A tetravalent nanovaccine that inhibits growth of HPV-associated head and neck carcinoma via dendritic and T cell activation

Summary The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qβ-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qβ-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.


INTRODUCTION
Head and neck cancer comprises a heterogeneous group of malignancies originating in the epithelial lining of the upper aerodigestive tract.The incidence of these cancers is on the rise globally, with a notable contributing factor being infection with human papillomavirus (HPV), particularly the high-risk subtype 16. 1,2 HPV-associated oropharyngeal carcinoma is more prevalent in younger patients, and they tend to have markedly better outcomes following standard of care treatment when compared to patients with HPV-unrelated head and neck cancer. 3owever, it is worth noting that conventional curative-intent therapies are associated with substantial morbidity. 4Furthermore, up to 36% of patients with HPV-associated head and neck cancer experience either locoregional relapse or distant metastasis within an eight-year timeframe. 5,6These challenges underscore the need for the development of more effective and less toxic immunotherapeutic approaches.
The objective of a therapeutic vaccine for HPV-associated head and neck cancer is to target and eliminate precancerous and cancerous lesions expressing the early oncogenic proteins E6 and E7. 7,8These proteins play a pivotal role in driving the proliferation of HPV-infected cells and continue to be expressed by the transformed cells. 1 Consequently, they represent suitable targets for therapeutic vaccines. 3A range of approaches has been explored to develop and assess the anti-tumor response of therapeutic head and neck cancer vaccines, both in preclinical models and in clinical trials. 9Examples of therapeutic vaccines for HPV-associated head and neck cancer tested in clinical trials include: peptide-based vaccines 10 (NCT02426892), an mRNA-based vaccine (NCT04534205), a DNA-based vaccine (NCT03162224), and a live-attenuated vaccine based on Listeria monocytogenes (NCT02002182).
Repetitive icosahedral virus-like particles (VLPs) represent a promising platform for the design and development of cancer vaccines.Their efficacy is attributed to several favorable characteristics, including safety, as these particles lack any replication competent viral genetic materials. 11The distinctive surface geometry of VLPs, representing a pathogen-associated structural pattern (PASP), further enhances their potential as vaccine candidates. 7Additionally, the ability to manipulate both the exterior and interior surfaces of VLPs as well as loading them with toll-like receptor (TLR) ligands provides a versatile framework for optimizing their immunogenicity and therapeutic potential in the ll context of cancer vaccination. 12VLPs have been specifically engineered for the development of effective therapeutic vaccines targeting HPV in preclinical models. 13][16][17] Dendritic cells (DCs) serve as critical antigen-presenting cells (APCs) responsible for bridging the innate and adaptive immune responses, and they play crucial roles in activating cytotoxic T cells.In fact, the effectiveness of cancer immunotherapy hinges on the interactions between DCs and CD8 + and CD4 + T cells, as well as other immune cells in the tumor microenvironment (TME).9][20][21][22] DCs are classified into distinct subsets, including cDC1 and cDC2, both originating from circulating precursors of conventional DCs (cDCs).cDC1s are particularly recognized for their pivotal role in cross-priming naive antigen-specific tumor CD8 + T cells within the tumor-draining lymph nodes (tdLNs) 23, 24.Indeed, studies have revealed that the extent of cDC1 infiltration in the TME is correlated with the efficacy of immunotherapy and prognosis in cancer patients. 23While cDC1s are specialized for activating CD8 + T cells, cDC2s exhibit functional and ontogenic heterogeneity.They are known to be essential for presenting exogenous antigens to CD4 + T cells and promoting the effective differentiation of T helper cells (T H cells). 24 Lastly, DC3s are likely derived from cDC1s or cDC2s and exhibit similarities to inflammatory DCs in inflamed tissues.However, the precise definition and nomenclature of DC subsets remain areas of ongoing research. 25he central objective of the present study is to provide a preclinical proof-of-concept for the therapeutic effectiveness of a tetravalent VLPbased vaccine loaded with a TLR 9-ligand (Qb-HPVag).This vaccine is designed to present four distinct elongated epitopes derived from HPV16 E6 and E7.Additionally, the study aims to explore the impact of VLP-based vaccines on reshaping the TME, particularly on enhancing the presence of DCs and promoting the infiltration of antigen-specific T cells.There is still limited understanding regarding the impact of VLPbased vaccines on enhancing DC subsets infiltration in the tumor.This knowledge gap signifies an opportunity for ongoing and future research, offering the potential for valuable insights to optimize VLP-based vaccines.

