A Rodent Model of Chikungunya Virus Infection in RAG1 -/- Mice,with Features of Persistence,for Vaccine Safety Evaluation

Chikungunya virus (CHIKV) is a positive sense, single stranded RNA virus in the genus Alphavirus, and the etiologic agent of epidemics of severe arthralgia in Africa, Asia, Europe and, most recently, the Americas. CHIKV causes chikungunya fever (CHIK), a syndrome characterized by rash, fever, and debilitating, often chronic arthritis. In recent outbreaks, CHIKV has been recognized to manifest more neurologic signs of illness in the elderly and those with co-morbidities. The syndrome caused by CHIKV is often self-limited; however, many patients develop persistent arthralgia that can last for months or years. These characteristics make CHIKV not only important from a human health standpoint, but also from an economic standpoint. Despite its importance as a reemerging disease, there is no licensed vaccine or specific treatment to prevent CHIK. Many studies have begun to elucidate the pathogenesis of CHIKF and the mechanism of persistent arthralgia, including the role of the adaptive immune response, which is still poorly understood. In addition, the lack of an animal model for chronic infection has limited studies of CHIKV pathogenesis as well as the ability to assess the safety of vaccine candidates currently under development. To address this deficiency, we used recombination activating gene 1 (RAG1^-/-) knockout mice, which are deficient in both T and B lymphocytes, to develop a chronic CHIKV infection model. Here, we describe this model as well as its use in evaluating the safety of a live-attenuated vaccine candidate.     

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.     

Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKV_mos) has slower replication than mammalian cell-derived CHIKV (CHIKV_vero), when tested in both human and murine cell lines. Consistent with this, CHIKV_mos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKV_mos produces a lower level of viremia and less severe footpad swelling when compared with CHIKV_vero. Interestingly, CHIKV_mos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKV_mos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKV_vero after a single passage in mammalian cells. Furthermore, CHIKV_vero and CHIKV_mos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.     

Characterization of Aedes aegypti Innate-Immune Pathways that Limit Chikungunya Virus Replication

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways.     

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1^−/− mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1^−/− mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.     

埃及伊蚊对登革Ⅱ型病毒感染Balb/C实验小鼠的增强

以Balb/C小鼠为实验动物,探讨了埃及伊蚊Aedes aegypti唾液对登革病毒感染宿主的影响。结果显示,在皮下接种剂量相同的情况下,如果Balb/C实验小鼠预先被一定数量的埃及伊蚊叮咬以后,实验小鼠感染病毒的程度有所提高,血清中的抗体滴度明显降低,腹腔巨噬细胞感染登革病毒的阳性率及感染的时间动态曲线也有明显差异,感染高峰期延迟。这些说明实验小鼠被媒介蚊虫叮咬以后变得相对容易被登革病毒感染,可能是媒介蚊虫叮咬小鼠时分泌的唾液对宿主的免疫反应系统有一定作用。因此可以初步肯定埃及伊蚊在登革病毒的感染传播过程中,影响了Balb/C实验小鼠的免疫功能,对登革病毒的感染有一定推动作用。     

Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice

Dengue viruses (DENV) are transmitted to humans by the bite of Aedes aegypti or Aedes albopictus mosquitoes, with millions of infections annually in over 100 countries. The diseases they produce, which occur exclusively in humans, are dengue fever (DF) and dengue hemorrhagic fever (DHF). We previously developed a humanized mouse model of DF in which mice transplanted with human hematopoietic stem cells produced signs of DENV disease after injection with low-passage, wild-type isolates. Using these mice, but now allowing infected A. aegypti to transmit dengue virus during feeding, we observed signs of more severe disease (higher and more sustained viremia, erythema, and thrombocytopenia). Infected mice mounted innate (gamma interferon [IFN-γ] and soluble interleukin 2 receptor alpha [sIL-2Rα]) and adaptive (anti-DENV antibodies) immune responses that failed to clear viremia until day 56, while a mosquito bite alone induced strong immunomodulators (tumor necrosis factor alpha [TNF-α], IL-4, and IL-10) and thrombocytopenia. This is the first animal model that allows an evaluation of human immunity to DENV infection after mosquito inoculation.     

