The interactions between PML nuclear bodies and small and medium size DNA viruses.

Promyelocytic leukemia nuclear bodies (PM NBs), often referred to as membraneless organelles, are dynamic macromolecular protein complexes composed of a PML protein core and other transient or permanent components. PML NBs have been shown to play a role in a wide variety of cellular processes. This review describes in detail the diverse and complex interactions between small and medium size DNA viruses and PML NBs that have been described to date. The PML NB components that interact with small and medium size DNA viruses include PML protein isoforms, ATRX/Daxx, Sp100, Sp110, HP1, and p53, among others. Interaction between viruses and components of these NBs can result in different outcomes, such as influencing viral genome expression and/or replication or impacting IFN-mediated or apoptotic cell responses to viral infection. We discuss how PML NB components abrogate the ability of adenoviruses or Hepatitis B virus to transcribe and/or replicate their genomes and how papillomaviruses use PML NBs and their components to promote their propagation. Interactions between polyomaviruses and PML NBs that are poorly understood but nevertheless suggest that the NBs can serve as scaffolds for viral replication or assembly are also presented. Furthermore, complex interactions between the HBx protein of hepadnaviruses and several PML NBs-associated proteins are also described. Finally, current but scarce information regarding the interactions of VP3/apoptin of the avian anellovirus with PML NBs is provided. Despite the considerable number of studies that have investigated the functions of the PML NBs in the context of viral infection, gaps in our understanding of the fine interactions between viruses and the very dynamic PML NBs remain. The complexity of the bodies is undoubtedly a great challenge that needs to be further addressed.

Pretransplantation seroreactivity in kidney donors and recipients as a predictive factor for posttransplant BKPyV-DNAemia.

BK polyomavirus (BKPyV) often reactivates after kidney transplantation, causing BKPyV-associated nephropathy (BKPyVAN) in 1%-10% of cases with a potential detrimental effect on allograft survival. Kidney transplant recipients are regularly screened for BKPyV DNA in plasma. As this strategy may not always reduce the risk of BKPyVAN, other predictive markers are needed. To evaluate the role of pretransplant BKPyV-specific antibody, 210 kidney transplant recipients and 130 donors were screened for BKPyV DNA and BKPyV-specific antibodies. We found that the donor BKPyV immunoglobulin G (IgG) seroprevalence and antibody level were strongly associated with BKPyV-DNAemia and BKPyVAN, although multivariant analysis found the presence of anti-BKPyV-specific antibodies as a predictive factor only for BKPyV-DNAemia. The pretransplant recipient status had no effect on posttransplant BKPyV-DNAemia and BKVAN. BKPyV IgG levels remained stable in BKPyV-negative recipients during 1-year follow-up, while a considerable increase was observed in BKPyV-positive patients. The presence of anti-BKPyV-specific antibodies in kidney allograft donors is a good and reliable predictive marker for posttransplant BKPyV replication with relevance to risk stratification in transplant recipients.

The Interplay between Viruses and Host DNA Sensors.

DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.

Pretransplant BK Virus-Specific T-Cell-Mediated Immunity and Serotype Specific Antibodies May Have Utility in Identifying Patients at Risk of BK Virus-Associated Haemorrhagic Cystitis after Allogeneic HSCT.

