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 100μg 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.
Graphical Abstract
One sentence summary:
Adjuvant vaccination with TA-CIN is well tolerated, and its administration in the thigh of HPV16+ cervical cancer patients elicited greater CD8 T cell and antibody responses than in the arm.
INTRODUCTION
An estimated 660,000 cervical cancer cases and 350,000 deaths occurred in 2022 [1]. While a dozen hrHPV types cause virtually all cases of cervical cancer, 50-60% are HPV16+ [2]. HPV16 also causes >80% of virally-driven anogenital (vaginal/vulval/anal/penile) and oropharyngeal cancers [3]. Adjuvant immunotherapy combining chemoradiation with PD-1 blockade can improve treatment outcomes for locally advanced cervical cancer or in combination with chemotherapy with or without bevacizumab in the metastatic setting [7, 8]. While this relieves immunosuppression or exhaustion, vaccination against HPV antigens might augment tumor-specific cytotoxic T-cell responses [9].
The HPV early proteins, E6 and E7, are necessary for malignant transformation [10], solely expressed in diseased cells, and non-self, i.e. logical targets for immunotherapies that induce T cell responses. Although capsid protein L2 is considered a protective antigen, it is a therapeutic target in some models and can elicit potent T helper responses [11]. The HPV16 L2, E7, and E6 fusion protein ‘TA-CIN’ is expressed in E. coli and purified from inclusion bodies [12]. It is administered as filterable aggregates, mainly 70-80 nm in diameter, with some larger forms, given their potential to enhance immunogenicity compared to a soluble product [13, 14]. Adjuvants can greatly strengthen immune responses to TA-CIN vaccination, but are associated with increased reactogenicity and may not be necessary [15-17].
In healthy volunteers, TA-CIN was well tolerated when administered intramuscularly thrice in the upper arm at monthly intervals. Antibodies and proliferative T cell responses to TA-CIN were evident in most patients at doses ≥128μg [18]. However, immunization with TA-CIN showed only modest clinical benefit against high-grade anogenital intraepithelial neoplasia (AGIN), regardless of either an additional booster or a priming dose with HPV16 E6/E7 recombinant vaccinia virus (TA-HPV) [19, 20]. In contrast in women with high-grade vulvar intraepithelial neoplasia (VIN) [21], the complete histologic response rate increased from 32% (6/19) after imiquimod treatment (week 10) to 58% (11/19) after completing three TA-CIN vaccinations (week 20). At week 52, 63% of (12/19) patients had a complete response and 79% (15/19) of the women were symptom-free. Additionally, 36% (5/14) of HPV16+ lesions showed clearance of the virus. In another study, boosting with TA-CIN (100μg) after two priming vaccinations with an E7-targetted DNA vaccine was associated with HPV16 clearance in 45% (5/11) of women with HPV16+ ASC-US or LSIL by month 6 [22]. These findings suggest that TA-CIN is well tolerated, potentially most effective against minimal disease and when combined with other standard of care treatment modalities, and that treatment effects may require months to observe.
Surgical treatment of localized cervical cancer is associated with a 91% 5-year survival rate [6]. Chemoradiation is used to treat more advanced cases, with a 61% 5-year survival rate for regional and 19% for distant disease [6]. Recently, the KEYNOTE-A18 study showed that PD-1 blockade treatment for advanced PD-L1+ cervical cancer delays progression and improves survival [7], but efficacy likely requires pre-existing antitumor immune responses.
TA-CIN is readily produced, requires few well-tolerated administrations, and its antitumor effects are augmented by cisplatin therapy or PD-1 blockade in the TC-1 mouse model of HPV16+ cervical cancer [9]. Radiotherapy was also synergistic with therapeutic HPV vaccination in this model [23]. Route of administration impact antitumor effects, with administration into tumors producing superior antitumor effects compared to intramuscular immunization and intramuscular vaccination in a limb proximal to the tumor being more effective than in a distal limb of mice [9, 24], suggesting that vaccination within or adjacent to the tumor may be more effective [25-27]. Prior surgical removal of the tumor-draining lymph nodes compromised these effects [24]. Furthermore, immunotherapy is most effective against low tumor burden after treatment with cisplatin or radiation [23]. In these preclinical studies and a phase II study of TA-CIN vaccination in patients with high-grade VIN, systemic cellular immune responses correlated with tumor control [21]. Here we compare the safety, feasibility and humoral and cellular immunogenicity of adjuvant TA-CIN vaccination of HPV16+ cervical cancer patients at a tumor-proximal site (upper thigh) versus a distal site (upper arm). Single cell T cell receptor sequencing (TCRseq) and RNAseq was used to evaluate immune phenotypes of CD8 T cells in depth and determine the impact of vaccination on these parameters.
