Mouse Memory CD8 T Cell Subsets Defined by Tissue-Resident Memory Integrin Expression Exhibit Distinct Metabolic Profiles.

Tissue-resident memory CD8 T cells (TRM) principally reside in peripheral nonlymphoid tissues, such as lung and skin, and confer protection against a variety of illnesses ranging from infections to cancers. The functions of different memory CD8 T cell subsets have been linked with distinct metabolic pathways and differ from other CD8 T cell subsets. For example, skin-derived memory T cells undergo fatty acid oxidation and oxidative phosphorylation to a greater degree than circulating memory and naive cells. Lung TRMs defined by the cell-surface expression of integrins exist as distinct subsets that differ in gene expression and function. We hypothesize that TRM subsets with different integrin profiles will use unique metabolic programs. To test this, differential expression and pathway analysis were conducted on RNA sequencing datasets from mouse lung TRMs yielding significant differences related to metabolism. Next, metabolic models were constructed, and the predictions were interrogated using functional metabolite uptake assays. The levels of oxidative phosphorylation, mitochondrial mass, and neutral lipids were measured. Furthermore, to investigate the potential relationships to TRM development, T cell differentiation studies were conducted in vitro with varying concentrations of metabolites. These demonstrated that lipid conditions impact T cell survival, and that glucose concentration impacts the expression of canonical TRM marker CD49a, with no effect on central memory-like T cell marker CCR7. In summary, it is demonstrated that mouse resident memory T cell subsets defined by integrin expression in the lung have unique metabolic profiles, and that nutrient abundance can alter differentiation.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Interferon alpha inducible protein 6 is a negative regulator of innate immune responses by modulating RIG-I activation.

Interferons (IFNs), IFN-stimulated genes (ISGs), and inflammatory cytokines mediate innate immune responses, and are essential to establish an antiviral response. Within the innate immune responses, retinoic acid-inducible gene I (RIG-I) is a key sensor of virus infections, mediating the transcriptional induction of IFNs and inflammatory proteins. Nevertheless, since excessive responses could be detrimental to the host, these responses need to be tightly regulated. In this work, we describe, for the first time, how knocking-down or knocking-out the expression of IFN alpha-inducible protein 6 (IFI6) increases IFN, ISG, and pro-inflammatory cytokine expression after the infections with Influenza A Virus (IAV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and Sendai Virus (SeV), or poly(I:C) transfection. We also show how overexpression of IFI6 produces the opposite effect, in vitro and in vivo, indicating that IFI6 negatively modulates the induction of innate immune responses. Knocking-out or knocking-down the expression of IFI6 diminishes the production of infectious IAV and SARS-CoV-2, most likely because of its effect on antiviral responses. Importantly, we report a novel interaction of IFI6 with RIG-I, most likely mediated through binding to RNA, that affects RIG-I activation, providing a molecular mechanism for the effect of IFI6 on negatively regulating innate immunity. Remarkably, these new functions of IFI6 could be targeted to treat diseases associated with an exacerbated induction of innate immune responses and to combat viral infections, such as IAV and SARS-CoV-2.

The Negative Effect of Preexisting Immunity on Influenza Vaccine Responses Transcends the Impact of Vaccine Formulation Type and Vaccination History.

The most effective measure to induce protection from influenza is vaccination. Thus, yearly vaccination is recommended, which, together with infections, establishes diverse repertoires of B cells, antibodies, and T cells. We examined the impact of this accumulated immunity on human responses in adults to split, subunit, and recombinant protein-based influenza vaccines. Enzyme-linked immunosorbent assay (ELISA) assays, to quantify serum antibodies, and peptide-stimulated CD4 T-cell cytokine ELISpots revealed that preexisting levels of hemagglutinin (HA)-specific antibodies were negatively associated with gains in antibody postvaccination, while preexisting levels of CD4 T cells were negatively correlated with vaccine-induced expansion of CD4 T cells. These patterns were seen independently of the vaccine formulation administered and the subjects' influenza vaccine history. Thus, although memory CD4 T cells and serum antibodies consist of components that can enhance vaccine responses, on balance, the accumulated immunity specific for influenza A H1 and H3 proteins is associated with diminished future responses.

