Laboratory Projects

Why does the influenza vaccine fail? In spite of substantial efforts to vaccinate, influenza epidemics remain a major public health threat. In the US, the currently licensed vaccines are very safe, but only partially effective at protecting from infection.  Though efficacy rates vary with age of the subjects and virus strains, estimates of protection range from 10-70%.  As our abilities to study influenza viruses at the molecular level increase, and the costs to do so decrease, there are more reports of a significant degree of sequence diversity in the viruses that circulate seasonally.  This raises the possibility that some of the vaccine failures may be due to antigenic drift in the viruses.  Unfortunately, there are few (to no) studies that have assayed the virus sequences and antigenicity, and the specificity or function of the antibodies and memory B cells present in the subject who is infected. As part of our surveillance activities, we will collect and analyze the viruses causing infections and match these to the individual’s antibody and B cell responses present before infection or at the time illness begins.  We will also study the impact of infections versus vaccination on the responding B cell and immunoglobulin (Ig) specificity and function, including development of memory.  We will also use in vitro approaches that drive antigenic drift in prototypical seasonal influenza viruses to better understand how differences in immune profiles affect virus evolution.  To take this further, we propose to use the information from circulating viruses and antibody/B cell specificity to develop computational tools that analyze and predict human immune responses to influenza. We will use these models to both better understand human immunity to flu, but to also simulate how prior immunity affects immune responses to various immunization strategies.  Our goal is to develop approaches to immunization that produce more broadly cross-reactive and even “universal” immunity to flu. To accomplish these lofty goals, we have assembled a team of the very best immunologists, virologists, computational biologists, and clinical investigators who will work together on this project using cutting edge technologies. These studies have the potential to change the way we think about influenza immunity and vaccination, and create a better understanding of immune protection and virus selection.

Project 1 will perform the following aims:

Aim 1: Test the hypothesis that microevolution and antigenic drift of human influenza viruses contributes to susceptibility to acute infection and failed vaccine-mediated protection. Hypothesis: Susceptibility to acute influenza is associated with deficiencies in protective (neutralizing) antibodies against the infecting virus.  These deficiencies arise from antigenic mutations in the virus or through functional deficits in the immunity of an individual’s antibodies and B cell memory to the virus. Subjects documented with acute influenza infection will be sampled for virus, serum, and mucosal antibodies at presentation of illness. We will antigenically type infecting viruses in parallel with interrogation of the serum and mucosal antibodies. Antibody assessment will be performed against the infecting virus as well as seasonal vaccine strains, and will include detailed determination of antibody epitope specificities and functions.

Aim 2: Compare host immunoglobulin adaptation to HA during infection and vaccination.

Hypothesis: Vaccination and infection drive adaptation of the antibody secreting and memory B cell populations, and differ based on the infecting virus and immune history of the subject. We propose to perform parallel assessment of antibody secreting cells and memory B cells over the course of an immune response to determine how infection and vaccination modify the B cell and antibody repertoires. As a measure of the cells responding to the infection or vaccination, our assays will focus on the acutely activated antibody-secreting B cells (ASC, plasmablasts) and antibodies secreted. Because these ASC can be short lived, we will also look at in vitro reactivated memory B cells and secreted antibodies as a measure of the adapted responses. The influenza viruses we see early in life have a strong effect on our subsequent immunity. Therefore we will assess how B cell memory established early in life affects current human immune responses to influenza.

Aim 3: Develop computational models and tools to predict likely changes resulting from antigenic drift and verify through biological testing. Hypothesis: Mutational hotspots that lead to antigenic drift can inform the development of novel computational prediction tools that can be tested and validated through in vitro approaches. We will develop novel computational approaches to measure antigenic differences in influenza viruses using data collected in our acute flu studies, as well as data in national and international databases. This information will be used to develop computational models to simulate human immunity to influenza based on differences in exposure history. This may lead to novel vaccination strategies to achieve broadly cross reactive and universal flu vaccine approaches using personalized approaches.

