Ad-E6/7-HR vaccine improves the prophylactic and therapeutic efficacy in HPV-associated cancers

2.1 Construction of Ad-E6/7-HR vaccine

We employed a replication-defective Ad5 vector to construct a novel vaccine named Ad-E6/7-HR, incorporating HPV16 E6 and E7, along with HR1 and HR2, arranged in tandem (Figure 1A and B). Ad-E6/7, based on the same Ad5 vector (Figure 1A and B), was designed concurrently as control to evaluate the superiority of the Ad-E6/7-HR. Ad-NC, a recombinant adenovirus vector devoid of target genes, serves as a negative control to exclude any immune responses that might arise from the vector itself (Figure 1A and B), rather than from fusion antigens. Ad-NC, Ad-E6/7, and Ad-E6/7-HR were then transduced into the HEK293A cells to detect the fusion protein expression. We observed protein expression in cells transduced with Ad-E6/7-HR and Ad-E6/7 analysed by Western blot. Notably, fusion proteins were expressed as trimers attributable to the self-assembly properties of HR1 and HR2, in line with previous studies (Figure 1C).³⁴ Moreover, muscle tissues from mice injected with PBS, Ad-NC, Ad-E6/7-HR, or Ad-E6/7 were isolated and analysed 48 h post-injection, demonstrating protein expression in vivo in Ad-E6/7-HR and Ad-E6/7 groups (Figure 1D). We proposed a hypothesis that the trimeric form of E6 and E7 could enhance the immunogenicity and antitumour efficacy of Ad-E6/7-HR. Although Ad-E6/7-HR and Ad-E6/7 didn't vary significantly in the antitumour efficacy in the prophylactic model (Figure S1A-C), Ad-E6/7-HR exhibited superior efficacy in the therapeutic model (Figure 1E-G), which suggested that Ad-E6/7-HR vaccine owned superior antitumour efficacy over conventional vaccine formulations, highlighting its prospect as a promising candidate for malignancies associated with HPV.

2.2 Trimeric structure enhances the immunogenicity of Ad-E6/7-HR

To assess whether the trimeric configuration augmented the immunogenicity of Ad-E6/7-HR, mice were euthanised two weeks following a single intramuscular administration of Ad-E6/7-HR, Ad-E6/7, Ad-NC, or PBS (Figure 2A). Compared with the other three groups, Ad-E6/7-HR considerably increased the proportion of antigen-specific CD8⁺ T cells in peripheral blood (Figure 2B and C). Further analysis of splenocytes revealed that Ad-E6/7-HR caused a pronounced increase in antigen-specific CD8⁺ T cells, indicative of robust systemic immune responses conferred by the trimeric structure (Figure 2D and E). Activation of CD4⁺ and CD8⁺ T cells was boosted (Figure 2F and G), confirming that Ad-E6/7-HR vaccine effectively primed the adaptive immune system. Notably, we found a sharp decrease in CD8⁺ naïve T cells (Figure 2I) along with a substantial expansion in CD8⁺ effector memory T cells (TEM, Figure 2J), pivotal for providing robust and timely protection when bodies were faced with the same attack.

To figure out the activity of splenic CD8⁺ T cells, we detected the cytokine secretion following stimulation with E6 peptide pool or E7 peptide respectively. Intracellular cytokine staining (ICS) manifested that after stimulated by E6 peptide pool, a 5.7-fold expansion in the ratio of tumour necrosis factor-α⁺ (TNF-α) CD8⁺ T cells was induced by Ad-E6/7-HR compared with the other groups (Figure 2K). Similarly, while interferon-γ⁺ (IFN-γ) CD8⁺ T cells were nearly undetectable in PBS and Ad-NC groups, Ad-E6/7-HR elicited a 2.1-fold higher frequency of these cells relative to Ad-E6/7 (Figure 2L). Meanwhile, multiple folds of CD8⁺ T cells secreting IFN-γ and TNF-α were fostered upon E7 peptide stimulation in the Ad-E6/7-HR group (Figure 2M and N). In conclusion, Ad-E6/7-HR not only evoked potent immediate immune responses but also generated a memory pool that could offer durable protection against HPV-associated cancers, underscoring the immunogenic superiority of Ad-E6/7-HR attributable to its trimeric confirmation.

