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Background A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical trials registration NCT02401828.

We have generated a recombinant influenza A virus with the HIV-1 p17(Gag) (rFlu-p17) gene inserted into the influenza virus neuraminidase (NA) gene. Expression of HIV-1 p17 protein was detected by conventional Western blot analysis and also by liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of rFlu-p17 infected cells. The latter method does not depend on protein-specific antibody preparations and proved to be a powerful tool to detect proteins of interest. Next, antigen presentation of p17 expressed after infection of antigen-presenting cells was determined. Cloned p17-specific CD8+ T-cells were co-cultured with rFlu-p17 infected B-cells and produced IFN-gamma upon stimulation. Furthermore, we showed that immunization with rFlu-p17 elicited a humoral immune response in mice. This study shows that replication-deficient rFlu-p17 is antigenic in vitro and immunogenic in vivo and warrants further development as a candidate vaccine vector. (C) 2009 Elsevier Ltd. All rights reserved.

Many HIV-2-infected individuals maintain low, often undetectable, viral loads for prolonged periods. Virus and/or host factors that contribute to this high level of virus control are largely unknown. Previously we demonstrated that HIV-2 variants from long-term aviremic individuals have relatively low replication kinetics in vitro in comparison to HIV-1 variants. We hypothesized that the relatively low replication rates of HIV-2 in vitro as well as the high level of virus control in vivo might be explained by HIV-2 replication being more sensitive to inhibitory host factors like beta-chemokines or other CD8(+) T cell-derived factors than HIV-1 replication. To test this we determined the effect of exogenously added beta-chemokines and healthy donor CD8 (+) T cells on the in vitro virus production of HIV-2 and HIV-1 variants from long-term nonprogressors (LTNPs). Contrary to expectations, HIV-2 replication was inhibited less efficiently by RANTES and MIP-1 alpha than HIV1 replication. CD8(+) T cells from 8 of 12 healthy donors reduced HIV replication minimally 2-fold. Interestingly, cells from five of these donors inhibited HIV-1 but hardly affected HIV-2 replication, while the reverse was observed for cells from one donor. For HIV-1, but not HIV-2, the magnitude of the antiviral effect of CD8(+) T cells correlated with their effect on RANTES levels in culture supernatants. Our findings indicate that RANTES is a more important factor of CD8 (+) T cell-associated anti-HIV-1 activity than it is of HIV-2 activity and that the benign clinical course of HIV-2 infection is not due to enhanced beta-chemokine sensitivity of HIV-2 variants.

To establish whether efficient suppression of virus replication in HIV-2-infected individuals is associated with low replicative capacity of HIV-2, replication kinetics of HIV-2 variants from long-term aviremic individuals was analyzed and compared with that of the relatively slow-replicating HIV-1 variants from asymptomatics and long-term nonprogressors (AS/LTNP). On average, HIV-2 from aviremic individuals had lower replication rates than HIV-1 variants from AS/LTNP in cells of 8 donors (0.45 log10 [range 0.14–0.77] vs. 0.58 log10 [range 0.32–0.99] pg RT/ml/day, P = 0.036). The relatively low replication rate of HIV-2 compared to HIV-1 variants was not related to different sensitivities to inhibition by CD8+ T cells or different degrees of infectivity. HIV-2 replication rates increased with progressive infection and with switch from CCR5 to CXCR4 usage.The relatively low replicative capacity of HIV-2 variants from aviremic individuals likely contributes to the low viral load and benign course of infection in these individuals.

Human immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associated with progressive infection could not be excluded. To test this, we determined the coreceptor usage of 43 HIV-2 variants isolated from six long-term-infected individuals with undetectable plasma viremia. Using GHOST indicator cells, we showed for the first time that the only coreceptors efficiently used by low-pathogenic HIV-2 variants are CCR5, GPR15 (BOB), and CXCR6 (BONZO). Surprisingly, control HIV-2 variants ( n = 45) isolated from seven viremic individuals also mainly used these three coreceptors, whereas use of CCR1, CCR2b, or CCR3 was rare. Nearly a quarter of all HIV-2 variants tested could infect the parental GHOST cells, which could be partially explained by CXCR4 usage. Use of CXCR4 was observed only for HIV-2 variants from viremic individuals. Thirty-eight variants from aviremic and viremic HIV-2-infected individuals were additionally tested in U87 cells. All except one were capable of infecting the parental U87 cells, often with high efficiency. When virus production in parental cells was regarded as background in the coreceptor-transduced cell lines, the results in U87 cells were largely in agreement with the findings in GHOST cells. HIV-2 isolates from aviremic individuals commonly use as coreceptors CCR5, GPR15, and CXCR6, as well as an unidentified receptor expressed by U87 cells. Broad coreceptor usage, therefore, does not appear to be associated with pathogenicity of HIV-2.

