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A rise in adiposity in the United States has resulted in more than 70% of adults being overweight or obese, and global obesity rates have tripled since 1975. Following the 2009 H1N1 pandemic, obesity was characterized as a risk factor that could predict severe infection outcomes to viral infection. Amidst the SARS-CoV-2 pandemic, obesity has remained a significant risk factor for severe viral disease as obese patients have a higher likelihood for developing severe symptoms and requiring hospitalization. However, the mechanism by which obesity enhances viral disease is unknown. In this study, we utilized a diet-induced obesity mouse model of West Nile virus (WNV) infection, a flavivirus that cycles between birds and mosquitoes and incidentally infects both humans and mice. Likelihood for severe WNV disease is associated with risk factors such as diabetes that are comorbidities also linked to obesity. Utilizing this model, we showed that obesity-associated chronic inflammation increased viral disease severity as obese female mice displayed higher mortality rates and elevated viral titers in the central nervous system. In addition, our studies highlighted that obesity also dysregulates host acute adaptive immune responses, as obese female mice displayed significant dysfunction in neutralizing antibody function. These studies highlight that obesity-induced immunological dysfunction begins at early time points post infection and is sustained through memory phase, thus illuminating a potential for obesity to alter the differentiation landscape of adaptive immune cells.

Powassan virus (POWV) is a tick-borne pathogen for which humans are an incidental host. POWV infection can be fatal or result in long-term neurological sequelae; however, there are no approved vaccinations for POWV. Integral to efficacious vaccine development is the identification of correlates of protection, which we accomplished in this study by utilizing a murine model of POWV infection. Using POWV lethal and sub-lethal challenge models, we show that (1) robust B and T cell responses are necessary for immune protection, (2) POWV lethality can be attributed to both viral- and host-mediated drivers of disease, and (3) knowledge of the immune correlates of protection against POWV can be applied in a virus-like particle (VLP)-based vaccination approach that provides protection from lethal POWV challenge. Identification of these immune protection factors is significant as it will aid in the rational design of POWV vaccines.

The 2015/2016 Zika virus epidemic in South and Central America left the scientific community urgently trying to understand the factors that contribute to Zika virus pathogenesis. Because multiple other flaviviruses are endemic in areas where Zika virus emerged, it is hypothesized that a key to understanding Zika virus disease severity is to study Zika virus infection in the context of prior flavivirus exposure. Human and animal studies have highlighted the idea that having been previously exposed to a different flavivirus may modulate the immune response to Zika virus. However, it is still unclear how prior flavivirus exposure impacts Zika viral burden and disease. In this murine study, we longitudinally examine multiple factors involved in Zika disease, linking viral burden with increased neurological disease severity, weight loss, and inflammation. We show that prior heterologous flavivirus exposure with dengue virus type 2 or 3 or the vaccine strain of yellow fever provides protection from mortality in a lethal Zika virus challenge. However, reduction in viral burden and Zika disease varies depending on the infecting primary flavivirus; with primary Zika virus infection being most protective from Zika virus challenge, followed by dengue virus 2, with yellow fever and dengue virus 3 protecting against mortality but showing more severe disease. This study demonstrates the variation in protective effects of prior flavivirus exposure on Zika virus pathogenesis and identifies distinct relationships between primary flavivirus infection and the potential for Zika virus disease. IMPORTANCE The emergence and reemergence of various vector-borne diseases in recent years highlights the need to understand the mechanisms of protection for each pathogen. In this study, we investigated the impact of prior exposure to Zika virus, dengue virus serotypes 2 or 3, or the vaccine strain of yellow fever on pathogenesis and disease outcomes in a mouse model of Zika virus infection. We found that prior exposure to a heterologous flavivirus was protective from mortality, and to varying degrees, prior flavivirus exposure was protective against neurological disease, weight loss, and severe viral burden during a lethal Zika challenge. Using a longitudinal and cross-sectional study design, we were able to link multiple disease parameters, including viral burden, with neurological disease severity, weight loss, and inflammatory response in the context of flavivirus infection. This study demonstrates a measurable but varied impact of prior flavivirus exposure in modulating flavivirus pathophysiology. Given the cyclic nature of most flavivirus outbreaks, this work will contribute to the forecasting of disease severity for future outbreaks.

