Vvax001, a Therapeutic Vaccine, for Patients with HPV16-Positive High-grade Cervical Intraepithelial Neoplasia: A Phase II Trial

Human papillomavirus (HPV) is the most prevalent sexually transmitted viral infection worldwide, with an estimated lifetime probability of HPV acquisition ranging from 84.6% to 91.3% (1). Persistent infection of the cervix with HPV high-risk (hr-HPV) subtypes can cause cervical intraepithelial neoplasia (CIN). CIN encompasses a spectrum of squamous cell precancerous lesions ranging from low- to high-grade lesions, including CIN grade 2 (CIN2) and CIN grade 3 (CIN3; refs. 2, 3). Left untreated, the likelihood of progression from CIN3 to cervical cancer is approximately 30.1% within 10 years and increases to 50.3% over a 30-year time frame (4). Of the hr-HPV types, HPV type 16 (HPV16) accounts for about 60% of the cases involving (pre)malignant disease of the cervix (5, 6). HPV can integrate into the host cell genome, resulting in constitutive overexpression of the oncogenic viral proteins E6 and E7. Currently, standard-of-care treatment of CIN3 is a large loop excision of the transformation zone (LLETZ). LLETZ, an invasive surgical procedure, can be associated with a higher risk of adverse obstetric outcomes (e.g., preterm birth), cervical stenosis, and postoperative complications (e.g., bleeding, vaginal discharge, and infection; refs. 7, 8). Therefore, therapeutic HPV vaccines targeting HPV E6 and E7 tumor-specific antigens represent an attractive noninvasive alternative to the current invasive treatment of CIN lesions.

Vvax001 (scientific name: rSFVeE6,7) is a therapeutic vector vaccine comprising replication-deficient recombinant Semliki Forest virus (rSFV) particles encoding a fusion protein of the oncogenic proteins and tumor-specific antigens E6 and E7 of HPV16 (9, 10). A translational enhancer is inserted upstream of the gene encoding the E6,7 fusion protein to increase translation. Forty-eight to seventy-two hours after vaccination, infected cells undergo apoptosis, forming apoptotic cell bodies containing high levels of the HPV16 E6,7 fusion protein. Dendritic cells take up these apoptotic cell bodies, mature, and migrate to the lymph nodes, initiating a robust cellular immune response against HPV16-transformed constitutive cells expressing the E6 and/or E7 proteins.

In a previous phase I clinical trial, Vvax001 was shown to be safe and to induce robust cellular immune responses in the majority of participants (9). The aim of this phase II trial is to investigate the clinical effectiveness of Vvax001 in patients with newly diagnosed untreated HPV16-positive CIN3 lesions.

This open-label phase II intervention study was conducted in the Netherlands at the University Medical Center Groningen (UMCG). The study protocol and all amendments were approved by the Central Committee on Research Involving Human Subjects of the Netherlands, the Dutch Ministry of Health (the Hague, the Netherlands), and the local ethics committee (METc) of the UMCG (EudraCT registration number: 2019-004050-29). The study was conducted in accordance with the protocol, the Declaration of Helsinki, and the International Conference on Harmonization Guidelines for Good Clinical Practice. All patients provided written informed consent before undergoing any study procedures. Funding was provided by the Dutch Cancer Society (KWF). The trial is registered at ClinicalTrial.gov (NCT06015854).

Eligible patients were 18 years of age and older and had a histopathologic confirmed primary HPV16-positive CIN3. CIN3 was diagnosed according to a routine pathologic workup on the diagnostic biopsy. Key exclusion criteria were history of an LLETZ, adenocarcinoma in situ within the lesion, immunosuppressive treatment, active autoimmune disease, other active cancer, immunodeficiency, and pregnancy. A summary of the representativeness of the study participants is provided in Supplementary Table S1.

The vaccine Vvax001 was produced as previously described at the Unit Biotech & Advanced Therapy Medicinal Products (ATMPs), Department of Clinical Pharmacy and Pharmacology of the UMCG, following good manufacturing practices (9). In short, Vvax001 (scientific name: rSFVeE6,7) is a sterile suspension of replication-deficient rSFV encoding a fusion protein of HPV16 E6 and E7 (Supplementary Fig. S1). Vvax001 contained 1.25 × 10⁸ infectious units (i.u.) per mL in a buffer containing 227 mmol/L sodium chloride, 19 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 1% human albumin and was stored at ≤−60°C. For administration to patients, the vaccine was diluted to a dose of 5 × 10⁷ i.u. per mL with the same buffer. This dose was selected based on the results of our phase I clinical trial as plateau immunogenicity was reached at 5 × 10⁷ i.u. per mL (9).