Use of Host Response to Refine the Diagnosis of Group A Streptococcal Pharyngitis.

BACKGROUND: Current diagnostic tests for pharyngitis do not distinguish between symptomatic group A Streptococcus (GAS) infection and asymptomatic colonization, resulting in over-diagnosis and unnecessary use of antibiotics. We assessed whether measures of host response could make this distinction. METHODS: We enrolled 18 children with pharyngitis having Centor scores of 4 or 5 and 21 controls without pharyngitis or other acute infections. Both groups had throat cultures, molecular tests for GAS and respiratory viruses and IgM serology for Epstein-Barr virus. Host response was evaluated with white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), and sequencing of RNA from peripheral blood leukocytes. RESULTS: Of 18 cases, 11 had GAS pharyngitis, 3 had adenovirus pharyngitis and 4 had other pharyngitis. Among asymptomatic controls, 5 were positive for GAS. WBC, CRP, and PCT were higher in subjects with pharyngitis compared to asymptomatic controls including those with GAS. Transcriptional profiles from children with symptomatic GAS were clearly distinct from those of children in all other groups. The levels of two genes, CD177 and TLR5 each individually accurately distinguished between symptomatic and asymptomatic GAS. Optimal diagnostic sensitivity and specificity were achieved by the combination of CRP and PCT, and by each of the two gene markers. CONCLUSION: In this exploratory study, we showed that traditional measures of inflammation and markers of host gene expression distinguish between symptomatic and asymptomatic GAS. These results point to future rapid molecular approaches for improving the diagnosis of GAS pharyngitis, that may help reduce unnecessary antibiotic use.

Aberrant newborn T cell and microbiota developmental trajectories predict respiratory compromise during infancy.

Neonatal immune-microbiota co-development is poorly understood, yet age-appropriate recognition of - and response to - pathogens and commensal microbiota is critical to health. In this longitudinal study of 148 preterm and 119 full-term infants from birth through one year of age, we found that postmenstrual age or weeks from conception is a central factor influencing T cell and mucosal microbiota development. Numerous features of the T cell and microbiota functional development remain unexplained; however, by either age metric and are instead shaped by discrete perinatal and postnatal events. Most strikingly, we establish that prenatal antibiotics or infection disrupt the normal T cell population developmental trajectory, influencing subsequent respiratory microbial colonization and predicting respiratory morbidity. In this way, early exposures predict the postnatal immune-microbiota axis trajectory, placing infants at later risk for respiratory morbidity in early childhood.

Formation and Expansion of Memory B Cells against Coronavirus in Acutely Infected COVID-19 Individuals.

Infection with the ß-coronavirus SARS-CoV-2 typically generates strong virus-specific antibody production. Antibody responses against novel features of SARS-CoV-2 proteins require naïve B cell activation, but there is a growing appreciation that conserved regions are recognized by pre-existing memory B cells (MBCs) generated by endemic coronaviruses. The current study investigated the role of pre-existing cross-reactive coronavirus memory in the antibody response to the viral spike (S) and nucleocapsid (N) proteins following SARS-CoV-2 infection. The breadth of reactivity of circulating antibodies, plasmablasts, and MBCs was analyzed. Acutely infected subjects generated strong IgG responses to the S protein, including the novel receptor binding domain, the conserved S2 region, and to the N protein. The response included reactivity to the S of endemic ß-coronaviruses and, interestingly, to the N of an endemic α-coronavirus. Both mild and severe infection expanded IgG MBC populations reactive to the S of SARS-CoV-2 and endemic ß-coronaviruses. Avidity of S-reactive IgG antibodies and MBCs increased after infection. Overall, findings indicate that the response to the S and N of SARS-CoV-2 involves pre-existing MBC activation and adaptation to novel features of the proteins, along with the potential of imprinting to shape the response to SARS-CoV-2 infection.

Association of Human Milk Antibody Induction, Persistence, and Neutralizing Capacity With SARS-CoV-2 Infection vs mRNA Vaccination.

