A Chlamydia trachomatis VD1-MOMP vaccine elicits cross-neutralizing and protective antibodies against C/C-related complex serovars

Abstract

Ocular and urogenital infections with Chlamydia trachomatis (C.t.) are caused by a range of different serovars. The first C.t. vaccine in clinical development (CTH522/CAF®01) induced neutralizing antibodies directed to the variable domain 4 (VD4) region of major outer membrane protein (MOMP), covering predominantly B and intermediate groups of serovars. The VD1 region of MOMP contains neutralizing B-cell epitopes targeting serovars of the C and C-related complex. Using an immuno-repeat strategy, we extended the VD1 region of SvA and SvJ to include surrounding conserved segments, extVD1^A and extVD1^J, and repeated this region four times. The extVD1^A*4 was most immunogenic with broad cross-surface and neutralizing reactivity against representative members of the C and C-related complex serovars. Importantly, in vitro results for extVD1^A*4 translated into in vivo biological effects, demonstrated by in vivo neutralization of SvA and protection/cross-protection against intravaginal challenge with both SvA and the heterologous SvIa strain.

Fig. 1. Immunogenicity of monomers (extVD1) and homologous immuno-repeats (extVD1*4).

Plasma samples (n = 5–6) were isolated 1 week post third subcutaneous (s.c.) vaccination of A/J mice with 10 µg of extVD1^A, extVD1^J, extVD1^A*4, or extVD1^J*4 emulsified in CAF01, serially diluted and added to extVD1^A (a), extVD1^J (d), intact C.t. SvA/HAR-13 (b), or intact C.t. SvJ (e) coated plates, and antigen-specific IgG was analyzed by ELISA. Each dot represents the median OD value with 25th and 75th percentiles at each titration step. In vitro neutralization of C.t. SvA/HAR-13 (c) and C.t. SvJ (f). Sera isolated 3 weeks post third vaccination were pooled for each group (n = 5–6), titrated, mixed with a fixed concentration of bacteria, inoculated onto a HaK cell monolayer, fixed, and inclusions counted. The dotted line indicates the reciprocal NT₅₀ titer. The individual experiments were repeated twice with similar results.

Fig. 2. Fine specificity of antibody and T-cell responses after extVD1^A*4 and extVD1^J*4 vaccination.

A/J mice were immunized 3 times s.c. with either 10 µg of extVD1^A*4/CAF01 or extVD1^J*4/CAF01. Three weeks after the last vaccination, sera from immunized mice were pooled (n = 5–6), diluted 1:200, and the fine specificity of the IgG antibody responses was studied using a panel of biotinylated overlapping peptides (9-mers with 8 amino acid overlap) representing the extVD1 regions from SvA (a) and SvJ (d). Each bar represents the mean OD value of two determinations. Competitive peptide inhibition of in vitro neutralization of C.t. SvA and SvJ with peptides representing the four identified B-cell epitope regions in extVD1^A (b) and extVD1^J (e), respectively. Spleen cells were used to investigate the specific IFN-γ responses using panels of 20–22-mer peptides with 10 amino acid overlap (Supplementary Table 2) spanning extVD1^A (c) and extVD1^J (f) regions. Cells from 6 mice/group were pooled and tested in triplicates. Each bar represents mean ± SEM. The individual experiment was repeated 2–3 times with similar results.

Fig. 3. Cross-neutralization of SvA and SvJ with extVD1^J*4 and extVD1^A*4-specific sera.

A/J mice were immunized with either 10 µg of extVD1^A*4/CAF01 or extVD1^J*4/CAF01. Sera were isolated 3 weeks post third s.c. vaccination and pooled for each group (n = 5–6), titrated, mixed with a fixed concentration of either C.t. SvA or SvJ, and inoculated onto a HaK cell monolayer, fixed, and inclusions counted. Cross-neutralization of C.t. SvJ with extVD1^A*4-specific serum (a). For comparison, the homologous neutralization using extVD1^J*4-specific serum is depicted (dotted gray line). Cross-neutralization of C.t. SvA with extVD1^J*4-specific serum (b). For comparison, the homologous neutralization using extVD1^A*4-specific serum is depicted (dotted gray line). Dotted black lines indicate the reciprocal 50% neutralization titers. The individual experiments have been repeated with similar results.

Fig. 4. Protective effect of extVD1^A*4/CAF01 induced immune responses.