Development and characterization of a tetravalent nanovaccine (Qb-HPVag) for targeted immunotherapy
To design a murine VLP-based nanovaccine capable of displaying HPV16-derived E6 and E7 epitopes, we conducted an extensive review of published data that reported antigen-specific T cell responses observed in C57BL/6 mice.Our epitope selection process was guided by several key criteria: (1) in vivo anti-tumor efficacy in wild-type mice, as reported in studies 12,[18][19][20][21][22]26,27 ; (2) immunodominance; (3) peptide length, with a preference for 9-10 amino acids (a.a.); and (4) high binding affinity to major histocompatibility complex-I (MHC-I) molecules, as determined by NetMHCpan 4.1. Ultimtely, we selected two peptides from E6 and two from E7 oncogenic proteins.The selected peptides were denoted as E6.1, E6.2, E7.1, and E7.2 (Figure 1A).A summary of these four chosen epitopes, along with their respective MHC-I binding strengths to H-2D b and H-2K b , is provided in Table 1.E6.2 (E6 48-57 ) and E7.1 (E 48-57 ) likely cover immunodominant cytotoxic T lymphocyte (CTL) epitopes.The CTL epitopes in E6.1 and E6.2 have high binding affinity to H-2K b molecules.The chosen E7.1 MHC-I epitope has high binding affinity to both H-2K b and H-2K d molecules.Despite not predicted to bind either H-2D b or H-2K b by the used prediction algorithm, E7.2 has been included as a fourth epitope due to favorable anti-tumor effects observed in preclinical HNC models.28,29 To achieve effective immunotherapy, it is imperative to mobilize DCs because of their crucial role in regulating the immune response.To ensure effective stimulation of the innate immune system, we loaded our Qb-VLPs (Figures 1B, 1C, and 1D) with type-B 1668 unmethylated  CG-motif-containing oligonucleotides (CpG), a well-known ligand for TLR 9 (Figure 1E).To assess the efficacy of our nanoparticles in inducing an enhanced innate immune response compared to spontaneously packaged ssRNA-Qb or the naked empty particle Qb(Ø), we conducted the experiment outlined in Figure 1F.We measured the secretion of IL-12 in the popliteal lymph node (LN) 24 h after subcutaneous injection into the mouse footpad of either naked Qb(Ø), Qb(ssRNA), or Qb packaged with oligonucleotides (Qb(CpGs)).The results clearly demonstrated a significant increase in IL-12 secretion by activated CD11b + DCs in the group that received Qb(CpGs) when compared to those that received Qb(ssRNA) (p = 0.0059) or the naked Qb(Ø) (p = 0.0008) (Figures 1G and 1H).
Building upon former research, 32 we have previously demonstrated that extending MHC-I epitopes by incorporating their natural flanking sequences can significantly enhance T cell activation and anti-tumor efficacy in comparison to shorter peptides. 33Accordingly, we designed peptides of approximately 30 a.a.length (Figure 1A).The elongated peptides were synthesized with an additional lysine (K) a.a. and an azide group (N3) at the C-terminus as shown in (Table 2).The coupling of each peptide to Qb was achieved through our well-established bioorthogonal Cu-free click chemistry method using (dibenzocyclooctyne NHS ester DBCO linker).Briefly, the NHS ester group of DBCO reacts with the K residue on the surface of Qb and incorporates cyclooctyne moiety which reacts-in a second step-with the (N3) labeled peptide to form a stable triazole linkage.The coupling was achieved through our well-established bio-orthogonal Cu-free click chemistry method, as outlined in the STAR methods section.Confirmation of the successful coupling of epitopes to Qb(CpGs)-VLPs was obtained through SDS-PAGE analysis (Figure 1I).The resulting tetravalent nanovaccine was given the name Qb-HPVag, as depicted in Figure 1J and the dosage was according to Table 3.

The tetravalent Qb-HPVag vaccine demonstrated significant anti-tumor efficacy in vivo
In the next phase of our study, we evaluated the therapeutic anti-tumor efficacy of the tetravalent Qb-HPVag in C57BL/6 mice bearing aggressive mEERL95 tumors.To do this, we subcutaneously inoculated 10 5 mEERL95 tumor cells mixed with Matrigel into the flanks of the mice.Subsequently, the mice were vaccinated with either Qb-HPVag or a control vaccine without peptide antigens (Qb-(CpGs), referred to as Qb), on days 3, 9, and 15, as shown in Figure 2A.
The administered doses were 100mg Qb-HPVag (25mg of Qb-E6.1, 25mg of Qb-E6.2, 25mg of Qb-E7.1 and 25mg of Qb-E7.2) and the control (100mg of Qb).We evaluated the systemic T cell responses eight days after the first immunization dose (day 11), which is typically the peak of the response.Our findings revealed a notable increase in both CD8 + and CD4 + T cell percentages in the group that received Qb-HPVag compared to the control group which received Qb (p = 0.0023 and p = 0.0324 respectively; Figures 2B-2E), suggesting induction of a peptide-specific response.
Tumor growth was notably delayed in the Qb-HPVag vaccinated mice (p < 0.0001; Figures 2F and 2G).Our prior research has revealed a significant correlation between the density of CD8 + T cells within the TME and the resulting inhibition of tumor growth, leading to improved survival rates among the mice. 33,34,35To validate the effectiveness of our vaccination strategy, we assessed the tumor-infiltrating lymphocytes (TILs) on day 24 after inoculation of the mEERL95 tumor cell line.We measured the densities of CD8 + and CD4 + T cell populations within each tumor.The densities were determined by calculating the ratio of the total number of the respective cell populations within each tumor to the tumor weight in (mg).This approach allows for a standardized measurement of cell density that accounts for variation in tumor sizes.In the group that received the tetravalent Qb-HPVag, we observed a noteworthy increase in CD8 + (Figures 2H  and 2J) and CD4 + (Figures 2I and 2K) T cell densities when compared to the control group (p = 0.0014 and p = 0.0009, respectively).These findings strongly suggest that Qb-HPVag has the capacity to enhance the infiltration of T cells and effectively restrain mEERL95 tumor growth.
TIL activity and functionality was assessed by harvesting tumors on day 24 following the administration of three doses of Qb-HPVag or the control vaccine.TILs were isolated and stimulated with E6 and E7 or peptides.CD8 + T cells in the vaccinated group with Qb-HPVag exhibited an augmented production of key cytokines, namely IFN-g and TNF-a (Figures 2L and 2M).However, no significant increase in cytokine production was observed in CD4 + T cells within this group (Figures 2N and 2O).To gain a more comprehensive understanding of CD4 + T cell functionality and exhaustion markers at different stages of tumor growth following vaccination with Qb-HPVag, future experiments should explore earlier time-points, aligning with the heightened T cell infiltration into the TME.This is an avenue that we are presently exploring.

