Formation and Expansion of Memory B Cells against Coronavirus in Acutely Infected COVID-19 Individuals.

Infection with the ß-coronavirus SARS-CoV-2 typically generates strong virus-specific antibody production. Antibody responses against novel features of SARS-CoV-2 proteins require naïve B cell activation, but there is a growing appreciation that conserved regions are recognized by pre-existing memory B cells (MBCs) generated by endemic coronaviruses. The current study investigated the role of pre-existing cross-reactive coronavirus memory in the antibody response to the viral spike (S) and nucleocapsid (N) proteins following SARS-CoV-2 infection. The breadth of reactivity of circulating antibodies, plasmablasts, and MBCs was analyzed. Acutely infected subjects generated strong IgG responses to the S protein, including the novel receptor binding domain, the conserved S2 region, and to the N protein. The response included reactivity to the S of endemic ß-coronaviruses and, interestingly, to the N of an endemic α-coronavirus. Both mild and severe infection expanded IgG MBC populations reactive to the S of SARS-CoV-2 and endemic ß-coronaviruses. Avidity of S-reactive IgG antibodies and MBCs increased after infection. Overall, findings indicate that the response to the S and N of SARS-CoV-2 involves pre-existing MBC activation and adaptation to novel features of the proteins, along with the potential of imprinting to shape the response to SARS-CoV-2 infection.

Association of Human Milk Antibody Induction, Persistence, and Neutralizing Capacity With SARS-CoV-2 Infection vs mRNA Vaccination.

Importance: Long-term effect of parental COVID-19 infection vs vaccination on human milk antibody composition and functional activity remains unclear. Objective: To compare temporal IgA and IgG response in human milk and microneutralization activity against SARS-CoV-2 between lactating parents with infection and vaccinated lactating parents out to 90 days after infection or vaccination. Design, Setting, and Participants: Convenience sampling observational cohort (recruited July to December 2020) of lactating parents with infection with human milk samples collected at days 0 (within 14 days of diagnosis), 3, 7, 10, 28, and 90. The observational cohort included vaccinated lactating parents with human milk collected prevaccination, 18 days after the first dose, and 18 and 90 days after the second dose. Exposures: COVID-19 infection diagnosed by polymerase chain reaction within 14 days of consent or receipt of messenger RNA (mRNA) COVID-19 vaccine (BNT162b2 or mRNA-1273). Main Outcomes and Measures: Human milk anti-SARS-CoV-2 receptor-binding domain IgA and IgG and microneutralization activity against live SARS-CoV-2 virus. Results: Of 77 individuals, 47 (61.0%) were in the infection group (mean [SD] age, 29.9 [4.4] years), and 30 (39.0%) were in the vaccinated group (mean [SD] age, 33.0 [3.4] years; P = .002). The mean (SD) age of infants in the infection and vaccinated group were 3.1 (2.2) months and 7.5 (5.2) months, respectively (P < .001). Infection was associated with a variable human milk IgA and IgG receptor-binding domain-specific antibody response over time that was classified into different temporal patterns: upward trend and level trend (33 of 45 participants [73%]) and low/no response (12 of 45 participants [27%]). Infection was associated with a robust and quick IgA response in human milk that was stable out to 90 days after diagnosis. Vaccination was associated with a more uniform IgG-dominant response with concentrations increasing after each vaccine dose and beginning to decline by 90 days after the second dose. Vaccination was associated with increased human milk IgA after the first dose only (mean [SD] increase, 31.5 [32.6] antibody units). Human milk collected after infection and vaccination exhibited microneutralization activity. Microneutralization activity increased throughout time in the vaccine group only (median [IQR], 2.2 [0] before vaccine vs 10 [4.0] after the first dose; P = .003) but was higher in the infection group (median [IQR], 20 [67] at day 28) vs the vaccination group after the first-dose human milk samples (P = .002). Both IgA and non-IgA (IgG-containing) fractions of human milk from both participants with infection and those who were vaccinated exhibited microneutralization activity against SARS-CoV-2. Conclusions and Relevance: In this cohort study of a convenience sample of lactating parents, the pattern of IgA and IgG antibodies in human milk differed between COVID-19 infection vs mRNA vaccination out to 90 days. While infection was associated with a highly variable IgA-dominant response and vaccination was associated with an IgG-dominant response, both were associated with having human milk that exhibited neutralization activity against live SARS-CoV-2 virus.

Broadly Reactive IgG Responses to Heterologous H5 Prime-Boost Influenza Vaccination Are Shaped by Antigenic Relatedness to Priming Strains.

