Amlashi, P.; Kim, J.; Mendis, N. N.; Wasdin, P. T.; Bass, L. E.; Jordaan, G.; Abu-Shmais, A. A.; Slein, M. D.; Johnson, N. V.; Bonami, R. H.; Leib, D. A.; Ackerman, M. E.; McLellan, J. S.; Georgiev, I.

Herpes simplex virus (HSV) can cause severe perinatal viral infections, leading to mortality or long-term neurological disability. To date, antivirals have limited efficacy, and no vaccines are approved. HSV entry requires the coordinated action of multiple viral glycoproteins. Glycoprotein B (gB), the most conserved glycoprotein among herpesviruses, mediates membrane fusion by undergoing a conformational transition from a metastable prefusion state to a stable postfusion state. Potently neutralizing human antibodies targeting prefusion-specific epitopes have been identified for many virus families, however, such antibodies for human herpesviruses are currently limited to an alpaca-derived nanobody and a murine monoclonal antibody. In this study, we used stabilized HSV-2 prefusion gB and LIBRA-seq single-cell sequencing technology to probe B cells from HSV-2 seropositive and healthy human donors, resulting in the isolation of four prefusion-specific and two prefusion-preferring human antibodies. Cryo-EM studies revealed that the four prefusion-specific antibodies bind distinct epitopes on prefusion gB and exhibit different mechanisms of prefusion specificity. Two antibodies, 5-18 and 3-6, showed potent cross-neutralizing activity against HSV-1 and HSV-2 and conferred protection in a neonatal mouse model of HSV-2 infection. These findings highlight the molecular and structural determinants of HSV-2 prefusion gB recognition by human antibodies and demonstrate the translational potential of prefusion-specific gB antibodies.