Between March 2021 and August 2023, 24 patients were recruited from the outpatient clinic of the UMCG, of whom 19 met our inclusion and exclusion criteria, and 18 patients completed all study visits (Fig. 1A). The first patient was enrolled on March 23, 2021, and the last patient on August 8, 2023.

Patients received three immunizations of Vvax001 (5 × 10⁷ i.u.) every 3 weeks at visits (V) 2, 3, and 4 (weeks 0, 3, and 6, respectively; Fig. 1B). Vvax001 was administered as two injections: one intramuscular injection of 1 mL into each upper leg (vastus lateralis muscle). Follow-up visits were done 1 week (V5), 3 weeks (V6), 11 weeks (V7), and 19 weeks (V8) after the last Vvax001 immunization to monitor regression and potential long-term effects, including physical examination and blood collections for safety values and colposcopic assessments for monitoring regression.

The primary endpoint was clinical efficacy, defined as histopathologic regression to CIN grade 1 (CIN1) or no dysplasia at V8 (19 weeks after the last Vvax001 injection). Posttreatment biopsies were assessed by an experienced gynecopathologist, employing the same procedures used during routine pathologic workups, according to a quality assurance system following ISO 15189:2012 requirements. Shortly, after collection, tissue samples were fixed in PBS-buffered formalin, dehydrated, and paraffin blocks created. Tissue slides were stained with hematoxylin and eosin following routine procedures. Slides were assessed using the Philips digital microscopy system. Additional samples were taken from the tissue blocks to examine the presence of HPV.

Secondary endpoints included HPV16 clearance, immunogenicity of Vvax001, and safety. HPV16 clearance was determined by genotyping for HPV16 at baseline and the last visit (V8). Immunogenicity was assessed by measuring HPV16 E6– and E7–specific T-cell responses in peripheral blood at baseline and at the follow-up visits V5 to V8 (1, 3, 11, and 19 weeks after the last Vvax001 immunization, respectively). Throughout the study, adverse events (AE) were monitored according to the standard Common Terminology Criteria for Adverse Events, version 4. All AE were followed up until they were abated or until a stable situation was reached. Safety was assessed by evaluation of treatment-related AE (trAE) and serious AE.

Lesion size on colposcopic images taken at baseline and the last study visit (V8) was measured using ImageJ (version 1.54g; RRID: SCR_003070) to determine the change in lesion size (11).

To assess treatment-induced systemic changes in immunity, peripheral blood mononuclear cells (PBMC) were collected from study participants at baseline (V1) and during follow-up (V5–V8). At each collection time point, 140 mL of peripheral blood was collected. PBMC were isolated from heparinized fresh blood by Ficoll (Ficoll-Paque PLUS; GE Healthcare) density gradient, cryopreserved in 90% FCS and 10% DMSO at a concentration of 5 to 20 × 10⁶ cells per cryovial, and stored at −180°C until further experiments. Mycoplasma testing was not conducted on these PBMC.

Vaccine-induced HPV16-specific T-cell responses were assessed by enzyme-linked immunosorbent spot (ELISpot) assay as previously reported for the phase I trial (9). Cryopreserved PBMC were thawed, rested for at least 12 hours in Iscove’s modified Dulbecco’s medium (Corning), supplemented with 10% human AB serum (Sigma; IMDM-AB), and resuspended at a concentration of 2 × 10⁶ cells/mL/well in 24-well plates. Then, cells were stimulated with 2 μg/mL of HPV16 E6- or E7-derived peptide pools (15-mer with 11-aa overlap; JPT Peptide Technologies) and cultured at 37°C and 5% CO₂. Medium-only served as a negative control, whereas the CEF pool (JPT Peptide Technologies), consisting of 23 different viral peptides originating from human cytomegalovirus, Epstein-Barr virus, and influenza A virus, was used as a positive control. After 3 days, cells were harvested, washed, and seeded in four replicate wells at a density of 1 × 10⁵ cells/well in 100-μL X-VIVO 15 (Lonza) in multiscreen 96-well ELISpot plates precoated with anti-human IFN-γ (5 μg/mL in PBS; Mab-1-D1K; Mabtech; RRID: AB_2877719). As a nonspecific positive control for IFN-γ production, the cells cultured with CEF were stimulated with phytohemagglutinin (2 μg/mL; Thermo Fisher Scientific). After 20 to 24 hours of incubation at 37°C and 5% CO₂, the plates were washed thoroughly with washing buffer, consisting of PBS supplemented with 0.05% Tween 20 (Sigma). Following incubation of the cells with biotinylated anti-human IFN-γ antibody (0.3 μg/mL; mAB-7-B6-1; Mabtech; RRID: AB_2877719) for 2 hours at room temperature in the dark, cells were washed and incubated with ExtrAvidin-alkaline phosphatase (1:1,000 in PBS; Sigma) for 1 hour at room temperature in the dark. After washing thoroughly, cells were incubated with a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium-alkaline phosphatase (Sigma) substrate for spot visualization. The plates were washed with demi water, and spots were counted using the AID ELISpot reader (Autoimmun Diagnostika).

