Modeling of Humoral Immune Response to Repeated Influenza A Virus Infections

Seasonal infections by Influenza A virus (IAV) causes hundreds of thousands of deaths worldwide each year, with most individuals being infected multiple times throughout their lifetimes. The relative impact of the components of the host immune system in controlling the severity and duration of repeated challenges from an IAV infection remains unclear. In particular, the differential contribution of the humoral immune response in primary and secondary challenges from IAV are relatively little explored. We develop a parsimonious mathematical model of the humoral immune response to IAV infection with biologically meaningful and identifiable parameters. We show the relative sensitivity of the viral load and antibody response to dynamics of B cell proliferation and antibody production. We relate immunoglobin class switching to a CD4+ T-cell driven process for the formation of humoral memory. Results of this study help to illuminate the relative contribution of CD4+ T-cells, B-cells, and antibody in the control of IAV infection and formation of humoral memory

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.<br /

Antitumor Immunity and Dietary Compounds

The mechanisms by which natural dietary compounds exert their antitumor effects have been the focus of a large number of research efforts in recent years. Induction of apoptosis by inhibition of cell proliferative pathways is one of the common means of cell death employed by these dietary compounds. However, agents that can activate an antitumor immune response in addition to a chemotherapeutic effect may be useful adjuvants or alternative therapies for the treatment of cancer. The focus of this review is to highlight representative dietary compounds, namely Withania somnifera, Panax ginseng, curcumin and resveratrol with special emphasis on their antitumor immune mechanism of action. Each of these dietary compounds and their sources has a history of safe human use as food or in herbal medicine traditions, potentially making them ideal therapeutics. Here we report the recent advances in the cellular immune mechanisms utilized by these compounds to induce antitumor immunity. Taken together, these findings provide a new perspective for exploiting novel dietary compounds as chemoimmunotherapeutic anti-cancer agents

The Hypoxia Inducible Factor 1 Alpha is Required for Optimal GammaHerpes Virus Lytic Replication and Reactivation from Latency

Lytic replication of Kaposi's Sarcoma Associated Herpes Virus (KSHV) exacerbates Kaposi's sarcoma (KS) progression, an angiogenic spindle‐cell sarcoma associated with AIDS. Current antiviral therapy targeting lytic replication can prevent KS in seropositive patients. KSHV lytic infection is not manifested in vitro, and currently there are no animal models that display pathogenic phenotypes. In this study, we employed a biological and genetically mouse pathogen similar to KSHV, the murine gammaherpes virus (gHV) 68 (MHV68), that readily infects laboratory mice providing a valuable small animal model. Moreover, MHV68 in vitro infection exhibits a default lytic infection. Several studies have shown that during infection KSHV employs several viral proteins to de‐regulate the host transcription factor hypoxia inducible factor 1 alpha (HIF1α) and KSHV‐induced activation of HIF1α is necessary for viral persistence and KS progression. However, little is known about the role of HIF1 α in gHV replication and pathogenesis. To further investigate the role of this host factor, we induced transcriptional inactivation of HIF1α in MHV68 virus‐specific cells in vivo. Conditional knock out of HIF1α was achieved by infecting transgenic mice with Cre‐recombinase (Cre) LoxP specific site within the functional domain of HIF1α gene using a recombinant MHV68 that expressing a CMV driven Cre expression. Our data demonstrated for the first time that HIF1α inactivation during gammaherpes virus infection in vivo affects viral gene transcription resulting in impaired virus expansion and early clearance of acute infection in a mouse model. In addition, our results from in vitro MHV68 lytic infection of mouse fibroblasts lacking HIF1α showed that lytic virus production and transcription is decreased. During latency establishment in vivo, the frequency of MHV68 latent splenocytes undergoing latent to lytic replication was largely decreased as well. Our data suggest that HIF1α is required for sustained acute infection since it's deletion in virus‐infected cells resulted in early clearance of productive infection and impairment in reactivation. Moreover, HIF1α is required for mRNA upregulation of glycolytic enzymes during MHV68 lytic infection suggesting a role for HIF1a as a modulator of cell energetics and viral gene expression. We conclude that gammaherpes viruses required the function of the cellular host factor HIF1α in order to effectively replicate and establish latency within its host. Support or Funding Information National Cancer Institute 3R01CA136387‐08S1 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal

Replication and interaction of herpes simplex virus and human papillomavirus in differentiating host epithelial tissue

AbstractWe have investigated the interactions and consequences of superinfecting and coreplication of human papillomavirus (HPV) and herpes simplex virus (HSV) in human epithelial organotypic (raft) culture tissues. In HPV-positive tissues, HSV infection and replication induced significant cytopathic effects (CPE), but the tissues were able to recover and maintain a certain degree of tissue integrity and architecture. HPV31b not only maintained the episomal state of its genomic DNA but also maintained its genomic copy number even during times of extensive HSV-induced CPE. E2 transcripts encoded by HPV31b were undetectable even though HPV31b replication was maintained in HSV- infected raft tissues. Expression of HPV31b oncogenes (E6 and E7) was also repressed but to a lesser degree than was E2 expression. The extent of CPE induced by HSV is dependent on the magnitude of HPV replication and gene expression at the time of HSV infection. During active HSV infection, HPV maintains its genomic copy number even though genes required for its replication were repressed. These studies provide new insight into the complex interaction between two common human sexually transmitted viruses in an in vitro system, modeling their natural host tissue in vivo

ERBB2 Overexpression Establishes ERBB3-Dependent Hypersensitivity of Breast Cancer Cells to Withaferin A

