Human papillomavirus prevalence among unvaccinated young female college students in Botswana: A cross-sectional study.

BACKGROUND: Human papillomavirus (HPV) is a sexually transmitted infection and a causative agent of cervical cancer. It is common in adolescent girls and young women, and the majority of infections are transient and asymptomatic. In Botswana, there are currently no data on the HPV prevalence against which the impact of prophylactic HPV vaccines can be measured. OBJECTIVES: To establish a baseline HPV prevalence in an unvaccinated cohort of young women. METHODS: Women aged ≥18 years were recruited from the University of Botswana between September 2016 and May 2020. Demographic and behavioural characteristics of participants were collected. Subsequently, cervicovaginal swabs were obtained and tested for HPV using polymerase chain reaction-restriction fragment length polymorphism. We determined the prevalent HPV types, and evaluated the risk factors associated with HPV positivity. RESULTS: A total of 978 young women were recruited. Overall, there were 589 (60.2%) participants with HPV infection and 12 (1.2%) with HIV. The median (interquartile range) age of the study participants was 19 (18 - 20) years. Multivariate logistic regression analysis showed that significant factors associated with HPV positivity were sexual activity (adjusted odds ratio (aOR) 2.06; 95% confidence interval (CI) 1.49 - 2.63; p<0.001), number of sex partners ≥3 (aOR 2.10; 95% CI 1.39 - 3.18; p<0.001), and smoking (aOR 2.00; 95% CI 1.26 - 3.20; p=0.004). CONCLUSION: Our results demonstrate for the first time the prevalence of HPV in unvaccinated young women in Botswana. We found a high prevalence of HPV infection, with statistical differences with different risk factors. This finding supports the need for HPV vaccination strategies for females prior to sexual debut to reduce the future burden of cervical cancer in Botswana.

EBV PCR in the diagnosis and monitoring of posttransplant lymphoproliferative disorder: results of a two-arm prospective trial.

While EBV PCR is used in the management of PTLD, the optimal primer set, relative importance of intracellular versus free plasma EBV, and the baseline profile in an organ transplant population remains unclear. We performed a prospective 2-arm trial utilizing an EBV PCR panel measuring LMP-1, EBER-1 and EBNA-1 in both free plasma as well as intracellular whole blood. Control Arm A consisted of 31 lung transplant patients and Arm B consisted of 35 transplant patients being evaluated for possible PTLD. In Arm A, 1/31 (3%) patients developed a transient plasma EBV load. Thirteen of 31 (42%) had detectable intracellular EBV. In Arm B, 17 (49%) patients were diagnosed with PTLD. Thirteen (76%) had EBV-positive PTLD with 12/13 (92%) having detectable EBV by PCR. The EBV PCR panel had a high sensitivity (92%), specificity (72%), positive predictive value (PPV) (71%) and negative predictive value (NPV) (93%) for diagnosing EBV-positive PTLD and followed patients' clinical course well (p < 0.001). Comparing the individual PCR assays, plasma EBNA PCR was superior with high sensitivity (77%), specificity (100%), PPV (100%) and NPV (86%). We conclude that EBV PCR is a useful test for managing PTLD patients. While plasma EBNA PCR is the best single assay for diagnosing and monitoring PTLD, the complete PCR panel is superior for ruling out its presence.

The latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus activates two major essential Epstein-Barr virus latent promoters.

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the -204 to +40 region had increased levels of activation compared with a larger region, -512 to +40, which contains two recombination signal-binding protein J kappa binding sites. The smaller, -204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the -204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.

An Epstein-Barr virus isolated from a lymphoblastoid cell line has a 16-kilobase-pair deletion which includes gp350 and the Epstein-Barr virus nuclear antigen 3A.

Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.

Latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus interacts with Tat and activates the long terminal repeat of human immunodeficiency virus type 1 in human cells.

