through extensive vaccination of dogs and pre/post-prophylaxis treatments in humans with inactivated rabies vaccines. The vaccine manufacture involves a series of quality control assays using laboratory animals, which are mandatory to exclude the presence of viable residual virus. The quality controls must be carried out in various steps during the vaccine production, which demands the use of a large number of animals. In this study, we standardized a real-time quantitative RT-PCR duplex assay to be used during intermediate stages of the vaccine production. This assay was done for the quantification of vaccine strain rabies virus, targeting rabies nucleoprotein, and ß-actin mRNA of BHK-21 cells as an internal endogenous control. The results showed specific amplification, with the analytical sensitivity ranged from 101 to 106 TCID50/mL with high repeatability rate for the quantification of rabies virus in inactivated vaccine samples. Global organizations are engaged to develop new approaches to determine viable residual virus, and this assay can be applied in combination with traditional in vitro methods for the release of intermediate batches of vaccines during the production process, keeping the in vivo tests only for final release.

Street rabies virus strains associated with insectivorous bats are less pathogenic than strains isolated from other reservoirs.

Rabies is a fatal and viral zoonosis that causes acute, progressive encephalitis and remains an important concern in public health. In the last few years, there has been a change in the epidemiological profile of rabies after implementing canine rabies control in the Americas, which has led to a significant increase in both human and pet cases of rabies associated with insectivorous bats. Thus, it is important to understand the pathogenesis caused by Rabies virus (RABV) isolates from insectivorous bats. Viral growth kinetics, cell-to-cell spread and virus uptake in vitro were analyzed for RABV isolates from Eptesicus furiralis and Myotis nigricans. For pathogenesis evaluation, mice were inoculated with RABV isolates from Eptesicus furiralis and Myotis nigricans, and clinical signs were observed for 40 days. We observed that the insectivorous bat strains showed a higher replication rate, faster cell-to-cell spread and delayed virus uptake in N2a cells. Furthermore, after the first sign of a clinical infection, mice infected with Myotis nigricans and Eptesicus furiralis isolates succumbed rapidly (6 ±â€¯9 days) compared with RABV strains associated with other reservoirs. Our results show that the insectivorous bat RABV strains are less pathogenic for mice than strains associated with other reservoirs. In addition, this study also indicates that the differences in the biological characteristics of the RABV strains are important to their pathogenicity. An enhanced understanding of rabies pathogenesis may be important for the development of novel therapies for humans and in the implementation of rabies control strategies.

Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8-3.8 EU) than those induced by Rabisin vaccine (0.9-6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.

Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-ß-d-Glucose Involves mTOR-Dependent Autophagy.

The compound 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), a gallotannin present in various plants such as Rhus chinensis Mill and Paeonia suffruticosa, has a broad spectrum of antiviral effects. The present study investigated its potency against infection of mice with rabies virus (RABV). Results demonstrated that PGG strongly inhibited virus titers (50-fold), viral mRNA expression (up to 90%), and protein synthesis in vitro. Importantly, we found that PGG not only suppressed viral adsorption and entry, but also directly inactivated RABV through suppression of autophagy by mediating activation of the mTOR-dependent autophagy signaling pathway. In vivo, PGG (10 mg/kg) alleviated the clinical symptoms and reduced the mortality of infected mice by 27.3%. Collectively, our results indicate that PGG has potent anti-RABV effect, and merits further investigation as an anti-RABV drug.

Rabies Outbreak among Livestock in a Pastoralist Community, Southern Ethiopia.

BACKGROUND: Rabies still poses a significant health problem in most of African countries, where the majority of the cases result from dog bites. The situations in the marginalized pastoral areas were not well documented. CASE: In September 2015, rabid wild fox entered the pastoralist village and bit a domestic dog. The victim dog had turned rabid after four months and bit livestock, and rabies outbreak occurred in the family livestock. Consequently, one bull, one lactating cow, one calf, two donkeys and one heifer died of outbreak. The head of one heifer was removed and transported within 24 hours to the Rabies Referral Laboratory of Ethiopian Public Health Institute in Addis Ababa. The sample was confirmed as strong positive for lyssa virus antigen by Direct Fluorescent Anti-Body Test. This was the first confirmed case report from southern Oromia pastoralists. The occurrence of rabies cases across the district was also reported by veterinary and human health officers. CONCLUSION: Integrated intervention strategy and collaboration of animal health, human health and wildlife authority is needed. To halt the ongoing outbreak in the district, immediate response from the Government is recommended.

