SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

SARS-CoV-2-infection- and vaccine-induced antibody responses are long lasting with an initial waning phase followed by a stabilization phase.

It is thought that mRNA-based vaccine-induced immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wanes quickly, based mostly on short-term studies. Here, we analyzed the kinetics and durability of the humoral responses to SARS-CoV-2 infection and vaccination using >8,000 longitudinal samples collected over a 3-year period in New York City. Upon primary immunization, participants with pre-existing immunity mounted higher antibody responses faster and achieved higher steady-state antibody titers than naive individuals. Antibody kinetics were characterized by two phases: an initial rapid decay, followed by a stabilization phase with very slow decay. Booster vaccination equalized the differences in antibody concentration between participants with and without hybrid immunity, but the peak antibody titers decreased with each successive antigen exposure. Breakthrough infections increased antibodies to similar titers as an additional vaccine dose in naive individuals. Our study provides strong evidence that SARS-CoV-2 antibody responses are long lasting, with initial waning followed by stabilization.

Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong1,2. Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events3, and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness2,4-6. Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Assessment of a quadrivalent nucleoside-modified mRNA vaccine that protects against group 2 influenza viruses.

Combined vaccine formulations targeting not only hemagglutinin but also other influenza virus antigens could form the basis for a universal influenza virus vaccine that has the potential to elicit long-lasting, broadly cross-reactive immune responses. Lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines can be utilized to efficiently target multiple antigens with a single vaccine. Here, we assessed the immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP vaccines that contain four influenza A group 2 virus antigens (hemagglutinin stalk, neuraminidase, matrix protein 2, and nucleoprotein) in mice. We found that all vaccine components induced antigen-specific cellular and humoral immune responses after administration of a single dose. While the monovalent formulations were not exclusively protective, the combined quadrivalent formulation protected mice from all challenge viruses, including a relevant H1N1 influenza virus group 1 strain, with minimal weight loss. Importantly, the combined vaccine protected from morbidity at a dose of 125 ng per antigen after a single vaccination in mice. With these findings, we confidently conclude that the nucleoside-modified mRNA-LNP platform can be used to elicit protection against a large panel of influenza viruses.

Immunity to Seasonal Coronavirus Spike Proteins Does Not Protect from SARS-CoV-2 Challenge in a Mouse Model but Has No Detrimental Effect on Protection Mediated by COVID-19 mRNA Vaccination.

Seasonal coronaviruses have been circulating widely in the human population for many years. With increasing age, humans are more likely to have been exposed to these viruses and to have developed immunity against them. It has been hypothesized that this immunity to seasonal coronaviruses may provide partial protection against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been shown that coronavirus disease 2019 (COVID-19) vaccination induces a back-boosting effects against the spike proteins of seasonal betacoronaviruses. In this study, we tested if immunity to the seasonal coronavirus spikes from OC43, HKU1, 229E, or NL63 would confer protection against SARS-CoV-2 challenge in a mouse model, and whether pre-existing immunity against these spikes would weaken the protection afforded by mRNA COVID-19 vaccination. We found that mice vaccinated with the seasonal coronavirus spike proteins had no increased protection compared to the negative controls. While a negligible back-boosting effect against betacoronavirus spike proteins was observed after SARS-CoV-2 infection, there was no negative original antigenic sin-like effect on the immune response and protection induced by SARS-CoV-2 mRNA vaccination in animals with pre-existing immunity to seasonal coronavirus spike proteins. IMPORTANCE The impact that immunity against seasonal coronaviruses has on both susceptibility to SARS-CoV-2 infection as well as on COVID-19 vaccination is unclear. This study provides insights into both questions in a mouse model of SARS-CoV-2.

Binding and Avidity Signatures of Polyclonal Sera From Individuals With Different Exposure Histories to Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Vaccination, and Omicron Breakthrough Infections.