METHODS
Clinical study execution
This was an open-label, single-arm, pilot trial (NCT02405221) in patients with a history of HPV16+ cervical cancer. The trial was conducted at the Johns Hopkins University (JHU) and University of Alabama-Birmingham (UAB) Medical Centers, in accordance with the Declaration of Helsinki, the International Conference on Harmonization Good Clinical Practice guidelines, and applicable regulatory requirements under a single IRB (JHU IRB Approval IRB00054202). Written informed consent was obtained from all patients prior to enrollment. Complete eligibility criteria and DLT definitions for the study are provided in the Supplementary Methods.
Subjects were eligible if diagnosed with a HPV16 E6/E7 mRNA+ cervical cancer, had completed primary standard of care (SOC) treatment within the previous year, and had no clinical evidence of disease. Eligibility was restricted to disease stages 1B1-IVA. Consequently, the participants had previously received surgery alone, chemo/radiation therapy, or a combination based upon SOC approaches for their stage of cervical cancer. To assess the relevance of the site of administration on immunogenicity in the absence of measurable tumor burden, participants in the study were randomized to receive 100μg TA-CIN intramuscularly either in the upper thigh or arm (deltoid) (Supplementary Table S1). The primary objective of this study was to determine the safety and feasibility of intramuscular administration of TA-CIN vaccine via the thigh or arm in patients with a history of HPV16-associated IB1-IV cervical cancer. The secondary objectives were to evaluate the levels in each study cohort of i) antibodies and ii) T cells specific to HPV16 E6, E7, and L2 in the peripheral blood before and after post-vaccination.
Formal hypothesis testing was not designed to determine the preferred vaccination site. However, a “winner” was selected based on the observed number of immune responses [28] and TA-CIN-specific antibody titer levels. With a 1:1 randomization of a total of 14 patients (n=7/per group), the study had approximately a 70% chance to select the correct injection site based on the observed number of responders in the two groups, assuming an immune response rate of 65% vs. 45% for thigh and arm administration, respectively.
Immunology assays
HPV16 E6, E7, L2 and TA-CIN ELISA were performed as previously described [29] and SARS-Cov-2 Spike ELISA per the manufacturer’s protocol (Thermo BMS2325). Bioplex bead array kits were used to quantify serum cytokines/chemokines (Bio-Rad). Overlapping peptide pools spanning the HPV proteins of interest were used to stimulate CD8+ T cells in the MANAFEST assay [30, 31]. T cell receptor (TCR) sequencing data were analyzed using R. For each sample, TCR sequences were first aggregated by their CDR3 amino acid sequences, with frequencies calculated as the proportion of total reads per sample. Clonality was calculated using the inverse normalized Shannon entropy: 1 - (-∑(pi × log2(pi))/log2(N)), where pi represents the frequency of clone i and N is the total number of unique clones [32]. Peripheral blood CD8+ T cells were subjected to single-cell TCRseq/RNAseq using the 10X Genomics 5’DGE+VDJ assay. Data analysis was performed using the Seurat software.
RESULTS
Demographics, safety, and tolerability
Eighteen patients provided informed consent between April 2019 and February 2022, and 15, median age of 44 (range 35-83), were eligible (Figure 1). Randomization resulted in two generally balanced populations based on relevant clinical parameters (Table 1). One patient experienced an unrelated serious adverse event (adhesion-related bowel obstruction) after one dose. The patient withdrew and was replaced. Fourteen patients received 3 doses (randomized: thigh, n=7; arm, n=7). Two patients were lost to follow-up, one after completing the 6-month visit, and the other after the 12-month visit, and 12 completed the 24-month study. A total of 41 AE were reported in 15 (100%) participants, per CTCAE4.0 (Supplementary Table S2).
Fig. 1. CONSORT diagram.
