Stalk-specific Antibodies Research Articles

A chimeric haemagglutinin-based universal influenza virus vaccine boosts human cellular immune responses directed towards the conserved haemagglutinin stalk domain and the viral nucleoprotein

The development of a universal influenza virus vaccine, to protect against both seasonal and pandemic influenza A viruses, is a long-standing public health goal. The conserved stalk domain of haemagglutinin (HA) is a promising vaccine target. However, the stalk is immunosubdominant. As such, innovative approaches are required to elicit robust immunity against this domain. In a previously reported observer-blind, randomised placebo-controlled phase I trial (NCT03300050), immunisation regimens using chimeric HA (cHA)-based immunogens formulated as inactivated influenza vaccines (IIV)-/+ AS03 adjuvant, or live attenuated influenza vaccines (LAIV), elicited durable HA stalk-specific antibodies with broad reactivity. In this study, we sought to determine if these vaccines could also boost T cell responses against HA stalk, and nucleoprotein (NP). We measured interferon-γ (IFN-γ) responses by Enzyme-Linked ImmunoSpot (ELISpot) assay at baseline, seven days post-prime, pre-boost and seven days post-boost following heterologous prime:boost regimens of LAIV and/or adjuvanted/unadjuvanted IIV-cHA vaccines. Our findings demonstrate that immunisation with adjuvanted cHA-based IIVs boost HA stalk-specific and NP-specific T cell responses in humans. To date, it has been unclear if HA stalk-specific T cells can be boosted in humans by HA-stalk focused universal vaccines. Therefore, our study will provide valuable insights for the design of future studies to determine the precise role of HA stalk-specific T cells in broad protection. Considering that cHA-based vaccines also elicit stalk-specific antibodies, these data support the further clinical advancement of cHA-based universal influenza vaccine candidates. This study was funded in part by the Bill and Melinda Gates Foundation (BMGF).

Group 1 and group 2 hemagglutinin stalk antibody response according to age.

Antibodies elicited by seasonal influenza vaccines mainly target the head of hemagglutinin (HA). However, antibodies against the stalk domain are cross-reactive and have been proven to play a role in reducing influenza disease severity. We investigated the induction of HA stalk-specific antibodies after seasonal influenza vaccination, considering the age of the cohorts. A total of 166 individuals were recruited during the 2018 influenza vaccine campaign (IVC) and divided into groups: <50 (n = 14), 50-64 (n = 34), 65-79 (n = 61), and ≥80 (n = 57) years old. Stalk-specific antibodies were quantified by ELISA at day 0 and day 28 using recombinant viruses (cH6/1 and cH14/3) containing an HA head domain (H6 or H14) from wild bird origin with a stalk domain from human H1 or H3, respectively. The geometric mean titer (GMT) and the fold rise (GMFR) were calculated, and differences were assessed using ANOVA adjusted by the false discovery rate (FDR) and the Wilcoxon tests (p <0.05). All age groups elicited some level of increase in anti-stalk antibodies after receiving the influenza vaccine, except for the ≥80-year-old cohort. Additionally, <65-year-old vaccinees had higher group 1 antibody titers versus group 2 before and after vaccination. Similarly, vaccinees within the <50-year-old group showed a higher increase in anti-stalk antibody titers when compared to older individuals (≥80 years old), especially for group 1 anti-stalk antibodies. Seasonal influenza vaccines can the induction of cross-reactive anti-stalk antibodies against group 1 and group 2 HAs. However, low responses were observed in older groups, highlighting the impact of immunosenescence in adequate humoral immune responses.

Influenza vaccination-induced H3 stalk-reactive memory B-cell clone expansion

Current vaccine formulations elicit a recall immune response against viruses by targeting epitopes on the globular head of hemagglutinin (HA), and stalk-reactive antibodies are rarely found. However, stalk-specific memory B-cell expansion after influenza vaccination is poorly understood. In this study, B cells were isolated from individuals immunized with seasonal tetravalent influenza vaccines at days 0 and 28 for H7N9 stimulation in vitro. Plasma and supernatants were collected for the analysis of anti-HA IgG using ELISA and a Luminex assay. Memory B cells were positively enriched, and total RNA was extracted for B cell receptor (BCR) H-CDR3 sequencing. All subjects displayed increased anti-H3 antibody secretion after vaccination, whereas no increase in cH5/3-reactive IgG levels was detected. The number of shared memory B-cell clones among individuals dropped dramatically from 593 to 37. Four out of 5 subjects displayed enhanced frequencies of the VH3-23 and VH3-30 genes, and one exhibited an increase in the frequency of VH1-18, which are associated with the stalk of HA. An increase in H3 stalk-specific antibodies produced by B cells stimulated with H7N9 viruses was detected after vaccination. These results demonstrated that H3 stalk-specific memory B cells can expand and secrete antibodies that bind to the stalk in vitro, although no increase in serum H3 stalk-reactive antibodies was found after vaccination, indicating potential for developing a universal vaccine strategy.

