Human anti-N1 monoclonal antibodies elicited by pandemic H1N1 virus infection broadly inhibit HxN1 viruses in vitro and in vivo.

Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

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.

Safety and Immunogenicity of mRNA-1010, an Investigational Seasonal Influenza Vaccine, in Healthy Adults: Final Results From a Phase 1/2 Randomized Trial.

BACKGROUND: Seasonal influenza remains a global public health concern. A messenger RNA (mRNA)-based quadrivalent seasonal influenza vaccine, mRNA-1010, was investigated in a 3-part, first-in-human, phase 1/2 clinical trial. METHODS: In Parts 1-3 of this stratified, observer-blind study, adults aged ≥18 years old were randomly assigned to receive a single dose (6.25 µg to 200 µg) of mRNA-1010 or placebo (Part 1) or an active comparator (Afluria; Parts 2-3). Primary study objectives were assessment of safety, reactogenicity, and humoral immunogenicity of mRNA-1010, placebo (Part 1), or active comparator (Parts 2-3). Exploratory endpoints included assessment of cellular immunogenicity (Part 1) and antigenic breadth against vaccine heterologous (A/H3N2) strains (Parts 1-2). RESULTS: In all study parts, solicited adverse reactions were reported more frequently for mRNA-1010 than placebo or Afluria and most were grade 1 or 2 in severity. No vaccine-related serious adverse events or deaths were reported. In Parts 1-2, a single dose of mRNA-1010 (25 µg to 200 µg) elicited robust Day 29 hemagglutination inhibition (HAI) titers that persisted through 6 months. In Part 3, lower doses of mRNA-1010 (6.25 µg to 25 µg) elicited Day 29 HAI titers that were higher or comparable to Afluria for influenza A strains. Compared with Afluria, mRNA-1010 (50 µg) elicited broader A/H3N2 antibody responses (Part 2). mRNA-1010 induced greater T-cell responses than placebo at Day 8 that were sustained or stronger at Day 29 (Part 1). CONCLUSIONS: Data support the continued development of mRNA-1010 as a seasonal influenza vaccine. CLINICALTRIALS.GOV IDENTIFIER: NCT04956575 (https://clinicaltrials.gov/study/NCT04956575).

Humoral correlates of protection against influenza A H3N2 virus infection.

BACKGROUND: Influenza virus remains a threat to human health, but gaps remain in our knowledge of the humoral correlates of protection against influenza virus A/H3N2, limiting our ability to generate effective, broadly protective vaccines. The role of antibodies against the hemagglutinin (HA) stalk, a highly conserved but immunologically sub-dominant region, has not been established for influenza virus A/H3N2. METHODS: Household transmission studies were conducted in Managua, Nicaragua across three influenza seasons. Household contacts were tested for influenza virus infection using RT-PCR. We compared pre-existing antibody levels against full-length hemagglutinin (FLHA), HA stalk, and neuraminidase (NA) measured by enzyme-linked immunosorbent assay (ELISA), along with HA inhibition assay (HAI) titers, between infected and uninfected participants. RESULTS: A total of 899 individuals participated in household activation, with 329 infections occurring. A four-fold increase in initial HA stalk titers was independently associated with an 18% decrease in the risk of infection (OR=0.82, 95%CI 0.68-0.98, p=0.04). In adults, anti-HA stalk antibodies were independently associated with protection (OR=0.72, 95%CI 0.54-0.95, p=0.02). However, in 0-14-year-olds, anti-NA antibodies (OR=0.67, 95%CI 0.53-0.85, p<0.01) were associated with protection against infection, but anti-HA stalk antibodies were not. CONCLUSIONS: The HA stalk is an independent correlate of protection against A/H3N2 infection, though this association is age dependent. Our results support the continued exploration of the HA stalk as a target for broadly protective influenza vaccines but suggest that the relative benefits may depend on age and influenza virus exposure history.

Characterization of non-neutralizing human monoclonal antibodies that target the M1 and NP of influenza A viruses.

IMPORTANCE: Currently, many groups are focusing on isolating both neutralizing and non-neutralizing antibodies to the mutation-prone hemagglutinin as a tool to treat or prevent influenza virus infection. Less is known about the level of protection induced by non-neutralizing antibodies that target conserved internal influenza virus proteins. Such non-neutralizing antibodies could provide an alternative pathway to induce broad cross-reactive protection against multiple influenza virus serotypes and subtypes by partially overcoming influenza virus escape mediated by antigenic drift and shift. Accordingly, more information about the level of protection and potential mechanism(s) of action of non-neutralizing antibodies targeting internal influenza virus proteins could be useful for the design of broadly protective and universal influenza virus vaccines.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

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.

Differential T-Cell Reactivity to Endemic Coronaviruses and SARS-CoV-2 in Community and Health Care Workers.

