Wide prevalence of heterosubtypic broadly neutralizing human anti-influenza A antibodies

Abstract

Background: Lack of life-long immunity against influenza viruses represents a major global health care problem with profound medical and economic consequences. A greater understanding of the broad-spectrum "heterosubtypic" neutralizing human antibody (BnAb) response to influenza should bring us closer toward a universal influenza vaccine.

Methods: Serum samples obtained from 77 volunteers in an H5N1 vaccine study were analyzed for cross-reactive antibodies (Abs) against both subtype hemagglutinins (HAs) and a highly conserved pocket on the HA stem of Group 1 viruses. Cross-reactive Abs in commercial intravenous immunoglobulin were affinity purified using H5-coupled beads followed by step-wise monoclonal antibody competition or acid elution. Enzyme-linked immunosorbent assays were used to quantify cross-binding, and neutralization activity was determined with HA-pseudotyped viruses.

Results: Prevaccination serum samples have detectable levels of heterosubtypic HA binding activity to both Group 1 and 2 influenza A viruses, including subtypes H5 and H7, respectively, to which study subjects had not been vaccinated. Two different populations of Broadly neutralizing Abs (BnAbs) were purified from intravenous immunoglobulin by H5 beads: ~0.01% of total immunoglobulin G can bind to HAs from both Group 1 and 2 and neutralize H1N1 and H5N1 viruses; ~0.001% is F10-like Abs directed against the HA stem pocket on Group 1 viruses.

Conclusion: These data--to our knowledge, for the first time--quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels.

Figure 1.

Serological study of 77 paired pre- and post-immune serum samples from H5N1 vaccinees. A–D, Enzyme-linked immunosorbent assays (ELISAs). Serum samples were serially diluted and applied to H5-VN04– (A and B), H3-A2/68– (C), or H7-NL03– (D) coated ELISA plates and the binding of immunoglobulin (Ig) M or IgG in serum samples to hemagglutinin (HA) proteins were determined using secondary Ab HRP-anti-human IgM (A) or IgG (B - –D). The binding levels are shown as optical density at 450 nm (OD 450). E, microneutralization assay (MN) titer against H5N1 virus; y-axis shows the Log₂ MN titer. F, Competition ELISA. Pre- and post-immune serum samples from H5N1 vaccinees were tested for their competition activity against a Group 1-specific BnAb, F10, binding to H5-VN04. Serially diluted serum samples were mixed with 3 ng/mL Bio-F10 and applied to H5-coated ELISA plates. The serum competition for binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin. The serum competition activity is shown as percentage of inhibition. For all panels except panel E, data at 1 representative serum dilution are shown, as follows: panel A, 1:270; panel B, 1:5120; panels C and D, 1:2430; and panel F, 1:90. For all panels, data are shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles, respectively. The dots above and below the whiskers are data points beyond the 95th and 5th percentiles.

Figure 2.

Heterosubtypic antibodies against influenza A viruses in intravenous immunoglobulin (IVIG). H5-VN04 was immobilized on magnetic beads (H5-beads) and the beads were used to affinity purify antibodies (Abs) from the IVIG sample. F10-like Abs and the remaining H5-bound Abs were purified separately. F10-like Abs were purified by Bio-F10 competition elution followed by multiple steps of streptavidin-beads absorption to eliminate Bio-F10 completely. The remaining H5-bound Abs on the H5 beads were eluted with standard acid elution method followed by a complete absorption of Bio-F10 using Streptavidin-beads as well.An enzyme-linked immunosorbent assay (ELISA) detecting Bio-F10 was used to confirm that no residual Bio-F10 remained in either Bio-F10-eluted or the acid-eluted samples. The binding activity of these purified Abs to H1-NY18 (A), H5-VN04 (B), H3-A2/68 (C), and H7-NL03 (D) was measured by ELISA at different serially diluted concentration. The neutralization activity of these samples was measured using neutralization assay with pseudotyped viruses of H1-1918 (E) and H5-TH04 (F); 80R was used as a negative control mAb that is specifically against the spike protein of severe acute respiratory syndrome coronavirus [24]. OD 450, optical density at 450 nm.