Table 1.
Demographics of patients enrolled in the phase I study (NCT02405221).
| Arm-vaccinated (n = 7) |
Thigh-vaccinated (n = 8) |
Overall (n = 15) |
p-value | |
|---|---|---|---|---|
| Age | 0.52 | |||
| Median (Range) | 58 (37, 83) | 43 (35, 67) | 44 (35, 83) | |
| Race | 0.49 | |||
| White | 3 (43%) | 6 (75%) | 9 (60%) | |
| Black | 3 (43%) | 1 (13%) | 4 (27%) | |
| Other | 1 (14%) | 1 (13%) | 2 (13%) | |
| Ethnicity | > 0.99 | |||
| Non-Hispanic | 7 (100%) | 7 (88%) | 14 (93%) | |
| Hispanic | 0 (0%) | 1 (13%) | 1 (6.7%) | |
| Primary Site Disease | > 0.99 | |||
| Cervix, NOS | 3 (43%) | 4 (50%) | 7 (47%) | |
| Endocervix | 4 (57%) | 4 (50%) | 8 (53%) | |
| Histology | 0.12 | |||
| Adenocarcinoma (ADC) | 1 (14%) | 5 (63%) | 6 (40%) | |
| Squamous cell (SCC) | 5 (71%) | 3 (38%) | 8 (53%) | |
| Small cell carcinoma+ADC | 1 (14%) | 0 (0%) | 1 (6.7%) | |
| Histology Grade | > 0.99 | |||
| Poorly differentiated | 1 (14%) | 0 (0%) | 1 (6.7%) | |
| Moderately differentiated | 2 (29%) | 3 (38%) | 5 (33%) | |
| Well-differentiated | 1 (14%) | 2 (25%) | 3 (20%) | |
| Unknown/No comment | 3 (43%) | 3 (38%) | 6 (40%) | |
| Stage | 0.41 | |||
| IB | 6 (86%) | 4 (50%) | 10 (67%) | |
| IIA/IIB | 1 (14%) | 3 (38%) | 4 (27%) | |
| IIIC | 0 (0%) | 1 (13%) | 1 (6.7%) | |
| Prior Treatment | > 0.99 | |||
| Surgery Only | 2 (29%) | 3 (38%) | 5 (33%) | |
| ChemoRadiation Only | 3 (43%) | 4 (50%) | 7 (47%) | |
| Surgery + ChemoRadiation | 1 (14%) | 0 (0%) | 1 (6.7%) | |
| Surgery + Radiation | 1 (14%) | 1 (13%) | 2 (13%) | |
| Adjuvant Chemo Regimen | 0.78 | |||
| None | 3 (43%) | 5 (63%) | 8 (53%) | |
| Cisplatin | 3 (43%) | 3 (38%) | 6 (40%) | |
| Cisplatin/Etoposide | 1 (14%) | 0 (0%) | 1 (6.7%) | |
| Adjuvant Radiation Site | 0.69 | |||
| No Radiation | 2 (29%) | 3 (37%) | 5 (33%) | |
| Cervix | 1 (14%) | 2 (25%) | 3 (20%) | |
| Pelvis Involved | 4 (57%) | 2 (25%) | 6 (40%) | |
| Syed brachytherapy | 0 (0%) | 1 (13%) | 1 (7%) | |
| Months from end of prior treatment until first vaccination | >0.99 | |||
| Median (Range) | 8.4 (4-10.9) | 6.5 (4.5-11.6) | 6.7 (4-11.6) |
Treatment-related adverse events were all grade 1, including 8 skin/injection site reactions and 1 episode each of arthralgia and myalgia (Supplementary Table S3). No dose delay was required. No treatment-related serious adverse events (SAE) were reported, no IND Safety Reports were filed, and no deaths occurred during the study period. Across all the complete blood count parameters at each available time point, there were no statistically significant differences between the treatment groups (thigh vs. arm). Similarly, there were no significant differences in the trend (i.e., slope) between the treatment arms.
