Characterization of immune responses following intramuscular DNA immunization with the MOMP gene of Chlamydia trachomatis mouse pneumonitis strain

Abstract

Studies were carried out to characterize the cellular and humoral immune responses evoked by intramuscular DNA vaccination with the major outer membrane protein (MOMP) gene of Chlamydia trachomatis mouse pneumonitis strain. The data demonstrate that DNA vaccinated mice develop antigen-specific delayed-type hypersensitivity, lymphocyte proliferation and interferon-gamma (IFN-gamma) production. Serum antibody responses (mainly immunoglobulin G2a; IgG2a) were evoked in two-thirds of the mice. We conclude that intramuscular DNA immunization with the MOMP gene evokes cellular and humoral immune responses suggestive of a T helper 1 (Th1) bias.

Figure 1

Protective immunity elicited by MOMP DNA (pMOMP) vaccination. BALB/c mice (4–5/group) were immunized with different amounts of the pMOMP DNA intramuscularly on four occasions at 0, 3, 6, 8 weeks. 100 μg of vector DNA (pcDNA3) was injected intramuscularly as a negative control on the same occasions. The mice were challenged with 5×10³ IFU of MoPn EB intranasally and killed on the 10th day postinfection. The MoPn growth in the lung was analysed by quantitative tissue culture. The data represents mean +SEM of the log₁₀ IFU per lung. *P < 0·05 when compared with pcDNA3 treated group.

Figure 2

(a) The DTH reaction in mice elicited by immunization by MOMP DNA immunization. BALB/c mice (four/group) were immunized by 200 μg of MOMP DNA (pMOMP) or vector DNA (– pcDNA3) in both quadriceps muscles (100 μg DNA/100 μl/injection site) on four occasions at 0, 3, 6, 8 weeks. A group of mice were also immunized intramuscularly with 5×10⁶ IFUs of UV-inactivated MoPn EBs on the same time schedule. DTH was evaluated 2 weeks after last immunization. UV-killed MoPn EB (25 μl; 2×10⁵ IFU) in SPG buffer was injected into a hind footpad. SPG buffer (25 μl) was injected into the other hind footpads as control. Footpad swelling was measured at 48 hr and 72 hr following the injection. The difference in thickness between the two footpads was used as a measure of the DTH. (b) Lymphocyte proliferation of the mice immunized with pMOMP. BALB/c mice (four to six/group) were immunized with pMOMP, pcDNA3 or MoPn EBs as described in (a). Mice were killed 2 weeks after the last immunization. 5×10⁵ cells/well of spleen cells in 200 μl of RPMI-1640 medium containing 10% FCS and 5×10⁻⁵ M 2ME were incubated with 1×10⁵ IFU of UV killed MoPn in 5% CO₂ at 37° for 96 hr. Negative control wells contained spleen cells without EB. ³H-Thymidine (0·25 μCi) was added 16 hr before harvest. The cells were harvested after incubation for 96 hr and ³H-thymidine uptake was counted in scintillation solution using a Beckman LS5000 B-counter. *P < 0·05, **P < 0·01, when compared with the pcDNA group.

Figure 3

Profile of IFN-γ and IL-10 production by spleen cells isolated from pMOMP-immunized mice. Mice (four to six) were immunized by pMOMP, pcDNA3 or MoPn EB as described in Fig. 2. Spleen cells were collected two weeks after the last immunization. Spleen cells (5×10⁶ cells/ml) were incubated with 2×10⁵ IFU of UV-killed MoPn EB for 96 hr. The amount of IFN-γ (a) and IL-10 (c) in the supernatant of the cell culture was measured by ELISA. The number of IFN-γ- (b) and IL-10- (d) producing cells per 2×10⁵ spleen cells was tested by ELISPOT. *P < 0·05, **P < 0·01, when compared with pcDNA3-immunized control group.

Figure 4

The profile of IgG subclass of MoPn-specific Ab responses in serum of mice immunized with pMOMP. Balb/c mice were immunized by pMOMP or pcDNA3 or MoPn EB as described in Fig. 2. Sera were collected from the immunized mice 2 weeks after last immunization. MoPn-specific IgG₁ and IgG_2a Abs were tested by ELISA. The cutoff point was 0·5 on OD 405 nm.

Figure 5

Detection of serum antibody against MoPn MOMP in pMOMP immunized mice by immunoblot. Balb/c mice were immunized with pMOMP (lane 1–12), or pcDNA3 (lane pc1 and pc2) or MoPn EB (lane EB) as described in Fig. 1(a). Lane N is the serum collected from a nonimmunized mouse. Sera collected from immunized mice at two weeks post last immunization were diluted at 1:100 and reacted with purified MoPn MOMP protein that had been separated in 10% SDS–polyacrylamide gel and transferred to a nitrocellulose membrane. The bands were developed by the ECL method.