Diet switch pre-vaccination improves immune response and metabolic status in formerly obese mice.

Metabolic disease is epidemiologically linked to severe complications upon influenza virus infection, thus vaccination is a priority in this high-risk population. Yet, vaccine responses are less effective in these same hosts. Here we examined how the timing of diet switching from a high-fat diet to a control diet affected influenza vaccine efficacy in diet-induced obese mice. Our results demonstrate that the systemic meta-inflammation generated by high-fat diet exposure limited T cell maturation to the memory compartment at the time of vaccination, impacting the recall of effector memory T cells upon viral challenge. This was not improved with a diet switch post-vaccination. However, the metabolic dysfunction of T cells was reversed if weight loss occurred 4 weeks before vaccination, restoring a functional recall response. This corresponded with changes in the systemic obesity-related biomarkers leptin and adiponectin, highlighting the systemic and specific effects of diet on influenza vaccine immunogenicity.

Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2.

SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection.

Mapping antibody footprints using binding profiles.

The increasing use of monoclonal antibodies (mAbs) in biology and medicine necessitates efficient methods for characterizing their binding epitopes. Here, we developed a high-throughput antibody footprinting method based on binding profiles. We used an antigen microarray to profile 23 human anti-influenza hemagglutinin (HA) mAbs using HA proteins of 43 human influenza strains isolated between 1918 and 2018. We showed that the mAb's binding profile can be used to characterize its influenza subtype specificity, binding region, and binding site. We present mAb-Patch-an epitope prediction method that is based on a mAb's binding profile and the 3D structure of its antigen. mAb-Patch was evaluated using four mAbs with known solved mAb-HA structures. mAb-Patch identifies over 67% of the true epitope when considering only 50-60 positions along the antigen. Our work provides proof of concept for utilizing antibody binding profiles to screen large panels of mAbs and to down-select antibodies for further functional studies.

Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2.

SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ markedly between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections to understand the correlates of disease severity and immune memory. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection. Teaser: A nasopharyngeal swab can be used to study the intranasal immune response and yields much more information than a simple viral diagnosis.

Correlates of protection for booster doses of the SARS-CoV-2 vaccine BNT162b2.

Vaccination, especially with multiple doses, provides substantial population-level protection against COVID-19, but emerging variants of concern (VOC) and waning immunity represent significant risks at the individual level. Here we identify correlates of protection (COP) in a multicenter prospective study following 607 healthy individuals who received three doses of the Pfizer-BNT162b2 vaccine approximately six months prior to enrollment. We compared 242 individuals who received a fourth dose to 365 who did not. Within 90 days of enrollment, 239 individuals contracted COVID-19, 45% of the 3-dose group and 30% of the four-dose group. The fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple VOCs reducing the risk of symptomatic infection by 37% [95%CI, 15%-54%]. However, a group of individuals, characterized by low baseline titers of binding antibodies, remained susceptible to infection despite significantly increased neutralizing antibody titers upon boosting. A combination of reduced IgG levels to RBD mutants and reduced VOC-recognizing IgA antibodies represented the strongest COP in both the 3-dose group (HR = 6.34, p = 0.008) and four-dose group (HR = 8.14, p = 0.018). We validated our findings in an independent second cohort. In summary combination IgA and IgG baseline binding antibody levels may identify individuals most at risk from future infections.

DRAK2 contributes to type 1 diabetes by negatively regulating IL-2 sensitivity to alter regulatory T cell development.

Drak2-deficient (Drak2-/-) mice are resistant to multiple models of autoimmunity yet effectively eliminate pathogens and tumors. Thus, DRAK2 represents a potential target to treat autoimmune diseases. However, the mechanisms by which DRAK2 contributes to autoimmunity, particularly type 1 diabetes (T1D), remain unresolved. Here, we demonstrate that resistance to T1D in non-obese diabetic (NOD) mice is due to the absence of Drak2 in T cells and requires the presence of regulatory T cells (Tregs). Contrary to previous hypotheses, we show that DRAK2 does not limit TCR signaling. Rather, DRAK2 regulates IL-2 signaling by inhibiting STAT5A phosphorylation. We further demonstrate that enhanced sensitivity to IL-2 in the absence of Drak2 augments thymic Treg development. Overall, our data indicate that DRAK2 contributes to autoimmunity in multiple ways by regulating thymic Treg development and by impacting the sensitivity of conventional T cells to Treg-mediated suppression.

Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report.

Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

FLU-LISA (fluorescence-linked immunosorbent assay): high-throughput antibody profiling using antigen microarrays.

Vaccination and natural infection both elicit potent humoral responses that provide protection from subsequent infections. The immune history of an individual following such exposures is in part encoded by antibodies. While there are multiple immunoassays for measuring antibody responses, the majority of these methods measure responses to a single antigen. A commonly used method for measuring antibody responses is ELISA-a semiquantitative assay that is simple to perform in research and clinical settings. Here, we present FLU-LISA (fluorescence-linked immunosorbent assay)-a novel antigen microarray-based assay for rapid high-throughput antibody profiling. The assay can be used for profiling immunoglobulin (Ig) G, IgA and IgM responses to multiple antigens simultaneously, requiring minimal amounts of sample and antigens. Using several influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen microarrays, we demonstrated the specificity and sensitivity of our novel assay and compared it with the traditional ELISA, using samples from mice, chickens and humans. We also showed that our assay can be readily used with dried blood spots, which can be collected from humans and wild birds. FLU-LISA can be readily used to profile hundreds of samples against dozens of antigens in a single day, and therefore offers an attractive alternative to the traditional ELISA.

SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8+ T cells.

Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7-CD45RA+ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.

Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity.

Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing variable influenza epitopes greatly outnumber antibodies reactive against conserved epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.

Pre-existing humoral immunity to human common cold coronaviruses negatively impacts the protective SARS-CoV-2 antibody response.

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells.

Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence and breakthrough infections have changed vaccine policy to include booster immunizations. However, the effect of diverse and repeated antigen exposures on SARS-CoV-2 memory T cells is poorly understood. Here, we utilize DNA-barcoded MHC-multimers combined with scRNAseq and scTCRseq to capture the ex vivo profile of SARS-CoV-2-responsive T cells within a cohort of individuals with one, two, or three antigen exposures, including vaccination, primary infection, and breakthrough infection. We found that the order of exposure determined the relative distribution between spike- and non-spike-specific responses, with vaccination after infection leading to further expansion of spike-specific T cells and differentiation to a CCR7-CD45RA+ effector phenotype. In contrast, individuals experiencing a breakthrough infection mount vigorous non-spike-specific responses. In-depth analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all types of exposures elicit diverse repertoires characterized by shared, dominant TCR motifs, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and that current vaccination protocols continue to expand and differentiate spike-specific memory responses.

Host Predictors of Broadly Cross-Reactive Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants of Concern Differ Between Infection and Vaccination.

BACKGROUND: Following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination there is significant variability between individuals in protective antibody levels against SARS-CoV-2, and within individuals against different virus variants. However, host demographic or clinical characteristics that predict variability in cross-reactive antibody levels are not well-described. These data could inform clinicians, researchers, and policymakers on the populations most likely to require vaccine booster shots. METHODS: In an institutional review board-approved prospective observational cohort study of staff at St. Jude Children's Research Hospital, we identified participants with plasma samples collected after SARS-CoV-2 infection, after mRNA vaccination, and after vaccination following infection, and quantitated immunoglobulin G (IgG) levels by enzyme-linked immunosorbent assay to the spike receptor binding domain (RBD) from 5 important SARS-CoV-2 variants (Wuhan Hu-1, B.1.1.7, B.1.351, P.1, and B.1.617.2). We used regression models to identify factors that contributed to cross-reactive IgG against 1 or multiple viral variants. RESULTS: Following infection, a minority of the cohort generated cross-reactive antibodies, IgG antibodies that bound all tested variants. Those who did had increased disease severity, poor metabolic health, and were of a particular ancestry. Vaccination increased the levels of cross-reactive IgG levels in all populations, including immunocompromised, elderly, and persons with poor metabolic health. Younger people with a healthy weight mounted the highest responses. CONCLUSIONS: Our findings provide important new information on individual antibody responses to infection/vaccination that could inform clinicians on populations that may require follow-on immunization.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation.

