Oncolytic virus-mediated remodeling of tumor microenvironment enhances efficacy of an HPV16 E6/E7 mRNA vaccine in an HPV-positive tumor

Abstract

Therapeutic vaccination targeting the viral oncoproteins E6 and E7 in HPV-positive tumors remains an attractive strategy; however, current HPV E6/E7 vaccines produced only limited clinical benefit. We developed mRNA-ubiquitin (UB)-E6/E7, an mRNA-based HPV16 E6/E7 construct incorporating an N-terminal UB tag to enhance antigen processing. UB tagging in the mRNA format significantly enhanced antigen-specific immune responses, leading to increased cytotoxic T lymphocyte (CTL) activity and higher frequencies of E7-specific CD8⁺ T cells. Consequently, mRNA-UB-E6/E7 exhibited enhanced antitumor activity compared with the control when administered as a monotherapy. However, tumor control remained incomplete. To improve therapeutic efficacy, we evaluated several combination strategies, including oncolytic virotherapy, immune modulator-encoding mRNAs, PD-1 checkpoint blockade, and a liver X receptor (LXR) antagonist. Among these approaches, only the combination with the oncolytic herpes simplex virus (FusOn-H2) showed improved therapeutic efficacy. This improvement was associated with increased intratumoral CD8⁺ T cell infiltration and enhanced CTL activity and IFN-γ production. These findings suggest that effective tumor vaccination requires not only robust systemic T cell responses but also efficient trafficking and function of effector T cells within tumor microenvironment. Collectively, our results support the combination of mRNA-UB-E6/E7 vaccination with FusOn-H2 as a promising strategy for enhancing therapeutic efficacy against HPV-associated malignancies.

Graphical abstract

Zhang and colleagues present mRNA-UB-E6/E7, an mRNA-based HPV16 E6/E7 construct incorporating an N-terminal ubiquitin tag to enhance antigen processing. This mRNA design enhances antitumor immune responses and, when combined with HSV-based oncolytic virotherapy, further improves therapeutic efficacy against cervical cancer. These findings support such a combinatorial treatment for cervical cancer.

Introduction

Human papillomavirus (HPV) is the most prevalent sexually transmitted infection worldwide and represents a major etiologic driver of several malignancies, including cervical, vulvar, anal, and penile cancers.¹ Cervical cancer remains the fourth most common cancer among women globally,² and persistent infection with high-risk HPV types accounts for more than 99% of cases.³ Although over 100 HPV genotypes have been identified, the vast majority of HPV-associated cancers are attributable to two high-risk strains, HPV16 and HPV18. HPV16 alone accounts for more than half of HPV-related malignancies, while HPV18 contributes to approximately 20% of cases.⁴ The progression from initial infection to invasive cancer is typically slow, often occurring over decades. Given the substantial global burden of HPV-driven cancers, there is an urgent need to develop improved therapeutic interventions.

HPV-driven carcinogenesis is primarily sustained by the viral oncogenes E6 and E7.⁵ These proteins are constitutively expressed in HPV-transformed cells but absent in normal tissues, making them essential for both tumor initiation and maintenance.⁶ Importantly, as foreign viral antigens, E6 and E7 are attractive targets for immunotherapy. Consequently, most therapeutic vaccine strategies for HPV-associated cancers focus on inducing robust T cell immunity against E6 and E7.⁷ By delivering these antigens, therapeutic vaccines aim to prime cytotoxic T lymphocytes (CTLs) and generate durable immune memory capable of eliminating HPV-infected and malignant cells.

A broad range of vaccine platforms targeting E6 and E7 have been explored, including viral and bacterial vectors, peptide- and protein-based formulations, cell-based approaches, and nucleic acid vaccines.^{8,9,10,11,12,13,14} Among these, DNA vaccines such as GVX-3100 have shown particular promise due to their stability, scalability, safety, and ease of antigen engineering.¹² Additionally, plasmid-based vaccines expressing HPV16 E7 fused to HSP70 have advanced into early phase clinical testing in cervical cancer patients, with second-generation variants also under evaluation.¹⁵ However, despite favorable safety profiles and measurable antigen-specific immune responses, DNA-based therapeutic vaccines have generally demonstrated limited clinical efficacy as monotherapies, suggesting that tumor-mediated immune evasion can blunt vaccine-induced antitumor immunity.¹⁶

To overcome the modest potency of DNA vaccination, mRNA-based vaccines have emerged as an attractive alternative, capable of producing stronger and instantaneous antigen expression and more robust immune activation.¹⁷ This is driven by the recent advances in mRNA vaccine technology, which leverage the inherent advantages of mRNA-lipid nanoparticle (LNP) delivery, including potent innate immune activation, more efficient in situ antigen production, and favorable safety profiles. However, most reported HPV E6/E7 mRNA vaccines have focused primarily on increasing antigen expression or enhancing immunogenicity through antigen reshuffling, secretion signals, or antigen-presenting cell (APC)-targeting domains.^18,19,20,21 These approaches optimize antigen abundance but do not directly control antigen fate or intracellular processing kinetics, critical determinants of epitope generation and MHC class I presentation. Ubiquitin (UB)-mediated proteasomal targeting has been previously used to enhance antigen processing and MHC class I presentation in DNA- and protein-based HPV vaccine platforms,^22,23 yet it has not been systematically implemented for HPV E6/E7 in an mRNA vaccine format. Incorporating UB into an E6/E7 mRNA construct is particularly compelling in light of the defining kinetics of mRNA-LNP vaccination: antigen expression is intense but transient, and therefore benefits from design features that convert short-lived protein production into a high-density “epitope burst.” Enhanced degradation is expected to increase epitope density on cells receiving the mRNA delivery, potentially facilitating cross-presentation via rapid generation and release of antigenic substrates, thereby promoting high-avidity CD8⁺ T cell priming, an essential determinant of therapeutic efficacy in HPV-driven malignancies. Moreover, UB-directed turnover minimizes intracellular accumulation of viral oncoproteins, improving the safety profile of an oncogene-encoding mRNA platform.

