Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

Peli1 signaling blockade attenuates congenital zika syndrome.

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.

Non-structural protein 1-specific antibodies directed against Zika virus in humans mediate antibody-dependent cellular cytotoxicity.

There is growing interest in understanding antibody (Ab) function beyond neutralization. The non-structural protein 1 (NS1) of Zika virus (ZIKV) is an attractive candidate for an effective vaccine as Abs against NS1, unlike the envelope or premembrane, do not carry the risk of mediating antibody-dependent enhancement. Our aim was to evaluate whether ZIKV NS1 Abs elicited following natural infection in humans can mediate antibody-dependent cellular cytotoxicity (ADCC). We evaluated the isotype specificity of ZIKV-specific Abs in immune sera and supernatants from stimulated immune PBMC and found that Abs against ZIKV NS1 and virus-like particles were predominantly of the IgG1 isotype. Using a recently developed FluoroSpot assay, we found robust frequencies of NS1-specific Ab-secreting cells in PBMC of individuals who were naturally infected with ZIKV. We developed assays to measure both natural killer cell activation by flow cytometry and target cell lysis of ZIKV NS1-expressing cells using an image cytometry assay in the presence of ZIKV NS1 Abs. Our data indicate efficient opsonization of ZIKV NS1-expressing CEM-NKR cell lines using ZIKV-immune but not ZIKV-naïve sera, a prerequisite of ADCC. Furthermore, sera from immune donors were able to induce both NK cell degranulation and lysis of ZIKV NS1 CEM-NKR cells in vitro. Our data suggest that ADCC is a possible mechanism for ZIKV NS1 Abs to eliminate virally infected target cells.

Multiplexed FluoroSpot for the Analysis of Dengue Virus- and Zika Virus-Specific and Cross-Reactive Memory B Cells.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

High seroprevalence of dengue virus indicates that dengue virus infections are frequent in central and eastern Sudan.

OBJECTIVES: To determine the seroprevalence of dengue in central and eastern Sudan and the breadth of neutralising antibody responses. METHODS: Blood was drawn from 483 patients with fever who visited outpatient clinics in Port Sudan, Red Sea state, in three towns in Kassala state and in El Obeid, North Kordofan, in December 2012 and January 2013. Sera were tested for dengue virus IgG and IgM by ELISA (Panbio) and sera without serologic evidence of acute infection (IgM negative) were used for the analysis of the seroprevalence. DENV neutralisation tests were performed to determine the specificity of the ELISA and to examine the degree of cross-neutralisation of multiple DENV serotypes. RESULTS: Sixty-seven per cent (302 of 448) of the sera were dengue virus IgG-positive. The seroprevalence in Port Sudan was 89% (106 of 119 sera), in Kassala 61% (128 of 209) and in North Kordofan 56.7% (68 of 120). Thirty-one of 32 ELISA-positive sera neutralised dengue viruses indicating that the ELISA was highly specific. The majority of the sera broadly neutralised all four dengue virus serotypes indicating multiple infections. CONCLUSIONS: The majority of the population in central and eastern Sudan has been infected with dengue viruses, many people repeatedly. The high seroprevalence underscores the need for extended dengue surveillance in Sudan, broad disease awareness in medical institutions and in the population and diagnostic capacity building for severe dengue infections.

A murine model of post-acute neurological sequelae following SARS-CoV-2 variant infection.

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.

A Murine Model of Post-acute Neurological Sequelae Following SARS-CoV-2 Variant Infection.

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. Surviving mice showed no detectable viral RNA in the brain and minimal neuroinflammation post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and reduced levels of genes associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent T helper 1 prone cellular immune responses and high neutralizing antibodies against Delta and Omicron variants in the periphery for months post-acute infection. Overall, infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long COVID patients and may be useful for future assessment of the efficacy of vaccines and therapeutics against SARS-CoV-2 variants.

A safe insect-based Chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques.

