Nanobodies targeting a conserved henipavirus G glycoprotein receptor-binding region potently protect against Nipah virus infection

Nipah virus (NiV) and Hendra virus (HeV) are highly lethal zoonotic henipaviruses with pandemic potential, yet no approved antiviral therapeutics are currently available. Here, we identified four nanobodies targeting the viral attachment glycoprotein (G) with strong neutralizing activity. All candidates potently neutralized NiV-Malaysia (NiVM) and NiV-Bangladesh (NiVB) in authentic virus assays, with IC50 values in the low ng/mL range (2.50-18.3 ng/mL). 2E2 shows the most potent activity, with an IC50 of 2.50 ng/mL. These candidates cross-neutralized HeV in pseudovirus assays at levels comparable to those against NiV, yet authentic HeV neutralization was reduced with the most potent clone reaching an IC₅₀ of 160 ng/mL and the remaining clones showing variable potency. In Syrian hamsters challenged with 1000 LD50 NiVM, prophylactic administration of 1C3, 2E2, or 5A3 provides complete protection. Notably, 2E2 confers superior therapeutic protection (66.7% survival) when administered 24 h post-infection. Additionally, 2E2 provides 50% prophylactic protection against lethal HeV. Alanine-scanning mutagenesis indicated that the nanobodies recognize a conserved region within the receptor-binding domain (RBD) of G, with distinct sensitivities to individual residue substitutions. Notably, 2E2 exhibited minimal sensitivity to the tested NiV G mutations and retained neutralizing activity against the m102.4 escape-associated substitutions NiV G-V507I and HeV G-D582N. These findings identify 2E2 as a candidate RBD-targeting nanobody with potent prophylactic and therapeutic efficacy against NiV and cross-neutralizing activity against HeV, warranting further evaluation as a countermeasure for NiV outbreaks.