BK polyomavirus (BKPyV) persists lifelong in renal and urothelial cells with asymptomatic urinary shedding in healthy individuals. In some immunocompromised persons after transplantation of hematopoietic stem cells (HSCT), the BKPyV high-rate replication is associated with haemorrhagic cystitis (HC). We tested whether the status of BKPyV immunity prior to HSCT could provide evidence for the BKPyV tendency to reactivate. We have shown that measurement of pretransplant anti-BKPyV 1 and 4 IgG levels can be used to evaluate the HC risk. Patients with anti-BKPyV IgG in the range of the 1st-2nd quartile of positive values and with positive clinical risk markers have a significantly increased HC risk, in comparison to the reference group of patients with "non-reactive" anti-BKPyV IgG levels and with low clinical risk (LCR) (p = 0.0009). The predictive value of pretransplant BKPyV-specific IgG was confirmed by determination of genotypes of the shed virus. A positive predictive value was also found for pretransplant T-cell immunity to the BKPyV antigen VP1 because the magnitude of IFN-γ T-cell response inversely correlated with posttransplant DNAuria and with HC. Our novel data suggest that specific T-cells control BKPyV latency before HSCT, and in this way may influence BKPyV reactivation after HSCT. Our study has shown that prediction using a combination of clinical and immunological pretransplant risk factors can help early identification of HSCT recipients at high risk of BKPyV disease.

Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells.

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

Immune sensing of mouse polyomavirus DNA by p204 and cGAS DNA sensors.

The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-ß) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.

Detection of human polyomaviruses MCPyV, HPyV6, and HPyV7 in malignant and non-malignant tonsillar tissues.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare skin malignancy. Human polyomavirus six and seven (HPyV6 and HPyV7) were identified on a skin but have not been associated with any pathology. The serology data suggest that infection with polyomaviruses occurs in childhood and they are widespread in population. However, the site of persistent infection has not been identified. Altogether, 103 formalin-fixed paraffin-embedded (FFPE) specimens and five fresh frozen tissues (FF) of non-malignant tonsils and 97 FFPE and 15 FF samples of tonsillar carcinomas were analyzed by qPCR for the presence of MCPyV, HPyV6, and HPyV7 DNA. All MCPyV DNA positive FF tissues were screened for the expression of early viral transcripts. Overall prevalence of MCPyV, HPyV6, and HPyV7 in non-malignant tonsillar tissues was 10.2%, 4.6%, and, 0.9%, respectively. The prevalence of MCPyV DNA in non-malignant tonsils increased with age (P < 0.05). While the prevalence of MCPyV DNA was significantly higher in the tumors than non-malignant tissues (35.7% vs. 10.2%) (P < 0.001), the prevalence of HPyV6 DNA (5.4% vs. 4.6%) and HPyV7 DNA (1.8% vs. 0.9%) were comparable. In all MCPyV DNA positive FF tissues early transcripts were detected. MCPyV, HPyV6, and HPyV7 DNAs were found in tonsils, suggesting that the tonsils may be a site of viral latency. The viral load was low indicating that only a fraction of cells are infected. The higher prevalence of MCPyV DNA was detected in tonsillar tumors but there was no difference in the viral load between tumor and healthy tissues.

Seroprevalence rates of HPyV6, HPyV7, TSPyV, HPyV9, MWPyV and KIPyV polyomaviruses among the healthy blood donors.

Human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV have been discovered between 2007 and 2012. TSPyV causes a rare skin disease trichodysplasia spinulosa in immunocompromised patients, the role of remaining polyomaviruses in human pathology is not clear. In this study, we assessed the occurrence of serum antibodies against above polyomaviruses in healthy blood donors. Serum samples were examined by enzyme-linked immunoassay (ELISA), using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, and KIPyV were found in 88.2%, 65.7%, 63.2%, 31.6%, 84.4%, and 58%, respectively, of this population. The seroprevalence generally increased with age, the highest rise we observed for HPyV9 and KIPyV specific antibodies. The levels of anti-HPyV antibodies remained stable across the age-groups, except for TSPyV and HPyV9, where we saw change with age. ELISAs based on VLP and GST-VP1 gave comparable seroprevalence for HPyV6 antibodies (88.2% vs.85.3%) but not for HPyV7 antibodies (65.7% vs. 77.2%), suggesting some degree of crossreactivity between HPyV6 and HPyV7 VP1 proteins. In conclusion, these results provide evidence that human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MwPyV, and KIPyV circulate widely in the Czech population and their seroprevalence is comparable to other countries.

Viral microRNA effects on pathogenesis of polyomavirus SV40 infections in syrian golden hamsters.