Importance: Long-term effect of parental COVID-19 infection vs vaccination on human milk antibody composition and functional activity remains unclear. Objective: To compare temporal IgA and IgG response in human milk and microneutralization activity against SARS-CoV-2 between lactating parents with infection and vaccinated lactating parents out to 90 days after infection or vaccination. Design, Setting, and Participants: Convenience sampling observational cohort (recruited July to December 2020) of lactating parents with infection with human milk samples collected at days 0 (within 14 days of diagnosis), 3, 7, 10, 28, and 90. The observational cohort included vaccinated lactating parents with human milk collected prevaccination, 18 days after the first dose, and 18 and 90 days after the second dose. Exposures: COVID-19 infection diagnosed by polymerase chain reaction within 14 days of consent or receipt of messenger RNA (mRNA) COVID-19 vaccine (BNT162b2 or mRNA-1273). Main Outcomes and Measures: Human milk anti-SARS-CoV-2 receptor-binding domain IgA and IgG and microneutralization activity against live SARS-CoV-2 virus. Results: Of 77 individuals, 47 (61.0%) were in the infection group (mean [SD] age, 29.9 [4.4] years), and 30 (39.0%) were in the vaccinated group (mean [SD] age, 33.0 [3.4] years; P = .002). The mean (SD) age of infants in the infection and vaccinated group were 3.1 (2.2) months and 7.5 (5.2) months, respectively (P < .001). Infection was associated with a variable human milk IgA and IgG receptor-binding domain-specific antibody response over time that was classified into different temporal patterns: upward trend and level trend (33 of 45 participants [73%]) and low/no response (12 of 45 participants [27%]). Infection was associated with a robust and quick IgA response in human milk that was stable out to 90 days after diagnosis. Vaccination was associated with a more uniform IgG-dominant response with concentrations increasing after each vaccine dose and beginning to decline by 90 days after the second dose. Vaccination was associated with increased human milk IgA after the first dose only (mean [SD] increase, 31.5 [32.6] antibody units). Human milk collected after infection and vaccination exhibited microneutralization activity. Microneutralization activity increased throughout time in the vaccine group only (median [IQR], 2.2 [0] before vaccine vs 10 [4.0] after the first dose; P = .003) but was higher in the infection group (median [IQR], 20 [67] at day 28) vs the vaccination group after the first-dose human milk samples (P = .002). Both IgA and non-IgA (IgG-containing) fractions of human milk from both participants with infection and those who were vaccinated exhibited microneutralization activity against SARS-CoV-2. Conclusions and Relevance: In this cohort study of a convenience sample of lactating parents, the pattern of IgA and IgG antibodies in human milk differed between COVID-19 infection vs mRNA vaccination out to 90 days. While infection was associated with a highly variable IgA-dominant response and vaccination was associated with an IgG-dominant response, both were associated with having human milk that exhibited neutralization activity against live SARS-CoV-2 virus.

A systems genomics approach uncovers molecular associates of RSV severity.

Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity.

MicroRNA-29 specifies age-related differences in the CD8+ T cell immune response.

MicroRNAs (miRNAs) have emerged as critical regulators of cell fate in the CD8+ T cell response to infection. Although there are several examples of miRNAs acting on effector CD8+ T cells after infection, it is unclear whether differential expression of one or more miRNAs in the naive state is consequential in altering their long-term trajectory. To answer this question, we examine the role of miR-29 in neonatal and adult CD8+ T cells, which express different amounts of miR-29 only prior to infection and adopt profoundly different fates after immune challenge. We find that manipulation of miR-29 expression in the naive state is sufficient for age-adjusting the phenotype and function of CD8+ T cells, including their regulatory landscapes and long-term differentiation trajectories after infection. Thus, miR-29 acts as a developmental switch by controlling the balance between a rapid effector response in neonates and the generation of long-lived memory in adults.

CD49a Identifies Polyfunctional Memory CD8 T Cell Subsets that Persist in the Lungs After Influenza Infection.