Prime-boost vaccinations of humans with different H5 strains have generated broadly protective antibody levels. However, the effect of an individual's H5 exposure history on antibody responses to subsequent H5 vaccination is poorly understood. To investigate this, we analyzed the IgG responses to H5 influenza A/Indonesia/5/2005 (Ind05) virus vaccination in three cohorts: (i) a doubly primed group that had received two H5 virus vaccinations, namely, against influenza A/Vietnam/203/2004 (Vie04) virus 5 years prior and A/Hong Kong/156/1997 (HK97) 11 years prior to the Ind05 vaccination; (ii) a singly primed group that had received a vaccination against Vie04 virus 5 years prior to the Ind05 vaccination; and (iii) an H5-naive group that received two doses of the Ind05 vaccine 28 days apart. Hemagglutinin (HA)-reactive IgG levels were estimated by a multiplex assay against an HA panel that included 21 H5 strains and 9 other strains representing the H1, H3, H7, and H9 subtypes. Relative HA antibody landscapes were generated to quantitatively analyze the magnitude and breadth of antibody binding after vaccination. We found that short-interval priming and boosting with the Ind05 vaccine in the naive group generated a low anti-H5 response. Both primed groups generated robust antibody responses reactive to a broad range of H5 strains after receiving a booster injection of Ind05 vaccine; IgG antibody levels persisted longer in subjects who had been doubly primed years ago. Notably, the IgG responses were strongest against the first priming H5 strain, which reflects influenza virus immune imprinting. Finally, the broad anti-H5 IgG response was stronger against strains having a small antigenic distance from the initial priming strain. IMPORTANCE The antigenic shift and draft of hemagglutinin (HA) in influenza viruses is accepted as one of the major reasons for immune evasion. The analysis of B cell immune responses to influenza infection and vaccination is complicated by the impact of exposure history and antibody cross-reactions between antigenically similar influenza strains. To assist in such analyses, the influenza "antibody landscape" method has been used to analyze and visualize the relationship of antibody-mediated immunity to antigenic distances between influenza strains. In this study, we describe a "relative antibody landscape" method that calculates the antigenic distance between the vaccine influenza strain and other H5 strains and uses this relative antigenic distance to plot the anti-H5 IgG levels postvaccination. This new method quantitatively estimates and visualizes the correlation between the humoral response to a particular influenza strain and the antigenic distance from other strains. Our findings demonstrate the effect of a subject's H5 exposure history on H5 vaccine responses quantified by the relative antibody landscape method.

Squalene-Based Influenza Vaccine Adjuvants and Their Impact on the Hemagglutinin-Specific B Cell Response.

Influenza infections continue to cause significant annual morbidity and mortality despite ongoing influenza vaccine research. Adjuvants are administered in conjunction with influenza vaccines to enhance the immune response and strengthen protection against disease. Squalene-based emulsion adjuvants including MF59, AS03, and AF03, are registered for administration with influenza vaccines and are widely used in many countries. Squalene-based emulsion adjuvants induce a strong innate immune response, enhancing antigen presentation both quantitively and qualitatively to generate strong B cell responses and antibody production. They also diversify the reactivity profiles and strengthen the affinities of antibodies against the influenza hemagglutinin, increasing protection across virus clades. In this review, we consider the mechanisms of the enhancement of innate and adaptive immune responses by squalene-based emulsionSE adjuvants and the resulting increase in magnitude and breadth of hemagglutinin-specific B cell responses. We relate observed effects of SE adjuvants and current mechanistic understandings to events in responding lymph nodes. These insights will guide the rational design and optimization of influenza vaccines to provide broad and effective protection.

Characterization of SARS-CoV-2 RNA, Antibodies, and Neutralizing Capacity in Milk Produced by Women with COVID-19.

Whether mother-to-infant SARS-CoV-2 transmission can occur during breastfeeding and, if so, whether the benefits of breastfeeding outweigh this risk during maternal COVID-19 illness remain important questions. Using RT-qPCR, we did not detect SARS-CoV-2 RNA in any milk sample (n = 37) collected from 18 women following COVID-19 diagnosis. Although we detected evidence of viral RNA on 8 out of 70 breast skin swabs, only one was considered a conclusive positive result. In contrast, 76% of the milk samples collected from women with COVID-19 contained SARS-CoV-2-specific IgA, and 80% had SARS-CoV-2-specific IgG. In addition, 62% of the milk samples were able to neutralize SARS-CoV-2 infectivity in vitro, whereas milk samples collected prior to the COVID-19 pandemic were unable to do so. Taken together, our data do not support mother-to-infant transmission of SARS-CoV-2 via milk. Importantly, milk produced by infected mothers is a beneficial source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.IMPORTANCE Results from prior studies assaying human milk for the presence of SARS-CoV-2, the causative virus of COVID-19, have suggested milk may act as a potential vehicle for mother-to-child transmission. Most previous studies are limited because they followed only a few participants, were cross-sectional, and/or failed to report how milk was collected and/or analyzed. As such, considerable uncertainty remains regarding whether human milk is capable of transmitting SARS-CoV-2 from mother to child. Here, we report that repeated milk samples collected from 18 women following COVID-19 diagnosis did not contain SARS-CoV-2 RNA; however, risk of transmission via breast skin should be further evaluated. Importantly, we found that milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness as milk likely provides specific immunologic benefits to infants.