Chikungunya virus (CHIKV), which induces chikungunya fever and chronic arthralgia, is an emerging public health concern. Safe and efficient vaccination strategies are needed to prevent or mitigate virus-associated acute and chronic morbidities for preparation of future outbreaks. Eilat (EILV)/CHIKV, a chimeric alphavirus which contains the structural proteins of CHIKV and the non-structural proteins of EILV, does not replicate in vertebrate cells. The chimeric virus was previously reported to induce protective adaptive immunity in mice. Here, we assessed the capacity of the virus to induce quick and durable protection in cynomolgus macaques. EILV/CHIKV protected macaques from wild-type (WT) CHIKV infection one year after a single dose vaccination. Transcriptome and in vitro functional analyses reveal that the chimeric virus triggered toll-like receptor signaling and T cell, memory B cell and antibody responses in a dose-dependent manner. Notably, EILV/CHIKV preferentially induced more durable, robust, and broader repertoire of CHIKV-specific T cell responses, compared to a live attenuated CHIKV 181/25 vaccine strain. The insect-based chimeric virus did not cause skin hypersensitivity reactions in guinea pigs sensitized to mosquito bites. Furthermore, EILV/CHIKV induced strong neutralization antibodies and protected cynomolgus macaques from WT CHIKV infection within six days post vaccination. Transcriptome analysis also suggest that the chimeric virus induction of multiple innate immune pathways, including Toll-like receptor signaling, type I IFN and IL-12 signaling, antigen presenting cell activation, and NK receptor signaling. Our findings suggest that EILV/CHIKV is a safe, highly efficacious vaccine, and provides both rapid and long-lasting protection in cynomolgus macaques.

Rational Development of Live-Attenuated Zika Virus Vaccines.

Zika virus (ZIKV), a re-emerging mosquito-borne flavivirus, has caused outbreaks in Africa, Asia, the Pacific, and, more recently, in the Americas. ZIKV has been associated with the neurological autoimmune disorder Guillain-Barre syndrome in adults and congenital Zika syndrome in fetuses and infants, including microcephaly, spontaneous abortion, and intrauterine growth restriction. It is considered to be a major threat to global public health due to its unprecedented clinical impact on humans. Currently, there are no specific prophylactics or therapeutics available to prevent or treat ZIKV infection. The development of a safe and efficacious ZIKV vaccine remains a global health priority. Since the recent outbreak, multiple platforms have been used in the development of candidate ZIKV vaccines. The candidate vaccines have been shown to elicit strong T cell and neutralization antibody responses and protect against ZIKV infection in animal models. Some candidates have progressed successfully to clinical trials. Live-attenuated vaccines, which induce rapid and durable protective immunity, are one of the most important strategies for controlling flavivirus diseases. In this review, we discuss recent progress in the development of candidate live-attenuated ZIKV vaccines.

Humoral and T-cell-mediated responses to a pre-clinical Zika vaccine candidate that utilizes a unique insect-specific flavivirus platform.

Vaccination is critical for the control and prevention of viral outbreaks, yet conventional vaccine platforms may involve trade-offs between immunogenicity and safety. Insect-specific viruses have emerged as a novel vaccine platform to overcome this challenge. Detailed studies of humoral and T-cell responses induced by new insect-specific flavivirus (ISFV)-based vaccine platforms are needed to better understand correlates of protection and improve vaccine efficacy. Previously, we used a novel ISFV called Aripo virus (ARPV) to create a Zika virus (ZIKV) vaccine candidate (designated ARPV/ZIKV). ARPV/ZIKV demonstrated exceptional safety and single-dose efficacy, completely protecting mice from a lethal ZIKV challenge. Here, we explore the development of immune responses induced by ARPV/ZIKV immunization and evaluate its correlates of protection. Passive transfer of ARPV/ZIKV-induced immune sera to naïve mice prior to challenge emphasized the importance of neutralizing antibodies as a correlate of protection. Depletion of T-cells in vaccinated mice and adoptive transfer of ARPV/ZIKV-primed T-cells to naïve mice prior to challenge indicated that ARPV/ZIKV-induced CD4 + and CD8 + T-cell responses contribute to the observed protection but may not be essential for protection during ZIKV challenge. However, vaccination of Rag1 KO, Tcra KO, and muMt - mice demonstrated the critical role for ARPV/ZIKV-induced T-cells in developing protective immune responses following vaccination. Overall, both humoral and T-cell-mediated responses induced by ISFV-based vaccines are important for comprehensive immunity, and ISFV platforms continue to be a promising method for future vaccine development.