In this study, we directly compared DNA- and mRNA-based therapeutic vaccination targeting HPV16 E6/E7 and explored strategies to overcome the immunosuppressive barriers imposed by HPV-driven tumors. We developed an LNP-formulated mRNA vaccine encoding an HPV16 E6/E7 fusion antigen incorporating an N-terminal UB tag to enhance antigen processing and presentation. We demonstrate that this UB-enhanced mRNA platform elicits markedly stronger CD8⁺ T cell immunity than an otherwise identical DNA vaccine, leading to a measurable antitumor effect in the murine HPV16 E6/E7-expressing TC-1 tumor model. We further evaluated multiple immune-modulating combination strategies and identified oncolytic virotherapy as a particularly effective partner. Notably, combining UB-tagged E6/E7 mRNA vaccination with the oncolytic herpes simplex virus FusOn-H2 produced a better therapeutic synergy, resulting in enhanced tumor regression and maximal cytotoxic T cell activity. Together, these findings provide a framework for optimizing therapeutic HPV vaccination and underscore the importance of combinatorial approaches to overcome immune evasion in HPV-driven cancers.

Results

HPV16 E6/E7 vaccine designs and characterization

The design of the HPV16 E6/E7 fusion vaccine constructs, delivered either as mRNA with or without an N-terminal UB tag, as well as the corresponding DNA plasmids, is summarized in Figure 1A. Briefly, the HPV16 E6 and E7 coding sequences were fused in-frame by removal of the native E6 stop codon and E7 start codon, generating a single continuous open reading frame encoding the E6/E7 fusion antigen. The resulting sequence was codon-optimized during synthesis to maximize mammalian expression while preserving antigenic integrity and minimizing unintended alterations to epitope composition.

Figure 1.

Design and characterization of HPV16 E6/E7 mRNA and DNA vaccine constructs

(A) Schematic of mRNA and DNA constructs encoding the HPV16 E6/E7 fusion gene, with key elements indicated. (B) Western blot analysis of E6/E7 expression in transfected HEK293 cells using anti-HPV16 E7 primary antibody and HRP-conjugated goat anti-mouse IgG. Total protein of 30 μg was loaded per lane to ensure comparable protein input across samples. Lane 1: mRNA-GFP; Lane 2: TC-1 cells; Lane 3: pcDNA-E6/E7 (DNA-E6/7); Lane 4: pcDNA-UB-E6/7 (DNA-UbE6/E7); Lane 5: mRNA-E6/E7 (RNA-E6/7; Lane 6: mRNA-UB-E6/E7 (RNA-UbE6/7).

To evaluate whether antigen processing and presentation following mRNA delivery could be further enhanced, we incorporated an N-terminal UB tag designed to function as a degron according to the N-end rule pathway. This modification promotes rapid ubiquitination and proteasomal degradation of the expressed fusion protein, thereby increasing the generation of peptide fragments available for MHC loading. In turn, this is expected to enhance cross-presentation efficiency and improve priming of antigen-specific CD8⁺ T cell responses. Parallel DNA plasmid constructs encoding the same fusion antigen, with or without the UB tag, was generated to enable direct comparison of platform-dependent differences in antigen expression, stability, and immunogenicity.

To confirm antigen expression, HEK293 cells were transfected with each vaccine construct, with an mRNA-GFP formulation included as a negative control. Cell lysates were prepared from transfected HEK293 cells, as well as from TC-1 cells, which served as a positive control due to their endogenous expression of HPV16 E6 and E7 (not as a fusion gene, but as separate proteins). Protein samples were analyzed by western blotting using an HPV16 E7-specific monoclonal antibody (Figure 1B). As expected, no E7-reactive signal was detected in lysates from the negative control cells (lane 1). In contrast, a distinct lower-molecular-weight band corresponding to E7 alone was clearly observed in TC-1 lysates (lane 2), confirming that the antibody reliably recognizes HPV16 E7. Importantly, lysates from cells transfected with either the DNA (lanes 3 and 4) or mRNA (lanes 5 and 6) vaccine constructs exhibited a higher-molecular-weight band consistent with expression of the E6/E7 fusion antigen. These results validate successful expression of the engineered fusion constructs in vitro.