Zika virus (ZIKV) is an arbovirus belonging to the flaviviridae family with a risk assessment that has been increasing in recent years and was labeled a global health emergency by the WHO in 2016. There are currently no FDA-approved treatment options available for ZIKV, so expeditious development of treatment options is urgent. To expedite this process, an on-market drug, tamoxifen (TAM), was selected as a promising candidate for repurposing due to its wide range of biological activities and because it has already been shown to possess activity against hepatitis C (HCV), a flavivirus in a separate genus. Anti-ZIKV activity of TAM was assessed by compound screens using infectious virus and mechanistic details were gleaned from time of addition and virucidal studies. TAM and an active metabolite, 4-hydroxytamoxifen (TAM-OH), both showed promising antiviral activity (EC50 ≈ 9 µM and 5 µM respectively) in initial compound screening and up to 8 hours post-infection, though the virucidal assay indicated that they do not possess any direct virucidal activity. Additionally, TAM was assessed for its activity against ZIKV in the human male germ cell line, SEM-1, due to the sexually transmitted nature of ZIKV owing to its extended survival times in germ cells. Virus titers show diminished replication of ZIKV over seven days compared to controls. These data indicate that TAM has the potential to be repurposed as an anti-ZIKV therapeutic and warrants further investigation. This article is protected by copyright. All rights reserved.

Zika virus (ZIKV) and dengue virus (DENV) are co-endemic in many parts of the world, but the impact of ZIKV infection on subsequent DENV infection is not well understood. Here we show in rhesus macaques that the time elapsed after ZIKV infection affects the immune response to DENV infection. We show that previous ZIKV exposure increases the magnitude of the antibody and T cell responses against DENV. The time interval between ZIKV and subsequent DENV infection further affects the immune response. A mid-convalescent period of 10 months after ZIKV infection results in higher and more durable antibody and T cell responses to DENV infection than a short period of 2 months. In contrast, previous ZIKV infection does not affect DENV viremia or pro-inflammatory status. Collectively, we find no evidence of a detrimental effect of ZIKV immunity in a subsequent DENV infection. This supports the implementation of ZIKV vaccines that could also boost immunity against future DENV epidemics., Here, the authors show that the time elapsed between Zika infection and subsequent dengue virus infection affects the magnitude and durability of the antibody and cell-mediated immune responses against dengue virus, but not viremia. This research in non-human primates has implications for co-endemic regions and vaccination.

The closely related flaviviruses, dengue and Zika, cause significant human disease throughout the world. While cross-reactive antibodies have been demonstrated to have the capacity to potentiate disease or mediate protection during flavivirus infection, the mechanisms responsible for this dichotomy are still poorly understood. To understand how the human polyclonal antibody response can protect against, and potentiate the disease in the context of dengue and Zika virus infection we used intravenous hyperimmunoglobulin (IVIG) preparations in a mouse model of the disease. Three IVIGs (ZIKV-IG, Control-Ig and Gamunex®) were evaluated for their ability to neutralize and/or enhance Zika, dengue 2 and 3 virusesin vitro. The balance between virus neutralization and enhancement provided by thein vitroneutralization data was used to predict the IVIG concentrations which could protect or enhance Zika, and dengue 2 diseasein vivo. Using this approach, we were able to define the uniquein vivodynamics of complex polyclonal antibodies, allowing for both enhancement and protection from flavivirus infection. Our results provide a novel understanding of how polyclonal antibodies interact with viruses with implications for the use of polyclonal antibody therapeutics and the development and evaluation of the next generation flavivirus vaccines.

Dengue virus (DENV) is the most common vector-borne viral disease with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly-neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding for the membrane and envelope structural proteins from DENV serotype 1 encapsulated into lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection with high levels of neutralizing antibody titers and antiviral CD4+ and CD8+ T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine, or a vaccine with mutations in the immunodominant fusion-loop epitope, elicited equivalent humoral and cell mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype specific immunity with minimal serum cross-reactivity and reduced ADE compared to a live DENV1 viral infection.IMPORTANCEWith 400 million worldwide infections each year, dengue is the most common vector-born viral disease. 40% of the world’s population is at risk with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing sub optimally, the need for an effective dengue vaccine is urgent. Here we develop and characterize a novel mRNA vaccine encoding for the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement. Importantly our vaccine only induced serotype specific immune responses and did not induce ADE.

