Lanes 5 and 6, UL128/UL130/UL131A recombinant protein purified using Cobalt affinity purification and followed by size exclusion chromatography. HCMV neutralizing activity analyzed with fibroblasts. These data suggest that the combination of HCMV core fusion machinery envelope proteins gB+gH/gL or the combination of gB and pentameric complex could be ideal vaccine candidates that would induce optimal immune responses against HCMV infection. 0.05 was considered significant. 3. Results 3.1. Production of HCMV gH/gL and UL128/UL130/UL131A Recombinant Proteins We previously produced in CHO cells the recombinant HCMV trimeric gB protein, Epstein Barr virus recombinant gB, gH/gL and gp350 proteins [54,57,61,62]. We took a similar approach to produce recombinant HCMV gH/gL and recombinant UL128/UL130/UL131A proteins in the current study. Specifically, a (Gly4Ser)3 linker coding sequence was inserted between the sequences encoding HCMV gL and gH to let both proteins fold properly, with an IgG ? leader coding sequence located at 5 for the protein to be secreted. Recombinant UL128/UL130/UL131A proteins were Saikosaponin B expressed similarly, where the coding sequences for UL128, UL130 and UL131A in tandem were separated by a (Gly4Ser)3 linker coding sequence in between. Synthesized DNA coding for recombinant HCMV gH/gL or UL128/UL130/UL131A protein was cloned into pOptiVEC vector and transfected CHO cells. Stable CHO cell lines expressing gH/gL or UL128/UL130/UL131A were generated by limiting dilution cloning. Recombinant proteins were purified from supernatants of CHO cell cultures using affinity and size exclusion chromatography. Purified gH/gL protein was analyzed by Western blot under reducing conditions using a monoclonal anti-HCMV gH antibody, and showed a single size ~110 kDa band, consistent with the predicted size of the heterodimeric gH/gL (Figure 1). Western blot analysis of the UL128/UL130/UL131A protein showed a band of ~57 kDa under reducing conditions, which was consistent with the predicted size of UL128/UL130/UL131A (Figure 2). Open in a separate window Figure 1 Human cytomegalovirus (HCMV) gH/gL recombinant protein expression and purification. (A) Coomassie brilliant blue stained polyacrylamide SDS gel under reducing conditions. Lanes 1 and 2, cell culture supernatant. Lanes 3 and 4, flow-through after Cobalt affinity purification. Lanes 5 and 6, HCMV gH/gL purified by Cobalt affinity purification and size exclusion chromatography. (B) Purified HCMV gH/gL recombinant protein was analyzed with Western blot under reducing conditions using an anti-gH monoclonal antibody. Lanes 1 and 2, purified proteins. Open in a separate window Figure 2 Expression and purification of HCMV UL128/UL130/UL131A recombinant protein. (A) Coomassie brilliant blue stained polyacrylamide SDS gel under reducing conditions. Lanes 1 and 2, cell culture supernatant. Lanes 3 and 4, flow-through after Cobalt affinity purification. Lanes 5 and 6, UL128/UL130/UL131A recombinant protein purified using Cobalt affinity purification and followed by size exclusion chromatography. (B) Western blot analysis of HCMV UL128/UL130/UL131A recombinant protein using anti-UL128 polyclonal antibodies under reducing conditions. Lanes 1 and 2, purified proteins. 3.2. Immunization of Rabbits with HCMV Trimeric gB, gH/gL or UL128/UL130/UL131A Recombinant Proteins Each Induced High Serum Titers of Antigen-specific IgG, with No Interference in the Induction of Individual Antigen-specific IgG Following Immunization with Protein Combinations Groups of adult rabbits, five in each group, were subcutaneously immunized with 25 g of HCMV trimeric gB, gH/gL or UL128/UL130/UL131A recombinant protein individually or in various combinations of these recombinant proteins (25 g each) using alum + CpG-ODN as adjuvants. Rabbits were then boosted on days 21 and 42 in a similar fashion. As shown in Figure 3ACC, immunization of rabbits with trimeric HCMV gB, HCMV gH/gL or UL128/UL130/UL131A individually induced high serum IgG titers (~1:100,000) of antigen-specific antibodies. The titers of anti-gB IgG and anti-gH/gL IgG induced by immunization with trimeric gB in combination with gH/gL were not significantly different from the IgG titers induced by immunization with trimeric gB or gH/gL alone (Figure 3A,B). The titers of anti-gH/gL IgG and anti-UL128/UL130/UL131A IgG induced by immunization with the combination of gH/gL and UL128/UL130/UL131A were not significantly different from the IgG titers induced by immunization with gH/gL or UL128/UL130/UL131A alone (Figure 3A,B). Using the trivalent combination of trimeric gB, Rabbit Polyclonal to ACHE gH/gL and UL128/UL130/UL131A for immunization induced high titers of antigen-specific IgG against gB, gH/gL and UL128/UL130/UL131A. The titers of anti-gB IgG, anti-gH/gL IgG or anti-UL128/UL130/UL131A IgG were not significantly different from that induced by immunization with corresponding proteins individually (Figure 3ACC). Open in a separate window Saikosaponin B Figure 3 Immunization of rabbits Saikosaponin B with HCMV trimeric gB, gH/gL and/or UL128/UL130/UL131A recombinant proteins induced high serum titers of antigen-specific IgG, without interference when used in combination. Groups of 12C15 week old rabbits (= 5),.