It has become more apparent in recent years that a universally protective, "one-shot" influenza vaccine may be possible. Every year we as a scientific community discover several new highly conserved epitopes that when targeted by antibodies can neutralize various influenza strains. Although promising, the practicality of effectively targeting these epitopes with a vaccine is almost entirely uncertain. In this project we study the human B cell response during influenza infection and after vaccination with newly licensed influenza vaccines. These cohorts will allow us to test several hypotheses central to understanding how a universal vaccine might be developed and function. In these efforts we will also develop and test a large collection of human monoclonal antibodies (mAbs) that will drive other projects in the center. The panel of mAbs to newly arising influenza strains will be quite valuable to inform on vaccine development or for development directly as therapeutics. Finally, key antibodies will be used to develop knock-in transgenic mouse models to directly test key mechanistic hypotheses that cannot be easily tested on human cohorts. Three specific aims are proposed:

For specific aim 1 we will characterize the mechanisms of immune escape during epidemic influenza infections in the young and old. Our methods provide a direct evaluation of the individual B cells activated by the ongoing infection. Thus by isolating the cognate infecting virus as well, we can directly determine the mechanisms of viral escape and neutralization in infected people at the monoclonal level. Further, we have found that after influenza vaccination people over 70 have responses reduced in magnitude and inefficient adaptation to drifted influenza strains, resulting in an original antigenic sin-like response. We will determine if these defects underlie susceptibility to infection in an aged cohort of patients.

For specific aim 2, we will determine if the subdominant responses to the highly conserved HA-stalk epitopes are due to the inaccessibility of these epitopes on whole virions. We have compelling preliminary data suggesting that biases in the HA-stalk specific antibody response are due to the inaccessible nature of this epitope.  In this aim we will compare the human antibody response to whole virion vaccines with the new FluBloc vaccine composed of rHA generated in baculovirus. We hypothesize that the rHA vaccine will more readily allow targeting of these important, broadly conserved epitopes. Further, we will compare the efficacy of the various current vaccines to induce protective and adaptive B cell responses in an aged cohort.

For the third specific aim  we will Characterize HA-head versus HA-stalk responses in immunoglobulin knock-in transgenic mice. We will generate HA—stalk versus HA–head reactive Ig transgenic mice and use them to test the mechanistic basis of epitope targeting and immune escape from these epitopes in mice chimeric for both epitopes. We will also determine the relative capacity of B cells to adapt to HA-stalk versus HA-head specificities on divergent influenza strains by Ig gene diversification. Finally, we will compare various vaccine compositions and strategies to determine how HA-stalk reactive B cells can be optimally induced in both young and aged mice.

The research in our laboratory focuses on the role of CD4 T cells in protective immunity to influenza.  One of the key distinctions between CD4 T cells and other cells in the adaptive immune response, such as B cells and CD8 T cells, is the multiplicity of functions that CD4 T cells contribute to protective immunity to influenza.  We study human responses to infection and vaccination and utilize mouse models that allow deeper mechanistic understanding in order gain maximal insight into the factors that control recruitment of CD4 T cells into the response to influenza and their ultimate effector function.

CD4 T cell help for antibody responses is the most generally acknowledged and essential contribution of CD4 T cell responses to protective immunity induced by influenza vaccines and infection. Our studies in both animal models and humans supports the conclusion that the specificity of CD4 T cells is critically important in their ability to help B cells and that the antigens that influenza-specific B cells take up after infection or vaccination through their immunoglobulin receptor will dictate the subsets of CD4 T cells that can be recruited to help.  For example, in human vaccine responses and in mouse models of infection, recruitment of memory CD4 T cells specific for HA, but not NP, is associated with earlier and more robust anti-HA antibody responses. We are currently using a human vaccine study to examine the impact on the formulation of licensed influenza vaccines on CD4 T cell responses and protective antibody responses. The comparison includes egg-based split vaccines, that contain HA, NA as well as some internal virion proteins, baculovirus-derived pure HA proteins and a mammalian cell based vaccine. We are characterizing many features of the elicited CD4 T cells responses and correlating this with the elicited antibody response.