Dengue virus (DENV) is the most common vector-borne viral disease, with nearly 400 million worldwide infections each year concentrated in the tropical and subtropical regions of the world. Severe dengue complications are often associated with a secondary heterotypic infection of one of the four circulating serotypes. In this scenario, humoral immune responses targeting cross-reactive, poorly neutralizing epitopes can lead to increased infectivity of susceptible cells via antibody-dependent enhancement (ADE). In this way, antibodies produced in response to infection or vaccination are capable of contributing to enhanced disease in subsequent infections. Currently, there are no available therapeutics to combat DENV disease, and there is an urgent need for a safe and efficacious vaccine. Here, we developed a nucleotide-modified mRNA vaccine encoding the membrane and envelope structural proteins from DENV serotype 1 encapsulated in lipid nanoparticles (prM/E mRNA-LNP). Vaccination of mice elicited robust antiviral immune responses comparable to viral infection, with high levels of neutralizing antibody titers and antiviral CD4(+) and CD8(+) T cells. Immunocompromised AG129 mice vaccinated with the prM/E mRNA-LNP vaccine were protected from a lethal DENV challenge. Vaccination with either a wild-type vaccine or a vaccine with mutations in the immunodominant fusion loop epitope elicited equivalent humoral and cell-mediated immune responses. Neutralizing antibodies elicited by the vaccine were sufficient to protect against a lethal challenge. Both vaccine constructs demonstrated serotype-specific immunity with minimal serum cross-reactivity and reduced ADE in comparison to a live DENV1 viral infection. IMPORTANCE With 400 million worldwide infections each year, dengue is the most common vector-borne viral disease. Forty percent of the world's population is at risk, with dengue experiencing consistent geographic spread over the years. With no therapeutics available and vaccines performing suboptimally, the need for an effective dengue vaccine is urgent. Here, we develop and characterize a novel mRNA vaccine encoding the dengue serotype 1 envelope and premembrane structural proteins that is delivered via a lipid nanoparticle. Our DENV1 prM/E mRNA-LNP vaccine induces neutralizing antibody and cellular immune responses in immunocompetent mice and protects an immunocompromised mouse from a lethal DENV challenge. Existing antibodies against dengue can enhance subsequent infections via antibody-dependent enhancement (ADE). Importantly our vaccine induced only serotype-specific immune responses and did not induce ADE.

The 2015/16 Zika virus epidemic in South and Central America left the scientific community urgently trying to understand the disease and the factors which modulate Zika virus pathogenesis. Multiple other flaviviruses are endemic in areas where Zika virus emerged in 2015/16. Therefore, it is hypothesized that a key to understanding how Zika virus infection and disease progresses, is to study Zika virus infection in the context of prior flavivirus exposure. Humans and animal studies have highlighted the idea that having been previously exposed to a heterologous flavivirus may modulate the immune response to Zika virus. However, it is still unclear 1) how this impacts viral burden and pathology, and 2) the factors which correlate with the multiple metrics of disease. In this murine study, we longitudinally examine multiple factors involved in Zika disease, linking viral burden over time with increased neurological disease severity and weight loss. We show that prior heterologous flavivirus exposure with dengue virus type 2 or 3, or the vaccine strain of yellow fever, provides protection from mortality in a lethal Zika challenge. Reduction in viral burden and Zika disease in the context of prior flavivirus exposure varies depending on the infecting primary virus; with primary Zika infection being most protective from Zika challenge, followed by dengue 2, yellow fever, and dengue 3. This study demonstrates a protective effect of prior heterologous flavivirus exposure on Zika virus pathogenesis, and defines the relationship between prior flavivirus exposure and the potential for Zika virus disease.IMPORTANCEThe emergence and re-emergence of various vector-borne diseases in recent years highlights the need to understand the mechanisms of protection for each pathogen. In this study, we investigated the impact of prior exposure to Zika, dengue serotypes 2, 3, and the vaccine strain of yellow fever on pathogenesis and disease outcomes in a mouse model of Zika virus infection. We found that prior exposure to a heterologous flavivirus was protective from mortality, neurological disease, weight loss, and severe viral burden during a lethal Zika challenge. Using a longitudinal study design, we were able to link multiple disease parameters including viral burden over time with neurological disease severity and weight loss in the context of heterologous infection. This study demonstrates a role for heterologous flavivirus exposure in modulating flavivirus pathophysiology. Given the cyclic nature of most flavivirus outbreaks, this work will contribute to the forecasting of disease severity for future outbreaks.

