scholarly journals Determination of a Statistically Valid Neutralization Titer in Plasma That Confers Protection against Simian-Human Immunodeficiency Virus Challenge following Passive Transfer of High-Titered Neutralizing Antibodies

2002 ◽  
Vol 76 (5) ◽  
pp. 2123-2130 ◽  
Author(s):  
Yoshiaki Nishimura ◽  
Tatsuhiko Igarashi ◽  
Nancy Haigwood ◽  
Reza Sadjadpour ◽  
Ron J. Plishka ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Lymph Node ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Passive Transfer ◽  
Neutralization Titer ◽  
Peripheral Blood Mononuclear ◽  
Vitro Assay ◽  
Immunodeficiency Virus

ABSTRACT We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (108 cells) plus 8 ml of whole blood from protected animals to naïve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.

scholarly journals MDL 74,968, a new acyclonucleotide analog: activity against human immunodeficiency virus in vitro and in the hu-PBL-SCID.beige mouse model of infection.

1996 ◽  
Vol 40 (5) ◽  
pp. 1072-1077 ◽  
Author(s):  
C G Bridges ◽  
D L Taylor ◽  
P S Ahmed ◽  
T M Brennan ◽  
J M Hornsperger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Placebo Control ◽  
Intravenous Route ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Orally Bioavailable

The novel acyclonucleotide derivative of guanine, 9-[2-methylidene-3-(phosphonomethoxy)propyl] guanine (MDL 74,968), had antiviral activity comparable to those of 9-(2-phosphonomethoxyethyl) adenine (PMEA) and 2',3'-dideoxyinosine against laboratory strains of both human immunodeficiency virus (HIV) types 1 and 2 cultured in MT-4 cells and several clinical HIV isolates cultured in human peripheral blood mononuclear cells (PBMCs). MDL 74,968 was at least fourfold less toxic than PMEA to MT-4 cells or PBMCs, thereby producing a more favorable in vitro selectivity index for the former compound. Studies of acute toxicity in CD-1 mice showed that MDL 74,968 was not toxic at doses of 1,600 mg/kg of body weight via the intraperitoneal route or at doses of 500 mg/kg via the intravenous route. Furthermore, no adverse effects of MDL 74,968 were apparent when mice were treated at doses of 200 mg/kg twice daily for 5 days. Treatment by continuous subcutaneous infusion of MDL 74,968 or PMEA at the daily dose of 20 mg/kg in the hu-PBL-SCID.beige murine model of HIV infection significantly reduced the severity of infection compared with that in placebo-treated controls. Quantitation of virus recovery by endpoint titration of spleen cells in coculture with mitogen-activated PBMCs demonstrated that MDL 74,968 as well as PMEA significantly reduced the amount of virus (P < 0.02). Moreover, by using DNA extracted from spleens, the mean HIV:HLA PCR product ratio, which takes into account individual variation in immune system reconstitution, were 0.50 and 0.40 for MDL 74,968 and PMEA treatments, respectively, whereas animals receiving the placebo control had significantly higher levels of HIV proviral DNA (mean 0.78; P < 0.02). Taken together, these promising findings suggest that an orally bioavailable prodrug of MDL 74,968 should be developed for the treatment of HIV infection.

scholarly journals Design and Profiling of GS-9148, a Novel Nucleotide Analog Active against Nucleoside-Resistant Variants of Human Immunodeficiency Virus Type 1, and Its Orally Bioavailable Phosphonoamidate Prodrug, GS-9131

2007 ◽  
Vol 52 (2) ◽  
pp. 655-665 ◽  
Author(s):  
Tomas Cihlar ◽  
Adrian S. Ray ◽  
Constantine G. Boojamra ◽  
Lijun Zhang ◽  
Hon Hui ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mononuclear Cells ◽  
Biological Properties ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (Ki = 0.8 μM) and exhibits low inhibitory potency against host polymerases including DNA polymerase γ. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 μM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

scholarly journals Induction of Murine Mucosal CCR5-Reactive Antibodies as an Anti-Human Immunodeficiency Virus Strategy

2005 ◽  
Vol 79 (11) ◽  
pp. 6848-6858 ◽  
Author(s):  
C. Barassi ◽  
E. Soprana ◽  
C. Pastori ◽  
R. Longhi ◽  
E. Buratti ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Flock House Virus ◽  
Peripheral Blood Mononuclear ◽  
Genital Mucosa ◽  
Conformationally Constrained ◽  
Main Site ◽  
Immunodeficiency Virus