Other research in our laboratory has probed the tropism of influenza virus in the infected lung, using a novel reporter virus.  Our studies have revealed that in addition to epithelial cells, a wide variety of myeloid cells become infected and are antigen-bearing.  Many of these cells in the infected lung express MHC class II molecules, thus serving as potential targets of virus specific CD4 T cells. We are now exploring the trafficking of CD4 T cells into the lung after infection, their viral epitope specificity and effector functions including production of cytokines and cytotoxic mediators.  In addition to seeking a better understanding of CD4 function and specificity in the lung, we designing and testing novel intranasal influenza vaccines in order to promote recruitment of the needed CD4 T cell effector functions into the lung.

Influenza viruses rapidly accumulate mutations in antibody binding sites within the hemagglutinin and neuraminidase proteins, a process termed ‘antigenic drift’.  Due to antigenic drift, humans are continuously re-infected with influenza viruses and vaccines must be updated frequently.  Seasonal influenza vaccines can be ineffective when vaccine strains are mismatched to circulating viral strains.  The process of choosing seasonal influenza vaccine strains is an imperfect process.  The decision of which strains to include in future vaccine formulations must be made months in advance of an influenza season.  This decision is based on antigenic and epidemiological data, but there is currently no way to reliably predict which viral strains will circulate in the future.

In order to predict how influenza viruses will change in the future, it is important to identify specific types of antibodies that are prevalent in the human population.  Our recent studies indicate that humans preferentially mount antibodies that recognize epitopes that are shared between current vaccine strains and viral strains that were present during each individual’s childhood.  Most vaccine efficacy studies do not take into account prior exposure histories or the precise specificity of antibodies elicited by vaccination.  Further, most models that are being developed to predict antigenic drift of influenza viruses do not account for different antibody signatures that are prevalent in specific age groups.  The Hensley laboratory is completing experiments that identify vaccine-elicited antibodies that are protective and non-protective and we are developing predictive models that take into account specific types of antibodies that are prevalent in different aged individuals.  These experiments will ultimately inform the process of selecting seasonal influenza vaccine strains.

As a component of this non-severable, immunology option (Option 16) the subcontracting organization will use information regarding the age-related antibody specificity of sera from various sources to derive new predictive models that incorporate antibody specificities that are common in certain age groups.  In contrast to traditional approaches, these models will not simply label individuals as ‘susceptible’ or ‘immune’, but rather will delineate the specific strains that each individual is susceptible or immune against.  The investigators will use large datasets to examine the relationship between antibody selection pressure, mutational fixation rates over time, and infection rates among different aged individuals.   Once developed, they will use these models to make viral strain predictions, and they we will make all of the computational tools developed freely available to the surveillance and public health communities. 

Our work uses computational and statistical models to identify the role of adaptive immunity in the epidemiology and evolution of influenza and to understand how these interactions shape vaccine effectiveness. We are tackling this problem from several complementary directions. In collaboration with Scott Hensley's lab, we are using cross-sectional serology to investigate the structure of herd immunity and test if serology can improve viral forecasting. We are also using cross-sectional and longitudinal serology from several labs to infer how primary infections, vaccination, and recent exposure shape infection risk in individuals and cohorts. More broadly, these models infer how variation in B cell specificity between people arises and evolves over time. A special modeling focus is how vaccination interacts with preexisting immune responses and relates to variation in vaccine effectiveness between age groups and over time. Our overarching aim is to integrate observations across multiple scales, from individual B cells to seasonal incidence, using diverse quantitative approaches.

The goal of this study is to understand how immune history and sequential vaccinations impact responses to seasonal influenza vaccines, in order to understand how immune history might shape immunity to universal influenza vaccines. For this the Wilson lab is sorting plasmablasts from vaccine recipients that have received annual vaccinations repeatedly over recent years compared to those that are receiving the vaccine for the first time in multiple years. The cohort is being recruited at Rochester University where blood is drawn. The blood is then sent to the Wilson laboratory where it is processed and used to sort single plasmablasts that are then being used to express monoclonal antibodies (mAbs). These antibodies will then be characterized by the Hensley laboratory for issues such as imprinting with past strain specificities and epitope focusing.

