Pandemic H1N1 Influenza Infection and Vaccination in Humans Induces Cross-Protective Antibodies that Target the Hemagglutinin Stem

Abstract

Most monoclonal antibodies (mAbs) generated from humans infected or vaccinated with the 2009 pandemic H1N1 (pdmH1N1) influenza virus targeted the hemagglutinin (HA) stem. These anti-HA stem mAbs mostly used IGHV1-69 and bound readily to epitopes on the conventional seasonal influenza and pdmH1N1 vaccines. The anti-HA stem mAbs neutralized pdmH1N1, seasonal influenza H1N1 and avian H5N1 influenza viruses by inhibiting HA-mediated fusion of membranes and protected against and treated heterologous lethal infections in mice with H5N1 influenza virus. This demonstrated that therapeutic mAbs could be generated a few months after the new virus emerged. Human immunization with the pdmH1N1 vaccine induced circulating antibodies that when passively transferred, protected mice from lethal, heterologous H5N1 influenza infections. We observed that the dominant heterosubtypic antibody response against the HA stem correlated with the relative absence of memory B cells against the HA head of pdmH1N1, thus enabling the rare heterosubtypic memory B cells induced by seasonal influenza and specific for conserved sites on the HA stem to compete for T-cell help. These results support the notion that broadly protective antibodies against influenza would be induced by successive vaccination with conventional influenza vaccines based on subtypes of HA in viruses not circulating in humans.

Keywords: competition for T-cell help; cross-protective antibodies; hemagglutinin; heterosubtypic; memory B cells; pandemic H1N1 influenza; plasmablasts; vaccines.

Figure 1

Infection or vaccination with pdmH1N1 induces a dominant antibody response that preferentially uses IGHV1-69 and that cross-reacts with the HA of the highly pathogenic avian H5N1 influenza virus. (A) Elispot assay showing spots of anti-pdmHA antibodies that had been secreted by individual PB. (B) Pie chart representing all 48 mAbs from Table 1. The 95% confidence intervals of IGHV1-69 usage were 38–66%. *8 of 9 mAbs using IGHV4-39 were from a single subject, V2. (C,D) Cross-reactivity of selected mAbs with the HA of influenza A/Hong Kong/156/197 (H5N1; Clade 0) expressed by the A549 human cell-line. Binding was quantified by indirect immunofluorescence and the Cellomics instrument. (C) Cellomics images of cells stained with either secondary antibodies alone or first with mAb I4-128 (5 μg/ml). (D) Percentage of cells stained with varying concentrations of the indicated mAbs. Note that anti-stem HA mAbs that did not use IGHV1-69, like V3-1E8 and I5-52 also bound to the H5 HA in this assay (data not shown). KE5, an human anti-HCMV monoclonal antibody (McLean et al., 2005), was used as a negative control with no significant binding observed (data not shown).

Figure 2

Infection or vaccination with pdmH1N1 induces a dominant antibody response targeting the HA stem. (A) mAbs to the HA stem fail to inhibit hemagglutination but a minority of mAbs inhibit hemagglutination by pdmH1N1. All mAbs were at 40 μg/ml and diluted 50% with virus in the first well. No detectable hemagglutination means that hemagglutination was not inhibited at 20 μg/ml. (B) Anti-HA stem mAbs inhibit binding of a mouse mAb (C179 (Okuno et al., 1993)) to the HA stem in a competition ELISA. Note that mAb I5-52, not using IGHV1-69, also inhibited C179 binding. (C) pH 5 pre-treatment of HA selectively decreases binding of anti-HA stem mAbs whether encoded by IGHV1-69 or other IGHV genes (like I5-52) but not anti-HA head mAbs like V2-36. (D) ELISA showing competitive inhibition of the indicated mAbs of the binding of biotinylated I5-24, which uses IGHV1-69 and binds to the HA stem. Note that V2-36 also inhibits binding of I5-24. Also shown is competitive inhibition of I5-24 binding by I14-2B7, which uses IGHV1-18 and IGKV2-30. KE5, an human anti-HCMV monoclonal antibody (McLean et al., 2005), was used as a negative control.

Figure 3

Vaccines for seasonal influenza or pdmH1N1 and purified recombinant HA from highly pathogenic avian H5N1 influenza exhibit the epitope targeted by anti-HA stem mAbs. ELISA reactivity with the 2009/2010 seasonal influenza vaccine, the pdmH1N1 vaccine, and purified recombinant ectodomain of the HA of an H5N1 avian influenza virus A/Vietnam 1203/2004 (Clade 1) of 5 anti-HA stem mAbs (V3-2G6, I4-128, I5-24, I8-1B6 using IGHV1-69 and I5-52 using IGHV1-18), with for comparison, three anti-HA head antibodies (I4-1G8, V2-36, and V4-17).

