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Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines., Importance: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines., Competing Interests: A.M.D., S.S.-C., S.M.W., K.B.W., and T.M.R. are co-inventors on patent serial number 63/435,053 entitled “Compositions and Methods for Self-Adjuvanting AAV Vaccines.”

It is clear that new approaches are needed to promote broadly protective immunity to viral pathogens, particularly those that are prone to mutation and escape from antibody-mediated immunity. Prototypic pathogens of this type are influenza and SARS-CoV-2, where the receptor-binding protein exhibits extremely high variability in its receptor-binding regions. T cells, known to target many viral proteins, and within these, highly conserved peptide epitopes, can contribute greatly to protective immunity through multiple mechanisms but are often poorly recruited by current vaccine strategies. Here, we have studied a promising novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP), which was previously recognized for its ability to generate anti-tumor immunity through the induction of potent cytotoxic CD8 T cells. Using a preclinical mouse model, we have assessed an R-DOTAP nanoparticle adjuvant system for its ability to promote CD4 T cell responses to vaccination with recombinant influenza protein. Our studies revealed that R-DOTAP consistently outperformed a squalene-based adjuvant emulsion, even when it was introduced with a potent TLR agonist CpG, in the ability to elicit peptide epitope-specific CD4 T cells when quantified by IFN-γ and IL-2 ELISpot assays. Clinical testing of R-DOTAP containing vaccines in earlier work by others has demonstrated an acceptable safety profile. Hence, R-DOTAP can offer exciting opportunities as an immune stimulant for next-generation prophylactic recombinant protein-based vaccines.

The most effective measure to induce protection from influenza is vaccination. Thus, yearly vaccination is recommended, which, together with infections, establishes diverse repertoires of B cells, antibodies, and T cells. We examined the impact of this accumulated immunity on human responses in adults to split, subunit, and recombinant protein-based influenza vaccines. Enzyme-linked immunosorbent assay (ELISA) assays, to quantify serum antibodies, and peptide-stimulated CD4 T-cell cytokine ELISpots revealed that preexisting levels of hemagglutinin (HA)-specific antibodies were negatively associated with gains in antibody postvaccination, while preexisting levels of CD4 T cells were negatively correlated with vaccine-induced expansion of CD4 T cells. These patterns were seen independently of the vaccine formulation administered and the subjects' influenza vaccine history. Thus, although memory CD4 T cells and serum antibodies consist of components that can enhance vaccine responses, on balance, the accumulated immunity specific for influenza A H1 and H3 proteins is associated with diminished future responses., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung-localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization, and persistence of the T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 T cells isolated from airway and lung tissue produce significantly more effector cytokines IFN-γ and TNF-α, as well as cytotoxic functionality. When adoptively transferred to naive recipients, CD4 T cells from NLP:NP-immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. IMPORTANCE Exploiting new, more efficacious strategies to potentiate influenza virus-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

Despite the benefits of yearly influenza vaccination, accumulating evidence suggests that diminished vaccine efficacy may be related to repeated vaccination. Although studied at the level of B-cell responses, CD4 T-cell responses have not yet been examined. In this study, we analyze CD4 T-cell responses to influenza vaccination in subjects who differ in their vaccine history. We find a striking disparity in their responses, with previously vaccinated subjects exhibiting significantly blunted CD4 T-cell responses and diminished antibody responses. These results suggest that limiting CD4 T-cell help mteaserrlie the diminished or altered antibody responses in repeatedly vaccinated subjects., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok) and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (MAbs) and 38 Flucelvax-induced MAbs for avidity, cross-reactivity, and any selectivity toward the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of MAbs targeting epitopes near the receptor-binding domain on HA head (hemagglutinin inhibition-positive MAbs) than Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive MAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive antibody-secreting cells, showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head. IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax, that use alternate production strategies can induce protective influenza-specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations., (Copyright © 2019 American Society for Microbiology.)