Comparative evaluation of mRNA and DNA vaccines encoding E6/E7 fusion antigens with or without UB modification for induction of antitumor immunity in vivo

To evaluate the ability of the different vaccine constructs to elicit HPV16 E6/E7-specific immune responses and confer therapeutic efficacy, C57BL/6 mice were immunized with either DNA- or mRNA-based vaccines encoding HPV16 E6/E7 fusion antigens, with or without UB fusion, according to the schedule shown in Figure 2A. Mice were assigned to five groups: pcDNA-E6/E7, pcDNA-UB-E6/E7, mRNA-E6/E7, mRNA-UB-E6/E7, and PBS control. DNA vaccines (25 μg/mouse) were administered intramuscularly three times at 7-day intervals, whereas LNP-encapsulated mRNA vaccines (10 μg/mouse) were delivered intramuscularly twice at 14-day intervals. Two weeks after the final immunization, all mice were challenged subcutaneously with 2 × 10⁵ TC-1 cells, a C57BL/6-derived tumor line stably expressing HPV16 E6 and E7.¹ Tumor growth was monitored regularly until it reached the humane endpoint defined by the approved animal protocol, at which point all remaining animals were euthanized. Mice that reached euthanasia criteria prior to study termination were recorded as events for survival analysis. Spleens were collected at the endpoint for immunological evaluation. While this design facilitated direct comparison of vaccine-induced immune responses across treatment groups, the downside is that it limits the interpretation of the Kaplan-Meier analysis as a measure of long-term survival benefit.

Figure 2.

In vivo evaluation of mRNA and DNA vaccines for antitumor immunity and efficacy

(A) Immunization and tumor challenge schedule showing vaccination timing and TC-1 tumor implantation (n = 6 mice/group). The study was terminated on day 26, when most PBS control mice reached the humane endpoint. (B) Tumor growth (showing individual tumor volumes). (C) Kaplan-Meier survival analysis. (D and E) CTL activity against TC-1 cells (D) and E6/E7-negative Panc02-H7 cells (E) at an effector-to-target ratio of 10:1. See materials and methods section for assay details. Statistical significance: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

As shown in Figure 2B, mRNA vaccination demonstrated superior antitumor efficacy compared with the control group. Importantly, incorporation of UB enhanced therapeutic activity in both platforms, resulting in significantly improved tumor control. Among all groups, mRNA-UB-E6/E7 (UB-tagged E6/E7 delivered via mRNA) exhibited the strongest antitumor effect. This therapeutic trend was mirrored in the survival analysis (Figure 2C), where the mRNA-UB-E6/E7 group showed the most prolonged survival.

Cell-mediated antitumor immunity was evaluated by cytotoxicity assays using splenocytes collected at the study endpoint or at the time of euthanasia for mice reaching humane criteria (Figure 2D). TC-1 cells were used as target cells for the cytotoxic T lymphocyte (CTL) activity assay, and syngeneic Panc02-H7 cells lacking E6/E7 expression served as specificity controls. All vaccinated groups exhibited significantly elevated tumor-specific CTL activity against TC-1 cells compared with the PBS control group, while demonstrating only background-level killing of Panc02-H7 cells (Figure 2E), confirming antigen-specific CTL responses. Fusion with UB significantly enhanced tumor cell killing in both vaccine platforms, with a more pronounced effect observed in the mRNA group. Tetramer staining also confirmed that mice immunized with mRNA-UB-E6/E7 induced E7 epitope-specific CD8 T cell response (Figure 3E). These results suggest that UB-mediated proteasomal targeting improves antigen processing and presentation, thereby augmenting CTL priming, particularly in the context of mRNA vaccination.

Figure 3.

Enhanced therapeutic efficacy of mRNA-UB-E6/E7 combined with oncolytic HSV

(A) Immunization, tumor challenge, and treatment schedule. Mice were vaccinated as described, followed by TC-1 tumor implantation. Oncolytic virus was administered intratumorally when tumors reached ∼5 × 5 mm. (B) Tumor growth of different treatment groups (showing individual volumes). (C) Kaplan-Meier survival analysis. (D) CTL activity against TC-1 cells. (E) IFN-γ release in the supernatants of the CTL assays as determined by ELISA. (F) Frequency of HPV16 E7 tetramer⁺ CD8⁺ T cells. Statistical significance: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

However, despite the marked enhancement in tumor-specific CTL activity induced by UB-tagged mRNA vaccination, this increase did not translate proportionally into improved tumor growth inhibition. Although the mRNA-UB-E6/E7 group showed a trend toward smaller tumor volumes, the difference was not statistically significant compared with the mRNA-E6/E7 group (Figure 2B). These findings suggest that the augmented antitumor T cell response did not fully manifest as effective tumor control in vivo, potentially due to suboptimal T cell trafficking to the tumor site or suppression within the immunosuppressive tumor microenvironment. This discrepancy prompted us to investigate combinatorial strategies aimed at enhancing the functional impact of RNA-UB-E6E7 elicited antitumor T cell responses.

Oncolytic HSV can potentiate the therapeutic of mRNA-UB-E6/E7-induced antitumor immunity

One possible explanation for the relatively limited therapeutic efficacy of the antitumor immunity induced by mRNA-UB-E6/E7 vaccination is the inefficient infiltration of immune cells into the tumor site. FusOn-H2, an oncolytic virus constructed from type 2 herpes simplex virus,²⁴ has been shown to promote the migration of immune cells to tumors.^25,26 Additionally, replication of oncolytic viruses within tumor cells can remodel the immunosuppressive tumor microenvironment, thereby enhancing the function of tumor-specific T cells.²⁷ To determine whether combining FusOn-H2 with mRNA-UB-E6/E7 vaccination could enhance therapeutic efficacy, we first immunized C57BL/6 mice with the mRNA-UB-E6/E7 vaccine, followed by TC-1 tumor cell implantation using the same immunization scheme as shown in Figure 3A. Once tumors reached approximately 5 mm in diameter, intratumoral (i.t.) injection of FusOn-H2 (2 × 10⁵ PFU) was administered once tumors reached the approximate size of 5 mm in diameter. Tumor growth was subsequently monitored. The results showed that FusOn-H2 alone had minimal antitumor activity in this mouse tumor model. In contrast, mRNA-UB-E6/E7 vaccination produced a significant antitumor effect, consistent with the results previously shown in Figure 2. Notably, the combination of FusOn-H2 virotherapy with mRNA-UB-E6/E7 vaccination further significantly enhanced the therapeutic effect, even with only a single administration of the virus (Figures 3B and 3C). This improved therapeutic efficacy was also reflected in increased survival of animals receiving the combination treatment.