Herein we measured CD4+ T-cell responses against common cold coronaviruses (CCC) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC-reactive T cells in SARS-CoV-2-seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 T-cell reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC T-cell reactivity was decreased in SARS-CoV-2-infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego. CD4+ T-cell responses against common cold coronaviruses (CCC) are elevated in SARS-CoV-2 seronegative high-risk health care workers (HCW) compared to COVID-19 convalescent HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses and/or cross-reactivity associated with a protective effect.

Pre-existing Antineuraminidase Antibodies Are Associated With Shortened Duration of Influenza A(H1N1)pdm Virus Shedding and Illness in Naturally Infected Adults.

BACKGROUND: Influenza causes a substantial burden worldwide, and current seasonal influenza vaccine has suboptimal effectiveness. To develop better, more broadly protective vaccines, a more thorough understanding is needed of how antibodies that target the influenza virus surface antigens, hemagglutinin (HA) (including head and stalk regions) and neuraminidase (NA), impact influenza illness and virus transmission. METHODS: We used a case-ascertained, community-based study of household influenza virus transmission set in Managua, Nicaragua. Using data from 170 reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed influenza virus A(H1N1)pdm infections and 45 household members with serologically confirmed infection, we examined the association of pre-existing NA, hemagglutination inhibiting, and HA stalk antibody levels and influenza viral shedding and disease duration using accelerated failure time models. RESULTS: Among RT-PCR-confirmed infections in adults, pre-existing anti-NA antibody levels ≥40 were associated with a 69% (95% confidence interval [CI], 34-85%) shortened shedding duration (mean, 1.0 vs 3.2 days). Neuraminidase antibody levels ≥80 were associated with further shortened shedding and significantly shortened symptom duration (influenza-like illness, 82%; 95% CI, 39-95%). Among RT-PCR-confirmed infections in children, hemagglutination inhibition titers ≥1:20 were associated with a 32% (95% CI, 13-47%) shortened shedding duration (mean, 3.9 vs 6.0 days). CONCLUSIONS: Our results suggest that anti-NA antibodies play a large role in reducing influenza illness duration in adults and may impact transmission, most clearly among adults. Neuraminidase should be considered as an additional target in next-generation influenza virus vaccine development.We found that antibodies against neuraminidase were associated with significantly shortened viral shedding, and among adults they were also associated with shortened symptom duration. These results support neuraminidase as a potential target of next-generation influenza virus vaccines.

Anti-Neuraminidase Antibodies Reduce the Susceptibility to and Infectivity of Influenza A/H3N2 Virus.

Immune responses against neuraminidase (NA) are of great interest for developing more robust influenza vaccines, but the role of anti-NA antibodies on influenza infectivity has not been established. We conducted household transmission studies in Managua, Nicaragua to examine the impact of anti-NA antibodies on influenza A/H3N2 susceptibility and infectivity. Analyzing these data with mathematical models capturing household transmission dynamics and their drivers, we estimated that having higher preexisting antibody levels against the hemagglutinin (HA) head, HA stalk, and NA was associated with reduced susceptibility to infection (relative susceptibility 0.67, 95% Credible Interval [CrI] 0.50-0.92 for HA head; 0.59, 95% CrI 0.42-0.82 for HA stalk; and 0.56, 95% CrI 0.40-0.77 for NA). Only anti-NA antibodies were associated with reduced infectivity (relative infectivity 0.36, 95% CrI 0.23-0.55). These benefits from anti-NA immunity were observed even among individuals with preexisting anti-HA immunity. These results suggest that influenza vaccines designed to elicit NA immunity in addition to hemagglutinin immunity may not only contribute to protection against infection but reduce infectivity of vaccinated individuals upon infection.

Safety and immunogenicity of a phase 1/2 randomized clinical trial of a quadrivalent, mRNA-based seasonal influenza vaccine (mRNA-1010) in healthy adults: interim analysis.

Despite vaccine availability, influenza remains a substantial global public health concern. Here, we report interim findings on the primary and secondary objectives of the safety, reactogenicity, and humoral immunogenicity of a quadrivalent messenger RNA (mRNA) vaccine against seasonal influenza, mRNA-1010, from the first 2 parts of a 3-part, first-in-human, phase 1/2 clinical trial in healthy adults aged ≥18 years (NCT04956575). In the placebo-controlled Part 1, a single dose of mRNA-1010 (50 µg, 100 µg, or 200 µg) elicited hemagglutination inhibition (HAI) titers against vaccine-matched strains. In the active-comparator-controlled Part 2, mRNA-1010 (25 µg, 50 µg, or 100 µg) elicited higher HAI titers than a standard dose, inactivated seasonal influenza vaccine for influenza A strains and comparable HAI titers for influenza B strains. No safety concerns were identified; solicited adverse reactions were dose-dependent and more frequent after receipt of mRNA-1010 than the active comparator. These interim data support continued development of mRNA-1010.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Titer Levels in Pregnant Individuals After Infection, Vaccination, or Both.

