To gain insight into how an adjuvant impacts vaccination responses, we use systems immunology to study human H5N1 influenza vaccination with or without the adjuvant AS03, longitudinally assessing 14 time points including multiple time points within the first day after prime and boost. We develop an unsupervised computational framework to discover high-dimensional response patterns, which uncover adjuvant- and immunogenicity-associated early response dynamics, including some that differ post prime versus boost. With or without adjuvant, some vaccine-induced transcriptional patterns persist to at least 100 days after initial vaccination. Single-cell profiling of surface proteins, transcriptomes, and chromatin accessibility implicates transcription factors in the erythroblast-transformation-specific (ETS) family as shaping these long-lasting signatures, primarily in classical monocytes but also in CD8+ naive-like T cells. These cell-type-specific signatures are elevated at baseline in high-antibody responders in an independent vaccination cohort, suggesting that antigen-agnostic baseline immune states can be modulated by vaccine antigens alone to enhance future responses.

The emergence of highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses and their transmission to dairy cattle and animals, including humans, poses a major global public health threat. Therefore, the development of effective vaccines and therapeutics against H5N1 clade 2.3.4.4b virus is considered a public health priority. In the United States, three H5N1 vaccines derived from earlier strains of HPAI H5N1 (A/Vietnam, clade 1, and A/Indonesia, clade 2.1) virus, with (MF59 or AS03) or without adjuvants, are licensed and stockpiled for pre-pandemic preparedness, but whether they can elicit neutralizing antibodies against circulating H5N1 clade 2.3.4.4b viruses is unknown. In this study, we evaluated the binding, hemagglutination inhibition and neutralizing antibody response generated after vaccination of adults with the three licensed vaccines. Individuals vaccinated with the two adjuvanted licensed H5N1 vaccines generated cross-reactive binding and cross-neutralizing antibodies against the HPAI clade 2.3.4.4b A/Astrakhan/3212/2020 virus. Seroconversion rates of 60-95% against H5 clade 2.3.4.4b were observed after two doses of AS03-adjuvanted-A/Indonesia or three doses of MF59-adjuvanted-A/Vietnam vaccine. These findings suggest that the stockpiled US-licensed adjuvanted H5N1 vaccines generate cross-neutralizing antibodies against circulating HPAI H5N1 clade 2.3.4.4b in humans and may be useful as bridging vaccines until updated H5N1 vaccines become available. Avian influenza vaccines in the US strategic stockpile elicit immune responses that recognize the clade of avian influenza circulating in cows and could be a resource before newer vaccines are approved and deployed.

Viral infections can have profound and durable functional impacts on the immune system. There is an urgent need to characterize the long-term immune effects of SARS-CoV-2 infection given the persistence of symptoms in some individuals and the continued threat of novel variants. Here we use systems immunology, including longitudinal multimodal single cell analysis (surface proteins, transcriptome, and V(D)J sequences) from 33 previously healthy individuals after recovery from mild, non-hospitalized COVID-19 and 40 age- and sex-matched healthy controls with no history of COVID-19 to comparatively assess the post-infection immune status (mean: 151 days after diagnosis) and subsequent innate and adaptive responses to seasonal influenza vaccination. Identification of both sex-specific and -independent temporally stable changes, including signatures of T-cell activation and repression of innate defense/immune receptor genes (e.g., Toll-like receptors) in monocytes, suggest that mild COVID-19 can establish new post-recovery immunological set-points. COVID-19-recovered males had higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy males and COVID-19-recovered females, partly attributable to elevated pre-vaccination frequencies of a GPR56 expressing CD8+ T-cell subset in male recoverees that are "poised" to produce higher levels of IFNγ upon inflammatory stimulation. Intriguingly, by day 1 post-vaccination in COVID-19-recovered subjects, the expression of the repressed genes in monocytes increased and moved towards the pre-vaccination baseline of healthy controls, suggesting that the acute inflammation induced by vaccination could partly reset the immune states established by mild COVID-19. Our study reveals sex-dimorphic immune imprints and in vivo functional impacts of mild COVID-19 in humans, suggesting that prior COVID-19, and possibly respiratory viral infections in general, could change future responses to vaccination and in turn, vaccines could help reset the immune system after COVID-19, both in an antigen-agnostic manner.

