Influenza viruses infect ten of millions of people each year. To conquer a flu infection, the human immune system develops antibodies that hasten recovery and prevent future flu infections. Unfortunately, flu is constantly changing in response to the human immune response, and antibodies induced by previous infection or vaccination provide partial protection, at best, against new strains.

An ideal flu vaccine would stimulate the immune system to produce antibodies that protect against all future strains of influenza. Most human antibodies that are induced by influenza target a part of the virus called the hemagglutinin, which attaches the virus to cells to start a flu infection. Some hemagglutinin-specific antibodies recognize many strains of influenza, but individuals do not produce enough of these antibodies to prevent infections with new strains. A basic understanding of what drives the production of different types of antibodies is important to devise vaccines that produce broadly effective antibodies for flu and for other viruses and pathogens that have proven to be difficult vaccine targets.

To better understand the rules of antibody generation, Altman et al. compared antibodies produced in response to flu in mice and lampreys. Lampreys are a primitive fish that branched off from other vertebrates (animals with a backbone, like you) 550 million years ago and developed their own system of antibody recognition based on a completely different template. Despite this, Altman et al. found that the antibody response of mice and lampreys to flu is remarkably similar. Of several potential viral targets, antibodies from both mice and lampreys were predominantly directed against the hemagglutinin. Of numerous potential locations on the hemagglutinin, mouse and lamprey antibodies predominantly recognized the same region.

These similarities suggest that the specificity of antibodies is based largely on the properties of the virus, and varies little with the properties of the responding organism. Most importantly, this supports the conclusion that studies in mice and other mammals are likely to accurately predict how humans will respond to vaccines for viruses and other pathogens.

A cornerstone of modern biology and medicine is that humans and other mammals generate a highly specific humoral immune response when confronted with microbes or toxins, providing the basis for vaccination against many pathogens. Originally defined as a functional principle (e.g., ability to protect animals against injection with a toxin), the responsible substance was termed an antibody (Ab), which we now know to consist of immunoglobulins (Igs) in jawed vertebrates. After more than a century of steady progress, a basic molecular understanding of Ig function is at hand (Ramaraj et al., 2012; Georgiou et al., 2014); surprisingly little is known, however, regarding the basic rules of immunogenicity. In responding to viruses, why are some proteins more immunogenic than others? Why do Ig responses focus on certain regions of proteins? To what extent is this due to the specific features of Ig structure and combining site chemistry or repertoire limitations imposed by self-tolerance?

Here we address these issues by comparing the Ab responses of lampreys and mice to influenza A virus (IAV), a model antigen of high practical relevance. IAV is composed of four major structural proteins: two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), embedded in a lipid envelope, lined by the matrix protein (M1), which encases the nucleoprotein (NP) coated viral genome. Due to its importance as the target of protective Igs (Couch and Kasel, 1983), HA is probably the most intensively characterized immunogen/antigen. Most HA-specific Igs with virus neutralizing activity bind to or bridge 5 antigenic regions in the globular domain (termed Sa, Sb, Ca1, Ca2, and Cb), which surround the sialic acid receptor site that attaches HA to host cells (Gerhard et al., 1981). Variation in these sites as IAV evolves in the human population (‘antigenic drift’) prevents effective IAV vaccination, necessitating frequent changes in vaccine formulation. The recent discovery that humans can generate protective Igs to conserved structures in the membrane-proximal HA stem have raised hopes of more effective vaccination if stem responses can be augmented using appropriately designed vaccines (Laursen and Wilson, 2013). Better understanding the rules of immunogenicity could inform these critical efforts.

We characterized the variable lymphocyte receptor B (VLRB) response of lampreys, which along with hagfish, are the only known living jawless vertebrates (cyclostomes). Cyclostomes branched evolutionarily from jawed vertebrates approximately 550 million years ago (Mya) (Figure 1). Pioneering studies from the Good laboratory established that lampreys generate Ab responses to protein and carbohydrate immunogens (Finstad and Good, 1964). More than 40 years later, Cooper and colleagues discovered that lamprey Abs, VLRBs, bear no relationship to Igs, but rather are structurally similar to Toll-like receptors (Pancer et al., 2004). In place of RAG-mediated V(D)J recombination, VLRB diversity is generated using a gene assembly mechanism reminiscent of the activation-induced cytidine deamine-catalyzed gene conversion mechanism used to diversify Ig genes in birds and some mammals (Figure 1).

