Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors.

Tumor infiltration by T cells profoundly affects cancer progression and responses to immunotherapy. However, the tumor immunosuppressive microenvironment can impair the induction, trafficking, and local activity of antitumor T cells. Here, we investigated whether intratumoral injection of virus-derived peptide epitopes could activate preexisting antiviral T cell responses locally and promote antitumor responses or antigen spreading. We focused on a mouse model of cytomegalovirus (CMV), a highly prevalent human infection that induces vigorous and durable T cell responses. Mice persistently infected with murine CMV (MCMV) were challenged with lung (TC-1), colon (MC-38), or melanoma (B16-F10) tumor cells. Intratumoral injection of MCMV-derived T cell epitopes triggered in situ and systemic expansion of their cognate, MCMV-specific CD4+ or CD8+ T cells. The MCMV CD8+ T cell epitopes injected alone provoked arrest of tumor growth and some durable remissions. Intratumoral injection of MCMV CD4+ T cell epitopes with polyinosinic acid:polycytidylic acid (pI:C) preferentially elicited tumor antigen-specific CD8+ T cells, promoted tumor clearance, and conferred long-term protection against tumor rechallenge. Notably, secondary proliferation of MCMV-specific CD8+ T cells correlated with better tumor control. Importantly, intratumoral injection of MCMV-derived CD8+ T cell-peptide epitopes alone or CD4+ T cell-peptide epitopes with pI:C induced potent adaptive and innate immune activation of the tumor microenvironment. Thus, CMV-derived peptide epitopes, delivered intratumorally, act as cytotoxic and immunotherapeutic agents to promote immediate tumor control and long-term antitumor immunity that could be used as a stand-alone therapy. The tumor antigen-agnostic nature of this approach makes it applicable across a broad range of solid tumors regardless of their origin.

Chondroitin Sulfate Proteoglycans Are De Facto Cellular Receptors for Human Papillomavirus 16 under High Serum Conditions.

Human papillomaviruses (HPVs) are nonenveloped double-stranded DNA viruses that utilize heparan sulfate proteoglycans (HSPGs) as initial attachment factors prior to cell entry and infection. While extensively characterized, the selective interaction between HPV and HSPGs is generally studied using standard in vitro conditions, which fail to account for the effects that media additives, such as fetal bovine serum (FBS), can have on viral binding. As environmental conditions and growth factors associated with wound healing are thought to play a role in natural HPV infection, we sought to investigate the effects that serum or platelet extracts could have on the binding and infectivity of HPV. Here, we demonstrate that high concentrations of FBS and human serum greatly inhibit HPV16 binding, and that for FBS, this effect results from the obstruction of cell surface HSPGs by serum-derived heparin-binding proteins (HBPs). Surprisingly, we found that under these conditions, HPV particles utilize 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as initial binding receptors prior to infection. These findings were corroborated by small interfering RNA (siRNA)-mediated knockdown experiments, as well as through a cancer cell line screen, where we identified a strong association between viral binding in high serum and the expression of chondroitin sulfate biosynthesis genes. Furthermore, HPV binding in the presence of human platelet lysate also demonstrated an increased dependance on CSPGs, suggesting a possible role for these receptor proteoglycans in active wound healing environments. Overall, this work highlights the significant influence that serum/platelet factors can have on virus binding and identifies CSPGs as alternative cell attachment receptors for HPV. IMPORTANCE Heparan sulfate proteoglycans (HSPGs) have previously been identified as primary attachment factors for the initial binding of human papillomaviruses (HPVs) prior to infection. Here, we demonstrate that in vitro, HPV binding to HSPGs is strongly dependent on the surrounding experimental conditions, including the concentration of fetal bovine serum (FBS). We found that high concentrations of FBS can block HSPG-binding sites and cause a dependence on 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as alternative initial viral receptors. Further, we demonstrate that use of a human-derived alternative to FBS, human platelet lysate, also occludes HSPG-dependent binding, causing a shift toward CSPGs for viral attachment. As HPV infection of basal epithelial cells is thought to occur at sites of microtrauma with exposure to high serum levels and platelet factors, these unexpected findings highlight a possible role for CSPGs as important cellular receptors for the binding and infectivity of HPV in vivo.