Augmented T cell infiltration and mitigated necrosis through Qb-HPVag immunotherapy
We investigated T cell infiltration in the tumor and the adjacent tumor border, examining their distribution in viable and necrotic tumor regions through Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC) staining.Employing a vaccination scheme according to Figure 2A, tumors were harvested on day 24.Consistent with earlier results, Qb-HPVag demonstrated a hindrance to the progression of mEERL95 tumors (Figure 3A). Figure 3B shows a representative visual colorization of CD8 + and CD4 + T cells in a control tumor tissue (left) and the group vaccinated with Qb-HPVag (right).In the control group (weighing 1404mg), CD4 + T cells exhibited overall low numbers, with some regional infiltration detected in the deepest marginal tumor areas, close to tumor necrosis.CD8 + T cells were uniformly distributed within the tumor, featuring small foci in various tumor regions.Contrastingly, in the vaccinated tumor with Qb-HPVag (weighing 385.4 mg), CD4 + T cells were predominantly situated in the tumor margin (peritumoral area), with a distinct focal accumulation in the apical and lateral tumor region.CD8 + T cells demonstrated clear infiltration into the tumor, showcasing multifocal intratumoral foci concentrated in the lower portion of the tumor.Similar findings were obtained in other assessed tissues.In the subsequent phase, we conducted a quantitative analysis of intratumoral and peritumoral CD8 + and CD4 + T cells in both the control and Qb-HPVag immunized groups, examining five tumors in each category.The intratumoral CD8 + T cell count exhibited a significant increase in the Qb-HPVag group (p = 0.0295; Figures 3C and 3H).However, the difference of peritumoral CD8 + T cells only demonstrated borderline statistical significance (p = 0.05761; Figure 3D), possibly because many CD8 + T cells may have infiltrated the TME which would explain the observed anti-tumor response in the treated group with Qb-HPVag.For CD4 + T cells, the peritumoral counts differed significantly between both groups (p = 0.0248; Figures 3F and 3I), while the intratumoral counts showed no significant disparity (Figure 3E).Afterward, we conducted a digital histological evaluation of the tumor tissues to measure the overall area of tumor necrosis.As previously demonstrated, tumor necrosis often attributed to hypoxia, indicates rapid cellular proliferation and correlates with cell proliferation in aggressive cancer. 34,36Our latest findings align with these earlier observations, revealing a notable reduction (p = 0.0491) in the tumor necrosis within the group treated with Qb-HPVag (Figure 3G).Collectively, these findings suggest the indispensable role of CD8 + T cells in instigating a potent anti-tumor response in the TME.The observed increased localization of CD4 + T cells at the tumor margin in the group receiving Qb-HPVag might be attributed again to the specific time point at which these tumors were collected.An examination at an earlier time point could therefore also be informative following vaccination with Qb-HPVag.