Figure 1

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The germline VLRB gene is incomplete because the invariant 5′ and 3′ coding sequences are separated by non-coding intervening sequences (Pancer et al., 2004). Several of the hundreds of leucine rich repeat (LRR)-encoding genes flanking the VRLB gene are copied into the gene to generate an in-frame, functional VLRB gene during lymphocyte development (Nagawa et al., 2007; Rogozin et al., 2007; Alder et al., 2008). This generates a VLRB repertoire with diversity comparable to Igs (Alder et al., 2005). VLRB encodes for single-chain, crescent-shaped proteins that bind to antigens with a concave surface composed of multiple LRR β-strands and a C-terminal variable loop (LRRCT) (Kim et al., 2007; Han et al., 2008; Herrin et al., 2008; Velikovsky et al., 2009; Kirchdoerfer et al., 2012; Deng et al., 2013; Luo et al., 2013). In contrast, Igs consist of a heavy and a light chain, each of which contributes three complementarity determining region loops to form a structurally distinct antigen-binding site (Figure 1).

To probe the VLRB response to IAV, we collected blood from lamprey larvae immunized three times with inactivated, purified prototypic H1N1 PR8 IAV. Polyclonal VLRB primarily migrates on an SDS-PAGE gel as disulfide-linked multimers under non-reducing conditions and as monomers in the presence of reducing agents (Alder et al., 2008; Herrin et al., 2008). As seen previously (Alder et al., 2005), monitoring plasma VLRB by immunoblotting revealed that unlike mammalian Ig, where immunization induces only minor increases in substantial serum levels, VLRB levels increase ∼sevenfold (Figure 2A). ELISAs revealed that each immunized lamprey generated VLRBs that bind PR8, but not a similar amount of plate-bound parainfluenza-3 virus, which is a genetically and serologically completely distinct enveloped virus, but similar in architecture and complexity to IAV (Figure 2B).

Figure 2

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To determine the immunogenicity of IAV structural proteins, we measured serum from PR8-immunized mice and lamprey via ELISA using either detergent soluble proteins from purified virus (HA, NA, M1), or the detergent insoluble core (NP, M1, small amounts of other non-glycoproteins [Hutchinson et al., 2014]) (Figure 3A and Figure 3—figure supplement 1). This revealed that in both mice and lamprey, more than 90% of the functional ELISA response is specific for HA and NA, as shown by the large difference in titers between detergent soluble proteins from PR8 (H1N1) vs X31 (H3N2), a reassortant virus with the PR8 internal proteins but serologically distinct HK68 glycoproteins. Genetically isolating HA from NA using the J1 (H3N1, PR8 internal proteins) and P50 (H1N2, HK internal proteins) reassortants shows that upwards of 80% of ELISA-detected Abs are specific for HA in lamprey and mice (Figure 3B). Low binding to X31 and HK soluble proteins, which contain significant amounts of M1 (Figure 3—figure supplement 1), indicate that M1 is negligibly immunogenic (note that internal viral proteins from H3 and H1 viruses are antigenically highly conserved). Further, the low serum titers against PR8 cores confirm that only a small fraction of Igs are specific for NP or a low abundance internal virion component.

Figure 3 with 3 supplements see all

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Reciprocal immunization of lampreys with HK virus (Figure 3C) confirmed the dominance of HA. This experiment also provides a direct control for the specificity of lamprey VLRB for H1N1 vs H3N2 glycoproteins. Flow cytometry of cells expressing either HA, NA, NP, M1/M2 or NS1 (which is present in virions [Hutchinson et al., 2014]) from transfected cDNAs stained with lamprey plasma showed that PR8 induced detectable VLRB responses to HA and NA but not NP, M1, M2, or NS1 (Figure 3—figure supplement 2). Similarly, mouse serum Ig was positive against HA and NA and negative for M1 + M2, although there was a response to NP. While lamprey plasma did not bind plasmid expressed NP by flow, in ELISA, both immune lamprey plasma and mouse sera bound plated NP, but neither bound M1 (Figure 3—figure supplement 3). The lack of NP binding in the flow assay is most likely spurious; due to limited VLRB access to NP within permeabilized cells, or low signal.

