Faculty Bibliography

SARS-CoV-2 infection is associated with platelet hyperreactivity and increased rates of arterial and venous thrombosis. SARS-CoV-2 mutations have resulted in several variants with differences in transmissibility, infectivity, and patient outcomes. This study investigates the effects of the ancestral strain of SARS-CoV-2 (WA1) and two variants of concern, Delta and Omicron, on the human megakaryocyte (MK) phenotype and transcriptome. Human CD34⁺-derived MKs were incubated with WA1, Delta or Omicron SARS-CoV-2 variants for 24 hours. MK activation markers were measured under resting and thrombin-stimulated conditions. RNA-seq and cytokine release in response to the viruses were assessed. Plasma cytokines were measured in hospitalized COVID-19 patients. Treatment of MKs with WA1, Delta or Omicron variants of SARS-CoV-2 resulted in similar increases in classical activation markers. However, SARS-CoV-2 variants mediated distinct transcriptomic changes. Across variants, 60 genes overlapped, including CXCL8. Consistent with transcriptomic changes, SARS-CoV-2-incubated MKs secreted significantly elevated levels of IL-8. Among hospitalized COVID-19 patients, plasma IL-8 levels were highest in COVID-19 patients who subsequently experienced thrombotic events or died. In conclusion, WA1, Delta, and Omicron similarly induce classical MK activation responses while mediating distinct transcriptomic changes. Increased IL-8 levels may serve as a biomarker to inform platelet hyperreactivity and thrombotic events associated with COVID-19.

Staphylococcus aureus is a global health concern, resulting in significant disease burden in both hospital and community settings. To establish infection, the bacteria must contend with a multitude of host defense mechanisms, including "nutritional immunity", in which nutrients are sequestered away from invading pathogens. Importantly, S. aureus requires iron for growth during infection, which it acquires through the lysis of erythrocytes (hemolysis). HlgAB, a secreted bi-component pore forming toxin, contributes to the ability of S. aureus to lyse erythrocytes to release heme iron. HlgAB consists of two subunits, the S-subunit HlgA and the F-subunit HlgB. Prior work has shown that the hemolytic activity of HlgAB is dependent on the binding of HlgA to the host receptor Duffy Antigen Receptor for Chemokines (DARC). Here we show that HlgB binds the surface of erythrocytes independently of DARC or HlgA. Our comparative genomic analysis reveals high conservation of hlgA and hlgB genes across S. aureus lineages. By performing structure-function studies, we identified a series of loops within the rim domain of HlgB that are required for the binding of HlgB to erythrocytes and erythrocyte lysis by HlgAB. The importance of HlgB-mediated host targeting was validated in a tissue culture model of S. aureus-mediated lysis of primary human erythrocytes, in an in vivo murine model of intoxication, and during in vivo systemic infection. Altogether, these findings expand our mechanistic insights into how S. aureus overcomes nutritional immunity, and the role of HlgB in S. aureus pathophysiology.

Snow Mountain Virus (SMV), the prototype of genogroup II and genotype II Norovirus (NoV), was used in human challenge studies to examine the infectivity, pathogenicity, and immune response to NoV. Clinical and laboratory data from two previously completed SMV human challenge trials using two different inocula (primary and secondary) were analyzed to compare the infectivity, illness, viral shedding, and serum IgG conversion. The primary and secondary SMV inocula were sequenced for detecting single nucleotide mutations. Of 15 subjects challenged with the primary inoculum between 2000 and 2002, nine were infected, and seven presented with acute gastroenteritis. Of 33 subjects challenged with the secondary inoculum between 2016 and 2018, 25 were infected, and nine presented with acute gastroenteritis. There were no statistically significant differences in overall infection and illness rates between subjects challenged with the primary inoculum versus the secondary inoculum. However, subjects infected with the primary inoculum experienced more severe clinical symptoms of acute gastroenteritis, showing higher severity scores (6.00 vs. 2.94, p = 0.003) compared with those infected with the secondary inoculum. We also observed that infection with the secondary inoculum resulted in longer viral shedding compared with the primary inoculum. Partial sequencing of the SMV genome identified three mutations in both inocula. Understanding the differences between these two SMV inocula is critical for NoV vaccine evaluation and using a less pathogenic inoculum for a vaccine trial will require more participants to meet the target reduction in illness when evaluating the efficacy of candidate vaccines.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

