Phosphate substitution in an AlOOH - TLR4 adjuvant system (SPA08) modulates the immunogenicity of Serovar E MOMP from Chlamydia trachomatis

Abstract

Chlamydia trachomatis is one of the most common sexually transmitted pathogens and the development of an effective vaccine is highly desirable. The Major Outer Membrane Protein (MOMP) is one of the most abundant and immunogenic chlamydial proteins. Here we investigated the effects of phosphate substitution on the physicochemical and immunochemical properties of an experimental vaccine composed of serovar E recombinant MOMP (rMOMP) and a proprietary adjuvant system SPA08, consisting of aluminum oxyhydroxide (AlOOH) containing the TLR4 agonist E6020.An increase in phosphate substitution in the AlOOH component of the adjuvant markedly decreased the adsorptive coefficient and adsorptive capacity for both Ser E rMOMP and E6020. In vaccine formulations used for immunizations, phosphate substitution induced a decrease in the % adsorption of Ser E rMOMP without affecting the % adsorption of E6020.Immunogenicity studies in CD1 mice showed that an increase in phosphate substitution of the SPA08 adjuvant resulted in an increase in Ser E rMOMP-specific serum total IgG and IgG1 but not IgG2a titers. The degree of phosphate substitution in SPA08 also significantly increased in vitro neutralization concomitant with a decrease in proinflammatory cytokines secreted by Ser E rMOMP-restimulated splenocytes.Taken together, the results of these studies suggest that the degree of phosphate substitution in AlOOH greatly affects the adsorption of E6020 and Ser E rMOMP to AlOOH resulting in significant effects on vaccine-induced cellular and humoral responses.

Protection of outbred mice against a vaginal challenge by a Chlamydia trachomatis serovar E recombinant major outer membrane protein vaccine is dependent on phosphate substitution in the adjuvant

“…Three SPA08 adjuvanted formulations containing 10 µg of Ser E rMOMP and 40 μg/mL E6020, 2.4 mg/mL elemental Al and no phosphate (SPA08-1), 88 mM (SPA08-3) or 176 mM (SPA08-4) phosphate were prepared as described previously. 32 The phosphate to aluminum molar ratio (P:Al) of each adjuvant formulation is reported in Table 1. The SPA08-2 formulation, with 8.8 mM phosphate, was not assessed in this study as its immunological characteristics were previously shown to be similar to those of SPA08-1.…”

“…The zeta potential, particle size distribution and percentage of MOMP adsorbed for the three SerE MOMP-SPA08 formulations were determined as previously described. 32 Mice Six-week-old outbred female CD-1 mice (Charles River Laboratories) were housed at the University of California, Irvine (UCI) in isolation cubicles at a constant temperature of 24°C with a 12 h light/12 h darkness cycle and fed chow and water ad libitum. The UCI Animal Care and Use Committee approved the animal protocols.…”

“…C. trachomatis Ser E MOMP specific IgG, IgG1, IgG2a titers were measured by an enzyme-linked immunosorbent assay (ELISA) as described previously. 32,44 Briefly, 96well plates were coated with EBs in PBS at a concentration of 10 µg per ml (1 μg/well), and blocked with PBS-Tween-milk (0.05% Tween, 1% (w/v) powdered skim milk). The following steps were then carried out in a final volume of 100 µL, followed by three washes with PBS-Tween.…”

“…In vitro sero-neutralization assays, performed in 96-well plates as described previously, were repeated three times on different days. 32 Briefly, two-fold serial dilutions of mouse sera in Ca 2+and Mg 2+ -free PBS, pH 7.2, supplemented with 5% guinea pig serum were incubated with C. trachomatis Ser E [1x10 4 infectious forming units (IFU)] for 45 min at 37°C. The mixtures were added to HeLa-229 cell monolayers, centrifuged for 1 h and incubated at 37°C for 48 h. After washing, the monolayers were fixed with methanol and stained with an in-house monoclonal antibody E4 to MOMP.…”

“…We recently reported that the cellular and humoral immune responses to a vaccine containing recombinant C. trachomatis serovar E MOMP, and a proprietary adjuvant (SPA08), composed of AIOOH and the TLR4 agonist, E6020, were dependent on the degree of phosphate substitution on the AlOOH component. 32 The use of inbred strains of mice has greatly facilitated initial testing of experimental vaccine formulations by providing invaluable data on the assessment of the immune responses of the host which can help improve the protective efficacy of vaccine formulations. Inbred animals only provide information on a limited range of the possible immune responses observed in humans following vaccination.…”