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

Seroprevalence rates of BKV, JCV, and MCPyV polyomaviruses in the general Czech Republic population.

JC and BK polyomaviruses (JCV and BKV) infect humans and can cause severe illnesses in immunocompromised patients. Merkel cell polyomavirus (MCPyV) can be found in skin carcinomas. In this study, we assessed the occurrence of serum antibodies against MCPyV, BKV, and JCV polyomaviruses in a healthy population of the Czech Republic. Serum samples from 991 healthy individuals (age range: 6-64 years) were examined by enzyme-linked immunoassay (ELISA) using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against MCPyV, JCV, and BKV were found in 63%, 57%, and 69%, respectively, of this population. For all three viruses, these rates were associated with age; the occurrence of antibodies against MCPyV and JCV was highest for those older than 59 years, while the occurrence of antibodies against BKV was highest in those aged 10-19 years and 20-29 years. This is the first large study to determine the seroprevalence rates for BKV, JCV, and MCPyV polyomaviruses in the general Czech Republic population.

Effects of route of inoculation and viral genetic variation on antibody responses to polyomavirus SV40 in Syrian golden hamsters.

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation-but not viral genetic variation-is an important factor in production of neutralizing antibody to SV40.

Lymphotropism of Merkel cell polyomavirus infection, Nova Scotia, Canada.

To test the hypothesis that Merkel cell polyomavirus (MCPyV) can infect cells of the lymphoid system, we analyzed 353 specimens, including 152 non-Hodgkin lymphomas, 44 Hodgkin lymphomas, 110 benign lymph nodes, 27 lymph nodes with metastasis, and 20 extranodal tissue samples. MCPyV DNA was detected by quantitative PCR in 13 (6.6%) of 196 lymphomas, including 5 (20.8%) of 24 chronic lymphocytic leukemia specimens, and in 11 (10%) of 110 benign lymph nodes, including 8 (13.1%) of 61 samples of reactive hyperplasia and 3 (10.3%) of 29 normal lymph nodes. Other samples were MCPyV negative. Sequence analysis of 9 virus-positive samples confirmed the identity of MCPyV; 3 viral strains were represented. Immunohistochemical testing showed that 1 T-cell lymphoma expressed MCPyV T-antigen. These findings suggest that the lymphoid system plays a role in MCPyV infection and may be a site for MCPyV persistence.

Variable frequency of polyomavirus SV40 and herpesvirus EBV in lymphomas from two different urban population groups in Houston, TX.

BACKGROUND: Studies have reported differing frequencies of detection of polyomavirus simian virus 40 (SV40) in association with human lymphomas. OBJECTIVE: We addressed the hypothesis that SV40 positivity in lymphomas can vary among sampled populations. STUDY DESIGN: Archival paraffin-embedded lymphoma specimens (n=171) from patients at two urban hospitals in Houston, TX, USA, were analyzed following a cross-sectional study design. Extracted DNAs were characterized by quantitative polymerase chain reaction for the cellular RNase P gene and for SV40 and herpesvirus Epstein-Barr virus (EBV) sequences. RESULTS: Patient characteristics of the two study populations differed significantly whereas the classification of tumor types studied did not. SV40 DNA was detected more frequently in lymphomas from the public hospital population (10/44, 23%) than in lymphomas from the veterans' hospital (VAMC) (4/127, 3%; P<0.0001). EBV detection in lymphomas also differed between the two groups (17/44, 39% vs. 23/127, 18%; P=0.01). SV40 positivity was associated with a younger age category of VAMC lymphoma patients (P=0.02). Expression of T-antigen was detected by immunohistochemistry in half of lymphomas that contained SV40 DNA. Variation was observed in the quality and quantity of DNA recovered from paraffin-embedded specimens, but there was no difference in recoveries of DNA from samples from the two hospitals. CONCLUSIONS: This study demonstrated that, in a direct comparison, the prevalence of SV40 DNA in lymphomas can differ significantly between groups with different demographic distributions.