The tetravalent Qb-HPVag activates dendritic cells within the tumor microenvironment
To explore the presence of DCs within the TME subsequent to vaccination with Qb-HPVag, we carried out additional experiments.Initially, we assessed the density of total CD11c + MHC-II + DCs in tumors from both vaccinated and control mice.The findings unveiled a noteworthy elevation in the density of activated DCs upon immunization with Qb-HPVag (p = 0.0313; Figure 4A).The migration and localization of activated DCs within organs are orchestrated by the chemokine receptor 7 (CCR7). 37,38Accordingly, we evaluated the density of CCR7-expressing CD11c + MHC-II + cells in the tumor.Intriguingly, a notable increase was observed in the group that received the tetravalent Qb-HPVag, underscoring the capacity of the vaccine to stimulate DCs within the TME (Figure 4B).
In the subsequent phase of our study, we conducted a more detailed analysis of the Qb-HPVag treated group, dividing it into responders and partial-responders to gain deeper insights into the TME composition of DC subsets using the same panel of markers mentioned above (CD45, CD24, CD11b, CD11c, CCR7, CD103, and MHC-II).The 'responder' group was defined by tumors weighing a maximum of 100 mg.To ensure robust and consistent comparisons of unknown cell populations between these groups, we employed an unbiased automated analysis method, utilizing a non-linear dimensionality reduction technique, t-stochastic neighbor embedding (tSNE), followed by FlowSOM analysis, which revealed five different clusters (Populations 0, 1, 2, 3, and 4) of DC subsets within the TME following Qb-HPVag treatment (Figure 4I).Notably, populations 1 and 3 displayed an approximately similar percentage in responders and partial-responders.The results indicated dissimilar percentage distributions among the other identified populations (0, 2, and 4).Population 0 exhibited an increase in the partialresponder group and demonstrated low expression of the utilized surface markers, underscoring the necessity for additional markers to investigate this population.Populations 2 and 4 exhibited considerably reduced size in the partial-responder group (Figure 4I).Population 2 exhibits the markers used to differentiate the DC3 subset and also shows overall increased expression of the migratory CCR7 receptor in the responder group.The generated tSNE heatmaps revealed increased expression of CD24 and CD11b and a slight expression of CD45, MHC-II, CCR7, and CD11c for population 2 (Figure 4K).Subsequently, we generated a histogram count analysis to validate these expression patterns in population 2 (Figure 4J).Population 4, reduced in the partial-responder group, displayed notably elevated expression solely in CD24, distinct from the other surface markers used.Overall, these findings indicate that DC subsets are critically affected in the TME following vaccination with Qb-HPVag, which may play a pivotal role in elucidating the differences between responder and partial-responder groups.
The tetravalent Qb-HPVag reduced postsurgical tumor recurrence, which is dependent on the presence of CD8 + T cells Encouraged by the observed anti-tumor efficacy of the Qb-HPVag vaccine, we embarked on an investigation into its potential for preventing tumor relapse following the surgical resection of the primary tumor.To explore this, we designed a new experiment where a mixture of 10 5 mEERL95 cells with Matrigel was subcutaneously inoculated followed by administration of a series of three vaccinations on days 3, 9 and 15.On day 24, we surgically removed the primary tumor as depicted in Figure 5A.One week after surgery, we subjected the mice to challenge with 10 5 mEERL95 cells, which were subcutaneously inoculated into the collateral flank.We diligently monitored the mice for an additional 56 days to detect any signs of tumor regrowth, without administering additional vaccinations.As anticipated, Qb-HPVag substantially curtailed tumor growth before resection (p = 0.0124; Figure 5B).Remarkably, Qb-HPVag vaccination resulted in a significant improvement in survival, showing a 70% tumor-free survival in contrast to approximately 30% rate in the control group (p = 0.0375), all of this despite the absence of further immunizations (Figure 5C).The experimental plan is depicted in the sketch.10 5 mEERL95 cells were formulated in Matrigel and inoculated subcutaneously in the mouse flank region.Two distinct groups were employed: the control group, represented by Qb(CpGs) and the experimental group, which received the Qb-HPVag vaccine.The treatment regimen involves the subcutaneous administration of a prime vaccination on day 3, followed by two subsequent booster vaccinations on days 9 and 15.Tumors were collected on day 24 and blood samples were collected on day 11.(B and C) Percentage of CD8 + and CD4 + T cells in the blood of mice on day 11, determined via flow cytometry (FACS) and analyzed in both groups.Each dot point on the plot represents an individual value.The gating strategy used involved a sequential selection of singlets, followed by lymphocytes, and further categorized into either CD8 + or CD4 + T cells.(D and E) Representative FACS plots illustrating the percentage of CD8 + or CD4 + T cells in blood in both groups.(F) Tumor size was evaluated by measuring tumor volume (mm 3 ) in both groups.It's worth noting that the control group reached the humane endpoint by day 24 due to the progression of tumor growth.(G) Tumor weight, measured in (mg), assessed on day 24 of the experiment, with each data point on the plot representing the weight of an individual tumor.(H and I) Densities of CD8 + and CD4 + T cells within the tumors.The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).(J and K) Representative FACS plots illustrating the total number of CD8 + or CD4 + T cells acquired from each tumor.The analysis was pregated on TILs.(L and M) Densities of CD8 + T cells producing IFN-g + or TNF-a + within the tumor.(N and O) Densities of CD4 + T cells producing IFN-g + or TNF-a + within the tumor.TILs isolated from mice vaccinated with Qb-HPVag and stimulated with a control peptide are included (a separate experiment).The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).To investigate the influence of CD8 + and CD4 + T cells on the outcome of Qb-HPVag immunization, we employed monoclonal antibodies (mAbs) to deplete these specific cell populations.This experiment encompassed four groups of mice: a control group treated with Qb, a group receiving 3 tetravalent Qb-HPVag injections over a 13-days period, and two additional groups (groups three and four) treated with the same Qb-HPVag vaccine, in combination with either anti-CD8 or anti-CD4 monoclonal antibodies (Figure 5D).The depleting mAbs were administered intravenously on a weekly basis, ensuring an efficient depletion of CD8 + or CD4 + T cells, confirmed at levels of approximately 99-100% in the blood via flow cytometry (Figures 5E-5H).It is worth noting that the successful depletion of CD8 + T cells lead to a modest depletion of CD4 + T cells but not the other way round (Figure 5F).This effect occurred despite the use of low/few doses for depletion.
Consistent with our prior findings, Qb-HPVag substantially inhibited tumor progression (p < 0.0001).Notably, the benefits of Qb-HPVag vaccination were entirely abrogated when CD8 + T cells were depleted, leading to increased tumor growth and tumor weight, as observed on day 24 (Figures 5I and 5J).Unexpectedly, the depletion of CD4 + T cells only partially reduced the impact on the anti-tumor efficacy of Qb-HPVag.This is consistent with the idea that CD4 + T cells promote the action of CD8 + T cells but show limited effector function themselves.In addition, the anti-CD4 mAb targets both conventional CD4 + T cells as well as T regulatory cells (Tregs), which will reduce tumor growth by itself.Taken together, these findings highlight the crucial role of CD8 + T cells in mediating the favorable anti-tumor effects of Qb-HPVag vaccination.

