Novel Epitopes of the Influenza Virus N1 Neuraminidase Targeted by Human Monoclonal Antibodies.

Influenza virus neuraminidase (NA)-targeting antibodies are an independent correlate of protection against influenza. Antibodies against the NA act by blocking enzymatic activity, preventing virus release and transmission. As we advance the development of improved influenza virus vaccines that incorporate standard amounts of NA antigen, it is important to identify the antigenic targets of human monoclonal antibodies (mAbs). Here, we describe escape mutants generated by serial passage of A/Netherlands/602/2009 (H1N1)pdm09 in the presence of human anti-N1 mAbs. We observed escape mutations on the head domain of the N1 protein around the enzymatic site (S364N, N369T, and R430Q) and also detected escape mutations located on the sides and bottom of the NA (N88D, N270D, and Q313K/R). This work increases our understanding of how human antibody responses target the N1 protein. IMPORTANCE As improved influenza virus vaccines are being developed, the influenza virus neuraminidase (NA) is becoming an important new target for immune responses. By identifying novel epitopes of anti-NA antibodies, we can improve vaccine design. Additionally, characterizing escape mutations in these epitopes aids in identifying NA antigenic drift in circulating viruses.

SARS-CoV-2 during pregnancy and associated outcomes: Results from an ongoing prospective cohort.

BACKGROUND: The COVID-19 pandemic is an ongoing global health threat, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Questions remain about how SARS-CoV-2 impacts pregnant individuals and their children. OBJECTIVE: To expand our understanding of the effects of SARS-CoV-2 infection during pregnancy on pregnancy outcomes, regardless of symptomatology, by using serological tests to measure IgG antibody levels. METHODS: The Generation C Study is an ongoing prospective cohort study conducted at the Mount Sinai Health System. All pregnant individuals receiving obstetrical care at the Mount Sinai Healthcare System from 20 April 2020 onwards are eligible for participation. For the current analysis, we included participants who had given birth to a liveborn singleton infant on or before 22 September 2020. For each woman, we tested the latest prenatal blood sample available to establish seropositivity using a SARS-CoV-2 serologic enzyme-linked immunosorbent assay. Additionally, RT-PCR testing was performed on a nasopharyngeal swab taken during labour. Pregnancy outcomes of interest (i.e., gestational age at delivery, preterm birth, small for gestational age, Apgar scores, maternal and neonatal intensive care unit admission, and length of neonatal hospital stay) and covariates were extracted from medical records. Excluding individuals who tested RT-PCR positive at delivery, we conducted crude and adjusted regression models to compare antibody positive with antibody negative individuals at delivery. We stratified analyses by race/ethnicity to examine potential effect modification. RESULTS: The SARS-CoV-2 seroprevalence based on IgG measurement was 16.4% (95% confidence interval 13.7, 19.3; n=116). Twelve individuals (1.7%) were SARS-CoV-2 RT-PCR positive at delivery. Seropositive individuals were generally younger, more often Black or Hispanic, and more often had public insurance and higher pre-pregnancy BMI compared with seronegative individuals. None of the examined pregnancy outcomes differed by seropositivity, overall or stratified by race/ethnicity. CONCLUSION: Seropositivity for SARS-CoV-2 without RT-PCR positivity at delivery (suggesting that infection occurred earlier during pregnancy) was not associated with selected adverse maternal or neonatal outcomes among live births in a cohort sample from New York City.

Identification and Characterization of Novel Antibody Epitopes on the N2 Neuraminidase.

The influenza virus neuraminidase (NA) is becoming a focus for novel vaccine designs. However, the epitopes of human anti-NA antibodies have been poorly defined. Using a panel of 10 anti-N2 monoclonal antibodies (MAbs) that bind the H3N2 virus A/Switzerland/9715293/2013, we generated five escape mutant viruses. These viruses contained mutations K199E/T, E258K, A272D, and S331N. We found that mutations at K199 and E258 had the largest impact on MAb binding, NA inhibition and neutralization activity. In addition, a natural isolate from the 2017-2018 season was found to contain the E258K mutation and was resistant to numerous antibodies tested. The mutation S331N, was identified in virus passaged in the presence of antibody; however, it had little impact on MAb activity and greatly decreased viral fitness. This information aids in identifying novel human MAb epitopes on the N2 and helps with the detection of antigenically drifted NAs.IMPORTANCE The influenza virus neuraminidase is an emerging target for universal influenza virus vaccines. However, in contrast to influenza virus hemagglutinin, we know little about antibody epitopes and antigenic sites on the neuraminidase. Characterizing and defining these sites is aiding vaccine development and helping to understand antigenic drift of NA.

