Nimbolide Inhibits SOD2 to Control Pancreatic Ductal Adenocarcinoma Growth and Metastasis.

Reactive oxygen species are frequently associated with various cancers including pancreatic ductal adenocarcinomas (PDACs). Superoxide dismutase 2 (SOD2) is an enzyme that plays an important role in reactive oxygen species (ROS) signaling. Investigating the molecular function and biological functions of SOD2 can help us develop new therapeutic options and uncover new biomarkers for PDAC diagnosis and prognosis. Here, we show that nimbolide (NB), a triterpene limonoid, effectively blocks the growth and metastasis of PDACs by suppressing the expression and activity of SOD2. To identify the role of SOD2 in NB-induced anticancer activity, we used RNA interference to silence and plasmid transfection to overexpress it. Silencing SOD2 significantly reduced the growth and metastatic characteristics like epithelial-to-mesenchymal transition, invasion, migration, and colony-forming capabilities of PDACs, and NB treatment further reduced these characteristics. Conversely, the overexpression of SOD2 enhanced these metastatic characteristics. ROS signaling has a strong feedback mechanism with the PI3K/Akt signaling pathway, which could be mediated through SOD2. Finally, NB treatment to SOD2-overexpressing PDAC xenografts resulted in significant inhibition of tumor growth and metastasis. Overall, this work suggests that NB, a natural and safe phytochemical that silences SOD2 to induce high levels of ROS generation, results in increased apoptosis and reduced growth and progression of PDACs. The role of SOD2 in regulating NB-induced ROS generation presents itself as a therapeutic option for PDACs.

Phytochemicals as an Alternative or Integrative Option, in Conjunction with Conventional Treatments for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most abundant form of liver cancer. It accounts for 75-85% of liver cancer cases and, though it ranks globally as the sixth most common cancer, it ranks second in cancer-related mortality. Deaths from HCC are usually due to metastatic spread of the cancer. Unfortunately, there are many challenges and limitations with the latest HCC therapies and medications, making it difficult for patients to receive life-prolonging care. As there is clearly a high demand for alternative therapy options for HCC, it is prudent to turn to plants for the solution, as their phytochemicals have long been used and revered for their many medicinal purposes. This review explores the promising phytochemical compounds identified from pre-clinical and clinical trials being used either independently or in conjunction with already existing cancer therapy treatments. The phytochemicals discussed in this review were classified into several categories: lipids, polyphenols, alkaloids, polysaccharides, whole extracts, and phytochemical combinations. Almost 80% of the compounds failed to progress into clinical studies due to lack of information regarding the toxicity to normal cells and bioavailability. Although large obstacles remain, phytochemicals can be used either as an alternative or integrative therapy in conjunction with existing HCC chemotherapies. In conclusion, phytochemicals have great potential as treatment options for hepatocellular carcinoma.

Enhancement of Zika virus infection by antibodies from West Nile virus seropositive individuals with no history of clinical infection.

BACKGROUND: Recent outbreaks of Zika Virus (ZIKV) infection and associated microcephaly has raised multiple scientific questions. The close antigenic relatedness between flaviviruses makes diagnosis of specific infection difficult. This relatedness also raises the potential of Antibody Dependent Enhancement (ADE) via cross reactive antibodies to flaviviruses like West Nile Virus (WNV) and Dengue Virus (DENV). Asymptomatic WNV infections are endemic throughout the US creating a large proportion of the population that is seropositive for WNV antibodies. Whether these sero-positive individuals potentially carry ZIKV enhancing antibodies remains unknown. RESULTS: Serum samples obtained from human subjects with symptomatic or asymptomatic WNV infection from a WNV endemic region in Texas were tested for their ability to enhance or neutralize ZIKV infection. Sero-surveillance data demonstrated a ~ 7% prevalence for WNV antibodies in the population. Sera from both symptomatic and asymptomatic WNV seropositive donors effectively neutralized WNV and to some extent DENV infection. Interestingly, WNV+ sera failed to inhibit ZIKV while significantly enhancing infection. Conversely, ZIKV specific sera effectively neutralized ZIKV, with ADE only evident at lower concentrations. The enhancement of ZIKV via WNV antibody positive sera was likely due to non-neutralizing Envelope (E) antibodies as seen with monoclonal ZIKV E antibodies. CONCLUSIONS: Overall, our findings suggest that WNV antibodies in the sera significantly enhance ZIKV infection in Fc receptor positive cells with limited neutralization activity. Further studies in more relevant models of ADE will be needed to confirm the relevance of these findings in vivo.

Combination gene therapy for HIV using a conditional suicidal gene with CCR5 knockout.