BACKGROUND: The number of exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to vaccine antigens affect the magnitude and avidity of the polyclonal response. METHODS: We studied binding and avidity of different antibody isotypes to the spike, the receptor-binding domain (RBD), and the nucleoprotein (NP) of wild-type (WT) and BA.1 SARS-CoV-2 in convalescent, mRNA vaccinated and/or boosted, hybrid immune individuals and in individuals with breakthrough cases during the peak of the BA.1 wave. RESULTS: We found an increase in spike-binding antibodies and antibody avidity with increasing number of exposures to infection and/or vaccination. NP antibodies were detectible in convalescent individuals and a proportion of breakthrough cases, but they displayed low avidity. Omicron breakthrough infections elicited high levels of cross-reactive antibodies between WT and BA.1 antigens in vaccinated individuals without prior infection directed against the spike and RBD. The magnitude of the antibody response and avidity correlated with neutralizing activity against WT virus. CONCLUSIONS: The magnitude and quality of the antibody response increased with the number of antigenic exposures, including breakthrough infections. However, cross-reactivity of the antibody response after BA.1 breakthroughs, was affected by the number of prior exposures.

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.

Real-Time Investigation of a Large Nosocomial Influenza A Outbreak Informed by Genomic Epidemiology.

BACKGROUND: Nosocomial respiratory virus outbreaks represent serious public health challenges. Rapid and precise identification of cases and tracing of transmission chains is critical to end outbreaks and to inform prevention measures. METHODS: We combined conventional surveillance with influenza A virus (IAV) genome sequencing to identify and contain a large IAV outbreak in a metropolitan healthcare system. A total of 381 individuals, including 91 inpatients and 290 healthcare workers (HCWs), were included in the investigation. RESULTS: During a 12-day period in early 2019, infection preventionists identified 89 HCWs and 18 inpatients as cases of influenza-like illness (ILI), using an amended definition without the requirement for fever. Sequencing of IAV genomes from available nasopharyngeal specimens identified 66 individuals infected with a nearly identical strain of influenza A H1N1pdm09 (43 HCWs, 17 inpatients, and 6 with unspecified affiliation). All HCWs infected with the outbreak strain had received the seasonal influenza virus vaccination. Characterization of 5 representative outbreak viral isolates did not show antigenic drift. In conjunction with IAV genome sequencing, mining of electronic records pinpointed the origin of the outbreak as a single patient and a few interactions in the emergency department that occurred 1 day prior to the index ILI cluster. CONCLUSIONS: We used precision surveillance to delineate a large nosocomial IAV outbreak, mapping the source of the outbreak to a single patient rather than HCWs as initially assumed based on conventional epidemiology. These findings have important ramifications for more-effective prevention strategies to curb nosocomial respiratory virus outbreaks.

The post-COVID-19 population has a high prevalence of cross-reactive antibodies to spikes from all Orthocoronavirinae genera.

IMPORTANCE: As demonstrated by severe acute respiratory syndrome coronavirus 2, coronaviruses pose a significant pandemic threat. Here, we show that coronavirus disease 2019 mRNA vaccination can induce significant levels of cross-reactive antibodies against diverse coronavirus spike proteins. While these antibodies are binding antibodies that likely have little neutralization capacity and while their contribution to cross-protection is unclear, it is possible that they may play a role in protection from progression to severe disease with novel coronaviruses.

SARS-CoV-2 BA.1 and BA.2 breakthrough infections boost antibody responses to early Omicron subvariants but not BQ.1.1 or XBB.1.5.

Subvariants of the Omicron lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) efficiently escape neutralizing antibody responses induced by both vaccination and infection with antigenically distinct variants. Here, we describe the potency and breadth of neutralizing and binding antibody responses against a large panel of variants following an Omicron BA.1 or BA.2 breakthrough infection in a heterogeneous cohort of individuals with diverse exposure histories. Both BA.1 and BA.2 breakthrough infections significantly boost antibody levels and broaden antibody reactivity. However, this broader immunity induced by BA.1 and BA.2 breakthrough infections does not neutralize Omicron BQ and XBB subvariants efficiently. While these subvariants are not neutralized well by post-breakthrough sera, suggesting escape, binding non-neutralizing antibody responses are sustained. In summary, our data suggest that while BA.1 and BA.2 breakthrough infections broaden the immune response to SARS-CoV-2 spike, the induced neutralizing antibody response is still outpaced by viral evolution.