A/J mice were immunized with extVD1^A*4/CAF01 or CAF01 alone using the SIM vaccination protocol and the results were pooled from two individual experiments (a–c). Vaginal wash samples were collected from 20 individual mice/group by flushing the vagina with 100 µl of sterile 1× PBS, diluted 15 times, and extVD1^A*4-specific IgG and IgA were measured by ELISA (a). Each line indicates median level with 25th and 75th percentiles. A Mann–Whitney test was used for comparison among groups ****p < 0.0001. ExtVD1^A*4/CAF01 (n = 19) or CAF01 alone (n = 20) vaccinated mice were challenged i.vag. 6 weeks post last vaccination with 1 × 10⁶ IFU/mouse of SvA/HAR-13 (b, c). Data are presented as median log₁₀ IFU with 25th and 75th percentiles recovered from vaginal swabs at day 3, 7, 10, 14, and 17 post infection (PID) (b). A Mann–Whitney test was used for comparison among groups. **p < 0.01 at PID3–PID10. Area under the curve (AUC) was calculated and the results presented for individual mice (c). Each line represents the median AUC with 25th and 75th percentiles. A Mann–Whitney test was used for comparison among groups **p = 0.0012. In vivo neutralization of SvA with extVD1^A*4-specific serum (d). C.t. SvA was incubated with heat-inactivated sera from vaccinated and control mice before i.vag. infection (1 × 10⁶ IFU/mouse) of C3H/HeN mice. In vivo neutralization was assessed by C.t. culture at day 3 and 7 post infection. ExtVD1^A*4 serum, n = 10, control serum, n = 10, control mice, n = 20. Data are presented as log₁₀ IFU. Each line represents the median number of IFU with 25th and 75th percentiles. Dunn’s multiple-comparison test was used for comparison among groups. *p < 0.05; **p < 0.01; ****p < 0.0001. The experiment was repeated once with similar results.

Fig. 5. Recognition of peptides representing the VD1 region of C.t. serovars with extVD1^A*4-specific serum.

A/J mice were immunized with extVD1^A*4/CAF01 (n = 20) or CAF01 alone (n = 20) using the SIM vaccination protocol. Three weeks after the 3rd vaccination, individual serum samples were pooled (n = 20), serially diluted in triplicates, and added to peptide-coated plates representing the major part of the VD1 regions of C.t. SvA, SvC/J, SvH, SvI, SvIa, SvK, SvB, SvD, SvE, SvF, and SvG. Sequences of VD1 peptides (a). Peptide-specific IgG was analyzed by ELISA (b). Each dot represents the mean OD value ± SD of triplicate readings at each titration step.

Fig. 6. Competitive inhibition of surface recognition and neutralization.

A/J mice were immunized with extVD1^A*4/CAF01 (n = 20) or CAF01 alone (n = 20) using the SIM vaccination protocol. ExtVD1^A*4-specific serum was pooled from 20 mice, prediluted, and mixed with 1 mg/ml of VD1^A/HAR-13 or 1× PBS buffer. After incubation, the mixture was added to C.t.-coated ELISA plates in duplicates and C.t.-specific IgG measured by ELISA (a). Each bar represents the mean of duplicate readings with and without the presence of the VD1^A/HAR-13 peptide. ExtVD1^A*4-specific serum was pooled from 20 mice, prediluted, and mixed with 1 mg/ml of VD1^A/HAR-13 or SPG buffer. After 45 min of incubation, the mixture was further incubated 1:1 with different C.t. serovars for 45 min before inoculation onto a HaK cell monolayer, incubated, fixed, and inclusions counted (b). Bar represents % neutralization with and without the presence of the VD1^A/HAR-13 peptide.

Fig. 7. Protective effect of extVD1^A*4/CAF01 induced immune responses against SvIa challenge.

A/J mice were immunized with extVD1^A*4/CAF01 (n = 8) or CAF01 alone (n = 9) using the SIM vaccination protocol. Six weeks post last vaccination, the mice were challenged i.vag. with 1 × 10⁶ IFU of SvIa/mouse. Data are presented as log₁₀ IFU. Each line represents the median number of IFU with 25th and 75th percentiles recovered from vaginal swabs at days 3, 7, and 10 post infection. A Mann–Whitney test was used for comparison among groups. ***p < 0.001.