A chimeric hemagglutinin-based universal influenza virus vaccine approach induces broad and long-lasting immunity in a randomized, placebo-controlled phase I trial.

Seasonal influenza viruses constantly change through antigenic drift and the emergence of pandemic influenza viruses through antigenic shift is unpredictable. Conventional influenza virus vaccines induce strain-specific neutralizing antibodies against the variable immunodominant globular head domain of the viral hemagglutinin protein. This necessitates frequent re-formulation of vaccines and handicaps pandemic preparedness. In this completed, observer-blind, randomized, placebo-controlled phase I trial (NCT03300050), safety and immunogenicity of chimeric hemagglutinin-based vaccines were tested in healthy, 18-39-year-old US adults. The study aimed to test the safety and ability of the vaccines to elicit broadly cross-reactive antibodies against the hemagglutinin stalk domain. Participants were enrolled into five groups to receive vaccinations with live-attenuated followed by AS03-adjuvanted inactivated vaccine (n = 20), live-attenuated followed by inactivated vaccine (n = 15), twice AS03-adjuvanted inactivated vaccine (n = 16) or placebo (n = 5, intranasal followed by intramuscular; n = 10, twice intramuscular) 3 months apart. Vaccination was found to be safe and induced a broad, strong, durable and functional immune response targeting the conserved, immunosubdominant stalk of the hemagglutinin. The results suggest that chimeric hemagglutinins have the potential to be developed as universal vaccines that protect broadly against influenza viruses.

Repeated cross-sectional sero-monitoring of SARS-CoV-2 in New York City.

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in China and has since caused a pandemic of coronavirus disease 2019 (COVID-19). The first case of COVID-19 in New York City was officially confirmed on 1 March 2020 followed by a severe local epidemic1. Here, to understand seroprevalence dynamics, we conduct a retrospective, repeated cross-sectional analysis of anti-SARS-CoV-2 spike antibodies in weekly intervals from the beginning of February to July 2020 using more than 10,000 plasma samples from patients at Mount Sinai Hospital in New York City. We describe the dynamics of seroprevalence in an 'urgent care' group, which is enriched in cases of COVID-19 during the epidemic, and a 'routine care' group, which more closely represents the general population. Seroprevalence increased at different rates in both groups; seropositive samples were found as early as mid-February, and levelled out at slightly above 20% in both groups after the epidemic wave subsided by the end of May. From May to July, seroprevalence remained stable, suggesting lasting antibody levels in the population. Our data suggest that SARS-CoV-2 was introduced in New York City earlier than previously documented and describe the dynamics of seroconversion over the full course of the first wave of the pandemic in a major metropolitan area.

Correctly folded - but not necessarily functional - influenza virus neuraminidase is required to induce protective antibody responses in mice.

The influenza virus neuraminidase (NA) plays an integral role in the influenza virus life cycle through the release of virions from infected cells. NA-specific antibodies can impede virus replication by binding to the NA and blocking its enzymatic activity, providing significant protection from influenza-associated morbidity and mortality. NA included in current seasonal influenza virus vaccines exhibits low immunogenicity, potentially caused by compromised antigenic integrity during vaccine production. To determine how certain types of "stress" could influence the antigenicity of NA we performed a series of in vitro experiments where we treated NA with formalin, EDTA or heat and measured the impact of these treatments on NA enzymatic activity and structural integrity. We found that increasing concentrations of formalin or EDTA and increasing temperature abolished the enzymatic activity of both H1N1, H3N2, and influenza B purified viruses and recombinant NA proteins. However, formalin and EDTA treatment did not drastically affect conformational epitopes found on the NA, whereas heat treatment abolished conformational epitopes. We next performed a vaccination experiment, where mice were vaccinated with recombinant N2 NA treated with 0.3% formalin or 0.125 M EDTA (which both inactivated NA activity) were protected from virus challenge while animals vaccinated with heat treated NA were not. We next tested the protective effect of monomeric (no enzymatic activity) versus tetrameric (highly active) N1 NA. Again, only the tetrameric form protected mice from challenge while the monomeric form did not. Together, our data demonstrate that enzymatically active NA is not required to induce protective antibody responses as a vaccine, however a correctly folded NA is essential.

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup.

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.