Determinants of health as predictors for differential antibody responses following SARS-CoV-2 primary and booster vaccination in an at-risk, longitudinal cohort.

Post vaccine immunity following COVID-19 mRNA vaccination may be driven by extrinsic, or controllable and intrinsic, or inherent health factors. Thus, we investigated the effects of extrinsic and intrinsic on the peak antibody response following COVID-19 primary vaccination and on the trajectory of peak antibody magnitude and durability over time. Participants in a longitudinal cohort attended visits every 3 months for up to 2 years following enrollment. At baseline, participants provided information on their demographics, recreational behaviors, and comorbid health conditions which guided our model selection process. Blood samples were collected for serum processing and spike antibody testing at each visit. Cross-sectional and longitudinal models (linear-mixed effects models) were generated to assess the relationship between selected intrinsic and extrinsic health factors on peak antibody following vaccination and to determine the influence of these predictors on antibody over time. Following cross-sectional analysis, we observed higher peak antibody titers after primary vaccination in females, those who reported recreational drug use, younger age, and prior COVID-19 history. Following booster vaccination, females and Hispanics had higher peak titers after the 3rd and 4th doses, respectively. Longitudinal models demonstrated that Moderna mRNA-1273 recipients, females, and those previously vaccinated had increased peak titers over time. Moreover, drug users and half-dose Moderna mRNA-1273 recipients had higher peak antibody titers over time following the first booster, while no predictive factors significantly affected post-second booster antibody responses. Overall, both intrinsic and extrinsic health factors play a significant role in shaping humoral immunogenicity after initial vaccination and the first booster. The absence of predictive factors for second booster immunogenicity suggests a more robust and consistent immune response after the second booster vaccine administration.

Examining the Effect of SARS-CoV-2 Pandemic-Induced Stress and Anxiety on Humoral Immunity in Health Care Workers.

OBJECTIVE: The effect of stress on vaccine-induced humoral immunity and therapeutic interventions to mitigate pandemic-related stress remain underexplored. METHOD: Participants in a longitudinal cohort study ( n = 189) completed a validated measure, GAD-7, and 10-instrument stress measure to assess stress and anxiety after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Serum was collected to obtain SARS-CoV-2 antibody titer levels. RESULTS: Participants experienced increased stress due to the SARS-CoV-2 pandemic with a positive correlation between GAD-7 scores and peak antibody titers overall; however, there was a negative association with scores commensurate with severe anxiety. Health care workers and younger participants were more significantly affected by anxiety. CONCLUSIONS: Mild anxiety levels may have immune-enhancing effects, whereas severe anxiety may cause antibody generation reduction. Mental health-focused interventions are imperative for younger adults and health care workers. Young adults may be more resilient to increased stress levels.

Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination.

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets.

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Maturation of germinal center B cells after influenza virus vaccination in humans.

Germinal centers (GC) are microanatomical lymphoid structures where affinity-matured memory B cells and long-lived bone marrow plasma cells are primarily generated. It is unclear how the maturation of B cells within the GC impacts the breadth and durability of B cell responses to influenza vaccination in humans. We used fine needle aspiration of draining lymph nodes to longitudinally track antigen-specific GC B cell responses to seasonal influenza vaccination. Antigen-specific GC B cells persisted for at least 13 wk after vaccination in two out of seven individuals. Monoclonal antibodies (mAbs) derived from persisting GC B cell clones exhibit enhanced binding affinity and breadth to influenza hemagglutinin (HA) antigens compared with related GC clonotypes isolated earlier in the response. Structural studies of early and late GC-derived mAbs from one clonal lineage in complex with H1 and H5 HAs revealed an altered binding footprint. Our study shows that inducing sustained GC reactions after influenza vaccination in humans supports the maturation of responding B cells.

SARS-CoV-2 infection, inflammation and birth outcomes in a prospective NYC pregnancy cohort.

