The Impact of Receptor Binding Avidity and Immune History on the Antigenic Determination of Influenza A Viruses

Most humans are repeatedly infected with new strains of influenza throughout their lifetime even though protective neutralizing antibodies against the viral hemagglutinin (HA) are generated after both natural infection and vaccination. This observed lack of protection against variant strains is largely attributed to a process termed `antigenic drift\u27, where accumulating mutations in HA quickly abrogate recognition by antibodies elicited by earlier strains. Consequently, current influenza vaccines must be updated frequently in an attempt to match the antigenic profiles of vaccine strains to those of circulating strains. However, the existing process of antigenic determination is imperfect: it fails to consider the effects of receptor binding avidity in the interpretation of hemagglutination inhibition (HAI) assays or the effects of pre-exposure history on how a novel virus is viewed antigenically by an altered immune system. Here, we designed a series of experiments to address these issues. First, we computationally modeled how variation in receptor binding avidity could affect the antigenic characterization of historic H3N2 strains and experimentally demonstrated that single point mutations in HA can skew HAI titers without actually affecting antibody binding. Additionally, using the same H3N2 system, we showed that a single amino acid mutation can significantly alter the immunodominance of the anti-HA antibody response. We then completed a series of studies to determine how immune history influences the specificity of antibody repertoires. In examining patient serology, we found that the specificity of the human antibody response against the 2009 pandemic H1N1 virus (pH1N1) was highly correlated with pre-exposure history to different seasonal H1N1 (sH1N1) strains during childhood. Using a ferret model, we demonstrate that the anti-pH1N1 antibody response can be shifted to highly conserved epitopes on HA when the animals were primed with sH1N1s that are otherwise antigenically distinct. Collectively, our studies demonstrate that accounting for receptor binding avidity and factors that alter antibody repertoires will improve influenza vaccination strategies in the future

Antibody-Based Strategies to Prevent and Treat Influenza

Passive immunization using antibodies is a promising alternative to other antiviral treatment options. The potential for seasonal protection arising from a single injection of antibodies is appealing and has been pursued for a number of infectious agents. However, until recently, antibody-based strategies to combat infectious agents have been hampered due to the fact that most antibodies have been found to be strain specific, with the virus evolving resistance in many cases. The discovery of broadly neutralizing antibodies (bNAbs) in influenza, dengue virus, and HIV, which bind to multiple, structurally diverse strains, has provided renewed interest in this area. This review will focus on new technologies that enable the discovery of bNAbs, the challenges and opportunities of immunotherapies as an important addition to existing antiviral therapy, and the role of antibody discovery in informing rational vaccine discovery – with agents targeting influenza specifically addressed. Multiple candidates have entered the clinic and raise the possibility that a single antibody or small combination of antibodies can effectively neutralize a wide variety of strains. However, challenges remain – including combating escape variants, pharmacodynamics of antibody distribution, and development of efficacy biomarkers beyond virologic endpoints.National Institutes of Health (U.S.) (Award 1R01AI111395)National Institutes of Health (U.S.) (Merit Award R37 GM057073-13)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Large-Scale Analysis of B-Cell Epitopes on Influenza Virus Hemagglutinin – Implications for Cross-Reactivity of Neutralizing Antibodies

Influenza viruses continue to cause substantial morbidity and mortality worldwide. Fast gene mutation on surface proteins of influenza virus result in increasing resistance to current vaccines and available antiviral drugs. Broadly neutralizing antibodies (bnAbs) represent targets for prophylactic and therapeutic treatments of influenza. We performed a systematic bioinformatics study of cross-reactivity of neutralizing antibodies (nAbs) against influenza virus surface glycoprotein hemagglutinin (HA). This study utilized the available crystal structures of HA complexed with the antibodies for the analysis of tens of thousands of HA sequences. The detailed description of B-cell epitopes, measurement of epitope area similarity among different strains, and estimation of antibody neutralizing coverage provide insights into cross-reactivity status of existing nAbs against influenza virus. We have developed a method to assess the likely cross-reactivity potential of bnAbs for influenza strains, either newly emerged or existing. Our method catalogs influenza strains by a new concept named discontinuous peptide, and then provide assessment of cross-reactivity. Potentially cross-reactive strains are those that share 100% identity with experimentally verified neutralized strains. By cataloging influenza strains and their B-cell epitopes for known bnAbs, our method provides guidance for selection of representative strains for further experimental design. The knowledge of sequences, their B-cell epitopes, and differences between historical influenza strains, we enhance our preparedness and the ability to respond to the emerging pandemic threats

Systematic Experimental Determination of Functional Constraints on Proteins and Adaptive Potential of Mutations: A Dissertation

