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Biomedical subjects

J P Moore

Publications and source records attributed to J P Moore.

At least 19 recordsLinked to original sources
PubMed2003

Human immunodeficiency virus type 1 Env with an intersubunit disulfide bond engages coreceptors but requires bond reduction after engagement to induce fusion.

A mutant human immunodeficiency virus (HIV) envelope protein (Env) with an engineered disulfide bond between the gp120 and gp41 subunits (SOS-Env) was expressed on cell surfaces. With the disulfide bond intact, these cells did not fuse to target cells expressing CD4 and CCR5, but the fusion process did advance to an intermediate state: cleaving the disulfide bond with a reducing agent after but not before binding to target cells allowed fusion to occur. Through the use of an antibody directed against CCR5, it was found that at the intermediate stage, SOS-Env had associated with coreceptors. Reducing the disulfide bond after this intermediate had been reached resulted in hemifusion at low temperature and fusion at physiological temperature. The addition of C34 or N36, peptides that prevent six-helix bundle formation, at the hemifused state blocked the fusion that would have resulted after raising the temperature. Thus, Env has not yet folded into six-helix bundles after hemifusion has been achieved. Because SOS-Env binds CCR5, it is suggested that the conformational changes in wild-type Env that result from this binding cause disengagement of gp120 from gp41 in the region of the engineered bond. It is proposed that this disengagement is the event that directly frees gp41 to undergo the conformational changes that lead to fusion. The intermediate state achieved prior to reduction of the disulfide bond was stable. The capture of this configuration of Env could yield a suitable antigen for vaccine development, and it may also be a target for pharmacological intervention against HIV-1 entry.

CD4 Antigens↗
PubMed2001

SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo.

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime-piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50-60% and a serum half-life of 5-6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed2001

A luciferase-reporter gene-expressing T-cell line facilitates neutralization and drug-sensitivity assays that use either R5 or X4 strains of human immunodeficiency virus type 1.

We describe the production and properties of a permanent cell line, CEM.NKR-CCR5-Luc. This line is a derivative of the CEM.NKR-CCR5 line, stably transfected to express the luciferase reporter gene under the transcriptional control of the HIV-2 LTR. Thus the cells respond to Tat expression during HIV-1 infection by producing luciferase, a protein that can be readily and accurately quantitated in a luminometer. The CEM.NKR-CCR5-Luc line expresses both the CCR5 and CXCR4 coreceptors and can therefore be infected with a range of HIV-1 isolates, irrespective of their tropism properties. This is true of HIV-1 isolates from several genetic subtypes and also of a group O isolate. Furthermore, luciferase expression is also activated by infection of the cells with SIVmac239 or SIVmac251. We show that the CEM.NKR-CCR5-Luc cells can be used in assays of HIV-1 neutralization and also for identifying inhibitors of HIV-1 entry targeted at the CCR5 and CXCR4 coreceptors. The luciferase end point simplifies the performance of neutralization and inhibitor-screening assays compared to the use of more conventional end points such as the detection of extracellular p24 antigen.

Amides↗
PubMed2001

HIV type 1 molecular clones able to use the Bonzo/STRL-33 coreceptor for virus entry.

We describe the cloning of env genes from the mother-infant HIV-1 isolate pair P6-v3 and M6-v3. These viruses are unusual in that they can use the coreceptor Bonzo/STRL33 as well as CCR5 and, in the case of M6, CXCR4, to enter transfected cell lines in vitro. The phenotype of the parental isolates is generally reflected by the properties of the cloned env genes, when these are used in an Env-complementation assay of virus entry. Chimeric viruses were also made that contain the env genes of P6-v3 and M6-v3 inserted into the background of the infectious molecular clone, HIV-1 NL4-3. Some of the chimeric viruses derived from HIV1 P6-v3 were able to use Bonzo for entry into transfected cell lines, albeit to a lesser extent than they could use CCR5. There are some indications that one of these chimeric viruses, P6-v3-22-1, can use a coreceptor other than CCR5, perhaps Bonzo, to enter mitogen-stimulated PBMC, although only weakly. However, formal proof that this virus can use Bonzo in primary cells has not been obtained. The P6-v3-22-1 chimeric virus was unable to infect CD4-negative, placental cell lines, in the presence or absence of soluble CD4. Env sequence analysis revealed several differences among viruses with different tropisms, most notably a four amino acid deletion in the central region of the V3 loop that distinguishes the R5 virus P6-v3-25-4 from the R5, Bonzo virus P6-v3-22-1.

Amino Acid Sequence↗
PubMed2001

Risk factors for post-ERCP pancreatitis: a prospective, multicenter study.

BACKGROUND: Post-ERCP pancreatitis is poorly understood. The goal of this study was to comprehensively evaluate potential procedure- and patient-related risk factors for post-ERCP pancreatitis over a wide spectrum of centers. METHODS: Consecutive ERCP procedures were prospectively studied at 11 centers (6 private, 5 university). Complications were assessed at 30 days by using established consensus criteria. RESULTS: Pancreatitis occurred after 131 (6.7%) of 1963 consecutive ERCP procedures (mild 70, moderate 55, severe 6). By univariate analysis, 23 of 32 investigated variables were significant. Multivariate risk factors with adjusted odds ratios (OR) were prior ERCP-induced pancreatitis (OR 5.4), suspected sphincter of Oddi dysfunction (OR 2.6), female gender (OR 2.5), normal serum bilirubin (OR 1.9), absence of chronic pancreatitis (OR 1.9), biliary sphincter balloon dilation (OR 4.5), difficult cannulation (OR 3.4), pancreatic sphincterotomy (OR 3.1), and 1 or more injections of contrast into the pancreatic duct (OR 2.7). Small bile duct diameter, sphincter of Oddi manometry, biliary sphincterotomy, and lower ERCP case volume were not multivariate risk factors for pancreatitis, although endoscopists performing on average more than 2 ERCPs per week had significantly greater success at bile duct cannulation (96.5% versus 91.5%, p = 0.0001). Combinations of patient characteristics including female gender, normal serum bilirubin, recurrent abdominal pain, and previous post-ERCP pancreatitis placed patients at increasingly higher risk of pancreatitis, regardless of whether ERCP was diagnostic, manometric, or therapeutic. CONCLUSIONS: Patient-related factors are as important as procedure-related factors in determining risk for post-ERCP pancreatitis. These data emphasize the importance of careful patient selection as well as choice of technique in the avoidance of post-ERCP pancreatitis.