TA-CIN-specific antibody responses
Nine of 14 patients had TA-CIN-specific serum IgG titers of ≥50 at ≥1 time point and were considered responders, and five as non-responders (Fig. 2A). None had a detectable response (titer <50) at baseline. Four (57.1%) of the arm-vaccinated patients had detectable responses (i.e., ≥50), and five (71.4%) of the thigh-vaccinated patients responded (p>0.99). Four responding arm-vaccinated patients reached their peak titer at month 3 (median=1478; Range 1104-3793). Likewise, four responding thigh-vaccinated patients reached peak titers at month 3, and one patient reached a peak titer at month 6. Thus, the overall titers of TA-CIN serum IgG peaked after the third vaccination (week 12) and waned thereafter. TA-CIN antibody titers tended to be higher in patients vaccinated in the thigh. The antibody response to TA-CIN did not correlate with the local skin reactogenicity or patient age.
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.
Regardless of the vaccination site, antibody responses were predominantly against L2, followed by E7, whereas E6-specific antibodies were less prominent (Fig. 2B), consistent with prior data [21]. To evaluate whether surgical lymphadenectomy affected immune responsiveness to TA-CIN vaccination, rates of antibody response between patients who had received nonsurgical treatment or surgery were compared and were not significantly different (Fig. 2C). Surgical treatment typically involves resection of tumor-draining lymph nodes, yet leg-vaccination with TA-CIN, which might be expected to share these lymph nodes, was not less effective at inducing an antibody response than arm vaccination.
To understand whether there was an underlying humoral immune system deficit in the serologic non-responders, we tested for antibodies to the SARS-CoV-2 Spike (S) protein. Of the poor responders and non-responders 7/8 were positive for SARS-CoV-2 antibodies (Supplementary Table S4).
TA-CIN elicited a robust IgG response, but minimal levels of IgM were detected, suggesting a memory response. The IgG responses were not consistently skewed to a particular subclass or impacted by vaccination site (Supplementary Fig. S1).
The serum levels of 40 chemo/cytokines were assessed longitudinally. Most were undetected and did not change after vaccination. Interestingly, higher TA-CIN antibody titers at week 12 were associated with lower serum IFN-γ levels at weeks 5 and 12 (Supplementary Fig. S2). Lower G-CSF levels at week 1 was correlated with higher TA-CIN antibody titers at week 12. Higher TA-CIN antibody titers at week 12 were also correlated with lower IL8, and higher IL12p70 at week 5. None of the levels of the 40 chemokines analyzed differed significantly between the study arms, in patients who received surgery alone versus chemo/radiotherapy (±surgery), or between TA-CIN ELISA responders and non-responders.
Impact of vaccination site on TCR repertoire metrics
Prior studies in VIN patients receiving TA-CIN vaccination showed no consistent responses using ELISPOT above their weak pre-existing HPV16-specific cellular immune responses, despite the clearance of their lesions [21]. Since these data and our in-house pilot testing suggested that ELISPOT lacked sensitivity for detection of HPV16-specific CD8 T cell responses to TA-CIN, we elected to use bulk TCR sequencing to compare the clonotype frequency and diversity in PBMC pre-vaccination and from week 12 for 13 patients [33]. Analysis of TCR sequencing data from 26 samples (pre- and post-vaccination pairs from 13 patients) yielded 145,648 unique TCR clones, with a total read count of 6,996,423. Examination of the overall TCR repertoire diversity revealed distinct patterns between the arm- and thigh-vaccinated patients. Arm-vaccination showed increased clonality post-vaccination, while thigh vaccination demonstrated a slight decrease in clonality, although this difference was not statistically significant (Fig. 3A, B; p = 0.101). Prior radiotherapy was not significantly associated with a difference in CD8 T cell response with respect to clonal space (Fig. S3A).
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.
We next identified TCR Vβ clones that increased in frequency in the periphery upon vaccination, and categorized these as ‘expanded’ (clones that were detected prior to vaccination) and ‘de novo’ (clones that were not detected prior to vaccination). Analysis of expanded clonal populations revealed significant differences in repertoire structures between vaccination sites. De novo clones from the arm-vaccinated group showed higher clonality than those from the thigh-vaccinated group (Fig. 3C; p = 0.014), suggesting a more focused, oligoclonal expansion that is often indicative of an antigen-specific response. Conversely, the clonality of pre-existing expanded clones did not differ significantly between vaccination sites (Fig. 3D; p = 0.366).
