Mouse Models of Heterologous Flavivirus Immunity: A Role for Cross-Reactive T Cells

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

Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus

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

The role of vaccination route with an adenovirus-vectored vaccine in protection, viral control, and transmission in the SARS-CoV-2/K18-hACE2 mouse infection model

IntroductionVaccination is the most effective mechanism to prevent severe COVID-19. However, breakthrough infections and subsequent transmission of SARS-CoV-2 remain a significant problem. Intranasal vaccination has the potential to be more effective in preventing disease and limiting transmission between individuals as it induces potent responses at mucosal sites.MethodsUtilizing a replication-deficient adenovirus serotype 5-vectored vaccine expressing the SARS-CoV-2 RBD (AdCOVID) in homozygous and heterozygous transgenic K18-hACE2, we investigated the impact of the route of administration on vaccine immunogenicity, SARS-CoV-2 transmission, and survival.ResultsMice vaccinated with AdCOVID via the intramuscular or intranasal route and subsequently challenged with SARS-CoV-2 showed that animals vaccinated intranasally had improved cellular and mucosal antibody responses. Additionally, intranasally vaccinated animals had significantly better viremic control, and protection from lethal infection compared to intramuscularly vaccinated animals. Notably, in a novel transmission model, intranasal vaccination reduced viral transmission to naïve co-housed mice compared to intramuscular vaccination.DiscussionOur data provide convincing evidence for the use of intranasal vaccination in protecting against SARS-CoV-2 infection and transmission

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus

CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.

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

Immunogenicity and Efficacy of a Recombinant Human Adenovirus Type 5 Vaccine against Zika Virus

Zika virus (ZIKV) is a significant public health concern due to the pathogen’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

mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2.

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

Effective control of early Zika virus replication by Dengue immunity is associated to the length of time between the 2 infections but not mediated by antibodies.

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