Potent Neutralization of Hendra and Nipah Viruses by Human Monoclonal Antibodies

Abstract

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 g/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 g/ml, and 98% neutralization required only 1.6 g/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.

Structure of Severe Acute Respiratory Syndrome Coronavirus Receptor-binding Domain Complexed with Neutralizing Antibody

“…This fragment was used as a selecting antigen for panning of a large (ϳ10 10 different antibodies) human antibody Fab library, which we constructed from the B lymphocytes of 10 healthy volunteers. Recently, this library was also used for the selection of potent nAbs against Hendra and Nipah viruses (23). The Fab with the strongest binding to the RBD, m396, was converted to full antibody (IgG1), expressed, and purified.…”

“…10 members) was constructed from peripheral blood B cells of 10 healthy donors 5 and used for selection of Fabs against purified, soluble, monomeric RBD, conjugated to magnetic beads (Dynabeads M-270 Epoxy, Dynal Inc., New Hyde Park, NY) following a previously described procedure (23). Briefly, amplified libraries of 10 12 phage-displayed Fabs were incubated with 5, 3, and 1 g of RBD in a 500-l volume for 2 h at room temperature during the first, second, an third rounds of biopanning, respectively.…”

Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers

“…M36, m36SAbp, m36CH3, and m36b0Fc were expressed in E. coli HB2151, as described previously (20). The bacterial pellet was collected after centrifugation at 5,000 ϫ g for 10 min and resuspended in PBS (pH 7.4) containing 0.5 million-unit polymixin B (SigmaAldrich).…”

“…1 Evans, currently at Novartis, Boston) (6). This dAb library was used for selection of VHs against HIV-1 antigens conjugated to magnetic beads (Dynabeads M-270 epoxy; DYNAL Inc., New Hyde Park, NY) as described previously (20). For sequential panning, 10 and 5 g of gp120Bal-CD4 was used in the first round and third round, respectively; antigens were alternated with 5 g of gp140R2 or gp140JRFL during the second round and fourth round.…”

“…For sequential panning, 10 and 5 g of gp120Bal-CD4 was used in the first round and third round, respectively; antigens were alternated with 5 g of gp140R2 or gp140JRFL during the second round and fourth round. Clones that bound to HIV-1 antigens were identified from the third and fourth round of biopanning by using monoclonal phage ELISA as described (20).…”