A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

Abstract

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E-mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro. Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-gamma, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.

Fig. 1

Construction of the vaccine vectors, pKS–MOMP and pKSM1–CTA2B. The omp1 cDNA was inserted between and in frame with the LacZ′ and L′ genes of vector pKSEL5-2 at the KpnI–PstI sites to generate plasmid pKS–MOMP. Also, the coding region of omp1: ctA2B was amplified by PCR from plasmid pUAB084 and inserted between and in frame with the LacZ′ and L′ anchor sequences at the KpnI and BamHI restriction sites of vector, pKSEL5-2, to generate plasmid, pKSM1–CTA2B. Expression of omp1 and omp1–ctA2B are under the transcriptional regulation of the lac promoter (lac_po).

Fig. 2

Expression of MOMP and MOMP–CTA2B. The expression of MOMP and MOMP/CTA2B by the Vibrio clones was evaluated by immunoblotting analysis. Mid-log phase cultures of H1 harboring the expression plasmids were induced with IPTG and cell lysates assayed for rMOMP or rMOMP/CTA2B expression by Western blot. MOMP and MOMP/CTA2B were detected using the mouse MOMP-specific monoclonal antibody MoPn 40. Lane 1, Vibrio cholerae HI cells harboring the recombinant plasmid, pKS–MOMP expressing rMOMP (54 kDa); lanes 2 and 3, H1 cells harboring the recombinant plasmid, pKSM1/CTA2B expressing rMOMP/CTA2B (74 kDa) at 1 and 2 h, respectively. Numbers to the left are approximate molecular masses, in kilodaltons (kDa).

Fig. 3

Expression of costimulatory molecules and cytokine production by DCs after treatment with rVCG–MOMP with and without CTA2B. BMDCs were isolated from mice by established procedures using IL-4 and GM-CSF and characterized as loosely adherent mononuclear cells expressing high levels of CD11c but lacking B220 surface antigens. Harvested cells were pulsed with rVCG vaccines for 24 h, stained with conjugated monoclonal antibodies against CD11C, CD40, CD80, CD83, CD86, 1Ab, CD14 and CD71 or isotype-matched controls and quantified in triplicate by flow cytometry. Also, supernatants from control and experimental cultures were assayed for the synthesis of cytokines by the Multiplex Luminex technique. The data show (a) the mean fluorescence intensity of staining of the indicated molecules and (b) the amount of Th1-promoting cytokines secreted after treatment. The experiments were repeated twice.

Fig. 4

Chlamydia-specific IFN-γ production by ILN and splenic T cells. Groups of mice were vaccinated by the IM, IVag or TC routes and boosted twice, 2 weeks apart, with the rVCG vaccine constructs. Two weeks after the last boost, Tcells were purified from the ILN and spleens of immunized mice and controls and restimulated in vitro with UV-irradiated Chlamydia trachomatis (a) or Chlamydia muridarum (b). Local mucosal and systemic IFN-γ response was assessed as a measure of Thl response. The amounts of IFN-γ contained in the supernatants of culture-stimulated cells were measured using the Bio-Plex cytokine assay kit in combination with the BIO-PLEX MANAGER software. The concentration of the cytokine in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Data were calculated as the mean values (±SD) for triplicate cultures for each experiment. The control cultures without antigen did not contain detectable levels of IFN-γ and so the data were excluded from the results. The results are from two independent experiments. IM, intramuscular; IVag, intravaginal; TC, transcutaneous.

Fig. 5

Genital mucosal and systemic Chlamydia-specific humoral immune response after immunization with rVCG vaccines. Groups of mice were immunized and boosted as described above and vaginal lavage and serum samples were obtained 2 weeks postimmunization. The amount of antibody elicited in genital lavage (a) and serum samples (b) was assessed by a modified antibody ELISA and expressed in ng mL⁻¹. Data were calculated and presented as the mean values ± SD of triplicate cultures for each experiment. The results are from two independent experiments. IM, intramuscular; IVag, intravaginal; TC, transcutaneous.

Fig. 6

Protection against genital chlamydial challenge after immunization with rVCG vaccines. Two weeks after the last immunization, mice immunized with rVCG chlamydial and control (rVCG–gD2) vaccines were challenged intravaginally with 10⁷ IFU of live Chlamydia muridarum (MoPn). One week before challenge, mice were administered Depo Provera to stabilize the estrous cycle and facilitate a productive infection. Infections were monitored by cervicovaginal swabbing of individual animals every 3 days and Chlamydia was isolated from swabs in tissue culture and enumerated. The data show the mean recoverable IFUs expressed as log₁₀ IFU mL⁻¹±SD, for mice immunized with (a) rVCG–MOMP and (b) rVCG–MOMP/CTA2B. Differences between the control and the experimental groups were compared with Student’s t-test at P < 0.05. The experiment was repeated to contain 10–12 mice per group. IM, intramuscular; IVag, intravaginal; TC, transcutaneous.