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.

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.

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.

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.

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.

Inhibition of Langerhans cell maturation by human papillomavirus type 16: a novel role for the annexin A2 heterotetramer in immune suppression.

High-risk human papillomaviruses (HPVs) are sexually transmitted viruses causally associated with several cancers. During its natural life cycle, HPV16, the most common high-risk genotype, infects the epithelial basal cells in a process facilitated through a recently identified receptor, the annexin A2 heterotetramer (A2t). During infection, HPV16 also interacts with Langerhans cells (LC), the APC of the epithelium, inducing immune suppression, which is mediated by the HPV16 L2 minor capsid protein. Despite the importance of these virus-immune cell interactions, the specific mechanisms of HPV16 entry into LC and HPV16-induced immune suppression remain undefined. An N-terminal peptide of HPV16 L2 (aa 108-126) has been shown to specifically interact with A2t. In this study, we show that incubation of human LC with this peptide blocks binding of HPV16. Inhibiting this interaction with an A2t ligand or by small interfering RNA downregulation of A2t significantly decreases HPV16 internalization into LC in an L2-dependent manner. A2t is associated with suppression of LC maturation as demonstrated through attenuated secretion of Th1-associated cytokines and decreased surface expression of MHC class II on LC exposed to A2t. Conversely, small molecule inhibition of A2t prevents HPV16-induced suppression of LC immune function as indicated by significantly increased secretion of inflammatory cytokines and surface expression of CD86 in HPV16 treated LC pre-exposed to A2t inhibitors. These results demonstrate that HPV16 suppresses LC maturation through an interaction with A2t, revealing a novel role for this protein.

Forced LIGHT expression in prostate tumors overcomes Treg mediated immunosuppression and synergizes with a prostate tumor therapeutic vaccine by recruiting effector T lymphocytes.

BACKGROUND: LIGHT, a ligand for lymphotoxin-ß receptor (LTßR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTßR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTßR has been previously shown to activate immune cells and result in tumor regression in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors. METHODS: Real Time PCR was used to evaluate expression of forced LIGHT and other immunoregulatory genes in prostate tumors samples. For in vivo studies, adenovirus encoding murine LIGHT was injected intratumorally into TRAMP-C2 prostate cancer cell tumor bearing mice. Chemokine and cytokine concentrations were determined by multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes and expression of LIGHT on the tumor cell surface. Tumor-specific lymphocytes were quantified via ELISpot assay. Treg induction and Treg suppression assays determined Treg functionality after LIGHT treatment. RESULTS: LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced tumor burden. LIGHT expression peaked within 48 hr of infection, recruited effector T cells that recognized mouse prostate stem cell antigen (PSCA) into the tumor microenvironment, and inhibited infiltration of Tregs. Tregs isolated from tumor draining lymph nodes had impaired suppressive capability after LIGHT treatment. CONCLUSION: Forced LIGHT treatment combined with PSCA TriVax therapeutic vaccination delays prostate cancer progression in mice by recruiting effector T lymphocytes to the tumor and inhibiting Treg mediated immunosuppression. Prostate 75:280-291, 2015. © 2014 Wiley Periodicals, Inc.

Langerhans cells from women with cervical precancerous lesions become functionally responsive against human papillomavirus after activation with stabilized Poly-I:C.

Human papillomavirus (HPV)-mediated suppression of Langerhans cell (LC) function can lead to persistent infection and development of cervical intraepithelial neoplasia (CIN). Women with HPV-induced high-grade CIN2/3 have not mounted an effective immune response against HPV, yet it is unknown if LC-mediated T cell activation from such women is functionally impaired against HPV. We investigated the functional activation of in vitro generated LC and their ability to induce HPV16-specific T cells from CIN2/3 patients after exposure to HPV16 followed by treatment with stabilized Poly-I:C (s-Poly-I:C). LC from patients exposed to HPV16 demonstrated a lack of costimulatory molecule expression, inflammatory cytokine secretion, and chemokine-directed migration. Conversely, s-Poly-I:C caused significant phenotypic and functional activation of HPV16-exposed LC, which resulted in de novo generation of HPV16-specific CD8(+) T cells. Our results highlight that LC of women with a history of persistent HPV infection can present HPV antigens and are capable of inducing an adaptive T cell immune response when given the proper stimulus, suggesting that s-Poly-I:C compounds may be attractive immunomodulators for LC-mediated clearance of persistent HPV infection.

Small molecule inhibitors of the annexin A2 heterotetramer prevent human papillomavirus type 16 infection.

OBJECTIVES: High-risk human papillomavirus (HPV) infection leads to the development of several human cancers that cause significant morbidity and mortality worldwide. HPV type 16 (HPV16) is the most common of the cancer-causing genotypes and gains entry to the basal cells of the epithelium through a non-canonical endocytic pathway that involves the annexin A2/S100A10 heterotetramer (A2t). A2t is composed of two annexin A2 monomers bound to an S100A10 dimer and this interaction is a potential target to block HPV16 infection. Here, recently identified small molecule inhibitors of A2t (A2ti) were investigated for their ability to prevent HPV16 infection in vitro. METHODS: A2ti were added to HeLa cells in increasing concentrations prior to the addition of HPV16. Cytotoxicity was evaluated via trypan blue exclusion. HPV16 pseudovirion infection and fluorescently labelled HPV16 capsid internalization was measured with flow cytometry. RESULTS: A2ti blocked HPV16 infection by 100% without substantial cellular toxicity or reduction in cell growth. Furthermore, A2ti blocked HPV16 entry into epithelial cells by 65%, indicating that the observed inhibition of HPV16 infection is in part due to a block in entry and that non-infectious entry may occur in the absence of A2t binding. CONCLUSIONS: These results demonstrate that targeting A2t may be an effective strategy to prevent HPV16 infection.

The evolving field of human papillomavirus receptor research: a review of binding and entry.

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs.

Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy.

Gamma delta (γδ) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, γδ T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of γδ T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-γδ T cells in support of therapeutic application. Engineered CAR-γδ T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-γδ T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-γδ T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-γδ T cell subsets to therapeutic efficacy in vivo.

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.

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.

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.

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.

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.

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.

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.