Complete protection of mice against lethal murine cytomegalovirus challenge by immunization with DNA vaccines encoding envelope glycoprotein complex III antigens gH, gL and gO

Abstract

Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines.

Fig 1. Expression of plasmid DNAs encoding gH, gL and gO of MCMV.

293T cells were transfected with either the vector DNA or co-transfected with gH/gL/gO pDNAs, and lysates were made 48 h later. 3T3 cells were infected with MCMV for 3 days and then lysates were made. Lysate proteins were resolved on a non-reducing SDS-10% PAGE gel and transferred to PVDF membranes and immunoblotted with polyclonal antisera to gH-gL-gO. The lanes labeled as 1^# and 2^# represented the samples prepared from cells transfected with 2 μg or 1μg each in the mixture of gH/gL/gO pDNAs, respectively.

Fig 2. Confocal microscope analyses of pgH, pgL, pgO-transfected (A, C) and MCMV strain Smith-infected 3T3 cells (B, D).

3T3 cells were transfected with empty vector, gH, gL, gO or co-transfected with gH/gL/gO pDNAs and harvested 48 h post-transfection. Strain Smith-infected 3T3 cells were harvested 3 days post-infection. Immunofluorescence assays were performed either with (A, B) or without permeabilization (C, D) to determine the intracellular and surface distribution of three glycoproteins, respectively. Anti-gH/gL/gO antibodies were detected with FITC-conjugated goat anti-mouse IgG (left columns). Nuclei were stained with Hoechst 33258 (middle columns). Bars, 10 μm in A and B, 12 μm in C, and 20 μm in D. Specific immunofluorescence was observed with a confocal laser scanning microscope.

Fig 3. Survival rates (A) and body weight changes (B) after the challenge in the mice immunized with pgH, pgL, pgO or various joint DNA vaccines.

Mice were immunized thrice with gB, gH, gL, gO, gH/gL, gH/gO, gL/gO and gH/gL/gO pDNAs at a dosage of 50 μg (25 μg each in the mixture of two DNAs, 16.7 μg each in the mixture of three DNAs). Control mice were immunized with 50 μg vector plasmid. Three weeks after the last immunization, all the mice were challenged with a lethal dose of SG-MCMV. Body weight losses and survival rates of mice were determined 21 days post-challenge. Data points represent mean ± SD in B.

Fig 4. Neutralization titers of mice sera against MCMV.

Mice were immunized thrice with gB, gH, gL, gO, gH/gL, gH/gO, gL/gO and gH/gL/gO pDNAs at a dosage of 50 μg (25 μg each in the mixture of two DNAs, 16.7 μg each in the mixture of three DNAs), respectively. Control mice were immunized with 50 μg vector plasmid. Sera were collected 3 weeks after the last immunization. Serum samples were diluted twofold serially. Neutralization titers shown were the highest sera dilutions at which 50% reduction of MCMV infection was achieved. Values represent the geometrical means ± SD of each group. ^a Significant difference compared to the mice in control group (p < 0.05). ^b Significant difference compared to the mice in gH, gL or gO single immunization groups (p < 0.05). ^c Significant difference compared to the mice in two gene co-immunization groups (p < 0.05).

Fig 5. Cellular immune responses of mice vaccinated with gH/gL/gO pDNAs by ELISPOT assay.

Mice were immunized with gH, gL, gO alone or a mixture of gH/gL/gO pDNAs at a dosage of 50 μg. Control group was inoculated with vector plasmid. Splenocytes were isolated 2 weeks later and stimulated in vitro with 10 μg/ml of the corresponding peptides. The numbers of IFN-γ secreting splenocytes were shown. Values represent the geometrical means ± SD of each group. ^a Significant difference compared to the mice in control group (p < 0.05). ^b Significant difference compared to the mice in gH, gL or gO single immunization groups (p < 0.05).