Lamprey VLRB response to influenza virus supports universal rules of immunogenicity and antigenicity

Abstract

Immunoglobulins (Igs) are a crown jewel of jawed vertebrate evolution. Through recombination and mutation of small numbers of genes, Igs can specifically recognize a vast variety of natural and man-made organic molecules. Jawless vertebrates evolved a parallel system of humoral immunity, which recognizes antigens not with Ig, but with a structurally unrelated receptor called the variable lymphocyte receptor B (VLRB). We exploited the convergent evolution of Ig and VLRB antibodies (Abs) to investigate if intrinsic chemical features of foreign proteins determine their antigenicity and immunogenicity. Surprisingly, we find lamprey VLRB and mouse Ig responses to influenza A virus are extremely similar. Each focuses ~80% of the response on hemagglutinin (HA), mainly through recognition of the major antigenic sites in the HA globular head domain. Our findings predict basic conservation of Ab responses to protein antigens, strongly supporting the use of animal models for understanding human Ab responses to viruses and protein immunogens.

Keywords: antibodies; antigenicity; hemagglutinin; immunodominance; immunology; infectious disease; influenza; lamprey; microbiology; mouse.

Figure 1.. Origin of variable lymphocyte receptor B (VLRB) in jawless vertebrates.

Jawless and jawed vertebrates last shared a common ancestor ∼550 Mya. VLR genes are only in jawless vertebrates, whereas Immunoglobulin (Ig) genes are only in jawed vertebrates. However, both jawed and jawless vertebrates have a lymphocyte-based adaptive immune system suggesting that the genetic programs necessary for lymphocyte development originated in a common ancestor before the antigen receptor genes. Cytidine deaminases are expressed by lymphocytes in both jawed and jawless vertebrates and may have originated in a common ancestor; activation-induced cytidine deaminase (AID) and cytosine deaminase (CDA). Structures of prototypic VLRB (Top, PDB: 3e6j) and IgG (Bottom, PDB: 1Igt) are shown to the right, along with cartoons of their secreted forms. Regions of antigen recognition are shaded in blue or green. In red are the concave antigen binding residues of VLR and the complementarity determining regions (CDRs) of Ig. DOI: http://dx.doi.org/10.7554/eLife.07467.003

Figure 2.. Lamprey make VLRBs specific for influenza A virus (IAV) after immunization with non-adjuvented, UV-inactivated virus.

(A) Left, whole lamprey plasma (5 µl of naïve or immunized three times with PR8 [L9]) electrophoresed on a 4–12% SDS PAGE gel probed with anti-VLR monoclonal Ab (mAb) by immunoblotting. VLR monomers (∼35–45 kDa) are naturally cross-linked by disulfide bonds to form VLR multimers >100 kDa. Right, lane intensity measured by ImageJ for immunoblots of 2 µl Naïve (3 animals) or PR8 immunized (8 animals) probed with anti-VLR 2° Ab. Each point represents one animal. Data were analyzed by two-tailed t-test using PRISM software (**p < 0.01). The mean signal from immunized plasma was 7.4 ± 1.8 × greater than the naïve mean. (B) Equal protein quantities of purified virus were adsorbed to ELISA plates and probed with lamprey plasma from either immunized (n = 9) or naïve (n = 2) animals. Data were analyzed by two-way ANOVA followed by Bonferroni multiple comparison using PRISM software (****p < 0.0001). DOI: http://dx.doi.org/10.7554/eLife.07467.004

Figure 3.. Immunodominance hierarchy against IAV for lamprey and mice is the same.

(A) Scheme depicting reassortant virus components used for experiments in this figure. (B) Equal protein quantities split (HA/NA/M1) and core (NP/M1) antigens bound to ELISA plates were tested for binding to anti-PR8 mouse sera or lamprey plasma. Mouse data are representative of two mice with n = 4 independent experiments. Lamprey data are from three pooled animals with n = 4 independent experiments. (C) Same as Figure 3B, but using anti-HK lamprey plasma. Data are from three pooled animals with n = 4 ELISA replicates. DOI: http://dx.doi.org/10.7554/eLife.07467.005

Figure 3—figure supplement 1.. Detergent-split reassorted viruses.

Western blot of reassorted virus components probed with mouse mAbs specific for H1N1 hemagglutinin (HA) + neuraminidase (NA) or cross-reactive nucleoprotein (NP), M1, and M2 dimer. DOI: http://dx.doi.org/10.7554/eLife.07467.006

Figure 3—figure supplement 2.. PR8 antibodies (Abs) bind HA and NA but not M influenza proteins.

HeLa cells transfected with pDZ control plasmid or PR8 HA, NA, M1/2, NP, or NS1 plasmids were stained with their respective mouse mAb, mouse sera, or lamprey plasma raised against PR8 virus. Both lamprey plasma and mouse sera bound HA and NA. The mouse sera bound NP, but the lamprey plasma did not. Neither bound M1/2 proteins. Neither immune sera stained NS1 protein. Note naïve lamprey plasma has less total VLRB and a lower background than immune lamprey plasma. Data is representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.07467.007

Figure 3—figure supplement 3.. PR8 immunized lamprey plasma binds purified NP protein, but not purified M1 by ELISA.

Purified NP or M1 protein were adsorbed to ELISA plates and probed with mouse sera (n = 2) or lamprey plasma from either immunized (n = 4) or naïve (n = 2) animals. Data were analyzed by two-way ANOVA followed by Bonferroni multiple comparison using PRISM software (*p < 0.0001). Both PR8-immune mouse sera and lamprey plasma bound NP better than unimmunized control. Neither bound M1 protein. mAbs M2-1C6 for M1 and HB65 for NP are shown to confirm proper folding of purified protein on plate. DOI: http://dx.doi.org/10.7554/eLife.07467.008

Figure 4.. Lamprey VLRBs bind to hemagglutinin and neutralize infection.

(A) Plasma from PR8-immunized lamprey inhibits PR8 hemagglutination at a 1:30 plasma dilution, but did not inhibit hemagglutination by either HK or B/Lee at any dilution. Data are representative of two experiments. (B) MDCK cells were infected with an MOI 0.07 of PR8 in the presence of titrated mAb supernatants (H17L2 against PR8 or control 1.2F4 against influenza B/Lee) or lamprey plasma (L9 vs Naïve). After 8 hr cells were fixed, double-stained with anti-HA and anti-NP Igs. Cells positive for either HA or NP by flow cytometry were considered infected. Data from four independent experiments were normalized to control for different percentages of infection between experiments and fit to a variable dose–response curve. The best-fit, calculated infectious dose 50 (ID50) was significantly lower for both the immunized plasma and PR8 specific Ig (***p < 0.001). DOI: http://dx.doi.org/10.7554/eLife.07467.009

Figure 5.. Paratope signature of VLRBs.

(A) Contact residues determined by the crystal structures of VLRBs in complex with their antigens are highlighted in orange. RBC36 against H trisaccharide (3E6J); aGPA.23 against TF disaccharide (4K79); VLR4 against BclA (3TWI); and VLRB.2D against HEL (3G39). (B) Enrichment or shortfall of each amino acid in the contact residues relative to the total amino acids found in the full VLRB was determined from the ratio of frequency of each amino acid in contact residues vs the frequency in the total VLRB sequence. Leucine was excluded from the analysis as it is the major structural amino acid of VLRBs. No M, K, or P were found among the contact residues. Shortfall was determined by estimating, based on total VLRB frequency, how many amino acids would be there if the amino acid distribution was even throughout the VLRB. DOI: http://dx.doi.org/10.7554/eLife.07467.018