Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

Abstract

Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

Fig. 1

The isolation of pandemic H1N1 HA-specific memory B cells. (A) Small lymphocytes were sorted from the human peripheral blood of a vaccinated individual. (B) Memory B cells were sorted using CD19 and IgG as markers. (C) Negative control: cells stained with an unrelated protein (RBD, receptor-binding domain of SARS-CoV). (D) Cells were sorted using the pandemic H1N1 HA protein. HA-specific memory B cells accounted for approximately 0.61% of the total memory B cells.

Fig. 2

An ELISA to determine the binding activities of the naturally isolated human monoclonal antibodies. (A) HA, heat-denatured (100 °C, 5 min) HA or the HA1 domain of the 2009 pandemic H1N1 influenza virus was used to coat the wells of the plates for binding activity tests of the 19 human monoclonal antibodies. (B) Influenza virus lysates from both the pandemic and the seasonal H1N1 viruses were used for binding activity tests of the 19 human monoclonal antibodies. Cal07: pandemic H1N1 virus lysate with HA from A/California/7/2009 (H1N1), Bri59: seasonal H1 influenza virus lysate with HA from A/Brisbane/59/2007 (H1N1). S-95-7: a mouse monoclonal antibody recognizing a specific conformational epitope in the HA1 domain of the 2009 pandemic H1N1 influenza virus. Vector: human antibody IgG and Igκ constant regions encoded by the antibody expression plasmid. OD 450 nm: optical density measured at 450 nm.

Fig. 3

Microneutralization assay of the human monoclonal antibodies. The neutralizing abilities of the seven selected human mAbs against several influenza virus strains of different subtypes, including SC09, H3N2, PR8-H5, PR8-H7, and H9N2, as listed in the “Viruses” section.

Fig. 4

Inhibition of cell–cell fusion by the broadly neutralizing mAbs. HEK293T cells were transfected with plasmids expressing the pandemic H1N1 HA0, then treated with trypsin and pH 5.0. (A) Treatment with no antibody added. (B–H) Treatment in the presence of 100 μg/ml of broadly neutralizing mAbs: 1C4, 3C4, 1E1, 3E1, 1F2, 1F4, and 1G1 represented by B to H in sequence. (I) Cells transfected with plasmids expressing enhanced green fluorescent protein following with the same treatment. (J) Treatment in the presence of 100 μg/ml of a control mAb.

Fig. 5

A competitive ELISA to detect the binding epitopes of the seven neutralizing mAbs. The percentage of competition between two mAbs in the ELISA binding assay is shown. The percentage of competition was calculated as the reduction in the absorbance when mAb binding competition occurred. Shadowed grids: complete competition (percentage of competition >90%). epit-1 to epit-4: four neutralizing epitopes. The vertical row: non-conjugated mAbs. The horizontal row: biotin-conjugated mAbs.

Fig. 6

A peptide ELISA to detect the binding epitopes of the seven neutralizing mAbs. A total of 19 overlapping peptides covering the HA2, as listed in Table 3, were tested. No. 1 to 19: the overlapping peptides. 0: biotin without peptide conjugation. vac: the 2009 pandemic H1N1 influenza vaccine. OD 450 nm: optical density measured at 450 nm.

Fig. 7

Sequence alignment of the linear peptide epitope (FIEGGWTGMVDGWYGYHH). SC09, H3N2, PR8-H5, PR8-H7, and H9N2 referred to the influenza viruses tested in the microneutralization assay as listed in the “Viruses” section. Underlined letters represent the conserved amino acids among the viruses.