We collected spleens from mice at the end of the experiment to assess tumor-specific immune responses. Splenocytes prepared from the harvested spleens were analyzed to evaluate antitumor immunity by measuring tumor-specific CTL activity, key cytokine release, and tetramer staining using an H-2Db tetramer loaded with the HPV16 E7 49–57 peptide RAHYNIVTF. The results demonstrated that vaccination with mRNA-UB-E6/E7 induced robust tumor-specific CTL activity, which was accompanied by interferon-gamma (IFN-γ) release (Figures 3D and 3E), consistent with findings from the previous experiment shown in Figure 2. Tetramer staining further revealed a significant population of E7 epitope-specific T cells (Figure 3F), indicating that at least part of the observed CTL activity is mediated through specific recognition of a dominant epitope within the E7 oncoprotein. Notably, combination therapy with FusOn-H2 virotherapy enhanced both CTL activity and IFN-γ production, although it did not lead to an increase in the percentage of tetramer-positive T cells, indicating that the enhanced functional immune response is likely due to increased activation or effector function of existing antigen-specific T cells rather than an expansion in their overall frequency.

Next, we performed immunohistochemical (IHC) staining on tumor tissues collected at the end of the experiment to assess the extent of immune cell infiltration. As shown in Figures 4A and 4B, tumor sections from mice treated with PBS control exhibited minimal CD8⁺ T cell infiltration, indicating that the TC-1 tumor model is immunologically “cold.” Tumors from mice vaccinated with mRNA-UB-E6/E7 alone showed noticeable CD8⁺ T cell infiltration, suggesting that the mRNA vaccination can promote T cell trafficking to the tumor site. Administration of FusOn-H2 induced appreciable CD8⁺ T cell infiltration, consistent with our previous single-cell RNA sequencing (scRNA seq) data and accompanying IHC analyses.²⁶ Notably, tumors from mice receiving the combination of mRNA-UB-E6/E7 vaccination and i.t. FusOn-H2 exhibited the highest levels of CD8⁺ T cell infiltration, suggesting possible local proliferation of T cells following their recruitment to the tumor. We also examined CD4⁺ T cell presence in tumors from the combination treatment group and found that CD4⁺ cells were only sparsely detected within the tumor tissues. Flow cytometric analysis of splenocytes revealed no significant differences in the frequencies of CD4⁺ or CD8⁺ T cells among the treatment groups (Figures 4C and 4D), indicating that the increased and preferred i.t. CD8⁺ T cell infiltration was not due to systemic alterations in T cell composition. Together, these data indicate that i.t. delivery of FusOn-H2 in TC-1 tumors not only promotes immune cell trafficking to the tumor site but may also support their expansion within the tumor microenvironment, which may be the key to potentiate the antitumor effect of the mRNA vaccine-induced antitumor immune responses.

Figure 4.

Characterization of tumor-infiltrating and splenic T cells following treatment

(A and B) Immunohistochemical staining of CD8⁺ and CD4⁺ T cells in tumor sections (20×). Representative images are shown for indicated treatments (A) and the quantification of positively stained cells by ImageJ. (B). (C and D) Flow cytometry analysis of CD8⁺ (C) and CD4⁺ (D) T cells in splenocytes, shown as percentage of total cells. Statistical significance: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Mechanistic dissection of the key determining factors for potentiating the antitumor effect of the mRNA-UB-E6/E7-induced antitumor immunity

As part of our effort to identify key factors that potentiate the antitumor activity of mRNA-UB-E6/E7-induced immunity, we conducted an additional in vivo study in which the mRNA vaccine was combined with i.t. delivery of three critical immune-modulating molecules known to support T cell activation and proliferation: CD28, 4-1BBL, and IL-15. Each of these molecules was delivered as mRNA formulated in LNPs to enable efficient local expression within the tumor microenvironment. The overall treatment scheme is illustrated in Figure 5A.

Figure 5.

In vivo evaluation of therapeutic efficacy and immune responses following combinatorial treatment with intratumoral delivery of immune modulator mRNAs

(A) Experimental design schematic. (B) CTL activity against TC-1 cells. (C) IFN-γ production in CTL assay supernatants (ELISA). (D) Frequency of HPV16 E7 tetramer⁺ CD8⁺ T cells. (E) Tumor growth curve. (F and G) Images (20×) of immunohistochemical staining of CD4⁺ and CD8⁺ T cells in tumors from IM-treated groups with or without mRNA-UB-E6/E7 vaccination, and the quantification of positively stained cells by ImageJ (G). Statistical significance: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

This combinatorial strategy enhanced tumor-specific immune responses at the functional level. Specifically, we observed increased CTL-mediated killing of tumor cells (Figure 5B), along with elevated IFN-γ release during CTL activity (Figure 5C), indicating heightened effector function. Furthermore, the treatment significantly increased the proportion of tetramer-positive CD8⁺ T cells (Figure 5D), suggesting an expansion of tumor antigen-specific T cell populations in response to the added immune stimulation.

