Influenza Virus–Specific Human Antibody Repertoire Studies

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Influenza Virus−Specific Human Antibody Repertoire Studies James E. Crowe, Jr. This information is current as J Immunol 2019; 202:368-373; ; of September 25, 2021. doi: 10.4049/jimmunol.1801459 http://www.jimmunol.org/content/202/2/368 Downloaded from References This article cites 44 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/202/2/368.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Influenza Virus–Specific Human Antibody Repertoire Studies James E. Crowe, Jr. The diversity of Ag-specific adaptive receptors on the by the error-prone nature of the viral RNA-dependent RNA surface of B cells and in the population of secreted polymerase, which frequently introduces missense mutations. Abs is enormous, but increasingly, we are acquiring This occurrence is challenging because antigenic drift is thus the technical capability to interrogate Ab repertoires caused by accumulating point mutations in the genes encoding in great detail. These Ab technologies have been espe- HA and NA. Some of these mutations encode escape mutations cially pointed at understanding the complex issues of for Abs, and these viruses can be selected over time and enriched immunity to infection and disease caused by influenza in the population. Gradual genetic drift in HA and NA genes virus, one of the most common and vexing medical causes the antigenic variation that reduces the protective effect Downloaded from of seasonal influenza vaccines. Genetic drift in influenza occurs problems in man. Influenza immunity is particularly in- in a direction over time (1), such that older individuals possess teresting as a model system because the antigenic diver- immunity to older strains, in patterns that can be recognized by sity of influenza strains and proteins is high and the decade of birth. Human repertoire studies suggest that the constantly evolving. Discovery of canonical features potential diversity of the human Ab repertoire far exceeds that in the subset of the influenza repertoire response that of the influenza Ag diversity, but the problem for vaccine pre- http://www.jimmunol.org/ is broadly reactive for diverse influenza strains has vention of new strains is a matter of timing. Our current in- spurred the recent optimism for creating universal influenza vaccine strategy is to make educated guesses about the fluenza vaccines. Using new technologies for sequencing likely dominant drifted strains based on molecular epidemiol- Ab repertoires at great depth is helping us to understand ogy studies and then to manufacture trivalent (with H1N1, the central features of influenza immunity. The Jour- H3N2, and B Ags) or quadrivalent (adding a second type B nal of Immunology, 2019, 202: 368–373. strainAg)vaccinesstarting∼6 mo before the season. In some cases, the dominant drifted strain can be mismatched antigen-

ically, leading to suboptimal efficacy. Broader human Ab re- by guest on September 25, 2021 he B cell repertoire diversity (D) gene studies with sponses are desirable. Understanding the genetic and structural influenza are interesting because of the wide extent of basis for broadly protective Abs is a major current goal of the T influenza variability. Influenza virus exhibits a signifi- influenza immune repertoire field. cant challenge to the human immune system because of antigenic variability in field strains. The virus achieves genetic and Concepts of repertoire antigenic D by two principal genetic mechanisms resulting in The vocabulary surrounding the concept of repertoire is antigenic shift and antigenic drift. Influenza causes periodic variously applied. In a broad sense, a repertoire is the collection human pandemics because the segmented viral genome allows of specificities that can comprise the variable (V) gene re- the creation of new viruses during coinfection of cells with ceptors in the adaptive immune system. Vaccine scientists and viruses of two different antigenic subtypes. Genetic reassortment infectious diseases investigators typically envision the adaptive of the segments mixed during coinfection with avian, swine, and immune repertoire as a functional system with diverse pat- human viruses allows complete changing of the surface proteins terns of antigenic recognition. In this sense, the influenza hemagglutinin (HA) and neuraminidase (NA) to subtypes never repertoire is the collection of clones that recognize particular seen by humans, resulting in antigenic shifts. Such shifts or influenza HA or NA molecules or collections of molecules adaptation of avian viruses for human transmission were asso- with varying patterns of epitope recognition, breadth, and ciated with large human pandemics caused by H1N1 in 1918, potency. This type of repertoire study is conducted mostly H2N2 in 1957, H3N2 in 1968, and a new H1N1 in 2009. with proteins and viruses, using binding and virus inhibition The virus has a second genetic mechanism for diversity caused assays and electron microscopic and crystallographic structural

Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN Address correspondence and reprint requests to Dr. James E. Crowe, Departments of 37232; Department of Pathology, Microbiology, and Immunology, Vanderbilt Univer- Pediatrics, and Pathology, Microbiology and Immunology, Vanderbilt Vaccine Center, sity Medical Center, Nashville, TN 37232; and Department of Pediatrics, Vanderbilt 11475 Medical Research Building IV, 2213 Garland Avenue, Nashville, TN 37232- University Medical Center, Nashville, TN 37232 0417. E-mail address: [email protected] ORCID: 0000-0002-0049-1079 (J.E.C.). Abbreviations used in this article: D, diversity; HA, hemagglutinin; indel, insertion/ deletion; J, joining; NA, neuraminidase; NGS, next generation sequencing; RBS, recep- Received for publication October 31, 2018. Accepted for publication November 20, tor binding site; V, variable. 2018. This work was supported by National Institutes of Health Grant U19 AI117905, U.S. Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 Department of Health and Human Services Contract HHSN272201400024C, and a grant from the Human Vaccines Project. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801459 The Journal of Immunology 369 determinations. From a more fundamental immunological HA head domain Abs. The most common human Ab responses viewpoint, the adaptive immune receptor repertoire alter- to influenza HA are directed to the head domain, especially the natively can be studied as a genetic system, comprising the RBS. Many head domain–specific mAbs with excellent neu- recombined gene sequences encoding the receptors. This tralizing potency have been isolated. The challenge for head type of repertoire study is conducted with DNA sequencing domain–based immunity is that the helices and loops sur- technologies, especially deep sequencing with next genera- rounding the RBS are hypervariable. Much of the head do- tion sequencing (NGS) techniques. In this article, we will main surface is very mutable, without disturbing the review both types of repertoire concepts, first the functional attachment and fusion functions mediated by HA. As above, concepts and second the genetic concepts. the antigenic drift is directional in both a genetic and an Functional classes of Abs revealed by recent influenza B cell studies. antigenic manner, and the effect of small numbers of amino Protective Abs for influenza are directed to the two surface acid changes can have outsized effects within an antigenic proteins HA and NA. There has been much more Ab and cluster of residues if they facilitate evasion of a dominant B cell work done on the response to the HA molecule than immune response (16). Some Abs bind to the RBS directly NA. The trimeric HA glycoprotein can be roughly thought and block attachment to sialic acid–bearing receptors (an ef- of in antigenic terms as comprising two major domains, the fect that can be mimicked in the hemagglutination inhibition membrane proximal stem domain and the membrane distal assay using RBCs, which are rich in surface sialic acid). a globular head domain. Neutralizing and protective Abs have Changes in HA receptor specificity (from the 2,3 sialic acid

a Downloaded from been isolated that recognize head or stem. binding preference of avian strains to the 2,6 sialic acid preference in human transmissible strains) drives some of the HA stem domain Abs more dramatic changes in the HA head domains, especially in The recognition of human Abs to the stem region is relatively the RBS. When a major shift in receptor specificity occurs, new. When the avian H5N1 viruscrossedspeciesfrombirds only a small subset of broadly neutralizing Abs is likely to to humans and revealed its high pathogenicity nature, many bind in such a way as to accommodate these changes (17). vaccinologists and immunologists committed to finding The driving forces for maintenance or shift of preferred sialic http://www.jimmunol.org/ prevention and treatment approaches for H5N1 viruses. acid receptor binding and the pressure on the HA RBS region Remarkably, some humans with prior exposure to seasonal to evade neutralizing Ab interact, exhibiting complex network influenza viruses but not avian viruses exhibited seropositivity