A chimeric thermostable M2e and H3 stalk-based universal influenza A virus vaccine

We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.

Detection of HA Stalk Specific Antibodies in Chicken Serum Immunized with Different Subtypes of Inactivated Avian Influenza Viruses

Detection of HA Stalk Specific Antibodies in Chicken Serum Immunized with Different Subtypes of Inactivated Avian Influenza Viruses

Influenza Vaccination Results in Differential Hemagglutinin Stalk-Specific Fc-Mediated Functions in Individuals Living With or Without HIV

Influenza virus hemagglutinin (HA) stalk-specific antibodies have been shown to potently induce Fc-mediated effector functions which are important in protection from disease. In placebo-controlled maternal influenza (MatFlu) vaccination trials of pregnant women living with or without HIV, reduced risk of influenza illness was associated with high HA stalk antibody titers following trivalent inactivated vaccination (TIV). However, the mechanisms of immunity conferred by the HA stalk antibodies were not well understood. Here, we investigated HA stalk-specific Fc effector functions including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent complement deposition (ADCD), and FcγRIIa and FcγRIIIa binding in response to seasonal influenza vaccination. These were measured pre- and 1-month post-vaccination in 141 HIV-uninfected women (67 TIV and 74 placebo recipients) and 119 women living with HIV (WLWH; 66 TIV and 53 placebo recipients). In contrast to HIV-uninfected women, where HA stalk-specific ADCP and FcγRIIa binding were significantly boosted, WLWH showed no increase in response to vaccination. HA stalk-specific ADCC potential and FcγRIIIa binding were not boosted regardless of HIV status but were higher in WLWH compared with HIV-uninfected women prior to vaccination. HA stalk-specific ADCD was significantly increased by vaccination in all women, but was significantly lower in the WLWH both pre- and post- vaccination. Co-ordination between HA stalk-specific ADCP and ADCD in WLWH was improved by vaccination. Fc polyfunctionality was enhanced by vaccination in HIV-uninfected women and driven by the HA stalk antibody titers. However, in the WLWH, higher pre-vaccination Fc polyfunctionality was maintained post-vaccination but was decoupled from titer. Overall, we showed differential regulation of Fc effector HA stalk responses, suggesting that HIV infection results in unique humoral immunity in response to influenza vaccination, with relevance for future strategies that aim to target the HA stalk in this population.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.

Mutations in the Hemagglutinin Stalk Domain Do Not Permit Escape from a Protective, Stalk-Based Vaccine-Induced Immune Response in the Mouse Model.

ABSTRACTCurrent seasonal influenza virus vaccines target regions of the hemagglutinin (HA) head domain that undergo constant antigenic change, forcing the painstaking annual reformulation of vaccines. The development of broadly protective or universal influenza virus vaccines that induce cross-reactive, protective immune responses could circumvent the need to reformulate current seasonal vaccines. Many of these vaccine candidates target the HA stalk domain, which displays epitopes conserved within and across influenza virus subtypes, including those with pandemic potential. While HA head-mediated antigenic drift is well understood, the potential for antigenic drift in the stalk domain is understudied. Using a panel of HA stalk-specific monoclonal antibodies (MAbs), we applied selection pressure to the stalk domain of A/Netherlands/602/2009 (pdmH1N1) to determine fitness and phenotypes of escape mutant viruses (EMVs). We found that HA stalk MAbs with lower cross-reactivity caused single HA stalk escape mutations, whereas MAbs with broader cross-reactivity forced multiple mutations in the HA. Each escape mutant virus greatly decreased mAb neutralizing activity, but escape mutations did not always ablate MAb binding or Fc-Fc receptor-based effector functions. Escape mutant viruses were not attenuated in vitro but showed attenuation in an in vivo mouse model. Importantly, mice vaccinated with a chimeric HA universal vaccine candidate were protected from lethal challenge with EMVs despite these challenge viruses containing escape mutations in the stalk domain. Our study indicates that while the HA stalk domain can mutate under strong MAb selection pressure, mutant viruses may have attenuated phenotypes and do not evade a polyclonal, stalk-based vaccine-induced response.