Despite these improvements in systemic and functional T cell responses, the combinatorial treatment did not translate into a significant enhancement of overall antitumor efficacy. While mRNA-UB-E6/E7 vaccination alone produced a clear antitumor effect, as observed in previous experiments, the addition of these immune modulators failed to significantly further improve therapeutic outcomes (Figure 5E). To better understand this discrepancy, we again performed IHC analysis on tumor tissues harvested from treated mice. Although i.t. delivery of these immune modulators increased overall T cell infiltration, the infiltrating population consisted predominantly of CD4⁺ T cells rather than CD8⁺ T cells (Figures 5F and 5G). This finding contrasts sharply with the combination treatment involving FusOn-H2, where the increased tumor-infiltrating lymphocytes were primarily CD8⁺ T cells.

Taken together, these results suggest that the superior ability of FusOn-H2 virotherapy to enhance mRNA-UB-E6/E7-induced antitumor immunity is likely driven by its capacity to promote robust infiltration—and potentially local expansion—of CD8⁺ cytotoxic T cells within the tumor microenvironment, rather than simply increasing overall tetramer-positive T numbers or their activation.

Effects of some other immune modulators on the antitumor effect of mRNA-UB-E6/E7-induced tumor-specific immunity

We also evaluated the effects of combining mRNA-UB-E6/E7 vaccination with two additional immune-modulating strategies. The first was a PD-1 checkpoint inhibitor, which functions by blocking inhibitory signaling in T cells, thereby reinvigorating exhausted T cells, enhancing CTL activity, and promoting the expansion and persistence of tumor-specific T cells.²⁸ The second approach targeted liver X receptors (LXRs; LXRα and LXRβ), nuclear receptors that sense cholesterol-derived oxysterols and regulate lipid metabolism and inflammatory pathways.²⁹ Emerging evidence indicates that LXR signaling plays an important role in shaping both the metabolic and immunologic landscape of the tumor microenvironment. In particular, activation of LXR pathways has been associated with the promotion of immunosuppressive myeloid populations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which can further impair effective T cell responses. Accordingly, inhibition of LXR signaling has been proposed as a strategy to relieve metabolic and myeloid-mediated immunosuppression and thereby improve antitumor T cell function.^30,31

Based on these considerations, we combined mRNA-UB-E6/E7 vaccination with either an anti-PD-1 monoclonal antibody or an LXR inverse agonist, SR9243,³² using the treatment regimen illustrated in Figure 6A. Immunological analyses revealed that, in contrast to the combinations involving oncolytic virotherapy or immune modulator-encoding mRNAs, neither PD-1 blockade nor SR9243 co-administration significantly enhanced CTL-mediated cytotoxicity or the associated IFN-γ release (Figures 6B–6E). Only SR9243 treatment resulted in an increased proportion of tetramer-positive CD8⁺ T cells (Figures 6F and 6G), suggesting a modest effect on the expansion of antigen-specific T cells by co-administering this molecule.

Figure 6.

In vivo evaluation of therapeutic efficacy and immune responses following combinatorial treatment with anti-PD-1 and LXR inverse agonist

(A) RNA-UbE6/7 immunization, tumor implantation, and treatment schedules. (B and C) Anti-PD-1 combinatorial group: CTL activity (B) and IFN-γ production (C). (D and E) LXR inverse agonist combinatorial group: CTL activity (D) and IFN-γ production (E). (F and G) Frequency of HPV16 E7 tetramer⁺ CD8⁺ T cells in anti-PD-1 (F) and LXR inverse agonist (G) group. (H and I) Tumor growth in anti-PD-1 (H) and LXR inverse agonist (I) group. Statistical significance: ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Consistent with these findings, neither PD-1 blockade nor SR9243 co-administration led to a significant improvement in overall antitumor efficacy compared with mRNA-UB-E6/E7 vaccination alone (Figures 6H and 6I). These results suggest that, despite their known immunomodulatory functions, these interventions may be insufficient to enhance therapeutic outcomes following mRNA-UB-E6/E7 vaccination in this model, likely due to limited effects on T cell trafficking, tumor infiltration, or local effector function within the tumor microenvironment.

Discussion

Persistent infection with high-risk HPV, particularly HPV16, is a near-universal driver of cervical cancer and contributes to a substantial fraction of other anogenital and head and neck malignancies.^33,34 The viral oncoproteins E6 and E7 are constitutively expressed in tumor cells and are essential for maintaining the malignant phenotype, making them highly attractive and tumor-specific targets for immunotherapy.³⁵ Despite this strong biological rationale, the development of effective therapeutic vaccines targeting E6 and E7 has proven challenging. A variety of platforms, including DNA and mRNA, peptide, protein, and viral vector and bacterial vector-based vaccines, have demonstrated the ability to induce antigen-specific immune responses.³⁶ Although VGX-3100, a DNA-based HPV16/18 E6 and E7 vaccine, has shown therapeutic benefit in patients with HPV16/18-associated CIN2/3 lesions,¹² the overall clinical efficacy of these vaccines as monotherapies in treating established cancers has generally been limited, likely due to the immunosuppressive tumor microenvironment and inadequate effector T cell function at tumor sites.