effects (18). to H5 Ags, which was unexpected. Soon thereafter, stem- Major antigenic sites on the HA head have been designated reactive human mAbs were reported that exhibited cross- based principally on early murine mAb epitope mapping. The reactivity for H5 and H1 HA molecules or broader (2–5). legacy nomenclatures for Ab recognition of these sites on HA The discovery of the stem epitope that is the target for uses slightly different numbering of amino acids for H1 and broadly protective Abs has excited the influenza research H3. The designations for antigenic sites also differ. For H1, the by guest on September 25, 2021 community because of the potential to achieve broadly sites were named during mapping of murine mAbs on the HA protective or universal immunity with a stem Ag vaccine. of the early prototype isolate H1N1 A/Puerto Rico/8/34 The stem region is relatively conserved across subtypes, and (A/PR/8/34) and the sites are designated Sa, Sb, Ca1, Ca2, also the stem domain evolves more slowly under immune and Cb (19). For H3N2 strains, the HA head domain prin- pressure than the head domain (6). The first cross-reactive cipal antigenic sites for murine mAbs were designated A, B, stem Abs to be described in 2009, F10 and CR6261, have C, D, and E (20, 21). The site designations oversimplify the been well characterized, and even broader stem Abs are modes of recognition to some extent, because most human continually reported. A very broad stem Ab that recognizes neutralizing mAbs recognize broad footprints that contact two both antigenic group 1 and 2 influenza type A viruses, FI6, or more of these sites. Our 1918 influenza mAb 1F1 contacts has been described (7). Many stem Abs appear to mediate residues in the Sa, Sb, and Ca2 sites and contacts the RBS protective effects in animal models using Ab-dependent (22). From those same studies of 1918 virus survivors, the cellular cytotoxicity activity, as the potency in virus neu- HA-specific Ab 2D1 that neutralizes the 1918 virus contacts tralization tests of stem Abs as a class tends to be lower than residues in diverse sites, including sites Sb and Ca1 (23). that of human Abs to the head domain especially to the Some HA Abs recognize quaternary structures that are formed receptor binding site (RBS). As a proof-of-principle for stem by a surface that is formed only by two HA protomers in the vaccine design, some human mAbs are in clinical testing in HA trimer (24). As the number of Ab epitopes and Ag–Ab therapeutic challenge studies, including the Crucell stem Abs complexes has proliferated, the use of structure-based descrip- CR6261 and CR8020. Stem vaccines have been developed tions has become a common practice. The immunodominant on these principles of immunity, using HA molecules that structural features around the RBS called the 130-loop, the are chimerized using head domains from virus subtypes that 150-loop, the 190-helix, and the 220-loop incorporate the do not circulate in humans with the stem domain from H1 number of a residue in that structural element to designate subtype, “headless” HA constructs, and other stem-focusing the antigenic site. Representative epitope designations with an- strategies (8–13). If the Abs being tested in clinical trials tigenic and structural nomenclatures for H1 are shown in Fig. 1. mediateasignificantviralreduction,theyalsocouldbe considered for development as prophylactic or therapeutic Canonical genetic features of influenza virus–specific repertoires biological drugs. Similarly, Baker and colleagues (14, 15) Expressed Ab genes are complex in nature because they in- used Rosetta software–enabled structure-based protein de- corporate two (L chain; V or joining [J]) or three (H chain; V, sign to develop small protein molecules that mimic the D, or J) germline genes during recombination at the genomic structure and inhibitory function of the stem Ab CR6261. DNA level. The junctions between V–D (nontemplated 1 or 370 BRIEF REVIEWS: INFLUENZA-SPECIFIC HUMAN ANTIBODY REPERTOIRES Downloaded from

FIGURE 1. Structural features and antigenic sites on inﬂuenza HA protein. Features are mapped onto the HA crystal structure of a 2009 H1N1 inﬂuenza virus (PDB 3LZG). HA is a trimer; the subunits of one protomer are shown on the left-most protomer in white (HA1) or gray (HA2). Conformational http://www.jimmunol.org/ antigenic sites Sa (red), Sb (green), Ca1 (dark blue), Ca2 (light blue), and Cb (purple) are mapped onto an adjacent protomer. The RBS is indicated in pink. The position of amino acids in the 130-loop, 190-helix, and 220-loop are indicated in yellow. Contact residues for a typical stem Ab are indicated in cyan.