DISCUSSION
In this study, we have designed a therapeutic tetravalent VLP-based vaccine (Qb-HPVag) that displays elongated MHC-I epitopes derived from the E6 and E7 proteins of HPV16.The rationale behind incorporating elongated epitopes is grounded in both previous research by others and our own investigations.Accumulating evidence suggests that elongated epitopes exhibit superior anti-tumor efficacy compared to shorter ones.This observation holds true across various solid tumors, as evidenced by both preclinical and clinical studies as CD4 + T cells can deliver help to CD8 + T cells, in part by activating DCs through the CD40 À CD40L signaling pathway. 32,33,39,40In the current preclinical research, E6 and E7 MHC-I murine epitopes have been specifically chosen.However, for humans, a broad range of human leukocyte antigens (HLAs) epitopes must be considered.Given the elongation of the MHC-I epitopes to approximately 32 a.a., it is anticipated that some sequences will also align with human HLAs.It is well-established that VLPs have an inherent capacity to provide additional T cell help beyond the primary benefits of the VLP itself, as they induce VLP-specific CD4 + T cells. 11Consequently, the activation of DCs through CD40L can also occur independently of tumor-specific T H cells. Additionally, encapsulation of nucleic acids such as CpGs in VLPs induces DC activation, regardless of the presence of T H cells.
The systemic increase in both CD8 + and CD4 + T cells in the blood stream after a prime and single boost administration of Qb-HPVag indicates the efficacy of our vaccine construct.The increased T cell infiltration of both CD8 + and CD4 + T cells into the TME reinforces this observation.Indeed, assessing the density of TILs in the TME has been established in prior studies as a correlate of improved prognosis. 41It is important to note that measuring TILs density via flow cytometry lacks the precision to allocate these cells within the tumor environment.This precision can be achieved through standard HE staining and IHC.Notably, our data reveals a predominant allocation of CD8 + T cells within the tumor bed, with fewer cells at the margins, suggesting that a substantial proportion of CD8 + T cells may have infiltrated the TME which may explain the correlation to the induced anti-tumor response in the Qb-HPVag group.This is further supported by the detrimental impact on tumor protection upon CD8 + T cell depletion.Contrastingly, when evaluating the localization of CD4 + T cells, opposite results emerge.Peritumoral CD4 + T cells were significantly higher in the Qb-HPVag group whereas the intratumoral cells were not.We hypothesize that the time point post-treatment plays a role in the infiltration of CD4 + T cells into the tumor.In addition, the density of MHC class II expressing APCs at the tumor border may be higher than within the tumor, attracting the CD4 + T cells.Although cytokine production from CD4-positive T cells in the tumor was deemed insignificant 24 days post-tumor inoculation, CD8-positive T cells exhibited a notably increased IFN-g and TNF-a production.The differences in cytokine production may also be attributed to the delayed assessment of CD4-specific T cell functionality.Future experiments should explore earlier time-points as well as different markers for effector function and exhaustion on CD4 + T cells.Our study further revealed that therapeutic vaccination using the tetravalent Qb-HPVag incorporating TLR 9 ligands leads to an elevated infiltration of diverse DC subsets within the TME.DCs in the Qb-HPVag group exhibit heightened activation and migration capabilities compared to those in the control group.This observation is likely relevant, as DCs proficiently transport tumor antigens from the TME to the tumor draining LNs in a CCR7-dependent manner, facilitating the effective priming of anti-tumor CD8 + and CD4 + T cells. 37,42,43otably, the cDC1 subset showed a significant increase in the Qb-HPVag group, and the elevation in cDC2 was even more pronounced.Given that cDC2 can present exogenous antigens to CD4 + T cells and facilitate their differentiation, we hypothesize that peptide elongation may contribute to augmenting the infiltration of this specific subset into the TME.Interestingly, through high-throughput multi-parameter analysis, we discerned a notable differentiation between responder and partial-responder mice.Population 2 in both tSNE and FlowSOM analyses displayed distinct characteristics, mostly aligning with the DC3 population, marked by the CCR7 marker.Intriguingly, this specific population was reduced in partial-responder mice, suggesting that it is not only crucial to induce DC infiltration in the TME but also imperative for these cells to gain the ability of migrating to the tdLN through CCR7 expression.This migration is posited to be pivotal in eliciting more robust systemic protection against relapse, an area that is currently under investigation in our research endeavors.The reduction in Population 4 observed in the partial-responders group is noteworthy, particularly due to its high expression of the cell surface glycoprotein CD24.CD24, known for its diverse implications in cell adhesion, migration, and signal transduction, is expressed on various immune cells within the TME, such as T cells, B cells, macrophages, NK cells as well as on Tregs. 44The observed reduction in Population 4 suggests a potential impact of CD24-expressing cells on the response to vaccination with Qb-HPVag.CD24's involvement in immune cell interactions and modulation of immune responses may influence the dynamic changes within the TME following vaccination.Further profiling of this population may hold promise for providing deeper insights into how tumors in the responders' cohort behave post-vaccination with VLP-based vaccines.
Surgical resection is a key element in cancer management, but the risk of recurrence remains.Cancer vaccines, especially for high-risk patients with solid tumors like head and neck carcinoma, are promising options. 45Our data a notable 70% tumor-free survival rate upon rechallenge in mice receiving the Qb-HPVag vaccine, indicating potential activation of memory T cells.Remarkably, this effect is achieved without additional dosing.Exploring combination therapy with checkpoint inhibitors may be a strategy to further enhance the tetravalent Qb-HPVag vaccine's efficacy, potentially with synergistic effects for improving immune responses and outcomes in cancer treatment.
In conclusion, we demonstrated the efficacy of a preclinical tetravalent Qb-HPVag vaccine in generating a potent anti-tumor response, preventing relapse in an aggressive murine head and neck cancer model.The vaccine enhanced T cell and DC subset infiltration in the TME, with a significant impact on local immune responses.Ongoing research aims to further optimize the vaccine's efficacy and translate the findings for clinical application.

