Vero (African green monkey kidney, American Type Culture Collection (ATTC), Manassas, VA, USA), VD60 [20] and HaCat (human keratinocyte (ATCC PCS-200-011)) cells were grown in Dulbecco modified Eagle medium (DMEM) (Thermo-Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) and 1% penicillin-streptomycin (Thermo-Fisher Scientific, Waltham, MA, USA). ΔgD-2 was propagated in VD60 cells and viral titers (PFU/mL) were quantified on complementing VD60 and non-complementing Vero cells [21]. HSV-1 (B3x1.1) [15], HSV-2(G) [22], HSV-2(333)ZAG [23] (which expresses a green fluorescence protein under control of the cytomegalovirus promoter inserted at an intergenic site) and HSV-2(4674) [24] were propagated on Vero cells. HSV-2 gD protein (gD-2) was synthesized by the Einstein Protein Core Facility and combined with 150 µg of alum (Imject-Thermo Scientific, Thermo-Fisher Scientific, Waltham, MA, USA) and 12.5 µg of monophosphoryl lipid A (MPL) (Invivogen, San Diego, CA, USA) (rgD-2/Alum-MPL) [15].

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