Priorização de doenças zoonóticas da One Health para o envolvimento multissectorial em Moçambique / One Health's prioritization of zoonotic diseases for multisectoral involvement in Mozambique

O objectivo desta oficina de trabalho de 2 dias sobre Priorização de Doenças Zoonóticas One Health (OHZDP) foi identificar doenças zoonóticas de maior preocupação nacional para Moçambique, usando contribuições iguais de representantes da saúde humana, gado, ambiente, vida selvagem, pesquisa, parceiros de desenvolvimento, sectores de ensino superior. Durante a oficina de trabalho, os representantes identificaram uma lista de doenças zoonóticas relevantes para Moçambique, definiram os critérios para priorização e determinaram perguntas e pesos relevantes para cada critério. Sete doenças zoonóticas foram identificadas como prioritárias pelos participantes usando a ferramenta OHZDP, uma ferramenta de selecção semi-quantitativa desenvolvida pelos Centros de Controlo e Prevenção de Doenças dos EUA (CDC) (Anexo A)1, 2. As doenças zoonóticas priorizadas para Moçambique são vírus da raiva, tuberculose zoonótica, salmonelose, vírus da gripe aviária zoonótica, tripanasomose, brucelose e febre hemorrágica da Crimeia-Congo (Tabela 1). Os resultados finais do processo de priorização One Health e os pesos normalizados para todas as doenças zoonóticas discutidas na oficina de trabalho da OHZDP em Moçambique são mostrados no Anexo C. Este relatório resume o processo One HeO objectivo desta oficina de trabalho de 2 dias sobre Priorização de Doenças Zoonóticas One Health (OHZDP) foi identificar doenças zoonóticas de maior preocupação nacional para Moçambique, usando contribuições iguais de representantes da saúde humana, gado, ambiente, vida selvagem, pesquisa, parceiros de desenvolvimento, sectores de ensino superior. Durante a oficina de trabalho, os representantes identificaram uma lista de doenças zoonóticas relevantes para Moçambique, definiram os critérios para priorização e determinaram perguntas e pesos relevantes para cada critério. Sete doenças zoonóticas foram identificadas como prioritárias pelos participantes usando a ferramenta OHZDP, uma ferramenta de selecção semi-quantitativa desenvolvida pelos Centros de Controlo e Prevenção de Doenças dos EUA (CDC) (Anexo A)1, 2. As doenças zoonóticas priorizadas para Moçambique são vírus da raiva, tuberculose zoonótica, salmonelose, vírus da gripe aviária zoonótica, tripanasomose, brucelose e febre hemorrágica da Crimeia-Congo (Tabela 1). Os resultados finais do processo de priorização One Health e os pesos normalizados para todas as doenças zoonóticas discutidas na oficina de trabalho da OHZDP em Moçambique são mostrados no Anexo C. Este relatório resume o processo One Health usado para priorizar as principais doenças zoonóticas em Moçambique que devem ser conjuntamente abordadas usando uma abordagem multissectorial One Health, incluindo ministérios da saúde humana, animal e ambiental e outros sectores relevantes para as doenças zoonóticas priorizadas.alth usado para priorizar as principais doenças zoonóticas em Moçambique que devem ser conjuntamente abordadas usando uma abordagem multissectorial One Health, incluindo ministérios da saúde humana, animal e ambiental e outros sectores relevantes para as doenças zoonóticas priorizadas.

Near-infrared fluorescent protein iRFP720 is optimal for in vivo fluorescence imaging of rabies virus infection.

In vivo imaging is a noninvasive method that enables real-time monitoring of viral infection dynamics in a small animal, which allows a better understanding of viral pathogenesis. In vivo bioluminescence imaging of virus infection is widely used but, despite its advantage over bioluminescence that no substrate administration is required, fluorescence imaging is not used because of severe autofluorescence. Recently, several far-red and near-infrared (NIR) fluorescent proteins (FPs) have been developed and shown to be useful for whole-body fluorescence imaging. Here, we report comparative testing of far-red and NIR FPs in the imaging of rabies virus (RABV) infection. Using the highly neuroinvasive 1088 strain, we generated recombinant RABV that expressed FPs such as Katushka2S, E2-Crimson, iRFP670 or iRFP720. After intracerebral inoculation to nude mice, the 1088 strain expressing iRFP720, the most red-shifted FP, was detected the earliest with the highest signal-to-noise ratio using a filter set for >700 nm, in which the background signal level was very low. Furthermore, we could also track viral dissemination from the spinal cord to the brain in nude mice after intramuscular inoculation of iRFP720-expressing 1088 into the hind limb. Hence, we conclude that the NIR FP iRFP720 used with a filter set for >700 nm is useful for in vivo fluorescence imaging not only for RABV infection but also for other virus infections. Our findings will also be useful for developing dual-optical imaging of virus-host interaction dynamics using bioluminescence reporter mice for inflammation imaging.

Neuronal LRP4 regulates synapse formation in the developing CNS.