Examination of the proportion of significantly expanded clones showed that the thigh-vaccinated group had a higher fraction of de novo expanded clones than the arm group (Fig. 3E; p = 0.022), while the proportion of pre-existing expanded clones was similar between sites (Fig. 3F; p = 0.534). Moreover, de novo TCRs from thigh-vaccinated patients occupied a greater clonal space than arm vaccinations (Fig. 3G; p = 0.014). In contrast, the difference in the clonal space of expanded clones according to vaccination site was not significant (Fig. 3H; p = 0.295). Likewise, prior radiotherapy was not significantly associated with a difference in the induction of de novo clones (Fig. S3B).
ViraFEST
ViraFEST, the viral-antigen-specific version of Mutation-Associated Neoantigen Functional Expansion of Specific T Cells (MANAFEST), was used to examine the antigen specificity of the identified TCRβ clonotypes [30]. Briefly, PBMC aliquots were stimulated in culture for 10 days, optimally in triplicate, with comprehensive overlapping 15mer peptide libraries derived from HPV16 E6, E7, or L2. Upon TCRseq analysis of each culture, the clonotypes significantly expanded by one particular pool of peptides were assigned the specificity of the antigen from which the peptide was derived [30]. ViraFEST was performed on both pre-vaccination and post-vaccination samples (typically 1 month after the second or third vaccination); however, because of the large requirement for cells, data on only a subset of patients/time points were obtained (Fig. 4).
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.
Post-vaccination, the specificity of clonotypes was dominated by L2, with similarly weak responses to E6 and E7. Since cervical cancer expresses E6 and E7 but typically no detectable L2, this might suggest an exhaustion phenomenon in the T cell response against these early antigens. In contrast, L2 is principally expressed in productive infections (i.e., CIN1), so the immune system was exposed, but the relevance of its continued presence in cervical cancer patients is unclear. Interestingly, patients also had rare but detectable L2-specific clonotypes in pre-immunization PBMC samples. When examining the frequency of these clonotypes of known specificity in the bulk TCRseq analyses, they were in the great majority present at <0.05% of the total, well below the sensitivity of ELISPOT, and only apparent post-vaccination. The low frequency of these HPV-specific clonotypes is consistent with our observation that there was little difference in clonal space between the cohorts that received TA-CIN in the thigh versus an arm above the 0.05% threshold.
Single cell RNA sequencing (scRNAseq) with single cell TCRseq (scTCRseq)
To study the range of, and changes in phenotype of T cells induced by TA-CIN vaccination, we performed single-cell TCR/RNA sequencing on CD8+ T cells isolated from peripheral blood before and after vaccination from four patients with a measurable TA-CIN antibody response. From an initial dataset of 82,103 cells across eight samples, quality control filtering (including mitochondrial/ribosomal RNA thresholds) yielded 55,902 high-quality cells for downstream analyses. Seurat clustering revealed eight transcriptionally distinct cell populations, which were annotated based on canonical T cell markers into major subsets, including naïve, terminal effector, MAIT cells, and various intermediate states (Fig. 5A). Naïve cells were characterized by high expression of CCR7 and SELL, while Terminal cells showed elevated cytotoxic markers, including GZMB, PRF1, and GZMH along with immune checkpoint genes (LAG3, TIGIT, and PDCD1). GZMK+ cells expressed moderate levels of cytotoxic markers, with distinctive GZMK expression. MAIT cells were identified using the canonical markers, TRAV1-2 and SLC4A10. Intermediate states (I, II, and III) showed transitional expression profiles between the naïve and terminal phenotypes, with varying levels of TCF7 and IL7R. The CD4+ CD8+ cell population contained cells expressing either CD4 or CD8, representing a distinct subset within the analysis (Fig. 5B).
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.
A comparison of cell type proportions between pre- and post-vaccination samples revealed several notable trends. The most substantial changes were observed in naïve cells, which showed a decrease in frequency post-vaccination (−5.02%, 95% CI [−10.13%, −0.25%]); terminal cells, which demonstrated a considerable increase (2.83%, 95% CI [0.88%, 5.07%]); and MAIT cells, which also increased in proportion post-vaccination (3.13%, 95% CI [0.70%, 5.46%]) (Fig. 5C, D). Other T cell subsets, including GZMK+ cells, CD4+ CD8+ cells, and the three intermediate cell states, did not show significant changes in proportion after vaccination. After FDR correction for multiple comparisons, none of the observed changes reached statistical significance at a threshold of p<0.05. The details of the data analyses are provided in Supplementary Table S5.