Humanized Mice Show Clinical Signs of Dengue Fever according to Infecting Virus Genotype

We demonstrated that the infection of humanized NOD-scid IL2rγ ^null mice with different strains (representing the four genotypes) of dengue virus serotype 2 (DEN-2) can induce the development of human-like disease, including fever, viremia, erythema, and thrombocytopenia. Newborn mice were irradiated and received transplants by intrahepatic inoculation of human cord blood-derived hematopoietic progenitor cells (CD34⁺). After 6 weeks, mouse peripheral blood was tested by flow cytometry to determine levels of human lymphocytes (CD45⁺ cells); rates of reconstitution ranged from 16 to 80% (median, 52%). Infection (with approximately 10⁶ PFU, the equivalent of a mosquito bite) of these humanized mice with eight low-passage-number strains produced a high viremia extending to days 12 to 18 postinfection. We observed a significant decrease in platelets at day 10 in most of the mice and an increase in body temperature (fever) and erythema (rash) in comparison with humanized mice inoculated with cell culture medium only. Comparison of Southeast (SE) Asian and other genotype viruses (American, Indian, and West African) in this model showed significant differences in magnitude and duration of viremia and rash, with the SE Asian viruses always being highest. Indian genotype viruses produced lower viremias and less thrombocytopenia than the others, and West African (sylvatic) viruses produced the shortest periods of viremia and the lowest rash measurements. These results correlate with virulence and transmission differences described previously for primary human target cells and whole mosquitoes and may correlate with epidemiologic observations around the world. These characteristics make this mouse model ideal for the study of dengue pathogenesis and the evaluation of vaccine attenuation and antivirals.Dengue viruses, which cause the disease dengue fever (DF) and its more severe form, dengue hemorrhagic fever (DHF), in humans, have been spreading to more areas of the world along with their mosquito (Aedes aegypti and Aedes albopictus) vectors. Now over 100 countries are affected, including some areas of the United States (Texas and Hawaii) (5, 26). Due to the fact that only humans show clinical signs and symptoms of disease, it has been difficult to directly test the mechanisms of pathogenesis of these viruses (4). Through decades of research, including clinical, epidemiologic, and laboratory studies, the factors involved in producing disease, whether it be DF or DHF, have remained unproved. However, there are many indications that both the virus and the host contribute to the occurrence and severity of disease: there are genetic differences in the virus and host immune response that can be measured in vitro, and these factors seem to lead to immunopathology in addition to the damage done by virus replication. Because there are four antigenically distinct dengue viruses (serotypes 1 to 4), humans can theoretically have dengue virus infections leading to clinical disease up to four times, and the immunity to the first virus enhances the probability of developing severe dengue after a subsequent infection. Thus, the development of vaccines has been hampered by the unknown effects of inoculating with a tetravalent preparation that might cause immunopathology or severe disease, and there are no appropriate animal models in which to test vaccine attenuation and efficacy for human applications.In 2005 we reported the development of humanized NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice that produced signs of DF upon infection with one strain of dengue virus (3). The mice were humanized by giving them transplants of purified hematopoietic stem cells from human umbilical cord blood (CB) samples taken from normal births. After subcutaneous infection with a low dose of a Southeast (SE) Asian virus, the viremia, rash, and thrombocytopenia were significantly higher, longer lasting, and more like human disease than in any other animal model described at the time. We concluded that this model could be used to test antiviral treatments, since these mice did not produce measurable human antibodies. Since then, many other immunodeficient mouse strains have been produced that can have enhanced human engraftment levels, and they develop functional human immune system cells, including some level of adaptive immunity (20). It has been reported that some of these mouse strains develop immunoglobulins specific for human immunodeficiency virus and dengue virus, albeit at low levels (14, 25).Here we present results of dengue virus pathogenesis studies in a new mouse strain, NOD-scid IL2rγ ^null, that has a much higher degree of human lymphocyte development (median of 52%, versus 14% previously). The comparison of viruses from different genetic subgroups of dengue serotype 2 has led us to conclude that this model is reflective of actual human dengue pathogenesis, and this development might bring us to a new era in testing the factors that contribute to dengue disease.     

Immunization of Mice with Recombinant Mosquito Salivary Protein D7 Enhances Mortality from Subsequent West Nile Virus Infection via Mosquito Bite

Background

Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.

Methodology/Principal Findings

We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.

Conclusions/Significance

Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.     

Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1–5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys.     

pH-Dependent entry of chikungunya virus fusion into mosquito cells

Background

Millions of human infections caused by arthropod-borne pathogens are initiated by the feeding of an infected mosquito on a vertebrate. However, interactions between the viruses and the mosquito vector, which facilitates successful infection and transmission of virus to a subsequent vertebrate host, are still not fully understood.

Finding

Here we describe early chikungunya virus (CHIKV) infectious events in cells derived from one of the most important CHIKV vectors, Aedes albopictus. We demonstrated that CHIKV infection of mosquito cells depended on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysosomotropic drugs chloroquine, ammonium chloride, and monensin, which is consistent with observations in mammalian cells. While all three agents inhibited CHIKV infection in C6/36 cells, ammonium chloride was less toxic to cells than the other agents.

Conclusion

The observation of similar mechanisms for inhibition of CHIKV infection in mosquito and mammalian cell lines suggests that conserved entry pathways are utilized by CHIKV for vertebrate and invertebrate cell types.

     

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.     

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

Background

Application of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus.

Findings

We identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singh''s cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways.