HPV-specific spots were calculated by subtracting the mean number of spots in quadruplicate wells + 2 × SD of the medium-only control from the mean number of spots in the experimental wells and shown as spot-forming units per 10⁶ PBMC. HPV-specific T-cell responses were considered positive when the mean number of spots of HPV-stimulated PBMC was 10 spots higher than that of the medium control per 10⁵ cells. A vaccine-induced response was defined as positive when T-cell frequencies were at least threefold higher after immunization compared with those responses before immunization.

For the selection of women with HPV16-positive CIN3 lesions, HPV16 and other high-risk HPV genotypes were detected in accordance with ISO 15189 as reported previously (12). In short, six 10-μm sections were cut from formalin-fixed, paraffin-embedded (FFPE) tissue biopsies of CIN3 lesions (in duplicate). The presence of CIN3 lesions was confirmed through pathologic assessment of adjacent hematoxylin and eosin–stained sections. Genomic DNA (gDNA) was extracted from the FFPE sections using the Maxwell RSC FFPE Plus DNA Kit (AS1720) on the Maxwell Clinical Sample Concentrator device (RRID: SCR_025867) according to the manufacturer’s instructions (Promega). Twenty-five nanograms of gDNA was analyzed by PCR for the presence of HPV16 DNA using HPV16-specific primers as described previously (13). In all tests, a serial dilution of DNA extracted from CaSki (ATCC; CRL1550; ∼500 integrated HPV16 copies; RRID: CVCL_1100), HeLa (ATCC; CCL2; 20–50 integrated HPV 18 copies; RRID: CVCL_0030), SiHa (ATCC; HTB35; 1–2 integrated HPV16 copies; RRID: CVCL_0032), CC10B (HPV45-positive cell line; RRID: CVCL_DF80), CC11 (HPV67-positive cell line; RRID: CVCL_DF81), and HPV-negative cell lines were included as controls for the analytic specificity and sensitivity of the HPV16-specific PCR that showed a minimal analytic sensitivity of 1 in 1,000 SiHa cells (12).

Contamination of amplification products was prevented by all standard precautions (12) using separate laboratories for pre- and post-PCR handling. Cross-contamination was prevented by using a new microtome blade any time a new case was sectioned. Two 10-μm sections were cut from an empty paraffin block before every tissue block and were analyzed in parallel with every case as a negative control, to ensure no cross-contamination had occurred. For quality control, gDNA was amplified in a multiplex PCR containing a control gene primer set resulting in products of 100, 200, 300, 400, and 600 bp according to the BIOMED-2/EuroClonality protocol (14). Only DNA samples with PCR products of 300 bp and larger were used for the detection of HPV. All samples were tested on DNA extracted from two independent sections (duplicates).

HPV clearance was evaluated using INNO-LiPA HPV Genotyping Extra II assay (Innogenetics N.V.; Fujirebio.com), which detects 32 different HPV genotypes individually with high analytic sensitivity (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 70, 73, 82, 6, 11, 40, 42, 43, 44, 54, 61, 81, 62, 67, 83, and 89). This assay involves an initial PCR amplification step, followed by a line probe assay based on reverse hybridization following manufacturer’s instructions. In short, for the gDNA used for the HPV16-specific PCR analysis of pre- and posttreatment FFPE tissue biopsies, 10 ng of gDNA was subjected to a short PCR fragment assay using biotinylated consensus primers (SPF10) amplifying a 65-bp fragment in the L1 region of multiple α-HPV types. To identify HPV type–specific sequences, the resulting biotinylated amplicons were denatured and hybridized with HPV-specific oligonucleotide probes. The inclusion of co-amplification of HLA-DPB1 sequences served as a DNA adequacy control. After performing the INNO-LiPA test, the results of each sample were scored manually using the INNO-LiPA HPV Genotyping Extra II reading card. If multiple infections were present within the same sample, each HPV genotype was assessed individually.