Myelodysplastic syndromes (MDS) are diseases of bone marrow failure that result in dysplasia of multiple hematopoietic lineages producing anemia, thrombocytopenia, and increased risk of infections. While progression to acute myeloid leukemia (AML) is a phenomenon that receives most of the focus in managing this disease, prior studies have shown that infection is the leading cause of mortality. This is true across all MDS risk groups and highlights the critical importance of research into the clinical management and prevention of infection in patients with MDS, especially in the age of emergent pandemics such as COVID-19. We have previously observed that patients with MDS exhibit decreased responses to vaccination against SARS-CoV-2 (Bellusci, et al., Blood, 2022). There remains a gap in our knowledge regarding the success of vaccination against common seasonal pathogens in patients with MDS, such as influenza. Although patients with MDS and AML are routinely offered yearly influenza vaccination during the flu season, the efficacy of vaccination in producing immunity in the context of the underlying disease biology and the impact of therapies used for treatment of the disease (azacitidine & decitabine (HMAs), venetoclax + HMA and cytarabine based intensive chemotherapy) remains understudied. We hypothesized that MDS/AML patients would exhibit a decreased response to vaccination against seasonal influenza compared to healthy age-matched controls. To test this hypothesis, we enrolled patients in an IRB-approved non-randomized study (I-54017) that spanned the 2017-2018 to 2020-2021 influenza seasons. This study examined 5 patient groups: Cohort 1: Patients with MDS on best supportive care (ESAs, growth factors, transfusions, antibiotics; no disease modifying therapy; n = 28); Cohort 2: Patients with MDS or AML early in HMA therapy (≤5 cycles; n = 26); Cohort 3: Patients with MDS or AML on longer term HMA (> 6 cycles; n = 28); Cohort 4: Patients with AML receiving cytarabine based intensive chemotherapy ("7+3", HIDAC, CLAG+/-M, FLAG+/-Ida, etc.). Patients in this arm should not have received an HMA in the 6 months prior to enrollment (n = 10); Cohort 5: Healthy volunteers of similar age profile without antecedent or concurrent hematologic disorders (n = 12). All subjects received the yearly Fluzone High-Dose vaccine (Sanofi). Serum samples were collected at 1) baseline; 2) days 25-95 after vaccination; and 3) days 115-185 after vaccination. Viral titers were measured using the viral microneutralization assay against seasonal influenza vaccine strains based upon the yearly vaccine product. Positive responses to vaccination were defined as a 4-fold increase in titer compared to baseline. For each subject, we calculated a strain score (range 0-1) based on the fraction of 4-fold changes across either 3 to 4 influenza strains depending upon the yearly vaccination product; a score of 1 indicates a 4-fold increase in titers against all tested strains. Strain score was modeled as a function of study year, disease cohort, and age; blood-type, gender, race, and IPSS-M were analyzed but were not statistically significant. The primary analysis was a generalized linear model (GLM). In concert with prior reports, we found that compared to healthy donors, AML patients receiving intensive chemotherapy showed decreased vaccine response (Fig.1). In contrast, those with MDS responded equivalently to vaccination compared with healthy donors. Overall, these results demonstrate that in contrast with vaccination against newly emergent diseases (COVID-19) against which MDS patients appear to have inferior immune response, anamnestic responses to vaccination against previously encountered pathogens (influenza) are normal. As reported previously, patients with AML receiving intensive chemotherapy exhibit reduced protection from vaccination against both emergent pathogens (COVID-19) and amnestic vaccines (influenza). These results indicate that vaccination boosters, such as those given yearly against influenza, against previously encountered pathogens are likely to provide protection in MDS patients irrespective of standard therapy, but the efficacy of vaccination against novel pathogens may be less effective. These data highlight the complex immune milieu in patients with MDS and emphasize the importance of careful individualized vaccination strategies in those with hematological malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Several vaccines are approved in the United States for seasonal influenza vaccination every year. Here we compare the impact of repeat influenza vaccination on hemagglutination inhibition (HI) titers, antibody binding and affinity maturation to individual hemagglutinin (HA) domains, HA1 and HA2, across vaccine platforms. Fold change in HI and antibody binding to HA1 trends higher for H1N1pdm09 and H3N2 but not against B strains in groups vaccinated with FluBlok compared with FluCelvax and Fluzone. Antibody-affinity maturation occurs against HA1 domain of H1N1pdm09, H3N2 and B following vaccination with all vaccine platforms, but not against H1N1pdm09-HA2. Importantly, prior year vaccination of subjects receiving repeat vaccinations demonstrated reduced antibody-affinity maturation to HA1 of all three influenza virus strains irrespective of the vaccine platform. This study identifies an important impact of repeat vaccination on antibody-affinity maturation following vaccination, which may contribute to lower vaccine effectiveness of seasonal influenza vaccines in humans.

