Elucidating the antigenic evolution of influenza to inform vaccine design

Project Overview

Seasonal influenza is estimated to cause 3 to 5 million cases of severe illness and 250,000 to 500,000 deaths globally. Influenza pandemics have also occurred four times since 1918. In 1918, the ‘Spanish flu’ pandemic killed 50-100 million people, in 1957 the ‘Asian flu’ pandemic caused 1 million deaths, in 1968 ‘Hong Kong’ flu pandemic causes 1 million deaths and in 2009 ‘swine flu’ caused an estimated 300,000 deaths worldwide.

The antigenic evolution of influenza is conventionally assumed to occur by ‘antigenic drift’ where new strains arise through the incremental addition of mutations in surface glycoproteins. However, the antigenic drift model can only explain the epidemiology and limited genetic diversity observed among influenza virus populations by imposing constraints on the mode and tempo of mutation. The Gupta group has demonstrated that an alternative model known as ‘antigenic thrift’ successfully models the epidemiology and genetic diversity of influenza by assuming that the antigenic evolution of the virus population is primarily driven by immune responses against epitopes of limited structural variability. Using a combination of bioinformatic, structural and serological analyses, we have now identified and characterised epitopes of limited variability that is under strong immune selection in the major influenza antigens H1 and H3 haemagglutinin (HA) (Thompson et al. 2018; Recker et al. 2007).

We propose to (i) identify such epitopes of limited variability in potential pandemic avian influenza strains, namely H5, H7 and H9 subtype strains, (ii) determine if epitopes of limited variability are shared between HA groups and can account for the cross-reactivity reported in the literature to H5, H7 and H9 potential pandemic avian influenza strains supposedly induced by infection with H1 and H3 seasonal strains (te Beest et al. 2017; Song et al. 2018; Guo et al. 2016; Gostic et al. 2016), and (iii) incorporate these epitopes of limited variability into a universal influenza vaccine that the we are assembling. This study will utilise neutralisation and ELISA assays to determine cross-reactivity between H5/H7 potential pandemic and H1/H3 seasonal influenza viruses. Bioinformatic and phylogenetic analyses combined with site-directed mutagenesis of the epitopes of interest displayed on pseudotyped influenza viruses will be used to identify and experimentally demonstrate epitope cross-reactivity within and between influenza subtypes. Vaccine design and analysis will involve vaccine production through E. coli-based protein production as well as vaccination studies using mice and leading to influenza challenge.

Training Opportunities

The outcome of this study is four-fold: to identify shared epitopes of limited variability in potential pandemic influenza strains, use these epitopes to understand the antigenic evolution of human and avian influenza, use the epitopes to inform vaccine design and to inform our understanding of potential pandemic influenza. We would expect these studies to produce at least one first author paper in a high impact journal as well as a patent outlining their use a vaccine candidates.


Immunology & Infectious Disease


Project reference number: 1028

Funding and admissions information


Name Department Institution Country Email
Professor Paul Klenerman Experimental Medicine Division Oxford University, Peter Medawar Building GBR paul.klenerman@medawar.ox.ac.uk
Professor Sunetra Gupta University of Oxford GBR sunetra.gupta@zoo.ox.ac.uk

Thompson CP, Lourenço J, Walters AA, Obolski U, Edmans M, Palmer DS, Kooblall K, Carnell GW, O'Connor D, Bowden TA, Pybus OG, Pollard AJ, Temperton NJ, Lambe T, Gilbert SC, Gupta S. 2018. A naturally protective epitope of limited variability as an influenza vaccine target. Nat Commun, 9 (1), pp. 3859. Read abstract | Read more

Current antigenic targets for influenza vaccine development are either highly immunogenic epitopes of high variability or conserved epitopes of low immunogenicity. This requires continuous update of the variable epitopes in the vaccine formulation or boosting of immunity to invariant epitopes of low natural efficacy. Here we identify a highly immunogenic epitope of limited variability in the head domain of the H1 haemagglutinin protein. We show that a cohort of young children exhibit natural immunity to a set of historical influenza strains which they could not have previously encountered and that this is partially mediated through the epitope. Furthermore, vaccinating mice with these epitope conformations can induce immunity to human H1N1 influenza strains that have circulated since 1918. The identification of epitopes of limited variability offers a mechanism by which a universal influenza vaccine can be created; these vaccines would also have the potential to protect against newly emerging influenza strains. Hide abstract

Huang KA, Rijal P, Jiang H, Wang B, Schimanski L, Dong T, Liu YM, Chang P, Iqbal M, Wang MC, Chen Z, Song R, Huang CC, Yang JH, Qi J, Lin TY, Li A, Powell TJ, Jan JT, Ma C, Gao GF, Shi Y, Townsend AR. 2019. Structure-function analysis of neutralizing antibodies to H7N9 influenza from naturally infected humans. Nat Microbiol, 4 (2), pp. 306-315. Read abstract | Read more