BACKGROUND: Gene therapy approaches using hematopoietic stem cells to generate an HIV resistant immune system have been shown to be successful. The deletion of HIV co-receptor CCR5 remains a viable strategy although co-receptor switching to CXCR4 remains a major pitfall. To overcome this, we designed a dual gene therapy strategy that incorporates a conditional suicide gene and CCR5 knockout (KO) to overcome the limitations of CCR5 KO alone. METHODS: A two-vector system was designed that included an integrating lentiviral vector that expresses a HIV Tat dependent Thymidine Kinase mutant SR39 (TK-SR39) and GFP reporter gene. The second non-integrating lentiviral (NIL) vector expresses a CCR5gRNA-CRISPR/Cas9 cassette and HIV Tat protein. RESULTS: Transduction of cells sequentially with the integrating followed by the NIL vector allows for insertion of the conditional suicide gene, KO of CCR5 and transient expression of GFP to enrich the modified cells. We used this strategy to modify TZM cells and generate a cell line that was resistant to CCR5 tropic viruses while permitting infection of CXCR4 tropic viruses which could be controlled via treatment with Ganciclovir. CONCLUSIONS: Our study demonstrates proof of principle that a combination gene therapy for HIV is a viable strategy and can overcome the limitation of editing CCR5 gene alone.

Virus Like Particles (VLP) as multivalent vaccine candidate against Chikungunya, Japanese Encephalitis, Yellow Fever and Zika Virus.

Mosquito borne viral diseases are an emerging threat as evident from the recent outbreak of Zika virus (ZIKV) as well as repeated outbreaks of Chikungunya (CHIKV), Yellow fever (YFV) and Japanese encephalitis (JEV) virus in different geographical regions. These four arboviruses are endemic in overlapping regions due to the co-prevalence of the transmitting mosquito vector species Aedes and Culex. Thus, a multivalent vaccine that targets all four viruses would be of benefit to regions of the world where these diseases are endemic. We developed a potential Virus Like Particle (VLP) based multivalent vaccine candidate to target these diseases by using stable cell lines that continuously secrete VLPs in the culture supernatants. Moreover, inclusion of Capsid in the VLPs provides an additional viral protein leading to an enhanced immune response as evident from our previous studies with ZIKV. Immunization of Balb/c mice with different combinations of Capsid protein containing VLPs either as monovalent, bivalent or tetravalent formulation resulted in generation of high levels of neutralizing antibodies. Interestingly, the potential tetravalent VLP vaccine candidate provided strong neutralizing antibody titers against all four viruses. The 293 T stable cell lines secreting VLPs were adapted to grow in suspension cultures to facilitate vaccine scale up. Our stable cell lines secreting individual VLPs provide a flexible yet scalable platform conveniently adaptable to different geographical regions as per the need. Further studies in appropriate animal models will be needed to define the efficacy of the multivalent vaccine candidate to protect against lethal virus challenge.

Capsid containing virus like particle vaccine against Zika virus made from a stable cell line.

Zika virus infection during pregnancy is associated with severe birth defects including microcephaly in the new born. The lack of specific treatment calls for the development of a safe and effective vaccine for use in pregnant women. We recently tested the efficacy of a Virus Like Particle (VLP) vaccine for Zika virus in mice and found that Capsid-preMembrane-Env (CprME) VLPs generated a better neutralizing antibody response than preMembrane-Env (prME) VLPs. The superiority of CprME VLPs suggested that inclusion of capsid in the vaccine may enhance the immune response. However, production of CprME VLPs requires co-expression of NS2B-3 protease, which creates a major hurdle for generation of stable cell lines. To overcome this limitation, we generated a bicistronic vector that expresses CprME and NS2B-3 using an IRES sequence. This bicistronic expression cassette, in a lentiviral vector, was used to create a stable cell line that constitutively secretes CprME VLPs. The expression of NS2B-3, presence of capsid in the secreted VLPs, efficiency of VLP release, and stability of the cell line was extensively tested. Antigen sparing studies in mice using prME and CprME VLPs, both derived from stable cell lines, confirmed the superiority of CprME VLPs in generation of neutralizing antibody response. Capsid specific antibodies were detected in CprME VLP immunized mice providing mechanistic insights into the superiority of these VLPs. Challenge of CprME VLP immunized mice with Zika PRVABC59 showed complete protection against day 3 viremia further validating the efficacy of the vaccine. Our study is the first to generate a stable cell line secreting Zika CprME VLPs via natural NS2B-3 cleavage, demonstrate incorporation of capsid in CprME VLPs and complete protection in challenge studies. This is a major advancement for the Zika vaccine platform that is safe for use in pregnant women and readily scalable for use in developing countries.