Background: Patients with myelodysplastic syndromes (MDS) present across a clinical spectrum from mild disease to profound bone marrow failure and transformation to acute myeloid leukemia (AML). Those with lower risk disease are generally managed with watchful waiting and best supportive care (growth factors, blood and platelet transfusions and iron chelation; BSC), while those with higher risk disease are treated with repeated cycles of low dose "hypomethylating" chemotherapy (such as azacitidine or decitabine; HMA). A majority of patients with MDS, even those with lower risk disease, are likely to die of complications related to their diagnosis, mostly infections and bleeding but also leukemic transformation. As part of our standard approach to infection prevention, current clinical guidelines suggest annual vaccination against influenza. Patients with these disorders and their family members are advised to receive inactivated protein based vaccines rather than live vaccination approaches to limit infection risk. Most will receive high dose trivalent vaccination due to age. Despite these recommendations, limited data exist on the ability of patients with MDS across the spectrum of risk groups to respond to standard seasonal influenza vaccination. In light of the growing literature suggesting that patients with MDS have an altered immune environment, we hypothesized that they would show inferior response to standard vaccination. We sought to determine the response to influenza vaccination in patients with MDS receiving standard therapeutic management. Methods: A non-randomized study is currently ongoing at the Roswell Park Comprehensive Cancer Center for patients with MDS. Age-relevant family members are enrolled as a comparator population for vaccine response. Cohorts were stratified into 3 groups: healthy volunteers (Cohort 1), MDS patients receiving BSC (Cohort 2) and MDS patients actively receiving HMA (Cohort 3; Table 1). All participants are administered the yearly preparation of Sanofi Pasteur's Fluzone High-Dose Vaccine (containing trivalent inactivated strains: Influenza virus A (H1N1 and H3N2) and Influenza virus B). Baseline blood samples were collected prior to vaccination (day 0), and between days 25-90 and 115-185 post-vaccination. Serological responses to vaccination were determined by viral-neutralizing activity analyzed via microneutralization assay. Neutralizing antibody titers for the first year of the study were measured against seasonal influenza vaccine strains based upon the 2017-2018 vaccine product. Samples from the 2018-19 flu season are being analyzed. Results: To date 56 individuals have been recruited to the study over 2 years. Neutralizing antibody titers following vaccination are available for 20 individuals vaccinated in the 2017-18 flu season. Humoral immune responses to vaccination against different strains of Influenza virus A (H1N1 and H3N2) and Influenza virus B were observed across all cohorts (Figure 1). Response was deemed adequate if the titer for any vaccine component increased by >4 fold comparing the baseline to the day 25-90 time point. Cohort 1: 4/4 responded (100%); cohort 2: 4/4 responded (100%); cohort 3: 11/12 responded (92%). To better understand the effect of standard treatment for MDS on influenza vaccine response we are currently profiling immune cells pre and post vaccination using multi-parameter flow cytometry. Additional analyses are planned based on the number of HMA cycles received (<6, or ≥6) and cycle timing relative to vaccination. Conclusion: Patients with MDS respond to vaccination with Fluzone High Dose. Responses in patients with MDS were not statistically different from those seen in an age-relevant population of healthy family members. Additional individuals are being enrolled in order to assess whether standard HMA therapy impacts the response to influenza vaccination. These data suggest that MDS patients receiving BSC respond adequately to viral vaccination. Our preliminary data also show that patients receiving HMA therapy respond adequately to influenza vaccination. These data support the value of influenza vaccination in all patients with MDS and highlight the potential for anti-MDS immunotherapeutic vaccination strategies. Disclosures Vachhani: Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Przespolewski:Jazz Pharmaceuticals: Other: PI on clinical trial. Thota:Incyte, Inc.: Speakers Bureau. Wang:Amgen: Other: Advisory role; Agios: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Jazz: Other: Advisory role; Abbvie: Other: Advisory role; Kite: Other: Advisory role. Griffiths:Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Abbvie, Inc.: Consultancy; Boston Scientific: Consultancy; Novartis Inc.: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; New Link Genetics: Consultancy; Genentech, Inc.: Research Funding; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Abbvie, Inc.: Consultancy, PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Persimmune: Consultancy; Partner Therapeutics: Consultancy; Persimmune: Consultancy; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; Celgene, Inc: Consultancy, Research Funding; Appelis Pharmaceuticals: Other: PI on a clinical trial; Partner Therapeutics: Consultancy; New Link Genetics: Consultancy.