The latency-associated nuclear antigen (LANA) is constitutively expressed in cells infected with the Kaposi's sarcoma (KS) herpesvirus (KSHV), also referred to as human herpesvirus 8. KSHV is tightly associated with body cavity-based lymphomas (BCBLs) in immunocompromised patients infected with human immunodeficiency virus (HIV). LANA, encoded by open reading frame 73 of KSHV, is one of a small subset of proteins expressed during latent infection and was shown to be important in tethering the viral episome to host chromosomes. Additionally, it has been shown that LANA can function as a regulator of transcription. However, its role in the progression of disease is still being elucidated. Since KS is one of the most common AIDS-associated cancers in the United States and BCBLs appear predominantly in AIDS patients, we examined whether LANA is able to regulate the HIV type 1 (HIV-1) long terminal repeat (LTR). Using luciferase-based transient transfection assays, we found that LANA was able to transactivate the HIV-1 LTR in the human B-cell line BJAB, human monocytic cell line U937, and the human embryonic kidney fibroblast cell line 293T. Moreover, we observed that the virus-encoded HIV transactivator protein Tat cooperated with LANA in activation of the LTR in a dose-response fashion with increasing amounts of LANA. Surprisingly, LANA alone was sufficient to transactivate the HIV-1 LTR in BJAB cells. In similar assays using a HIV-1 LTR construct with the core enhancer elements deleted; the activity of LANA was diminished but not abolished, indicating a mechanism which involves the cooperation of the core enhancer elements and downstream elements which include Tat. Furthermore, transient transfection of an infectious clone of HIV with LANA demonstrated effects similar to those seen in the reporter assays based on Western blot analysis of HIV Gag polypeptide p24. Interestingly, we also demonstrated that the carboxy terminus of LANA associates with Tat in cells and in vitro. These experiments suggest a role for LANA in activating the HIV-1 LTR through association with cellular molecules targeting the core enhancer elements and Tat and may have important consequences in increasing the levels of HIV in infected individuals and, hence, the disease state.

The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus transactivates the telomerase reverse transcriptase promoter.

Telomerase is a multi-subunit ribonucleoprotein holoenzyme that stabilizes telomere length through the addition of new repeat sequence to the ends of chromosomes. Telomerase reverse transcriptase is the subunit of this complex responsible for the enzymatic activity of telomerase. Expression of the reverse transcriptase is regulated at the level of transcription through the action of transcription factors that target its promoter. Most Kaposi's sarcoma tumor cells are latently infected with the Kaposi's sarcoma-associated herpesvirus, and the constitutive expression of a viral-encoded latency-associated nuclear antigen has been shown to be important for the maintenance of the viral episome. The proliferative nature of Kaposi's sarcoma suggests that this antigen may also play a critical role in viral-mediated oncogenesis. In this study telomerase reverse transcriptase promoter elements cloned into a luciferase reporter plasmid were analyzed to determine the ability of the latency-associated nuclear antigen to regulate transcription. The latency-associated nuclear antigen transactivated the full-length promoter in 293T, 293, and BJAB cell lines. Furthermore, truncation promoter studies implicated sequence from -130 to +5 in viral-mediated activation. This region contains five Sp1 transcription factor-binding sites. Electrophoretic mobility shift assays indicated that the latency-associated nuclear antigen targets and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.

Epstein-Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: a molecular link to cancer metastasis.

Epstein-Barr virus (EBV) is an oncogenic virus associated with a number of human malignancies including Burkitt lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease and, though still debated, breast carcinoma. A subset of latent EBV antigens is required for mediating immortalization of primary B-lymphocytes. Here we demonstrate that the carboxy-terminal region of the essential latent antigen, EBNA-3C, interacts specifically with the human metastatic suppressor protein Nm23-H1. Moreover, EBNA-3C reverses the ability of Nm23-H1 to suppress the migration of Burkitt lymphoma cells and breast carcinoma cells. We propose that EBNA-3C contributes to EBV-associated human cancers by targeting and altering the role of the metastasis suppressor Nm23-H1.

Prothymosin alpha functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro.

Prothymosin alpha (ProTalpha), a cellular molecule known to be associated with cell proliferation, is transcriptionally up-regulated on expression of c-myc and interacts with histones in vitro and associates with histone H1 in cells. Previous studies have also shown that ProTalpha is involved in chromatin remodeling. Recent studies have shown that ProTalpha interacts with the acetyl transferase p300 and an essential Epstein-Barr virus protein, EBNA3C, involved in regulation of viral and cellular transcription. These studies suggest a potential involvement in regulation of histone acetylation through the association with these cellular and viral factors. In the current studies, we show that heterologous expression of ProTalpha in the Rat-1 rodent fibroblast cell line results in increased proliferation, loss of contact inhibition, anchorage-independent growth, and decreased serum dependence. These phenotypic changes seen in transfected Rat-1 cells are similar to those observed with a known oncoprotein, Ras, expressed under the control of a heterologous promoter and are characteristic oncogenic growth properties. These results demonstrate that the ProTalpha gene may function as an oncogene when stably expressed in Rat-1 cells and may be an important downstream cellular target for inducers of cellular transformation, which may include Epstein-Barr virus and c-myc.

The Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen binds to specific sequences at the left end of the viral genome through its carboxy-terminus.