A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8+ Memory T Cells.

Recent insight into the mechanisms of induction of tissue-resident memory (TRM) CD8+ T cells (CD8+ TRM) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8+ T cells against vaccine Ags, we assessed combinations of i.m. and intravaginal routes in heterologous prime-boost immunization regimens with unrelated viral vectors. Only i.m. prime followed by intravaginal boost induced concomitant strong systemic and intraepithelial genital-resident CD8+ T cell responses. Intravaginal boost with vectors expressing vaccine Ags was far superior to intravaginal instillation of CXCR3 chemokine receptor ligands or TLR 3, 7, and 9 agonists to recruit and increase the pool of cervicovaginal CD8+ TRM Transient Ag presentation increased trafficking of cognate and bystander circulating activated, but not naive, CD8+ T cells into the genital tract and induced in situ proliferation and differentiation of cognate CD8+ TRM Secondary genital CD8+ TRM were induced in the absence of CD4+ T cell help and shared a similar TCR repertoire with systemic CD8+ T cells. This prime-pull-amplify approach elicited systemic and genital CD8+ T cell responses against high-risk human papillomavirus type 16 E7 oncoprotein and conferred CD8-mediated protection to a vaccinia virus genital challenge. These results underscore the importance of the delivery route of nonreplicating vectors in prime-boost immunization to shape the tissue distribution of CD8+ T cell responses. In this context, the importance of local Ag presentation to elicit genital CD8+ TRM provides a rationale to develop novel vaccines against sexually transmitted infections and to treat human papillomavirus neoplasia.

Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection.

Infectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occur in vivo in the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCE Papillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.

Adenovirus vector-based prime-boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins.

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV-infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus-specific antigens for vaccine-induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV-specific CD8+ T cells producing IFN-γ and TNF-α to the cervicovaginal tract. Importantly, the cervicovaginal CD8+ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue-resident memory CD8+ T cells. This prime-boost strategy targeting heterologous locations also induced circulating HPV-specific CD8+ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.

Interferon Gamma Prevents Infectious Entry of Human Papillomavirus 16 via an L2-Dependent Mechanism.

In this study, we report that gamma interferon (IFN-γ) treatment, but not IFN-α, -ß, or -λ treatment, dramatically decreased infection of human papillomavirus 16 (HPV16) pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus types, we found that many, but not all, PsV types were also inhibited by IFN-γ. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-γ, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN-γ treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN-γ treatment was mapped to the L2 protein, as determined with infectious hybrid PsV, in which the L1 protein was derived from an IFN-γ-sensitive HPV type and the L2 protein from an IFN-γ-insensitive type or vice versa.IMPORTANCE A subset of HPV are the causative agents of many human cancers, most notably cervical cancer. This work describes the inhibition of infection of multiple HPV types, including oncogenic types, by treatment with IFN-γ, an antiviral cytokine that is released from stimulated immune cells. Exposure of cells to IFN-γ has been shown to trigger the expression of proteins with broad antiviral effector functions, most of which act to prevent viral transcription or translation. Interestingly, in this study, we show that infection is blocked at the early step of virus entry into the host cell by retention of the minor capsid protein, L2, and the viral genome instead of trafficking into the nucleus. Thus, a novel antiviral mechanism for IFN-γ has been revealed.

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging.

UNLABELLED: We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure. IMPORTANCE: Little is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for antiviral therapy, and allow for the development of new gene delivery systems based on in vitro-generated papillomavirus vectors.

Human papillomavirus capsids preferentially bind and infect tumor cells.