Viral regulatory region effects on vertical transmission of polyomavirus SV40 in hamsters.

Viral strain differences influence the oncogenic potential of polyomavirus simian virus 40 (SV40). We hypothesized that viral strain differences might also affect vertical transmission of SV40 in susceptible hosts. Pregnant Syrian golden hamsters were inoculated intraperitoneally with 10(7) plaque-forming units of SV40 and offspring were sacrificed post-delivery (1-21 days, 6 months). Organ extracts were analyzed for SV40 DNA by polymerase chain reaction assay. Transmission of SV40 from mother to offspring was detected in over half of litters. Most placentas were virus-positive. Mothers inoculated with SV40 strains containing complex regulatory regions transmitted virus more frequently than those infected with simple enhancer viruses (p<0.001). Virus was detected more often in progeny brain than in spleen (p<0.05). Several progeny were virus-positive at 6 months of age, suggesting viral persistence. Maternal animals retained virus in several tissues through day 21 and developed T-antigen antibodies. These results indicate that SV40 replicates in hamsters, vertical transmission of SV40 can occur, and the viral regulatory region influences transmission.

SV40 lymphomagenesis in Syrian golden hamsters.

Simian virus 40 (SV40) isolates differ in oncogenic potential in Syrian golden hamsters following intraperitoneal inoculation. Here we describe the effect of intravenous exposure on tumor induction by SV40. Strains SVCPC (simple regulatory region) and VA45-54(2E) (complex regulatory region) were highly oncogenic following intravenous inoculation, producing a spectrum of tumor types. Three lymphoma cell lines were established; all expressed SV40 T-antigen, were immortalized for growth in culture, and were tumorigenic following transplantation in vivo. New monoclonal antibodies directed against hamster lymphocyte surface antigens are described. The cell lines expressed MHC class II and macrophage markers and were highly phagocytic, indicating a histiocytic origin. Many hamsters that remained tumor-free developed SV40 T-antigen antibodies, suggesting that viral replication occurred. This study shows that route of exposure influences the pathogenesis of SV40-mediated carcinogenesis, that SV40 strain VA45-54(2E) is lymphomagenic in hamsters, that hamster lymphoid cells of histiocytic origin can be transformed in vivo and established in culture, and that reagents to hamster leukocyte differentiation molecules are now available.

Detection of polyomavirus SV40 in tonsils from immunocompetent children.

BACKGROUND: BK virus (BKV), JC virus (JCV) and simian virus 40 (SV40) are nonenveloped DNA viruses, members of the family Polyomaviridae. BK and JC viruses establish persistent infections in humans, and evidence suggests that SV40 can infect humans, as well. Whether persistence occurs in the lymphoid system is unknown. METHODS: Paraffin-embedded tonsils from 220 immunocompetent children (mean age 9.3 years) were examined by polymerase chain reaction (PCR) to detect viral DNA of BKV, JCV, SV40, and Epstein-Barr virus (EBV). RESULTS: Polyomavirus-specific DNA sequences were detected in 8.3% (29/351) of specimens collected from 220 children. Twenty-one (9.5%) children had polyomavirus DNA present in at least one tonsil, with sequences identified as SV40 (n=20) and BKV (n=1). Polyomavirus JCV was not detected. Among patients positive for SV40, 8 of 14 (57%) contained viral DNA in both available tonsils. EBV DNA was detected in 99 (28.2%) samples from 67 (30.5%) patients. Eleven samples (3.1%) from 8 (3.6%) children were positive for both polyomavirus and EBV. SV40-positive children were significantly older than the SV40-negative subjects (P<0.001). T-antigen expression was detected in an SV40 DNA-positive tonsil by immunohistochemistry. CONCLUSIONS: These results suggest that SV40 can infect tonsils, that lymphoid tissue may represent a site for polyomavirus persistence, and that immunohistochemistry is not a useful detection assay when there are very few virus-infected cells in a tissue.