Differential Pathogenicity of SHIV KB9 and 89.6 Env Correlates with Bystander Apoptosis Induction in CD4+ T cells.

SHIV variants KB9 and 89.6 show differential pathogenesis in primate models with KB9 causing rapid CD4 decline while 89.6 failing to induce disease. We attempted to determine whether the differential pathogenicity of KB9 versus 89.6 was a result of differential bystander apoptosis inducing potential (AIP) of the Env glycoproteins from these viruses. We find that the KB9 Env was highly potent at inducing bystander apoptosis in CD4+ target cells compared to 89.6 Env. Cell death induction by KB9 showed classical signs of apoptosis including mitochondrial depolarization, caspase activation and PARP cleavage. Inhibiting Env mediated fusion by T20 peptide inhibited KB9 mediated bystander apoptosis. KB9 and 89.6 differed in terms of co-receptor usage with 89.6 preferring CXCR4 while KB9 using both CXCR4 and CCR5 with equal efficiency. Our study suggests that higher bystander AIP of KB9 Env compared to 89.6 may be the basis for the differential pathogenesis of these viruses.

Anti-Zika Activity of a Library of Synthetic Carbohydrate Receptors.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders. Currently, there are no antiviral therapies that have been specifically approved to treat ZIKV, and there is an urgent need to develop effective anti-ZIKV agents. Here, we report anti-ZIKV activity of 16 synthetic carbohydrate receptors (SCRs) that inhibit ZIKV infection in Vero and HeLa cells. Using a ZIKV reporter virus particle-based infection assay, our data demonstrates these SCRs are highly potent with IC50s as low as 0.16 µM and negligible toxicity at several-fold higher concentrations. Time-of-addition studies showed that these SCRs inhibit the early stages of the virus infection, which is consistent with the proposed mode of action, where the SCRs likely inhibit binding between the virus and cell-surface glycans, thereby preventing viral entry into the cells and, as such, this study demonstrates a potential new strategy against ZIKV.

TLR9 polymorphism correlates with immune activation, CD4 decline and plasma IP10 levels in HIV patients.

BACKGROUND: The mechanism behind HIV mediated immune activation remains debated, although the role of virus replication in this process is increasingly evident. Toll like Receptor 9 (TLR9) has been implicated in HIV mediated immune activation via sensing of viral CpG DNA. Polymorphisms in the TLR9 gene and promoter region including TLR9 1635A/G and 1486C/T have been found to be associated with multiple infectious diseases and cancers. METHODS: In the current study, we looked at the correlation of TLR9 polymorphisms 1635A/G and 1486C/T with key hallmarks of HIV disease in a cohort of 50 HIV infected patients. We analyzed CD4 counts, T cell immune activation characterized by upregulation of CD38 and HLA-DR and upregulation of plasma biomarkers of inflammation like LPS, sCD14, IL-6 and IP10 in the HIV patient cohort and compared it to healthy controls. RESULTS: We found that TLR9 1635AA genotype was associated with lower CD4 counts and significantly higher immune activation in both CD4+ and CD8+ T cells. Analysis of HIV associated plasma biomarkers including LPS, sCD14, IL-6 and IP10 revealed a strong correlation between IP10 and immune activation. Interestingly, IP10 levels were also found to be higher in HIV patients with the 1635AA genotype. Furthermore, the TLR9 1486C/T polymorphism that is in linkage disequilibrium with 1635A/G was weakly associated with lower CD4 counts, higher CD8 immune activation and higher IP10 levels. CONCLUSIONS: As TLR9 stimulation is known to induce IP10 production by dendritic cells, our findings provide new insights into HIV mediated immune activation and CD4 loss. TLR9 stimulation by viral CpG DNA may be important to HIV immunopathogenesis and the TLR9 polymorphisms 1635A/G and 1486C/T may be associated with disease progression.

Recent Advances in Zika Virus Vaccines.

The recent outbreaks of Zika virus (ZIKV) infections and associated microcephaly in newborns has resulted in an unprecedented effort by researchers to target this virus. Significant advances have been made in developing vaccine candidates, treatment strategies and diagnostic assays in a relatively short period of time. Being a preventable disease, the first line of defense against ZIKV would be to vaccinate the highly susceptible target population, especially pregnant women. Along those lines, several vaccine candidates including purified inactivated virus (PIV), live attenuated virus (LAV), virus like particles (VLP), DNA, modified RNA, viral vectors and subunit vaccines have been in the pipeline with several advancing to clinical trials. As the primary objective of Zika vaccination is the prevention of vertical transmission of the virus to the unborn fetus, the safety and efficacy requirements for this vaccine remain unique when compared to other diseases. This review will discuss these recent advances in the field of Zika vaccine development.