Human leukocyte antigen (HLA) typing was done on PBMC of all participating patients. DNA was extracted from PBMC using the fully automated QIAcube (Qiagen; RRID: SCR_018618), according to the manufacturer’s instructions. HLA class I (HLA-A, HLA-B, and HLA-C) alleles were genotyped on the extracted DNA by next-generation sequencing, using NGSgo-MX11-3 kit (GenDx) based on paired-end sequencing on the Illumina iSeq platform (Illumina), according to the manufacturer’s instructions.

Allele frequencies were calculated using the formula $n2N×100%$⁠, in which n represents the number of alleles found in the cohort and N is the total number of individuals in the cohort. The percentage of individuals positive for a specific allele is calculated as $nN × 100%$⁠, in which n represents the number of individuals positive for the specific allele.

IHC analysis for CD3, CD8, and CD19 were performed on matched pre- and posttreatment biopsies, where available. IHC staining was carried out on the fully automated BenchMark ULTRA platform (Roche, Ventana Medical Systems, RRID: SCR_025506), under standardized laboratory conditions, accredited following the ISO 15189 quality system. In short, paraffin tissue sections (3 μm) were incubated with antibodies against CD3 (remote terminal unit; Ventana Medical Systems, cat. No. 790-4341, RRID: AB_2335978), CD8 (1:20, Agilent, cat. No. M7103; RRID: AB_2075537), and CD19 (1:1,600, Thermo Fisher Scientific, cat. No. MA1-81723; RRID: AB_927800). Each slide contained a suitable tissue section, serving as an external control. The localization of the lymphocytic infiltrate was characterized as intraepithelial and/or subepithelial. Based on the localization of the lymphocytic infiltrate, patients were classified as immune “desert” (lack of lymphocytes intra- and subepithelial), “excluded” (lymphocyte infiltration is predominantly concentrated in the subepithelial regions), or “inflamed” (significant intraepithelial infiltration of lymphocytes) for T- and B-cell subtypes. The density of the lymphocytic infiltrate was categorized by lymphocyte subtype as absent (0), sparse (+), moderate (++), dense (+++), or extremely dense (++++).

The sample size of this trial was 18 patients. This sample size was determined using a fully sequential one-sided one-group log-rank test in which events were failures with an expected failure rate of 75% for untreated patients and 37.5% for treated patients. The significance level for this study was set at 95% and power at 90%.

Statistical analysis was performed using GraphPad Prism software (version 8.4.2; RRID: SCR_002798) and SPSS (version 28.0; RRID: SCR_002865). Differences in clinicopathologic characteristics between groups were evaluated using Mann-Whitney U tests for numerical variables and the Fisher exact test for categorical variables (Figs. 2 and 3). Clinicopathologic variables were entered into a univariate analysis using the Cox proportional hazards model (Supplementary Tables S2 and S3). A P value of <0.05 was considered statistically significant.

The data generated in this study are available within the article and its Supplementary Materials. Additional data may contain personally identifying information and are thus not openly available under current General Data Protection Regulation (GDPR) guidelines. Data can be requested from the corresponding author. Data are stored in a controlled access data facility at UMCG, the Netherlands.

A total of 24 patients were screened for enrollment between March 2021 and August 2023. Nineteen patients met the inclusion criteria, of whom one patient withdrew participation after screening. Therefore, 18 patients were treated with three immunizations of Vvax001 (Fig. 1A and B). Table 1 summarizes the baseline characteristics of the patients. The median age was 32.5 years (range, 29–59 years), in line with previously reported data on the median age of CIN3 in the Netherlands (Supplementary Table S1; ref. 15). Twelve of the 18 (67%) patients had a single HPV16 infection at baseline. Small cervical lesion sizes have been linked to higher response rates to therapeutic vaccination in CIN3 lesions (16, 17). We therefore subclassified CIN3 lesions according to cervical lesion size by colposcopy (lesions that cover ≤2 quadrants vs. >2 quadrants of the cervix). Four of the eighteen (22%) patients had a baseline lesion size involving >2 quadrants of the cervix. Most patients were nonsmokers (67%), 9/18 (50%) patients had a history of CIN1 to 2 for which active surveillance was indicated without treatment, and the median body mass index (BMI) was 24.36 (range, 20.2–48.99 kg/m²). All 18 patients completed all study visits and were evaluated for efficacy and translational endpoints.

