Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

BACKGROUND: Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. RESULTS: We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i) two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii) the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV) efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39 mutant). CONCLUSION: Our Flip-Flop HSV-BAC system enables rapid generation of HSV vectors carrying transgene inserts. By introducing a tumor-specific-promoter-driven ICP4 cassette into pM24-BAC using this system, one should be able to generate transcriptionally-targeted oncolytic HSV vectors. We believe this system will greatly facilitate the screening of a plethora of clinically useful tumor-specific promoters in the context of oncolytic HSV vectors

Combination of vinblastine and oncolytic herpes simplex virus vector expressing IL-12 therapy increases antitumor and antiangiogenic effects in prostate cancer models

Oncolytic herpes simplex virus-1 (oHSV)–based vectors selectively replicate in tumor cells causing direct killing, ie., oncolysis, while sparing normal cells. oHSV’sare promising anticancer agents, but their efficacy, when used as single agents, leaves room for improvement. We hypothesized that combining the direct oncolytic and antiangiogenic activities of the IL-12 secreting NV1042 oHSV with microtubule disrupting agents (MDA’s) would be an effective means to enhance antitumor efficacy. Vinblastine (VB) was identified among several MDA’s screened that displayed consistent and potent cytotoxic killing of both prostate cancer and endothelial cell lines. In matrigel tube forming assays, VB was found to be highly effective at inhibiting tube formation of HUVEC cells. The combination of VB with NV1023 (the parental virus lacking IL-12) or NV1042 showed additive or synergistic activity against prostate cancer cell lines and was not due to increased oHSV replication by VB. In athymic mice bearing CWR22 prostate tumors, VB in combination with NV1042 was superior to the combination of VB plus NV1023 in reducing tumor burden, appeared to be nontoxic and resulted in a statistically significant diminution in the number of CD31+ cells as compared to other treatment groups. In human organotypic cultures using surgical samples from radical prostatectomies, both NV1023 and NV1042 were localized specifically to the epithelial cells of prostatic glands but not to the surrounding stroma. These data highlight the therapeutic advantage of combining the dual-acting anti-tumor and anti-angiogenic activities of oHSV’s and MDA’s

Modification of Extracellular Matrix Enhances Oncolytic Adenovirus Immunotherapy in Glioblastoma

Purpose: Extracellular matrix (ECM) component hyaluronan (HA) facilitates malignant phenotypes of glioblastoma (GBM), however, whether HA impacts response to GBM immunotherapies is not known. Herein, we investigated whether degradation of HA enhances oncolytic virus immunotherapy for GBM. Experimental design: Presence of HA was examined in patient and murine GBM. Hyaluronidase-expressing oncolytic adenovirus, ICOVIR17, and its parental virus, ICOVIR15, without transgene, were tested to determine if they increased animal survival and modulated the immune tumor microenvironment (TME) in orthotopic GBM. HA regulation of NF-κB signaling was examined in virus-infected murine macrophages. We combined ICOVIR17 with PD-1 checkpoint blockade and assessed efficacy and determined mechanistic contributions of tumor-infiltrating myeloid and T cells. Results: Treatment of murine orthotopic GBM with ICOVIR17 increased tumor-infiltrating CD8+ T cells and macrophages, and upregulated PD-L1 on GBM cells and macrophages, leading to prolonged animal survival, compared with control virus ICOVIR15. High molecular weight HA inhibits adenovirus-induced NF-κB signaling in macrophages in vitro, linking HA degradation to macrophage activation. Combining ICOVIR17 with anti-PD-1 antibody further extended the survival of GBM-bearing mice, achieving long-term remission in some animals. Mechanistically, CD4+ T cells, CD8+ T cells, and macrophages all contributed to the combination therapy that induced tumor-associated proinflammatory macrophages and tumor-specific T-cell cytotoxicity locally and systemically. Conclusions: Our studies are the first to show that immune modulatory ICOVIR17 has a dual role of mediating degradation of HA within GBM ECM and subsequently modifying the immune landscape of the TME, and offers a mechanistic combination immunotherapy with PD-L1/PD-1 blockade that remodels innate and adaptive immune cells

BAZ1B in Nucleus Accumbens Regulates Reward-Related Behaviors in Response to Distinct Emotional Stimuli

ATP-dependent chromatin remodeling proteins are being implicated increasingly in the regulation of complex behaviors, including models of several psychiatric disorders. Here, we demonstrate that Baz1b, an accessory subunit of the ISWI family of chromatin remodeling complexes, is upregulated in the nucleus accumbens (NAc), a key brain reward region, in both chronic cocaine-treated mice and mice that are resilient to chronic social defeat stress. In contrast, no regulation is seen in mice that are susceptible to this chronic stress. Viral-mediated overexpression of Baz1b, along with its associated subunit Smarca5, in mouse NAc is sufficient to potentiate both rewarding responses to cocaine, including cocaine self-administration, and resilience to chronic social defeat stress. However, despite these similar, proreward behavioral effects, genome-wide mapping of BAZ1B in NAc revealed mostly distinct subsets of genes regulated by these chromatin remodeling proteins after chronic exposure to either cocaine or social stress. Together, these findings suggest important roles for BAZ1B and its associated chromatin remodeling complexes in NAc in the regulation of reward behaviors to distinct emotional stimuli and highlight the stimulus-specific nature of the actions of these regulatory proteins

Correlation of pulse wave velocity with left ventricular mass in patients with hypertension once blood pressure has been normalized