Next we examined the functionality of the lamprey anti-HA response as revealed by hemagglutination inhibition (HI) or infectivity neutralization assays. HI measures the ability of Abs to block HA-mediated IAV attachment to erythrocyte surface terminal sialic acids. PR8-immunized lamprey plasma gave HI titers of 1:30 against PR8, but <1:5 against an H3N2 IAV and B/Lee, an influenza B virus, which is serologically totally distinct from IAV (Figure 4A). Immune lamprey plasma also significantly inhibited PR8 infectivity in MDCK cells relative to naïve plasma (Figure 4B).

Figure 4

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The vast majority of Igs that inhibit IAV hemagglutination and viral infectivity bind the HA globular domain. To test if this is also the major target of lamprey VLRBs, we used a panel of PR8 viruses with 3, 6, 9, or 12 amino acid substitutions located among the five defined antigenic sites (Das et al., 2013). ELISAs using intact wild-type or mutant viruses as immunoadsorbents show that lamprey plasma similarly detect antigenic drift in the globular domain, with a significant loss of binding with six substitutions and a loss of ∼60% of binding with 12 substitutions (Table 1). Similar binding is seen with mouse, guinea pig, and chicken PR8 immune seras (Table 1—source data 1). Factoring in the contribution of anti-NA Abs to the signal, this indicates that the bulk of mouse, guinea pig, chicken and lamprey HA-specific Ig and VLRB recognize the globular head domain, and therefore that the globular domain is the immunodominant antigen for both mice and lamprey. As predicted, anti-PR8 VLRBs could easily distinguish antigenic drift in H1N1 isolates from the 1940s and 1950s (Table 1 and Table 1—source data 2).

The loss of lamprey VLRB and mouse Ig binding in similar proportions to the PR8 HA-head domain mutants implies that each recognizes similar epitopes and is subject to similar physicochemical rules of binding. To compare Ig and VLRB footprints, we competed lamprey plasma against mouse monoclonal Abs (mAbs) specific for defined HA antigenic sites for binding to PR8-coated ELISA wells. Although IAV immunization elicited VLRB responses in all lampreys tested, we selected lampreys with the highest titers for the competition experiments because these experiments require larger amounts of VLRB. Comparing the relative titers of inhibitory activity provides an approximate measure of binding proximity. Such competition assays are complicated by steric hindrance between Abs binding physically adjacent epitopes as well as more subtle positive and negative conformational effects that occur upon Ig binding to HA (Lubeck and Gerhard, 1982).

Anti-PR8 plasma from lampreys 7 and 9 (L7, L9) competed with the mAbs tested, whereas neither naïve lamprey plasma nor mAbs to an irrelevant antigen competed with the mAbs (Table 2 and Table 2—source data 1). Also, while both L7 and L9 have a similar ELISA titer to whole virus, L9 plasma competes with mAbs specific for each of the five sites, but L7 fails to compete with Sa and Sb mAbs. This demonstrates that VLRB binding to the HA head does not uniformly block binding of all head-specific Igs and, importantly, indicates that fine antigen specificity varies among individual lampreys. We also infer this from the different titers observed against the various mAbs, an effect that is unlikely to be based on mAb affinity, since VLRBs are allowed to bind to HA prior to mAb addition.

To minimize steric effects, we extended these findings using Fab mAb fragment (25 kDa vs 150 kDa for intact Ig). The patterns observed with L7 and L9 were highly similar to those obtained with intact Igs. Plasma from an additional lamprey (L29) also effectively blocked each of the four Fabs tested (Table 2 and Table 2—source data 2). L29 plasma did not, however, block binding of either a representative mouse or human mAb specific for the stem region with broadly neutralizing activity, suggesting that, as in mammals, the stem region is poorly immunogenic in lampreys (Table 2 and Table 2—source data 3). Rather, as in man and mouse, the lamprey immune system focuses on the globular domain, and further, recognizes highly similar epitopes.

