scholarly journals Analysis of the S3 and S3' subsite specificities of feline immunodeficiency virus (FIV) protease: Development of a broad-based protease inhibitor efficacious against FIV, SIV, and HIV in vitro and ex vivo

1998 ◽  
Vol 95 (3) ◽  
pp. 939-944 ◽  
Author(s):  
T. Lee ◽  
G. S. Laco ◽  
B. E. Torbett ◽  
H. S. Fox ◽  
D. L. Lerner ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Immunodeficiency Virus

scholarly journals Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

2006 ◽  
Vol 80 (16) ◽  
pp. 7832-7843 ◽  
Author(s):  
Ying-Chuan Lin ◽  
Ashraf Brik ◽  
Aymeric de Parseval ◽  
Karen Tam ◽  
Bruce E. Torbett ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Expression System ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Virus Mutant ◽  
Substrate Inhibitor ◽  
Hiv 1

ABSTRACT We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.

scholarly journals AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Homologous Erythrocytes as a Delivery System for Preferential Immunization with Putative Protective Antigens

1998 ◽  
Vol 5 (2) ◽  
pp. 235-241 ◽  
Author(s):  
Laura Chiarantini ◽  
Donatella Matteucci ◽  
Mauro Pistello ◽  
Umberto Mancini ◽  
Paola Mazzetti ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Vaccine Development ◽  
Ex Vivo ◽  
Degree Of Protection ◽  
Aids Vaccine ◽  
Protective Antigens ◽  
Homologous Virus ◽  
Immunodeficiency Virus ◽  
Specific Pathogen

ABSTRACT Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 μg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

scholarly journals AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Protection from an Intraclade Challenge Administered Systemically or Mucosally by an Attenuated Vaccine

2003 ◽  
Vol 77 (20) ◽  
pp. 10740-10750 ◽  
Author(s):  
Mauro Pistello ◽  
Donatella Matteucci ◽  
Francesca Bonci ◽  
Patrizia Isola ◽  
Paola Mazzetti ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Field Studies ◽  
Vaccine Virus ◽  
Low Grade ◽  
Challenge Virus ◽  
Natural Host Species ◽  
Immunodeficiency Virus ◽  
Aids Vaccines

ABSTRACT Feline immunodeficiency virus (FIV) infection of domestic cats represents a valuable system through which to investigate criteria for antilentiviral vaccines in a natural host species. Here, we examined whether vaccination with a strain of FIV attenuated as a result of prolonged growth in vitro could protect against a fully virulent, highly heterologous intraclade challenge. The results indicated that the vaccine virus produced a low-grade infection with no detectable pathological effects and afforded a long-lasting sterilizing immunity if the challenge was delivered intraperitoneally as cell-free virus but not against a cell-associated intravaginal challenge. In the latter case, however, the replication and pathological consequences of the challenge virus were markedly suppressed. Together with similar results obtained in rhesus monkey models, these findings should give impulse to the development of attenuated FIV vaccines to be tested in controlled studies in field cats. Field studies may provide answers to some of the existing safety concerns surrounding attenuated AIDS vaccines in humans.

scholarly journals Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

2010 ◽  
Vol 84 (13) ◽  
pp. 6799-6809 ◽  
Author(s):  
Ying-Chuan Lin ◽  
Bruce E. Torbett ◽  
John H. Elder
Keyword(s):  
Human Immunodeficiency Virus ◽  
Feline Immunodeficiency Virus ◽  
Ex Vivo ◽  
Structural Parameters ◽  
Amino Acid Identity ◽  
Inhibitor Specificity ◽  
3 Dimensional ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteases (PRs) share only 23% amino acid identity and exhibit distinct specificities yet have very similar 3-dimensional structures. Chimeric PRs in which HIV residues were substituted in structurally equivalent positions in FIV PR were prepared in order to study the molecular basis of PR specificity. Previous in vitro analyses showed that such substitutions dramatically altered the inhibitor specificity of mutant PRs but changed the rate and specificity of Gag cleavage so that chimeric FIVs were not infectious. Chimeric PRs encoding combinations of the I37V, N55M, M56I, V59I, L97T, I98P, Q99V, and P100N mutations were cloned into FIV Gag-Pol, and those constructs that best approximated the temporal cleavage pattern generated by wild-type FIV PR, while maintaining HIV-like inhibitor specificity, were selected. Two mutations, M56I and L97T, were intolerant to change and caused inefficient cleavage at NC-p2. However, a mutant PR with six substitutions (I37V, N55M, V59I, I98P, Q99V, and P100N) was selected and placed in the context of full-length FIV-34TF10. This virus, termed YCL6, had low-level infectivity ex vivo, and after passage, progeny that exhibited a higher growth rate emerged. The residue at the position of one of the six mutations, I98P, further mutated on passage to either P98H or P98S. Both PRs were sensitive to the HIV-1 PR inhibitors lopinavir (LPV) and darunavir (DRV), as well as to the broad-based inhibitor TL-3, with 50% inhibitory concentrations (IC50) of 30 to 40 nM, consistent with ex vivo results obtained using mutant FIVs. The chimeras offer an infectivity system with which to screen compounds for potential as broad-based PR inhibitors, define structural parameters that dictate specificity, and investigate pathways for drug resistance development.

scholarly journals AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Reevaluation of Neutralizing Antibody Levels Elicited by a Protective and a Nonprotective Vaccine after Removal of Antisubstrate Cell Antibodies

