Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Precision Enhancement of CAR-NK Cells through Non-Viral Engineering and Highly Multiplexed Base Editing.

Natural killer (NK) cells' unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However, NK cells have demonstrated only moderate responses against cancer in clinical trials and likely require advanced genome engineering to reach their full potential as a cancer therapeutic. Multiplex genome editing with CRISPR/Cas9 base editors (BE) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here, we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations. We observed highly efficient single and multiplex base editing, resulting in significantly enhanced NK cell function. Next, we combined multiplex BE with non-viral TcBuster transposon-based integration to generate IL-15 armored CD19 CAR-NK cells with significantly improved functionality in a highly suppressive model of Burkitt's lymphoma both in vitro and in vivo. The use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

CRISPR-Cas9 base editors and their current role in human therapeutics.

BACKGROUND: Consistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification. METHODS: This review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes. RESULTS: CRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction. CONCLUSION: As such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.

Targeted genome editing for the correction or alleviation of primary Immunodeficiencies.

Primary immunodeficiencies (PID) are a growing list of unique disorders that result in a failure of the innate/adaptive immune systems to fully respond to disease or infection. PIDs are classified into five broad categories; B cell disorders, combined B and T cell disorders, phagocytic disorders, complement disorders, and disorders with recurrent fevers and inflammation. Many of these disorders, such as X-SCID, WAS, and CGD lead to early death in children if intervention is not implemented. At present, the predominant method of curative therapy remains an allogeneic transplant from a healthy donor, however many complications and limitations exist with his therapy such as availability of donors, graft vs host disease, graft rejection, and infection. More recently, gene therapy using viral based complementation vectors have successfully been implemented to functionally correct patient cells in an autologous transplant, but these methods carry significant risks, including insertional mutagenesis, and provide non-physiological gene expression. For these reasons, gene-editing reagents such as targeted nucleases, base editors (BE), and prime editors (PE) are being explored. The BE and PE tools, sometimes referred to as digital editors, are of very high interest as they provide both enhanced molecular specificity and do not rely on DNA repair pathways after DSBs to change individual base pairs or directly replace DNA sequences responsible for pathogenic phenotypes. With this in mind the purpose of this chapter is to highlight some of the most common PIDs found within the human population, discuss successes and shortcomings of previous intervention strategies, and highlight how the next generation of gene-editing tools may be deployed to directly repair the underlying genetic causes of this class of disease.

Theta-Defensins Inhibit High-Risk Human Papillomavirus Infection Through Charge-Driven Capsid Clustering.

Persistent infection with high-risk human papillomavirus (hrHPV) genotypes results in a large number of anogenital and head and neck cancers worldwide. Although prophylactic vaccination coverage has improved, there remains a need to develop methods that inhibit viral transmission toward preventing the spread of HPV-driven disease. Defensins are a class of innate immune effector peptides that function to protect hosts from infection by pathogens such as viruses and bacteria. Previous work utilizing α and ß defensins from humans has demonstrated that the α-defensin HD5 is effective at inhibiting the most common high-risk genotype, HPV16. A third class of defensin that has yet to be explored are θ-defensins: small, 18-amino acid cyclic peptides found in old-world monkeys whose unique structure makes them both highly cationic and resistant to degradation. Here we show that the prototype θ-defensin, rhesus theta defensin 1, inhibits hrHPV infection through a mechanism involving capsid clustering that inhibits virions from binding to cell surface receptor complexes.

Immune Activation in Patients with Locally Advanced Cervical Cancer Treated with Ipilimumab Following Definitive Chemoradiation (GOG-9929).

PURPOSE: A phase I clinical trial (GOG-9929) examined the safety and efficacy of adjuvant immune-modulation therapy with the checkpoint inhibitor ipilimumab [anti-CTL antigen-4 (anti-CTLA-4)] following chemoradiation therapy (CRT) for newly diagnosed node-positive human papillomavirus (HPV)-related cervical cancer. To better understand the mechanism of action and to identify predictive biomarkers, immunologic and viral correlates were assessed before, during, and after treatment. PATIENTS AND METHODS: Twenty-one patients who received CRT and ≥2 doses of ipilimumab and 5 patients who received CRT only were evaluable for translational endpoints. Circulating T-cell subsets were evaluated by multiparameter flow cytometry. Cytokines were evaluated by multiplex ELISA. HPV-specific T cells were evaluated in a subset of patients by IFNγ ELISpot. RESULTS: Expression of the activation markers ICOS and PD-1 significantly increased on T-cell subsets following CRT and were sustained or increased following ipilimumab treatment. Combined CRT/ipilimumab treatment resulted in a significant expansion of both central and effector memory T-cell populations. Genotype-specific E6/E7-specific T-cell responses increased post-CRT in 1 of 8 HPV16+ patients and in 2 of 3 HPV18+ patients. Elevation in levels of tumor-promoting circulating cytokines (TNFα, IL6, IL8) post-CRT was significantly associated with worse progression-free survival. CONCLUSIONS: Our data indicate that CRT alone and combined with ipilimumab immunotherapy show immune-modulating activity in women with locally advanced cervical cancer and may be a promising therapeutic option for the enhancement of antitumor immune cell function after primary CRT for this population at high risk for recurrence and metastasis. Several key immune biomarkers were identified that were associated with clinical response.