Individuals first encounter influenza virus as children, with an initial encounter either through a natural infection or inactivated influenza vaccine.  Recent data suggests that these early exposures have a substantial impact on the long-term development of influenza-specific immunity, yet how anti-influenza immune response develops in early childhood remains a critical void in our knowledge.  Multiple factors, including the route of influenza exposure, the age of a child at the time of a given exposure, and the history (or lack thereof) of previous influenza confrontations could substantially impact both CD4 T cell and B cell mediated immunity.  The goal of this project is to gain insight into how these different factors influence the anti-influenza immune response through the direct study of children of different ages who are either vaccinated or with IIV or contract a natural influenza infection.

The core hypothesis of this study is that the age of a child and history of previous exposures to live viral infection in comparison to vaccination will impact the induction of broadly protective anti-influenza immunity, leading to distinct changes in the phenotype and specificity of both the CD4 T cell and B cell compartments.  Subsequent exposures will result in remodeling of this repertoire, with alterations in cytokine production and increased expression of markers and transcriptional factors associated with a negative regulatory CD4 T cell phenotype.  To test this hypothesis, we are going to evaluate CD4 T cell and B cell responses in cohorts of children of different ages following either natural infection or IIV immunization.  These children will then be followed through to subsequent influenza seasons, with examination of the CD4 T cell response using multiparameter flow cytometry and the B cell response by characterization of the plasmablast and antigen-specific memory B cell response.  Serological reactivity will also be determined through measurement of neutralizing as well as non-neutralizing antibody production.  Through these comprehensive evaluations of the development of the anti-influenza immune response in children, we will gain new insights on the early establishment of immunologic memory necessary for the development of more broadly protective influenza vaccines. 

Strategies for generating Ab-mediated protection against influenza have largely focused on responses to the hemagglutinin (HA). The conserved HA stalk is currently receiving much attention as a key target of broadly protective Abs and a basis for universal vaccine development. In addition, conserved HA head epitopes have been identified that deserve continued investigation as targets of broadly reactive Abs that are more efficiently neutralizing than are stalk-specific Abs. Recently, several studies have demonstrated the broadly protective value of Abs against the neuraminidase (NA), and this molecule is now widely seen as an important component of improved influenza vaccines. Our goal is to investigate expansion of HA-reactive and NA-reactive memory B cells (MBCs) following administration of the inactivated influenza vaccine (IIV). In particular, we will determine the breadth of reactivity of HA- and NA-specific MBCs expanded by IIV. Findings will be compared to the response to influenza infection to determine how the form of antigen exposure influences the breadth of reactivity of expanded MBC populations and thus the breadth of protection conferred.

Aim 1: To measure the expansion of HA- and NA-reactive MBCs following seasonal IIV. The analysis will include investigation of MBC focusing after IIV that limits breadth of reactivity to particular epitopes or molecular domains.

Aim 2: To compare HA- and NA-reactive MBC expansion following seasonal IIV to that following infection, particularly with respect to the magnitude and breadth of MBC expansion and the potential to enhance broad protection.

To date, H3N2 or H3N8 Canine Influenza Viruses (CIVs) are only circulating in dogs and there is no evidence of transmission of these viruses from dogs to humans. However, influenza viruses are constantly evolving and it is possible they could cross this specie barrier and start infecting and circulating between humans. Therefore, considering the impact that H3N2 or H3N8 CIVs might have on public health, pandemic preparedness and identification of potential treatments for this scenario are needed.

