Immune responses in a phase 2 clinical trial of peptide-based therapeutic human papillomavirus vaccine, PepCan, versus Candida adjuvant alone in treating cervical intraepithelial neoplasia 2/3.
Authors: Nakagawa Mayumi , Evans Teresa , Bimali Milan , Coleman Hannah , Crane Jasmine , Darwish Nadia , Lu Yong-Chen , Nookaew Intawat , Marsh Keanna , Robeson Michael Scott , Shibata Takeo , Ussery David , Greenfield William 
Published: June 2024

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Background: A non-surgical alternative for treating cervical intraepithelial neoplasia (CIN2/3) would be desirable due to a risk of cervical incompetency in subsequent pregnancies. PepCan consists of four HPV 16 E6 peptides and a Candida skin testing reagent (Candin, Nielsen Biosciences),because of Candida’s ability to regress common warts in humans, and to promote T cell proliferation and interleukin-12 secretion in vitro. We previously demonstrated safety and efficacy of Candida alone in comparison to a historical placebo for regressing CIN2/3 to no CIN ( p=0.0004 for intention-to-treat and p=0.0002 for per-protocol analyses). Methods: In this single center, randomized, double-blind Phase II study (NCT02481414), four intradermal injections of PepCan or Candida were given 3 weeks apart which were followed with two visits 6 months apart. Immune responses were assessed using interferon-g enzyme-linked immunospot (ELISPOT) assay for human papillomavirus (HPV), fluorescent-activated cell sorter (FACS) analysis for peripheral immune cells, and plasma measurements of 51 cytokines and metabolomics in patients who completed the 6-month visit (n=76). Cervical microbiome was also examined. For subjects who were HPV 16, 18, 35, or 52-positive at entry, epitope spreading was examined (n=32). In patients with sustained ELISPOT responses, bulk T cell receptor (TCR) b deep sequencing was performed (n=15). If the sustained ELISPOT responses were significant, single-cell RNA-seq and TCR sequencing were performed (n=5) for vaccine-induced HPV-specific T cells. Results: Following vaccination, new CD3 T cell responses to at least one region of the HPV 16 E6 protein were detected in 18 of 33 patients (54.5%) in the PepCan group and in 17 of 38 (44.7%) patients in the Candida group. Therefore, exogenous HPV antigens were not necessary to induce anti-HPV T cell responses. The histological responders demonstrated new E6 response in 14 of 31 (45.2%) and non-responders had it in 21 of 40 (52.5%). Pre-existing E6 responses were present in 12 of 31 (38.7%) of responders and 8 of 40 (20%) of non-responders. Epitope spreading was demonstrated in 7 of 18 patients (38.9%) in the PepCan group, in 8 of 14 (57.1%) patients in the Candida group, 6 of 10 (60%) histological responders and 9 of 22 (40.9%) histological non-responders. Plasma RANTES/CCL5 level was significantly and consistently decreased after vaccination ( p=0.004). Single-cell RNA-seq revealed increased expression of granzymes, CCR5, and EOMES in a histological responder, compared to non-responders. Conclusions: Administration of immune-enhancing adjuvant alone is sufficient for inducing anti-HPV immune responses. Consistent systemic decrease in RANTES/CCL5 was demonstrated suggesting possible mechanism in which Candida works through reducing RANTES/CCL5. Clinical trial information: NCT02481414 .