Vaccination is widely used to generate protective immunity against influenza virus. CD4 + T cells contribute in diverse ways to protective immunity, most notably, in the provision of help for the production of neutralizing antibodies. Several recent reports have suggested that influenza virus infection elicits CD4 + T cells whose specificity only partially overlaps that of T cells elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza virus vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4 + T cell repertoire, we evaluated protein vaccine- versus infection-generated CD4 + T cell epitopes. Our studies revealed that all the infection-elicited CD4 + T cell epitope specificities are also elicited by protein vaccination, although the immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4 + memory T cells are recalled and boosted after infection and provide early help to accelerate hemagglutinin (HA)-specific antibody responses. The early CD4 + T cell response and HA-specific antibody production are associated with lowered viral titers during the infection challenge. Our data lend confidence to the ability of current protein-based vaccines to elicit influenza virus-specific CD4 + T cells that can potentiate protective immunity upon influenza virus infection. IMPORTANCE Most current and new influenza vaccine candidates consist of a single influenza virus protein or combinations of influenza virus proteins. For these vaccines to elicit CD4 + T cells that can be recalled after infection, the peptide epitopes should be shared between the two modes of confrontation. Recently, questions regarding the relatedness of epitope selection by influenza virus infection and protein vaccination have been raised. However, the studies reported here show that the specificity of CD4 + T cells elicited by protein-based vaccines overlaps that of T cells elicited by infection and that CD4 + T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection., (Copyright © 2019 American Society for Microbiology.)

Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Immunity to influenza is unique among pathogens, in that immune memory is established both via intermittent lung localized infections with highly variable influenza virus strains and by intramuscular vaccinations with inactivated protein-based vaccines. Studies in the past decades have suggested that the B cell responses to influenza infection and vaccination are highly biased by an individual's early history of influenza infection. This reactivity likely reflects both the competitive advantage that memory B cells have in an immune response and the relatively limited diversity of epitopes in influenza hemagglutinin that are recognized by B cells. In contrast, CD4 T cells recognize a wide array of epitopes, with specificities that are heavily influenced by the diversity of influenza antigens available, and a multiplicity of functions that are determined by both priming events and subsequent confrontations with antigens. Here, we consider the events that prime and remodel the influenza-specific CD4 T cell response in humans that have highly diverse immune histories and how the CD4 repertoire may be edited in terms of functional potential and viral epitope specificity. We discuss the consequences that imprinting and remodeling may have on the potential of different human hosts to rapidly respond with protective cellular immunity to infection. Finally, these issues are discussed in the context of future avenues of investigation and vaccine strategies.

Potentially pandemic strains of influenza pose an undeniable threat to human populations. Therefore, it is essential to develop better strategies to enhance vaccine design and predict parameters that identify susceptible humans. CD4 T cells are a central component of protective immunity to influenza, delivering direct effector function and potentiating responses of other lymphoid cells. Humans have highly diverse influenza-specific CD4 T-cell populations that vary in stimulation history, specificity, and functionality. These complexities constitute a formidable obstacle to predicting immune responses to pandemic strains of influenza and derivation of optimal vaccine strategies. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors of immunity in the CD4 T-cell compartment will aid in both predicting susceptible hosts and in development of vaccination strategies that will poise most human subjects to respond to pandemic influenza strains with protective immune responses., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

CD4 T cells play a multiplicity of roles in protective immunity to influenza. Included in these functions are help for high affinity antibody production, enhancement of CD8 T cell expansion, function and memory, acceleration of the early innate response to infection and direct cytotoxicity. The influenza-specific CD4 T cell repertoire in humans established through exposures to infection and vaccination has been found to be highly variable in abundance, specificity and functionality. Deficits in particular subsets of CD4 T cells recruited into the response result in diminished antibody responses and protection from infection. Therefore, improved strategies for vaccination should include better methods to identify deficiencies in the circulating CD4 T cell repertoire, and vaccine constructs that increase the representation of CD4 T cells of the correct specificity and functionality., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA., Competing Interests: Conflict of interest statement: S.J.Z., K.P., M.E.G., K.K., S.D.P., P.C.W., A.J.S., S.C., and S.E.H. have no conflicts of interest. J.J.T. is an advisor (nonpaid) for Protein Sciences and is on the scientific advisory board or received consulting payments for Sequiris, Medicago, Takeda, and Flugen. J.J.T.’s laboratory has also received support from Sanofi., (Copyright © 2017 the Author(s). Published by PNAS.)