Overall, Vvax001 immunizations were well tolerated and safe, with no grade ≥3 trAE (Table 2). TrAE were consistent with our phase I clinical trial. Besides local injection site reactions such as pain, swelling, erythema, and bruising, swollen lymph nodes in groins, malaise/fatigue, and myalgia were reported as well (9). All trAE resolved without intervention within a median time of 3 days (range, 1–52 days), depending on the AE.

The clinical efficacy of Vvax001 was assessed by evaluating changes in lesion size following Vvax001 immunization using colposcopy conducted at baseline and after treatment, as well as by analyzing histopathologic regression (Fig. 2A and B). All 18 patients were assessed for clinical efficacy. Reduction in lesion size on colposcopy, which was already evident at 3 to 11 weeks after the last immunization, was observed in 17/18 (94%) patients (Fig. 2A and C). Histopathologic regression, defined as regression of CIN3 lesions to no dysplasia or CIN1 (≤CIN1), occurred in 9/18 (50%) patients (Fig. 2B–D). Of the regressors, three (33%) patients had a complete regression (no dysplasia), and six patients (67%) had a regression to CIN1. All nonregressors (NR) underwent an LLETZ as the standard of care. Of note, among these patients, four (44%) patients had ≤CIN1 in the tissue derived from the LLETZ (Fig. 2D). Disease progression to invasive cancer during the trial was not observed.

To explore potential associations between histopathologic regression and clinicopathologic features, subsequent analyses were performed. No significant differences were found between histopathologic regressors and NR with regard to age, BMI, changes in lesion size upon Vvax001 immunizations, single versus multiple potential hr-HPV [(p)hr-HPV] infections at baseline, smoking status, history of CIN, and baseline lesion size (Fig. 2E–K). In addition, univariate analysis confirmed that none of the aforementioned clinicopathologic features and/or colposcopically assessed lesion size reduction were predictors for response in our cohort (Supplementary Table S2).

No disease recurrences were observed among both histopathologic regressors and NR who underwent an LLETZ, with a median and longest disease-free survival of 20 and 30 months, respectively (Supplementary Fig. S2; cutoff date May 1, 2024).

HPV16 clearance and the presence of HPV genotypes other than HPV16 were assessed by INNO-LiPA HPV genotyping on both the matched pre- and posttreatment biopsies. Sixteen of the eighteen patients could be evaluated for HPV16 clearance: eight histopathologic regressors and eight NR (Fig. 3A). Of the two patients that were not evaluable, one patient’s pre- and posttreatment biopsy tissue was not available at the time of analysis (Vvax-19) and from one patient’s posttreatment biopsy, not enough tissue could be retrieved (Vvax-12). Ten of the 16 (63%) patients exhibited HPV16 clearance 19 weeks after the last vaccination (V8; Fig. 3A and B). HPV16 clearance was strongly associated with histopathologic regression at V8 (P < 0.01; Fig. 3C). All patients (100%) with histopathologic regression exhibited HPV16 clearance at V8 (Fig. 3C). Of the NR, two (25%) patients exhibited HPV16 clearance at V8, but two other HPV types (HPV52 and HPV31) were detected in their posttreatment biopsies (Fig. 3C). Of note, multiple (p)hr-HPV types, distinct from HPV16, were more common in the posttreatment biopsies of NR when compared with the histopathologic regressors, respectively, 5/8 (63%) versus 2/8 (25%; Fig. 3A) patients. In addition, two patients with multiple (p)hr-HPV infections (one with HPV16 and HPV52 and another with HPV16 and HPV66 infections) in their pretreatment biopsy cleared both (p)hr-HPV types following Vvax001 immunizations.

No significant differences were found between HPV16 clearance and HPV persistence with regard to clinicopathologic features including the presence of single versus multiple (p)hr-HPV types at baseline, cervical lesion size, smoking status, history of CIN, change in lesion size upon Vvax001 immunization, age, and BMI (Fig. 3D–J). Additionally, univariate analysis revealed that HPV16 clearance was not correlated with any of the clinicopathologic features and/or colposcopically assessed lesion size reduction (Supplementary Table S3). The frequencies of HLA-A, HLA-B, and HLA-C alleles, across both regressors and NR, as well as among patients who cleared HPV16 and who did not, were consistent with the reference population (Leiden Eurotransplant cohort; Supplementary Table S4). Specifically, no clear over- or underexpression of specific HLA alleles was noted in any of the subgroups, and HLA-A*02:01 was present in the treated cohort.

