Faculty Bibliography

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

BACKGROUND:Haemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to determine the prevalence of vaginal carriage of Hi in reproductive age women and to examine behavioral and demographic characteristics associated with its carriage. METHODS:) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. Behavioral and demographic characteristics associated with vaginal carriage of Hi were examined. RESULTS:415 samples were available. 315 (75.9%) had sufficient bacterial DNA and were included. 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without. There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus in women with and without vaginal carriage of Hi. CONCLUSION:Hi was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences.

INTRODUCTION/BACKGROUND:There are vaccines in clinical trials that target the bacterium Group B Streptococcus (GBS). When approved, GBS vaccines will be intended for administration to pregnant women to prevent infection in their infants. The success of any vaccine will depend on its' uptake in the population. Experience with prior maternal vaccines, e.g. influenza, Tdap and COVID-19 vaccines, teaches us that acceptance of vaccines, especially if novel, is challenging for pregnant women, and that provider recommendation is a key driver of vaccine uptake. METHODS:This study investigated attitudes of maternity care providers towards the introduction of a GBS vaccine in three countries (the United States (US), Ireland, and the Dominican Republic (DR)) with different GBS prevalence and prevention practices. Semi-structured interviews with maternity care providers were transcribed and coded for themes. The constant comparative method, and inductive theory building were used to develop conclusions. RESULTS:Thirty-eight obstetricians, 18 general practitioners and 14 midwives participated. There was variability in provider attitudes towards a hypothetical GBS vaccine. Responses ranged from enthusiasm to doubts over the need for a vaccine. Attitudes were influenced by perceived additional benefits of a vaccine over current strategy and confidence in the safety of vaccines during pregnancy. Knowledge, experience and approaches to GBS prevention differed geographically and according to provider type, and influenced how participants assessed the risks and benefits of a GBS vaccine. CONCLUSION/CONCLUSIONS:Maternity care providers are engaged in the topic of GBS management and there is opportunity to leverage attitudes and beliefs that will support a strong recommendation for a GBS vaccine. However, knowledge of GBS, and of the limitations of current prevention strategies vary among providers in different regions, and between different provider types. Targeted educational efforts with antenatal providers should focus on highlighting safety data the potential benefits of vaccination over current strategies.

Group B Streptococcus (GBS) is a pathobiont that can ascend to the placenta and cause adverse pregnancy outcomes, in part through production of the toxin β-hemolysin/cytolysin (β-h/c). Innate immune cells have been implicated in the response to GBS infection, but the impact of β-h/c on their response is poorly defined. We show that GBS modulates innate immune cell states by subversion of host inflammation through β-h/c, allowing worse outcomes. We used an ascending mouse model of GBS infection to measure placental cell state changes over time following infection with a β-h/c-deficient and isogenic wild type GBS strain. Transcriptomic analysis suggests that β-h/c-producing GBS elicit a worse phenotype through suppression of host inflammatory signaling in placental macrophages and neutrophils, and comparison of human placental macrophages infected with the same strains recapitulates these results. Our findings have implications for identification of new targets in GBS disease to support host defense against pathogenic challenge.

BACKGROUND:In 2018-2019, large outbreaks of measles occurred in Israel and in New York City, driven in part by travel of unimmunized children between the 2 communities. METHODS:A retrospective chart review was conducted for children tested for measles (March 2018-September 2019) at NYU Langone Health in New York, NY, and in Ramla subdistrict, Israel. Vaccination records were reviewed to determine vaccination status for measles, mumps, and rubella (MMR) at the time of measles testing and 1-year post-testing. RESULTS:A total of 264 children were tested for measles, and 102 (38.6%) had confirmed measles. Only 20 (19.6%) of measles-positive cases received a full 2-dose course of vitamin A. 82.4% of children with measles were ≥1 year at the time of diagnosis and fully eligible for MMR vaccine. Of the 100 measles-positive cases with available vaccine records, 63 were unvaccinated at testing, and 27 remained unimmunized against MMR 1 year later. At testing, measles-negative children were significantly more likely to have received MMR than measles-positive children (65.4% vs 37%, P < .01). One year later, 70.4% of measles-negative cases and only 57.1% of measles-positive cases had received MMR vaccine (P = .18). CONCLUSIONS:The majority of measles cases occurred in unimmunized children eligible for vaccination, and >25% of children in both measles-positive and -negative groups remained unimmunized for MMR 1-year post-outbreak. Our results suggest the need for novel, longitudinal vaccination strategies and increased awareness of the role of vitamin A.

Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT β-hemolysin/cytolysin (βhc). Little is understood about the cellular factors involved in βhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to βhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for βhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of βhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on βhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that βhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to βhc, a toxin required for GBS virulence. These data suggest that βhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.

BACKGROUND:Starting in November 2020, the US Food and Drug Administration (FDA) has issued Emergency Use Authorizations (EUAs) for multiple novel virus-neutralizing monoclonal antibody therapies, including bamlanivimab monotherapy (now revoked), bamlanivimab and etesivimab, casirivimab and imdevimab (REGEN-COV), and sotrovimab, for treatment or postexposure prophylaxis of Coronavirus disease 2019 (COVID-19) in adolescents (≥12 years of age) and adults with certain high-risk conditions. Previous guidance is now updated based on new evidence and clinical experience. METHODS:A panel of experts in pediatric infectious diseases, pediatric infectious diseases pharmacotherapy, and pediatric critical care medicine from 18 geographically diverse US institutions was convened. Through a series of teleconferences and web-based surveys, a guidance statement was developed and refined based on a review of the best available evidence and expert opinion. RESULTS:The course of COVID-19 in children and adolescents is typically mild, though more severe disease is occasionally observed. Evidence supporting risk stratification is incomplete. Randomized controlled trials have demonstrated the benefit of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific monoclonal antibody therapies in adults, but data on safety and efficacy in children or adolescents are limited. Potential harms associated with infusion reactions or anaphylaxis are reportedly low in adults. CONCLUSIONS:Based on evidence available as of August 31, 2021, the panel suggests a risk-based approach to administration of SARS-CoV-2 monoclonal antibody therapy. Therapy is suggested for the treatment of mild to moderate COVID-19 in adolescents (≥12 years of age) at the highest risk of progression to hospitalization or severe disease. Therapeutic decision-making about those at moderate risk of severe disease should be individualized. Use as postexposure prophylaxis could be considered for those at the highest risk who have a high-risk exposure but are not yet diagnosed with COVID-19. Clinicians and health systems should ensure safe and timely implementation of these therapeutics that does not exacerbate existing healthcare disparities.