Limitations of the study
The study uses a subcutaneous tumor flank model to study the effects of a cancer vaccine.While recognizing the advantages of this model, we acknowledge that it may not completely mimic the complex tumor microenvironment present in orthotopic settings.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  (G) Immunohistochemistry of tumors in the control and the treated group, the cryosections were probed with antibodies detecting CD11b + cells.(H) Detailed gating strategy for DC subsets (cDC1: CD11c + , MHC-II + , CD24 + , CD11b À , CD103 + ; cDC2: CD11c + , MHC-II + , CD24 + , CD11b + , CD103 -; and DC3: CD11c + , MHC-II + , CD11b + , CCR7 + ).The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).
Tumor cell line mEERL95 cell line was kindly provided by Genrich Tolstonog 47 (University of Lausanne, Lausanne, Switzerland).The mEERL95 was cultured in Gibco Dulbecco's Modified Eagle Medium (DMEM) F-12 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1x human keratinocyte growth supplement (HKGS).When cells6reached 80% confluency the growth media was aspirated, and cells were washed three times with 1x phosphate buffered saline (PBS).After washing, 1x trypsin was added to the T75 Flask and incubated at 37 C and 5% CO 2 for 7 min.Cells were either resuspended in a growth medium for continued culture or in 1x PBS admixed with Matrigel (Corning) for cell injection.The cell line was tested several times for Mycoplasma infection using Microsart AMP Mycoplasma Kit.

Tumor model and treatment schedule
The mEERL95 tumor cells were resuspended in a mixture of 30 mL of PBS and 20 mL of Matrigel (Corning).Subsequently, 10 5 tumor cells were subcutaneously injected into the left flank of each mouse.The specific doses for both the control and the tetravalent vaccine can be found in Table 3. Tumor size was assessed every other day using a caliper, and the tumor volume was calculated using the formula 0.5 3 (length 3 width 2 ).To adhere to ethical guidelines, mice were euthanized before the tumor reached a humane-end point volume of 1 cm 3 , in accordance with the animal license (BE43/21).After 24 days, the experiment was concluded, and the tumors were excised for further analysis.The doses mentioned in Table 3 represent the concentrations of VLPs, either in isolation (control) or in conjunction with various peptides.

Single-cell isolation from peripheral blood samples
Blood samples were obtained from the tail vein of tumor-bearing mice and collected in Eppendorf tubes containing 0.5M EDTA (Fluka, Biochemika).To isolate white blood cells, red blood cells (RBCs) were lysed using ACK-Buffer (0.155M ammonium chloride, 0.01M potassium hydrogen carbonate, pH 7.2).Following a 5-7 min incubation on ice with ACK buffer, cells were resuspended in FACS buffer (PBS +2% FBS) within 96-well U-bottom plates.For optimal staining, cells were pre-incubated with FcR-block (CD16/CD32, clone 2.4G2, BD Biosciences) for 15 min at 4 C. Subsequently, cells were stained with anti-CD8a (PE-Cy7, clone 53-6.7,BD Biosciences) and anti-CD4 (PE, clone H129.19,BD Biosciences) for 20 min at 4 C.After staining, the cells underwent three washes and were resuspended in 400mL of FACS-buffer.The gating strategy on lymphocytes is illustrated in Figure S1A.

Intracellular cytokine staining
For intracellular cytokine staining (ICS), TILs were resuspended in DMEM (+10% FBS, 1% PenStrep) and transferred to 96-well plates.A mixture containing 100 U/ml of mouse interleukin-2 (mIL-2, Roche) and 1 mg/mL of each peptide was added, followed by incubation for 24 h at 37 C. On the subsequent day, the media was changed, and the stimulation cocktail was mixed with GolgiPlug (BD Biosciences, 1:1000) and Monensin (BioLegend, 1:1000).After another 16 h of incubation at 37 C, cells were washed three times using DMEM medium and resuspended in FACS Buffer.For optimal staining, single-cell suspensions were treated with FcR-Block (CD16/CD32, clone 2.4G2, BD Biosciences) for 15 min at 4 C, followed by centrifugation for 5 min at 300g.Subsequently, cell surface anti-mouse fluorophore monoclonal antibodies (mAbs) CD8 (APC-Cy7, clone 53-6.7,BD Biosciences) and CD4 (PE, clone H129.19,BD Biosciences) were used for 30 min at 4 C in the dark.After surface staining, cells were washed once with FACS buffer and then fixed with cytofix buffer (BD Biosciences) for 15 min at 4 C. Following fixation, cells were washed with FACS buffer and permeabilized using 13 perm buffer (Invitrogen).Subsequently, cells were centrifuged at 300g for 5 min and treated with the intracellular staining cocktail, which included anti-mouse fluorophore mAbs IFN-g (APC, clone XMG1.2, BD Biosciences) and TNF-a (Percep-Cy5.5,clone MP6-XT22, BioLegend) resuspended in 1x perm buffer, and incubated for 20 min at 4 C.After intracellular staining, cells underwent three washes and were resuspended in 400 mL of FACS buffer.Samples were analyzed using a BD LSRII, and data analysis was performed using Flojo (V.10) and GraphPad Prism (V10).In an additional experiment, mice were vaccinated with the vaccine Qb-HPVag and tumors were collected for TILs isolation.A similar protocol as described above was performed and the TILs were stimulated with a cocktail containing 1 mg/ml of an elongated control peptide derived from the 4T1 mammary carcinoma cell line (CFSAFGNRKN LKYNAVPTVFAFQNPTEVCPEK(N3)), 33 GolgiPlug (BD Biosciences, 1:1000) and Monensin (BioLegend, 1:1000).