Influence of the viral regulatory region on tumor induction by simian virus 40 in hamsters.

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10(7) PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.

Adjuvant effect of dendritic cells transduced with recombinant vaccinia virus expressing HPV16-E7 is inhibited by co-expression of IL12.

Dendritic cells (DC) enhanced the immunogenicity of recombinant vaccinia viruses (rVV) expressing the E7 protein of HPV16, a tumor-associated antigen (TAA). Immunization with DC transduced by rVV generated from strain Praha or MVA induced better protection against the growth of transplanted TC-1 tumors in C57Bl/6 mice than did immunization with either of these rVVs administered alone by the same route. Interestingly, DC transduced with a double recombinant vaccinia virus expressing E7 protein together with the Th1-polarizing cytokine IL12, which has been shown to enhance the cellular response in several other systems, induced lower anti-tumor immunity than DC transduced with rVV expressing E7 protein alone. The inhibitory effect mediated by IL12 on immunization with rVV-infected DC was dose-dependent and was observed after immunization with DC transduced with IL12-expressing rVV even at low multiplicity.

Experimental therapy of HPV16 induced tumors with IL12 expressed by recombinant vaccinia virus in mice.

Recombinant vaccinia viruses derived from strain Praha, clone P13, and strain MVA were used for intratumoral delivery and expression of IL12 genes in tumors induced by HPV16 E6+E7 oncogenes in mice. Intratumoral injection of 10(3) PFU of P13-IL12 virus resulted in an increase of intra-tumoral IL12 on days 6-13, while only low levels of IL12 were found in sera. After the inoculation of 10(6) PFU of MVA-IL12, the same levels of IL12 were found as in animals injected with control virus. The intratumoral inoculation of 10(3) PFU P13-IL12 resulted in only approximately 30% of the tumors being virus positive, which was a consequence of reduced multiplication of the recombinant virus in vivo. The number of virus-positive tumors was not increased by repeated inoculations on three consecutive days. Intratumoral therapy with a dose of 10(3) PFU of P13-IL12 slowed down the growth of TC1 tumors, but never caused their regression. When local P13-IL12 treatment was combined with antigen-specific, DNA-vaccination therapy, no synergy between the two treatments was observed. The treatment with IL12-expressing virus retarded tumor growth to some degree, but did not change the number of regressing tumors. The highest efficacy of intra-tumoral P13-IL12 therapy was observed when the TC-1/A9 cell subline, with downregulated MHC class I expression, was used. TC-1/A9 tumors are less refractory to treatment with 10(3) P13-IL12/EL than are parental TC-1 cells.

Immune response to E7 protein of human papillomavirus type 16 anchored on the cell surface.

To target the E7 protein of human papilloma virus 16 to the cell surface, a fusion gene was constructed. It encodes the signal peptide, part of the immunoglobulin (IgG)-like domain, the transmembrane anchor of vaccinia virus (VV) hemagglutinin (HA), and the complete E7-coding sequence. The fusion gene was expressed under the HA late promoter by a recombinant VV, designated VV-E7-HA. The E7-HA protein was displayed on the surface of cells infected with the recombinant virus and was more stable than unmodified E7. The biological properties of the VV-E7-HA virus were compared with those of a VV-E7 virus that expressed the unmodified E7 and with a VV expressing the Sig-E7-LAMP fusion protein. While the first two of these recombinants were based on VV strain Praha, the third was derived from the WR strain of VV. Infection of mice with the VV-E7-HA virus induced the formation of E7-specific antibodies with the predominance of the IgG2a isotype, whereas the other two viruses did not induce the formation of E7-specific antibodies. Unlike the other two viruses, VV-E7-HA did not induce a response of cytotoxic T lymphocytes or Th1 cells and did not protect mice against the growth of E7-expressing tumors. Thus, VV-E7-HA induced a differently polarized immune response to the E7 protein than the other two viruses.