2.3 Ad-E6/7-HR exhibits prophylactic efficacy in TC-1 models

A prophylactic murine model was employed to detect the antitumour potential of Ad-E6/7-HR. A single dose of 5× 10⁹ viral particles (VP) of Ad-E6/7-HR, Ad-NC, or PBS was administered intramuscularly to mice. Subsequent to immunisation, 2× 10⁵ TC-1 cells were subcutaneously inoculated 7 days later (Figure 3A). Tumour growth was monitored. In contrast to other groups, all mice in the Ad-E6/7-HR group remained tumour-free and survived throughout the study (Figure 3B and C). Tumour weights of the Ad-E6/7-HR group were substantially reduced relative to other groups (Figure 3D and E). Mice were sacrificed to investigate the cellular immune responses on Day 28. A pronounced increase in antigen-specific CD8⁺ T cells was demonstrated, both in peripheral blood and spleen (Figure 3F-I). A modest rise in overall CD8⁺ T cells indicated early lymphocyte activation (Figure 3J).³⁵ Additionally, Ad-E6/7-HR induced the elevated generation of CD8⁺ central memory T cells (TCM) and CD8⁺ TEM subsets (Figure 3K and L), which could provide long-lasting protection by swiftly generating activated cells, secreting cytokines, and recalling immune responses upon antigen re-exposure.³⁶ Ratios of CD8⁺ T cells expressing cytokines were considerably enhanced post-treatment (Figure 3M-O), with a more than 3-fold increase after E7 peptide stimulation (Figure 3N and O). Consistent with results of ICS, higher levels of IFN-γ stimulated by E6 peptide pool (Figure 3P) or E7 peptide (Figure 3Q) were observed in the Ad-E6/7-HR group than the other groups in the enzyme-linked immunospot (ELISPOT) analysis. In a word, Ad-E6/7-HR could elicit robust cellular immune responses and provide antitumour protection in response to HPV-associated cancers.

2.4 Ad-E6/7-HR provides long-lasting protection

To appraise the sustained antitumour protection provided by Ad-E6/7-HR, mice were rechallenged with 2× 10⁵ TC-1 cells in the contralateral flank of dorsum on Day 70 (Figure 4A). Notably, Ad-E6/7-HR completely rescued mice from rechallenge and all mice remained tumour-free (Figure 4B-E), underscoring the enduring protective ability of the vaccine. Fourteen days post-rechallenge, a subset of mice was euthanised, while the remaining mice were monitored till the end of the experiment. To elucidate the function of CD8⁺ T cells in the long-lasting protection, immune responses in rechallenged mice were appraised. Immunised mice exhibited a nearly 4-fold rise in E6 tetramer⁺ CD8⁺ T cells (Figure 4F), and a striking 35-fold boost in E7 tetramer⁺ CD8⁺ T cells (Figure 4G). Ad-E6/7-HR evidently attenuated the ratio of CD8⁺ naïve T cells (Figure 4I), reflecting CD8⁺ T-cell activation following tumour rechallenge. Concomitantly, CD8⁺ TCM and CD8⁺ TEM were substantially expanded, with an over 3-fold increase in CD8⁺ TEM (Figure 4J and K), suggesting the establishment of a durable memory pool capable of immune surveillance. Moreover, stimulated by E6 peptide pool, the frequency of IFN-γ⁺ CD8⁺ T cells was raised almost 5 times (Figure 4L), while E7 peptide stimulation resulted in a 3-fold increase analysed by ICS (Figure 4M), which was further corroborated by ELISPOT assays (Figure 4N and O). In general, all these results implied that Ad-E6/7-HR could elicit compelling cellular immunity and offer long-term protection against HPV-associated malignancies.

2.5 Ad-E6/7-HR exhibits therapeutic efficacy in established tumours

We next evaluated the therapeutic efficacy of Ad-E6/7-HR in murine models. TC-1 cells were subcutaneously inoculated and Ad-E6/7-HR, Ad-NC, or PBS were intramuscularly injected 3 days later (Figure 5A). Tumour growth was markedly slowed in the Ad-E6/7-HR group, with smaller sizes of tumour and extended survival witnessed (Figure 5B-E). To assess the immunological potential induced by Ad-E6/7-HR, peripheral blood and splenic cells were collected and analysed. Ratios of E6 tetramer⁺ CD8⁺ T cells in the Ad-E6/7-HR group was almost 3-fold greater than those in the PBS group and nearly 2-fold over those in the Ad-NC group (Figure 5F). With respect to E7 tetramer⁺ CD8⁺ T cells, the proportion was 10 times over that of the PBS group and 5 times compared with the Ad-NC group after treatment (Figure 5G). In accordance with what we found in the blood, levels of E6 tetramer⁺ CD8⁺ T cells were approximately twice higher than those in PBS and Ad-NC groups (Figure 5H), whilst the ratio of E7 tetramer⁺ CD8⁺ T cells was almost 10-fold larger (Figure 5I). Despite the minor rise of CD4⁺ T and CD8⁺ T cells (Figure 5J and K), Ad-E6/7-HR led to a notable expansion of CD8⁺ TEM in the spleen (Figure 5L). To evaluate the antigen specificity and functional capacity of T cells, splenic lymphocytes were isolated and restimulated with E6 peptide pool or E7 peptide respectively. As anticipated, multiple folds of IFN-γ- and TNF-α-expressing CD8⁺ T cells were observed, correlated with the antitumour efficacy exertion (Figure 5M-P). The ELISPOT assay further confirmed the IFN-γ secretion, whose result was consistent with that of ICS (Figure 5Q and R). These findings highlighted the therapeutic potential of the Ad-E6/7-HR in halting tumour progression.

