Faculty Bibliography

BACKGROUND:Respiratory viral infections account for a substantial fraction of pediatric emergency department (ED) visits. We examined the epidemiological patterns of seven common respiratory viruses in children presenting to EDs with influenza-like illness (ILI). Additionally, we examined the co-occurrence of viral infections in the accompanying adults and risk factors associated with the acquisition of these viruses. METHODS:Nasopharyngeal swab were collected from children seeking medical care for ILI and their accompanying adults (Total N = 1315). Study sites included New York Presbyterian, Bellevue, and Tisch hospitals in New York City. PCR using a respiratory viral panel was conducted, and data on symptoms and medical history were collected. RESULTS:Respiratory viruses were detected in 399 children (62.25%) and 118 (17.5%) accompanying adults. The most frequent pathogen detected was human rhinovirus (HRV) (28.81%). Co-infection rates were 14.79% in children and 8.47% in adults. Respiratory syncytial virus (RSV) and parainfluenza infections occurred more often in younger children. Influenza and HRV occurred more often in older children. Influenza and coronavirus were mostly isolated in winter and spring, RSV in fall and winter and HRV in fall and spring. Children with HRV were more likely to have history of asthma. Adults with the same virus as their child often accompanied ≤ 2-year-old-positive children and were more likely to be symptomatic compared to adults with different viruses. CONCLUSIONS:Respiratory viruses, while presenting the same suite of symptoms, possess distinct seasonal cycles and affect individuals differently based on a number of identifiable factors, including age and history of asthma.

Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

BACKGROUND:Respiratory viral infections are a leading cause of disease worldwide. However, the overall community prevalence of infections has not been properly assessed, as standard surveillance is typically acquired passively among individuals seeking clinical care. METHODS:We conducted a prospective cohort study in which participants provided daily diaries and weekly nasopharyngeal specimens that were tested for respiratory viruses. These data were used to analyze healthcare seeking behavior, compared with cross-sectional ED data and NYC surveillance reports, and used to evaluate biases of medically attended ILI as signal for population respiratory disease and infection. RESULTS:The likelihood of seeking medical attention was virus-dependent: higher for influenza and metapneumovirus (19%-20%), lower for coronavirus and RSV (4%), and 71% of individuals with self-reported ILI did not seek care and half of medically attended symptomatic manifestations did not meet the criteria for ILI. Only 5% of cohort respiratory virus infections and 21% of influenza infections were medically attended and classifiable as ILI. We estimated 1 ILI event per person/year but multiple respiratory infections per year. CONCLUSION/CONCLUSIONS:Standard, healthcare-based respiratory surveillance has multiple limitations. Specifically, ILI is an incomplete metric for quantifying respiratory disease, viral respiratory infection, and influenza infection. The prevalence of respiratory viruses, as reported by standard, healthcare-based surveillance, is skewed toward viruses producing more severe symptoms. Active, longitudinal studies are a helpful supplement to standard surveillance, can improve understanding of the overall circulation and burden of respiratory viruses, and can aid development of more robust measures for controlling the spread of these pathogens.

Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Free-living mammals, such as humans and wild mice, display heightened immune activation compared with artificially maintained laboratory mice. These differences are partially attributed to microbial exposure as laboratory mice infected with pathogens exhibit immune profiles more closely resembling that of free-living animals. Here, we examine how colonization by microorganisms within the natural environment contributes to immune system maturation by releasing inbred laboratory mice into an outdoor enclosure. In addition to enhancing differentiation of T cell populations previously associated with pathogen exposure, outdoor release increased circulating granulocytes. However, these "rewilded" mice were not infected by pathogens previously implicated in immune activation. Rather, immune system changes were associated with altered microbiota composition with notable increases in intestinal fungi. Fungi isolated from rewilded mice were sufficient in increasing circulating granulocytes. These findings establish a model to investigate how the natural environment impacts immune development and show that sustained fungal exposure impacts granulocyte numbers.

Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.