The catalytically deficient ERBB3 strongly synergizes with the receptor tyrosine kinase ERBB2, and elevated levels represent an overall risk factor for unfavorable disease outcomes in breast cancer. Although itself not a target of pan-ERBB kinase inhibitors, it contributes to resistance in ERBB2-targeted treatment regiments. The steroidal lactone Withaferin A (WA) has established broad anticancer properties through several modes of action and was shown to be effective against triple-negative breast cancers at elevated concentrations. We found that ERBB2 overexpression does render cells hypersensitive to WA. Although ERBB2 downregulation is one aspect of WA treatment at high concentrations, it is not causal for the elevated sensitivity at lower dosages. Instead, WA targets the ability of ERBB3 to amplify ERBB2 signaling. ERBB3 receptor levels, constitutive phosphorylation of both ERBB3 and ERBB2, as well as signaling through AKT are eliminated by WA treatment. By targeting ERBB2/ERBB3 as a functional unit, it is also effective in cases in which ERBB2-directed inhibitors, such as lapatinib, alone show reduced potency. Hence, WA or derivatives thereof may present a low toxicity addition to ERBB2-targeting therapeutics, especially in cases in which ERBB3 involvement is driving resistance or reduced overall sensitivity. Mol Cancer Ther; 15(11); 2750-7. ©2016 AACR

Abstract A115: Withaferin A downregulates her-2/neu protein expression in human breast cancer cells

Abstract HER-2/neu protein is over-expressed in approximately 20-30% of breast cancers. Overexpression of this oncoprotein results in a more aggressive tumor phenotype and poor prognosis for patients. Current treatment strategies include passive administration of anti-HER-2/neu monoclonal antibody Trastuzumab as well as the small molecule inhibitor lapatinib. Both of these molecules function at the receptor level to block HER-2/neu signaling resulting in inhibition of cell growth and tumor regression in the majority of patients. However, a significant number of patients relapse or develop progressive disease due to acquired mechanisms of resistance to current therapy. Alternative approaches, that utilize multiple modes of action, are therefore necessary to combat this severe form of breast cancer. Withaferin A (WA) is a natural compound isolated from the Indian ayurvedic medicinal plant Withania Somnifera. Natural compounds are notorious for their multiple mechanisms of action, which make them ideal therapeutics for complex diseases such as cancer. Recent research has demonstrated that WA is effective in inducing cell death in several cancer cell lines by inhibiting molecular pathways such as Akt and MAPK proliferative cascades which are downstream of her-2/neu activation. Additionally, WA has also been shown to inhibit function of the molecular chaperone heat shock protein 90 (HSP90) in pancreatic carcinoma cells. Since HER-2/neu is a client protein of HSP90 and the chaperone functions to stabilize HER-2/neu protein expression on the cell surface, our goal was to determine whether WA would directly affect HER-2/neu expression. We initially established the cytotoxic activity of WA on HER-2/neu expressing human breast cancer cell lines such as SKBr3, MCF-7her-2neu and BT474 by MTT assay. We report that WA exhibits potent cytotoxic activity in these cells with IC50 concentrations of 1.6, 2.1 and 0.6 μM respectively after 48hr treatment. Comparison of WA sensitivity in a low HER-2/neu expressing cell line MCF-7 generated an IC50 of 5.1μM, indicating that cell lines that overexpress HER-2/neu may be more susceptible to the compound. Analysis of HER-2/neu expression both at the cell surface and intracellular localization were next performed in response to WA. WA treatment completely downregulated the expression of HER-2/neu protein from cell surface by 24hr and was both time and concentration dependent. Abrogation of intracellular protein expression also correlated to the downregulation from the cell surface. We determined that WA does not alter transcription of HER-2/neu, as there was no change in the gene expression with WA treatment compared to untreated cells. Further experiments are ongoing to address whether WA specifically targets signaling pathways that alter HER-2/neu signaling. Understanding the mechanisms by which WA induces down-regulation of HER-2/neu would serve as a means of developing WA as an alternative therapeutic strategy for HER-2/neu cancers not amenable to standard treatments. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A115. Citation Format: Ananlise Smith, Sarah Poliquin, Darlah Lopez-Rodriguez, Samita Andreansky. Withaferin A downregulates her-2/neu protein expression in human breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A115

Reduced Functional Capacity of CD8 +

Mice (I-A(b-/-)) that lack CD4(+) T cells remain healthy for at least three months after respiratory exposure to the murine gamma-herpesvirus 68 (gammaHV68), then succumb with symptoms of chronic wasting disease. Postexposure challenge of gammaHV68-infected I-A(b+/+) and I-A(b-/-) mice with a recombinant vaccinia virus (Vacc-p56) expressing an antigenic gammaHV68 peptide caused a massive increase in the numbers of D(b)p56-specific CD8(+) T cells. Previous experiments showed that, despite the large numbers of potential CTL effectors, there was little effect on the long-term survival of the CD4-deficient group and no diminution in the level of persistent virus shedding and latency. Comparison of the expanded CD8(+)D(b)p56(+) sets in the I-A(b+/+) and I-A(b-/-) mice indicated that these two T cell populations were not identical. More CD69(high)CD8(+) D(b)p56(+) T cells were found in the CD4-deficient mice, an effect that might be thought to reflect higher Ag load. By contrast, the mean fluorescence intensity of staining for the CD44 glycoprotein was diminished on CD8(+)D(b)p56(+) T cells from the I-A(b-/-) group, the level of CTL activity was lower on a per cell basis, and the relative prevalence of IFN-gamma(+)TNF-alpha(+) T cells detected after in vitro stimulation with the p56 peptide was decreased. Given that this experimental system provides an accessible model for evaluating postexposure vaccination protocols that might be used in diseases like HIV/AIDS, the further need is to clarify the underlying molecular mechanisms and the relative significance of lack of CD4(+) T help vs higher Ag load for these expanded CD8(+) effector populations