Immune responses to inactivated vaccines against avian influenza are poor due in part to lack of immune memory. Adjuvants significantly increased virus neutralizing titers. We performed comprehensive analyses of polyclonal antibody responses following FDA-approved adjuvanted H5N1-A/Indonesia vaccine, administered in presence or absence of AS03. Using Whole Genome Fragment Phage Display Libraries, we observed that AS03 induced antibody epitope diversity to viral hemagglutinin (HA) and neuraminidase compared with unadjuvanted vaccine. Furthermore, AS03 promoted significant antibody affinity maturation to properly folded H5-HA1 (but not to HA2) domain, which correlated with neutralization titers against both vaccine and heterologous H5N1 strains. However, no increase in heterosubtypic cross-neutralization of Group1-H1N1 seasonal strains was observed. AS03-H5N1 vaccine also induced higher neuraminidase inhibition antibody titers. This study provides insight into the differential impacts of AS03 adjuvant on H5N1 vaccine-induced antibody responses that may help optimize vaccine platforms for future vaccines with improved protection against seasonal and pandemic influenza strains.

The H7N9 influenza virus causes high-mortality disease in humans but no effective therapeutics are available. Here we report a human monoclonal antibody, m826, that binds to H7 hemagglutinin (HA) and protects against H7N9 infection. m826 binds to H7N9 HA with subnanomolar affinity at acidic pH and 10-fold lower affinity at neutral pH. The high-resolution (1.9 Å) crystal structure of m826 complexed with H7N9 HA indicates that m826 binds an epitope that may be fully exposed upon pH-induced conformational changes in HA. m826 fully protects mice against lethal challenge with H7N9 virus through mechanisms likely involving antibody-dependent cell-mediated cytotoxicity. Interestingly, immunogenetic analysis indicates that m826 is a germline antibody, and m826-like sequences can be identified in H7N9-infected patients, healthy adults, and newborn babies. These m826 properties offer a template for H7N9 vaccine immunogens, a promising candidate therapeutic, and a tool for exploring mechanisms of virus infection inhibition by antibodies.