Influenza hemagglutinin-specific IgA Fc-effector functionality is restricted to stalk epitopes

In this study, we utilized a panel of human immunoglobulin (Ig) IgA monoclonal antibodies isolated from the plasmablasts of eight donors after 2014/2015 influenza virus vaccination (Fluarix) to study the binding and functional specificities of this isotype. In this cohort, isolated IgA monoclonal antibodies were primarily elicited against the hemagglutinin protein of the H1N1 component of the vaccine. To compare effector functionalities, an H1-specific subset of antibodies targeting distinct epitopes were expressed as monomeric, dimeric, or secretory IgA, as well as in an IgG1 backbone. When expressed with an IgG Fc domain, all antibodies elicited Fc-effector activity in a primary polymorphonuclear cell-based assay which differs from previous observations that found only stalk-specific antibodies activate the low-affinity FcγRIIIa. However, when expressed with IgA Fc domains, only antibodies targeting the stalk domain showed Fc-effector activity in line with these previous findings. To identify the cause of this discrepancy, we then confirmed that IgG signaling through the high-affinity FcγI receptor was not restricted to stalk epitopes. Since no corresponding high-affinity Fcα receptor exists, the IgA repertoire may therefore be limited to stalk-specific epitopes in the context of Fc receptor signaling.

Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies.

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1.

Induction of cross-group broadly reactive antibody response by natural H7N9 avian influenza virus infection and immunization with inactivated H7N9 vaccine in chickens.

Pre-existing immunity against the conserved haemagglutinin (HA) stalk underlies the elicitation of cross-group antibody induced by natural H7N9 virus infection and immunization in humans. However, whether broadly reactive antibodies can be induced by H7N9 infection and immunization in the absence of pre-existing stalk-specific immunity is unclear. In this study, antibody response induced by H7N9 virus infection and immunization with inactivated and viral-vectored H7N9 vaccines in naïve chickens was analysed. The results showed that H7N9 infection and immunization with inactivated vaccine resulted in potent induction of haemagglutination-inhibition (HI), virus neutralization (VN) and HA-binding antibodies, whereas Newcastle disease virus (NDV)-vectored H7N9 vaccine induced marginal HI and VN titres but high levels of HA-binding antibody. In addition, H7N9 infection and immunization induced stalk-specific antibodies in naïve chickens and these antibodies recognized different epitopes in the stalk. Virus infection and immunization with inactivated vaccine elicited antibodies cross-reactive with both group 1 and group 2 HAs, while antibodies induced by NDV-H7N9 vaccination showed a narrower cross-reactivity within group 2. Moreover, only homologous neutralizing activity of the sera against H7N9 virus was observed, and cross-binding antibodies did not show heterosubtypic neutralizing activity. Our results indicated that cross-group binding but non-neutralizing antibodies primarily targeting the stalk can be induced by natural H7N9 infection and immunization with inactivated vaccine in naïve chickens. This suggests that at least in a naïve chicken model, pre-existing stalk-specific immunity is not required for induction of broadly reactive antibodies. Additionally, H7N9-based immunogens may be explored as vaccine candidates or as a prime component to induce broadly protective influenza immunity.

The Transcription Factor T-bet Resolves Memory B Cell Subsets with Distinct Tissue Distributions and Antibody Specificities in Mice and Humans

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.

Pandemic influenza virus vaccines boost hemagglutinin stalk-specific antibody responses in primed adult and pediatric cohorts

Licensed influenza virus vaccines target the head domain of the hemagglutinin (HA) glycoprotein which undergoes constant antigenic drift. The highly conserved HA stalk domain is an attractive target to increase immunologic breadth required for universal influenza virus vaccines. We tested the hypothesis that immunization with a pandemic influenza virus vaccine boosts pre-existing anti-stalk antibodies. We used chimeric cH6/1, full length H2 and H18 HA antigens in an ELISA to measure anti-stalk antibodies in recipients participating in clinical trials of A/H1N1, A/H5N1 and A/H9N2 vaccines. The vaccines induced high titers of anti-H1 stalk antibodies in adults and children, with higher titers elicited by AS03-adjuvanted vaccines. We also observed cross-reactivity to H2 and H18 HAs. The A/H9N2 vaccine elicited plasmablast and memory B-cell responses. Post-vaccination serum from vaccinees protected mice against lethal challenge with cH6/1N5 and cH5/3N4 viruses. These findings support the concept of a chimeric HA stalk-based universal influenza virus vaccine. clinicaltrials.gov: NCT02415842.

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.

Novel correlates of protection against pandemic H1N1 influenza A virus infection.

