Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-inflammatory Cytokine Response

Mayaro virus (MAYV), an alphavirus similar to chikungunya virus (CHIKV), causes an acute debilitating disease which results in the development of long-term arthralgia in more than 50% of infected individuals. Currently, the immune response and its role in the development of MAYV-induced persistent arthralgia remain unknown. In this study, we evaluated the immune response of individuals with confirmed MAYV infection in a one-year longitudinal study carried out in Loreto, Peru. We report that MAYV infection elicits robust immune responses that result in the development of a strong neutralizing antibody response and the secretion of pro-inflammatory immune mediators. The composition of these inflammatory mediators, in some cases, differed to those previously observed for CHIKV. Key mediators such as IL-13, IL-7 and VEGF were strongly induced following MAYV infection and were significantly increased in subjects that eventually developed persistent arthralgia. Although a strong neutralizing antibody response was observed in all subjects, it was not sufficient to prevent the long-term outcomes of MAYV infection. This study provides initial immunologic insight that may eventually contribute to prognostic tools and therapeutic treatments against this emerging pathogen.     

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.     

Development of a next-generation chikungunya virus vaccine based on the HydroVax platform

Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including β-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O’nyong’nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4⁺ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.     

Effective Chikungunya Virus-like Particle Vaccine Produced in Insect Cells

The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV) causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.     

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.     

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical “macro” (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD–ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP‐r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure–function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.     

Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms

Dengue is a mosquito-borne viral disease of expanding geographical range and incidence. The existence of four viral serotypes and the association of prior dengue virus infection with an increased risk for more severe disease have presented significant obstacles to vaccine development. An increased understanding of the adaptive immune response to natural dengue virus infection and candidate dengue vaccines has helped to define the specific antibody and T cell responses that are associated with either protective or pathological immunity during dengue infection. Further characterization of immunological correlates of disease outcome and the validation of these findings in vaccine trials will be invaluable for developing effective dengue vaccines.     

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.     

Vaccine-Induced Protection of Rhesus Macaques against Plasma Viremia after Intradermal Infection with a European Lineage 1 Strain of West Nile Virus

The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques (Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.     

Chikungunya: A Potentially Emerging Epidemic?

Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.     

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.     

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.     

Immunization of mice against West Nile virus with recombinant envelope protein.

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.     

An ecological niche model to predict the geographic distribution of Haemagogus janthinomys,Dyar, 1921 a yellow fever and Mayaro virus vector,in South America

Yellow fever virus (YFV) has a long history of impacting human health in South America. Mayaro virus (MAYV) is an emerging arbovirus of public health concern in the Neotropics and its full impact is yet unknown. Both YFV and MAYV are primarily maintained via a sylvatic transmission cycle but can be opportunistically transmitted to humans by the bites of infected forest dwelling Haemagogus janthinomys Dyar, 1921. To better understand the potential risk of YFV and MAYV transmission to humans, a more detailed understanding of this vector species’ distribution is critical. This study compiled a comprehensive database of 177 unique Hg. janthinomys collection sites retrieved from the published literature, digitized museum specimens and publicly accessible mosquito surveillance data. Covariate analysis was performed to optimize a selection of environmental (topographic and bioclimatic) variables associated with predicting habitat suitability, and species distributions modelled across South America using a maximum entropy (MaxEnt) approach. Our results indicate that suitable habitat for Hg. janthinomys can be found across forested regions of South America including the Atlantic forests and interior Amazon.     

Immunogenicity and protective efficacy of recombinant M2e.Hsp70c（Hsp70359–610） fusion protein against influenza virus infection in mice

New strategies in vaccine development are urgently needed to combat emerging influenza viruses and to reduce the risk of pandemic disease surfacing. Being conserved, the M2 e protein, is a potential candidate for universal vaccine development against influenza A viruses. Mycobacterium tuberculosis Hsp70（mHsp70） is known to cultivate the function of immunogenic antigen-presenting cells, stimulate a strong cytotoxic T lymphocyte（CTL） response, and stop the induction of tolerance. Thus, in this study, a recombinant protein from the extracellular domain of influenza A virus matrix protein 2（M2e）, was fused to the C-terminus of Mycobacterium tuberculosis Hsp70（Hsp70c）, to generate a vaccine candidate. Humoral immune responses, IFN-γ-producing lymphocyte, and strong CTL activity were all induced to confirm the immunogenicity of M2 e.Hsp70c（Hsp70359–610）. And challenge tests showed protection against H1N1 and H9N2 strains in vaccinated groups. Finally these results demonstrates M2 e.Hsp70c fusion protein can be a candidate for a universal influenza A vaccine.     

The anti-tumor effect of Newcastle disease virus HN protein is influenced by differential subcellular targeting

Background  

Immunotherapy is emerging as a major player in the current standard of care for aggressive cancers such as non-small cell lung cancer (NSCLC). The Newcastle disease virus with its tumor-specific replicative and oncolytic abilities is a promising immunotherapeutic candidate. A DNA vaccine expressing the major immunogenic hemagglutinin-neuraminidase (HN) protein of this virus has shown promising results as an immunotherapeutic agent.     

Electroporation-Mediated Immunization of a Candidate DNA Vaccine Expressing Dengue Virus Serotype 4 prM-E Antigen Confers Long-Term Protection in Mice

Dengue fever, caused by dengue viruses(DENVs), is a widespread mosquito-borne zoonotic disease; however, there is no available anti-dengue vaccine for worldwide use. In the current study, a DNA vaccine candidate(pV-D4 ME) expressing prM-E protein of DENV serotype 4(DENV-4) was constructed, and its immunogenicity and protection were evaluated in immunocompetent BALB/c mice. The pV-D4 ME candidate vaccine induced effective humoral and cellular immunity of mice against DENV-4 in vivo when administered both at 50 μg and 5 μg through electroporation. Two weeks after receiving three immunizations, both doses of pV-D4 ME DNA were shown to confer effective protection against lethal DENV-4 challenge. Notably, at 6 months after the three immunizations, 50 μg, but not 5 μg, of pV-D4 ME could provide stable protection(100% survival rate) against DENV-4 lethal challenge without any obvious clinical signs. These results suggest that immunization with 50 μg pV-D4 ME through electroporation could confer effective and long-term protection against DENV-4, offering a promising approach for development of a novel DNA vaccine against DENVs.     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.     

Dengue epidemiology and pathogenesis:images of the future viewed through a mirror of the past

Every year, millions of individuals throughout the world are seriously affected by dengue virus. The unavailability of a vaccine and of anti-viral drugs has made this mosquito-borne disease a serious health concern. Not only does dengue cause fatalities but it also has a profoundly negative economic impact. In recent decades, extensive research has been performed on epidemiology, vector biology, life cycle, pathogenesis, vaccine development and prevention. Although dengue research is still not at a stage to suggest definite hopes of a cure, encouraging significant advances have provided remarkable progress in the fight against infection. Recent developments indicate that both anti-viral drug and vaccine research should be pursued, in parallel with vector control programs.