Sequence-function relationship is a fundamental question for many branches of modern biomedical research. It connects the primary sequence of proteins to the function of proteins and fitness of organisms, holding answers for critical questions such as functional consequences of mutations identified in whole genome sequencing and adaptive potential of fast evolving pathogenic viruses and microbes. Many different approaches have been developed to delineate the genotype-phenotype map for different proteins, but are generally limited by their throughput or precision. To systematically quantify the fitness of large numbers of mutations, I modified a novel high throughput mutational scanning approach (EMPIRIC) to investigate the fitness landscape of mutations in important regions of essential proteins from the yeast or RNA viruses. Using EMPIRIC, I analyzed the interplay of the expression level and sequence of Hsp90 on the yeast growth and revealed latent effect of mutations at reduced expression levels of Hsp90. I also examined the functional constraint on the receptor binding site of the Env of Human Immunodeficiency Virus (HIV) and uncovered enhanced receptor binding capacity as a common pathway for adaptation of HIV to laboratory conditions. Moreover, I explored the adaptive potential of neuraminidase (NA) of influenza A virus to a NA inhibitor, oseltamivir, and identified novel oseltamivir resistance mutations with distinct molecular mechanisms. In summary, I applied a high throughput functional genomics approach to map the sequence-function relationship in various systems and examined the evolutionary constraints and adaptive potential of essential proteins ranging from molecular chaperones to drug-targetable viral proteins

Mutations in influenza A virus neuraminidase and hemagglutinin confer resistance against a broadly neutralizing hemagglutinin stem antibody

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting influenza hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly-conserved stem region of influenza hemagglutinin (HA) can neutralize many influenza strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations with whole genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function

Universal Swine Influenza Antigen and Vaccine

Swine influenza A viruses pose a serious concern for global health and worldwide swine industry considering their evasion from vaccine-induced immune responses. Formulation of effective influenza vaccines can be complicated by both antigenic shift to a different subtype of hemagglutinin (HAs) and antigenic drift within a particular HA subtype. To address these concerns, we employed a systematic approach to determine the antigenic determinants of swine influenza A viruses by focusing on H1 and H3 subtypes. In parallel, we used a cross-disciplinary approach including structural biology and modeling, virology, and immunology to identify swine influenza A virus-derived HA fusion intermediates broadly reactive to antibodies elicited from various strains or subtypes of swine influenza A viruses. Finally, we conducted a swine influenza vaccine study to evaluate the protective efficacy of a newly identified HA fusion intermediate. The vaccine candidate, when administered into pigs, protected swine against infections by swine influenza H1N1 and H3N2 viruses. For the antigenicity study, we performed a detailed analysis of the antigenic determinants of the Hemagglutinin (HA) protein of pdm09 strain (subtype H1N1) by the HA peptide array in Enzyme-linked immunosorbent assay with immune serum from experimental infected pigs by swine influenza A viruses (SIVs). The peptide array contains 139 peptides spanning the HA protein of H1N1pdm09 and peptides are 14- or 15-mers with 11 amino acid overlaps. A panel of swine antisera against SIV H1 clusters α, β, γ, δ-A, δ-B, H1N1pdm09, and H3 cluster was used in this study and SIVs used for swine hyperimmune sera production are representative subtypes and clusters of SIV circulating in North America. The results of these studies identified three immunodominant peptides, one located in the HA1 (amino acids 57-71) and two in the HA2 (amino acids 481-495 and 553-566), reactive to its homologous antisera (H1N1pdm09). Analysis of peptide reactivity with heterologous serum samples revealed that antibody responses to two HA2 peptides (amino acids 481-495 and 553-566) are completely conserved among antisera of all H1 clusters. In addition to H1N1pdm09, HA1 peptide (amino acids 57-71) was reactive to SIV H1 γ antisera Interestingly, none of peptides in HA peptide array of H1N1pdm09 were reactive to SIV H3 antisera, despite that this antisera has a HI antibody titer similar to those of H1 clusters antisera. Structural modeling localized two conserved immunedominant determinants to the membrane proximal external region (MPER) (amino acids 481-495) and the intracytoplasmic tail (amino acids 553-566) of the HA molecule. For the HA fusion intermediate project, through a structure-guided sitespecific mutagenesis targeting HA2 A and CD helical domains, we identified a mutation, named N439G, that attenuated viral replication but the resultant virus was still recognized by antiviral antibodies. The attenuation was mediated probably through the disruption of triple CD helix formation in the viruses. In light of the fact that N439G virus is highly attenuated in that it may prolong the exposure of HA fusion intermediate to host B cells toward antibody production, we next evaluated its vaccination efficacy in pigs. Significantly, pigs receiving N439G immunizations were substantially protected from both swine influenza A H1N1 and H3N2. In summary, the results of our antigenicity study provide an important foundation for further analyzing the immune response against these B cell epitopes residing in the HA protein during natural SIV infection and also provide potential peptide substrates for diagnostic assays and for vaccine strategies. Our universal vaccine candidate identified from this work should pave the way for further studies toward developing a universal swine influenza vaccine and defining a protective mechanism