In the present study, we developed an mRNA-based HPV16 E6/E7 vaccine and demonstrated that incorporation of an N-terminal UB tag significantly enhances its immunogenicity and antitumor activity. This effect is consistent with the known role of UB in targeting proteins for proteasomal degradation.³⁷ By accelerating antigen turnover, UB tagging likely increases the generation of peptide fragments available for MHC class I presentation, thereby improving CD8⁺ T cell priming. This interpretation aligns with established principles of UB-dependent degradation pathways, including the N-end rule, which links protein stability to N-terminal residues and UB-mediated processing.³⁸ In the context of antigen presentation, enhanced proteasomal processing can increase peptide supply for MHC-I loading, ultimately strengthening CTL responses.³⁹ Indeed, adding UB to E7 has been shown to enhance T cell responses in both DNA and viral vector-based vaccines.^40,41 Our data show that this strategy also works in mRNA-based HPV16 E6/E7 fusion vaccine, in the mRNA-UB-E6/E7. Most importantly, mRNA-UB-E6/E7 demonstrated superior potency than the DNA vaccine that contains the same UB-tagged E6/E7 fusion antigen. These findings are consistent with the growing body of evidence demonstrating that mRNA vaccines are inherently more potent than DNA-based counterparts, likely due to more efficient antigen expression and immune activation.

However, despite the enhanced immunogenicity achieved through UB modification and the inherent advantages of the mRNA platform, the mRNA-UB-E6/E7 vaccination alone was insufficient to achieve robust tumor control. This highlights a critical limitation of therapeutic cancer vaccines: the generation of systemic tumor-specific T cell responses does not necessarily translate into effective tumor eradication. One major barrier is the failure of activated T cells to efficiently traffic to, infiltrate, and function within the tumor microenvironment. Consistent with this, our data showed that although mRNA-UB-E6/E7 vaccination induced robust CTL activity, the corresponding improvement in tumor growth inhibition was modest, suggesting that additional strategies are required to fully unleash the therapeutic potential of vaccine-induced immunity.

Combination approaches have therefore been actively explored to enhance the efficacy of HPV-targeted vaccines. Clinically, encouraging results have been achieved with combinations such as HPV vaccination plus PD-1 blockade. For example, combining ISA101 vaccine, which is composed of 12 E6 and E7 peptides, with nivolumab has produced meaningful responses in HPV16-positive cancers.^42,43 Preclinical studies have also demonstrated synergy between HPV E6/E7 vaccination and PD-1/PD-L1 blockade in TC-1 and related models, particularly when specific vaccine formats and delivery strategies, such as i.t. vaccination, are used to coordinate local immune activation with checkpoint inhibition.⁴⁴ In our studies, however, the addition of anti-PD-1 mAb to mRNA-UB-E6/E7 vaccination did not improve tumor control or antigen-specific killing beyond vaccination alone, despite evidence that PD-1 blockade alone induced measurable CTL activity. Some non-mutually exclusive explanations may account for this discrepancy. For example, the mRNA-UB-E6/E7 vaccine may already induce near-maximal systemic cytotoxic T cell responses under our vaccination and early intervention schedule, thereby limiting the dynamic range for further enhancement by PD-1 blockade.

Among the combinatorial strategies evaluated in this study, FusOn-H2 oncolytic virotherapy demonstrated the most pronounced enhancement of antitumor efficacy. Mechanistically, its superiority appears to be driven by its unique ability to markedly increase CD8⁺ T cell infiltration into tumors and to remodel the tumor microenvironment to support effective T cell responses.²⁶ This effect is particularly important given that HPV-associated tumors often evade immune surveillance by suppressing chemokine signaling and innate immune activation, thereby limiting immune cell recruitment.^45,46,47 By reversing this immune-excluded phenotype, FusOn-H2 enables vaccine-induced tumor-specific T cells to access the tumor and exert their cytotoxic effects. Notably, the increased density of CD8⁺ T cells within tumors occurred without a corresponding rise in tetramer-positive T cells in the periphery, suggesting that FusOn-H2 primarily enhances local recruitment and/or in situ expansion rather than systemic T cell priming. In contrast, strategies that primarily enhanced systemic T cell activation or expansion, such as co-delivery of CD28, 4-1BBL, and IL-15, failed to improve tumor control. Notably, these approaches led predominantly to CD4⁺ T cell infiltration within tumors rather than CD8⁺ T cell accumulation, further emphasizing that the phenotype and localization of tumor-infiltrating lymphocytes are critical determinants of therapeutic efficacy. Similarly, LXR pathway modulation did not yield significant therapeutic benefit, reinforcing the idea that overcoming immunosuppressive mechanisms alone is insufficient without effective recruitment and function of cytotoxic T cells. This distinction underscores the importance of tumor-local immune modulation as a key determinant of therapeutic efficacy.

Taken together, our findings highlight the central importance of CD8⁺ T cell infiltration within the tumor microenvironment as a key determinant of effective antitumor immunity. While systemic immune activation is necessary, it is not sufficient in the absence of efficient trafficking and local effector function. The ability of FusOn-H2 virotherapy to selectively promote CD8⁺ T cell infiltration, and potentially their local proliferation, distinguishes it from other immunomodulatory approaches and likely underlies its superior capacity to potentiate mRNA-UB-E6/E7-induced antitumor responses. Oncolytic virotherapy has shown considerable clinical promise, both as a standalone treatment and in combination with other modalities, particularly immunotherapies. In this context, our findings demonstrating strong synergy between FusOn-H2 and mRNA-UB-E6/E7 vaccination provide a compelling rationale for further development of this combinatorial approach and support its potential for clinical translation in the treatment of HPV-associated malignancies.