ABSTRACT The genital mucosa is the main site of initial human immunodeficiency virus type 1 (HIV-1) contact with its host. In spite of repeated sexual exposure, some individuals remain seronegative, and a small fraction of them produce immunoglobulin G (IgG) and IgA autoantibodies directed against CCR5, which is probably the cause of the CCR5-minus phenotype observed in the peripheral blood mononuclear cells of these subjects. These antibodies recognize the 89-to-102 extracellular loop of CCR5 in its native conformation. The aim of this study was to induce infection-preventing mucosal anti-CCR5 autoantibodies in individuals at high risk of HIV infection. Thus, we generated chimeric immunogens containing the relevant CCR5 peptide in the context of the capsid protein of Flock House virus, a presentation system in which it is possible to engineer conformationally constrained peptide in a highly immunogenic form. Administered in mice via the systemic or mucosal route, the immunogens elicited anti-CCR5 IgG and IgA (in sera and vaginal fluids). Analogous to exposed seronegative individuals, mice producing anti-CCR5 autoantibodies express significantly reduced levels of CCR5 on the surfaces of CD4+ cells from peripheral blood and vaginal washes. In vitro studies have shown that murine IgG and IgA (i) specifically bind human and mouse CD4+ lymphocytes and the CCR5-transfected U87 cell line, (ii) down-regulate CCR5 expression of CD4+ cells from both humans and untreated mice, (iii) inhibit Mip-1β chemotaxis of CD4+ CCR5+ lymphocytes, and (iv) neutralize HIV R5 strains. These data suggest that immune strategies aimed at generating anti-CCR5 antibodies at the level of the genital mucosa might be feasible and represent a strategy to induce mucosal HIV-protective immunity.

scholarly journals Determination of ddATP Levels in Human Immunodeficiency Virus-Infected Patients Treated with Dideoxyinosine

2004 ◽  
Vol 48 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Cecile Le Saint ◽  
Raphael Terreux ◽  
Daniele Duval ◽  
Jacques Durant ◽  
Helene Ettesse ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Highly Active Antiretroviral Therapy ◽  
Mononuclear Cells ◽  
Exchange Chromatography ◽  
Therapeutic Monitoring ◽  
Target Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

ABSTRACT Clinical failures of the highly active antiretroviral therapy could result from inefficient intracellular concentrations of antiviral drugs. The determination of drug contents in target cells of each patient would be useful in clinical investigations and trials. The purpose of this work was to quantify the intracellular concentration of ddATP, the active metabolite of dideoxyinosine (ddI), in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)-infected patients treated with ddI. We have raised antibodies against ddA-citrate, a stable isostere of ddATP selected on the basis of its structural and electronic analogies with ddATP. The anti-ddA-citrate antibodies recognized ddATP and ddA with nanomolar affinities and cross-reacted neither with any of the nucleotide reverse transcriptase inhibitors used in HIV therapy nor with their phosphorylated metabolites. The three phosphorylated metabolites of ddI (ddAMP, ddADP, and ddATP) were purified by anion exchange chromatography and the amount of each metabolite was determined by radioimmunoassay with or without prior phosphatase treatment. The intracellular levels of the three ddI metabolites were measured both in an in vitro model and in PBMCs of HIV-infected patients under ddI treatment. The possibility to measure intracellular levels of ddATP from small blood samples of HIV-infected patients treated with ddI could be exploited to develop individual therapeutic monitoring.

Immunophenotypic Analysis of Peripheral Blood Mononuclear Cells Undergoing In Vitro Apoptosis After Isolation From Human Immunodeficiency Virus–Infected Children

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4230-4237 ◽  
Author(s):  
Thomas W. McCloskey ◽  
Saroj Bakshi ◽  
Soe Than ◽  
Parisa Arman ◽  
Savita Pahwa
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Costimulatory Molecule ◽  
T Cell Subsets ◽  
Live Cells ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficiency Virus