Background Changes in adaptive immune cells after chemotherapy in adult acute myeloid leukemia (AML) may have implications for the success of immunotherapy. This study was designed to determine the functional capacity of the immune system in adult patients with AML who have completed chemotherapy and are potential candidates for immunotherapy. Methods We used the response to seasonal influenza vaccination as a surrogate for the robustness of the immune system in 10 AML patients in a complete remission post-chemotherapy and performed genetic, phenotypic, and functional characterization of adaptive immune cell subsets. Results Only 2 patients generated protective titers in response to vaccination, and a majority of patients had abnormal frequencies of transitional and memory B-cells. B-cell receptor sequencing showed a B-cell repertoire with little evidence of somatic hypermutation in most patients. Conversely, frequencies of T-cell populations were similar to those seen in healthy controls, and cytotoxic T-cells demonstrated antigen-specific activity after vaccination. Effector T-cells had increased PD-1 expression in AML patients least removed from chemotherapy. Conclusion Our results suggest that while some aspects of cellular immunity recover quickly, humoral immunity is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. The observed B-cell abnormalities may explain the poor response to vaccination often seen in AML patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immunity and increased PD-1 expression shortly after chemotherapy might have implications for the success of several modalities of immunotherapy.

Objective: The purpose of this study was to assess the potential immunosuppressive role of daclizumab, a humanized monoclonal antibody against the α chain of the interleukin 2 receptor, in vivo, by comparing immune responses to the 2013 seasonal influenza vaccination between patients with multiple sclerosis (MS) on long-term daclizumab therapy and controls. Methods: Previously defined subpopulations of adaptive immune cells known to correlate with the immune response to the influenza vaccination were evaluated by 12-color flow cytometry in 23 daclizumab-treated patients with MS and 14 MS or healthy controls before (D0) and 1 day (D1) and 7 days (D7) after administration of the 2013 Afluria vaccine. Neutralizing antibody titers and CD4+, CD8+ T cell, B cell, and natural killer cell proliferation to 3 strains of virus contained in the Afluria vaccine were assessed at D0, D7, and 180 days postvaccination. Results: Daclizumab-treated patients and controls demonstrated comparable, statistically significant expansions of previously defined subpopulations of activated CD8+ T cells and B cells that characterize the development of effective immune responses to the influenza vaccine, while proliferation of T cells to influenza and control antigens was diminished in the daclizumab cohort. All participants fulfilled FDA criteria for seroconversion or seroprotection in antibody assays. Conclusion: Despite the mild immunosuppressive effects of daclizumab in vivo demonstrated by an increased incidence of infectious complications in clinical trials, patients with MS under daclizumab therapy mount normal antibody responses to influenza vaccinations.

A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

In a previously reported phase I clinical trial, subjects vaccinated with two doses of an unadjuvanted H7N9 virus like particle (VLP) vaccine responded poorly (15.6% seroconversion rates with 45μg hemagglutinin (HA) dose). In contrast, 80.6% of subjects receiving H7N9 VLP vaccine (5μg HA) with ISCOMATRIX™ adjuvant developed hemagglutination-inhibition (HI) responses. To better understand the role of adjuvant, complete antibody epitope repertoires of post-vaccination sera were investigated using Whole Genome Fragment Phage Display Library (GFPDL). In addition, antibody affinity maturation following vaccination was measured against HA1 and HA2 antigenic domains using real time Surface Plasmon Resonance (SPR) based kinetic assays. Unadjuvanted H7N9-VLP vaccine generated primarily antibodies targeting the C-terminus of the HA1 domain, predicted to be mostly buried on the native HA spikes, while adjuvanted VLP vaccine generated antibodies against large epitopes in the HA1 spanning the receptor binding domain (RBD). SPR analysis using a functional H7-HA1 domain demonstrated that sera from adjuvanted H7N9-VLP vaccine induced higher total binding antibodies and significantly higher antibody affinity maturation to HA1 compared to sera from unadjuvanted vaccine. Total antibody binding and affinity to the HA1 (but not HA2) domain correlated with HI and neutralization titers. This study demonstrates that ISCOMATRIX™ adjuvanted vaccine promotes higher quality antibody immune response against avian influenza in naïve humans.