In response to the NYICE Pandemic Response Program, our lab has started working with H3N2 and H3N8 CIVs since May 2017. In line with the overall goal of the pandemic response program, we are implementing new experimental tools and technical approaches to study H3N2 and H3N8 CIV infections. These new experimental approaches will allow us to rapidly respond to the emergence of H3N2 and H3N8 CIVs, including the identification of potential antivirals that can be used for the treatment of CIVs as well as neutralizing antibodies that could be implemented for the prophylactic or therapeutic treatment of CIV infections in humans.  Moreover, during this time, we have also developed two different approaches for the development of attenuated forms of these CIVs that can be used as live-attenuated influenza vaccines (LAIVs) for the prevention and control of H3N2 and H3N8 CIVs in dogs and, if needed, in humans. Below we summarize our results for this pandemic response program:

1. Development of LAIVs for the treatment of CIVs: To date, only inactivated influenza vaccines (IIVs) are available for the prevention of H3N2 and H3N8 CIV infections. However, LAIVs have been shown to provide better protection because of their ability to induce more robust humoral and cell-mediated immune responses against subsequent exposure to influenza viruses. We employed two different experimental approaches for the development of LAIVs for the treatment of CIVs. In the first approach, we generated, using reverse genetics techniques, recombinant H3N8 CIVs with a deletion (DNS1) or with truncations (NS1-73, NS1-99 and NS1-126) in the non-structural 1 (NS1) protein. We have shown that H3N8 CIVs with either a deletion or with truncations in NS1 were able to replicate at similar levels than wild-type (WT) H3N8 CIV in vitro, important for vaccine production, but were attenuated and able to confer, upon a single intranasal administration, protection against challenge with WT H3N8 CIV, important for their implementation as LAIVs. Concomitantly, we also generated a H3N8 CIV LAIV based on the introduction of one mutation in the viral polymerase basic protein 2 (PB2; N265S) and three mutations in the viral polymerase basic protein 1 (PB1; K391E, E581G, A661T) responsible for the temperature sensitive (ts), cold-adapted (ca) and attenuated (att) phenotype of the human LAIV A/Ann Arbor/6/60 H2N2 (FluMist). In vitro studies demonstrated that introduction of these mutation in the H3N8 CIV allow the virus to grow at 33°C, important for vaccine production, but severely (37°C) or completely (39°C) attenuated the virus at non-permissive temperatures. Importantly, in vivo results indicate that our ts, ca and att H3N8 CIV was attenuated but able to confer, upon a single intranasal dose, protection against subsequent infection with WT H3N8 CIV, demonstrating the feasibility of implementing this approach as a LAIV for the treatment of H3N8 CIV infections. Notably, we used the safe backbone of our ts, ca and att H3N8 CIV as a Master Donor Virus (MDV) to develop a LAIV against the newly introduced (2015-2016) in the United States H3N2 CIV. To that end, we generated, using reverse genetics, a recombinant virus containing the six internal genes (PB2, PB1, PA, NP, M and NS) of our CIV H3N8 LAIV and the external genes (HA and NA) of the H3N2 CIV. We have been able to demonstrate that this H3N2 CIV LAIV is safe, immunogenic and able to protect against challenge with WT H3N2 CIV. Finally, based on the safety profile of our individual H3N8 and H3N2 CIV LAIVs, we mixed both monovalent H3N2 and H3N8 CIV LAIVs for the development of a bivalent LAIV for the treatment of both CIVs. This was the first description of a bivalent LAIV for the treatment of H3N2 and H3N8 CIVs. More recently, we have implemented the same ts, ca and att approach to generate a LAIV for the treatment of equine influenza virus (EIV) infections.

References:

1. Nogales A, Huang K, Chauche C, DeDiego ML, Murcia PR, Parrish CR, et al. Canine influenza viruses with modified NS1 proteins for the development of live-attenuated vaccines. Virology. 2017;500:1-10.

2. Nogales A, Rodriguez L, Chauche C, Huang K, Reilly EC, Topham DJ, et al. Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine. J Virol. 2017;91(4).

3. Rodriguez L, Nogales A, Reilly EC, Topham DJ, Murcia PR, Parrish CR, et al. A live-attenuated influenza vaccine for H3N2 canine influenza virus. Virology. 2017;504:96-106.

4. Rodriguez L, Nogales A, Murcia PR, Parrish CR, Martinez-Sobrido L. A bivalent live-attenuated influenza vaccine for the control and prevention of H3N8 and H3N2 canine influenza viruses. Vaccine. 2017;35(34):4374-81.