CD8 T cell memory offers critical antiviral protection, even in the absence of neutralizing antibodies. The paradigm is that CD8 T cell memory within the lung tissue consists of a mix of circulating TEM cells and non-circulating TRM cells. However, based on our analysis, the heterogeneity within the tissue is much higher, identifying TCM, TEM, TRM, and a multitude of populations which do not perfectly fit these classifications. Further interrogation of the populations shows that TRM cells that express CD49a, both with and without CD103, have increased and diverse effector potential compared with CD49a negative populations. These populations function as a one-man band, displaying antiviral activity, chemokine production, release of GM-CSF, and the ability to kill specific targets in vitro with delayed kinetics compared with effector CD8 T cells. Together, this study establishes that CD49a defines multiple polyfunctional CD8 memory subsets after clearance of influenza infection, which act to eliminate virus in the absence of direct killing, recruit and mature innate immune cells, and destroy infected cells if the virus persists.

Squalene-Based Influenza Vaccine Adjuvants and Their Impact on the Hemagglutinin-Specific B Cell Response.

Influenza infections continue to cause significant annual morbidity and mortality despite ongoing influenza vaccine research. Adjuvants are administered in conjunction with influenza vaccines to enhance the immune response and strengthen protection against disease. Squalene-based emulsion adjuvants including MF59, AS03, and AF03, are registered for administration with influenza vaccines and are widely used in many countries. Squalene-based emulsion adjuvants induce a strong innate immune response, enhancing antigen presentation both quantitively and qualitatively to generate strong B cell responses and antibody production. They also diversify the reactivity profiles and strengthen the affinities of antibodies against the influenza hemagglutinin, increasing protection across virus clades. In this review, we consider the mechanisms of the enhancement of innate and adaptive immune responses by squalene-based emulsionSE adjuvants and the resulting increase in magnitude and breadth of hemagglutinin-specific B cell responses. We relate observed effects of SE adjuvants and current mechanistic understandings to events in responding lymph nodes. These insights will guide the rational design and optimization of influenza vaccines to provide broad and effective protection.

Airway gene-expression classifiers for respiratory syncytial virus (RSV) disease severity in infants.

BACKGROUND: A substantial number of infants infected with RSV develop severe symptoms requiring hospitalization. We currently lack accurate biomarkers that are associated with severe illness. METHOD: We defined airway gene expression profiles based on RNA sequencing from nasal brush samples from 106 full-tem previously healthy RSV infected subjects during acute infection (day 1-10 of illness) and convalescence stage (day 28 of illness). All subjects were assigned a clinical illness severity score (GRSS). Using AIC-based model selection, we built a sparse linear correlate of GRSS based on 41 genes (NGSS1). We also built an alternate model based upon 13 genes associated with severe infection acutely but displaying stable expression over time (NGSS2). RESULTS: NGSS1 is strongly correlated with the disease severity, demonstrating a naïve correlation (ρ) of ρ = 0.935 and cross-validated correlation of 0.813. As a binary classifier (mild versus severe), NGSS1 correctly classifies disease severity in 89.6% of the subjects following cross-validation. NGSS2 has slightly less, but comparable, accuracy with a cross-validated correlation of 0.741 and classification accuracy of 84.0%. CONCLUSION: Airway gene expression patterns, obtained following a minimally-invasive procedure, have potential utility for development of clinically useful biomarkers that correlate with disease severity in primary RSV infection.

Gaps in Serologic Immunity against Contemporary Swine-Origin Influenza A Viruses among Healthy Individuals in the United States.

Influenza A Viruses (IAV) in domestic swine (IAV-S) are associated with sporadic zoonotic transmission at the human-animal interface. Previous pandemic IAVs originated from animals, which emphasizes the importance of characterizing human immunity against the increasingly diverse IAV-S. We analyzed serum samples from healthy human donors (n = 153) using hemagglutination-inhibition (HAI) assay to assess existing serologic protection against a panel of contemporary IAV-S isolated from swine in the United States (n = 11). Age-specific seroprotection rates (SPR), which are the proportion of individuals with HAI ≥ 1:40, corresponded with lower or moderate pandemic risk classifications for the multiple IAV-S examined (one H1-δ1, one H1-δ2, three H3-IVA, one H3-IVB, one H3-IVF). Individuals born between 2004 and 2013 had SPRs of 0% for the five classified H3 subtype IAV-S, indicating youth may be particularly predisposed to infection with these viruses. Expansion of existing immunologic gaps over time could increase likelihood of future IAV-S spillover to humans and facilitate subsequent sustained human-to-human transmission resulting in disease outbreaks with pandemic potential.

