Sphingosine 1-phosphate-induced nitric oxide production simultaneously controls endothelial barrier function and vascular tone in resistance arteries.

The regulations of endothelial permeability and vascular tone by sphingosine 1-phosphate (S1P) have been well-studied independently. Little is known about whether the effects of S1P on endothelial permeability can directly influence vascular tone in resistance arteries, which impact blood flow. The endothelium forms a partial barrier that regulates access of circulating agonists to underlying vascular smooth muscle cells (VSMCs). We hypothesized that physiological concentrations of circulating S1P simultaneously control endothelial barrier function and vascular tone through endothelial production of nitric oxide (NO). We adapted the pressure myograph system to simultaneously measure both functions in pressurized mesenteric compared to uterine resistance arteries from wild-type and eNOS KO mice. We established that: 1) S1P interacting directly with the endothelium inside pressurized arteries generates NO that limits endothelial permeability; 2) an intact endothelium forms a partial physical barrier that regulates access of intraluminal S1P to the underlying VSMCs and 3) S1P infused lumenally also generates NO through eNOS that counterbalances the constriction induced by S1P that is able to access VSMCs and this is critical to control vascular tone. We conclude that targeting the S1P signaling system, particularly the capacity to produce NO could be clinically important in the treatment of vascular diseases.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

Harnessing co-stimulatory TNF receptors for cancer immunotherapy: Current approaches and future opportunities.

Co-stimulatory tumor necrosis factor receptors (TNFRs) can sculpt the responsiveness of T cells recognizing tumor-associated antigens. For this reason, agonist antibodies targeting CD137, CD357, CD134 and CD27 have received considerable attention for their therapeutic utility in enhancing anti-tumor immune responses, particularly in combination with other immuno-modulatory antibodies targeting co-inhibitory pathways in T cells. The design of therapeutic antibodies that optimally engage and activate co-stimulatory TNFRs presents an important challenge of how to promote effective anti-tumor immunity while avoiding serious immune-related adverse events. Here we review our current understanding of the expression, signaling and structural features of CD137, CD357, CD134 and CD27, and how this may inform the design of pharmacologically active immuno-modulatory antibodies targeting these receptors. This includes the integration of our emerging knowledge of the role of Fcγ receptors (FcγRs) in facilitating antibody-mediated receptor clustering and forward signaling, as well as promoting immune effector cell-mediated activities. Finally, we bring our current preclinical and clinical knowledge of co-stimulatory TNFR antibodies into the context of opportunities for next generation molecules with improved pharmacologic properties.

Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission.

UNLABELLED: To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies/inhibitors. IMPORTANCE: Prevention of the transmission of human immunodeficiency virus type 1 (HIV-1) remains a prominent goal of HIV research. The relative contribution of HIV-1 within an infected cell versus cell-free HIV-1 to virus transmission remains debated. It has been suggested that cell-associated virus is more efficient at transmitting HIV-1 and more difficult to neutralize than cell-free virus. Several broadly neutralizing antibodies and retroviral inhibitors are currently being studied as potential therapies against HIV-1 transmission. The present study demonstrates a decrease in neutralizing antibody and inhibitor efficiencies against cell-associated compared to cell-free HIV-1 transmission among different strains of HIV-1. We also observed a significant reduction in virus transmission using a combination of two different neutralizing antibodies that target specific sites on the outermost region of HIV-1, the virus envelope. Therefore, our findings support the use of antibody combinations against both cell-free and cell-associated virus in future candidate therapy regimens.

Efficiency of cell-free and cell-associated virus in mucosal transmission of human immunodeficiency virus type 1 and simian immunodeficiency virus.

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.

Impact of local endothelial challenge with cytomegalovirus or glycoprotein B on vasodilation in intact pressurized arteries from nonpregnant and pregnant mice.

Cytomegalovirus (CMV) infections are associated with vascular diseases in the human population. We have previously shown vascular dysfunction in systemic and uterine arteries dissected from nonpregnant (NP) mouse CMV (mCMV)-infected mice that was further impaired during late pregnancy (LP). CMV attachment alone through glycoprotein B (GB) can generate signals that impact vascular tone regulation. However, the contribution of direct virus interactions with endothelium to the vascular dysfunction we previously observed after in vivo mCMV infection is not known. We used a pressure myograph system to infuse GB or whole intact mCMV inside arteries dissected from uninfected mice and assessed vasodilation to methacholine infused inside pressurized arteries rather than applied abluminally. These results were compared to those observed after methacholine infusion into untreated arteries dissected from mCMV-infected mice. In mesenteric arteries, vasodilation to infused methacholine did not differ among treatments in NP or LP groups in contrast to previously published studies. However, increased vasoconstrictor activity was unmasked after blocking thromboxane receptors or prostaglandin production. Vasodilation in uterine arteries from uninfected NP mice to infused methacholine was increased by both GB and whole intact mCMV pretreatment. Untreated uterine arteries from mCMV-infected NP mice showed even greater vasodilation. There was no effect of GB or whole intact mCMV pretreatment in uterine arteries from uninfected LP mice, whereas vasodilation to infused methacholine was reduced in untreated uterine arteries from mCMV-infected LP mice. CMV exerts direct effects on vascular function which should be considered during viral reactivation leading to viremia and during GB-based vaccine administration.

Differential effects on nitric oxide-mediated vasodilation in mesenteric and uterine arteries from cytomegalovirus-infected mice.

Chronic cytomegalovirus (CMV) infections are implicated in vascular diseases. Recently, we showed that an active mouse CMV (mCMV) infection in nonpregnant mice increased endothelial-dependent vasodilation in isolated mesenteric and uterine arteries. In late pregnancy, while increased vasodilation was found in mesenteric arteries from infected mice, there was a dramatic decrease in uterine arteries. Understanding the mechanisms for these vascular changes during CMV infections is important for pregnancy outcomes and long-term consequences of this chronic infection. Increased nitric oxide (NO) is implicated in CMV-associated atherosclerosis, and CMV replication is dependent on prostaglandin H synthase (PGHS) activity. Alternatively, CMV infections decrease NO under inflammatory conditions. We therefore hypothesized that changes in the contribution by NO or PGHS-induced vasodilators would explain the increased or decreased endothelial-dependent vasodilation in arteries from nonpregnant and late pregnant mice, respectively. We found that the contribution by NO to methacholine-induced vasodilation was significantly increased in mesenteric, but not uterine, arteries isolated from nonpregnant and pregnant mCMV-infected mice. Prostaglandin inhibition did not affect endothelial-dependent vasodilation in any group. Vasodilation responses to sodium nitroprusside, an NO donor, were increased in mesenteric and uterine arteries isolated only from mCMV-infected nonpregnant mice. These results explain the increased vasodilation responses observed in mesenteric arteries from mCMV-infected mice; however, the decreased vasodilation in uterine arteries from pregnant mice could not be explained by these mechanisms. Thus CMV infection affects the contribution of NO differently in endothelial-dependent vasodilation in pregnant compared with nonpregnant mice and also in the mesenteric compared with the uterine vascular bed.