N1 region) and D–J (nontemplated 2 or N2 region) joins are hypervariable because isoforms of the enzyme TdT can FIGURE 2. Schematic representation of genetic repertoire patterns typical by guest on September 25, 2021 remove some 39 end V, 59 or 39 end D, or 59 end J gene in clonal lineages. (A) Clonal divergence from the unmutated common an- segment nucleotides or add on nucleotides to V, D, or J gene cestor sequence from the original recombination occurs by the addition of segment ends that are nontemplated (N additions) or copied somatic mutations and indels. (B) Convergence occurs in clonal lineages when as palindromic sequences to the ends of the V (derived two different clonotypes (which differ in V, junction, and/or J genes at the original recombination) achieve a similar sequence or structure by introduc- from the ends of V, called P additions). The collection of tion of somatic mutations. (C) Both lineage divergence and subsequent features encoded by the choice of V, D, and J segments and convergence can occur within a single clonotype. the nontemplated N1 and N2 regions could generate com- binatorial and junctional diversity on the order of ∼1 3 1011 Ab H chains alone. Ab genes also undergo somatic hyper- AVH chain gene that is associated with influenza responses. When mutation, especially during memory responses in the germinal frequent use of an Ab H chain V gene is associated with centers following secondary exposure to Ags, resulting in Ag specificity, this observation typically suggests that the point mutations or insertions/deletions (indels) that encode germline-encoded HCDR2 loop is inherently fit for interacting somatic variants of the original recombination. Clearly, shared with the target Ag. VH1-69 is an unusual Ab H chain gene (or “public”) Ab V gene usage is important in some responses segment, because some alleles of this gene encode for a that are commonly observed, but also somatic hypermutation loop that is unusually hydrophobic for a loop projecting into plays an important role in achieving Ag-specific responses. solute and thus are inherently capable of interacting Interestingly, somatic mutations cause genetic divergence with recessed hydrophobic pockets on Ags. VH1-69–encoded within one clonal lineage, but also mutations can create Abs are identified frequently in virus-specific repertoires for similar Ab gene sequences in independent clonal lineages diverse viruses because of this property. The influenza HA through convergent evolution (25) (Fig. 2). Subjects vacci- stem region possesses a shallow hydrophobic pocket, and the nated against influenza virus show convergent Ab rearrange- prototypic stem Abs that launched the current “universal flu” ments (26). This type of sequence convergence from diverse movement were encoded by VH1-69 with an allele encoding a clonotypes into common sequence motifs in sequence from Phe residue at the tip of CDR2. Use of the VH1-69 gene individuals who have received the same Ag exposure also has segment is not sufficient to identify HA stem Abs as been reported for other Ags. Interestingly, each of these ge- additional residues are required in other regions of the Ab to netic features (common V or D gene usage and convergent interact with the stem. Also, now broad stem Abs encoded by amino acid motifs and indels) has been described to con- other VH genes have been reported, so also it can be said that tribute to common features of influenza-specific repertoires, as VH1-69 is not necessary to make a stem Ab. Nevertheless, the discussed below. use of this gene is a very common feature in many stem Abs. The Journal of Immunology 371

A DH chain gene that is associated with influenza responses. The group. One Ab, F045-092, was reported that uses both the central loop structure of the H chain CDR3 is encoded by the Phe aromatic residue interaction and carboxylate side chain of D gene. In many memory B cells, the sequence of the D gene an Asp residue to interact with the RBS (37, 38). from the original recombination is so mutated that a D gene is One way to achieve breadth of interaction with the HA is to no longer recognizable. Therefore, HCDR3 aa motifs reduce the contact region of the Ab with HA. The Ab C05 is an (discussed below) are more recognizable than a common D Ab that accomplishes this feat using an especially long HCDR3 gene. Still, there are likely to be a number of D germline loop that interacts with