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

A single center, open label study of intradermal administration of an inactivated purified chick embryo cell culture rabies virus vaccine in adults.

In the USA, rabies vaccines (RVs) are licensed for intramuscular (IM) use only, although RVs are licensed for use by the intradermal (ID) route in many other countries. Recent limitations in supplies of RV in the USA reopened discussions on the more efficient use of available biologics, including utilization of more stringent risk assessments, and potential ID RV administration. A clinical trial was designed to compare the immunogenic and adverse effects of a purified chicken embryo cell (PCEC) RV administered ID or IM. Enrollment was designed in four arms, ID Pre-Exposure Prophylaxis (Pre-EP), IM Pre-EP, ID Booster, and IM Booster vaccination. Enrollment included 130 adult volunteers. The arms with IM administration received vaccine according to the current ACIP recommendations: Pre-EP, three 1mL (2.5 I.U.) RV doses, each on day 0, 7, and 21; or a routine Booster, one 1ml dose. The ID groups received the same schedule, but doses administered were in a volume of 0.1mL (0.25 I.U.). The rate of increase in rabies virus neutralizing antibody titers 14-21days after vaccination were similar in the ID and correspondent IM groups. The GMT values for ID vaccination were slightly lower than those for IM vaccination, for both naïve and booster groups, and these differences were statistically significant by t-test. Fourteen days after completing vaccination, all individuals developed RV neutralizing antibody titers over the minimum arbitrary value obtained with the rapid fluorescent focus inhibition test (RFFIT). Antibodies were over the set threshold until the end of the trial, 160days after completed vaccination. No serious adverse reactions were reported. Most frequent adverse reactions were erythema, induration and tenderness, localized at the site of injection. Multi use of 1mL rabies vaccine vials for ID doses of 0.1 was demonstrated to be both safe and inmunogenic.

Susceptibility of neuroblastoma cells to rabies virus may be affected by passage number.

Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral neutralizing antibodies. Individuals who routinely work with rabies virus, such as rabies laboratory employees, or those who may have a high potential for exposure to rabies virus, including veterinarians, should be tested for the presence of anti-rabies viral neutralizing antibodies (VNA) every 6-24 months, depending on potential exposure level. The gold standard for serum neutralization assays require the use of live rabies virus and cells that are sensitive to rabies virus infection. Additionally, virus isolation assays are routinely performed in rabies laboratories as a back-up for the direct fluorescent antibody test (dFAT). Currently there are no guidelines or publications recommending the use of low, intermediate, or high passage cell lines in rabies assays. In this study, we compared the sensitivity of intermediate, high, and extremely high passaged neuroblastomas to rabies virus using virus isolation, serum neutralization, and real time RT-PCR techniques. Additionally, cells were examined microscopically to determine changes in morphology and dissemination of rabies virus antigen between intermediate, high, and extremely high passage cells. No significant difference was found between cell passage numbers and viral susceptibility between intermediate and high passaged cells. However, extremely high passaged cells (≥1200 passages) were less susceptible to viral infection and/or produced less virus following inoculation. As a result, rabies laboratories that use viral isolation and serum neutralization assays should regularly assess cell susceptibility to ensure the integrity and repeatability of the test.

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.

Expression of interleukin-6 by a recombinant rabies virus enhances its immunogenicity as a potential vaccine.

Several studies have confirmed that interleukin-6 (IL6) mediates multiple biological effects that enhance immune responses when used as an adjuvant. In the present study, recombinant rabies virus (RABV) expressing canine IL6 (rHEP-CaIL6) was rescued and its pathogenicity and immunogenicity were investigated in mice. We demonstrated that mice received a single intramuscular immunization with rHEP-CaIL6 showed an earlier increase and higher maximum titres of virus-neutralizing antibody (VNA) as well as anti-RABV antibodies compared with mice immunized with the parent strain. Moreover, survival rates of mice immunized with rHEP-CaIL6 were higher compared with mice immunized with parent HEP-Flury according to the challenge assay. Flow cytometry further confirmed that immunization with rHEP-CaIL6 induced the strong recruitment of mature B cells and CD8+ T cells to lymph nodes, which may partially explain the high levels of VNA and enhanced cellular immunity. Quantitative real-time PCR indicated that rHEP-CaIL6 induced stronger inflammatory and immune responses in the central nervous system, which might have allowed virus clearance in the early infection phase. Furthermore, mice infected intranasally with rHEP-CaIL6 developed no clinical symptoms while mice infected with HEP-Flury showed piloerection. In summary, these data indicate that rHEP-CaIL6 induces a strong, protective immune response with a good safety profile. Therefore, a recombinant RABV strain expressing canine IL6 may aid the development of an effective, safe attenuated rabies vaccine.

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.