To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

An Assessment of Serological Assays for SARS-CoV-2 as Surrogates for Authentic Virus Neutralization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.

Cross-reactive Antibody Response to mRNA SARS-CoV-2 Vaccine After Recent COVID-19-Specific Monoclonal Antibody Therapy.

The efficacy of coronavirus disease 2019 (COVID-19) vaccines administered after COVID-19-specific monoclonal antibody is unknown, and "antibody interference" might hinder immune responses leading to vaccine failure. In an institutional review board-approved prospective study, we found that an individual who received mRNA COVID-19 vaccination <40 days after COVID-19-specific monoclonal antibody therapy for symptomatic COVID-19 had similar postvaccine antibody responses to SARS-CoV-2 receptor binding domain (RBD) for 4 important SARS-CoV-2 variants (B.1, B.1.1.7, B.1.351, and P.1) as other participants who were also vaccinated following COVID-19. Vaccination against COVID-19 shortly after COVID-19-specific monoclonal antibody can boost and expand antibody protection, questioning the need to delay vaccination in this setting. TRIAL REGISTRATION: The St. Jude Tracking of Viral and Host Factors Associated with COVID-19 study; NCT04362995; https://clinicaltrials.gov/ct2/show/NCT04362995.

Broadly Reactive Influenza Antibodies Are Not Limited by Germinal Center Competition with High-Affinity Antibodies.

Enhancing the generation of broadly reactive antibodies against influenza A virus (IAV) is a pertinent goal toward developing a universal IAV vaccine. While antibodies that bind conserved IAV epitopes have been identified in humans, antibodies specific for the variable epitopes are much more prevalent than antibodies recognizing conserved epitopes. It is important to define the factors that limit the generation of broadly reactive IAV antibodies in order to develop an effective universal IAV vaccine. The predominant theory is that competition within germinal centers favors the synthesis of high-affinity antibodies specific for the variable region of the virus, and limits antibodies specific for conserved IAV epitopes. Here, we show that reducing germinal center formation and removing competition with high-affinity antibodies was not sufficient to increase broadly reactive IAV antibodies or enhance protection against distinct IAV subtypes. These data disprove the prevailing hypothesis that broadly reactive IAV antibodies are rare due to competition within germinal centers, and reveal the critical need to further investigate factors that limit broadly reactive IAV antibodies. Additionally, our data show that IAV-specific IgM antibodies persist in mice in the absence of germinal centers, highlighting the protective capacity of germinal center-independent IgM antibodies, which are not typically considered when testing correlates of protection, and offer an alternate target for delivering a universal IAV vaccine.IMPORTANCE It is estimated that 250,000 to 650,000 individuals worldwide die each year from seasonal influenza A virus (IAV) infections. Current vaccines provide little protection against newly emerging strains. Thus, considerable effort is focused on enhancing the generation of broadly reactive IAV antibodies in order to develop a universal IAV vaccine. However, broadly reactive IAV antibodies are rare and the factors that limit their generation are not completely understood. Our data disprove the prevailing hypothesis that broadly reactive IAV antibodies are uncommon due to competition in the germinal centers with antibodies specific for the variable, hemagglutinin (HA) head. Understanding the factors that constrain development of antibodies specific for conserved regions of IAV is imperative for developing an effective universal IAV vaccine, which could potentially circumvent a catastrophic pandemic. These findings are significant as they highlight the importance of investigating other mechanisms that contribute to the paucity of broadly reactive IAV antibodies.

Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function.

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.

Beta cell-specific CD8+ T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes.

The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.

Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway.

Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.