In conclusion, our study demonstrates that UB-modified mRNA vaccines targeting HPV16 E6/E7 can elicit robust antigen-specific immune responses, including strong cytotoxic T-lymphocyte activity in ex vivo co-culture assays. However, these immune responses did not consistently translate into proportional improvements in tumor control or survival in vivo. This discrepancy likely reflects the greater complexity of the tumor setting, where effective antitumor immunity depends not only on the generation of tumor-reactive T cells but also on their trafficking to the tumor site, persistence and expansion within the tumor microenvironment, sustained effector function, adequate antigen presentation, and the ability to overcome local immunosuppressive mechanisms. To address these barriers, we evaluated several combinatorial treatment strategies aimed at enhancing the in vivo antitumor activity of vaccine-induced immune responses. Among the approaches tested, combination therapy with the oncolytic virus FusOn-H2 produced the most pronounced therapeutic benefit, potentially through promoting CD8⁺ T cell infiltration and function within tumors. These findings support the continued development of combinatorial strategies that pair potent antigen-specific vaccination with interventions designed to improve immune cell trafficking, persistence, and activity within the tumor microenvironment for the treatment of HPV-associated malignancies.

Materials and methods

Mice

Five-to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Houston, TX, USA). Animals were housed under specific-pathogen-free conditions at the animal care facility at University of Houston (Houston, TX, USA) at a density of no more than five mice per cage and maintained on a 12-h light/dark cycle at 68 to 75°F and 30% to 70% humidity. Mice received purified water and Envigo Teklad-certified rodent chow ad libitum. All animals were quarantined for 1 week before use and individually identified by numerical ear tags. All procedures were performed in accordance with protocols approved by the University of Houston Institutional Animal Care and Use Committee (IACUC) and NIH guidelines for the care and use of laboratory animals. Animal weights were monitored during the entire experiment (Figure S1B).

Cells

The TC-1 cell line, derived from C57BL/6 mouse lung epithelial cells expressing HPV16 E6 and E7, was obtained from Dr. TC Wu at Johns Hopkins University. PancO2-H7 (H7) cells were obtained from Dr. Cathy Yao at Baylor College of Medicine. The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1× nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin. To generate TC-1-GFP-Luc and H7-GFP-Luc cells for cytotoxicity assays, lentiviral particles encoding a GFP-luciferase fusion reporter were used to transduce the cells. Following transduction, cells were expanded and then sorted. TC-1-GFP-Luc and H7-GFP-Luc cells were maintained in the same culture medium.

HEK293T cells were used for construct validation studies. For DNA-based validation, cells were transfected with the indicated plasmids. For mRNA-based validation, mRNA that was prepared via in vitro transcription was transfected into HEK293T cells using polyethyleneimine (PEI) at a 4:1 PEI-to-nucleic acid ratio. At 24 h post-transfection, cells were harvested in RIPA buffer, and total protein lysates were collected for downstream purification and quantification.

DNA and mRNA preparation

DNA sequences encoding a UB tag and the HPV16 E6/E7 fusion antigen were codon-optimized for murine expression and synthesized by GenScript (Piscataway, NJ, USA). For DNA constructs, the synthesized gene was inserted into the pcDNA3.1. For mRNA production, it was cloned into a pUC-based plasmid in vitro transcription vector containing a T7 promoter, optimized 5′- and 3′-untranslated regions (UTRs), and a poly(A) tail. All plasmids were verified by restriction digestion and Sanger sequencing. Expression of the resulting DNA- and mRNA-based constructs was confirmed by transfection into HEK293T cells followed by western blot analysis. The mRNA constructs encoding CD28, 4-1BBL, and IL-15 were constructed, prepared, and characterized in the same way.

In vitro transcription and mRNA-LNP formulation

mRNA was generated by in vitro transcription from the indicated DNA templates using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer’s instructions. Transcripts were co-transcriptionally capped using a CleanCap reagent. Following synthesis, mRNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs) to remove residual reaction components. The purified mRNA was then subjected to cellulose-based purification to deplete double-stranded RNA contaminants.⁴⁸ Subsequently, mRNA was encapsulated into LNPs using a NanoAssemblr Spark system (Precision NanoSystems) via microfluidic mixing, according to the manufacturer’s protocol. Freshly prepared mRNA-LNP formulations were used for downstream in vitro and in vivo studies. Particle size was measured by dynamic light scattering using a Zetasizer (Malvern Panalytical), with LNP diameters ranging from 80 to 120 nM.

Western blot analysis of construct expression

HEK293T cells were transfected with DNA plasmids or mRNAs using PEI as the transfection reagent. At 24 h post-transfection, cells were lysed in RIPA buffer, and total protein concentrations were determined using a bicinchoninic acid (BCA) assay. Equivalent amounts of protein (30 μg) were loaded for SDS-PAGE electrophoresis, which was subsequently transferred onto nitrocellulose membranes. Membranes were probed with an anti-HPV16 E7 antibody (NM2; Santa Cruz Biotechnology). This was followed by incubation with goat anti-mouse IgG-HRP and signal was detected using an LI-COR Odyssey Fc imaging system via chemiluminescence.

