The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar-/- mice completely lacking type I IFN signaling. In Mavs-/-×Ifnar-/- myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar-/- and CD11c Cre+Ifnarf/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.

Head and neck squamous cell carcinomas (HNSCC) are one of the leading causes of cancer deaths world wide. Up-regulation of the epidermal growth factor receptor (EGFR) and BCL-2 family anti-apoptosis proteins in these cancers is linked to aggressive tumor growth, metastasis and chemoresistance. Infection of two HNSCC cell lines, SCC25 and CAL27 by an Ad5 mutant (lp11w) defective in coding for the viral anti-apoptosis protein, E1B-19K efficiently induced apoptotic cell death. In cells infected with lp11w there was a dramatic down-regulation of EGFR by apoptosis-dependent and -independent mechanisms. The levels of the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 were also down-regulated in lp11w-infected cells compared to uninfected or Ad5-RM infected cells. Infection with lp11w also enhanced sensitivity of the HNSCC cells to the chemotherapeutic drug cisplatin. Our results suggest that adenoviral vectors defective in E1B-19K would be valuable for efficient down-regulation of cell survival proteins and EGFR in epithelial cancers and could be exploited as oncolytic agents to treat HNSCCs.

The development of high-throughput assays measuring Powassan virus (POWV) lineage I and II represents an important step in virological and immunological studies. By adapting focus-forming assays previously optimized for West Nile virus and Zika virus, this protocol is able to determine viral load, evaluate antivirals, and measure neutralizing antibodies. Although limited by its requirement of a detection antibody, this protocol includes a rapid and high-throughput assay for measuring viral titer. By utilizing a baby hamster kidney cell line and a 96-well plate format, this protocol allows for more sensitivity in the detection of POWV lineage I. For complete details on the use and execution of this protocol, please refer to Stone et al. (2022).

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. We also report that serum neutralization capacity correlates with IgG titers, wherein IgG1 was the predominant isotype (62.71%), followed by IgG4 (15.25%), IgG3 (13.56%), and IgG2 (8.47%) at the earliest tested timepoint. IgA titers were detectable in just 28.81% of subjects, and only 62.71% of subjects had detectable IgM in the first sample despite confirmation of infection by a molecular diagnostic assay. Our data suggests that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from 10 out of the 59 subjects which had received an initial first dose of mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for induction of neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern seen after natural infection, after the second vaccine dose, the total anti-S antibodies titers declined, however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. The decline in anti-S antibody titer, however, was significantly less in pre-exposed individuals, highlighting the potential for natural infection to prime a more robust immune response to the vaccine. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. However, this difference was significant only when neutralizing antibody titers were compared among the two groups. No differences were observed between naturally infected and vaccinated individuals when total anti-S antibodies and IgG titers were measured. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that a functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies. In this context, our results also support standardizing methods of assessing the humoral response to SARS-CoV-2 when determining vaccine efficacy and describing the immune correlates of protection for SARS-CoV-2.

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes.

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.

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination.

Murine norovirus (MNV) is the most common gastrointestinal pathogen of research mice and can alter research outcomes in biomedical mouse models of inflammatory bowel disease (IBD). Despite indications that an altered microbiota is a risk factor for IBD, the response of the murine intestinal microbiota to MNV infection has not been examined. Microbiota disruption caused by MNV infection could introduce the confounding effects observed in research experiments. Therefore, this study investigated the effects of MNV infection on the intestinal microbiota of wild-type mice.

C-terminal binding protein (CtBP) family transcriptional corepressors include CtBP1 and CtBP2. While CtBP1 and CtBP2 share significant amino acid sequence homology, CtBP2 possesses a unique N-terminal domain that is modified by acetylation and contributes to exclusive nuclear localization. Although CtBP1 and CtBP2 are functionally redundant for certain activities during vertebrate development, they also perform unique functions. Previous studies have identified several CtBP1-interacting proteins that included other transcriptional corepressors, DNA-binding repressors and histone modifying enzymatic components such as the histone deacetylases and the histone demethylase LSD-1. Here, we carried out an unbiased proteomic analysis of CtBP2-associated proteins and discovered the association of several components of the CtBP1 proteome as well as novel interactions. The CtBP2 proteome contained components of the NuRD complex and the E2F family member E2F7. E2F7 interacted with the hydrophobic cleft region of CtBP1 and CtBP2 through a prototypical CtBP binding motif, PIDLS. E2F7 repressed E2F1 transcription, inhibited cell proliferation in a CtBP-dependent fashion. Our study identified CtBP as a corepressor of E2F7 and as a regulator of DNA damage response.

Although the transcription factor c-Myc is essential for the establishment of a metabolically active and proliferative state in T cells after priming, its expression is transient. It remains unknown how T cell activation is maintained after c-Myc expression is downregulated. Here we identified AP4 as the transcription factor that was induced by c-Myc and sustained activation of antigen-specific CD8+ T cells. Despite normal priming, AP4-deficient CD8+ T cells failed to continue transcription of a broad range of c-Myc-dependent targets. Mice lacking AP4 specifically in CD8+ T cells showed enhanced susceptibility to infection with West Nile virus. Genome-wide analysis suggested that many activation-induced genes encoding molecules involved in metabolism were shared targets of c-Myc and AP4. Thus, AP4 maintains c-Myc-initiated cellular activation programs in CD8+ T cells to control microbial infection.

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.

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.

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).

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.

Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), beta interferon (IFN-β), or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR than in wild-type (WT) cells. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death, whereas WT congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre(+) Ifnar(f/f) or LysM Cre(+) Ifnar(f/f)) did not sustain enhanced disease with OROV or a selective (flox/flox) deletion La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar(-/-) mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar(-/-) bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV infection and tissue injury and suggest that IFN signaling in nonmyeloid cells contributes to the host defense against orthobunyaviruses.

A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event.

Human adenovirus (HAdV) vectors are intensely investigated for virotherapy of a wide variety of human cancers. Here, we have evaluated the effect of two apoptogenic HAdV5 vectors in an immunocompetent Syrian hamster animal model of head and neck cancer. We established two cell lines of hamster cheek pouch squamous cell carcinomas, induced by treatment with 9,10-dimethyl-1,2-benzanthracene. These cell lines, when infected with HAdV5 mutants lp11w and lp11w/Δ55 K (which are defective in the expression of either E1B-19 K alone or both E1B-19 K and E1B-55 K proteins) exhibited enhanced apoptotic and cytotoxic responses. The cheek pouch tumor cells transplanted either subcutaneously at the flanks or in the cheek pouches of hamsters readily formed tumors. Intratumoral administration of HAdV5-E1B mutants efficiently suppressed the growth of tumors at both sites. Histological examination of orthotopic tumors revealed reduced vascularity and the expression of the viral fiber antigen in virus-administered cheek pouch tumors. These tumors also exhibited increased caspase-3 levels, suggesting that virus-induced apoptosis may contribute to tumor growth suppression. Our results suggest that the apoptogenic HAdV5 vectors may have utility for the treatment of human head and neck cancers.

Proteins of the C-terminal binding protein (CtBP) family, CtBP1 and CtBP2 are closely related transcriptional regulators that are coded by two different gene loci in the vertebrate genomes. They perform redundant and unique functions during animal development. CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs. The N-terminal region of CtBP1/2 forms a hydrophobic cleft and is involved in interaction with both PLDLS-containing factors and non-PLDLS factors. CtBP proteins function as dimers to mediate transcriptional repression and dimerization is modulated by specific binding to NAD/NADH.

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.