5. Rodriguez L, Reedy S, Nogales A, Murcia PR, Chambers TM, Martinez-Sobrido L. Development of a novel equine influenza virus live-attenuated vaccine. Virology. 2018 Mar;516:76-85. doi: 10.1016/j.virol.2018.01.005.

2. Development of fluorescent-expressing CIVs: We have generated and characterized fluorescent-expressing H3N2 and H3N8 CIVs that display similar growth kinetics and plaque phenotype to their WT counterparts. These fluorescent-expressing recombinant H3N2 and H3N8 CIVs will allow us to easily monitoring viral infection without the need of secondary procedures to detect the presence of the virus in infected cells. We have also implemented our recently described fluorescent-based microneutralization assay for the rapid identification of antivirals or neutralizing antibodies against CIVs.  We are currently evaluating the antiviral activity of Food and Drug Administration (FDA)-approved antivirals (Oseltamivir, NA inhibitor; and Amantadine, M2 inhibitor) against H3N2 and H3N8 CIVs. Likewise, we are using the fluorescent-based microneutralization assay to screen a panel of human monoclonal antibodies (hmAbs) for their neutralizing activity against H3N2 and H3N8 CIVs for their potential use as prophylactic and/or therapeutics against H3N2 and H3N8 CIV infections.

References:

1. Nogales A, Baker SF, Martínez-Sobrido L. Replication-competent influenza A viruses expressing a red fluorescent protein. Virology. 2015 Feb;476:206-16. doi: 10.1016/j.virol.2014.12.006.

2. Nogales A, Rodríguez-Sánchez I, Monte K, Lenschow DJ, Perez DR, Martínez-Sobrido L. Replication-competent fluorescent-expressing influenza B virus. Virus Res. 2016 Feb 2;213:69-81. doi: 10.1016/j.virusres.2015.11.014.

3. Breen M, Nogales A, Baker SF, Perez DR, Martínez-Sobrido L. Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies. PLoS One. 2016 Jan 25;11(1):e0147723. doi: 10.1371/journal.pone.0147723.

 4. Breen M, Nogales A, Baker SF, Martínez-Sobrido L. Replication-Competent Influenza A Viruses Expressing Reporter Genes. Viruses. 2016 Jun 23;8(7). pii: E179. doi: 10.3390/v8070179. Review.

5. DiPiazza A, Nogales A, Poulton N, Wilson PC, Martínez-Sobrido L, Sant AJ. Pandemic 2009 H1N1 Influenza Venus reporter virus reveals broad diversity of MHC class II-positive antigen-bearing cells following infection in vivo. Sci Rep. 2017 Sep 7;7(1):10857. doi: 10.1038/s41598-017-11313-x.

3. Understanding the mechanism(s) of CIV pathogenesis. During the last year we have also studied the mechanisms by which influenza viruses adapt to new hosts to understand the mechanism of viral infection and pathogenesis. We focused our studies on the NS1 protein, a virulence factor essential for counteracting the host immune response and specially the production of type I interferon (IFN). Unlike other influenza viruses that impair IFN response by blocking general gene expression, we have shown that H3N8 CIV NS1 specifically counteract the IFN response without blocking general gene expression. However, the presence of a Glutamate (E) in the residue 186 of the H3N8 CIV NS1 protein restored the ability of H3N8 CIV NS1 to block general host gene expression. Notably, and contrary to the situation of H3N8 CIV NS1, the NS1 protein of H3N2 CIV has the ability to inhibit host protein expression. More recently we have confirmed the importance of this residue 186 in NS1 evolution during adaptation of H3N8 EIV, an avian-origin virus. Evolution of influenza NS1 protein, the mutations that sequentially appear during evolution and their effect on immune evasion are important to understand influenza virus adaptation to mammals.

References:

1. Nogales A, Chauche C, DeDiego ML, Topham DJ, Parrish CR, Murcia PR, et al. The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression. J Virol. 2017 Oct 27;91(22). pii: e00877-17. doi: 10.1128/JVI.00877-17. 

2. Chauche C, Nogales A, Zhu H, Goldfarb D, Ahmad Shanizza AI, Gu Q, et al. Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1. J Virol. 2018 Feb 12;92(5). pii: e01875-17. doi: 10.1128/JVI.01875-17.