Immunofluorescence of tumor sections
Tumors were carefully excised with adequate margin from the mice on day 24 as illustrated in Figure 2A, embedded in Tissue-Tek OCT compound (Sakura, 4583), and promptly frozen on dry ice.The OCT-embedded tumors were then sectioned into 12mm slices using a cryostat.These sections were fixed with ice-cold pure acetone for 10 min and air-dried for an additional 15 min.To ensure optimal preparation, the sections were prewetted three times in PBS for 5 min each.Subsequently, slides were blocked with 1% BSA and 1% normal mouse serum in PBS for 1 h.Following the blocking step, the sections were directly stained with a CD11b antibody (CD11b, APC, Clone M1/70, BD Biosciences) diluted to a concentration of 1:500 in the blocking buffer.Staining was carried out for 1 h, followed by three washes with PBS for 5 min each.Finally, sections were mounted with a single drop of Fluoroshield with Dapi (Merck, F6057), and images were captured using a Zeiss Axio Imager.A2 microscope.

High dimensional analysis of flow cytometry data
High dimensional analysis of flow cytometry data was performed using t-distributed stochastic neighbor embedding (tSNE) and FlowSOM clustering and visualizing technique.FCS files of TILs were used following compensation and gating on single cells and live cells using FlowJo X version 10.4.2.FCS files (8 FCS files from each group) were exported and then concatenated; each used file included a consistent number of events (5000 events), total concatenated events was 40,000.Five clusters were applied as mentioned in the figure legends.The resulting subpopulations were further analyzed for specific marker expression (CD45, CD24, CD11b, CD11c, CCR7, CD103 and MHC-II).In FlowSOM analysis, five clusters were applied, as indicated in the figure legends.The resulting subpopulations were then subjected to further analysis to assess specific marker expression using histogram count.

T cell depletion
In experiments involving the depletion of CD8 + or CD4 + cells, mice received intravenous injections of either 50mg of anti-CD8b antibodies (clone 53-5.8,BioXcell) or 50mg of anti-CD4 antibodies (clone GK1.5, BioXcell) on a weekly basis.The efficiency of depletion was evaluated by assessing peripheral blood samples 48 h after the initial depletion.Tumor growth was assessed using the method described above.

Survival experiments
In the survival experiment, the primary tumor was surgically removed under anesthesia 24 days after inoculation with the cell line.The wound closure was performed using a 9mm autoclip applier (Clay Adams 9mm autoclip applicator, Vet-Tech).Subsequently, mice received an antidote mixture (Revertor 0.22 mg/mL, Anexate 0.09 mg/mL, Temgesic 0.015 mg/mL) in accordance with our BE43/21 license.Following the surgery, mice were allowed one week to recover, after which they were subcutaneously re-challenged in the collateral flank with 10 5 mEERL95 cells in Matrigel.Euthanasia of animals occurred upon the observation of local recurrence or collateral tumor growth in accordance with the criteria outlined in our BE43/21 license.

Histology
Formalin-fixed tumors collected on day 24 as illustrated in Figure 2A were used for histology, n = 5 from each group.Tumor tissue was histologically examined by a board-certified veterinary pathologist (SdB).Of each tumor, a complete cross section was trimmed and routinely processed for histologic evaluation.Tissue sections were stained for hematoxylin and eosin (HE) as well as for CD4 (clone 4SM95, rat, Thermo F. Scientific, 14-9766) and CD8 (clone 4SM15, rat, Thermo F. Scientific, 14-0808-82) immunohistochemistry.Both antibodies were diluted 1:200.Detection and quantification of tumor tissue, tumor necrosis, and CD4 + and CD8 + cells was performed digitally using Visiopharm software (Visiopharm, Hørsholm, Denmark).The number of CD4 + and CD8 + cells was calculated per mm (tumor periphery) and per mm 2 (intratumoral area).Based on CD4 + and CD8 + cell counts, visual colorization were generated in order to determine the spatial distribution of T cells.