We examined differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses in pregnant individuals with natural, vaccine-induced, or combined immunity. Participants had live or nonlive births between 2020 and 2022, were seropositive (SARS-CoV-2 spike protein, anti-S), and had available mRNA vaccination and infection information (n=260). We compared titer levels among three immunity profiles: 1) natural immunity (n=191), 2) vaccine-induced immunity (n=37), and 3) combined immunity (ie, natural and vaccine-induced immunity; n=32). We applied linear regression to compare anti-S titers between the groups, controlling for age, race and ethnicity, and time between vaccination or infection (whichever came last) and sample collection. Anti-S titers were 57.3% and 94.4% lower among those with vaccine-induced and natural immunity, respectively, compared with those with combined immunity ( P <.001, P =.005).

Transcriptome signatures preceding the induction of anti-stalk antibodies elicited after universal influenza vaccination.

A phase 1 clinical trial to test the immunogenicity of a chimeric group 1 HA (cHA) universal influenza virus vaccine targeting the conserved stalk domain of the hemagglutinin of influenza viruses was carried out. Vaccination with adjuvanted-inactivated vaccines induced high anti-stalk antibody titers. We sought to identify gene expression signatures that correlate with such induction. Messenger-RNA sequencing in whole blood was performed on the peripheral blood of 53 vaccinees. We generated longitudinal data on the peripheral blood of 53 volunteers, at early (days 3 and 7) and late (28 days) time points after priming and boosting with cHAs. Differentially expressed gene analysis showed no differences between placebo and live-attenuated vaccine groups. However, an upregulation of genes involved in innate immune responses and type I interferon signaling was found at day 3 after vaccination with inactivated adjuvanted formulations. Cell type deconvolution analysis revealed a significant enrichment for monocyte markers and different subsets of dendritic cells as mediators for optimal B cell responses and significant increase of anti-stalk antibodies in sera. A significant upregulation of immunoglobulin-related genes was only observed after administration of adjuvanted vaccines (either as primer or booster) with specific induction of anti-stalk IGVH1-69. This approach informed of specific immune signatures that correlate with robust anti-stalk antibody responses, while also helping to understand the regulation of gene expression induced by cHA proteins under different vaccine regimens.

The antibody landscapes following AS03 and MF59 adjuvanted H5N1 vaccination.

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers.

Antibodies targeting the neuraminidase active site inhibit influenza H3N2 viruses with an S245N glycosylation site.

Contemporary influenza A H3N2 viruses circulating since 2016 have acquired a glycosylation site in the neuraminidase in close proximity to the enzymatic active site. Here, we investigate if this S245N glycosylation site, as a result of antigenic evolution, can impact binding and function of human monoclonal antibodies that target the conserved active site. While we find that a reduction in the inhibitory ability of neuraminidase active site binders is measurable, this class of broadly reactive monoclonal antibodies maintains protective efficacy in vivo.

Characterization of SARS-CoV-2 Spike mutations important for infection of mice and escape from human immune sera.

Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in "mouse-adapted" SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.

The E484K mutation in the SARS-CoV-2 spike protein reduces but does not abolish neutralizing activity of human convalescent and post-vaccination sera.

One year in the coronavirus disease 2019 (COVID-19) pandemic, the first vaccines are being rolled out under emergency use authorizations. It is of great concern that newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can escape antibody-mediated protection induced by previous infection or vaccination through mutations in the spike protein. The glutamate (E) to Lysine (K) substitution at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein is present in the rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. We performed in vitro microneutralization assays with both the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus that is identical to USA-WA1/2020 except for the E484K mutation introduced in the spike RBD. We selected 34 sera from study participants based on their SARS-CoV-2 spike ELISA antibody titer (negative [N=4] versus weak [N=8], moderate [N=11] or strong positive [N=11]). In addition, we included sera from five individuals who received two doses of the Pfizer SARS-CoV-2 vaccine BNT162b2. Serum neutralization efficiency was lower against the E484K rSARS-CoV-2 (vaccination samples: 3.4 fold; convalescent low IgG: 2.4 fold, moderate IgG: 4.2 fold and high IgG: 2.6 fold) compared to USA-WA1/2020. For some of the convalescent donor sera with low or moderate IgG against the SARS-CoV-2 spike, the drop in neutralization efficiency resulted in neutralization ID50 values similar to negative control samples, with low or even absence of neutralization of the E484K rSARS-CoV-2. However, human sera with high neutralization titers against the USA-WA1/2020 strain were still able to neutralize the E484K rSARS-CoV-2. Therefore, it is important to aim for the highest titers possible induced by vaccination to enhance protection against newly emerging SARS-CoV-2 variants. Two vaccine doses may be needed for induction of high antibody titers against SARS-CoV-2. Postponing the second vaccination is suggested by some public health authorities in order to provide more individuals with a primer vaccination. Our data suggests that this may leave vaccinees less protected against newly emerging variants.