OBJECTIVE: The purpose if this study was to assess immunosuppressive role of daclizumab in-vivo, by comparing immune responses to 2013 seasonal influenza vaccination between MS patients on long-term daclizumab (DAC) therapy and control subjects. BACKGROUND: Daclizumab, a humanized monoclonal antibody against the α-chain of the IL-2 receptor, has a unique mechanism of action with multiple effects on innate immunity, including T cell activation by dendritic cells (DCs). DESIGN/METHODS: Previously-defined subpopulations of adaptive immune cells, known to correlate with the immune response to influenza vaccination, were evaluated by 12 color flow cytometry in 22 DAC MS patients and 13 untreated MS or healthy subjects (controls) before (Day 0), one day after (Day 1), and seven days after (Day 7) administration of the 2013 Afluria vaccine. Ab titers to all 3 strains of virus contained in Afluria vaccine were assessed at Day 0, Day 7 and Day 180. RESULTS: Both DAC patients and controls demonstrated comparable, statistically-significant expansions of previously-defined subpopulations of activated CD4+ and CD8+ T cells and B cells that characterize development of effective immune responses to influenza vaccine. Correspondingly, all subjects fulfilled FDA definitions for seroconversion or seroprotection in antibody assays. CONCLUSIONS: Although daclizumab has some immunosuppressive effects in-vivo, as demonstrated by increased incidence of infections complications in Phase III trials, MS patients under DAC therapy mount normal immune responses to influenza vaccinations. Because most of the studied subjects had pre-existing immunity to the A/H1N1, A/H3N2 and B strains contained in 2013 seasonal influenza vaccine, we conclude that daclizumab does not affect activation of memory immune responses to strong environmental antigens. The effect of daclizumab on mounting naïve immune responses to environmental antigens are currently being investigated in pneumococcal vaccination study.

A WHO workshop organized following the 2009 H1N1 pandemic recommended development of alternative influenza vaccine potency assays as high priority that could expedite the release of vaccine lots in the face of future influenza pandemics. We have developed an antibody independent, simple, high throughput receptor-binding SPR-based potency assay, which does not require any reference antisera and could be used for rapid HA quantitation and vaccine release in pandemic scenarios. The assay utilizes synthetic glycans with sialic acid (SA) of either α-2,6 or α-2,3 linkage to galactose. Only functionally active forms of HA (trimers and oligomers) recognize the SA-glycans and are quantified in this receptor-binding SPR assay. The SA-glycan SPR assay demonstrated broad dynamic range for quantitation of HA content in influenza vaccines from different manufacturers for both seasonal (A/H1N1, A/H3N2, B lineages) and pandemic influenza (A/H5N1, A/H7N9) strains with high reproducibility and low variability across multiple assays. In addition, the SA-glycan SPR assay is indicative of active HA stability, and can accurately quantify HA content in alum and oil-in-water adjuvanted influenza vaccines. Importantly, there was a good agreement between HA content determined by the SPR-based potency assay and the traditional SRID assay.

Initiation of mass vaccination is critical in response to influenza pandemic. There is an urgent need of a simple, rapid method for production of influenza vaccine that is more effective than current traditional influenza vaccines. Recent H7N9 transmissions to humans in China with high morbidity/mortality initiated extensive vaccine evaluation. We produced the HA1 domains (amino acids 1-320) from H7N9 and H7N7 strains in E. coli. Both were found to contain primarily monomers/trimers with low oligomeric content. However, when residues from the N-terminal β sheet (first 8 amino acid) of H7 HA1 domains were swapped with the corresponding amino acids from H5N1, functional oligomeric H7 HA1 were produced (HA1-DS), demonstrating strong receptor binding and hemagglutination. In rabbits, the HA1-DS from either H7N9 or H7N7 generated high neutralization titers against both homologous and heterologous H7 strains, superior to the unmodified H7 HA1 proteins. In ferrets, HA1-DS from H7N7 elicited higher (and faster) HI titers, better protected ferrets from lethality, weight loss, and reduced viral loads following challenge with wild-type highly pathogenic H7N7 virus compared with inactivated H7N7 subunit vaccine. HA1-DS vaccinated ferrets were also better protected from weight loss after challenge with the heterologous H7N9 virus compared with inactivated H7N7 subunit vaccine. Importantly, the H7N7 HA1-DS vaccine induced antibody affinity maturation far superior to the inactivated H7N7 subunit vaccine, which strongly correlated with control of viral loads in the nasal washes after challenge with either H7N7 or H7N9 strains. We conclude that N-terminus β sheet domain-swap can be used to produce stable functional oligomeric forms of better recombinant HA1 vaccines in simple, inexpensive bacterial system for rapid response to emerging pandemic threat for the global population.