To determine whether the observed histopathologic regression was associated with vaccine-induced T-cell responses, an IFN-γ ELISpot assay was performed in all nine patients showing histopathologic regression (Fig. 4A and B). Isolated PBMC from these patients collected at baseline (V1) and at follow-up visits (V5–V8; 1, 3, 11, and 19 weeks after the last immunization, respectively) were used. Nine of the nine (100%) regressors exhibited HPV16 E6 or E7 vaccine-induced responses (≥threefold increase in IFN-γ T-cell response) compared with baseline in at least one time point after the last immunization (Fig. 4B). In four of nine (44%) patients, the HPV16 E6– and E7–specific T-cell responses were long-lasting and still detectable 19 weeks after the last immunization.

T- and B-cell infiltration was evaluated by IHC analysis of CD3, CD8, and CD19 in both intra- and subepithelial regions of matched pretreatment (diagnostic) and posttreatment biopsy samples (Supplementary Fig. S3A; Supplementary Tables S5–S7). CD19⁺ cells were largely absent or present in minimal numbers at baseline and posttreatment (Supplementary Fig. S3B; Supplementary Table S5). When present, they were confined to the subepithelial regions.

At baseline, CD3⁺ and CD8⁺ T cells, when present, were more abundant in the subepithelial regions than in the intraepithelial regions for most patients (Supplementary Tables S6 and S7). Most pathologic regressors exhibited an immune “excluded” phenotype, whereas pathologic NR displayed either an immune “excluded” or “desert” phenotype (Supplementary Fig. S3C and S3D). Approximately 50% of intraepithelial CD3⁺ T cells were CD8⁺ at baseline, regardless of whether patients were R or NR or whether they cleared HPV16 or not (Supplementary Fig. S4A and S4B; Supplementary Table S7). In the subepithelial regions, around 20% to 60% of CD3⁺ T cells were CD8⁺ in the majority of regressors and patients who cleared HPV16, compared with 0% to 40% in most NR and patients who did not show HPV16 clearance (Supplementary Table S7).

Following immunizations, regressors and patients who cleared HPV16 generally exhibited no change or a decrease in subepithelial CD3⁺ and CD8⁺ T cells. In contrast, NR and those who did not clear HPV16 showed increased subepithelial CD3⁺ and CD8⁺ T-cell infiltration (Supplementary Fig. S3C and S3D; Supplementary Tables S6 and S7). Changes in intraepithelial immune cells were more difficult to evaluate, as half of the regressors had no residual dysplastic tissue. In the normal cervical epithelium, CD3⁺ and CD8⁺ T cells were absent. Among NR, intraepithelial CD3⁺ and CD8⁺ T-cell infiltration remained unchanged after Vvax001 immunizations. When CD3⁺ and CD8⁺ T cells were present after immunization, an increase in the proportion of CD8⁺ T cells relative to CD3⁺ T cells was observed in subepithelial regions in both regressors and NR, as well as in patients who cleared HPV16 and those who did not (Supplementary Fig. S4A and S4B).

In this open-label phase II trial, the clinical efficacy and safety of a therapeutic vaccine based on rSFV were determined in women with HPV16-positive CIN3 lesions. Vaccine-induced clearance of the underlying chronic HPV infection was evaluated. Three intramuscular immunizations with Vvax001 resulted in lesion size reduction in 94% of patients, histopathologic regression in 50% of patients, and HPV16 clearance in 63% of patients. Vaccination was well tolerated and safe without any grade >2 trAE. Immunogenicity of Vvax001 was confirmed with HPV16 E6– and E7–specific cellular immune responses in peripheral blood.

To the best of our knowledge, Vvax001 is one of the most effective therapeutic vaccines for HPV16-associated CIN3 lesions thus far. We observed a regression rate of 50%, which far exceeds the spontaneous regression rates reported for CIN3, ranging from 1.3% to 30% within 2 to 24 months after the initial biopsy (18–22). Furthermore, previous studies with therapeutic immunizations targeting E6 and E7 proteins in patients with high-grade CIN yielded histopathologic regression rates, varying from 22% to 52%, within a time frame of 1 to 6 months (16, 19–21, 23–27). In contrast to our trial, the majority of these trials included both patients with CIN2 and CIN3, with the spontaneous regression rate of CIN3 being significantly lower compared with that of CIN2 (18–23, 25–28).