Development of reagents for ferret studies: We have also been tasked with the development of appropriate monoclonal antibodies to perform flow cytometry studies in ferrets as part of a CEIRS-wide effort to improve the ability to use this animal model to study the immunology and host response to influenza and influenza vaccination.

The subcontracting organization will perform mutagenesis of the major influenza viral antigen, the hemagglutinin protein, in order to generate novel universal influenza virus vaccine candidates. Standard influenza virus vaccines predominantly elicit antibody responses directed at the immunodominant hemagglutinin head domain. The head domain however, is easily mutable, and viruses can escape neutralization via mutation. In this work, we will perform mutagenesis of the hemagglutinin head domain in order to decrease its inherent immune-dominance. Candidate mutant antigens will then be used to vaccinate animals and the relative antibody response to the head and stalk domains will be quantified. Antibodies against the stalk domain are thought to be an important component of more universal influenza vaccine. Once developed and characterized, these H1 HA-based antigens can be immediately advanced for further testing. Additionally, this approach, once validated, can be utilized to develop other hemagglutinin based universal vaccine antigens for group 2 IAV HA proteins and HAs derived from influenza B viruses.

In this study, we will:

1. Develop assays delineating the effector functions of a set of anti-influenza virus broadly-neutralizing antibodies depending on complement receptors (CRs) from those depending on FcɣR receptors

2. Quantitatively asses the contribution of complement activation versus the contribution of other effector functions to provide broad, antibody based protection in vivo

3. Address the questions about the original isotypes of these antibodies

As a component of this non-severable, immunology option (Option 16) the subcontracting organization will perform studies to understand mechanisms by which the frequency and type of vaccine exposures influence the development of CD4 T cell mediated immunity in children and determine the effect this has on the development B-cell mediated antibody response.  Specifically, the subcontractor will evaluate the B cell specificity of cells from children with or without previous LAIV exposure and before and after vaccination.  The Wilson laboratory will isolate plasmablasts (day 7) and antigen-specific memory B cells (day 28) by flow cytometry. Monoclonal antibodies will be expressed from the cloned variable genes of single cells and the antibodies and serum will be tested for binding, neutralization, and epitope specificity. 

Dr. Langlois will be the Principal Investigator at the University of Minnesota. Under this award, he and his lab personnel will determine the impact of TCR signal strength on longevity tissue resident memory T cells during influenza virus infections. Additionally, they will Generate an atlas of transcriptional signatures and chromatin landscape for both long- and short-lived TRM after IAV infection. This will help elucidate a detailed understanding of what drives long-lasting TRM responses in the lung. This work will address critical components of the NIAID’s Universal influenza vaccine strategic plan, which specifically highlighted that: “The role of T-cell–mediated immunity in influenza virus infection and disease has historically received little research attention…” and that “…universal influenza vaccine strategies may need to incorporate this component of the immune response to be fully successful.” The annual CEIRS meeting in November 2017 also highlighted the need to “identify antigenic targets and/or adjuvant candidates that induce long-lived immunity.” And to define the “requirements for human protective immunity (e.g. humoral and cell mediated correlates of protection), including tissue-specific vs systemic immune responses.” This proposal will directly address these core features of the strategic plan.

In this study we will:

• Define the cross-reactivity of antibody responses induced by various influenza B viruses (IBV) after seasonal vaccination.
• Characterize protective epitopes targeted by human HA-reactive monoclonal antibodies and the frequency at which they are targeted.
• Study antibody responses to other conserved proteins induced by IBV; neuraminidase (NA) and nucleoprotein (NP).

The overall goal is to determine the best strategy to target IBV to develop a universal influenza virus vaccine.