Immunity to Influenza Infection in Humans.

This review discusses the human immune responses to influenza infection with some insights from studies using animal models, such as experimental infection of mice. Recent technological advances in the study of human immune responses have greatly added to our knowledge of the infection and immune responses, and therefore much of the focus is on recent studies that have moved the field forward. We consider the complexity of the adaptive response generated by many sequential encounters through infection and vaccination.

Analysis of Antigen-Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation.

Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells Basic Protocol 2: ELISpot assay for enumeration of memory B cell-derived antibody-secreting cells.

Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients.

Detection of antibodies to upper respiratory pathogens is critical to surveillance, assessment of the immune status of individuals, vaccine development, and basic biology. The urgent need for antibody detection tools has proven particularly acute in the COVID-19 era. We report a multiplex label-free antigen microarray on the Arrayed Imaging Reflectometry (AIR) platform for detection of antibodies to SARS-CoV-2, SARS-CoV-1, MERS, three circulating coronavirus strains (HKU1, 229E, OC43) and three strains of influenza. We find that the array is readily able to distinguish uninfected from convalescent COVID-19 subjects, and provides quantitative information about total Ig, as well as IgG- and IgM-specific responses.

COVID-19 and human milk: SARS-CoV-2, antibodies, and neutralizing capacity.

Background: It is not known whether SARS-CoV-2 can be transmitted from mother to infant during breastfeeding, and if so whether the benefits of breastfeeding outweigh this risk. This study was designed to evaluate 1) if SARS-CoV-2 RNA can be detected in milk and on the breast of infected women, 2) concentrations of milk-borne anti-SARS-CoV-2 antibodies, and 3) the capacity of milk to neutralize SARS-CoV-2 infectivity. Methods: We collected 37 milk samples and 70 breast swabs (before and after breast washing) from 18 women recently diagnosed with COVID-19. Samples were analyzed for SARS-CoV-2 RNA using RT-qPCR. Milk was also analyzed for IgA and IgG specific for the nucleocapsid protein, receptor binding domain (RBD), S2 subunit of the spike protein of SARS-CoV-2, as well as 2 seasonal coronaviruses using ELISA; and for its ability to neutralize SARS-CoV-2. Results: We did not detect SARS-CoV-2 RNA in any milk sample. In contrast, SARS-CoV-2 RNA was detected on several breast swabs, although only one was considered conclusive. All milk contained SARS-CoV-2-specific IgA and IgG, and levels of anti-RBD IgA correlated with SARS-CoV-2 neutralization. Strong correlations between levels of IgA and IgG to SARS-CoV-2 and seasonal coronaviruses were noted. Conclusions: Our data do not support maternal-to-child transmission of SARS-CoV-2 via milk; however, risk of transmission via breast skin should be further evaluated. Importantly, milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness.

S Protein-Reactive IgG and Memory B Cell Production after Human SARS-CoV-2 Infection Includes Broad Reactivity to the S2 Subunit.

The high susceptibility of humans to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the cause of coronavirus disease 2019 (COVID-19), reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and peripheral blood mononuclear cells (PBMCs) from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBC levels. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG, and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 were higher in convalescent subjects than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane.IMPORTANCE The recent rapid worldwide spread of SARS-CoV-2 has established a pandemic of potentially serious disease in the highly susceptible human population. Key issues are whether humans have preexisting immune memory that provides some protection against SARS-CoV-2 and whether SARS-CoV-2 infection generates lasting immune protection against reinfection. Our analysis focused on pre- and postinfection IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, we demonstrate that infection generates both IgG and IgG MBCs against the novel receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein. Thus, even if antibody levels wane, long-lived MBCs remain to mediate rapid antibody production. Our study results also suggest that SARS-CoV-2 infection strengthens preexisting broad coronavirus protection through S2-reactive antibody and MBC formation.

Implementing sequence-based antigenic distance calculation into immunological shape space model.