2001 ◽  
Vol 75 (9) ◽  
pp. 4424-4429 ◽  
Author(s):  
Simone Giannecchini ◽  
Daniela Del Mauro ◽  
Donatella Matteucci ◽  
Mauro Bendinelli
Keyword(s):  
Infected Cell ◽  
Feline Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Neutralizing Antibody ◽  
Cell Vaccine ◽  
Immunodeficiency Virus ◽  
Fixed Cell ◽  
Antibody Levels

ABSTRACT In the feline immunodeficiency virus system, immunization with a fixed-infected-cell vaccine conferred protection against virulent homologous challenge but the immune effectors involved remained elusive. In particular, few or no neutralizing antibodies were detected in sera from vaccinated cats. Here we show that, when preadsorbed with selected feline cells, the same sera revealed clearly evident virus-neutralizing activity. Because high titers of neutralizing antibody in cell-adsorbed sera from 23 cats immunized with fixed-infected-cell or whole-inactivated-virus vaccines correlated with protection, it is likely that they were more important for protection than formerly realized. In vitro, the fixed-cell vaccine efficiently removed neutralizing antibody from immune sera while the whole-inactivated-virus vaccine was much less effective.

scholarly journals During Readaptation In Vivo, a Tissue Culture-Adapted Strain of Feline Immunodeficiency Virus Reverts to Broad Neutralization Resistance at Different Times in Individual Hosts but through Changes at the Same Position of the Surface Glycoprotein

2001 ◽  
Vol 75 (10) ◽  
pp. 4584-4593 ◽  
Author(s):  
Mauro Bendinelli ◽  
Mauro Pistello ◽  
Daniela Del Mauro ◽  
Giancarlo Cammarota ◽  
Fabrizio Maggi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Amino Acid ◽  
Feline Immunodeficiency Virus ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Surface Glycoprotein ◽  
Intermediate Phenotype ◽  
Immunodeficiency Virus

ABSTRACT The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys→Asn or →Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser→Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.

scholarly journals AIDS Vaccination Studies Using an Ex Vivo Feline Immunodeficiency Virus Model: Failure To Protect and Possible Enhancement of Challenge Infection by Four Cell-Based Vaccines Prepared with Autologous Lymphoblasts

2002 ◽  
Vol 76 (14) ◽  
pp. 6882-6892 ◽  
Author(s):  
Simone Giannecchini ◽  
Patrizia Isola ◽  
Olimpia Sichi ◽  
Donatella Matteucci ◽  
Mauro Pistello ◽  
...  
Keyword(s):  
Feline Immunodeficiency Virus ◽  
Low Dose ◽  
Ex Vivo ◽  
Virus Dose ◽  
Immunodeficiency Virus ◽  
Virus Model ◽  
Infected Cells ◽  
Failure To Protect ◽  
Model Failure

ABSTRACT Immunogenicity and protective activity of four cell-based feline immunodeficiency virus (FIV) vaccines prepared with autologous lymphoblasts were investigated. One vaccine was composed of FIV-infected cells that were paraformaldehyde fixed at the peak of viral expression. The other vaccines were attempts to maximize the expression of protective epitopes that might become exposed as a result of virion binding to cells and essentially consisted of cells mildly fixed after saturation of their surface with adsorbed, internally inactivated FIV particles. The levels of FIV-specific lymphoproliferation exhibited by the vaccinees were comparable to the ones previously observed in vaccine-protected cats, but antibodies were largely directed to cell-derived constituents rather than to truly viral epitopes and had very poor FIV-neutralizing activity. Moreover, under one condition of testing, some vaccine sera enhanced FIV replication in vitro. As a further limit, the vaccines proved inefficient at priming animals for anamnestic immune responses. Two months after completion of primary immunization, the animals were challenged with a low dose of homologous ex vivo FIV. Collectively, 8 of 20 vaccinees developed infection versus one of nine animals mock immunized with fixed uninfected autologous lymphoblasts. After a boosting and rechallenge with a higher virus dose, all remaining animals became infected, thus confirming their lack of protection.

scholarly journals Novel bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI) UIC-94017 (TMC114) with Potent Activity against Multi-PI-Resistant Human Immunodeficiency Virus In Vitro

2003 ◽  
Vol 47 (10) ◽  
pp. 3123-3129 ◽  
Author(s):  
Yasuhiro Koh ◽  
Hirotomo Nakata ◽  
Kenji Maeda ◽  
Hiromi Ogata ◽  
Geoffrey Bilcer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitor ◽  
Therapeutic Agent ◽  
Close Contact ◽  
Wide Spectrum ◽  
Antiviral Agents ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (50% inhibitory concentration [IC50], ∼0.003 μM; IC90, ∼0.009 μM) with minimal cytotoxicity (50% cytotoxic concentration for CD4+ MT-2 cells, 74 μM). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected for resistance to saquinavir, indinavir, nelfinavir, or ritonavir at concentrations up to 5 μM (IC50s, 0.003 to 0.029 μM), although it was less active against HIV-1NL4-3 variants selected for resistance to amprenavir (IC50, 0.22 μM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active-site amino acids (Asp-29 and Asp-30) is important for its potency and wide spectrum of activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for the treatment of primary and multi-PI-resistant HIV-1 infections.

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