TCR clonotype-paired analyses showed that T cells with public virus-specific TCRs (including those specific to EBV, influenza A, and SARS-CoV2 identified by VDJdb) were distributed across clusters ranging from naïve to more effector-like CD8+ T cells in the GZMKhi clusters (Fig. 5E). Unfortunately, owing to the low frequency of uncultured peripheral blood of the antigen-specific clones identified by ViraFEST, only 14 TCRs corresponding to 136 cells were detected in the single-cell data, whereas TCRs specific for CMV, EBV, and influenza (CEF) identified by ViraFEST were localized to the same regions as the public virus-specific TCRs (Fig. 5F).
We then queried our single-cell data for expanded and de novo clones identified by bulk TCR sequencing. After excluding MAIT cells owing to their invariant TCR nature, we detected 94 expanded clonotypes in 7,354 cells and 135 de novo clonotypes in 334 cells. Expanded clones were predominantly localized to terminal clusters, whereas de novo clones were more distributed across all clusters (Fig. 5G).
DISCUSSION
This pilot study adds to the evidence that TA-CIN vaccination is well tolerated, not only by healthy volunteers, VIN patients, and AGIN patients, but also by cervical cancer patients. Most cervical cancer patients are long-term survivors, and in our two-year study none developed recurrence. While available therapies are potentially curative, minimal residual disease, especially in advanced stage, remains a significant concern. Immunotherapy has great promise for controlling chemo/radiation-resistant disease. Since a high tumor burden is associated with immune suppression, vaccination to treat minimal residual disease has significant promise [9]. The immunosuppressive milieu of the tumor microenvironment can be disrupted by chemo/radiation therapy [23, 25, 34], and its modification is likely important in the benefits of topical imiquimod treatment of VIN. Indeed, TA-CIN vaccination of VIN patients after topical treatment of lesions with imiquimod was associated with a delayed but high rate of clearance in a small phase II study [21]. While none of the patients in our study had progression of disease over the course of the 2-year follow-up, further studies are necessary to evaluate the impact of TA-CIN vaccination on clinical outcomes after standard of care therapy.
Pembrolizumab, used for the treatment of locally advanced cervical cancer, acts by relieving the suppression of prior anti-tumor immunity [7]. In a murine study, TA-CIN vaccination resulted in the upregulation of PD-L1 expression in the HPV16+ TC-1 tumor model driven by IFNγ from HPV16-specific CD8 T cell responses [9]. Combining TA-CIN vaccination with PD-1 antibody blockade synergistically controlled TC-1 tumors, and warrants study in the adjuvant setting.
Direct intratumoral administration of TA-CIN, either with or without adjuvant, in a mouse model, induced more potent antitumor immune responses and tumor control than intramuscular vaccination. However, this is not possible in patients with occult disease. In mice bearing vaginal TC-1 tumors, vaccination of the hind leg provided better tumor control than vaccination of the foreleg [24]. This suggests that while most vaccinations are given in the arm, TA-CIN vaccination in the thigh might also be more effective for cervical cancer patients. Conversely, we speculate that TA-CIN vaccination in the arm might be better for patients with HPV16+ oropharyngeal cancer. This phenomenon could be partly explained by the presence of local tumor-draining lymph nodes (tdLNs), allowing the vaccine to effectively stimulate T cell responses [24]. Earlier studies have shown evidence for the importance of the vaccination site, where tdLN-targeting vaccination elicited stronger local and systematic CD8+ T cell responses compared to non-tdLN-targeting vaccination, despite the immune-suppressed state of the tdLN [35, 36].
