ORF6 and ORF61 Expressing MVA Vaccines Impair Early but Not Late Latency in Murine Gammaherpesvirus MHV-68 Infection

Gammaherpesviruses (gamma HV) are important pathogens causing persistent infections which lead to several malignancies in immunocompromised patients. Murine gamma HV 68 (MHV-68), a homolog to human EBV and KSHV, has been employed as a classical pathogen to investigate the molecular pathogenicity of gamma HV infections. gamma HV express distinct antigens during lytic or latent infection and antigen-specific T cells have a significant role in controlling the acute and latent viral infection, although the quality of anti-viral T cell responses required for protective immunity is not well-understood. We have generated recombinant modified vaccinia virus Ankara (recMVA) vaccines via MVA-BAC homologous recombination technology expressing MHV-68 ORF6 and ORF61 antigens encoding both MHC class I and II-restricted epitopes. After vaccination, we examined T cell responses before and after MHV-68 infection to determine their involvement in latent virus control. We show recognition of recMVA- and MHV-68-infected APC by ORF6 and ORF61 epitope-specific T cell lines in vitro. The recMVA vaccines efficiently induced MHV-68-specific CD8+ and CD4+ T cell responses after a single immunization and more pronounced after homologous prime/boost vaccination in mice. Moreover, we exhibit protective capacity of prophylactic recMVA vaccination during early latency at day 17 after intranasal challenge with MHV-68, but failed to protect from latency at day 45. Further T cell analysis indicated that T cell exhaustion was not responsible for the lack of protection by recMVA vaccination in long-termlatency at day 45. The data support further efforts aiming at improved vaccine development against gamma HV infections with special focus on targeting protective CD4+ T cell responses

Effect of combined siRNA of HCV E2 gene and HCV receptors against HCV

<p>Abstract</p> <p>Background/Aim</p> <p>Hepatitis C virus (HCV) is a major threat as almost 3% of the world's population (350 million individual) and 10% of the Pakistani population is chronically infected with this virus. RNA interference (RNAi), a sequence-specific degradation process of RNA, has potential to be used as a powerful alternative molecular therapeutic approach in spite of the current therapy of interferon-α and ribavirin against HCV which has limited efficiency. HCV structural gene E2 is mainly involved in viral cell entry via attachment with the host cell surface receptors i.e., CD81 tetraspanin, low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), and Claudin1 (CLDN1). Considering the importance of HCV E2 gene and cellular receptors in virus infection and silencing effects of RNAi, the current study was designed to target the cellular and viral factors as new therapeutic options in limiting HCV infection.</p> <p>Results</p> <p>In this study the potential of siRNAs to inhibit HCV-3a replication in serum-infected Huh-7 cells was investigated by combined treatment of siRNAs against the HCV E2 gene and HCV cellular receptors (CD81 and LDLR), which resulted in a significant decrease in HCV viral copy number.</p> <p>Conclusion</p> <p>From the current study it is concluded that the combined RNAi-mediated silencing of HCV E2 and HCV receptors is important for the development of effective siRNA-based therapeutic option against HCV-3a.</p

Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery

Author summary In order to repel viral infections, cells activate stress responses. One such response involves inhibition of translation and restricted availability of the translation machinery via the formation of stress granules. However, the host translation machinery is absolutely essential for synthesis of viral proteins and consequently viruses have developed a broad spectrum of strategies to circumvent this restriction. Old World alphaviruses, such as Semliki Forest virus (SFV) and chikungunya virus (CHIKV), interfere with stress granule formation by sequestration of G3BP, a stress granule nucleating protein, mediated by the viral non-structural protein 3 (nsP3). Here we show that nsP3:G3BP complexes engage factors of the host translation machinery, which during the course of infection accumulate in the vicinity of viral replication complexes. Accordingly, we demonstrate that the nsP3:G3BP interaction is required for high localized translational activity around viral replication complexes. We find the RGG domain of G3BP to be essential for the recruitment of the host translation machinery. In cells expressing mutant G3BP lacking the RGG domain, SFV replication was attenuated, but detectable, while CHIKV was essentially non-viable. Our data demonstrate a novel mechanism by which viruses can recruit factors of the translation machinery in a G3BP-dependent manner.Peer reviewe

Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option.</p> <p>Results</p> <p>The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and <it>in </it><it>vitro </it>transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed.</p> <p>Conclusions</p> <p>Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype.</p

Characterizing the Inflammatory Profile of Neutrophil-Rich Triple-Negative Breast Cancer

Breast cancer (BC) is one of the most common types of cancer in women in the United Arab Emirates. Immunogenic tumours, such as triple-negative breast cancer (TNBC), show increased neutrophil infiltration, which is associated with poor prognosis and limited efficacy of immunotherapy. This study aims to investigate in vitro the bidirectional effect of neutrophils on metastatic TNBC (MDA-MB-231) compared to less-metastatic luminal breast cancer (MCF-7) cell lines. We found that BC cells or their conditioned medium (CM) reduced the viability of neutrophil-like cells (HL60). This was supported by increased cellular stress and NETosis in differentiated HL60 cells (dHL60) upon exposure to MDA-MB-231 compared to MCF-7-CM using nucleic acid staining essays. Flow cytometry showed comparable expression of inflammatory markers by polymorphonuclear cells (PMN) when treated with MDA-MB-231-CM and standard polarizing cocktails. Furthermore, MDA-MB-231-CM triggered an inflammatory pattern with evidence of stronger adhesion (CD62L) and degranulation (CD11b and CD66b) phenotypes. The proinflammatory polarization of dHL60 by MDA-MB-231-CM was additionally confirmed by the elevated CD54 expression, myeloperoxidase, and CD11b protein levels, which matched an increased transwell migratory capacity. In conclusion, BC might use neutrophils to their benefit through NETosis and complement system activation, which makes this crosstalk a potential mechanism for understanding tumour progression