Little is known about the specificities and neutralization breadth of the H7-reactive antibody repertoire induced by natural H7N9 infection in humans. We have isolated and characterized 73 H7-reactive monoclonal antibodies from peripheral B cells from four donors infected in 2013 and 2014. Of these, 45 antibodies were H7-specific, and 17 of these neutralized the virus, albeit with few somatic mutations in their variable domain sequences. An additional set of 28 antibodies, isolated from younger donors born after 1968, cross-reacted between H7 and H3 haemagglutinins in binding assays, and had accumulated significantly more somatic mutations, but were predominantly non-neutralizing in vitro. Crystal structures of three neutralizing and protective antibodies in complex with the H7 haemagglutinin revealed that they recognize overlapping residues surrounding the receptor-binding site of haemagglutinin. One of the antibodies, L4A-14, bound into the sialic acid binding site and made contacts with haemagglutinin residues that were conserved in the great majority of 2016-2017 H7N9 isolates. However, only 3 of the 17 neutralizing antibodies retained activity for the Yangtze River Delta lineage viruses isolated in 2016-2017 that have undergone antigenic change, which emphasizes the need for updated H7N9 vaccines. Hide abstract

Recker M, Pybus OG, Nee S, Gupta S. 2007. The generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types. Proc. Natl. Acad. Sci. U.S.A., 104 (18), pp. 7711-6. Read abstract | Read more

It is commonly believed that influenza epidemics arise through the incremental accumulation of viral mutations, culminating in a novel antigenic type that is able to escape host immunity. Successive epidemic strains therefore become increasingly antigenically distant from a founding strain. Here, we present an alternative explanation where, because of functional constraints on the defining epitopes, the virus population is characterized by a limited set of antigenic types, all of which may be continuously generated by mutation from preexisting strains and other processes. Under these circumstances, influenza outbreaks arise as a consequence of host immune selection in a manner that is independent of the mode and tempo of viral mutation. By contrast with existing paradigms, antigenic distance between epidemic strains does not necessarily accumulate with time in our model, and it is the changing profile of host population immunity that creates the conditions for the emergence of the next influenza strain rather than the mutational capabilities of the virus. Hide abstract

Guo L, Wang D, Zhou H, Wu C, Gao X, Xiao Y, Ren L, Paranhos-Baccalà G, Shu Y, Jin Q, Wang J. 2016. Cross-reactivity between avian influenza A (H7N9) virus and divergent H7 subtypic- and heterosubtypic influenza A viruses. Sci Rep, 6 pp. 22045. Read abstract | Read more

The number of human avian H7N9 influenza infections has been increasing in China. Understanding their antigenic and serologic relationships is crucial for developing diagnostic tools and vaccines. Here, we evaluated the cross-reactivities and neutralizing activities among H7 subtype influenza viruses and between H7N9 and heterosubtype influenza A viruses. We found strong cross-reactivities between H7N9 and divergent H7 subtypic viruses, including H7N2, H7N3, and H7N7. Antisera against H7N2, H7N3, and H7N7 could also effectively neutralize two distinct H7N9 strains. Two-way cross-reactivities exist within group 2, including H3 and H4, whereas one-way cross-reactivities were found across other groups, including H1, H10, H9, and H13. Our data indicate that the hemaglutinins from divergent H7 subtypes may facilitate the development of vaccines for distinct H7N9 infections. Moreover, serologic diagnoses for H7N9 infections need to consider possible interference from the cross-reactivity of H7N9 with other subtype influenza viruses. Hide abstract

Gostic KM, Ambrose M, Worobey M, Lloyd-Smith JO. 2016. Potent protection against H5N1 and H7N9 influenza via childhood hemagglutinin imprinting. Science, 354 (6313), pp. 722-726. Read abstract | Read more

Two zoonotic influenza A viruses (IAV) of global concern, H5N1 and H7N9, exhibit unexplained differences in age distribution of human cases. Using data from all known human cases of these viruses, we show that an individual's first IAV infection confers lifelong protection against severe disease from novel hemagglutinin (HA) subtypes in the same phylogenetic group. Statistical modeling shows that protective HA imprinting is the crucial explanatory factor, and it provides 75% protection against severe infection and 80% protection against death for both H5N1 and H7N9. Our results enable us to predict age distributions of severe disease for future pandemics and demonstrate that a novel strain's pandemic potential increases yearly when a group-mismatched HA subtype dominates seasonal influenza circulation. These findings open new frontiers for rational pandemic risk assessment. Hide abstract

Te Beest DE, de Bruin E, Imholz S, Koopmans M, van Boven M. 2017. Heterosubtypic cross-reactivity of HA1 antibodies to influenza A, with emphasis on nonhuman subtypes (H5N1, H7N7, H9N2). PLoS ONE, 12 (7), pp. e0181093. Read abstract | Read more

Epidemics of influenza A vary greatly in size and age distribution of cases, and this variation is attributed to varying levels of pre-existing immunity. Recent studies have shown that antibody-mediated immune responses are more cross-reactive than previously believed, and shape patterns of humoral immunity to influenza A viruses over long periods. Here we quantify antibody responses to the hemagglutinin subunit 1 (HA1) across a range of subtypes using protein microarray analysis of cross-sectional serological surveys carried out in the Netherlands before and after the A/2009 (H1N1) pandemic. We find significant associations of responses, both within and between subtypes. Interestingly, substantial overall reactivity is observed to HA1 of avian H7N7 and H9N2 viruses. Seroprevalence of H7N7 correlates with antibody titers to A/1968 (H3N2), and is highest in persons born between 1954 and 1969. Seroprevalence of H9N2 is high across all ages, and correlates strongly with A/1957 (H2N2). This correlation is most pronounced in A/2009 (H1N1) infected persons born after 1968 who have never encountered A/1957 (H2N2)-like viruses. We conclude that heterosubtypic antibody cross-reactivity, both between human subtypes and between human and nonhuman subtypes, is common in the human population. Hide abstract