Advanced stage cervical cancer patients can receive high doses of radiation in the pelvic area, raising concerns that vaccination in the thigh might be compromised due to radiation damage to the local draining lymph nodes. Additionally, removal of lymph nodes during surgery might compromise responses to vaccination in the thigh [24]. Results here allay these concerns. Antibody responses trended higher for TA-CIN administered in the thigh than in the arm, and CD8 T cell responses were significantly greater. Nevertheless, antibody responses to vaccination were barely detectable or absent in multiple patients. Since these patients responded to SARS-CoV2 S antigen, this likely does not reflect a broad underlying immune deficit. This further suggests that TA-CIN presented as a filterable aggregate is not sufficiently immunogenic alone and that it should be tested further in combination with a potent adjuvant [15, 16] like GPI-0100 or a heterologous boost [12]. A single TA-CIN dose after two priming DNA vaccinations in the arm was associated with HPV16 clearance in 5/11 patients with ASC-US or LSIL [22], perhaps reflecting lower grade disease without immunosuppression and their expression of L2 [37, 38].
Additionally, we investigated the potential adjuvant effects of radiation on the immune response to vaccination. Murine studies using the TC-1 tumor model suggest that low-dose radiation enhances the anti-tumor effects of vaccination [34]. Although radiation therapy is known to damage secondary lymphoid tissues at the treatment site, the subsequent immune recovery phase may provide an optimal window for vaccination efficacy. A retrospective study of patients with stage IB2-IVA cervical cancer who underwent chemoradiation documented lymphopenia within 2 months after radiation, but lymphocyte counts gradually increased and stabilized over 12 months [39]. The gradual regeneration of lymphocytes in these patients suggests that this recovery phase might potentially affect systemic T cell responses to TA-CIN vaccination. In this pilot study, neither radiation therapy nor time since treatment correlated with response to TA-CIN vaccination, suggesting that pelvic radiation had not disabled the draining lymph nodes proximal to the thigh and negatively influenced the immune response to vaccination.
Detection of weak immune responses to TA-CIN vaccination, which historically relied on T cell proliferation assays and ELISPOT, has proven to be challenging. The advent of next-generation sequencing of TCR and immune cell transcriptomes, even at the single-cell level, provides dramatically greater sensitivity and additional phenotypic information [33]. Its application in ex vivo expansion using viral peptides (ViraFEST) also identifies the specificity of individual TCR clonotypes [30]. This information will be invaluable for improving vaccine design, choosing adjuvant strategies, and gaining a better understanding of clinical responses. For example, using ELISPOT and T cell proliferation assays, the circulating HPV-specific immune response to TA-CIN vaccination was not clearly different in AGIN patients who failed to clear their disease [19] compared to VIN patients who cleared their lesions after local imiquimod therapy [21]. This also suggests the importance of examining the local immune response in the tumor and its environs, including neighboring tertiary lymphoid sites. For example, analysis of systemic T cells does not capture their ability to traffic to the tumor site, infiltrate the tumor, and effectively kill the tumor cells, as well as the presence of numerous types of suppressor cells [33]. This is challenging in the case of occult/minimal residual disease that escapes current imaging modalities, and integrating sequence analysis of circulating tumor DNA could be informative.
The limitations of this pilot study are its small sample size, missing or limited amounts of specimens or incomplete testing due to the costs of sequencing, lack of a placebo, limited responses to TA-CIN, and short study duration to determine time to recurrence and thus possible clinical benefit. Furthermore, differences in treatment regimens reflected the stage of the disease at diagnosis, and vaccination was initiated at different times after the completion of SOC treatment. Nevertheless, our findings indicate that the delivery site should be considered when seeking to optimize therapeutic immunization against HPV+ cancer.
Supplementary Material
HIGHLIGHTS.
Adjuvant vaccination of HPV16+ cervical cancer patients with HPV16 L2E7E6 fusion protein is safe and well tolerated
Vaccination increased E6-, E7-, and L2-specific antibody and T cell levels, although L2-specific responses were predominant
Vaccination with TA-CIN in the upper thigh elicited higher CD8 T cell and antibody responses than in the arm
ACKNOWLEDGEMENTS
General: Papivax Biotech Inc. (Taipei, Taiwan) provided TA-CIN vaccine. We thank Peter Stern and Henry Kitchener for helpful discussions and encouragement.
Funding:
This work was supported by the following statements:
Commonwealth Foundation (RBSR, TCW),
V foundation (RBSR, TCW),
The Charles T. Bauer Charitable Foundation (RBSR)
National Institutes of Health (P50CA098252 for SG, TCW, WKH, RV, ANF, RCA, CAL, KNS, and R01CA237067 for TCW, RBSR). Production and testing of the TA-CIN vaccine were supported in part by the National Cancer Institute’s PREVENT and NeXT programs (to RBSR). The project was supported in part by P30CA006973 to Sidney Kimmel Comprehensive Cancer Center Core at Johns Hopkins (RBSR).
