Unraveling of a neutralization mechanism by two human antibodies against conserved epitopes in the globular head of H5 hemagglutinin.

The rapid spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral treatment. Previously, we developed human monoclonal antibodies 65C6 and 100F4 that neutralize almost all (sub)clades of HPAI H5N1. The conserved 65C6 epitope was mapped to the globular head of HA. However, neither the 100F4 epitope nor the neutralization mechanism by these antibodies was known. In this study, we determined the 100F4 epitope and unraveled a neutralization mechanism by antibodies 65C6 and 100F4.

Potent and broad anti-HIV-1 activity exhibited by a glycosyl-phosphatidylinositol-anchored peptide derived from the CDR H3 of broadly neutralizing antibody PG16.

PG9 and PG16 are two recently isolated quaternary-specific human monoclonal antibodies that neutralize 70 to 80% of circulating HIV-1 isolates. The crystal structure of PG16 shows that it contains an exceptionally long CDR H3 that forms a unique stable subdomain that towers above the antibody surface to confer fine specificity. To determine whether this unique architecture of CDR H3 itself is sufficient for epitope recognition and neutralization, we cloned CDR H3 subdomains derived from human monoclonal antibodies PG16, PG9, b12, E51, and AVF and genetically linked them to a glycosyl-phosphatidylinositol (GPI) attachment signal. Each fusion gene construct is expressed and targeted to lipid rafts of plasma membranes through a GPI anchor. Moreover, GPI-CDR H3(PG16, PG9, and E51), but not GPI-CDR H3(b12 and AVF), specifically neutralized multiple clades of HIV-1 isolates with a great degree of potency when expressed on the surface of transduced TZM-bl cells. Furthermore, GPI-anchored CDR H3(PG16), but not GPI-anchored CDR H3(AVF), specifically confers resistance to HIV-1 infection when expressed on the surface of transduced human CD4(+) T cells. Finally, the CDR H3 mutations (Y100HF, D100IA, and G7) that were previously shown to compromise the neutralization activity of antibody PG16 also abolished the neutralization activity of GPI-CDR H3(PG16). Thus, we conclude that the CDR H3 subdomain of PG16 neutralizes HIV-1 when targeted to the lipid raft of the plasma membrane of HIV-1-susceptible cells and that GPI-CDR H3 can be an alternative approach for determining whether the CDR H3 of certain antibodies alone can exert epitope recognition and neutralization.