Abstract Lymphocytes of human immunodeficiency virus (HIV)-infected individuals undergo accelerated apoptosis in vitro, but the subsets of cells affected have not been clearly defined. This study examined the relationship between lymphocyte phenotype and apoptotic cell death in HIV-infected children by flow cytometry. Direct examination of the phenotype of apoptotic lymphocytes was accomplished using a combination of surface antigen labeling performed simultaneously with the Tdt mediated Utp nick end-labeling (TUNEL) assay. In comparison to live cells, apoptotic lymphocytes displayed an overrepresentation of CD45RO and HLA-DR expressing cells, while CD28 and CD95 expressing cells were underrepresented. Lymphocytes expressing CD4, CD8, and CD38 were equally represented in apoptotic and live populations. When percent lymphocyte apoptosis follow- ing culture was examined independently with lymphocyte subsets in fresh blood, apoptosis was negatively correlated with the percentage of CD4 cells, but not with specific CD4 T-cell subsets. Although not correlated with the percentage of total CD8 cells, apoptosis was positively correlated with specific CD8 T-cell subsets expressing CD45RO and CD95 and negatively correlated for CD8 T cells expressing CD45RA. These results provide direct evidence that a population of activated lymphocytes with the memory phenotype lacking the costimulatory molecule CD28 are especially prone to undergo apoptosis. The findings related to CD95 expression in fresh and apoptotic cells implicate Fas-dependent and Fas-independent pathways of apoptosis in HIV disease in children.

scholarly journals Identification of Mutations that Encode Drug Resistance in the Polymerase Gene of the Human Immunodeficiency Virus

1994 ◽  
Vol 5 (suppl e) ◽  
pp. 29E-33E
Author(s):  
Zhengxian Gu ◽  
Hengsheng Fang ◽  
Horacio Salomon ◽  
Qing Gao ◽  
Mark A Wainberg
Keyword(s):  
Human Immunodeficiency Virus ◽  
In Vitro Selection ◽  
Mononuclear Cells ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Cross Resistance ◽  
Peripheral Blood Mononuclear ◽  
Replication Assay ◽  
Immunodeficiency Virus

In vitro selection in MT-4 cells was used to generate human immunodeficiency virus-type 1 (HIV 1) variants that are resistant to 2',3'-dideoxycytidine (ddC), 2',3'-didcoxyinosine (ddI) and the (-) enantiomer of 2' ,3'-dideoxy-3'-thiacytidine (3TC). The complete reverse transcriptase open reading frames of these viruses, and portions of flanking protease and integrase within the pol gene, were cloned and sequenced by polymerase chain reaction (PCR) techniques. Mulalions were observed at each of amino acid sites 65 (Lys → Arg: AAA → AGA) and 184 (Met → Val: ATG → GTG) when ddC was used in this protocol, and at site 184 only when either 3TC or ddl was employed. These mutations were introduced into the pol gene of infectious recombinant HXB2-D DNA by site-directed mutagenesis to confirm, by viral replication assay, their importance in conferring resistance against these drugs. A recombinant virus containing the site 65 mutation only possessed greater than 10-fold resistance against ddC compared with parental HXB2-D. Moreover, cross-resistance of about 20-fold and threefold, respectively, was delectable against 3TC and dell but not against 3'-azido-3'-deoxthymidine (AZT). When the 65 and 184 mutations were combined into HXB2-D, the resultant construct did not possess higher levels of resistance lo any of these drugs than observed with the site 65 or 184 mutation alone. These mutations were further demonstrated by PCH analysis of peripheral blood mononuclear cells from 10 patients on long term ddC therapy, although variable patterns were observed in terms of which of the two mutations or both were present. Sometimes, the wild-type site 65 codon was also detected, indicating the presence of mixtures of viral quasi-species. Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from patients on prolonged ddC therapy.

scholarly journals Multispecific Vaccine-Induced Mucosal Cytotoxic TLymphocytes Reduce Acute-Phase Viral Replication but Fail inLong-Term Control of Simian Immunodeficiency VirusSIVmac239

2003 ◽  
Vol 77 (24) ◽  
pp. 13348-13360 ◽  
Author(s):  
Thorsten U. Vogel ◽  
Matthew R. Reynolds ◽  
Deborah H. Fuller ◽  
Kathy Vielhuber ◽  
Tim Shipley ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Replication ◽  
Acute Phase ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Chronic Phase ◽  
High Dose ◽  
Peripheral Blood Mononuclear ◽  
Virus Challenge ◽  
Immunodeficiency Virus

ABSTRACT Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01+ macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4+ helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01+ controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.

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