the conserved residues in the base of genes that encode sequences that enable initial low-affinity the RBS but not the V amino acids around the rim of the binding in the germline configuration to HA or NA. One RBS (39). example that has been recognized is the use of the D3-9 gene in broadly neutralizing Abs that recognize the HA The occurrence of indels in clonal lineages stem (27). Much of the interaction surface of some stem Insertions and deletions in Ab gene sequences occur in a subset Abs is encoded by D3-9, including in Abs that bind the of sequences in Ab genes from memory B cells. These sequence dominant stem epitope in differing orientations and alterations result from DNA duplication events following binding poses. Remarkably, some stem Abs that incorporate DNA strand breaks that occur during the somatic hyper- D3-9 use the gene segment in differing reading frames in the mutation process. Many insertions occur at mutational hot recombined sequence. spots for error-prone DNA polymerases. In some cases, the

HCDR3 amino acid motifs that are associated with influenza insertion enhances the affinity of binding of an influenza Ab. Downloaded from responses. In some respects, the Ab fold that forms the frame- For example, we isolated the influenza H1 HA-specific mAb work and even the CDR loops serve simply as scaffolds to 2D1 from a circulating B cell in the peripheral blood of a 1918 deliver a very small number of amino acids that function as the influenza pandemic survivor (40). In addition to point mu- effector molecules for virus binding and inhibition. Patterns of tations, the Ab gene has a 9-bp insertion in the framework 3 preferred amino acids in the Ag binding surface of Abs most of the H chain, adjacent to the HCDR2. We showed that often has been identified in Abs that insert a CDR loop into the the structure of 2D1 in complex with the HA of the 1918 http://www.jimmunol.org/ RBS. Contact residues in the RBS exhibit a high level of pandemic H1N1 influenza virus exhibited an unusual con- conservation because this region must preserve the capacity to formation and relative orientation of the HCDR1 and bind sialic acid–bearing receptors. Because the RBS has a HCDR2 loops in this neutralizing mAb (23). The insertion limited diversity of residues in positions that contact sialic causes displacement of the HCDR1, resolving a steric conflict acid, it stands to reason that only a few selected amino acids in the parental Ab–Ag interaction by moving a CDR away on the Ab loops can mediate the interaction with this area of from the region of interaction, rather than creating a new the RBS, and the Ab loops should in some way mimic the interaction in the Ab–Ag interface (41). chemical or structural features of sialic acid. Indeed, several by guest on September 25, 2021 specific amino acid patterns have been identified in Ab CDRs The coming wave of influenza-specific genetic repertoires based on as comprising canonical motifs for interaction with the in- NGS studies fluenza HA RBS. One of these canonical modes of interaction Bulk Ab V gene repertoire sequencing is interesting for is the display of an aspartate residue on a CDR that provides a comparing individuals with healthy versus disease states, but favorable charge interaction with amino acids in the RBS. The also now we are deploying approaches for using Ab NGS backbone atoms of aspartic acid can mimic the acetamido repertoire technologies to study Ag-specific responses, such as groups of the RBS, and a carboxylic acid mimics the car- those to influenza virus vaccination or infection. NGS data boxylate of sialic acid. Many mAbs the interact with the RBS from B cells in the circulating peripheral blood samples of possess an aspartate or dipeptide with an aspartic acid–hy- recently vaccinated subjects show that certain Ab sequences are drophobic motif positioned properly to interact with the RBS overrepresented in circulation at those time points, and thus position (28). A number of H1 HA-specific neutralizing Abs these clonotypes are likely to be specific for the recent exposure. have been described with this feature, including the CH65 Plasmablasts circulate briefly for a few days during a period (29, 30) and 5J8 (31, 32) Abs, which are especially interesting about a week after influenza vaccination. These cells have a very as a pair because the binding pose of each on the HA high copy number of Ab mRNA per cell, and