Figure 2 .
Figure2.Anti-tumor efficacy of Qb-HPVag (A) The experimental plan is depicted in the sketch.10 5 mEERL95 cells were formulated in Matrigel and inoculated subcutaneously in the mouse flank region.Two distinct groups were employed: the control group, represented by Qb(CpGs) and the experimental group, which received the Qb-HPVag vaccine.The treatment regimen involves the subcutaneous administration of a prime vaccination on day 3, followed by two subsequent booster vaccinations on days 9 and 15.Tumors were collected on day 24 and blood samples were collected on day 11.(B and C) Percentage of CD8 + and CD4 + T cells in the blood of mice on day 11, determined via flow cytometry (FACS) and analyzed in both groups.Each dot point on the plot represents an individual value.The gating strategy used involved a sequential selection of singlets, followed by lymphocytes, and further categorized into either CD8 + or CD4 + T cells.(D and E) Representative FACS plots illustrating the percentage of CD8 + or CD4 + T cells in blood in both groups.(F) Tumor size was evaluated by measuring tumor volume (mm 3 ) in both groups.It's worth noting that the control group reached the humane endpoint by day 24 due to the progression of tumor growth.(G) Tumor weight, measured in (mg), assessed on day 24 of the experiment, with each data point on the plot representing the weight of an individual tumor.(H and I) Densities of CD8 + and CD4 + T cells within the tumors.The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).(J and K) Representative FACS plots illustrating the total number of CD8 + or CD4 + T cells acquired from each tumor.The analysis was pregated on TILs.(L and M) Densities of CD8 + T cells producing IFN-g + or TNF-a + within the tumor.(N and O) Densities of CD4 + T cells producing IFN-g + or TNF-a + within the tumor.TILs isolated from mice vaccinated with Qb-HPVag and stimulated with a control peptide are included (a separate experiment).The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).Statistical analysis (mean G SEM) by Student's t test in B, C, F, G, H and I. One-way ANOVA in L-O.The sample size was n = 6 in B, C, H, and I, n = 26 for Qb and 29 for Qb-HPVag group in G (2 experiments were combined), n = 9 in L-O.Significance levels are denoted as follows: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.
Figure2.Anti-tumor efficacy of Qb-HPVag (A) The experimental plan is depicted in the sketch.10 5 mEERL95 cells were formulated in Matrigel and inoculated subcutaneously in the mouse flank region.Two distinct groups were employed: the control group, represented by Qb(CpGs) and the experimental group, which received the Qb-HPVag vaccine.The treatment regimen involves the subcutaneous administration of a prime vaccination on day 3, followed by two subsequent booster vaccinations on days 9 and 15.Tumors were collected on day 24 and blood samples were collected on day 11.(B and C) Percentage of CD8 + and CD4 + T cells in the blood of mice on day 11, determined via flow cytometry (FACS) and analyzed in both groups.Each dot point on the plot represents an individual value.The gating strategy used involved a sequential selection of singlets, followed by lymphocytes, and further categorized into either CD8 + or CD4 + T cells.(D and E) Representative FACS plots illustrating the percentage of CD8 + or CD4 + T cells in blood in both groups.(F) Tumor size was evaluated by measuring tumor volume (mm 3 ) in both groups.It's worth noting that the control group reached the humane endpoint by day 24 due to the progression of tumor growth.(G) Tumor weight, measured in (mg), assessed on day 24 of the experiment, with each data point on the plot representing the weight of an individual tumor.(H and I) Densities of CD8 + and CD4 + T cells within the tumors.The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).(J and K) Representative FACS plots illustrating the total number of CD8 + or CD4 + T cells acquired from each tumor.The analysis was pregated on TILs.(L and M) Densities of CD8 + T cells producing IFN-g + or TNF-a + within the tumor.(N and O) Densities of CD4 + T cells producing IFN-g + or TNF-a + within the tumor.TILs isolated from mice vaccinated with Qb-HPVag and stimulated with a control peptide are included (a separate experiment).The densities were determined by calculating the ratio of the total number of the specific-cell population within each tumor to the tumor weight in (mg).Statistical analysis (mean G SEM) by Student's t test in B, C, F, G, H and I. One-way ANOVA in L-O.The sample size was n = 6 in B, C, H, and I, n = 26 for Qb and 29 for Qb-HPVag group in G (2 experiments were combined), n = 9 in L-O.Significance levels are denoted as follows: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 3 .Figure 4 .
Figure 3. Histological assessment of mEERL95 tumors (A) The experimental plan is depicted in the sketch in Figure 2A.Tumor weight, measured in (mg), assessed on day 24 of the experiment, with each data point on the plot representing the weight of an individual tumor.(B) Visual colorization depicting CD8 + and CD4 + T cells in representative tumors for both control and vaccinated tissues is illustrated.The tumor border is delineated by a dashed black line, while areas of tumor necrosis are marked in white.Viable tumor regions are labeled in dark blue, featuring a visible heatmap determined by the count of CD8 or CD4 positive cells.Regions with the highest positive cell counts ('hot') are represented in red, while areas with the lowest positive cell counts are displayed in light blue ('cold').(C and D) Quantification of the number of intratumoral (expressed as cells/mm 2 ) and peritumoral CD8 + T cells (expressed as cells/mm).(E and F) Quantification of the number of intratumoral (expressed as cells/mm 2 ) and peritumoral CD4 + T cells (expressed as cells/mm).(G) The total area of tumor necrosis is measured in square millimeters (mm 2 ), divided by the tumor weight, with each dot on the graph representing an individual tumor.(H) IHC staining of intratumoral CD8 + T cells in mice treated with either the Qb control or Qb-HPVag vaccine is depicted.CD8 + T cells are identified by black arrows (stained pink).(I) IHC staining of peritumoral (marginal) CD4 + T cells in mice treated with either the Qb control or Qb-HPVag vaccine is depicted.Circles highlight CD4 + T cell infiltration (stained pink).Statistical analysis (mean G SEM) by Student's t test in A, C, D, E, F and G.The sample size was n = 5.Significance levels are denoted as follows: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Table 1 .
Selected E6 and E7 epitopes and their binding to MHC-I

Table 2 .
Synthesized elongated peptides with natural flanking regions, additional lysine and an azide group (N3) at the C-terminus

Table 3 .
Groups, doses, and treatment schedule

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS B In vivo experiments d METHOD DETAIL