Latent infection by members of the gammaherpesvirus family is typically characterized by stable episomal maintenance of genomic viral DNA. In the case of Epstein--Barr virus (EBV), this is dependent upon binding of the Epstein-Barr nuclear antigen 1 (EBNA1) to sites which lie within the origin of plasmid replication (OriP). The recently discovered Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the latency-associated nuclear antigen (LANA), which appears to be important for supporting the latent infection of human cells by KSHV. The present work describes site-specific binding of the LANA protein to multiple different elements at the left end of the genome, a region which appears to be critical for maintenance of KSHV episomes. Of the three sites, terminal LANA-binding region 4 (TLBR4) binds LANA with the highest affinity when compared to the other sites. Further characterization of this cis-acting element by mutagenesis studies indicates that the minimal TLBR4-binding sequence is represented by a 13-bp sequence 5prime prime or minute CGCCCGGGCATGG 3prime prime or minute. Furthermore, this specific binding to TLBR4 was mediated by the distal 200 amino acid C-terminus of the LANA protein.

Modulation of histone acetyltransferase activity through interaction of epstein-barr nuclear antigen 3C with prothymosin alpha.

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for EBV-dependent immortalization of human primary B lymphocytes. Genetic analysis indicated that amino acids 365 to 992 are important for EBV-mediated immortalization of B lymphocytes. We demonstrate that this region of EBNA3C critical for immortalization interacts with prothymosin alpha (ProTalpha), a cellular protein previously identified to be important for cell division and proliferation. This interaction maps to a region downstream of amino acid 365 known to be involved in transcription regulation and critical for EBV-mediated transformation of primary B lymphocytes. Additionally, we show that EBNA3C also interacts with p300, a cellular acetyltransferase. This interaction suggests a possible role in regulation of histone acetylation and chromatin remodeling. An increase in histone acetylation was observed in EBV-transformed lymphoblastoid cell lines, which is consistent with increased cellular gene expression. These cells express the entire repertoire of latent nuclear antigens, including EBNA3C. Expression of EBNA3C in cells with increased acetyltransferase activity mediated by the EBV transactivator EBNA2 results in down-modulation of this activity in a dose-responsive manner. The interactions of EBNA3C with ProTalpha and p300 provide new evidence implicating this essential EBV protein EBNA3C in modulating the acetylation of cellular factors, including histones. Hence, EBNA3C plays a critical role in balancing cellular transcriptional events by linking the biological property of mediating inhibition of EBNA2 transcription activation and the observed histone acetyltransferase activity, thereby orchestrating immortalization of EBV-infected cells.

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.

Intracellular forms of human NOTCH1 interact at distinctly different levels with RBP-jkappa in human B and T cells.

The cellular transcriptional repressor RBP-Jkappa associates with the Epstein-Barr virus nuclear antigens (EBNAs) determined to be essential for transformation of human primary B lymphocytes. It was demonstrated through genetic analysis that interaction between the viral transactivator EBNA2 and RBP-Jkappa is essential for EBV immortalization of primary B lymphocytes. We have shown that the association of RBP-Jkappa with intracellular NOTCH1 differs significantly in B and T cells. Immunoprecipitation analyses with antibodies to both the intracellular forms of NOTCH1 and to RBP-Jkappa demonstrated that little or no RBP-Jkappa is associated with NOTCH1 in B cell lines compared to the RBP-Jkappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-Jkappa in vitro. Northern blot for the cellular gene hairy enhancer of split (HES1) demonstrated that HES1 is upregulated in the EBV transformed lymphoblastoid cells expressing high levels of EBNA2 and in a T cell line SupT1 overexpressing intracellular activated NOTCH1. Hence, EBNA2 may be able to compete for the available pool of RBP-Jkappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to potently and efficiently infect and immortalize human B cells. Leukemia (2000) 14, 84-92.

The latency-associated nuclear antigen tethers the Kaposi's sarcoma-associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells.

Viruses that establish latent infection must maintain their DNA in the host nucleus through many cellular generations. Here we identify a novel mechanism by which the gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) may achieve this persistence in latently infected body cavity-based lymphoma (BCBL) cells. We find that KSHV genomic DNA is associated with host chromosomes and colocalizes with the latency-associated nuclear antigen (LANA). Furthermore, a region at the left end of the KSHV genome binds strongly to LANA and can colocalize to the host chromosomes with LANA. Additionally, we found that LANA associates with histone H1 in KSHV-infected BCBL cells. We propose that this chromosomal association of the KSHV genome is mediated by LANA and involves a tethering mechanism by which viral episomes are linked to host chromatin through simultaneous interaction with host chromosomal proteins including histone H1 and cis-acting KSHV DNA elements. This strategy may be employed by other viruses in establishment of latency in the infected cells.

Characterization of intertypic recombinants of the Epstein-Barr virus from the body-cavity-based lymphomas cell lines BC-1 and BC-2.