Introduction: Previous priming with avian influenza vaccines results in more rapid and more robust neutralizing antibody responses upon revaccination, but the role CD4(+) T cells play in this process is not currently known., Methods: Human subjects previously enrolled in trials of inactivated influenza A(H5N1) vaccines and naive subjects were immunized with an inactivated subunit influenza A/Indonesia/5/05(H5N1) vaccine. Neutralizing antibody responses were measured by a microneutralization assay, and hemagglutinin (HA)-specific and nucleoprotein (NP)-specific CD4(+) T-cell responses were quantified using interferon γ enzyme-linked immunosorbent spot assays., Results: While vaccination induced barely detectable CD4(+) T-cell responses specific for HA in the previously unprimed group, primed subjects had readily detectable HA-specific memory CD4(+) T cells at baseline and mounted a more robust response to HA-specific epitopes after vaccination. There were no differences between groups when conserved NP-specific CD4(+) T-cell responses were examined. Interestingly, neutralizing antibody responses following revaccination were significantly higher in individuals who mounted a CD4(+) T-cell response to the H5 HA protein, a correlation not observed for NP-specific responses., Conclusions: These findings suggest that prepandemic vaccination results in an enriched population of HA-specific CD4(+) T cells that are recruited on rechallenge with a drifted vaccine variant and contribute to more robust and more rapid neutralizing antibody responses., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The ability of influenza vaccines to elicit CD4(+) T cells and the relationship between induction of CD4(+) T cells and vaccine-induced neutralizing antibody responses has been controversial. The emergence of swine-origin 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) provided a unique opportunity to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to probe the relationship between B-cell and T-cell responses to vaccination., Methods: We tracked CD4(+) T-cell and antibody responses of human subjects vaccinated with monovalent subunit A(H1N1)pdm09 vaccine. The specificity and magnitude of the CD4(+) T-cell response was evaluated using cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influenza virus proteins., Results: Our studies revealed that vaccination induced readily detectable CD4(+) T cells specific for conserved portions of hemagglutinin (HA) and the internal viral proteins. Interestingly, expansion of HA-specific CD4(+) T cells was most tightly correlated with the antibody response., Conclusions: These results indicate that CD4(+) T-cell expansion may be a limiting factor in development of neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate CD4(+) T-cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza strains.

There are a number of related goals of influenza vaccination, including elicitation of protective antibodies and induction of cellular CD4 and CD8+ T cell responses. Because CD4+ T cell expansion and functionality are influenced by peptide specificity and T cell gene expression can be modified by repeated re-stimulations, it is important to evaluate how frequent influenza vaccinations affect CD4+ T cell dependent functions in protective immunity to influenza. Trivalent influenza vaccines (TIV) have production of neutralizing antibodies to HA as their primary goal and main criteria for efficacy. Accordingly, they are not characterized for any other viral components. In the current study, we evaluated whether other influenza virus proteins were present in commercial TIV at levels sufficient for immunogenicity in vivo. Mice that differed with regard to their expressed class II molecules were used in concert with peptide-stimulated cytokine ELISPOT assays to comprehensively evaluate the CD4+ T cell antigen specificity induced by the TIV. Our studies revealed that NA, NP, M1 and NS1 were present in sufficient quantities in the TIV to prime and boost CD4+ T cells. These results suggest that in humans, the broad CD4+ T cell repertoire induced by live infection is continually boosted and maintained throughout life by regular vaccination with licensed intramuscular split vaccines. The implications raised by our findings on CD4+ T cell functionality in influenza are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the current study, we demonstrate that the pattern of secretion of IL-2, IFN-γ, and IL-4 by CD4 T cells activated by NC infection is largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The response in B10 mice featured predominantly IFN-γ-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8 generated a response that was comparable to that in B10 mice, with predominantly IFN-γ-secreting CD4 T cells and greater numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.