2.6 Ad-E6/7-HR modulates the tumour microenvironment

To elucidate the mechanisms underlying tumour growth inhibition mediated by Ad-E6/7-HR, we extracted tumour tissues and explored cell infiltration in the tumour microenvironment (TME). The TME revealed a conspicuous rise of total CD8⁺ T-cell infiltration as judged by immunofluorescence staining and flow cytometry (Figure 6A and B), implying that T-cell accumulation probably mediated the effectiveness. By tetramer staining, we investigated that tetramer⁺ CD8⁺ T cells were dramatically increased (Figure 6C and D). Mice immunised with Ad-E6/7-HR generated a 10-fold and 2-fold expansion in E6 tetramer⁺ CD8⁺ T cells relative to PBS and Ad-NC groups. Meanwhile, E7 tetramer⁺ CD8⁺ T cells were elevated by 1000-fold and 390-fold in the Ad-E6/7-HR group compared with PBS and Ad-NC groups, respectively. Moreover, CD8⁺ TEM in the Ad-E6/7-HR group was almost 4-fold greater than that in the other groups (Figure 6E), suggesting enhanced immune surveillance and long-lasting protection. Corresponding with the efficacy of suppressing tumour growth, multiple times of functional CD8⁺ T cells were demonstrated (Figure 6F and G), highlighting the induction of robust antitumour immunity. Myeloid-derived suppressor cells (MDSC) and M2 macrophages, related to immune suppression,^{37, 38} were considerably decreased in the Ad-E6/7-HR group (Figure 6H and I). To conclude, Ad-E6/7-HR contributed to the inhibition of tumour progression by inducing cellular immune responses and reshaping the TME conducive to tumour control.

4.1 Design of the vaccine

Sequences including the sequences of HPV16 E6 and E7 along with HR1 (916-966 aa) and HR2 (1157-1203 aa) were cloned into shuttle plasmids pDC516. Site directed mutation was used to fuse the E6 and E7 proteins. Moreover, sequence of E7 contains two mutations (C24G and E26G) to modify the Rb binding sites of E7. HEK293A cells were cotransfected with shuttle plasmids pDC516 containing HPV16 E6/7-HR or HPV16 E6/7 gene and backbone plasmid pBHGfrtdelE13FLP. Ad-E6/7-HR and Ad-E6/7 were prepared through an array of procedures including amplification, concentration, purification, and characterisation of Ad vectors.

4.2 Mice

Vital River (Beijing, China) supplied female C57BL/6 mice (6-8 weeks old). Mice were reared under specific-pathogen-free conditions.

4.3 Cell lines

HEK293A were purchased from ATCC. TC-1 cells, established through genetic modification, incorporating HPV16 E6/E7 and c-ras oncogenes, were bought from Xiamen Immocell Biotechnology Company. HEK293A and TC-1 were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) and RPMI 1640 (Gibco) with 10% fetal bovine serum (FBS; Corning), penicillin (100 U/mL), and streptomycin (100 mg/mL). All cells were cultured at 37°C and 5% carbon dioxide.

4.4 Tumour challenge and treatment experiments

For therapeutic tumour models, 2× 10⁵ TC-1 cells were subcutaneously injected in the right flank of dorsum on Day 0, 5× 10⁹ VP vaccine was injected intramuscularly on the right leg of mice 3 days later, when the tumour was palpable but not measurable. For prophylactic model, on Day 0, 5× 10⁹ VP vaccine was injected intramuscularly, followed by subcutaneous implantation of 2× 10⁵ TC-1 cells 7 days later. For tumour rechallenging model, 2× 10⁵ TC-1 cells were injected subcutaneously 70 days post-vaccination. Tumour sizes were measured every 3 days. Tumour volumes were calculated using the equation: volume (mm³) = width× width× length× 0.52. 21 days post-tumour inoculation, mice were euthanised.

