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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.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2018., Influenza A viruses continue to pose a threat to human health worldwide and virusspecific CD4 T cells are key to protective immunity. The goal of the experiments within this thesis was to provide novel insight into elements within CD4 T cell responses that influence CD4 T cell specificity and function. Using a combination of novel reporter viruses, intravascular labeling in vivo, multi-parameter flow cytometry, and cytokine ELISpot procedures in two mammalian models, this work examined various stages of the developing immune response and the relationship between antigen acquisition, CD4 T cell fate choice, trafficking and compartmentalization within the inflamed lung, antigen specificity, and cytokine functionality. We found that pandemic 2009 H1N1 virus infection resulted in a broad pattern of viral antigen acquisition in the lung. Complex subpopulations of MHC class II-positive cells from both the hematopoietic and non-hematopoietic lineages were identified, with dynamic shifts over time. Our results demonstrated that antigen access occurs by uptake of exogenous antigen as well as infection. Finally, our results confirmed that many different cell types displayed peptide:MHC class II complexes in situ and possessed the capacity to stimulate influenza-specific CD4 T cells. These results correlated with differences in infection status, cell lineage, and MHC class II expression. These data suggest that there are many different types of antigen presenting cells in the lung that may impose additional checkpoints of CD4 T cell functionality and regulation. At the peak of the immune response, we found that lung CD4 T cells compartmentalized to vasculature and tissue. Antigen-experienced CD4 T cells were enriched in the tissue and expressed higher levels of molecules involved in trafficking to sites of pulmonary infection, damage, and repair. Surprisingly, we found similar representation in the fine specificity, immunodominance hierarchy, and polyfunctional potential of ef

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.

Effect of selectively priming HA CD4 T cells, A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016., We have sought to understand the role that epitope specificity in the CD4 T cell repertoire has on the functional outcome of both the CD4 T cell response and ultimately the B cell response to influenza infection. Protection against influenza infection, provided by immune memory elicited from either infection or vaccination, is primarily mediated by neutralizing antibody. The humoral response is in turn dependent on the follicular helper T cell (Tfh) response for the provision of cognate T cell help in the germinal center reaction. Accordingly, we envision several scenarios where T cell specificity may be affecting the immune response, including the selection of which B cells receive help, and which CD4 T cells become Tfh. To this end, this thesis work has taken a twopronged approach to elucidate the effects of specificity in shaping the adaptive immune response. First, we employed a strategy of peptide priming to establish memory CD4 T cells so that we could directly query the effects of T cell specificity on the primary B cell response to influenza infection. We discovered that only T cells of matched protein specificity were capable of helping antigen-specific B cells. Nucleoprotein-specific CD4 T cell memory was uniquely positioned to provide help to NP-specific B cells, but were unable to boost the HA-specific B cell response, indicating that T cell help was both limiting and selective for the primary B cell response. The opposite was true of HA specific CD4 cells. This work underscored the importance of CD4 T cell memory in shaping novel B cell responses to infection. To understand the role of specificity in selecting CD4 T cells into a pathway of Tfh differentiation, we studied the repertoire of Tfh and NonTfh cells generated after influenza infection. Surprisingly, we delineated distinct epitopes in two different influenza proteins that were more proficient at driving a Tfh-biased effector response. For many epitopes, these preferences persisted in responses

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2013., CD4 T cells are a key subset of leukocytes that help orchestrate adaptive immunity. Several lineages comprise the CD4 T cell compartment, each with unique abilities to coordinate immune responses. Our investigations have focused on one particular lineage of CD4 T cells that are specially programmed to support the B cell immune response, T follicular helper (Tfh) cells. Upon immunological challenge, Tfh cells help B cells to initiate and maintain germinal center reactions within B cell follicles of the lymph node. The result of the germinal center reaction is the generation of memory B cells and long-lived antibody secreting plasma cells that produce class-switched, highaffinity antibodies. Titers of high-affinity antibodies are the most common indication of human vaccine efficacy. Therefore, understanding the processes and cells that contribute to their generation are of great importance to human health. To generate Tfh cells, naïve CD4 T cells undergo an initial differentiation step through antigen-specific interactions with dendritic cells (DC) that poise CD4 T cells for secondary interactions with B cells. These secondary antigen-specific interactions with B cells are required for CD4 T cells to fully initiate and maintain the Tfh program. Since DC and B cells are known to differ in the way they acquire, process and present antigen, we were intrigued by the possibility that these distinct types of antigen presenting cells would present different repertoires of peptide-MHC class II complexes that would be reflected in their immunodominance hierarchy. Herein, we develop methods to purify Tfh cells from mouse and human tissues and blood. Using several different methods, we have evaluated the distribution of the hierarchy of antigen-specific responses of Tfh and conventional T cells generated from the endogenous CD4 T repertoire of the host. In our mouse model of protein immunization, peptide-specific immunodominance hierarchies were similar between Tfh and conventio

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2009., Establishment of an immune response that is focused on a limited number of peptide determinants expressed by a complex antigen or pathogen is a phenomenon known as immunodominance. For CD4 T cells, many of the mechanisms used to explain this selectivity suggest that events related to antigen processing play a major role in determining a peptide’s ability to recruit CD4 T cells. However, more recent data have revealed that an intrinsic biochemical property of the peptide – the kinetic stability of the peptide:MHC class II complex – both predicts and controls CD4 T cell immunodominance. At the initiation of these studies, we hypothesized that peptide stability from MHC class II is the principle factor in determining the immunogenicity of a given peptide. Those peptides that have a very stable interaction with its presenting MHC class II molecule will not only have an advantage during the processing of antigen but also in the events after peptide presentation at the surface of the antigen-bearing cell. Thus selecting for CD4 T cells that recognize only very persistent peptide:class II complexes for the final specificity of the CD4 T cell repertoire after antigen challenge. The results herein show that the immunodominant heirarchy of peptides contained in complex antigens is independent of molecular context, competing peptides and epitope availability and is principally due to an intrinsic factor of the peptide, based upon the stability of that peptide for MHC class II molecules. Additionally, when antigen processing is bypassed, low stability peptide:class II complexes were capable of supporting the initial priming and expansion of CD4 T cells but the expansion becomes strikingly aborted in the presence of competitive T cell responses to unrelated dominant peptides. These findings suggest that the immune system has adopted multiple independent mechanisms to select a CD4 T cell repertoire that is focused on long-lived peptide:class II complexes during a response to inva