Original antigenic sin priming of influenza virus hemagglutinin stalk antibodies Claudia P. Arevaloa, Valerie Le Sageb, Marcus J. Boltona, Theresa Eilolaa, Jennifer E. Jonesb, Karen A. Kormuthb, Eric Nturibib, Angel Balmasedac, Aubree Gordond, Seema S. Lakdawalab,e, and Scott E. Hensleya,1 aDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219; cCentro Nacional de Diagnóstico y Referencia, Ministry of Health, Managua, Nicaragua 16064; dDepartment of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109; and eCenter for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219 Edited by Michael B.A. Oldstone, Scripps Research Institute, La Jolla, CA, and approved May 27, 2020 (received for review November 18, 2019) Immunity to influenza viruses can be long-lived, but reinfections but there are more conserved epitopes in the HA stalk domain. with antigenically distinct viral strains and subtypes are common. Antibodies that recognize the HA stalk of group 1 or group 2 Reinfections can boost antibody responses against viral strains viruses have been well characterized, but antibodies that recog- first encountered in childhood through a process termed “original nize the HA stalk of both group 1 and group 2 viruses are rare antigenic sin.” It is unknown how initial childhood exposures af- (15, 16). Interestingly, childhood influenza A virus exposures are fect the induction of antibodies against the hemagglutinin (HA) associated with heterosubtypic protection against distinct stalk domain of influenza viruses. This is an important consider- emerging influenza virus subtypes later in life (13). For example, ation since broadly reactive HA stalk antibodies can protect early childhood exposures to H1N1 and H2N2 (both group 1 against infection, and universal vaccine platforms are being de- viruses) are associated with protection from H5N1 (also a group veloped to induce these antibodies. Here we show that experi- 1 virus), whereas childhood exposures to H3N2 (a group 2 virus) mentally infected ferrets and naturally infected humans establish strong “immunological imprints” against HA stalk antigens first are associated with protection from H7N9 (a group 2 virus) (13). encountered during primary influenza virus infections. We found This apparent cross-subtype protection is possibly mediated by that HA stalk antibodies are surprisingly boosted upon subsequent antibodies targeting conserved epitopes in the HA stalk; how- infections with antigenically distinct influenza A virus subtypes. ever, it is unclear how initial childhood influenza exposures af- MICROBIOLOGY Paradoxically, these heterosubtypic-boosted HA stalk antibodies fect the development of HA stalk antibody responses. do not bind efficiently to the boosting influenza virus strain. Our Here, we set out to determine how initial childhood influenza results demonstrate that an individual’s HA stalk antibody re- virus infections impact antibody responses against subsequent sponse is dependent on the specific subtype of influenza virus that heterosubtypic influenza virus infections. We first examined they first encounter early in life. We propose that humans are antibody responses elicited in ferrets sequentially exposed to susceptible to heterosubtypic influenza virus infections later in life different influenza virus subtypes. We then examined anti- since these viruses boost HA stalk antibodies that do not bind bodies elicited in children who had different prior influenza efficiently to the boosting antigen. virus exposures. influenza virus | hemagglutinin | original antigenic sin Significance ost humans are infected with influenza viruses early in life Humans are typically infected with influenza viruses in child- M(1) and then subsequently reinfected with antigenically hood and then continuously exposed to antigenically distinct distinct viral strains every 5 to 10 y (2). In a classic essay pub- influenza virus strains throughout life. Antibody responses lished in 1960, Thomas Francis Jr. reported that humans typi- elicited by initial influenza virus infections can be boosted cally have high antibody titers against influenza viruses that they upon subsequent exposures with antigenically drifted in- first encountered in childhood and that these antibodies are fluenza virus strains. Here, we examined how initial influenza boosted upon subsequent exposures to antigenically drifted in- virus infections affect antibody responses against subsequent fluenza virus strains (3). Francis coined the term “original anti- infections with an unrelated influenza virus subtype. We show genic sin” to describe this phenomenon. In 1966, Robert that heterosubtypic