The Mark Foundation for Cancer Research (KNS),
Cancer Research Institute (KNS).
The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.
Footnotes
Data and materials availability: Upon publication, the data collected for this study, including individual de-identified participant data and a data dictionary defining each field in the set, along with the study protocol, will be made available to others with investigator support and upon execution of a data access agreement. Likewise, materials will be made available upon execution of a material transfer agreement.
Conflict of Interest Statement: We declare the following:
Stéphanie Gaillard: Research funding from AstraZeneca, Beigene, Blueprint, Compugen, Clovis/Pharma, Immunogen, Genentech/Roche, Volastra, Verastem, Tesaro/GSK. Royalties from UpToDate, Wolters Kluwer Health. Honoraria from OncLive, Medscape. Participation in Data Safety Monitoring Boards of SignPath Pharma, Verastem, Astra Zeneca. Committee member NRG Oncology.
Jade Alvarez: None.
Tianbei Zhang: None.
Hao Wang: None.
Hua-Ling Tsai: None.
Leslie Cope: None.
Amy Deery: None.
Vikrant Palande: None.
Chi-Fen Lee: None.
Amanda N. Fader: Associate Editor, Gynecologic Oncology.
Warner K. Huh: Consultant to Pinion. Deputy Editor, Gynecologic Oncology.
Rebecca C. Arend: Research funding from Immunogen, Inc., National/International Principal Investigator; AbbVie, National/International Principal Investigator; Champions Oncology, Inc., Institutional Principal Investigator; GSK, National/International Principal Investigator; Merck Sharp and Dohme, LLC, National/International Principal Investigator; Merck Sharp and Dohme, LLC, Consultant; Exelixis Inc., National/International Principal Investigator; Regeneron Pharmaceuticals, Inc., Institutional Principal Investigator; Artera, Investigator; Faeth Therapeutics, Institutional Principal Investigator; LEAP Therapeutics, Institutional Principal Investigator; AstraZeneca Pharmaceuticals LP, Advisory Board Member; Daiichi Sankyo, Advisory Board Member; Merck Sharp and Dohme, LLC, Advisory Board Member.
Margaret I. Liang: Research funding from Merck and Foundation from Women’s Cancer unrelated to this work. Editorial Board Member for Gynecologic Oncology Reports (not Gynecologic Oncology). Spouse is employed by and owns stock in GoodRx unrelated to this work.
J. Michael Straughn, Jr.: None.
Russell Vang: None.
Darin Ostrander: None.
Karen Horner: None.
Li Zhang: None.
Dipika Singh: None.
Kellie N. Smith: Dr. Smith has filed for patent protection on the MANAFEST technology described herein, has received research funding from BMS, Abbvie, and AstraZeneca, and owns founders’ equity in Clasp Therapeutics.
T.C. Wu: Dr. Wu is a co-founder of and has an equity ownership interest in Papivax LLC. Also, he owns Papivax Biotech Inc. stock options and is a member of Papivax Biotech Inc.'s Scientific Advisory Board. Additionally, under a licensing agreement between Papivax Biotech Inc. and the Johns Hopkins University, Dr. Wu is entitled to royalties on an invention described in this article. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies.
Charles A. Leath, III: Contract Research with Agenus and Seattle Genetics. Lecture Honorarium and travel support from Merck. Scientific Advisory Boards for Merck and Seattle Genetics. Editorial Board, Gynecologic Oncology.
Richard B. S. Roden: Dr. Roden is a co-founder of and has an equity ownership interest in Papivax LLC. Dr. Roden owns Papivax Biotech Inc. stock options, and Dr. Roden is a member of Papivax Biotech Inc.'s Scientific Advisory Board. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict of interest policies. Scientific Advisory Board and membership of Up Therapeutics LLC. Scientific Advisory Board, royalty income (patent US-10046026; US-9388221) and membership of PathoVax LLC. Royalty income (patent US-9149517) from BravoVax Ltd. Scientific Advisor to Quince Therapeutics.
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