thus these se- determines the breadth of molecular recognition for diverse quences are overrepresented in bulk sequence repertoires at H1 HA molecules. The complementary breadth of these two those time points. Analysis of NGS libraries made from serial Abs determined by the angle of approach to the RBS likely blood samples taken at time points before and soon after would cover most H1 strains (33). The presence of an aspartate vaccination are especially helpful for this type of analysis, residue on a CDR inserting into the RBS is not sufficient to because the presence of clonally expanded B cells can be mediate the interaction, as structure of the H5.3 Ab with H5 inferred by the presence of the same Ab gene rearrangements HA shows that this CDR orients the aspartate away from the in libraries from sequential time points (26, 42). So, over- RBS even though it is near the tip of the RBS (34, 35). representation (unexpectedly high frequency) is a principal A second canonical mode of interaction with the RBS is tool for identifying putative Ag-specific clones in such rep- mediated by display of a Phe or Tyr aromatic residue on an Ab ertoire studies. A second tool is the identification of sequence loop, which can form p–p interactions between the HA and similarity or “convergence” of Ab gene rearrangements in mAb CDR (36). This interaction also uses a CDR to mimic repertoires from different individuals with the same exposure, sialic acid binding to HA. The Ab protein remarkably mimics such as two individuals who have received the same seasonal the interaction of the carbohydrate sialic acid by placing its influenza vaccine. If two H chain Ab V gene sequences are amino acid structure into the RBS with a backbone carbonyl encoded by the same inferred germline VH and JH gene 372 BRIEF REVIEWS: INFLUENZA-SPECIFIC HUMAN ANTIBODY REPERTOIRES segments and have identical length CDR3s with identical or humans, and the use of NGS to define genetic repertoires, very similar CDR3 sequences, these sequences can be con- have revealed that the human immune system can generate sidered members of a single clonotype. Each somatic variant very broad and potent Abs. Repertoire and human mAb of the clonotype may be considered a different clone. Lineages studies now enable researchers to isolate tens of thousands of of clones that are assigned to a single clonotype can be con- naturally occurring human Abs that inhibit influenza. By structed by alignments, which may suggest these clones were searching through these repertoires and using new technolo- derived from a single B cell with an unmutated sequence (the gies in synthetic genomics, we are increasingly able to express computationally inferred sequence is sometimes termed the and characterize thousands of Abs to identify broad and po- reverted unmutated ancestor or unmutated common ances- tent Abs that could be used for prophylaxis or therapy against tor). When sequences from two different individuals with a a broad array of influenzas. Furthermore, computational de- common exposure share the same inferred germline VH and sign of novel Ags, based on the plethora of Ag–Ab complexes JH gene segments and have identical length CDR3s with that are being reported, allows us to recapitulate the con- identical or very similar CDR3 sequences, we can term these formational of minimal protective epitopes with synthetic sequences to be members of a “public clonotype” (43). We vaccine construct. Such findings point the way toward de- believe such sequences are of particular interest because they velopment and testing of broader and more potent experi- can reveal canonical features of common responses in pop- mental influenza vaccines. ulations, and influenza-specific public clonotypes could be interesting to target with structure-based reverse vaccine de- Disclosures Downloaded from sign programs for universal influenza vaccines that would be J.E.C. has served as a consultant for Sanofi, Novavax, and Takeda and is on the effective in a broad distribution of subjects. Proof-of-principle Scientific Advisory Boards of CompuVax, GigaGen, and Meissa Vaccines, is a has been established for this reverse vaccinology approach in recipient of previous research grants from Moderna and Sanofi, and is founder previous studies of another respiratory pathogen, respiratory of IDBiologics, Inc. syncytial virus (44).

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