• HIV envelope glycoprotein-Fc fusion proteins target Fc gamma receptors and elicit effector antibody responses in rhesus macaques

      Shubin, Zhanna; Pauza, C. David (2016)
      A goal for HIV prevention programs is to develop safe and effective vaccines that elicit durable and broadly protective antibodies against both sexual and blood-borne HIV transmission. Many vaccine programs focus on the immune responses to critical epitopes in the HIV envelope glycoprotein (Env) and seek to improve the quality and quantity of antibodies by altering the conformation, oligomerization or glycosylation of Env gp120. As a complementary strategy, we envisioned that attaching an Fc moiety from IgG to immunogens would allow binding to neonatal Fc receptor (FcRn) for FcRn-mediated mucosal delivery. We reasoned that Fc fusion protein immunogens may also mimic immune complexes by binding Fc gamma receptors (Fc?R) on immune cells and would increase the potency of antibody responses. We developed Fc fusion immunogens consisting of either HIVBaL gp120 (Env-Fc) or critical epitopes in the V1V2 region of Env expressed within a carrier protein that provides highly multivalent epitope display (Gag-V1V2-Fc). Fc fusion proteins were attached at their carboxyl terminus to a Gly/Ser linker that is in turn fused to each half of the dimeric Fc domain from rhesus macaque IgG1 (Env-Fc). Env-Fc retained a capacity to bind both cell surface CD4 and Fc?Rs, which led to protein internalization and accumulation in cytoplasmic vesicles. In a rhesus macaque immunization study, Env-Fc was more potent compared to Env (gp120 monomer) in several ways. Env-Fc elicited higher gp120 binding antibody titers with increased breadth, including the capacity for recognizing CD4-induced epitopes, neutralizing activity against Tier 1A HIV pseudotyped viruses, and antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). Serum antibodies produced in Env-Fc immunized macaques had increased durability compared to Env monomer immunization. A Gag-V1V2-Fc fusion protein was constructed to test whether a self-assembling macromolecular structure was a more effective method for directing antibody responses to specific V1V2 regions of the Envelope glycoprotein. This immunogen crossed nasal epithelium barriers more efficiently than HIV Gag (p24 monomer), and elicited V1V2-specific IgG responses. Our work suggests that adding IgG1 Fc to engineered monomeric or oligomeric Env-based immunogens may improve the protective serum antibody response and contribute to the development of a protective HIV vaccine.
    • In vivo insulin action on skeletal muscle and adipose tissue glycogen synthase and phosphorylase activities in rhesus monkeys with varying degrees of spontaneous insulin resistance

      Ortmeyer, Heidi Karen; Hansen, Barbara C. (1992)
      Resistance to the action of insulin, particularly in the glycogen storage pathway, is one of the earliest detectable defects in the development of non-insulin-dependent diabetes mellitus (NIDDM) in humans and in monkeys, but the nature of the underlying defect and its role in the pathogenesis of diabetes are unknown. The present studies were carried out in monkeys (Macaca mulatta) who develop adult-onset obesity-associated NIDDM which is remarkably similar to human NIDDM. These experiments examined, under basal and insulin-stimulated conditions (euglycemic hyperinsulinemic clamp), the in vivo action of insulin on glycogen synthase (GS), the rate-limiting enzyme for glycogenesis, and on glycogen phosphorylase (GP), the key enzyme in glycogenolysis. To determine possible tissue specificity in the development of such defects, both muscle and adipose tissue biopsies were studied. Additional experiments addressed the possibility that in insulin resistance, a component of a putative mediator of insulin action, chiroinositol (CI), might be reduced or absent, and if restored, might improve insulin action. Urinary CI excretion rates, previously shown to be abnormally low in both monkeys and humans with NIDDM, were also determined. Twenty-seven monkeys chosen for study consisted of three groups: normal (nondiabetic), hyperinsulinemic (pre-diabetic), and impaired glucose tolerant/NIDDM. Results showed that covalent activation of muscle GS was significantly reduced in the insulin-resistant (pre-diabetic) monkeys and further reduced in the diabetic monkeys as compared to the normal monkeys. The covalent activation of adipose tissue GS was absent in both the insulin-resistant and diabetic groups. The independent activity and the percent independent to total activity (activity ratio) of muscle and adipose tissue GS in response to insulin were significantly related to whole-body insulin-mediated glucose disposal rate (M). Insulin activation of muscle GS was shown to be significantly inversely correlated with insulin activation of muscle GP. Urinary CI excretion rate was significantly inversely related to in vivo insulin resistance, and positively related to insulin activation of muscle and adipose tissue GS, and was inversely related to insulin activation of muscle GP. In addition, the intravenous administration of CI significantly increased the activity of muscle GS and inhibited the activity of muscle GP. We conclude that a reduced activation of GS by insulin is a major contributor to insulin resistance and NIDDM in monkeys, and this defect may be due to reduced amounts of chiroinositol-containing insulin mediator.
    • Spatial encoding of visual and somatosensory stimuli by single neurons in area 7B of the macaque parietal cortex

      Field, Patrick Roy; Olson, Carl, Ph.D. (1997)
      Lesions in certain areas of the parietal cortex in Macaque monkeys produces deficits in visual and somatosensory perception, as well as, eye and arm activity associated with exploration of extrapersonal space. This study focused on recording from neurons in area 7b of the posterior parietal association cortex with visual or somatosensory receptive fields (RFs) that were modulated by eye or arm position. Two Macaque (Maccaca mulatta) monkeys were trained to maintain a steady gaze and arm position while a visual stimulus (illuminated circle) or a tactile stimulus (brushing of the dorsal side of the hand) was delivered. Microelectrodes were advanced into cortical area 7b of the monkeys and action potentials were recorded extracellularly from neurons while the stimuli were being delivered. When neurons with visual or tactile RFs were identified, the eye or arm position was altered, and the phasic responses to sensory stimuli and tonic activity was evaluated. Modulated phasic and tonic activity in neurons provide information on the spatial encoding of stimuli in extrapersonal space. Previous studies of area 7b have documented a sparse number of neurons with visual RFs, but in the present study, fifty percent of the neurons in the middle and posterior thirds of area 7b had visual RFs. During the eye position task, neurons with craniocentric responses, visual RFs that were dependent upon the head's position, were observed, and in more than half of the trials, neurons with tonic angle of gaze activity discharged when the eyes were directed into the contralateral hemi-field. Eighty-six percent of the neurons with tactile RFs, sensitive to passive arm positioning, increased activity when the hand moved into the contralateral hemi-field. The majority of those neurons increased activity when the arm angle approached forty-five degrees contralateral, a position that coincides with one of the most responsive visual angles in the eye position task. In contrast, fifty-six percent of the neurons with tonic angle of arm signals increased activity when the arm was moving further into ipsilateral hemi-space. All of these neuronal types describe a region of the brain that contributes to the spatial encoding of stimuli in extrapersonal space.