Oral Supplementation with AHCC®, a Standardized Extract of Cultured Lentinula edodes Mycelia, Enhances Host Resistance against SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global public health safety and the economy. Multiple antiviral drugs have been developed, and some have received regulatory approval and/or authorization. The use of nutraceuticals can be beneficial for preventing and treating COVID-19 complications. AHCC is a standardized, cultured extract of an edible mushroom Lentinula edodes of the Basidiomycete family of fungi that is enriched in acylated α-1,4-glucans. Here, we evaluated the effects of the oral administration of AHCC on the host response to SARS-CoV-2 infection in two murine models, K18-hACE2 transgenic mice and immunocompetent BALB/c mice. Oral administration of AHCC every other day for one week before and one day post SARS-CoV-2 infection in both strains of mice decreased the viral load and attenuated inflammation in the lungs. AHCC treatment also significantly reduced SARS-CoV-2-induced lethality in the K18-hACE2 mice. AHCC administration enhanced the expansion of γδ T cells in the spleen and lungs before and after viral infection and promoted T helper 1-prone mucosal and systemic T cell responses in both models. In AHCC-fed BALB/c mice, SARS-CoV-2 specific IgG responses were also enhanced. In summary, AHCC supplementation enhances host resistance against mild and severe COVID-19 infection primarily via the promotion of innate and adaptive T cell immune responses in mice.

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity.

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity.

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

West Nile Virus Seroprevalence and Cross-Neutralization in Sera from Eastern and Central Sudan.

Objectives: In regions with co-existing flaviviruses, the diagnosis of previous West Nile virus (WNV) infections is challenging due to cross-reacting antibodies. The aim of the study was to determine the frequency of previous WNV infections in sera from three Sudanese states by excluding potentially dengue virus (DENV) and ZIKV cross-reacting sera and to determine the percentage of WNV cross-neutralizing sera from individuals with previous DENV infection. Methods: Serum samples from Kassala, North Kordofan, and Red Sea state were screened for antibodies against DENV by ELISA. Sera without DENV antibodies (N = 106) and a matched set of sera with DENV antibodies (N = 108) was selected. In all blood samples the frequency of WNV-neutralizing antibodies and the antibody titers were measured with microplate neutralization assays. DENV and Zika virus (ZIKV) microplate neutralization assays were performed with all WNV neutralizing sera of the DENV negative group. Results: A fraction of 30.2% of the DENV antibody negative sera neutralized WNV. The seroprevalence increased with age from 9.5% to 41.7%. Men and women were equally affected. The percentage of DENV positive sera that neutralized WNV was 83.3%. DENV positive sera had higher WNV neutralization titers than DENV negative sera. Conclusions: A significant fraction of the DENV antibody negative sera from three regions in Sudan showed serologic evidence of previous WNV infection. In comparison, the large majority of DENV antibody positive sera had WNV neutralizing antibodies. Studies are needed to identify clinical cases of WNV infection and to determine whether individuals with cross-neutralizing antibodies are protected from WNV disease.

A modified porous silicon microparticle potentiates protective systemic and mucosal immunity for SARS-CoV-2 subunit vaccine.

Development of optimal SARS-CoV-2 vaccines to induce potent, long-lasting immunity and provide cross-reactive protection against emerging variants remains a high priority. Here, we report that a modified porous silicon microparticle (mPSM) adjuvant to SARS-CoV-2 receptor-binding domain (RBD) vaccine activated dendritic cells and generated more potent and durable systemic humoral and type 1 helper T (Th) cell- mediated immune responses than alum-formulated RBD following parenteral vaccination, and protected mice from SARS-CoV-2 and Beta variant challenge. Notably, mPSM facilitated the uptake of SARS-CoV-2 RBD antigens by nasal and airway epithelial cells. Parenteral and intranasal prime and boost vaccinations with mPSM-RBD elicited stronger lung resident T and B cells and IgA responses compared to parenteral vaccination alone, which led to markedly diminished viral loads and inflammation in the lung following SARS-CoV-2 Delta variant challenge. Overall, our results suggest that mPSM is effective adjuvant for SARS-CoV-2 subunit vaccine in both systemic and mucosal vaccinations.

Changes of Small Non-coding RNAs by Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. In the context of viral infections, small non-coding RNAs (sncRNAs) are known to play important roles in regulating the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK-RNA-seq, a modified next-generation sequencing (NGS), we found that sncRNA profiles in human nasopharyngeal swabs (NPS) samples are significantly impacted by SARS-CoV-2. Among impacted sncRNAs, tRFs are the most significantly affected and most of them are derived from the 5'-end of tRNAs (tRF5). Such a change was also observed in SARS-CoV-2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several small virus-derived ncRNAs (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3'-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.

Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models.

Emergence of SARS-CoV-2 variants of concern (VOCs), including the highly transmissible Omicron and Delta strains, has posed constant challenges to the current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified messenger RNA (mRNA) vaccine that expresses the more conserved viral nucleoprotein (mRNA-N) and show that mRNA-N vaccination alone can induce modest control of SARS-CoV-2. Critically, combining mRNA-N with the clinically proven S-expressing mRNA vaccine (mRNA-S+N) induced robust protection against both Delta and Omicron variants. In the hamster models of SARS-CoV-2 VOC challenge, we demonstrated that, compared to mRNA-S alone, combination mRNA-S+N vaccination not only induced more robust control of the Delta and Omicron variants in the lungs but also provided enhanced protection in the upper respiratory tract. In vivo CD8+ T cell depletion suggested a potential role for CD8+ T cells in protection conferred by mRNA-S+N vaccination. Antigen-specific immune analyses indicated that N-specific immunity, as well as augmented S-specific immunity, was associated with enhanced protection elicited by the combination mRNA vaccination. Our findings suggest that combined mRNA-S+N vaccination is an effective approach for promoting broad protection against SARS-CoV-2 variants.

Epidemiology and Laboratory Diagnostics of Dengue, Yellow Fever, Zika, and Chikungunya Virus Infections in Africa.

Arbovirus infections are widespread, and their disease burden has increased in the past decade. In Africa, arbovirus infections and fever with unknown etiology are common. Due to the lack of well-established epidemiologic surveillance systems and accurate differential diagnosis in most African countries, little is known about the prevalence of human arbovirus infections in Africa. The aim of this review is to summarize the available epidemiological data and diagnostic laboratory tools of infections with dengue, yellow fever, Zika, and chikungunya viruses, all transmitted by Aedes mosquitoes. Studies indicate that these arboviral infections are endemic in most of Africa. Surveillance of the incidence and prevalence of the infections would enable medical doctors to improve the diagnostic accuracy in patients with typical symptoms. If possible, arboviral diagnostic tests should be added to the routine healthcare systems. Healthcare providers should be informed about the prevalent arboviral diseases to identify possible cases.

Memory B cell and antibody responses to flavivirus infection and vaccination.

Flaviviruses are a group of mosquito- or tick-borne single-stranded RNA viruses that can cause a wide range of clinical manifestations in humans and animals, including asymptomatic, flu-like febrile illness, hemorrhagic fever, encephalitis, birth defects, and death. Many of them have no licensed vaccines available for human use. Memory B cell development and induction of neutralizing antibody responses, which are important for the control of flavivirus infection and dissemination, have been used as biomarkers for vaccine efficacy. In this review, we will discuss recent findings on memory B cells and antibody responses from studies in clinical specimen and animal models of flavivirus infection and vaccination with a focus on several clinically important flaviviruses, including dengue, West Nile, yellow fever, and Zika viruses.

Optimized production and immunogenicity of an insect virus-based chikungunya virus candidate vaccine in cell culture and animal models.

A chimeric Eilat/ Chikungunya virus (EILV/CHIKV) was previously reported to replicate only in mosquito cells but capable of inducing robust adaptive immunity in animals. Here, we initially selected C7/10 cells to optimize the production of the chimeric virus. A two-step procedure produced highly purified virus stocks, which was shown to not cause hypersensitive reactions in a mouse sensitization study. We further optimized the dose and characterized the kinetics of EILV/CHIKV-induced immunity. A single dose of 108 PFU was sufficient for induction of high levels of CHIKV-specific IgM and IgG antibodies, memory B cell and CD8+ T cell responses. Compared to the live-attenuated CHIKV vaccine 181/25, EILV/CHIKV induced similar levels of CHIKV-specific memory B cells, but higher CD8+ T cell responses at day 28. It also induced stronger CD8+, but lower CD4+ T cell responses than another live-attenuated CHIKV strain (CHIKV/IRES) at day 55 post-vaccination. Lastly, the purified EILV/CHIKV triggered antiviral cytokine responses and activation of antigen presenting cell (APC)s in vivo, but did not induce APCs alone upon in vitro exposure. Overall, our results demonstrate that the EILV/CHIKV vaccine candidate is safe, inexpensive to produce and a potent inducer of both innate and adaptive immunity in mice.