A randomized pilot study of HPV16 L2E7E6 fusion protein vaccination site post-treatment for HPV16+ cervical cancer

Abstract

Objective: Since anti-tumor immunity is enhanced by vaccination of mice adjacent to human papillomavirus type 16 (HPV16+) tumors, we examined whether HPV16 L2E7E6 fusion protein (TA-CIN) vaccination in the thigh of HPV16+ cervical cancer patients would be more immunogenic than their arm.

Methods: HPV16+ cervical cancer (stage IB1-IVA) patients, who had completed standard-of-care treatment within the past year and absent evidence of disease (NED), were enrolled in a pilot study (NCT02405221). Participants were randomized 1:1 to receive three 100 μg TA-CIN monthly intramuscular immunizations either in the arm or thigh and followed for two years for safety (CTCAEv4.0), immune response, and recurrence.

Results: Fifteen patients were enrolled (median age 44, range 35-83 years); one patient experienced a non-vaccine-related adverse event after one vaccination and withdrew. Treatment-related adverse events (n = 8) were grade 1, primarily at the injection site, and self-resolved. No recurrence was observed. TA-CIN-specific antibody titers tended to be higher in thigh-vaccinated patients. Bulk TCRseq revealed significant increases in expanded and de novo T cell clones following thigh-vaccination compared with the arm. No correlation with prior treatment modality was observed. E6-, E7-, and L2-specific TCR clones expanded, although L2-specific T cell responses were predominant. One month post-vaccination, scRNAseq revealed significant expansion of MAIT and cytotoxic CD8+ T cells, and both expanded and novel TCR clonotypes were identified in the latter.

Conclusions: Thigh or arm vaccination with TA-CIN was well tolerated, but the former elicited higher CD8 T cell and antibody responses in HPV16+ cervical cancer patients with NED after primary therapy.

Keywords: Cervical cancer; E6; E7; HPV16; L2; immunotherapy.

Fig. 1.. CONSORT diagram.

Fig. 2.. Serology analysis following TA-CIN vaccination.

A) TA-CIN specific antibody titers measured by ELISA at every available time point. Titers were calculated based on IC50 values derived from absorbance curves starting at 1:25 dilution of the sera. B) Analysis of HPV16 E6, E7, and L2-specific serum antibodies represented by the difference in absorbance signal between 12-week or 6-month readings and those at the screening time point at a 1:200 dilution of the sera. C) Proportion of TA-CIN antibody responders (blue) and non-responders (yellow) at 1:200 dilution in each cohort: individuals with no prior surgery and those with prior surgery. Percentages and absolute counts are indicated within each bar.

Fig. 3.. T cell receptor repertoire metrics comparing arm and thigh vaccination sites.

A) Clonality measurements before and after vaccination. Individual lines represent paired samples from each patient, with color indicating vaccination site (Arm: orange, Thigh: blue). B) Change in clonality (post-pre vaccination) between arm and thigh vaccination sites. Wilcoxon test was used for statistical comparison. C) Clonality of de novo expanded T cell clones in post-vaccination samples by vaccination site. D) Clonality of pre-existing expanded T cell clones in post-vaccination samples by vaccination site. E) Proportion of de novo expanded T cell clones relative to the total TCR repertoire in post-vaccination samples. F) Proportion of pre-existing expanded T cell clones relative to the total TCR repertoire in post-vaccination samples. G) Clonal space occupied by de novo expanded T cell clones in post-vaccination samples. H) Clonal space occupied by pre-existing expanded T cell clones in post-vaccination samples.

Fig. 4:. Representative graphs of HPV-specific CD8+ T cell clones as identified by ViraFEST.

The ViraFEST assay was performed using PBMC collected A) pre- and B) post-TA-CIN vaccination from patient 1.002. Antigen-specific clonotypes were identified to significantly expand in at least one well by a single peptide relative to the no peptide control (NP) at an FDR of 0.001 and a p-value <0.001. Cytomegalovirus, Epstein Bar virus, and Flu viruses (CEF) peptide pools were used as positive control while HIV or no peptide (NP) were used as a negative control. Only HPV-specific expanded TCR clones are shown. C) TCR Vβ CDR3 sequences generated after stimulation with E6, E7, and L2 peptide pools in MANAFEST were used to determine clonotype frequencies identified by bulk TCR sequencing dataset of patient’s unstimulated PMBC. The y-axis represents the log frequency of TCR clones post-vaccination, while the x-axis shows pre-vaccination clone frequencies.

Fig. 5.. Single cell RNA sequencing (scRNAseq) with single cell TCRseq (scTCRseq) Analysis.

A) UMAP visualization of CD8+ T cell clusters from integrated single-cell RNA sequencing data. Eight distinct cell populations were identified: Naïve, Terminal, GZMK+, MAIT, Intermediate I, CD4+ CD8+, Intermediate II, and Intermediate III. Analysis included 55,902 cells after quality control and outlier removal. B) Expression of T cell subset-defining genes (CD8A, CD4), naïve T cell markers (CCR7, SELL), immune checkpoint (LAG3), cytotoxic cell markers (PRF1, GZMB, TCF7, IL7R, TIGIT), and MAIT cell markers (TRAV1-2, SLC4A10, PDCD1) displayed on UMAP projection. C) Cell type proportions (%) before and after vaccination across different T cell subsets, with data points colored by patient. Boxplots show the distribution of values, and lines connect paired samples from the same patient. D) Forest plot showing effect estimates from linear mixed effects models of treatment on cell type proportions. Points represent the coefficient estimate for the treatment effect, and horizontal lines represent 95% confidence intervals. Red indicates statistically significant changes, while black indicates non-significant changes. Abbreviated labels are used: "Int. I" (Intermediate I), "Int. II" (Intermediate II), and "Int. III" (Intermediate III). E) Distribution of T cells with public virus-specific TCRs identified by VDJdb across the UMAP projection. Colors indicate different antigen specificities: EBV (blue), influenza A (red), and SARS-CoV-2 (purple). F) Distribution of T cells with antigen specificities identified by ViraFEST across the UMAP projection. Colors indicate different antigen specificities: CEF (blue), L2 (green), and E6 (red). G) Distribution of T cell clones identified from bulk TCR sequencing across the UMAP projection. Red indicates pre-existing expanded clones (significantly increased from pre to post-vaccination) and blue indicates de novo expanded clones (not detected pre-vaccination but present post-vaccination). MAIT cells were excluded from this visualization due to their invariant TCR nature.