TNFSF14: LIGHTing the Way for Effective Cancer Immunotherapy.

Tumor necrosis factor superfamily member 14 (LIGHT) has been in pre-clinical development for over a decade and shows promise as a modality of enhancing treatment approaches in the field of cancer immunotherapy. To date, LIGHT has been used to combat cancer in multiple tumor models where it can be combined with other immunotherapy modalities to clear established solid tumors as well as treat metastatic events. When LIGHT molecules are delivered to or expressed within tumors they cause significant changes in the tumor microenvironment that are primarily driven through vascular normalization and generation of tertiary lymphoid structures. These changes can synergize with methods that induce or support anti-tumor immune responses, such as checkpoint inhibitors and/or tumor vaccines, to greatly improve immunotherapeutic strategies against cancer. While investigators have utilized multiple vectors to LIGHT-up tumor tissues, there are still improvements needed and components to be found within a human tumor microenvironment that may impede translational efforts. This review addresses the current state of this field.

The Essential Role of anxA2 in Langerhans Cell Birbeck Granules Formation.

Langerhans cells (LC) are the resident antigen presenting cells of the mucosal epithelium and play an essential role in initiating immune responses. LC are the only cells in the body to contain Birbeck granules (BG), which are unique cytoplasmic organelles comprised of c-type lectin langerin. Studies of BG have historically focused on morphological characterizations, but BG have also been implicated in viral antigen processing which suggests that they can serve a function in antiviral immunity. This study focused on investigating proteins that could be involved in BG formation to further characterize their structure using transmission electron microscopy (TEM). Here, we report a critical role for the protein annexin A2 (anxA2) in the proper formation of BG structures. When anxA2 expression is downregulated, langerin expression decreases, cytoplasmic BG are nearly ablated, and the presence of malformed BG-like structures increases. Furthermore, in the absence of anxA2, we found langerin was no longer localized to BG or BG-like structures. Taken together, these results indicate an essential role for anxA2 in facilitating the proper formation of BG.

Therapeutic efficacy of a human papillomavirus type 16 E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a preclinical mouse model for HPV-associated disease.

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8+ T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.

Annexin A2 in Virus Infection.

Viral life cycles consist of three main phases: (1) attachment and entry, (2) genome replication and expression, and (3) assembly, maturation, and egress. Each of these steps is intrinsically reliant on host cell factors and processes including cellular receptors, genetic replication machinery, endocytosis and exocytosis, and protein expression. Annexin A2 (AnxA2) is a membrane-associated protein with a wide range of intracellular functions and a recurrent host factor in a variety of viral infections. Spatially, AnxA2 is found in the nucleus and cytoplasm, vesicle-bound, and on the inner and outer leaflet of the plasma membrane. Structurally, AnxA2 exists as a monomer or in complex with S100A10 to form the AnxA2/S100A10 heterotetramer (A2t). Both AnxA2 and A2t have been implicated in a vast array of cellular functions such as endocytosis, exocytosis, membrane domain organization, and translational regulation through RNA binding. Accordingly, many discoveries have been made involving AnxA2 in viral pathogenesis, however, the reported work addressing AnxA2 in virology is highly compartmentalized. Therefore, the purpose of this mini review is to provide information regarding the role of AnxA2 in the lifecycle of multiple epithelial cell-targeting viruses to highlight recurrent themes, identify discrepancies, and reveal potential avenues for future research.

Validity and prognostic significance of sperm protein 17 as a tumor biomarker for epithelial ovarian cancer: a retrospective study.