Our findings support the conclusion that Ig and VLRB demonstrate similar specificity for HA head epitopes. Although steric issues limit interpretation of the competition experiments, the common effect of amino acid substitution on VLRB and Ig binding argues strongly for convergent recognition of highly overlapping epitopes. Equally surprising is the similarity of the overall immunodominance profile of IAV immunogens in mammals and lamprey responding to inactivated intact virus: the HA globular domain is favored over the stem; HA is favored over NA; and internal proteins, despite their relative abundance in the virion, are weakly (NP) or undetectably (M1) immunogenic.

Our findings extend understanding of the cyclostome VLRB response. We confirmed the Pancer group's finding that total plasma VLRB concentration increases after immunization (Alder et al., 2005). Total Ig can also increase in jawed fish after immunization (Castro et al., 2013; Xu et al., 2013). This differs from mammals, where high levels are maintained constitutively. The ability of lampreys to respond to purified IAV without additional adjuvant indicates that lampreys recognize viral RNA or other viral innate immune activating molecules. We also show in competition assays that individual lampreys mount VLRB responses against different antigenic sites. Whether this is due to genetic, environmental, or stochastic factors is a question for further study. It is well worth noting that there is little information regarding how individuals among outbred groups vary in their Ig response to viruses or other complex antigens.

It is surprising that such structurally disparate molecules as Ig and VLRB receptors recognize the same proteins with overlapping epitopes. These recognition systems have evolved independently for >500 Mya, presumably shaped by different selective pressures from the environment, self-tolerance, and microorganisms. These differences notwithstanding, the four available VLRB-antigen crystal structures reveal many similarities in Ig and VLRB antigen recognition.

In the two VLRB structures with protein antigens (hen egg lysozyme and anthrax coat protein, BclA), the contact area (∼1500 Ų) is in the same range as reported for Igs (1400–2300 Ų), and utilizes the same non-covalent forces (salt bridges, hydrogen bonds, and van der Waals contacts) in similar proportions to mediate binding, which requires close shape complementarity to the antigen (Velikovsky et al., 2009; Kirchdoerfer et al., 2012; Deng et al., 2013). In the VLRB-HEL structure, the relatively small, crescent-shaped VLRB binds to an epitope in the catalytic cleft, whereas larger, dimeric Ig VHVL Abs bind to flatter epitopes away from the catalytic site. Interestingly, structures of single-chain camelid VHH and shark IgNAR have revealed that they also favor the catalytic cleft of HEL that is presumably sterically inaccessible to dimeric VHVL Abs (Velikovsky et al., 2009).

Analysis of hundreds of VLRB sequences has previously revealed a bias towards aromatic amino acids at the variable positions on the concave surface (Velikovsky et al., 2009). In these analyses, less than half of the variable position residues contact antigen. When only the antigen contacting residue frequency is quantified, the amino acids are biased towards Tyr, Trp, Asn, and Asp residues (Figure 5). A similar bias towards these residues in antigen-contacting positions of Ig has also been observed (Mian et al., 1991; Davies and Cohen, 1996; Ramaraj et al., 2012; Robin et al., 2014). These similarities may account for the recognition of similar epitopes. If so, this may also represent the optimal general solution for producing a single family of receptors capable of recognizing what is essentially an infinite array of antigens with high specificity and affinity.

Figure 5

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The universality of Ig and VLRB antigenicity and immunogenicity illuminated by our findings provides strong support for the utility of animal models for understanding human Ab responses to vaccines and other medically relevant immunogens. Perhaps, when it comes to Ab responses, neither mice nor lamprey lie, after all.

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Author details

1. Meghan O Altman

   Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   MOA, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

2. Jack R Bennink

   Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   JRB, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Jonathan W Yewdell

   Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   JWY, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   For correspondence

   jyewdell@nih.gov

   Competing interests

   The authors declare that no competing interests exist.

4. Brantley R Herrin

   Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, United States

   Contribution

   BRH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

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