infections in ferrets and humans boost Webster and Stephen Fazekas de St. Groth demonstrated that hemagglutinin stalk antibodies that paradoxically do not bind original antigenic sin can be recapitulated in rabbits and ferrets effectively to the boosting influenza virus strain. We propose that are sequentially infected with different H1N1 strains (4, 5). that hemagglutinin stalk antibody repertoires are shaped by Recent human (6–9) and animal (9–12) serological studies have the specific subtype of influenza virus that an individual en- confirmed and extended these findings. Most studies of original counters early in life, and that this affects susceptibility to antigenic sin have analyzed immune responses elicited by se- heterosubtypic infections later in life. quential exposures with drifted versions of the same influenza virus subtype. Much less is known about how prior exposures Author contributions: C.P.A., S.S.L., and S.E.H. designed research; C.P.A., V.L.S., M.J.B., T.E., J.E.J., K.A.K., and E.N. performed research; A.B. and A.G. contributed new re- affect immune responses against heterosubtypic influenza virus agents/analytic tools; C.P.A., S.S.L., and S.E.H. analyzed data; and C.P.A. and S.E.H. infections. This is relevant since three different influenza A virus wrote the paper. subtypes (H1N1, H2N2, and H3N2) have circulated in humans Competing interest statement: S.E.H. reports receiving consulting fees from Sanofi Pas- over the past century, and most humans alive today have been teur, Lumen, Novavax, and Merck for work unrelated to this manuscript. infected with more than one of these subtypes at some point in This article is a PNAS Direct Submission. their lives (13). Published under the PNAS license. Different influenza A virus subtypes can be broadly split into 1To whom correspondence may be addressed. Email: [email protected]. two phylogenetic groups (“group 1” and “group 2”) based on This article contains supporting information online at https://www.pnas.org/lookup/suppl/ conservation in the hemagglutinin (HA) protein (14). The doi:10.1073/pnas.1920321117/-/DCSupplemental. globular head of HA differs substantially among these viruses, www.pnas.org/cgi/doi/10.1073/pnas.1920321117 PNAS Latest Articles | 1of7 Downloaded by guest on September 23, 2021 Results that recognized H3 antigens (Fig. 1 D–F). Similarly, animals HA Stalk Antibodies Elicited by Initial Influenza Virus Infections in initially infected with H3N2 produced H3-reactive antibodies Ferrets Are Boosted upon Subsequent Heterosubtypic Infections. To (Fig. 1D) that targeted both the H3 head (Fig. 1E) and H3 stalk better understand how initial influenza virus exposures shape (Fig. 1F), but did not produce antibodies that recognized H1 antibody responses against subsequent infections with distinct antigens (Fig. 1 A–C). For H1N1 and H3N2 primary infections, influenza virus subtypes, we completed a series of infection ex- total HA antibodies (Fig. 1 A and D) and HA stalk antibodies periments in ferrets that had never previously encountered in- (Fig. 1 C and F) remained at similar levels between 28 and 84 d fluenza virus. We infected ferrets with H1N1 (A/California/07/ after infection, while HA head antibodies peaked 14 d after in- 2009) and then reinfected the same ferrets 84 d later with H3N2 fection and then gradually declined over time (Fig. 1 B and E). (A/Perth/16/2009). We infected other ferrets with H3N2 and Animals initially infected with H1N1 and then subsequently then reinfected them with H1N1 84 d later. As controls, we in- infected with H3N2 mounted H3 antibody responses that were fected two additional groups of ferrets with either H1N1 or similar to those elicited by primary H3N2 infections (Fig. 1 D–F). H3N2 (both influenza A viruses) and then reinfected with in- Surprisingly, we found that secondary infections with H3N2 fluenza B virus (B/Brisbane/60/2008). We quantified total serum boosted H1 stalk-reactive antibodies in animals that were ini- HA antibody levels using ELISAs coated with full-length HA tially infected with H1N1 (4.2 μg/mL before and 81.0 μg/mL 14 d proteins, serum HA head-specific antibodies by hemagglutina- after secondary H3N2 infection; P = 0.0013 comparing titers; tion inhibition (HAI) assays, and serum HA stalk-reactive anti- Fig. 1C). We found similar results when the order of H1N1 and bodies using ELISAs coated with “headless” HA proteins. H3N2 infections was reversed. Animals initially infected with As expected, animals initially infected with H1N1 produced H3N2 and subsequently infected with H1N1 mounted H1 anti- H1-reactive antibodies (Fig. 1A) that targeted both the H1 head body responses