Powassan virus (POWV) is a tick-borne pathogen for which humans are an incidental host. POWV infection can be fatal or result in long-term neurological sequelae; however, there are no approved vaccinations for POWV. Integral to efficacious vaccine development is the identification of correlates of immune protection, which we accomplished in this study by utilizing a murine model of POWV infection. Using POWV lethal and sub-lethal challenge models, we show that 1) robust B and T cell responses are necessary for immune protection, 2) POWV lethality can be attributed to both viral- and host-mediated drivers of disease and 3) knowledge of the immune correlates of protection against POWV can be applied in a virus-like particle (VLP)-based vaccination approach that provides protection from lethal POWV challenge. We also provide evidence that POWV-experienced CD8+ T cells may potentiate immune-mediated pathogenesis during infection. Identification of beneficial and detrimental aspects of POWV-immune protection is significant as it will aid in the rational design of POWV vaccines. This research was supported in part by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID): 5R01AI152192-02, 5R01AI137424-03 and F31AI152460-01. This research was also supported in part by the Department of Defense (Award#: PR192269). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Natural infection of SARS-CoV-2 in humans leads to the development of a strong neutralizing antibody response, however the immunodominant targets of the polyclonal neutralizing antibody response are still unknown. Here, we functionally define the role SARS-CoV-2 spike plays as a target of the human neutralizing antibody response. In this study, we identify the spike protein subunits that contain antigenic determinants and examine the neutralization capacity of polyclonal sera from a cohort of patients that tested qRT-PCR-positive for SARS-CoV-2. Using an ELISA format, we assessed binding of human sera to spike subunit 1 (S1), spike subunit 2 (S2) and the receptor binding domain (RBD) of spike. To functionally identify the key target of neutralizing antibody, we depleted sera of subunit-specific antibodies to determine the contribution of these individual subunits to the antigen-specific neutralizing antibody response. We show that epitopes within RBD are the target of a majority of the neutralizing antibodies in the human polyclonal antibody response. These data provide critical information for vaccine development and development of sensitive and specific serological testing.

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection., Author summary The development of tractable small animal models is critical to gain an understanding of the immune response to the novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for the evaluation of vaccines against the virus. However, the development of murine models of infection has been hindered due to the lack of expression of the human angiotensin-converting enzyme 2 (hACE2), which is the receptor required for entry of SARS-CoV-2. In this study, we cloned the hACE2 gene into an mRNA expression vector and demonstrated that transfection with this mRNA allowed for SARS-CoV-2 entry and replication. We utilized this novel method of hACE2 expression in mice by in vivo mRNA transfection to characterize the adaptive immune response to SARS-CoV-2. This unique and tractable model allowed for the first ever characterization of the murine SARS-CoV-2 specific T cell response. This information will be critical to determining the correlates of protection against the virus and for the evaluation of vaccines.

Little is known about the contribution of virus-specific and cross-reacting antibodies (Abs) or the cellular immune response generated by a primary dengue (DENV) infection on the course of a secondary zika (ZIKV) infection in vivo. Here we show that the length of time between DENV/ZIKV infections has a qualitative impact on controlling early ZIKV replication. Depletion of DENV2-specific Abs in sera confirmed that those type-specific Abs do not contribute to ZIKV control. We show that the magnitude and durability of the neutralizing antibodies (nAbs) induced by a secondary ZIKV infection is modest compared to the response induced after a secondary heterologous DENV infection. Our in vivo results are showing a complex interplay between the cellular and innate immune responses characterized by a high frequency of plasmacytoid dendritic cells (pDC) correlating with an increase in the frequency of DENV antigen specific T cells and a significant control of ZIKV replication which is time dependent. Taken together, our results suggest that early after ZIKV infection other mechanisms such as the innate and cellular immune responses may play a predominant role in controlling ZIKV replication. Regardless of the time elapsed between infections there was no evidence of in vivo antibody-dependent enhancement (ADE) of ZIKV by DENV immunity. These findings have pivotal implications while interpreting ZIKV pathogenesis in flavivirus-experimented populations, diagnostic results interpretation and vaccine designs and schedules among others., Author summary From our previous work in non-human primates and others using humans, we believe that previous DENV immunity confers some degree of protection against ZIKV infection. However, at least two highly relevant questions remain unanswered. One is precisely if the time between primary DENV and a subsequent ZIKV infections may play a role in the degree of protection conferred by DENV immunity. The second question is related to the mechanisms of cross-protection. In this work we provide evidences that a period of 12 months between DENV and ZIKV infections has a significant impact controlling ZIKV replication compared to a shorter period of 3 months. We also provide evidences that the pre-existing DENV Abs play no role controlling early ZIKV replication. Our results strongly suggest that the mechanisms controlling ZIKV replication are related to the complex interaction between the innate and the cellular immune responses. Our results have significant implications for vaccine design and schedules.