BACKGROUND: In 2009, a novel influenza vaccine was distributed worldwide to combat the H1N1 influenza "swine flu" pandemic. However, antibodies induced by the vaccine display differences in their specificity and cross-reactivity dependent on pre-existing immunity. Here, we present a computational model that can capture the effect of pre-existing immunity on influenza vaccine responses. The model predicts the region of the virus hemagglutinin (HA) protein targeted by antibodies after vaccination as well as the level of cross-reactivity induced by the vaccine. We tested our model by simulating a scenario similar to the 2009 pandemic vaccine and compared the results to antibody binding data obtained from human subjects vaccinated with the monovalent 2009 H1N1 influenza vaccine. RESULTS: We found that both specificity and cross-reactivity of the antibodies induced by the 2009 H1N1 influenza HA protein were affected by the viral strain the individual was originally exposed. Specifically, the level of antigenic relatedness between the original exposure HA antigen and the 2009 HA protein affected antigenic-site immunodominance. Moreover, antibody cross-reactivity was increased when the individual's pre-existing immunity was specific to an HA protein antigenically distinct from the 2009 pandemic strain. Comparison of simulation data with antibody binding data from human serum samples demonstrated qualitative and quantitative similarities between the model and real-life immune responses to the 2009 vaccine. CONCLUSION: We provide a novel method to evaluate expected outcomes in antibody specificity and cross-reactivity after influenza vaccination in individuals with different influenza HA antigen exposure histories. The model produced similar outcomes as what has been previously reported in humans after receiving the 2009 influenza pandemic vaccine. Our results suggest that differences in cross-reactivity after influenza vaccination should be expected in individuals with different exposure histories.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Role of Memory B Cells in Hemagglutinin-Specific Antibody Production Following Human Influenza A Virus Infection.

When influenza A virus infects an immune individual, preexisting memory B cell (MBC) activation and rapid anamnestic antibody production plays a key role in viral clearance. The most effective neutralizing antibodies target the antigenically variable head of the viral hemagglutinin (HA); antibodies against the conserved HA stalk provide broader but less potent protection. In this review, we provide a comprehensive picture of an adult's HA-specific antibody response to influenza virus infection. The process is followed from preexisting HA-specific MBC activation and rapid production of anti-HA antibodies, through to germinal center seeding and adaptation of the response to novel features of the HA. A major focus of the review is the role of competition between preexisting MBCs in determining the character of the HA-reactive antibody response. HA novelty modifies this competition and can shift the response from the immunodominant head to the stalk. We suggest that antibodies resulting from preexisting MBC activation are important regulators of anti-HA antibody production and play a role in positive selection of germinal center B cells reactive to novel HA epitopes. Our review also considers the role of MBCs in the effects of early-life imprinting on HA head- and stalk-specific antibody responses to influenza infection. An understanding of the processes described in this review will guide development of vaccination strategies that provide broadly effective protection.

Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus.

Memory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection "imprints" for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCE Rapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

Pandemic influenza vaccines: what they have taught us about B cell immunology.

The emergence of avian influenza viruses stimulated pandemic concerns and efforts to develop protective vaccines. Studies of the immune responses to experimental vaccines for pandemic influenza have taught us lessons about human immunity to influenza in general that can be applied to seasonal, pandemic, and even universal vaccine responses. For example, the concepts of targeting the hemagglutinin stalk and elicitation of stalk reactive antibodies grew out of studies of the 2009 pandemic H1N1 vaccines. More recently, the phenomenon of imprinting, the influence of early life exposure to influenza modifying responses to the viruses or vaccines later in life, has been reinforced through the study of potential pandemic influenza virus vaccines such as H7N9. These studies have also revealed potential strategies to improve responses to novel influenza strains and produce more broadly cross-reactive B cell and antibody responses. These concepts are discussed in detail in this review.

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice.

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Generation and Protective Ability of Influenza Virus-Specific Antibody-Dependent Cellular Cytotoxicity in Humans Elicited by Vaccination, Natural Infection, and Experimental Challenge.

BACKGROUND: Nonneutralizing antibodies (Abs) involved in antibody-dependent cellular cytotoxicity (ADCC) may provide some protection from influenza virus infection. The ability of influenza vaccines to induce ADCC-mediating Abs (ADCC-Abs) in adults and children is unclear. METHODS: We quantified ADCC-Abs in serum samples from adults who received a dose of inactivated subunit vaccine (ISV) targeting monovalent 2009 pandemic influenza A(H1N1) virus or live-attenuated influenza vaccine (LAIV) or who had laboratory-confirmed influenza A(H1N1) virus infection. We also measured ADCC-Abs in children who either received a dose of trivalent seasonal ISV followed by trivalent seasonal LAIV or 2 doses of LAIV. Finally, we assessed the ability of low and high ADCC-Ab titers to protect adults from experimental challenge with influenza A/Wisconsin/67/131/2005(H3N2) virus. RESULTS: Adults and children who received a dose of ISV had a robust increase in ADCC-Ab titers to both recombinant hemagglutinin (rHA) protein and homologous virus-infected cells. There was no detectable increase in titers of ADCC-Abs to rHA or virus-infected cells in adults and children who received LAIV. Higher titers (≥320) of preexisting ADCC-Abs were associated with lower virus replication and a significant reduction in total symptom scores in experimentally infected adults. CONCLUSIONS: ADCC-Ab titers increased following experimental influenza virus infection in adults and after ISV administration in both children and adults.