Vascular stiffness has been proposed as a simple method to assess arterial loading conditions of the heart which induce left ventricular hypertrophy (LVH). There is some controversy as to whether the relationship of vascular stiffness to LVH is independent of blood pressure, and which measurement of arterial stiffness, augmentation index (AI) or pulse wave velocity (PWV) is best. Carotid pulse wave contor and pulse wave velocity of patients (n=20) with hypertension whose blood pressure (BP) was under control (<140/90 mmHg) with antihypertensive drug treatment medications, and without valvular heart disease, were measured. Left ventricular mass, calculated from 2D echocardiogram, was adjusted for body size using two different methods: body surface area and height. There was a significant (P<0.05) linear correlation between LV mass index and pulse wave velocity. This was not explained by BP level or lower LV mass in women, as there was no significant difference in PWV according to gender (1140.1+67.8 vs 1110.6+57.7 cm/s). In contrast to PWV, there was no significant correlation between LV mass and AI. In summary, these data suggest that aortic vascular stiffness is an indicator of LV mass even when blood pressure is controlled to less than 140/90 mmHg in hypertensive patients. The data further suggest that PWV is a better proxy or surrogate marker for LV mass than AI and the measurement of PWV may be useful as a rapid and less expensive assessment of the presence of LVH in this patient population

Interleukin-1 polymorphisms associated with increased risk of gastric cancer

Helicobacter pylori infection is associated with a variety of clinical outcomes including gastric cancer and duodenal ulcer disease. The reasons for this variation are not clear, but the gastric physiological response is influenced by the severity and anatomical distribution of gastritis induced by H. pylori. Thus, individuals with gastritis predominantly localized to the antrum retain normal (or even high) acid secretion, whereas individuals with extensive corpus gastritis develop hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer. Here we report that interleukin-1 gene cluster polymorphisms suspected of enhancing production of interleukin-1-beta are associated with an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer. Two of these polymorphism are in near-complete linkage disequilibrium and one is a TATA-box polymorphism that markedly affects DNA-protein interactions in vitro. The association with disease may be explained by the biological properties of interleukin-1-beta, which is an important pro-inflammatory cytokine and a powerful inhibitor of gastric acid secretion. Host genetic factors that affect interleukin-1-beta may determine why some individuals infected with H. pylori develop gastric cancer while others do no

Impact of Antiretroviral Therapy on Incidence of Pregnancy among HIV-Infected Women in Sub-Saharan Africa: A Cohort Study

A multicountry cohort study in sub-Saharan Africa by Landon Myer and colleagues reveals higher pregnancy rates in HIV-infected women on antiretroviral therapy (ART)

Epidemiology and awareness of hypertension in a rural Ugandan community: a cross-sectional study

BACKGROUND: Hypertension is one of the largest causes of preventable morbidity and mortality worldwide. There are few population-based studies on hypertension epidemiology to guide public health strategies in sub-Saharan Africa. Using a community-based strategy that integrated screening for HIV and non-communicable diseases, we determined the prevalence, awareness, treatment rates, and sociodemographic factors associated with hypertension in rural Uganda. METHODS: A household census was performed to enumerate the population in Kakyerere parish in Mbarara district, Uganda. A multi-disease community-based screening campaign for hypertension, diabetes, and HIV was then conducted. During the campaign, all adults received a blood pressure (BP) measurement and completed a survey examining sociodemographic factors. Hypertension was defined as elevated BP (≥140/≥90 mmHg) on the lowest of three BP measurements or current use of antihypertensives. Prevalence was calculated and standardized to age distribution. Sociodemographic factors associated with hypertension were evaluated using a log-link Poisson regression model with robust standard errors. RESULTS: Community participation in the screening campaign was 65%, including 1245 women and 1007 men. The prevalence of hypertension was 14.6%; awareness of diagnosis (38.1%) and current receipt of treatment (20.6%) were both low. Age-standardized to the WHO world standard population, hypertension prevalence was 19.8%, which is comparable to 21.6% in the US and 18.4% in the UK. Sociodemographic factors associated with hypertension included increasing age, male gender, overweight, obesity, diabetes, alcohol consumption, and family history. Prevalence of modifiable factors was high: 28.3% women were overweight/obese and 24.1% men consumed ≥10 alcoholic drinks per month. CONCLUSIONS: We found a substantial burden of hypertension in rural Uganda. Awareness and treatment of hypertension is low in this region. Enhanced community-based education and prevention efforts tailored to addressing modifiable factors are needed

Delayed-type hypersensitivity in classic Kaposi sarcoma patients and controls

BACKGROUND: Immune perturbation likely affects the development of Kaposi sarcoma (KS) among people infected with the KS-associated herpesvirus (KSHV). We tested whether KSHV-seropositive individuals or cases of classic KS (cKS), which typically originates in the leg, had differing delayed-type hypersensitivity (DTH) in the forearm or leg. METHODS: Mantoux DTH with three antigens (Candida, tetanus, PPD) was performed on the forearm and leg of 15 cKS cases, 14 KSHV-positives without KS, and 15 KSHV-negative controls. The diameters of induration responses were compared by group and body site. RESULTS: Leg DTH was greater than forearm DTH among controls (mean difference 5.6 mm, P\ubc0.0004), whereas this was not observed in cKS cases ( 2.2 mm, P\ubc0.32) or KSHV-positives (0.5 mm, P\ubc0.56). Leg-minus-forearm DTH difference was greater in controls compared with cKS cases (P\ubc0.004) and KSHV-positives (P\ubc0.002). Leg-plus-forearm DTH was similar in controls (mean 28.2 mm) and cKS cases (24.5 mm, P\ubc0.60), but it was reduced in KSHV-positives (11.8 mm, P\ubc0.02), particularly in the leg (P\ubc0.004) and marginally in the forearm (P\ubc0.07). CONCLUSION: KS cases had weaker DTH only in the leg, whereas both body sites appeared weaker in KSHV-positives without KS. Both systemic and regional immune alterations may influence the development of this malignancy