BACKGROUNDHighly pathogenic avian influenza A(H5N1) causes severe infections in humans. We generated 2 influenza A(H5N1) live attenuated influenza vaccines for pandemic use (pLAIVs), but they failed to elicit a primary immune response. Our objective was to determine whether the vaccines primed or established long-lasting immunity that could be detected by administration of inactivated subvirion influenza A(H5N1) vaccine (ISIV).METHODSThe following groups were invited to participate in the study: persons who previously received influenza A(H5N1) pLAIV; persons who previously received an irrelevant influenza A(H7N3) pLAIV; and community members who were naive to influenza A(H5N1) and LAIV. LAIV-experienced subjects received a single 45-μg dose of influenza A(H5N1) ISIV. Influenza A(H5N1)- and LAIV-naive subjects received either 1 or 2 doses of ISIV.RESULTSIn subjects who had previously received antigenically matched influenza A(H5N1) pLAIV followed by 1 dose of ISIV compared with those who were naive to influenza A(H5N1) and LAIV and received 2 doses of ISIV, we observed an increased frequency of antibody response (82% vs 50%, by the hemagglutination inhibition assay) and a significantly higher antibody titer (112 vs 76; P = .04). The affinity of antibody and breadth of cross-clade neutralization was also enhanced in influenza A(H5N1) pLAIV-primed subjects.CONCLUSIONSISIV administration unmasked long-lasting immunity in influenza A(H5N1) pLAIV recipients, with a rapid, high-titer, high-quality antibody response that was broadly cross-reactive across several influenza A(H5N1) clades.CLINICAL TRIALS REGISTRATIONNCT01109329.

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu(-)/nu(-)) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads. Treatment for 2 to 5 days of normal BALB/c mice with ST-246 at 100 mg/kg starting 24 h postchallenge conferred 100% protection and reduced viral loads in four organs compared to control mice. Mice also survived after 5 days of treatment with ST-246 at 30 mg/kg, and yet the viral loads and poxes were higher in these mice compared to 100-mg/kg treatment group. Nude mice were not protected by ST-246 alone or by 10 million adoptively transferred T cells. In contrast, nude mice that received T cells and 7-day treatment with ST-246 survived infection and exhibited reduced viral loads compared to nonreconstituted and ST-246-treated mice after ST-246 was stopped. Similar protection of nude mice was achieved using adoptively transferred 1.0 and 0.1 million, but not 0.01 million, purified T cells or CD4(+) or CD8(+) T cells in conjunction with ST-246 treatment. These data suggest that ST-246 protects immunocompetent mice from lethality and reduces viral dissemination in internal organs and poxvirus lesions. Furthermore, immune-deficient animals with partial T cell reconstitution can control virus replication after a course of ST-246 and survive lethal vaccinia virus challenge.

DNA priming improves the response to inactivated influenza A(H5N1) vaccination. We compared the immunogenicity of an H5 DNA prime (using strain A/Indonesia/5/2005) followed by an H5N1 monovalent inactivated vaccine boost at 4, 8, 12, 16, or 24 weeks to that of 2 doses of H5N1 monovalent inactivated vaccine in adults. Antibody epitope repertoires were elucidated by genome-fragment phage-display library analysis, and antibody avidities for HA1 and HA2 domains were measured by surface plasmon resonance. H5 DNA priming expanded the H5-specific antibody epitope repertoire and enhanced antibody avidity to the HA1 (but not the HA2) domain in an interval-dependent manner. Enhanced HA1 binding and avidity after an interval of ≥12 weeks between prime and boost correlated with improved neutralization of homologous and heterologous H5N1 strains. Clinical trials registration NCT01086657.

Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.