In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs). The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690. We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC 50 ) and IC 90 , PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M. Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC 50 s, >0.1 M). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T.

We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stromaderived factor 1␤ (SDF-1␤), Met-SDF-1␤, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1␤ had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.In 1995, Cocchi et al. reported potent in vitro human immunodeficiency virus type 1 (HIV-1) inhibition by three chemokines secreted by CD8 ϩ T lymphocytes (4) and focused attention on this class of molecules with low molecular masses (8 to 12 kDa). The chemokines described, RANTES, macrophage inflammatory protein-1␣ (MIP-1␣), and MIP-1␤, belong to the group of C-C chemokines that block HIV-1 entry into cells (5).Relationships among membrane coreceptors, chemokines, and cellular tropism were further defined in 1996 by Feng et al., who described a novel molecule which acted as a cofactor for T-cell-tropic HIV-1 isolates but not for macrophage-tropic isolates (14). This receptor, which was already known but did not have an identified natural ligand, belongs to the C-X-C chemokine receptor superfamily and was named "fusin," or CXCR4. The receptor for HIV-1 macrophage-tropic isolates was subsequently identified and named C-C chemokine receptor 5 (CCR5). CCR5 reacts with the chemokines RANTES, MIP-1␣, and MIP-1␤ (9, 12). The natural ligand for CXCR4 is stroma derived factor-1 (SDF-1), an ␣-chemokine with chemotactic properties for T lymphocytes and a developmental role in B lymphocyte maturation (2,25).During the early phases of HIV-1 infection, R5 viral strains usually predominate, whereas X4 strains frequently emerge in the late stages of HIV-1 infection, accompanied by a decline in peripheral blood CD4 lymphocytes and a clinical progression toward AIDS (7,34). Viral isolates with a dual tropism could represent a transition phase between viral R5 and X4 phenotypes, or they may represent X4 viruses that have maintained an ability to infect macrophages (31, 33).The aim of our study was to evaluate the interactions between attachment and entry inhibitors of HIV-1 infection. Our experiments suggest that the use of combined inhibitors of R5 and X4 viruses may be useful in inhibiting mixed infections. Analysis of cellular tropism of the different viral isolates on transformed cell lines. The two viral isolates examined in this study, RM and DK, were derived from two patients with primary HIV-1 infection acute syndrome (29), and the isolates were used to infect U87MG-transformed CD4 ϩ cells transfected with CCR5 or CXCR4 coreceptors (8), provided by Dan R. Littman (The Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New Yo...

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drugresistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions.

sequencing (NGS) technology. However, success of this approach depends on low prevalence of circulating antibodies against common AAV serotypes such as AAV2 and AAV9 because these serotypes serve as internal reference controls in this system. Therefore, it is essential to investigate the prevalence of neutralizing antibodies against various serotypes in cats for applying the AAV Barcode-Seq to preclinical studies and ultimately for successful translation of AAV vector-mediated gene therapy approaches into the veterinary clinic.Here we report the results of the initial screening of 30 cat serum samples obtained from the Liberty Research Inc. cat colony for the presence of antibodies against a total of 11 different AAV serotypes (AAV1 through 11). Such a study is imperative because a vaccine against feline panleukopenia virus (FPV), a feline parvovirus, is widely used to immunize cats and all the cats to be used for our study will have received that vaccine. In the study, serum samples were incubated in ELISA plates coated with serotype-specific particles followed by addition of an HRP-conjugated anti-cat IgG antibody. Anti-FPV antibody titers were determined by a hemagglutination inhibition test (Cornell University, Animal Health Diagnostic Center). The results showed that the majority of the 30 cats did not harbor any detectable antibodies against AAV1, 2, 3, 4, 5, 7, 8, 9, 10 or 11, while modest levels of anti-AAV6 antibodies were detected in two-thirds of the animals. Spearman's rank correlation coefficient between anti-AAV6 and anti-FPV antibody levels was 0.40, showing a positive correlation. In addition, the anti-FPV and anti-AAV6 antibody levels were significantly higher in the cats that had been vaccinated within 99 days prior to the blood sample collection (p=0.005 and p=0.008, respectively). These observations indicate that the prevalence of antibodies against the common AAV serotypes is very low in the population of cats we investigated even though the cats had developed high titers of anti-FPV antibodies, which appear to only cross-react with AAV6. We are currently investigating whether anti-AAV6 antibodies found in cat sera exhibit neutralizing activity.

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