Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages

Abstract

An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles. IMPORTANCE Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the bloodto produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

“…This mutation has previously been shown to have the greatest impact in lowering sialic acid binding activity (34,35), although it is important to keep in mind that the receptor-binding domain is composed of several residues that contribute to receptor recognition, including W166, analysis. An anti-mouse F(ab′) 2 -specific secondary antibody conjugated to HRP was used to visualized the antibody isoforms.…”

“…The coding regions of the FLAG-tagged HAs were subsequently subcloned into the ambisense expression plasmid pDZ (55). The HA of A/California/04/09 (H1) virus with a defective receptor-binding site (Y108F) was generated by mutating the tyrosine at site 108 (methionine, residue 1) into a phenylalanine and was subcloned into pDZ (34).…”

“…3E). Finally, we constructed an HA from the A/California/04/09 (H1) influenza A virus with a point mutation (tyrosine to phenylalanine at residue 108) in its receptor-binding site (Y108F HA), which was previously shown to affect sialic acid binding, to assess whether the HA mutant would affect activation by a stalk-specific antibody (34,35). the ability of the stalk-specific mAb 6F12 to activate FcγRIV against Y108F HA was reduced by >75% compared with that against WT HA (Fig.…”

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

“…Flow cytometry-based probes have recently been developed or optimized to evaluate memory B cells specific to antigens of common vaccines, including influenza (38) and tetanus (39). We took advantage of these highly specific probes to compare IgG + B cell reactivities against HIV and non-HIV antigens among 12 HIV-viremic individuals selected on the basis of their immunization histories (see details in Methods).…”

Focused antibody response to influenza linked to antigenic drift

“…We have previously confirmed that influenza virus-specific plasmablasts are induced transiently in large number in the peripheral blood during the first week after vaccination with inactivated former seasonal H1N1 and H1N1pdm09 viruses and the frequency of influenza virus-specific plasmablasts significantly correlated with the level of neutralizing antibody response (16)(17)(18) Binding of recombinant HA to transmembrane Ig G1. In these experiments, the HA molecules did not have any mutations to abolish sialic acid binding, as described by Whittle et al (19). In contrast to Whittle's method, in which the trimeric HA was combined in excess with streptavidin to form oligomers prior to staining, we stained sequentially with hemagglutinin followed by ExtrAvidin.…”