Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Background

Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses.

Methodology/Principal Findings

Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized.

Conclusions/Significance

Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.     

Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.     

Safe and effective two-in-one replicon-and-VLP minispike vaccine for COVID-19: Protection of mice after a single immunization

Vaccines of outstanding efficiency, safety, and public acceptance are needed to halt the current SARS-CoV-2 pandemic. Concerns include potential side effects caused by the antigen itself and safety of viral DNA and RNA delivery vectors. The large SARS-CoV-2 spike (S) protein is the main target of current COVID-19 vaccine candidates but can induce non-neutralizing antibodies, which might cause vaccination-induced complications or enhancement of COVID-19 disease. Besides, encoding of a functional S in replication-competent virus vector vaccines may result in the emergence of viruses with altered or expanded tropism. Here, we have developed a safe single round rhabdovirus replicon vaccine platform for enhanced presentation of the S receptor-binding domain (RBD). Structure-guided design was employed to build a chimeric minispike comprising the globular RBD linked to a transmembrane stem-anchor sequence derived from rabies virus (RABV) glycoprotein (G). Vesicular stomatitis virus (VSV) and RABV replicons encoding the minispike not only allowed expression of the antigen at the cell surface but also incorporation into the envelope of secreted non-infectious particles, thus combining classic vector-driven antigen expression and particulate virus-like particle (VLP) presentation. A single dose of a prototype replicon vaccine complemented with VSV G, VSVΔG-minispike-eGFP (G), stimulated high titers of SARS-CoV-2 neutralizing antibodies in mice, equivalent to those found in COVID-19 patients, and protected transgenic K18-hACE2 mice from COVID-19-like disease. Homologous boost immunization further enhanced virus neutralizing activity. The results demonstrate that non-spreading rhabdovirus RNA replicons expressing minispike proteins represent effective and safe alternatives to vaccination approaches using replication-competent viruses and/or the entire S antigen.     

Extended Preclinical Safety,Efficacy and Stability Testing of a Live-attenuated Chikungunya Vaccine Candidate

We recently described a new, live-attenuated vaccine candidate for chikungunya (CHIK) fever, CHIKV/IRES. This vaccine was shown to be well attenuated, immunogenic and efficacious in protecting against CHIK virus (CHIKV) challenge of mice and nonhuman primates. To further evaluate its preclinical safety, we compared CHIKV/IRES distribution and viral loads in interferon-α/β receptor-incompetent A129 mice to another CHIK vaccine candidate, 181/clone25, which proved highly immunogenic but mildly reactive in human Phase I/II clinical trials. Compared to wild-type CHIK virus, (wt-CHIKV), both vaccines generated lower viral loads in a wide variety of tissues and organs, including the brain and leg muscle, but CHIKV/IRES exhibited marked restrictions in dissemination and viral loads compared to 181/clone25, and was never found outside the blood, spleen and muscle. Unlike wt-CHIKV, which caused disrupted splenic architecture and hepatic lesions, histopathological lesions were not observed in animals infected with either vaccine strain. To examine the stability of attenuation, both vaccines were passaged 5 times intracranially in infant A129 mice, then assessed for changes in virulence by comparing parental and passaged viruses for footpad swelling, weight stability and survival after subcutaneous infection. Whereas strain 181/clone25 p5 underwent a significant increase in virulence as measured by weight loss (from <10% to >30%) and mortality (from 0 to 100%), CHIKV/IRES underwent no detectible change in any measure of virulence (no significant weight loss and no mortality). These data indicate greater nonclinical safety of the CHIKV/IRES vaccine candidate compared to 181/clone25, further supporting its eligibility for human testing.     

Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Complete Protection against Severe Acute Respiratory Syndrome Coronavirus-Mediated Lethal Respiratory Disease in Aged Mice by Immunization with a Mouse-Adapted Virus Lacking E Protein

Zoonotic coronaviruses, including the one that caused severe acute respiratory syndrome (SARS), cause significant morbidity and mortality in humans. No specific therapy for any human coronavirus is available, making vaccine development critical for protection against these viruses. We previously showed that recombinant SARS coronavirus (SARS-CoV) (Urbani strain based) lacking envelope (E) protein expression (rU-ΔE) provided good but not perfect protection in young mice against challenge with virulent mouse-adapted SARS-CoV (MA15). To improve vaccine efficacy, we developed a second set of E-deleted vaccine candidates on an MA15 background (rMA15-ΔE). rMA15-ΔE is safe, causing no disease in 6-week-, 12-month-, or 18-month-old BALB/c mice. Immunization with this virus completely protected mice of three ages from lethal disease and effected more-rapid virus clearance. Compared to rU-ΔE, rMA15-ΔE immunization resulted in significantly greater neutralizing antibody and SARS-CoV-specific CD4 and CD8 T cell responses. After challenge, inflammatory cell infiltration, edema, and lung destruction were decreased in the lungs of rMA15-ΔE-immunized mice compared to those in rU-ΔE-immunized 12-month-old mice. Collectively, these results show that immunization with a species-adapted attenuated coronavirus lacking E protein expression is safe and provides optimal immunogenicity and long-term protection against challenge with lethal virus. This approach will be generally useful for development of vaccines protective against human coronaviruses as well as against coronaviruses that cause disease in domestic and companion animals.     

A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.     

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8⁺ T cell responses. Moreover, E2-E1-LNP induced CD8⁺ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.     

Cross-Inhibition of Chikungunya Virus Fusion and Infection by Alphavirus E1 Domain III Proteins

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (K_d [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.     

Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection

In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.     

A Neutralizing Monoclonal Antibody Targeting the Acid-Sensitive Region in Chikungunya Virus E2 Protects from Disease

The mosquito-borne alphavirus, chikungunya virus (CHIKV), has recently reemerged, producing the largest epidemic ever recorded for this virus, with up to 6.5 million cases of acute and chronic rheumatic disease. There are currently no licensed vaccines for CHIKV and current anti-inflammatory drug treatment is often inadequate. Here we describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody and a biosensor antibody binding study demonstrated it recognized residues on intact CHIKV VLPs. Shotgun mutagenesis alanine scanning of 98 percent of the residues in the E1 and E2 glycoproteins of CHIKV envelope showed that the epitope bound by C9 included amino-acid 162 in the acid-sensitive region (ASR) of the CHIKV E2 glycoprotein. The ASR is critical for the rearrangement of CHIKV E2 during fusion and viral entry into host cells, and we predict that C9 prevents these events from occurring. When used prophylactically in a CHIKV mouse model, C9 completely protected against CHIKV viremia and arthritis. We also observed that when administered therapeutically at 8 or 18 hours post-CHIKV challenge, C9 gave 100% protection in a pathogenic mouse model. Given that targeting this novel neutralizing epitope in E2 can potently protect both in vitro and in vivo, it is likely to be an important region both for future antibody and vaccine-based interventions against CHIKV.     

Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus

Background

Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.

Methodology/Principal Findings

We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.

Conclusion/Significance

These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.     

Role of Pentraxin 3 in Shaping Arthritogenic Alphaviral Disease: From Enhanced Viral Replication to Immunomodulation

The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV) and Ross River virus (RRV), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3) was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3^-/-) mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis.     

Non-replicating adenovirus based Mayaro virus vaccine elicits protective immune responses and cross protects against other alphaviruses

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1^-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1^-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses–chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1^-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.     

Myeloid Cell Arg1 Inhibits Control of Arthritogenic Alphavirus Infection by Suppressing Antiviral T Cells

Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.     

Expression of Plasmid-Based shRNA against the E1 and nsP1 Genes Effectively Silenced Chikungunya Virus Replication

Background

Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection.

Methods

Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection.

Results

Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1.

Conclusion

Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.     

IgY antibodies against Ebola virus possess post-exposure protection in a murine pseudovirus challenge model and excellent thermostability

Ebola virus (EBOV) is one of the most virulent pathogens that causes hemorrhagic fever and displays high mortality rates and low prognosis rates in both humans and nonhuman primates. The post-exposure antibody therapies to prevent EBOV infection are considered effective as of yet. However, owing to the poor thermal stability of mammalian antibodies, their application in the tropics has remained limited. Therefore, a thermostable therapeutic antibody against EBOV was developed modelled on the poultry(chicken) immunoglobulin Y (IgY). The IgY antibodies retaining their neutralising activity at 25°C for one year, displayed excellent thermal stability, opposed to conventional polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs). Laying hens were immunised with a variety of EBOV vaccine candidates and it was confirmed that VSVΔG/EBOVGP encoding the EBOV glycoprotein could induce high titer neutralising antibodies against EBOV. The therapeutic efficacy of immune IgY antibodies in vivo was evaluated in the newborn Balb/c mice who have been challenged with the VSVΔG/EBOVGP model. Mice that have been challenged with a lethal dose of the pseudovirus were treated 2 or 24 h post-infection with different doses of anti-EBOV IgY. The group receiving a high dose of 10⁶ NAU/kg (neutralising antibody units/kilogram) showed complete protection with no symptoms of a disease, while the low-dose group was only partially protected. Conversely, all mice receiving naive IgY died within 10 days. In conclusion, the anti-EBOV IgY exhibits excellent thermostability and protective efficacy. Anti-EBOV IgY shows a lot of promise in entering the realm of efficient Ebola virus treatment regimens.     

A Combination of Doxycycline and Ribavirin Alleviated Chikungunya Infection

Lack of vaccine and effective antiviral drugs against chikungunya virus (CHIKV) outbreaks have led to significant impact on health care in the developing world. Here, we evaluated the antiviral effects of tetracycline (TETRA) derivatives and other common antiviral agents against CHIKV. Our results showed that within the TETRA derivatives group, Doxycycline (DOXY) exhibited the highest inhibitory effect against CHIKV replication in Vero cells. On the other hand, in the antiviral group Ribavirin (RIBA) showed higher inhibitory effects against CHIKV replication compared to Aciclovir (ACIC). Interestingly, RIBA inhibitory effects were also higher than all but DOXY within the TETRA derivatives group. Docking studies of DOXY to viral cysteine protease and E2 envelope protein showed non-competitive interaction with docking energy of -6.6±0.1 and -6.4±0.1 kcal/mol respectively. The 50% effective concentration (EC50) of DOXY and RIBA was determined to be 10.95±2.12 μM and 15.51±1.62 μM respectively, while DOXY+RIBA (1:1 combination) showed an EC₅₀ of 4.52±1.42 μM. When compared, DOXY showed higher inhibition of viral infectivity and entry than RIBA. In contrast however, RIBA showed higher inhibition against viral replication in target cells compared to DOXY. Assays using mice as animal models revealed that DOXY+RIBA effectively inhibited CHIKV replication and attenuated its infectivity in vivo. Further experimental and clinical studies are warranted to investigate their potential application for clinical intervention of CHIKV disease.