Figure 4

Neutralization of pdmH1N1 and the highly pathogenic avian H5N1 influenza A viruses by anti-HA stem mAbs. (A) Neutralization of pdmH1N1 by mAbs present for the entire assay. Titrations commenced at a concentration of 5 μg/ml of each mAb. The most potent mAbs at neutralization were V2-36 (against the HA head) and V3-2G6 (against the HA stem), which inhibited infectivity completely at ∼40 ng/ml and ∼80 ng/ml. Anti-HA stem mAbs not using IGHV1-69 like I14-2B7, using IGHV1-18, and V3-1B9, using IGHV3-11, also completely neutralized pdmH1N1. Neutralizing titers against influenza virus A/Brisbane/59/07(H1N1) were 2- to 32-fold lower in all cases (data not shown). No neutralization activity against Brisbane influenza A/Brisbane/10/2007 (H3N2) by any mAb tested was observed (data not shown), consistent with previous observations on mAbs using IGHV1-69 (Throsby et al., ; Sui et al., 2009). (B,C) Neutralization of infectivity of the highly pathogenic Influenza A/Goose/Ger/R1400/07 (H5N1) avian virus. (B) Shown are representative images of the plaque reduction assay performed with influenza A virus A/Goose/Ger/R1400/07 (H5N1) and mAb I8-1B6 (5 μg/ml). (C) Only V2-36 (which failed to bind to H5 HA, Figure 1) failed to neutralize infectivity. (D) Anti-HA stem mAbs encoded by IGHV1-69 inhibit H5 HA-mediated fusion, correlating with low binding to the HA of highly pathogenic avian influenza A/Hong Kong/156/197 (H5N1) virus (Figure 1). Note the decreased syncytia formation in wells treated by the anti-stem mAb, C179, or the IGHV1-69-using mAbs, I4-128, I8-1B6, and V3-2G6, in contrast to the control with no antibody added (no Ab) and the anti-HA head mAb, V2-36, which did not bind to this H5 HA.

Figure 5

Therapeutic effects of mAbs on lethal infections with a human isolate of pdmH1N1. (A,B) Groups of 5 CD-1 mice were infected intranasally with 2 × 10⁵ PFU of A/Halifax/210/2009 (pdmH1N1). Twenty-four hours post-infection, the mice were treated IP with (A) 300 μg of the indicated anti-HA stem mAbs (the survival curves for V3-2G6 and I8-1B6 are superimposed and mainly superimposed with the curve of I4-128) or (B) with the indicated doses of the anti-HA head mAb, V2-36.

Figure 6

Evidence that most mAbs originated from memory B cells that were activated by pdmH1N1. Shown are the number of somatic mutations in IGHV genes of mAbs generated from PB and memory B cells. Whiskers show the 5/95th percentile. Mann–Whitney Rank Sum Test of the number of somatic mutations in IGHV genes of mAbs generated from memory B cells (N = 23) versus PB (N = 25) demonstrates a significant difference (p < 0.001). Note that more than 50% of the mAbs generated from PB (which all but one cross-reacted with seasonal influenza vaccine) exhibited IGHV genes with more than 10% of their nucleotides mutated (>28 mutations), with the median number of mutations being 29.

Figure 7

The dominant clonotype of mAbs against the HA head generated from subject V2. (A) Shown are alignments of the amino acid sequences of the variable region of the H chain of the dominant clonotype from V2 using the IGHV4-39, another mAb against the HA head from V2 also using IGHV4-39, and a mAb against the HA head from V4 also using IGHV4-39. We also included the amino acid sequence encoded by the germline IGHV4-39*01 allele to indicate somatic mutations. Note that the mAbs in red belong to the same clonotype as seen by the common IGHD-6-13-, IGHJ-5-encoded residues (and the similar light chains, Table 1). Shown in (B) is a phylogram using the Clustal-W website.

Figure 8

Prophylactic effects of human plasma from a vaccinated subject against lethal infections with H5N1 avian influenza virus. (A) Twenty-four hours before intranasal infection of BALB/c mice with 2 × 10⁵ PFU of A/Hong Kong/213/2003 (H5N1) virus, groups of five mice were treated with a 400 μl intraperitoneal injection of PBS as a control, or 400 μl of plasma collected from subject V3 either 14 days after pdmH1N1 vaccination or 1 year after pdmH1N1 vaccination, and as a control, plasma from a young individual unvaccinated and uninfected with pdmH1N1, collected in 2006. Another group of five mice were pre-treated 72, 48, and 24 h prior to infection with 400 μl of plasma from V3 collected 1 year after pdmH1N1 vaccination. (B) pH 5 treatment of H9 HA drastically decreases the binding of V3 sera compared with binding of H9 HA treated at pH 7.4.

Figure 9

Therapeutic and prophylactic effects of purified mAb against lethal infections with H5N1 avian influenza virus. (A) Twenty-four hours before intranasal infection with 2 × 10⁵ PFU of A/Hong Kong/213/2003 (H5N1) virus, three groups (five mice each) of BALB/c mice were injected intraperitoneally with PBS as a control, or 15 or 5 μg of V3-2G6 generated from a subject vaccinated with the pdmH1N1 vaccine. The data shows survival rates and average weight at over 14 days. (B,C) Groups of five mice were infected intranasally with 2 × 10⁵ PFU of A/Hong Kong/213/2003 (H5N1) virus and (B) treated after 24 h with 150 or 300 μg of V3-2G6 or after 48 h with 300 or 600 μg of V3-2G6 and (C) treated after 24 h with 150, 75 or 37.5 μg of V3-2G6.