Immunity to Influenza Infection in Humans.

This review discusses the human immune responses to influenza infection with some insights from studies using animal models, such as experimental infection of mice. Recent technological advances in the study of human immune responses have greatly added to our knowledge of the infection and immune responses, and therefore much of the focus is on recent studies that have moved the field forward. We consider the complexity of the adaptive response generated by many sequential encounters through infection and vaccination.

Temporal Dysbiosis of Infant Nasal Microbiota Relative to Respiratory Syncytial Virus Infection.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of infant respiratory disease. Infant airway microbiota has been associated with respiratory disease risk and severity. The extent to which interactions between RSV and microbiota occur in the airway, and their impact on respiratory disease susceptibility and severity, are unknown. METHODS: We carried out 16S rRNA microbiota profiling of infants in the first year of life from (1) a cross-sectional cohort of 89 RSV-infected infants sampled during illness and 102 matched healthy controls, and (2) a matched longitudinal cohort of 12 infants who developed RSV infection and 12 who did not, sampled before, during, and after infection. RESULTS: We identified 12 taxa significantly associated with RSV infection. All 12 taxa were differentially abundant during infection, with 8 associated with disease severity. Nasal microbiota composition was more discriminative of healthy vs infected than of disease severity. CONCLUSIONS: Our findings elucidate the chronology of nasal microbiota dysbiosis and suggest an altered developmental trajectory associated with RSV infection. Microbial temporal dynamics reveal indicators of disease risk, correlates of illness and severity, and impact of RSV infection on microbiota composition.

Airway Gene Expression Correlates of Respiratory Syncytial Virus Disease Severity and Microbiome Composition in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants. The causes and correlates of severe illness in the majority of infants are poorly defined. METHODS: We recruited a cohort of RSV-infected infants and simultaneously assayed the molecular status of their airways and the presence of airway microbiota. We used rigorous statistical approaches to identify gene expression patterns associated with disease severity and microbiota composition, separately and in combination. RESULTS: We measured comprehensive airway gene expression patterns in 106 infants with primary RSV infection. We identified an airway gene expression signature of severe illness dominated by excessive chemokine expression. We also found an association between Haemophilus influenzae, disease severity, and airway lymphocyte accumulation. Exploring the time of onset of clinical symptoms revealed acute activation of interferon signaling following RSV infection in infants with mild or moderate illness, which was absent in subjects with severe illness. CONCLUSIONS: Our data reveal that airway gene expression patterns distinguish mild/moderate from severe illness. Furthermore, our data identify biomarkers that may be therapeutic targets or useful for measuring efficacy of intervention responses.

Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Understanding of the fundamental processes underlying the versatile clinical manifestations of COVID-19 is incomplete without comprehension of how different immune cells are recruited to various compartments of virus-infected lungs, and how this recruitment differs among individuals with different levels of disease severity. As in other respiratory infections, leukocyte recruitment to the respiratory system in people with COVID-19 is orchestrated by specific leukocyte trafficking molecules, and when uncontrolled and excessive it results in various pathological complications, both in the lungs and in other organs. In the absence of experimental data from physiologically relevant animal models, our knowledge of the trafficking signals displayed by distinct vascular beds and epithelial cell layers in response to infection by SARS-CoV-2 is still incomplete. However, SARS-CoV-2 and influenza virus elicit partially conserved inflammatory responses in the different respiratory epithelial cells encountered early in infection and may trigger partially overlapping combinations of trafficking signals in nearby blood vessels. Here, we review the molecular signals orchestrating leukocyte trafficking to airway and lung compartments during primary pneumotropic influenza virus infections and discuss potential similarities to distinct courses of primary SARS-CoV-2 infections. We also discuss how an imbalance in vascular activation by leukocytes outside the airways and lungs may contribute to extrapulmonary inflammatory complications in subsets of patients with COVID-19. These multiple molecular pathways are potential targets for therapeutic interventions in patients with severe COVID-19.