For therapeutic tumour models in the pulmonary metastasis model, on Day 0, 3× 10⁵ TC-1 cells were injected intravenously, 5× 10⁹ VP vaccine was injected intramuscularly on the right leg of mice on the next day. For prophylactic model, 5× 10⁹ VP vaccine was injected intramuscularly on Day 0 and 3× 10⁵ TC-1 cells were implanted intravenously 7 days later. Sixteen days post-tumour implantation, mice were sacrificed.

4.5 Tissue preparation

Tumour tissues were digested by 1 mg/mL collagenase Type I as well as 0.5 mg/mL collagenase Type IV (all from Sigma-Aldrich) in RPMI 1640 basic medium, and incubated for 1 h at 37 °C. Cell suspension was filtered through 70 µM strainer (Falcon) and lysed by red blood cell (RBC) lysis buffer. After washes, cells were maintained in phosphate-buffered solution (PBS). Spleens were mechanically disrupted through a 70 µM strainer. Lymphocytes from spleens were isolated by density gradient centrifugation. Subsequent to RBC lysis and washes, cells were resuspended in PBS. Lymphocytes derived from whole blood were isolated in RBC lysis buffer for several times and resuspended in PBS.

4.6 Flow cytometry

Lymphocytes from tumours were incubated with antibodies (1:100 dilution, BioLegend) at 4°C for 30 min. For ICS, cells were stained for surface markers at first. Then, cells were fixed and permeabilised with BD cytofix/Cytoperm™ Fixation/Permeabilization Kit (BD Bioscience) and stained with antibodies for 1 h at room temperature.

To evaluate cellular immune responses, splenic lymphocytes were isolated and cultivated. E7-derived peptide (2 µg/mL, RAHYNIVTF) and E6-derived peptide pool (4 µg/mL) were added respectively to stimulate cells. Brefeldin A (BD Biosciences) was added to impede cytokine secretion 6 h before staining. Upon completion of surface marker staining, cells were fixed, permeabilised, and incubated with intracellular antibodies. For tetramer staining, cells from spleens were stained with PE-conjugated E6-Tetramer and PE-conjugated E7-Tetramer (all from BetterGen) respectively. Cells were stained at 4°C for 1 h.

Flow cytometry was performed and analysed by NovoExpress® software (1.3.0, ACEA Biosciences, Inc., San Diego, CA).

4.7 ELISPOT assay

Lymphocytes were harvested from spleens and seeded (1.5× 10⁵ cells per well) in nitrocellulose-bottom plates (MABTECH) and incubated with E7-derived peptide (2 µg/mL) and E6-derived peptide pool (4 µg/mL) respectively for at least 12 h in vitro. The detection of secreted IFN-γ was conducted following protocols. Spots were then quantified and analysed.

4.8 Western blot analysis

HEK293A cells were transduced with adenovirus for 48 h. Then cell lysis buffer (Beyotime Institute of Biotechnology) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich) were used to lyse samples. Moreover, muscle tissues at the injection site were isolated and extracted 48 h post-vaccination for sample preparation. Proteins were separated by polyacrylamide gel and non-denaturing electrophoresis separately, and transferred to polyvinylidene difluoride membranes. After blocked with 5% non-fat milk, membranes were incubated with antibodies overnight at 4°C: rabbit anti-HPV16-E6 (GeneTex, 1:1000), rabbit anti-HPV16-E7 (GeneTex, 1:1000), mouse anti-actin (HUABIO, 1:1000), and mouse anti-vinculin (Sigma, 1:1000). Secondary antibody incubation was performed using horseradish peroxidase-conjugated goat anti-rabbit (Abcam, 1:5000) and goat anti-mouse (Abcam, 1:10 000). Plots were shown by ChemiDoc Touch (Bio-Rad).

4.9 Immunofluorescence staining

Sections of tumour tissues were used for immunofluorescence staining. After deparaffinisation, hydration via alcohol gradient, antigen retrieval, and blocking, rabbit anti-CD8 antibody (Cell Signaling Technology, 1:250) was utilised to incubate sections at 4°C overnight. Sections were then exposed to Alexa Fluor™ 488-labelled goat anti-rabbit secondary antibody (Invitrogen, 1:1000) for 1 h at ambient temperature.

4.10 Statistical analysis

Statistics were analysed by GraphPad Prism 9.4.0 (GraphPad, San Diego, CA). One-way analysis of variance, two-way analysis of variance, Student–Newman–Keuls test, and log rank tests were used and data was shown as mean ± SEM. Statistical significance was determined at p < .05.