Conclusions

In our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.     

Prior exposure to uninfected mosquitoes enhances mortality in naturally-transmitted West Nile virus infection

Background

The global emergence of West Nile virus (WNV) has highlighted the importance of mosquito-borne viruses. These are inoculated in vector saliva into the vertebrate skin and circulatory system. Arthropod-borne (arbo)viruses such as WNV are transmitted to vertebrates as an infectious mosquito probes the skin for blood, depositing the virus and saliva into the skin and circulation. Growing evidence has demonstrated that arthropod, and recently mosquito, saliva can have a profound effect on pathogen transmission efficiency, pathogenesis, and disease course. A potentially important aspect of natural infections that has been ignored is that in nature vertebrates are typically exposed to the feeding of uninfected mosquitoes prior to the mosquito that transmits WNV. The possibility that pre-exposure to mosquito saliva might modulate WNV infection was explored.

Principal Findings

Here we report that sensitization to mosquito saliva exacerbates viral infection. Prior exposure of mice to mosquito feeding resulted in increased mortality following WNV infection. This aggravated disease course was associated with enhanced early viral replication, increased interleukin-10 expression, and elevated influx of WNV-susceptible cell types to the inoculation site. This exacerbated disease course was mimicked by passive transfer of mosquito-sensitized serum.

Significance

This is the first report that sensitization to arthropod saliva can exacerbate arthropod-borne infection, contrary to previous studies with parasite and bacteria infections. This research suggests that in addition to the seroreactivity of the host to virus, it is important to take into account the immune response to vector feeding.     

ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L^−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15^−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15^−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.     

Proteomics Profiling of Chikungunya-Infected Aedes albopictus C6/36 Cells Reveal Important Mosquito Cell Factors in Virus Replication

Chikungunya virus (CHIKV) is the only causative agent of CHIKV fever with persistent arthralgia, and in some cases may lead to neurological complications which can be highly fatal, therefore it poses severe health issues in many parts of the world. CHIKV transmission can be mediated via the Aedes albopictus mosquito; however, very little is currently known about the involvement of mosquito cellular factors during CHIKV-infection within the mosquito cells. Unravelling the neglected aspects of mosquito proteome changes in CHIKV-infected mosquito cells may increase our understanding on the differences in the host factors between arthropod and mammalian cells for successful replication of CHIKV. In this study, the CHIKV-infected C6/36 cells with differential cellular proteins expression were profiled using two-dimensional gel electrophoresis (2DE) coupled with the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 2DE analysis on CHIKV-infected C6/36 cells has shown 23 mosquito cellular proteins that are differentially regulated, and which are involved diverse biological pathways, such as protein folding and metabolic processes. Among those identified mosquito proteins, spermatogenesis-associated factor, enolase phosphatase e-1 and chaperonin-60kD have been found to regulate CHIKV infection. Furthermore, siRNA-mediated gene knockdown of these proteins has demonstrated the biological importance of these host proteins that mediate CHIKV infection. These findings have provided an insight to the importance of mosquito host factors in the replication of CHIKV, thus providing a potential channel for developing novel antiviral strategies against CHIKV transmission.     

Host Immune Response to Mosquito-Transmitted Chikungunya Virus Differs from That Elicited by Needle Inoculated Virus

Background

Mosquito-borne diseases are a worldwide public health threat. Mosquitoes transmit viruses or parasites during feeding, along with salivary proteins that modulate host responses to facilitate both blood feeding and pathogen transmission. Understanding these earliest events in mosquito transmission of arboviruses by mosquitoes is essential for development and assessment of rational vaccine and treatment strategies. In this report, we compared host immune responses to chikungunya virus (CHIKV) transmission by (1) mosquito bite, or (2) by needle inoculation.

Methods and Findings

Differential cytokine expression was measured using quantitative real-time RT-PCR, at sites of uninfected mosquito bites, CHIKV-infected mosquito bites, and needle-inoculated CHIKV. Both uninfected and CHIKV infected mosquitoes polarized host cytokine response to a T_H2 profile. Compared to uninfected mosquito bites, expression of IL-4 induced by CHIKV-infected mosquitoes were 150 fold and 527.1 fold higher at 3 hours post feeding (hpf) and 6 hpf, respectively. A significant suppression of T_H1 cytokines and TLR-3 was also observed. These significant differences may result from variation in the composition of uninfected and CHIKV-infected mosquito saliva. Needle injected CHIKV induced a robust interferon-γ, no detectable IL-4, and a significant up-regulation of TLR-3.

Conclusions

This report describes the first analysis of cutaneous cytokines in mice bitten by CHIKV–infected mosquitoes. Our data demonstrate contrasting immune activation in the response to CHIKV infection by mosquito bite or needle inoculation. The significant role of mosquito saliva in these earliest events of CHIKV transmission and infection are highlighted.     

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.