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. 2025 Jan 1;43(Pt 1):126526.
doi: 10.1016/j.vaccine.2024.126526. Epub 2024 Nov 12.

Intranasal immunization with CPAF combined with ADU-S100 induces an effector CD4 T cell response and reduces bacterial burden following intravaginal infection with Chlamydia muridarum

Taylor B Poston  1 , Jenna Girardi  2 , Marie Kim  3 , Peter Zwarycz  3 , A Grace Polson  2 , Kacy S Yount  2 , Courtne Hanlan  2 , Ian Jaras Salas  2 , Sarah Mae Lammert  4 , Daisy Arroyo  5 , Tony Bruno  5 , Manhong Wu  5 , James Rozzelle  5 , Jeff Fairman  5 , Aaron P Esser-Kahn  3 , Toni Darville  2
Affiliations

Intranasal immunization with CPAF combined with ADU-S100 induces an effector CD4 T cell response and reduces bacterial burden following intravaginal infection with Chlamydia muridarum

Taylor B Poston et al. Vaccine. .

Abstract

Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection globally, and a vaccine is urgently needed to stop transmission and disease. Chlamydial Protease Activity Factor (CPAF) is an immunoprevalent and immunodominant antigen for CD4 T cells and B cells, which makes it a strong vaccine candidate. Due to the tolerogenic nature of the female genital tract (FGT) and its lack of secondary lymphoid tissue, effective induction of protective cell-mediated immunity will likely require potent and safe mucosal adjuvants. To address this need, we produced CPAF in a cell-free protein synthesis platform and adjuvanted it with the TLR9-agonist CpG1826, a synthetic cyclic-di-AMP (CDA) STING (stimulator of interferon genes) agonist ADU-S100, and/or the squalene oil-in-water nanoemulsion, AddaS03. We determined that intranasal immunization with CPAF plus ADU-S100 was well tolerated in female mice, induced CD4 T cells characterized by TNFα production alone or in combination with IL-17 A or IFNγ, significantly reduced bacterial shedding, and shortened the duration of infection in mice intravaginally challenged with Chlamydia muridarum. These data demonstrate the potential for CDA as a mucosal adjuvant for vaccines against Chlamydia genital tract infection.

Keywords: ADU-S100; AddaS03; Chlamydia; CpG1826; Mucosal; STING; T cell.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daisy Arroyo reports financial support was provided by Vaxcyte, Inc. Tony Bruno reports financial support was provided by Vaxcyte, Inc. Manhong Wu reports financial support was provided by Vaxcyte, Inc. James Rozzelle reports financial support was provided by Vaxcyte, Inc. Jeff Fairman reports financial support was provided by Vaxcyte, Inc. Taylor B. Poston has patent Chlamydial Protease-Like Activity Factor and Adjuvant Compositions And Uses Thereof pending to Assignee. Toni Darville has patent Chlamydial Protease-Like Activity Factor and Adjuvant Compositions And Uses Thereof pending to Assignee. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.. Recombinant Cm CPAF S491A expression in a cell-free protein synthesis platform.
(A) SDS-PAGE showing full-length inactive CPAF and N-terminal and C-terminal protein fragments. (B) SDS-PAGE showing unclipped full-length K232Q inactive CPAF. (C) Analytical size-exclusion chromatography for both CPAF variants.
Figure 2.
Figure 2.. Intranasal administration of adjuvanted CPAF does not induce inflammation or gross pathology in the lung.
Levels of IFNγ, IL-6, and TNFα in the lung 3 hours (A), 24 hours (B), and 1 week (C) post immunization. H&E staining of lungs from immunized mice (n=5/group) after 3 h hours (D), 24 hours (E), and 1 week (F) post-immunization. Group 1: CPAF only, Group 2: CPAF + CpG + CDA, Group 3: CPAF + CpG + CDA + AS03, Group 4: CpG + CDA + AS03, Group 5: CPAF-2Bxy-dopa (TLR7/8 agonist).
Figure 3.
Figure 3.. Cellular and humoral responses after intranasal CPAF immunization.
(A) Schematic for immunogenicity and challenge experiments. (B) Antibody titers in mice immunized with CPAF plus CDA alone, triple adjuvant combination, or antigen alone. p=NS for all comparisons by one-way ANOVA (n=5/group). (C) IFNγ ELISpot responses in immunized mice for all adjuvant iterations (n=4–5/group). ****p<0.0001 by one-way ANOVA (D) Representative plots for CPAF-specific cytokine responses in CPAF + CDA immunized mice by ICS (n=5). (E) Frequency of CPAF-specific mono- and poly-functional memory (CD44hi CD62L−) CD4 and CD8 T cell responses in CPAF + CDA immunized mice (n=5/group). **p<0.01 by Wilcoxon rank-sum test. All data are representative of two independent experiments.
Figure 4.
Figure 4.. CPAF plus CDA intranasal immunization reduces burden in intravaginally challenged mice and does not enhance oviduct pathology.
(A) Course of C. muridarum challenge infection in immunized mice and controls (n=9–10 mice/group). rCPAF + CDA vs. PBS (−1.51 log, ****p<0.0001), rCPAF + CDA vs. rCPAF (−0.85 log, ****p<0.0001), rCPAF + CDA vs. rCPAF + CpG (−1.10 log, ****p<0.0001), rCPAF + CDA vs. rCPAF + AS03 (−0.84 log, ****p<0.0001), rCPAF + CDA vs. rCPAF + CpG + AS03 (−0.69 log, ****p<0.0001), p=NS for rCPAF + CDA vs. all vaccine combinations incorporating CDA. Significance determined by two-way repeated-measures ANOVA. (B) Frequency of oviduct hydrosalpinx after challenge of mice immunized with CPAF plus CDA alone, triple adjuvant combination, CPAF alone, or PBS. Dotted line set to oviduct hydrosalpinx frequency in PBS controls. (C) Oviduct dilatation scores of immunized mice and controls. Significance determined by Kruskal-Wallis test. *p<0.05, **p<0.01
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