It is interesting to note that four of nine NR (44%) had no residual disease in their LLETZ and showed a clear decrease in lesion size on colposcopy. It could be that in these patients, the time after the last immunization was too short to eradicate the full lesion. Recent work has shown that extending the follow-up period after three immunizations from 5 to 9 months resulted in an increased regression rate from 52% at the 5-month mark to 67% at the 9-month mark (16). Furthermore, the administration of a booster dose after the third immunization in our study could have enhanced histopathologic regression as well. A previous study, which included a booster dose 10 weeks after the third immunization, observed an increase in histopathologic regression rates, from 38% following three immunizations to 59% following four immunizations (17). However, caution is warranted in comparing these results with our results, as—unlike our trial—this trial conducted multiple biopsies throughout its duration, potentially affecting regression rates.

HPV clearance plays a crucial role in the effectiveness of CIN3 management. Data on recurrences after excision of CIN2 to 3 indicate that instances of recurrent ≥CIN2 lesions primarily occurred in women with persistent hr-HPV infection within 4 to 10 months after treatment, with HPV16 being the predominant persisting hr-HPV type (29, 30). Hence, the value of prophylactic HPV vaccination (e.g., Gardasil 9), after LLETZ to reduce the risk of recurrence, is currently under investigation in the NOVEL and VACCIN study. In our cohort, all the evaluable regressors and 25% of the evaluable NR showed HPV16 clearance. Therefore, by eliminating HPV16, our vaccine does not only have the potential to prevent recurrence among regressors but also among NR by eliminating HPV16. The observed rate of 100% HPV16 clearance among histopathologic regressors underscores that HPV16 clearance plays an important role in effective treatment response and exceeds those documented in prior trials, which ranged from 49% to 80% (16, 17, 21, 25, 26). This discrepancy may be attributed to the fact that (p)hr-HPV infection driving the CIN3 lesion in our regressors is likely HPV16, whereas in other trials, regressors might have harbored multiple (p)hr-HPV infections, with a distinct hr-HPV type acting as the primary driver that was (spontaneously) cleared while HPV16 persisted.

No recurrences were detected in either regressors or NR with the longest follow-up extending to 30 months after treatment. This suggests that Vvax001 promotes the generation of long-lasting HPV-specific memory T cells, which was confirmed by responses up to 6 months after the initiation of treatment in our IFN-γ ELISpot assay.

Although a subset of patients did not show a histopathologic response, Vvax001 significantly decreased cervical lesion size in 17 of 18 patients, including in all histopathologic NR. A smaller lesion size in the NR resulted in the removal of smaller amounts of cervical tissue by the LLETZ procedure, thereby limiting the risk of potential complications accompanied by this procedure, such as cervical insufficiency.

Five of the nine (56%) NR were found to have other (p)hr-HPV types, next to HPV16, in their pre- or posttreatment lesions. It is conceivable that a (p)hr-HPV type other than HPV16 may have been the predominant factor driving the development of the CIN lesion in these women, potentially explaining why two of the nonregressing patients managed to clear HPV16 but maintained their lesion following immunization with Vvax001, which exclusively targets HPV16. Another less likely explanation could be that the nonregressing patients had a more persistent HPV16 variant in their lesions. HPV16 variants exhibiting heightened persistence have been described in literature (31). However, most of these variants are classified within non-European lineages, diminishing the likelihood of their presence in our cohort. At baseline, nonregressing patients had a more desert phenotype characterized by a lack or scarcity of immune cells, compared with regressing patients. In high-grade vulvar squamous intraepithelial lesions, it has been demonstrated that a preexisting inflamed immune contexture within the tumor microenvironment, containing activated type 1 CD8⁺ T cells, Th cells (CD3⁺CD8⁻Foxp3⁻), and myeloid cells (HLA⁻DR⁺CD14⁺), is required for effective therapeutic immunization (32). A way to reconstruct the immune microenvironment might be imiquimod, a topical immune modulator. Imiquimod is a Toll-like receptor 7 and 8 agonist, which exerts its effect via the upregulation of IFN-α, thereby triggering a robust local inflammation, marked by local infiltration of CD4⁺ and CD8⁺ T cells, and activation of dendritic cells (33). Hence, combination treatment, in which patients with CIN3 are pretreated with imiquimod followed by the Vvax001 vaccine, could further enhance response rates.