The unexpected emergence of pandemic H1N1 influenza has generated significant interest in understanding immunological memory to influenza and how previous encounters with seasonal strains influence our ability to respond to novel strains. In this study, we evaluate the memory T cell repertoire in healthy adults to determine the abundance and protein specificity of influenza-reactive CD4 T cells, using an unbiased and empirical approach, and assess the ability of CD4 T cells to recognize epitopes naturally generated by infection with pandemic H1N1 virus. Our studies revealed that most individuals have abundant circulating CD4 T cells that recognize influenza-encoded proteins and that a strikingly large number of CD4 T cells can recognize autologous cells infected with live H1N1 virus. Collectively, our results indicate that a significant fraction of CD4 T cells generated from priming with seasonal virus and vaccines can be immediately mobilized upon infection with pandemic influenza strains derived from antigenic shift.

CD4 T cells play a primary role in regulating immune responses to pathogenic organisms and to vaccines. Antigen-specific CD4 T cells provide cognate help to B cells, a requisite event for immunoglobulin switch and affinity maturation of B cells that produce neutralizing antibodies and also provide help to cytotoxic CD8 T cells, critical for their expansion and persistence as memory cells. Finally, CD4 T cells may participate directly in pathogen clearance via cell-mediated cytotoxicity or through production of cytokines. Understanding the role of CD4 T-cell immunity to viruses and other pathogens, as well as evaluation of the efficacy of vaccines, requires insight into the specificity of CD4 T cells. This review focuses on the events within antigen-presenting cells that focus CD4 T cells toward a limited number of peptide antigens within the pathogen or vaccine. The molecular events are discussed in light of the special challenges that the influenza virus poses, owing to the high degree of genetic variability, unpredictable pathogenicity and the repeated encounters that human populations face with this highly infectious pathogenic organism.

Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells. Influenza vaccines: Multiple viral proteins in licensed vaccines Licensed influenza virus vaccines are evaluated for their ability to elicit neutralizing antibodies specific for hemagglutinin (HA), but the manufacturing process does not completely exclude other virion components from the formulations. Andrea Sant and colleagues now report the presence of several viral proteins, such as M1, NA, H3, and HA-B, in licensed formulations from different manufacturers and spanning stocks from several years. These viral proteins form heterologous complexes, and immunization of mice with some of the formulations analyzed elicited antibody responses specific to these viral proteins. These findings reveal heterogeneity across licensed influenza virus vaccine formulations, potentially due to variations in production processes, and raise the possibility that the presence of these additional viral protein complexes could influence the elicited immune responses following immunization, particularly in the context of multivalent strategies involving mixing of different formulations. [ABSTRACT FROM AUTHOR]

Vaccination is widely used to generate protective immunity against influenza virus. CD4+ T cells contribute in diverse ways to protective immunity, most notably, in the provision of help for the production of neutralizing antibodies. Several recent reports have suggested that influenza virus infection elicits CD4+ T cells whose specificity only partially overlaps that of T cells elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza virus vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4+ T cell repertoire, we evaluated protein vaccine-versus infection-generated CD4+ T cell epitopes. Our studies revealed that all the infection-elicited CD4+ T cell epitope specificities are also elicited by protein vaccination, although the immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4+ memory T cells are recalled and boosted after infection and provide early help to accelerate hemagglutinin (HA)-specific antibody responses. The early CD4+ T cell response and HA-specific antibody production are associated with lowered viral titers during the infection challenge. Our data lend confidence to the ability of current protein-based vaccines to elicit influenza virus-specific CD4+ T cells that can potentiate protective immunity upon influenza virus infection. [ABSTRACT FROM AUTHOR]