Most of the world is at risk of being infected with a flavivirus such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and Zika virus, significantly impacting millions of lives. Importantly, many of these genetically similar viruses co-circulate within the same geographic regions, making it likely for individuals living in areas of high flavivirus endemicity to be infected with multiple flaviviruses during their lifetime. Following a flavivirus infection, a robust virus-specific T cell response is generated and the memory recall of this response has been demonstrated to provide long-lasting immunity, protecting against reinfection with the same pathogen. However, multiple studies have shown that this flavivirus specific T cell response can be cross-reactive and active during heterologous flavivirus infection, leading to the question: How does immunity to one flavivirus shape immunity to the next, and how does this impact disease? It has been proposed that in some cases unfavorable disease outcomes may be caused by lower avidity cross-reactive memory T cells generated during a primary flavivirus infection that preferentially expand during a secondary heterologous infection and function sub optimally against the new pathogen. While in other cases, these cross-reactive cells still have the potential to facilitate cross-protection. In this review, we focus on cross-reactive T cell responses to flaviviruses and the concepts and consequences of T cell cross-reactivity, with particular emphasis linking data generated using murine models to our new understanding of disease outcomes following heterologous flavivirus infection.

Prior exposure to a single serotype of dengue virus (DENV) predisposes individuals to severe disease upon secondary heterologous DENV infection. Here we show that the length of time between DENV/Zika (ZIKV) infections has a qualitative impact on controlling ZIKV replication. We identified limited but significant differences in the magnitude of the early humoral immune response associated with a period of twelve months but not three months of DENV convalescence. However, their role limiting ZIKV replication is not conclusive. There was no evidence of in vivo antibody-dependent amplification of ZIKV by DENV immunity in any group. We are also showing that the significant differences among groups may be linked to a pre-existing polyfunctional CD4+ T cells response (increased IFN-g and Cd107a before ZIKV infection) and to an early and continuous expansion of the CD4+ effector memory cells early on after ZIKV infection. Those significant differences were associated with a period of 12 months after DENV infection that were not observed in a span of 3-months. These results suggest that there is a window of optimal cross-protection between ZIKV and DENV with significant consequences. These results have pivotal implications while interpreting ZIKV pathogenesis in flavivirus-experimented populations, diagnostic results interpretation and vaccine designs among others.Author SummarySince its introduction in the Americas region ZIKV virus has been associated to severe birth defects. One of the questions that remains open is the role of previous dengue or any other flavivirus immunity in the pathogenesis of ZIKV and more important, if the time elapse between DENV and ZIKV play a role enhancing ZIKV pathogenesis as it is the case for subsequent DENV infections. On this work, using NHP as a model we compared the effect of a period of 12 months vs. a period of 3 months of DENV immunity in the outcome of ZIKV infection. We found that previous DENV infection, at any of the tested period of time do not induce ZIKV enhancement. More relevant are showing that when the two infection occurs at least one year apart the preexisting DENV immunity is better at controlling ZIKV replication and that the role of the neutralizing antibodies is very limited. On the contrary our results suggest that early after ZIKV infection the cellular immune response, may plays a predominant role. Our findings have critical relevance to understand the dynamic interaction between these two flavivirus, their pathogenies, diagnosis and vaccine design.