To date, therapeutic HPV vaccines have demonstrated limited efficacy in cervical cancer largely because of the immunosuppressive tumor microenvironment, reduced uniformity of antigen expression, downregulation of MHC molecules, and impaired antigen processing. To overcome these immunoevasive features, therapeutic vaccination should be combined with other treatment modalities in cervical cancer. Preclinical studies using murine tumor models expressing HPV16 E6/E7–related tumors have shown significant efficacy of therapeutic HPV vaccination when combined with immune checkpoint inhibition (34, 35). Our study is limited by the relatively short duration of postimmunization follow-up. Although a 6-month follow-up period was chosen to enhance patient adherence and minimize loss to follow-up, trials with an extended follow-up have shown higher histopathologic response rates (16, 17). Another limitation of our study is the small sample size of 18 patients, particularly with respect to the high spontaneous regression rates observed in some studies for CIN3. To further evaluate the therapeutic effectiveness of Vvax001, larger studies incorporating a control or placebo group, along with an extension cohort with a minimum follow-up duration of 9 to 12 months, are of interest.

In conclusion, our findings demonstrate that the therapeutic vaccine Vvax001 is safe, well tolerated, and effective in eradicating HPV16-associated CIN3 lesions and clearing the underlying persistent HPV16 infection in newly diagnosed untreated patients. Therefore, evaluation of Vvax001 in a phase III trial is warranted to further explore its therapeutic efficacy and application as nonsurgical therapy for premalignant cervical lesions.

E. Schuuring reports grants from Abbott, Biocartis, SNN/EFRO, Bio-Rad, and Invitae/Archer; grants and personal fees from MSD/Merck, CC Diagnostics, Agena Bioscience, Roche, Bayer, and AstraZeneca; and personal fees from Novartis, Bristol Myers Squibb, Amgen, Illumina, Janssen Cilag (Johnson & Johnson), Astellas Pharma, GSK, Sinnovisionlab, Sysmex, and Protyon outside the submitted work. J.G.W. Kosterink reports grants from the Dutch Cancer Society (KWF) during the conduct of the study. J.C. Wilschut reports grants from the Dutch Cancer Society during the conduct of the study and is the cofounder and shareholder of Vicinivax, a Dutch clinical-stage biopharmaceutical company. Vicinivax is a spin-off of the University Medical Center Groningen, Groningen, the Netherlands, which develops therapeutic cancer vaccines. T. Daemen reports grants from the Dutch Cancer Society (KWF) during the conduct of the study; holds a patent for US2004005711A1 licensed to Vicinivax and a patent for US2024392255A1 issued; and is the cofounder of Vicinivax. J. Bart reports grants from the Dutch Cancer Society and AstraZeneca outside the submitted work. H.W. Nijman reports grants and other support from Vicinivax and grants from the Dutch Cancer Society (KWF) during the conduct of the study, as well as grants from MSD, Mendus, IMMIOS, and Sairopa outside the submitted work. M. de Bruyn reports grants from the Dutch Cancer Society and Vicinivax during the conduct of the study; grants from the European Research Council, Health Holland, Mendus, BioNovion, Aduro Biotech, Genmab, and IMMIOS; nonfinancial support from BioNTech, Surflay Nanotec, and MSD; and other support from Sairopa outside the submitted work. No disclosures were reported by the other authors.

A.L. Eerkens: Data curation, formal analysis, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. M.D. Esajas: Data curation, investigation, visualization, methodology, project administration. K. Brummel: Investigation, project administration. A. Vledder: Investigation, project administration. N. van Rooij: Data curation, formal analysis, investigation, methodology, project administration. A. Plat: Investigation, methodology, project administration. S.B. Avalos Haro: Data curation, formal analysis, investigation, project administration. S.T. Paijens: Conceptualization, methodology. L. Slagter-Menkema: Investigation, methodology, data acquisition (processing patient samples and HPV tests). E. Schuuring: Data curation, formal analysis, visualization, methodology. N. Werner: Investigation, visualization, methodology, data acquisition (assessment of posttreatment biopsies for histopathological response). J.G.W. Kosterink: Methodology, GMP vaccine development. B.-J. Kroesen: Investigation, visualization, methodology, HLA typing. J.C. Wilschut: Conceptualization, methodology. T. Daemen: Conceptualization, visualization, methodology. J. Bart: Data curation, formal analysis, investigation, visualization, methodology, writing–review and editing. H.W. Nijman: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, visualization, methodology, writing–review and editing. M. de Bruyn: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, visualization, methodology, writing–review and editing. R. Yigit: Conceptualization, data curation, formal analysis, supervision, funding acquisition, validation, visualization, methodology, writing–review and editing.

We thank all patients, their families, caregivers, and clinical investigators for participating in the Vvax phase II trial. The present trial was funded by the Dutch Cancer Society (KWF; grant number 12389). Vicinivax provided the Vvax001 vaccines. The funding source played no role in the execution of the trial, data analysis, and writing of the manuscript.