We previously determined that human papillomavirus (HPV) virus-like particles (VLPs) and pseudovirions (PsV) did not, respectively, bind to or infect intact epithelium of the cervicovaginal tract. However, they strongly bound heparan sulfate proteoglycans (HSPG) on the basement membrane of disrupted epithelium and infected the keratinocytes that subsequently entered the disrupted site. We here report that HPV capsids (VLP and PsV) have the same restricted tropism for a wide variety of disrupted epithelial and mesothelial tissues, whereas intact tissues remain resistant to binding. However, the HPV capsids directly bind and infect most tumor-derived cell lines in vitro and have analogous tumor-specific properties in vivo, after local or intravenous injection, using orthotopic models for human ovarian and lung cancer, respectively. The pseudovirions also specifically infected implanted primary human ovarian tumors. Heparin and ι-carrageenan blocked binding and infection of all tumor lines tested, implying that tumor cell binding is HSPG-dependent. A survey using a panel of modified heparins indicates that N-sulfation and, to a lesser degree, O-6 sulfation of the surface HSPG on the tumors are important for HPV binding. Therefore, it appears that tumor cells consistently evolve HSPG modification patterns that mimic the pattern normally found on the basement membrane but not on the apical surfaces of normal epithelial or mesothelial cells. Consequently, appropriately modified HPV VLPs and/or PsV could be useful reagents to detect and potentially treat a remarkably broad spectrum of cancers.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

UNLABELLED: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.

Strain-specific properties and T cells regulate the susceptibility to papilloma induction by Mus musculus papillomavirus 1.

The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×1010 MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated "MmuPV1"), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.

Identification of a role for the trans-Golgi network in human papillomavirus 16 pseudovirus infection.

Human papillomavirus 16 (HPV16) enters its host cells by a process that most closely resembles macropinocytosis. Uncoating occurs during passage through the endosomal compartment, and the low pH encountered in this environment is essential for infection. Furin cleavage of the minor capsid protein, L2, and cyclophilin B-mediated separation of L2 and the viral genome from the major capsid protein, L1, are necessary for escape from the late endosome (LE). Following this exodus, L2 and the genome are found colocalized at the ND10 nuclear subdomain, which is essential for efficient pseudogenome expression. However, the route by which L2 and the genome traverse the intervening cytoplasm between these two subcellular compartments has not been determined. This study extends our understanding of this phase in PV entry in demonstrating the involvement of the Golgi complex. With confocal microscopic analyses involving 5-ethynyl-2'-deoxyuridine (EdU)-labeled pseudogenomes and antibodies to virion and cellular proteins, we found that the viral pseudogenome and L2 travel to the trans-Golgi network (TGN) following exit from the LE, while L1 is retained. This transit is dependent upon furin cleavage of L2 and can be prevented pharmacologically with either brefeldin A or golgicide A, inhibitors of anterograde and retrograde Golgi trafficking. Additionally, Rab9a and Rab7b were determined to be mediators of this transit, as expression of dominant negative versions of these proteins, but not Rab7a, significantly inhibited HPV16 pseudovirus infection.

Characterization of Mus musculus papillomavirus 1 infection in situ reveals an unusual pattern of late gene expression and capsid protein localization.

Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.

Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan.

Genital human papillomavirus (HPV) infection is the most common sexually transmitted infection, and virtually all cases of cervical cancer are attributable to infection by a subset of HPVs (reviewed in ref. 1). Despite the high incidence of HPV infection and the recent development of a prophylactic vaccine that confers protection against some HPV types, many features of HPV infection are poorly understood. It remains worthwhile to consider other interventions against genital HPVs, particularly those that target infections not prevented by the current vaccine. However, productive papillomavirus infection is species- and tissue-restricted, and traditional models use animal papillomaviruses that infect the skin or oral mucosa. Here we report the development of a mouse model of cervicovaginal infection with HPV16 that recapitulates the establishment phase of papillomavirus infection. Transduction of a reporter gene by an HPV16 pseudovirus was characterized by histology and quantified by whole-organ, multispectral imaging. Disruption of the integrity of the stratified or columnar genital epithelium was required for infection, which occurred after deposition of the virus on the basement membrane underlying basal keratinocytes. A widely used vaginal spermicide, nonoxynol-9 (N-9), greatly increased susceptibility to infection. In contrast, carrageenan, a polysaccharide present in some vaginal lubricants, prevented infection even in the presence of N-9, suggesting that carrageenan might serve as an effective topical HPV microbicide.