Many flaviviruses including dengue (DENV), and Zika (ZIKV) have attracted significant attention in the past few years. As many flaviviruses are spread by arthropods, most of the world's population is at risk of encountering a flavivirus, and infection with these viruses has created a significant disease burden worldwide. Vaccination against flaviviruses is thought to be one of the most promising avenues for reducing the disease burden associated with these viruses. The optimism surrounding a vaccine approach is supported by the highly successful vaccines for yellow fever and Japanese encephalitis. Central to the development of new successful vaccines is the understanding of the correlates of protection that will be necessary to engineer into new vaccines. To aid in this endeavor we have directed our efforts to identify correlates of protection that will reduce the disease burden associated with ZIKV and DENV. Within this study we have identified a novel murine ZIKV specific CD8+ T cell epitope, and shown that the ZIKV epitope specific CD8+ T cell response has a distinct immunodominance hierarchy present during acute infection and is detectible as part of the memory T cell responses. Our studies confirm that ZIKV-specific CD8+ T cells are an important correlate of protection for ZIKV and demonstrate that both naïve and ZIKV immune CD8+ T cells are sufficient for protection against a lethal ZIKV infection. Overall this study adds to the body of literature demonstrating a role for CD8+ T cells in controlling flavivirus infection.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis thaliana has 10 ARGONAUTE (AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1,AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggered TAS3 pathway functions in timing and polarity.

Flaviviruses constitute a genus of closely related arthropod-borne viruses including Zika virus, the four serotypes of dengue virus, and yellow fever virus, which circulate in the same geographic regions. These viruses share a substantial degree of genetic similarity and consequently, antigenic overlap has been reported by evaluating T cell and antibody responses in humans, non-human primates, and mice. However, it has not been determined how existing immunity to a heterologous flavivirus impacts functional immune responses to virus-specific and cross-reactive epitopes. We hypothesize that cross-reactive T cells from prior flavivirus exposures will expand robustly during a heterologous challenge and that these cells primed during a heterologous infection will be functionally different from cells of the same specificity primed during a homologous infection. Using various mouse models of flavivirus infection, we have identified a pan-flavivirus reactive CD8+ T cell epitope. We show in a heterologous infection model in which dengue virus exposure precedes Zika virus infection, T cell responses to the cross-reactive epitope dominate at the expense of the Zika-specific T cell responses. These cross-reactive T cells display enhanced killing capacity, in addition to other functional changes. The culmination of these features drive altered protective capacity when compared to homologously primed T cells in our mouse model of Zika infection. Our findings provide a mechanistic understanding of cross-reactive T cell control during heterologous infection and have important implications for vaccine design, as these results define the functional consequences of priming a cross-reactive T cell response for a pan-flavivirus vaccine.

The plasmablast B cell (PB) response to viruses provides the first wave of functional virus specific antibodies. PBs may undergo immunoglobulin class switch recombination (CSR), giving these antibodies a diverse array of properties and functions that contribute to their mechanisms of viral clearance. Dendritic cells (DCs) can prime a PB response through native antigen production, cell surface interactions, and soluble mediators including cytokines. Additionally, we know that type I interferon (IFN) has dynamic regulatory functions by inducing expression of interferon stimulated genes (ISGs) in addition to having potent anti-proliferative and anti-viral effects. We hypothesize that signaling of type I IFN inhibits the PB response induced by virial infection by impacting the cytokine milieu, magnitude and diversity of the PB response, and mechanisms of antibody mediated protection. As DCs are potent producers of type I IFN and mediate direct interactions with B cells, we employed a DC specific knockout of Ifnar1 from the type I IFN receptor, CD11c Cre X Ifnarfl/fl, to challenge our hypothesis. Overall, we show that ablation of type I IFN signaling followed by infection with Zika virus (ZIKV) results in an increase in PB frequency in lymphoid tissue, increase in CSR, and potent increase in the neutralization capacity of polyclonal sera. The results of these studies can be leveraged to increase immunogenicity of mRNA and attenuated vaccines while targeting for a quicker antibody response.

