Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine

Abstract

The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1-H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid-sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus.

Figure 1. Vaccine-binding and H5 pseudotype-neutralizing antibodies in plasma samples collected before and after seasonal influenza vaccination.

Volunteers (A to X) were immunized with seasonal influenza vaccine in 2 consecutive seasons. Plasma and PBMC samples were collected before and 2 weeks after vaccination. (A and B) Vaccine-specific IgG was measured in plasma by ELISA using the homologous vaccine as antigen. Reciprocal EC₅₀ values before (white bars) and after vaccination (black bars) in 2007 (A) and 2008 seasons (B) are shown. (C and D) H5-specific neutralizing titers were measured in the same plasma samples by a pseudotyped neutralization assay against an H5 pseudovirus (A/VN/1194/04). Reciprocal ID₅₀ values before (white bars) and after vaccination (black bars) in 2007 (C) and 2008 (D) are shown. (E and F) Correlation between the increase of vaccine binding titers (E) and H5-neutralizing titers (F) following vaccination in 2007 (x axis) and 2008 (y axis) in the 9 donors that received the seasonal influenza vaccine for the 2 consecutive years.

Figure 2. Detection of heterosubtypic antibodies in plasma samples following seasonal vaccination.

ELISA plates were coated with vaccine or H5 HAs and incubated with serial plasma dilutions, followed by biotinylated FE17 or FE43 mAbs, which were then detected using enzyme-conjugated streptavidin. Shown is the reciprocal plasma dilution that blocks 80% binding (BD80) of heterosubtypic mAbs FE17 and FE43 to homologous HA (A) or heterologous H5 HA (B). Each symbol represents a different individual from Figure 1, A and C. Lines indicate geometric mean values.

Figure 3. Broad neutralization of H1N1 viruses including A/California/07/09 by selected human mAbs.

Serial dilutions of the indicated mAbs (starting at 1 mg/ml) were tested for the capacity to neutralize a panel of H1N1 viruses spanning more than 70 years of antigenic evolution. Shown is the dilution providing 50% protection (ID₅₀).

Figure 4. Prophylactic efficacy of human mAbs in vivo.

5 BALB/c mice per group were passively injected i.p. with 25 or 2.5 mg/kg of mAbs FE17 or FE43 or a control (ctr) mAb and challenged 24 hours later with 50 MLD₅₀ of the following influenza viruses: (A) A/PR/8/34(H1N1); (B) A/VN/1203/04 (H5N1); (C) A/INA/5/05(H5N1); (D) A/teal/Hong Kong/W312/97 (H6N1); or (E) A/Netherlands/219/03 (H7N7). mAb efficacy was measured as percentage survival after 14 days.

Figure 5. Histologic sections (H&E) of lungs infected with 2009 H1N1.

Original magnification, ×10. (A) Prominent necrotizing bronchointerstitial pneumonia in the lungs of control animals; red asterisks denote necrotic airways. (B and C) Lungs of animals treated with either FB110 (B) or FE43 (C) show focal areas of interstitial inflammation, edema, and congestion (black asterisks).