BACKGROUND AND OBJECTIVE/OBJECTIVE:We describe the kinetics of maternally derived antibodies in infants in the first 6 months of life following 2- or 3-dose maternal vaccination during pregnancy or postpartum. METHODS:This prospective, multicenter cohort study enrolled infants born to mothers vaccinated with 2- (n = 280) or 3-dose (boosted) monovalent messenger RNA vaccines in pregnancy (n = 202) or to mothers vaccinated postpartum (n = 36) from July 2021 to January 2022. Binding (immunoglobulin G to S and receptor-binding domain), pseudovirus, and live neutralizing antibody (nAb) geometric mean titers (GMTs) to vaccine and Omicron BA.1/BA.5 strains were measured at birth and 2 and 6 months of age. Antibody half-life and the effect of maternal or infant COVID-19 infection were assessed. RESULTS:Significantly higher GMTs of binding antibody and nAb to all antigens were present at birth and 2 months in infants of boosted mothers (P < .01) and higher titers to the vaccine strain, but not Omicron BA.1 and BA.5, persisted up to 6 months of age in infants of boosted mothers compared with the other groups (P < .01). Higher infant antibody titers at delivery and 6 months of age were associated with a booster dose during pregnancy and maternal prenatal and infant COVID-19 infection. Maternal infection status or vaccine regimen did not influence the half-life of infant antibodies. CONCLUSIONS:A maternal COVID-19 booster in pregnancy results in significantly higher functional antibody titers in infants compared with 2 doses in pregnancy or postpartum. High titers at birth and maternal hybrid immunity result in persistently elevated titers in infants for 6 months.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1^T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1^T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1^T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1^T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

BACKGROUND:Mucosal immunity plays a critical role in preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and replication. Understanding the capacity of coronavirus disease 2019 (COVID-19) vaccines to elicit both mucosal and systemic antibodies could help optimize vaccination strategies. METHODS:We conducted an open-label, phase 1/2 adaptive-design clinical trial to evaluate the safety and immunogenicity of COVID-19 immunizations. Healthy adults received 2 priming doses of mRNA-1273, a booster dose of mRNA-1273, and a second booster of bivalent (WA-1 and BA.4/BA.5) mRNA-1273.222. Adverse event data were collected. Serum and mucosal immunity were evaluated. RESULTS:One hundred six persons were enrolled. Thirty received all 4 study-related vaccine doses. All vaccines were well tolerated, with injection site pain, malaise, myalgias, and headache being the most frequently reported symptoms. Among those who received a second booster, 24 of 30 (80%) had serological evidence of SARS-CoV-2 infection. Following the second booster, increases in geometric mean binding and pseudovirus neutralization antibody titers to the ancestral strain and BA.1 and BA.5 variants were observed. Increases in mucosal immunoglobulin G and immunoglobulin A (IgA) antibodies in nasal and salivary samples were observed in both previously infected and infection-naive participants, although prior infection markedly boosted virus-specific mucosal IgA responses. CONCLUSIONS:The mRNA-1273.222 booster vaccine was safe and immunogenic and induced mucosal antibody responses in previously infected and infection-naive persons. CLINICAL TRIALS REGISTRATION/BACKGROUND:NCT04889209.

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

BACKGROUND/UNASSIGNED:Global study data show injection drug use is driving upwards of 79% of all new hepatitis C virus (HCV) cases in high-income countries. Low-threshold models can engage vulnerable populations in treatment to achieve HCV elimination targets. We examined the implementation of low-threshold models for HCV care in New York State, which has a robust HCV elimination program. METHODS/UNASSIGNED:We conducted semi-structured interviews with 16 healthcare providers in 2022. Included providers either self-described as "low-threshold," had a clinical focus on marginalized populations, or practiced in non-traditional settings. Interviews focused on the implementation of low-threshold HCV care. Transcripts were analyzed using thematic analysis and were categorized into themes guided by the Exploration, Preparation, Implementation, Sustainment (EPIS) framework. RESULTS/UNASSIGNED:Providers implemented low-threshold HCV care by facilitating access (e.g., having walk-in or telemedicine HCV services). Point-of-care testing and peer support were other important features. The inner context was driven by provider and organization values and involved providing low-threshold HCV care within health systems that were not themselves "low-threshold." Adequate staffing was crucial for the extensive care coordination and outreach activities needed to engage persons who inject drugs (PWID). The outer context was characterized by a limited funding environment, restrictive insurance policies, and the high impact of patients' unmet social needs. Providers relied on care coordination and integrated care models to overcome these barriers. CONCLUSIONS/UNASSIGNED:Low-threshold HCV care incorporates operational flexibility and patient navigation but is challenged by patients' unmet social needs. Jurisdictions can support implementation by providing adequate funding for substantial outreach activities needed to engage vulnerable populations.