Cholangiopancreatography, Endoscopic Retrograde↗
PubMed2001

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

Administration, Intravaginal↗
PubMed2001

Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140.

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.

Amides↗
PubMed2001

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41.

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

Amino Acid Motifs↗
PubMed2001

Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

AIDS Vaccines↗
PubMed2001

Mosquitoes of Grand Teton National Park, Teton County, Wyoming, USA.

An inventory of the mosquitoes of Grand Teton National Park and the John D. Rockefeller, Jr. Memorial Parkway was conducted during 1998 and 2000. Twenty-five culicid species belonging to 3 genera and 5 subgenera were recorded. This is the 1st substantive effort to record the mosquito fauna of this national park since its establishment in 1929. Collection of specimens of Ochlerotatus communis and Ochlerotatus nevadensis from the same larval site supports the species status of Oc. nevadensis.

Aedes↗
PubMed2001

New Nebraska mosquito distribution records.

Larval collections in Nebraska during 2000 established 2 new state distribution records for Ochlerotatus epactius and Anopheles perplexens and confirmed the presence of Culiseta melanura, bringing the total number of mosquito species reported from Nebraska to 50. Fifteen new county records for 10 species are noted, including the 1st report of Aedes albopictus from Lancaster County, marking its westernmost collection in Nebraska to date.

Aedes↗
PubMed2000

The effect of highly active antiretroviral therapy on binding and neutralizing antibody responses to human immunodeficiency virus type 1 infection.

The effect on humoral immune responses of highly active antiretroviral therapy (HAART) commenced during primary or chronic human immunodeficiency virus type 1 (HIV-1) infection was investigated. HAART inhibited the development of anti-gp120 antibodies when initiated during primary infection and could sometimes reduce antibody titers in patients treated within 2 years of HIV-1 infection. Conversely, antibody responses in patients infected for several years were less sensitive to HAART. Administering HAART during primary infection usually did not substantially affect the development of weak neutralizing antibody responses against autologous virus. However, 2 patients treated very early after infection did not develop neutralizing responses. In contrast, 3 of 4 patients intermittently adherent to therapy developed autologous neutralizing antibodies of unusually high titer, largely coincident with brief viremic periods. The induction of strong neutralizing antibody responses during primary HIV-1 infection might require the suppression of virus replication by HAART, to allow for the recovery of immune competency, followed by exposure to native envelope glycoproteins.

Anti-HIV Agents↗
PubMed2000

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5.

HIV-1 entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Amides↗
PubMed2000

Passive infusion of immune serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia.

Antibody responses are often considered to play only a limited role in controlling viremia during chronic infections with human or simian immunodeficiency virus (SIV). We investigated this by determining the effect of passively infused antibody on plasma viremia in infected rhesus macaques. The emphasis of the study was to understand the mechanism(s) underlying any observed effects. We infused serum immunoglobulins (SIVIG) purified from SIV(mac)251-infected macaques into other SIV(mac)251-infected macaques. The rapid progressor recipients had high viral loads but negligible titers of antibodies to SIV. Thus, we could significantly increase antibody titers with exogenous SIVIG. Despite restoring anti-SIV titers to levels typical of macaques with a normal disease course, SIVIG had only a modest effect on plasma SIV RNA and cell-associated viral load; the maximum, transient, reduction was threefold. The decrease in plasma RNA commenced within 1-2 h of SIVIG infusion, the nadir was at 12 h, and then a rebound occurred. A two- to threefold drop in cell-associated viral RNA was simultaneous with the decrease in plasma RNA. The kinetics of the viremia changes are inconsistent with neutralization of new cycles of infection. More likely, perhaps unexpectedly, is that infused antibodies killed SIV-infected cells, via an effector mechanism such as antibody-dependent cellular cytotoxicity.

Animals↗
PubMed2000

The relationship between T cell proliferative responses and plasma viremia during treatment of human immunodeficiency virus type 1 infection with combination antiretroviral therapy.

The relationship between human immunodeficiency virus (HIV) type 1 replication and CD4+ T cell function was examined. T lymphocyte proliferation in response to both HIV-1 antigens and recall antigens was measured in HIV-1-infected individuals before and after they received highly active antiretroviral therapy (HAART). No correlation was observed between baseline viral load or CD4+ T cell count and the T cell proliferative response to HIV-1 Gag. Suppression of viremia was not associated with an increase in T cell proliferative responses. Emergence of viral replication during short periods of intermittent therapy promoted generalized activation of T helper lymphocytes, manifested by increased T cell proliferative responses to HIV-1 Gag and recall antigens. Recovery of CD4+ T cell responses occurred in some individuals who initiated HAART years after infection and who were intermittently adherent to drug treatment. Thus, CD4+ T cell responses can sometimes be regenerated if viral load is suppressed to allow some immune recovery and if antigenic stimulation is later provided.

Ambulatory Care Facilities↗