Associations between antenatal SARS-CoV-2 infection and pregnancy outcomes have been conflicting and the role of the immune system is currently unclear. This prospective cohort study investigated the interaction of antenatal SARS-CoV-2 infection, changes in cytokine and HS-CRP levels, birthweight and gestational age at birth. 2352 pregnant participants from New York City (2020-2022) were included. Plasma levels of interleukin (IL)-1ß, IL-6, IL-17A and high-sensitivity C-reactive protein (HS-CRP) were quantified in blood specimens obtained across pregnancy. Quantile and linear regression models were conducted to 1) assess the impact of antenatal SARS-CoV-2 infection, overall and by timing of detection of SARS-CoV-2 positivity (< 20 weeks versus ≥ 20 weeks), on birthweight and gestational age at delivery; 2) examine the relationship between SARS-CoV-2 infection and maternal immune changes during pregnancy. All models were adjusted for maternal demographic and obstetric factors and pandemic timing. Birthweight models were additionally adjusted for gestational age at delivery and fetal sex. Immune marker models were also adjusted for gestational age at specimen collection and multiplex assay batch. 371 (15.8%) participants were infected with SARS-CoV-2 during pregnancy, of which 98 (26.4%) were infected at < 20 weeks gestation. Neither SARS-CoV-2 infection in general nor in early or late pregnancy was associated with lower birthweight nor earlier gestational age at delivery. Further, we did not observe cytokine or HS-CRP changes in response to SARS-CoV-2 infection and thus found no evidence to support a potential association between immune dysregulation and the diversity in pregnancy outcomes following infection.

Comparative analysis of SARS-CoV-2 neutralization titers reveals consistency between human and animal model serum and across assays.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires ongoing monitoring to judge the ability of newly arising variants to escape the immune response. A surveillance system necessitates an understanding of differences in neutralization titers measured in different assays and using human and animal serum samples. We compared 18 datasets generated using human, hamster, and mouse serum and six different neutralization assays. Datasets using animal model serum samples showed higher titer magnitudes than datasets using human serum samples in this comparison. Fold change in neutralization of variants compared to ancestral SARS-CoV-2, immunodominance patterns, and antigenic maps were similar among serum samples and assays. Most assays yielded consistent results, except for differences in fold change in cytopathic effect assays. Hamster serum samples were a consistent surrogate for human first-infection serum samples. These results inform the transition of surveillance of SARS-CoV-2 antigenic variation from dependence on human first-infection serum samples to the utilization of serum samples from animal models.

Bioprocess development for universal influenza vaccines based on inactivated split chimeric and mosaic hemagglutinin viruses.

Seasonal influenza viruses account for 1 billion infections worldwide every year, including 3-5 million cases of severe illness and up to 650,000 deaths. The effectiveness of current influenza virus vaccines is variable and relies on the immunodominant hemagglutinin (HA) and to a lesser extent on the neuraminidase (NA), the viral surface glycoproteins. Efficient vaccines that refocus the immune response to conserved epitopes on the HA are needed to tackle infections by influenza virus variants. Sequential vaccination with chimeric HA (cHA) and mosaic HA (mHA) constructs has proven to induce immune responses to the HA stalk domain and conserved epitopes on the HA head. In this study, we developed a bioprocess to manufacture cHA and mHA inactivated split vaccines and a method to quantify HA with a prefusion stalk based on a sandwich enzyme-linked immunosorbent assay. Virus inactivation with beta-propiolactone (ßPL) and splitting with Triton X-100 yielded the highest amount of prefusion HA and enzymatically active NA. In addition, the quantity of residual Triton X-100 and ovalbumin (OVA) was reduced to very low levels in the final vaccine preparations. The bioprocess shown here provides the basis to manufacture inactivated split cHA and mHA vaccines for pre-clinical research and future clinical trials in humans, and can also be applied to produce vaccines based on other influenza viruses.

Mucosal antibody responses to SARS-CoV-2 booster vaccination and breakthrough infection.

IMPORTANCE: Antibodies on mucosal surfaces of the upper respiratory tract have been shown to be important for protection from infection with SARS-CoV-2. Here we investigate the induction of serum IgG, saliva IgG, and saliva sIgA after COVID-19 mRNA booster vaccination or breakthrough infections.