Influenza viruses remain a severe threat to human health, causing up to 650,000 deaths annually1,2. Seasonal influenza virus vaccines can prevent infection, but are rendered ineffective by antigenic drift. To provide improved protection from infection, novel influenza virus vaccines that target the conserved epitopes of influenza viruses, specifically those in the hemagglutinin stalk and neuraminidase, are currently being developed3. Antibodies against the hemagglutinin stalk confer protection in animal studies4-6. However, no data exist on natural infections in humans, and these antibodies do not show activity in the hemagglutination inhibition assay, the hemagglutination inhibition titer being the current correlate of protection against influenza virus infection7-9. While previous studies have investigated the protective effect of cellular immune responses and neuraminidase-inhibiting antibodies, additional serological correlates of protection from infection could aid the development of broadly protective or universal influenza virus vaccines10-13. To address this gap, we performed a household transmission study to identify alternative correlates of protection from infection and disease in naturally exposed individuals. Using this study, we determined 50% protective titers and levels for hemagglutination inhibition, full-length hemagglutinin, neuraminidase and hemagglutinin stalk-specific antibodies. Further, we found that hemagglutinin stalk antibodies independently correlated with protection from influenza virus infection.

A Universal Influenza Virus Vaccine Candidate Tested in a Pig Vaccination-Infection Model in the Presence of Maternal Antibodies.

The antigenically conserved hemagglutinin stalk region is a target for universal influenza virus vaccines since antibodies against it can provide broad protection against influenza viruses of different subtypes. We tested a universal influenza virus vaccination regimen based on sequential immunization with chimeric hemagglutinin (HA) containing viruses in a swine influenza virus pig model with maternal antibodies against pandemic H1N1. Vaccines were administered as live attenuated virus or inactivated influenza virus split vaccine (+/− Emulsigen adjuvant). As controls, we included groups that received trivalent inactivated influenza vaccine that contained pandemic H1N1 antigens, inactivated adjuvanted H1N2 vaccine (control group for vaccine associated enhanced respiratory disease in the pig model) or mock-vaccination. No induction of H1 head or stalk-specific antibody responses was observed upon vaccination, while responses against H3 and influenza B HA were elicited in the group vaccinated with the trivalent vaccine. Four weeks post vaccination, pigs were intratracheally challenged with pandemic H1N1 virus and euthanized 5 days after challenge. Despite the lack of detectable anti-stalk immunity, the chimeric hemagglutinin vaccine resulted in better clinical outcomes compared to control groups.

Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.

Impact of pre-existing immunity on the induction of functional cross-reactive anti-hemagglutinin stalk antibodies following vaccination with an AS03 adjuvanted pandemic H1N1 vaccine

The 2009 pandemic H1N1 (A(H1N1)pdm09) virus had a highly divergent hemagglutinin (HA) compared to pre-2009 seasonal H1N1 strains. Most peoples were immunologically naïve to the A(H1N1)pdm09, although hospital workers were exposed early in the pandemic before pandemic vaccines became available. Here, we evaluated how pre-existing antibodies influence the induction of cross-functional HA stalk antibodies following A(H1N1)pdm09 vaccination.Fifty-six healthcare workers vaccinated with AS03 adjuvanted A(H1N1)pdm09 vaccine were chosen by their pre-vaccination priming status (primed HI titers ≥ 40 or unprimed < 40). We analyzed the HA head- and stalk-specific serum IgG subclasses at pre- and 21 days post-vaccination. We also assessed the functionality of the HA stalk-specific antibodies to neutralize virus and mediate antibody dependent cellular cytotoxicity (ADCC).Primed individuals had higher pre-existing HA head- and stalk-specific IgG1, as well as higher ADCC functionality of stalk antibodies. However, following vaccination with the adjuvanted pandemic vaccine, only the quantity of HA head specific IgG1 antibodies were significantly higher than in unprimed individuals. The priming status did not impact upon the cross-reactive HA stalk specific IgG antibodies or their ability to neutralize virus or induce ADCC post-vaccination. In conclusion, a single dose of AS03 adjuvanted pandemic vaccine elicited similar levels of functional antibodies in naïve and primed individuals. These findings are important for understanding the immunological factors that impact or modulate pandemic vaccine responses in humans.

Assessment of Influenza Virus Hemagglutinin Stalk-Specific Antibody Responses.

Animal models are essential to examine the pathogenesis and transmission of influenza viruses and for preclinical evaluation of influenza virus vaccines. Among the animal models used in influenza virus research, the domestic ferret (Mustela putorius furo) is the gold standard. As seen in humans, infection with influenza virus or immunization with an influenza virus vaccine induces humoral and cellular immunity in ferrets that provides protection against infection by an antigenically similar influenza virus. Antibodies against the globular head domain of the influenza hemagglutinin can provide sterilizing immunity against virus infection by blocking receptor binding. However, antibodies that bind the stalk region of the hemagglutinin also confer protection by several mechanisms including antibody-dependent cellular cytotoxicity or phagocytosis. Recently, the antigenically and structurally conserved hemagglutinin stalk has become an attractive target for the development of universal influenza virus vaccines that hold the promise to provide protection against influenza epidemics and pandemics. Herein, in vivo and in vitro assays, including optimization of assay conditions to examine hemagglutinin stalk-specific antibody responses in small animal models, are described.