Reduced cellular binding affinity has profoundly different impacts on the spread of distinct poxviruses.

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae

Chimeric tumor modeling reveals role of partial PDL1 expression in resistance to virally induced immunotherapy

Abstract Expression of PDL1 on the surface of tumor cells can blunt the efficacy of many cancer immunotherapies. For example, our lab has previously shown that tumors derived from malignant cells incapable of expressing PDL1 are highly susceptible to immunotherapy induced by oncolytic virus treatment while tumors derived from PDL1 capable cells are highly resistant. In patient biopsies, however, expression of PDL1 on malignant cells is often not uniform with some cells expressing PDL1 while others do not. Importantly, how this partial PDL1 positivity influences the outcomes of immunotherapy remains largely unknown. In the current work, we expand on our previous findings by generating partially PDL1 positive tumors in immune competent animals and asking what percentage of tumor cells must express PDL1 for a tumor to become functionally resistant to oncolytic treatment. Our results indicate that the responsiveness of partially PDL1+ tumors correlates linearly with the percentage of PDL1 capable cells present at the initiation of treatment. Additionally, we observe that tumors which relapse after treatment display a significant increase in the numbers of PDL1 capable cells present suggesting that specific editing of mixed tumors might play a role in disease relapse. These data indicate that varying levels of PDL1 expression can play a significant role in the outcomes of oncolytic immunotherapy and challenges the concept that tumors should be viewed as simply PDL1+ or PDL1−

Defects in intratumoral arginine metabolism attenuate the replication and therapeutic efficacy of oncolytic myxoma virus

Background Arginine (Arg) is a semiessential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. In vivo, Arg bioavailability is regulated by a combination of dietary intake, protein catabolism, and limited biosynthesis through portions of the urea cycle. Interestingly, despite the importance of bioavailable Arg to support cellular proliferation, many forms of cancer are functionally auxotrophic for this amino acid due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate. The impact of this silencing on oncolytic virotherapy (OV), however, has never been examined.Methods To address this gap in knowledge, we generated tumor cells lacking ASS1 and examined how loss of this enzyme impacted the in vivo replication and therapeutic efficacy of oncolytic myxoma virus (MYXV). We also generated a series of recombinant MYXV constructs expressing exogenous ASS1 to evaluate the therapeutic benefit of virally reconstituting Arg biosynthesis in ASS1−/− tumors.Results Our results show that the in vitro replication of oncolytic MYXV is dependent on the presence of bioavailable Arg. This dependence can be overcome by the addition of the metabolic precursor citrulline, however, this rescue requires expression of ASS1. Because of this, tumors formed from functionally ASS1−/− cells display significantly reduced MYXV replication as well as poorer therapeutic responses. Critically, both defects could be partially rescued by expressing exogenous ASS1 from recombinant oncolytic MYXVs.Conclusions These results demonstrate that intratumoral defects to Arg metabolism can serve as a novel barrier to virally induced immunotherapy and that the exogenous expression of ASS1 can improve the efficacy of OV in Arg-auxotrophic tumors

Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro.

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury

Viral Cross-Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro

<div><p>Poxviruses express highly active inhibitors, including <em>ser</em>ine <em>p</em>roteinase <em>in</em>hibitors (<em>serpins</em>), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models <em>in vivo</em>. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis <em>in vitro</em> of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either <em>in vitro</em> with GzmB antibody or <em>in vivo</em> in ApoE/GzmB double knockout mice. <b><em>Conclusions</em></b> The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion <em>in vivo</em>, and inhibits human T cell apoptosis <em>in vitro</em>. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.</p> </div

Animal models.

<p>SD – Sprague Dawley; ApoE^−/− – Apolipoprotein E; GzmB^−/− – Granzyme B (B6.129S2–<i>Gzmbtm1Ley</i>/J); PAI-1^−/− – Plasminogen Activator Inhibitor-1 (B6.129S2-<i>Serpine1^tm1Mlg).</i></p

Blockade of granzyme B reduces viral cross-class serpin inhibition of T cell induced T cell apoptosis.

<p>Jurkat T cells were treated with PMA and ionophore (PI) and the level of granzyme B expressed was measured by IEPDase assay and caspase 3 and 7 activity by DEVDase assay (<b>A</b>). Increased granzyme B (GzmB, p<0.001) secreted by these cells and was inhibited by treating the cells with an intracellular inhibitor of granzyme B, ZAAD-CMK (p<0.001) or anti-granzyme B antibody (p<0.001) (<b>A</b>). The medium containing granzyme B from PI treated T cells (CTLm) was applied to naive HUVECs to induce apoptosis (<b>B</b>). Treatment with Serp-2, but not CrmA, reduced caspase activity in CTLm treated HUVECs (<b>B</b>). The CTLm was also applied to naive T cells in culture to induce apoptosis and increased levels of caspase 3 and granzyme B were observed as IEPDase activity (<b>C</b>, p<0.0001) and DEVDase activity (<b>D</b>, p<0.0001) respectively. Treatment with Serp-2 reduced both granzyme B (<b>C</b>, p<0.01) and caspase 3 (<b>D</b>, p<0.0009) activities significantly. Antibody to granzyme B (GzmB) blocked Serp-2 mediated reductions in CTLm induced granzyme B (<b>C)</b> when compared to Serp-2 treatment alone (p<0.0004), but with a still significant decrease (p<0.005) when compared to CTLm treatment alone. Antibody to granzyme B also blocked the Serp-2 mediated decrease in caspase 3 (<b>D</b>, p<0.149) when compared to CTLm activation. This Serp-2 mediated inhibition of CTLm induced granzyme B activity was also blocked by incubation of cells with antibody to perforin (<b>C</b>, p = 0.412) but not the caspase 3 activity (<b>D</b>, p<0.0193). The results shown here represent mean ± SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.</p