Flaviviruses constitute a genus of closely related arthropod-borne viruses including Zika virus, the four serotypes of dengue virus, and yellow fever virus, which circulate in the same geographic regions. These viruses share a substantial degree of genetic similarity and consequently, antigenic overlap has been reported by evaluating T cell and antibody responses in humans, non-human primates, and mice. However, it has not been determined how existing immunity to a heterologous flavivirus impacts functional immune responses to virus-specific and cross-reactive epitopes. We hypothesize that cross-reactive T cells from prior flavivirus exposures will expand robustly during a heterologous challenge and that these cells primed during a heterologous infection will be functionally different from cells of the same specificity primed during a homologous infection. Using various mouse models of flavivirus infection, we have identified a pan-flavivirus reactive CD8+ T cell epitope. We show in a heterologous infection model in which dengue virus exposure precedes Zika virus infection, T cell responses to the cross-reactive epitope dominate at the expense of the Zika-specific T cell responses. These cross-reactive T cells display enhanced killing capacity, in addition to other functional changes. The culmination of these features drive altered protective capacity when compared to homologously primed T cells in our mouse model of Zika infection. Our findings provide a mechanistic understanding of cross-reactive T cell control during heterologous infection and have important implications for vaccine design, as these results define the functional consequences of priming a cross-reactive T cell response for a pan-flavivirus vaccine.

Zika virus (ZIKV) is a significant public health concern due to the pathogen&rsquo, s ability to be transmitted by either mosquito bite or sexual transmission, allowing spread to occur throughout the world. The potential consequences of ZIKV infection to human health, specifically neonates, necessitates the development of a safe and effective Zika virus vaccine. Here, we developed an intranasal Zika vaccine based upon the replication-deficient human adenovirus serotype 5 (hAd5) expressing ZIKV pre-membrane and envelope protein (hAd5-ZKV). The hAd5-ZKV vaccine is able to induce both cell-mediated and humoral immune responses to ZIKV epitopes. Importantly, this vaccine generated CD8+ T cells specific for a dominant ZIKV T cell epitope and is shown to be protective against a ZIKV challenge by using a pre-clinical model of ZIKV disease. We also demonstrate that the vaccine expresses pre-membrane and envelope protein in a confirmation recognized by ZIKV experienced individuals. Our studies demonstrate that this adenovirus-based vaccine expressing ZIKV proteins is immunogenic and protective in mice, and it encodes ZIKV proteins in a conformation recognized by the human antibody repertoire.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner.Arabidopsis thalianahas tenARGONAUTE(AGO) genes, with specialized roles in RNA-directed DNA methylation, post-transcriptional gene silencing, and antiviral defense. To better understand specialization amongAGOgenes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters ofAGO1, AGO10, andAGO7using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of theAGO7promoter by a number of proteins in two families: the miR156-regulated SPL family and the miR319-regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern ofAGO7, nor for the function ofAGO7in leaf shape. NormalAGO7transcription levels and function appear to depend instead on an adjacent 124-bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7-triggeredTAS3pathway functions in timing and polarity.

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis., Antibodies against dengue virus (DENV) can increase Zika virus (ZIKV) infection in vitro, but their role in vivo remains largely unknown. Here, the authors show that pre-existing immunity from a 2.8 years earlier DENV infection does not affect ZIKV pathogenesis in macaques but instead shortens Zika viremia.

Zika virus (ZIKV) has gained worldwide attention since it emerged, and a global effort is underway to understand the correlates of protection and develop diagnostics to identify rates of infection. As new therapeutics and vaccine approaches are evaluated in clinical trials, additional effort is focused on identifying the adaptive immune correlates of protection against ZIKV disease. To aid in this endeavor we have begun to dissect the role of CD4+T cells in the protection against neuroinvasive ZIKV disease. We have identified an important role for CD4+T cells in protection, demonstrating that in the absence of CD4+T cells mice have more severe neurological sequela and significant increases in viral titers in the central nervous system (CNS). The transfer of CD4+T cells from ZIKV immune mice protect type I interferon receptor deficient animals from a lethal challenge; showing that the CD4+T cell response is necessary and sufficient for control of ZIKV disease. Using a peptide library spanning the complete ZIKV polyprotein, we identified both ZIKV-encoded CD4+T cell epitopes that initiate immune responses, and ZIKV specific CD4+T cell receptors that recognize these epitopes. Within the ZIKV antigen-specific TCRβ repertoire, we uncovered a high degree of diversity both in response to a single epitope and among different mice responding to a CD4+T cell epitope. Overall this study identifies a novel role for polyfunctional and polyclonal CD4+T cells in providing protection against ZIKV infection and highlights the need for vaccines to develop robust CD4+T cell responses to prevent ZIKV neuroinvasion and limit replication within the CNS.