In vivo studies

Mice were randomized into treatment groups. DNA vaccines were administered intramuscularly into the hindlimb (25 μg per dose) every 7 days for a total of three immunizations. mRNA-LNP formulations were administered intramuscularly (10 μg per dose) twice at a 17-day interval. Seven days after the final immunization, 2 × 10⁵ TC-1 cells were implanted subcutaneously into the right flank. Tumor growth was measured with digital calipers and monitored until tumor burden in the control group reached the humane endpoint, at which point mice were euthanized and spleens and tumors collected. Tumor volume was calculated as (length/2) × (width^2).

For combination studies, treatments were initiated after mRNA-UB-E6/E7 vaccination and tumor implantation. Anti-PD-1 antibody was administered intraperitoneally (200 μg per dose) on days 3, 5, and 7 post-implantation. The LXR inverse agonist SR9243 (MedChemExpress, HY-101442) was administered daily by intraperitoneal (i.p.) injection (60 mg/kg), as previously described.³⁰ For virotherapy, we used FusOn-H2, an HSV-2-based oncolytic virus originally constructed in our laboratory by deleting the N-terminal domain of the ICP10 gene and replacing it with the GFP gene. FusOn-H2 has been shown to induce direct oncolysis and to remodel the tumor immune microenvironment, thereby enhancing antitumor immune responses.^25,26 A single i.t. injection (2 × 10⁵ PFU) was administered when tumors reached ∼5 × 5 mm (approximately day 7 post-implantation). For immune modulation, mRNAs encoding CD28, 4-1BBL, and IL-15 (10 μg each) were administered intratumorally once tumors reached ∼5 × 5 mm.

Splenocyte preparation, tetramer staining, and other characterizations

Spleens were dissociated through a 50-μm cell strainer in RPMI-based T cell medium using a syringe plunger. Cells were pelleted by centrifugation, and erythrocytes were lysed with ACK buffer for 1 min at room temperature. Splenocytes were washed, resuspended in fluorescence-activated cell sorting (FACS) buffer, filtered, and counted for downstream analysis.

For tetramer staining, splenocytes (1 × 10⁶) were incubated with H-2D^b tetramer loaded with the HPV16 E7₄₉–₅₇ peptide (RAHYNIVTF; NIH Tetramer Core Facility) at 1:100 dilution for 30 min at 4°C in the dark. Cells were washed and stained with fluorochrome-conjugated antibodies against CD4 (clone RM4-5), CD8a (clone 53–6.7), CD44 (clone IM7), and CD62L (clone MEL-14) (all from Cytek) for 30 min at 4°C. After washing, cells were fixed, permeabilized, and analyzed by flow cytometry (BD FACSMelody). The gating strategy is shown in Figure S1A. Data were analyzed using FlowJo.

For cytotoxicity assays, TC-1-GFP-Luc cells were cocultured with splenocytes at an effector-to-target ratio of 10:1. H7-GFP-Luc cells served as antigen-negative controls. Target cell viability was assessed after 48 h using a luciferase-based assay, as previously described.⁴⁹ Supernatants collected at 48 h were analyzed for IFN-γ by ELISA. Plates were coated overnight with anti-IFN-γ capture antibody (AN-18; BioLegend), blocked, and incubated with samples and standards for 2 h. After washing, biotinylated detection antibody and streptavidin-horseradish peroxidase (HRP) were added. Plates were developed with TMB (3,3′,5,5′-tetramethylbenzidine) substrate, stopped with 1 N sulfuric acid, and read at 450 nm with background correction at 540 and 570 nm.

Immunohistochemistry

Tumors were fixed in 10% neutral buffered formalin for 48 h, transferred to 70% ethanol for 1 week, and paraffin-embedded. Sections were prepared and stained for CD4 and CD8 by immunohistochemistry. Briefly, sections were deparaffinized, rehydrated, and subjected to endogenous peroxidase quenching and blocking. Slides were incubated with primary antibodies against CD4 (EPR19514; Abcam) and CD8α (CAL38; Abcam), followed by HRP-conjugated goat anti-rabbit IgG. Signals were developed chromogenically, and slides were counterstained with hematoxylin. For quantification of positive stained cells, multiple histological sections were analyzed via ImageJ. Images were acquired at 20× magnification, and multiple fields were selected from each slide for quantification. Positive staining was quantified by color deconvolution to isolate 3,3′-diaminobenzidine (DAB)-positive signal, followed by thresholding and particle analysis to identify and count positively stained cells.

Statistical analysis

Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Group comparisons were performed using two-way ANOVA or mixed-effects analysis, as appropriate, with Tukey’s multiple-comparisons test for post hoc analysis. Tumor growth curves were analyzed longitudinally. For Kaplan-Meier analysis, survival/time-to-endpoint curves were compared using log-rank analysis with correction for multiple comparisons against the control group using the Holm-Sidak method. A p value of less than 0.05 was considered statistically significant.

Data and code availability

The data that support the findings of this study are contained in the supplementary files or available from the authors on request.

Acknowledgments

This work was supported in part by NIH grant R01CA269002 (to S.X.Z.). We thank Dr. Tzyy-Choou Wu from Johns Hopkins School of Medicine for the gift of TC-1 cells and Dr. Jiakai Hou for assistance with the flow cytometry.

Author contributions

K.J. and S.X.Z. conceived and designed the experiments; K.J. executed most of the experiments in the study with assistance from J.J.L and N.T.; W.W. conducted the IHC staining and data collection; K.J. and S.X.Z. wrote the manuscript.

Declaration of interests

The authors declare no competing interests.