Antiviral Immune Responses Against Murine Cytomegalovirus Induced by an Oral Salmonella-Based Vaccine Expressing Viral M33 Protein

Abstract

Human cytomegalovirus (CMV) is the leading cause of congenital infections, often leading to mental retardation and neurological disorders. It is a major public health priority to develop a vaccine for preventing and controlling human CMV infection. In this report, we generated an oral Salmonella-based vaccine to express the M33 protein of murine cytomegalovirus (MCMV) and investigated the anti-MCMV immune responses induced in mice immunized with this vaccine. Compared to those administered with phosphate-buffered saline (PBS) or a control vaccine without M33 expression, mice immunized with the vaccine expressing the M33 protein exhibited a remarkable induction of antiviral serum IgG and mucosal IgA humoral responses and a significant elicitation of antiviral T cell responses. Successful inhibition of viral growth in lungs, spleens, livers, and salivary glands was also found in the vaccinated animals compared to the PBS-treated animals or those immunized with the control vaccine without M33 expression. Furthermore, substantial protection against MCMV challenge was observed in mice immunized with the vaccine. Thus, Salmonella-based vaccine expressing MCMV M33 can induce anti-MCMV effective immune responses and protection. Our study implies that attenuated Salmonella expressing human CMV antigens, including its homologue to M33, may represent promising oral anti-CMV vaccine candidates.

Keywords: Salmonella; cytomegalovirus; herpesvirus; immune responses; murine cytomegalovirus; oral vaccine; vaccine.

Figure 1

In vitro growth of Salmonella clinical strain ST14028s, mutant strain SL7207, empty vector-containing vaccine SL-vec, and M33-containing vaccine SL-M33 in LB broth. Data and error bars represent mean values ± standard deviations (SDs) calculated from the results of three independent experiments, with each independent experiment performed in duplicate. Experimental procedures and data analyses are described in the Section 2.

Figure 2

(Left panel) Viability of mice (five animals per group) intragastrically infected with 2 × 10³ CFU of Salmonella ST14028 per mice or 1 × 10⁹ CFU of SL7207, SL-vec, and SL-M33 per mice. (Right panel) Viability of mice (five mice per group) intragastrically infected or administered with Salmonella ST14028 (2 × 10³ CFU per mice) once on day 0 or with phosphate-buffered saline (PBS), SL-vec (1 × 10⁹ CFU per administration per animal), or SL-M33 (1 × 10⁹ CFU for per administration per animal) three times on days 0, 14, and 28.

Figure 3

Serum IgG and mucosal IgA titers from pooled serum or mucosal wash samples collected at 42 days post immunization from mice that were administered with PBS only, SL-vec, or SL-M33 at days 0, 14, and 28. We conducted ELISA assays to obtain serum IgG titers against antigen samples from Smith-infected (A) and m-M33-infected cells (B) and obtain mucosal IgA titers against antigen samples from Smith-infected (C) and m-M33-infected cells (D). Data and error bars represent mean values ± standard deviations (SD) calculated from the results of three independent experiments with each independent experiment performed in duplicate. Experimental procedures and data analyses are described in the Section 2. ** p < 0.05. NS, not significant.

Figure 4

T cell responses elicited by the Salmonella vaccines. We harvested splenocytes (n = 5) at 42 days post immunization from mice that were administered with PBS, SL-vec, or SL-M33 at days 0, 14, and 28, and then exposed the splenocytes to antigen samples from Smith-infected (A) or m-M33-infected cells (B) for 48 h. The number of IFN-γ-producing T cells, shown as spot-forming cells (SFCs), per 1 × 10⁶ cells, was obtained by ELISPOT assays. Data and error bars represent mean values ± standard deviations (SDs) calculated from the results of three independent experiments, with each independent experiment performed in duplicate. Experimental procedures and data analyses are described in the Section 2. ** p < 0.05. NS, not significant.

Figure 5

Viability of mice upon MCMV challenge. Mice (10 animals per group) were administered with PBS only, SL-vec, or SL-M33 at days 0, 14, and 28, and then intraperitoneally (A) or intranasally (B) infected with salivary gland-passaged MCMV Smith strain (1 × 10⁶ PFU per mice) at 42 days post immunization; they were monitored daily for their viability.

Figure 6

MCMV titers in lungs (A), livers (B), spleens (C), and salivary glands (D) in the vaccinated mice on day 5 after intraperitoneal viral challenge. We administered mice (10 animals per group) with PBS only, SL-vec, or SL-M33 at days 0, 14, and 28, and at 42 days post immunization, we infected them intraperitoneally with MCMV Smith strain (5 × 10⁴ PFU). Data and error bars represent mean values ± standard deviations (SDs) calculated from the results of three independent experiments, with each independent experiment performed in duplicate. Experimental procedures and data analyses are described in the Section 2. ** p < 0.05. NS, not significant.

Figure 7

MCMV titers in lungs (A), livers (B), spleens (C), and salivary glands (D) in the vaccinated mice at day 5 after intranasal viral challenge. We administered mice (10 animals per group) with PBS only, SL-vec, or SL-M33 at days 0, 14, and 28, and at 42 days post immunization and infected them intranasally with MCMV Smith (5 × 10⁴ PFU). Data and error bars represent mean values ± standard deviations (SDs) calculated from the results of three independent experiments, with each independent experiment performed in duplicate. Experimental procedures and data analyses are described in the Section 2. ** p < 0.05. NS, not significant.