Epstein-Barr Virus (EBV) can infect and transform human B-lymphocytes and has been associated with numerous human malignancies. Two distinct types of EBV have been described, EBV-1 and EBV-2. Whereas type 1 is known to be most widespread throughout the healthy adult population, type 2 EBV has been shown to be significantly present in certain T-cell immunocompromised patients. Some evidence also suggests that such immune impairment promotes coinfection with multiple strains of EBV and fosters the development of intertypic recombinant viruses. In this work, we have analyzed two established body-cavity-based lymphoma or primary effusion lymphoma cell lines, BC-1 and BC-2, for the presence of intertypic EBV recombinants. Using PCR primers to amplify across several markers in the genome, we have typed the BC-1 and BC-2 EBV at these loci. Immunoblot analysis of the EBNA1 protein expressed by these cell lines also suggests a change in EBV typing at this locus in these genomes. Additionally, we have analyzed the expression patterns of the latent EBNA proteins from these viruses and performed Southern blot analysis of the BamHI- and EcoRI-digested genomes to detect variations occurring from type I and II genomes. On the basis of these data, we suggest that the genomes of EBV in BC-1 and BC-2 are intertypic recombinants of type 1 and type 2 EBV genomes. This work corroborates other reports that intertypic EBV recombinants occur in the immunocompromised population. It is likely that intertypic recombination is a mechanism by which novel variants of EBV emerge having selective advantages over a strictly type 1 or type 2 strain.

Distinct patterns of viral antigen expression in Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus coinfected body-cavity-based lymphoma cell lines: potential switches in latent gene expression due to coinfection.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens approximately 110 and 41-42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.

Molecular genetic analysis of herpesviruses and their potential use as vectors for gene therapy applications.

Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses. Some of these cells include neuronal cells and B-lymphocytes infected with herpes simplex virus (HSV) and Epstein-Barr virus (EBV), respectively as well as human T-lymphocytes with herpesvirus saimiri. These large DNA viruses have the potential to deliver fragments of human heterologous DNA > 150 kbp to specific cells. Additionally, EBV recombinants can maintain these large pieces of DNA in the infected B-cells as episomal DNA. The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 150 kbp and to infect cells that can stably maintain DNA as episomes. Moreover, other herpesvirus vector systems can be utilized as a source for the propagation of specific DNA fragments of interest, including wild-type genes to correct genetic defects in human cells. Other herpesvirus systems include defective infectious single cycle (DISC-HSV) mutants of HSV, which are now undergoing trials for the treatment of genital herpesvirus infections and certain types of cancers. The use of herpesviruses as an agent to deliver heterologous gene products has the potential to make a significant difference to the development of therapeutic approaches to human diseases.

Technology evaluation: DISC.

Cantab is developing its DISC technology as a potential gene therapy product for cancer (DISC-Onc) and neurological and blood disorders (DISC-GT). Clinical trials are expected to commence in early 1999 [296831]. The DISC technology utilizes a herpes virus that has had a gene removed to prevent it from replicating [250526]. Phase I trials in leukemia were scheduled to commence in 1998 [250526], however, it was decided that although DISC-Onc is capable of carrying genes into leukemic cells, the levels of immunomodulator genes did not meet the target initially set for the commencement of trials. Hence, Cantab turned its attention to other cancers, and hoped to identify an alternative target for phase I trials in the first half of 1998 [279798]. Additional preclinical work using a murine version of the lead construct produced a significant therapeutic effect in animal tumor models [289716]. DISC-Onc is envisaged to deliver immunogenic genes such as cytokine or stimulatory protein genes [275129]. Cantab, in collaboration with Nottingham Trent University and Birmingham University, has shown that the DISC-Onc has delivered genes effectively to human colorectal, gastric and ovarian cancer tumors. Transfection rates have been shown to be favorable and have been proven to be at least as good as, if not better than, other vectors [279798]. Also, DISC-Onc carrying a functional GM-CSF, has antitumor activity in mouse models of renal cancer and leukemia [250526], [261768]. The DISC Neurology technology (DISC-GT), for gene therapy of neurological disease, is being developed in collaboration with Cambridge University, and enables HSV-driven long-term gene expression in nerve cells [279798].

Prevention of acute allograft rejection by antibody targeting of TIRC7, a novel T cell membrane protein.

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

Oncogenic forms of NOTCH1 lacking either the primary binding site for RBP-Jkappa or nuclear localization sequences retain the ability to associate with RBP-Jkappa and activate transcription.

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (DeltaE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jkappa response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jkappa in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jkappa-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jkappa-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jkappa, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, DeltaE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound DeltaE. Transcriptional activation by this mutant form of membrane-bound DeltaE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jkappa at promoter sites.