The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding.

Using a murine challenge model, we previously determined that human papillomavirus (HPV) pseudovirions initially bind preferentially to the cervicovaginal basement membrane (BM) at sites of trauma. We now report that the capsids undergo a conformational change while bound to the BM that results in L2 cleavage by a proprotein convertase (PC), furin, and/or PC5/6, followed by the exposure of an N-terminal cross-neutralization L2 epitope and transfer of the capsids to the epithelial cell surface. Prevention of this exposure by PC inhibition results in detachment of the pseudovirions from the BM and their eventual loss from the tissue, thereby preventing infection. Pseudovirions whose L2 had been precleaved by furin can bypass the PC inhibition of binding and infectivity. Cleavage of heparan sulfate proteoglycans (HSPG) with heparinase III prevented infection and BM binding by the precleaved pseudovirions, but did not prevent them from binding robustly to cell surfaces. These results indicate that the infectious process has evolved so that the initial steps take place on the BM, in contrast to the typical viral infection that is initiated by binding to the cell surface. The data are consistent with a dynamic model of in vivo HPV infection in which a conformational change and PC cleavage on the BM allows transfer of virions from HSPG attachment factors to an L1-specific receptor on basal keratinocytes migrating into the site of trauma.

Virus-Like Particle-Drug Conjugates Induce Protective, Long-lasting Adaptive Antitumor Immunity in the Absence of Specifically Targeted Tumor Antigens.

This study examined the ability of a papillomavirus-like particle drug conjugate, belzupacap sarotalocan (AU-011), to eradicate subcutaneous tumors after intravenous injection and to subsequently elicit long-term antitumor immunity in the TC-1 syngeneic murine tumor model. Upon in vitro activation with near-infrared light (NIR), AU-011-mediated cell killing was proimmunogenic in nature, resulting in the release of damage-associated molecular patterns such as DNA, ATP, and HMGB-1, activation of caspase-1, and surface relocalization of calreticulin and HSP70 on killed tumor cells. A single in vivo administration of AU-011 followed by NIR caused rapid cell death, leading to long-term tumor regression in ∼50% of all animals. Within hours of treatment, calreticulin surface expression, caspase-1 activation, and depletion of immunosuppressive leukocytes were observed in tumors. Combination of AU-011 with immune-checkpoint inhibitor antibodies, anti-CTLA-4 or anti-PD-1, improved therapeutic efficacy, resulting in 70% to 100% complete response rate that was durable 100 days after treatment, with 50% to 80% of those animals displaying protection from secondary tumor rechallenge. Depletion of CD4+ or CD8+ T cells, either at the time of AU-011 treatment or secondary tumor rechallenge of tumor-free mice, indicated that both cell populations are vital to AU-011's ability to eradicate primary tumors and induce long-lasting antitumor protection. Tumor-specific CD8+ T-cell responses could be observed in circulating peripheral blood mononuclear cells within 3 weeks of AU-011 treatment. These data, taken together, support the conclusion that AU-011 has a direct cytotoxic effect on tumor cells and induces long-term antitumor immunity, and this activity is enhanced when combined with checkpoint inhibitor antibodies.

Arrangement of L2 within the papillomavirus capsid.

Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or "split GFP" technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.

Carrageenan is a potent inhibitor of papillomavirus infection.

Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Our recent development of high-titer HPV pseudoviruses has made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan, a type of sulfated polysaccharide extracted from red algae, is an extremely potent infection inhibitor for a broad range of sexually transmitted HPVs. Although carrageenan can inhibit herpes simplex viruses and some strains of HIV in vitro, genital HPVs are about a thousand-fold more susceptible, with 50% inhibitory doses in the low ng/ml range. Carrageenan acts primarily by preventing the binding of HPV virions to cells. This finding is consistent with the fact that carrageenan resembles heparan sulfate, an HPV cell-attachment factor. However, carrageenan is three orders of magnitude more potent than heparin, a form of cell-free heparan sulfate that has been regarded as a highly effective model HPV inhibitor. Carrageenan can also block HPV infection through a second, postattachment heparan sulfate-independent effect. Carrageenan is in widespread commercial use as a thickener in a variety of cosmetic and food products, ranging from sexual lubricants to infant feeding formulas. Some of these products block HPV infectivity in vitro, even when diluted a million-fold. Clinical trials are needed to determine whether carrageenan-based products are effective as topical microbicides against genital HPVs.

Efficient Production of Papillomavirus Gene Delivery Vectors in Defined In Vitro Reactions.

Papillomavirus capsids can package a wide variety of nonviral DNA plasmids and deliver the packaged genetic material to cells, making them attractive candidates for targeted gene delivery vehicles. However, the papillomavirus vectors generated by current methods are unlikely to be suitable for clinical applications. We have developed a chemically defined, cell-free, papillomavirus-based vector production system that allows the incorporation of purified plasmid DNA (pseudogenome) into high-titer papillomavirus L1/L2 capsids. We investigated the incorporation of several DNA forms into a variety of different papillomavirus types, including human and animal types. Our results show that papillomavirus capsids can package and transduce linear or circular DNA under defined conditions. Packaging and transduction efficiencies were surprisingly variable across capsid types, DNA forms, and assembly reaction conditions. The pseudoviruses produced by these methods are sensitive to the same entry inhibitors as cell-derived pseudovirions, including neutralizing antibodies and heparin. The papillomavirus vector production systems developed in this study generated as high as 1011 infectious units/mg of L1. The pseudoviruses were infectious both in vitro and in vivo and should be compatible with good manufacturing practice (GMP) requirements.

Mouse model of cervicovaginal papillomavirus infection.

Virtually all cervical cancers are caused by human papillomavirus infections. The efficient assembly of pseudovirus (PsV) particles incorporating a plasmid expressing a reporter gene has been an invaluable tool in the development of in vitro neutralization assays and in studies of the early mechanisms of viral entry in vitro. Here, we describe a mouse model of human papillomavirus PsV infection of the cervicovaginal epithelium that recapitulates the early events of papillomavirus infection in vivo.

Involvement of Nucleophosmin (NPM1/B23) in Assembly of Infectious HPV16 Capsids.

We report that during assembly of HPV16 pseudovirus (PsV) the minor capsid protein, L2, interacts with the host nucleolar protein nucleophosmin (NPM1/B23). Exogenously-expressed L2 colocalized with NPM1, a complex containing both proteins could be immunoprecipitated, and L2 could redirect to the nucleus NPM1 that was pharmacologically or genetically restricted to the cytoplasm. Coexpression of the major capsid protein, L1, prevented both the colocalization and the biochemical association, and L1 pentamers could displace L2 from L2/NPM1 complexes attached to a nuclear matrix. HPV16 PsV that was produced in a cell line with reduced NPM1 levels had significantly lower infectivity compared to PsV produced in the parental cell line, although the PsV preparations had comparable L1 and L2 ratios and levels of encapsidated DNA. The PsV produced in NPM1-deficient cells showed increased trypsin sensitivity and exhibited decreased L2 levels during endocytosis. These results suggest a critical role for NPM1 in establishing the correct interactions between L2 and L1 during HPV capsid assembly. A decrease in cellular levels of NPM1 results in the formation of seemingly normal, but unstable, capsids that result in a premature loss of L2, thus inhibiting successful infection. No role for NPM1 in HPV infectious entry was found.