Background. Colonization with Staphylococcus aureus, particularly MRSA, is a crucial risk factor for subsequent infection. Decolonization measures are often undertaken to prevent recurrent MRSA infection and transmission; however, increasing rate of resistance to the gold standard mupirocin has been noted globally. At our institution, there is >85% high-level resistance to mupirocin among strains from a geographically defined genotypic cluster of CA-MRSA in children from Orthodox communities in Brooklyn. Retapamulin is a topical bacteriostatic pleuromutilin antibiotic that has demonstrated excellent in vitro activity against mupirocin-resistant isolates from pediatric patients with MRSA infection presenting to our institution suggesting that it may be a promising alternative decolonization therapy. We sought to determine the efficacy of retapamulin as a topical decolonizing agent against mupirocin-resistant MRSA among the identified high-risk Brooklyn cluster via a randomized, placebo-controlled, double-blinded phase three trial. Methods. Children aged 9 months-17 years who resided in high-risk zip codes used as a proxy for Orthodox Jewish predominant neighborhoods were recruited either from inpatient units at NYU Langone or at a partnered community clinic. Participants were screened via nasal and rectal culture to detect MRSA colonization. Enrolled participants were randomized to receive either retapamulin or placebo and instructed to apply the ointment nasally and rectally twice a day for 5 days. Repeat nasal and rectal swab cultures were collected one week and one month after completion of topical therapy to assess MRSA colonization status. The change in colonization rates was assessed via Fisher's exact test. Results. 173 participants were screened from December 2017 to March 2019 in which 47 ultimately underwent randomization (23 in the retapamulin group and 24 in the placebo group). The median age was 3.9 years (SD 3.5 years). Children in the placebo group were 15.2 times more likely to be colonized with MRSA after one week of the decolonization protocol compared with the retapamulin group (OR 15.2, CI 2.8-81, P = 0.0004). However, children in the placebo group were only 1.1 times more likely to be colonized with MRSA after one month compared with the retapamulin group (OR 1.1, CI 0.3-3.9, P = 1). (*Full data analysis currently in progress with additional results available soon.) Conclusion. In this small pilot randomized trial, children who received retapamulin had a significantly lower rate of MRSA colonization and higher rates of clearance compared with placebo at one week post decolonization, but no significant difference at the one month mark. These data suggest that retapamulin is a promising alternative short-term nasal and peri-rectal decolonzing therapy in order to prevent infections and the spread of this mupirocin-resistant MRSA clone among pediatric patients in this affected community and our hospital

Background. Methicillin-resistant staphylococcus aureus (MRSA) colonization of hospitalized patients is associated with higher readmission rates and increased morbidity. Depending on the mechanisms of transmission, numerous potential control interventions exist to reduce the burden of disease. However, given the preponderance of asymptomatic colonization, it is challenging to quantify the relative importance of different transmission mechanisms and assess control efficacy. By identifying clusters of transmission, whole-genome sequencing (WGS) provides an opportunity to overcome these challenges. Methods. We sought to apply cluster analysis techniques to WGS data for MRSA, in order to assess MRSA prevalence, transmissibility, the degree of transmission heterogeneity and the potential effectiveness of control. Our model builds upon previous work that showed a direct relationship between the size distribution of infection clusters, the effective reproduction number (R) and the dispersion parameter (k). To demonstrate its functionality, our model was applied to existing WGS data for MRSA isolates collected during a 12 month period in the East of England (DOI: 10.1126/scitranslmed.aak9745) Results. The effective reproduction number for the East of England data is 0.29 (95% CI: 0.24-0.36). The dispersion parameter is 0.09 (0.03-0.33) reflecting a high degree of transmission heterogeneity. This implies all transmission is caused by just 12% of the cases. Targeted control of these cases could have decreased overall burden of MRSA colonization by 29% during the time period of the study. Conclusion. The high degree of transmission heterogeneity seen in MRSA transmission suggests that the risk for infection is variable.This observation motivates the need for more detailed mechanistic modeling of hospital-based MRSA transmission that integrates patients-specific factors, movement data and genome sequencing. Such models could be used to forecast which patients are at greatest risk for either acquiring or transmitting MRSA, thereby improving targeted control

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.