BACKGROUND: Prior small studies have shown increased expression of sperm protein 17 (Sp17) in epithelial ovarian cancer (EOC) tissue and suggest Sp17 as a potential biomarker for EOC. However, how Sp17 expression varies with histology, grade, and stage of EOC and its expression in other ovarian neoplasms has not been defined. It is unknown whether patients with EOC have elevated serum Sp17 levels or if Sp17 expression is associated with survival outcomes. METHODS: The study included 982 patients with benign, borderline, and malignant ovarian neoplasms and normal ovary. There were 878 patients with tissue only, 39 with serum only, and 65 with matching serum and tissue. Immunohistochemical (IHC) staining with anti-Sp17 antibody was performed on tissue specimens and the intensity scored as weak, moderate, or strong. A sandwich enzyme-linked immunosorbent assay (ELISA) was performed to measure Sp17 sera concentrations. RESULTS: Sp17 expression was most commonly seen in serous cystadenomas (83%) and serous borderline tumors (100%). Of the 773 EOC specimens, 223 (30%) expressed Sp17. Grade and histology were significantly associated with Sp17 expression among EOC specimens (p < 0.001) on both univariate and multivariable analysis, with grade 1 serous adenocarcinomas showing the highest expression (51%). Sp17 expression was limited in other benign and non-epithelial malignant neoplasms. Neither Sp17 tissue expression nor serum concentration correlated with survival outcomes. Serum concentrations were higher in patients with Sp17 tissue expression, and the highest concentrations were noted among patients with serous and clear cell adenocarcinomas. CONCLUSIONS: Sp17 is highly expressed in benign, borderline, and low grade malignant serous ovarian neoplasms and can be quantified in serum. Sp17 expression may have diagnostic significance in this subset of patients.

Heterotetrameric annexin A2/S100A10 (A2t) is essential for oncogenic human papillomavirus trafficking and capsid disassembly, and protects virions from lysosomal degradation.

Human papillomavirus (HPV) entry into epithelial cells is independent of canonical endocytic pathways. Upon interaction with host cells, HPV establishes infection by traversing through an endocytic pathway that is clathrin- and caveolin-independent, but dependent on the annexin A2/S100A10 heterotetramer (A2t). We examined the contribution of monomeric annexin A2 (AnxA2) vs. A2t in HPV infection and endocytosis, and further characterized the role of these molecules in protein trafficking. We specifically show that cell surface A2t is not required for HPV attachment, and in the absence of A2t virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. These findings suggest that A2t is a central mediator of high-risk HPV intracellular trafficking post-entry and pre-viral uncoating.

Nanobody-Antigen Conjugates Elicit HPV-Specific Antitumor Immune Responses.

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor-infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870-80. ©2018 AACR.

Nano-Pulse Stimulation induces immunogenic cell death in human papillomavirus-transformed tumors and initiates an adaptive immune response.

Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. In vitro NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8+ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8+ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8+ T cells capable of protecting individuals from disease recurrence.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Human Immunodeficiency Virus Immune Cell Receptors, Coreceptors, and Cofactors: Implications for Prevention and Treatment.

In the last three decades, extensive research on human immunodeficiency virus (HIV) has highlighted its capability to exploit a variety of strategies to enter and infect immune cells. Although CD4(+) T cells are well known as the major HIV target, with infection occurring through the canonical combination of the cluster of differentiation 4 (CD4) receptor and either the C-C chemokine receptor type 5 (CCR5) or C-X-C chemokine receptor type 4 (CXCR4) coreceptors, HIV has also been found to enter other important immune cell types such as macrophages, dendritic cells, Langerhans cells, B cells, and granulocytes. Interestingly, the expression of distinct cellular cofactors partially regulates the rate in which HIV infects each distinct cell type. Furthermore, HIV can benefit from the acquisition of new proteins incorporated into its envelope during budding events. While several publications have investigated details of how HIV manipulates particular cell types or subtypes, an up-to-date comprehensive review on HIV tropism for different immune cells is lacking. Therefore, this review is meant to focus on the different receptors, coreceptors, and cofactors that HIV exploits to enter particular immune cells. Additionally, prophylactic approaches that have targeted particular molecules associated with HIV entry and infection of different immune cells will be discussed. Unveiling the underlying cellular receptors and cofactors that lead to HIV preference for specific immune cell populations is crucial in identifying novel preventative/therapeutic targets for comprehensive strategies to eliminate viral infection.

T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation.

Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8+ T cells receiving signal 1 + 2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.

Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases.

Carcinomas of the anogenital tract, in particular cervical cancer, remains one of the most common cancers in women, and represent the most frequent gynecological malignancies and the fourth leading cause of cancer death in women worldwide. Human papillomavirus (HPV)-induced lesions are immunologically distinct in that they express viral antigens, which are necessary to maintain the cancerous phenotype. The causal relationship between HPV infection and anogenital cancer has prompted substantial interest in the development of therapeutic vaccines against high-risk HPV types targeting the viral oncoproteins E6 and E7. This review will focus on the most recent clinical trials for immunotherapies for mucosal HPV-induced lesions as well as emerging therapeutic strategies that have been tested in pre-clinical models for HPV-induced diseases. Progress in peptide- and protein-based vaccines, DNA-based vaccines, viral/bacterial vector-based vaccines, immune checkpoint inhibition, immune response modifiers, and adoptive cell therapy for HPV will be discussed.

Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.