The subcontracting organization will utilize a recently developed influenza virus genetic platform to generate improved seasonal influenza virus vaccines. Standard influenza virus vaccines are susceptible to mutations in viral glycoproteins acquired during vaccine propagation in embryonated chicken eggs. These mutations are thought to be contribute to decreased influenza vaccine efficacy. In this work, dual-hemagglutinin influenza viruses will be generated in which a “helper” hemagglutinin will prevent mutations in the clinically relevant antigen. The antigen yield and stability of these novel vaccine strains will be compared to standard reassortant vaccines after growth in chicken eggs. Finally, the protective efficacy of antigenically matched vaccines will be compared to egg adapted vaccines in animal models. Once developed and characterized, this dual-HA vaccine technology has the potential to be used for multiple influenza virus strains and could be utilized to improve the protection afforded by influenza virus vaccines.

Dr. Langlois will be the Principal Investigator at the University of Minnesota. Under this award, he and his lab personnel will generate a detailed assessment of the humoral immune response to influenza infection in SPF and dirty mice by analyzing antibody and the HA-specific B cells directly. Additionally, they will develop an atlas of the transcriptional signatures of HA-specific B cells in SPF and dirty mice. This proposal will address critical components of the NIAID’s Universal influenza vaccine strategic plan, which specifically highlighted the need to “improve animal models and reagents to advance vaccine development”. The annual CEIRS meeting in November 2017 also highlighted the need to define the “requirements for human protective immunity (e.g. humoral and cell mediated correlates of protection), including tissue-specific vs systemic immune responses.” This proposal will directly address these critical components of the strategic plan.

As a component of this non-severable, pilot option (16J) the subcontracting organization aims to develop candidate vaccine viruses (CVVs) and an isolation procedure(s) for increasing NA yields and antigenicity, which will be evaluated for efficacy in collaboration the University of Rochester.

In this study, we will characterize the immune response to influenza virus infection and seasonal influenza vaccination in 60 children who had documented prior natural infection, documented prior vaccination or neither. We will enroll six cohorts of 10 children each (aged 6 months to 10 years); cohorts 1-5 will be followed for two years and will be vaccinated in both years with the seasonal quadrivalent influenza vaccine (QIV). Cohort 6 will be enrolled in year 2 and will receive QIV only in year 2. Study subjects will be enrolled at two clinical sites. The laboratory work will be completed at a single site.

Scope of Work: 

The Hensley laboratory will define the specificity of protective antibodies elicited by influenza vaccines in humans The Hensley laboratory will develop new methods of predicting influenza virus evolution that taken into account immune repertoires that are prevalent in humans. We will particularly focus on antibodies that are affected by egg-adapted mutations in this project.

In this study, we will:

The Bloom lab will use deep mutational scanning to completely map the mutations selected by protective and non‐protective antibodies to influenza virus hemagglutinin and neuraminidase. These results will inform the rational design of a new influenza vaccine intended to preferentially elicit antibodies that target broadly protective epitopes.

Scope of Work:

The Hensley laboratory will define the specificity of protective antibodies elicited by influenza vaccines in humans The Hensley laboratory will develop new methods of predicting influenza virus evolution that taken into account immune repertoires that are prevalent in humans. We will particularly focus on antibodies that are elicited by H5 and H7 vaccination in this project.

The Krammer laboratory will: 

Design, produce and provide recombinant HA for analysis of the anti-stalk response after H5N1 and H7N9 vaccination

We have begun vaccinating several different strains of mice with the different HA constructs obtained from the VRC. Intact HA proteins without the modifications to the carboxytermii are being examined in parallel. CD4 T cell specificity in the respiratory tract, draining lymph node and in the spleen are being examined in parallel.

Potentiating broadly protective local immunity to influenza virus through a novel vaccine platform

PI: Andrea Sant, Ph. D. (University of Rochester) and Amy Rasley, Ph. D. (Lawrence Livermore National Laboratory​) 

Our goal is to explore the potential of NLP-based vaccines for influenza to provide broadly protective immunity in vivo.

Drs. Fischer and Rasley will design nanolipoprotein vaccine constructs comprised of influenza viral proteins NA, NP and HA along with various adjuvant molecules including single and double stranded RNA and novel synthetic adjuvants. All vaccine constructs will be produced at LLNL and characterized using techniques well established in our laboratories. Vaccine formulations will be shipped to URMC for testing in vivo.