BALB/c mice immunized with a combination of virus-like particles incorporating Kaposi sarcoma-associated herpesvirus (KSHV) envelope glycoproteins gpK8.1, gB, and gH/gL induced comparable serum neutralizing antibody activity to UV-inactivated KSHV

Abstract

Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is estimated to account for over 44,000 new cases of Kaposi sarcoma annually, with 84% occurring in Africa, where the virus is endemic. To date, there is no prophylactic vaccine against KSHV. KSHV gpK8.1, gB, and gH/gL glycoproteins, implicated in the virus entry into host cells, are attractive vaccine targets for eliciting potent neutralizing antibodies (nAbs) against virus infection. We incorporated gpK8.1, gB, or gH/gL on the surface of virus-like particles (VLPs) and characterized these VLPs for their composition, size, and functionality. To determine which viral glycoprotein(s) elicit the most effective serum-nAbs, we immunized BALB/c mice with gpK8.1, gB, or gH/gL VLPs individually or in combination. Neutralizing antibody assay revealed that sera from mice immunized with the VLPs inhibited KSHV infection of HEK-293 cells in a dose-dependent manner. As a single immunogen, gpK8.1 VLPs stimulated comparable nAb activity to that of UV-inactivated KSHV (UV-KSHV). In contrast, UV-KSHV stimulated higher titers of nAb compared to gB (p = 0.0316) or gH/gL (p = 0.0486). Mice immunized with the combination of gB and gH/gL VLPs had a better nAb response than those immunized with either gB (p = 0.0268), or gH/gL (p = 0.0397) as single VLP immunogens. Immunization with any VLP combination stimulated comparable nAb activity to UV-KSHV serum. Our data provide the first evidence that KSHV gpK8.1, gB, and gH/gL glycoproteins can be incorporated onto the surface of VLPs and used as prophylactic vaccine candidates, with potential to prevent KSHV infection.

The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) gH/gL Complex Is the Predominant Neutralizing Antigenic Determinant in KSHV-Infected Individuals

“…However, since the input plasmids transfected into the cells was based on total DNA amount, rather than equal copy number, there is some variation in expression among different glycoproteins. For most of the KSHV glycoproteins, their corresponding bands, as reported by other groups, were detected by immunoblot in the total cell lysates [17,20,22,35]. However, the molecular weight of the gM protein in our study is higher than its theoretical size of 45 kDa.…”

“…Since KSHV envelope glycoproteins are incorporated into the viral surface and are critical for viral entry into permissive cells, they are the candidate antigens for eliciting nAbs through vaccination. In recent years, several studies have shown that immunizing mice or rabbits with Newcastle disease virus-like particles (VLP) expressing KSHV gpK8.1, gB and gH/gL, either alone or together, can elicit nAbs responses against KSHV [16,17]. However, whether these glycoprotein-targeted responses are consistent with those in KSHV-infected individuals with strong neutralizing responses has not yet been investigated.…”

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types

“…Plasmids and mutagenesis of KSHV WT-eGFP genome. To generate plasmid for qPCR standardization, full-length WT K8.1 was individually cloned into the pCAGGS mammalian expression vector as described previously (70). To construct gH/gL Fc-6ϫHis-tagged plasmids for protein production, a single transcript expressing WT gL (gL-WT; aa 1 to 139) and the gH ectodomain (aa 1 to 702) was synthesized by Genewiz (South Plainfield, NJ).…”

“…Samples were analyzed in triplicate. An eight-series of 10-fold dilutions of plasmid pCAGGS-K8.1 (70) was used as a standard for absolute quantification of viral genome copies. The sequences of reverse transcriptase qPCR primer sets for amplification of viral ORF targets are provided in Table 1.…”

A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits

“…Purified EBV, purified EBV-LPs, cells, or purified protein were prepared for SDS-PAGE as described [59]. Prepared samples were loaded onto a 4-12% polyacrylamide gel for protein separation using 1×MES SDS running buffer (ThermoFisher Scientific).…”