Using an optimized HIV co-culture protocol it was possible to isolate infectious HIV-2 variants from 6 HIV-2-infected individuals who had undetectable plasma viremia and maintained high CD4 + T-cell numbers for prolonged periods. This shows for the first time that HIV-2-infected individuals with no demonstrable in vivo virus production carry replication-competent virus in peripheral blood mononuclear cells (PBMCs). The frequency of PBMCs with infectious virus was low, ranging from 0.01-0.9 infectious units per million (IUPM) CD4' T cells with a median value of 0.2 IUPM. In comparison, viremic HIV-2-infected individuals had a 2-log higher median infectious load (36 IUPM, range 1-673; P = 0.003). HIV-2 infectious load correlated with CD4 counts (r s = -0.88, P < 0.0001). The low infectious load in aviremic HIV-2-infected persons is reminiscent of what has been observed for HIV-1 infection controlled by highly active antiretroviral therapy.

In this study, we characterized the viral determinants of coreceptor usage in relation to susceptibility to antibody-mediated neutralization or enhancement of infectivity by using chimeras of three highly related human immunodeficiency virus type 1 (HIV-1) isolates of different phenotypes. We found that the V3 region was the main determinant of antibody-mediated enhancement and coreceptor specificity but that the overall structure of gp120 was also important for these properties. Constructs susceptible to antibody-mediated enhancement preferentially use CCR5 as a coreceptor, in contrast to constructs that were neutralized or not affected. Using monoclonal antibodies directed against CD4 or CCR5, we were able to show that antibody-mediated enhancement was CD4 dependent. Altogether, our results suggest that the modulation of the interaction of gp120 with CCR5 is the mechanism underlying antibody-mediated enhancement of HIV-1 infectivity.

textabstractObjective: To study phenotypic and genotypic resistance of HIV-2 against nucleoside reverse transcriptase inhibitors (NRTI). Methods: Biologic HIV-2 clones were generated from 3 patients before and after initiation of antiretroviral therapy with zidovudine (AZT) in patient RH2-7, AZT and didanosine (ddI) in patient PH2-1, and after addition of lamivudine (3TC) to AZT monotherapy in patient RH2-5. The sensitivity to NRTI of the virus clones, as defined by the 50% inhibitory concentration (IC50), was determined in vitro. The predicted amino acid sequences of the reverse transcriptase proteins from these clones were determined. Results: Comparing the sensitivity of the biologic HIV-2 clones obtained after start of therapy with those from antiviral naive patients, resistance had developed to AZT (patients RH2-7 and RH2-5) and 3TC (patient PH2-1 and RH2-5). No resistance to AZT was observed in the biologic clone from PH2-1 obtained after start of therapy. The resistant clones from RH2-5 and PH2-1, but not RH2-7, contained amino acid mutations at positions where HIV-1 has been shown to mutate after AZT and 3TC treatment. Conclusions: Phenotypic resistance of HIV-2 to nucleoside analogues, which developed in HIV-2-infected patients treated with NRTIs, was associated with genotypic changes. Some of the mutations at amino acid positions in the HIV-2 reverse transcriptase gene corresponded with those involved in HIV-1 resistance, although no conventional mutations associated with resistance to AZT were observed.

The pathogenic properties of four primary human immunodeficiency virus type 2 (HIV-2) isolates and two primary HIV-2 biological clones were studied in an in vivo human-to-mouse chimeric model. The cell-associated viral load and the ability to reduce the severity of the induced graft-versus-host disease symptoms, the CD4/CD8 ratio and the level of repopulation of the mouse tissues by the graft, were determined. All HIV-2 strains, irrespective of their in vitro biological phenotype, replicated to high titres and significantly reduced graft-versus-host disease symptoms as well as the CD4/CD8 ratios. Reduction of graft repopulation caused by infection with the respective HIV-2 strains showed that the in vitro replication rate, syncytium-inducing capacity and ability to infect human macrophages did influence the in vivo pathogenic potential whereas broadening of coreceptor usage did not.The pathogenic properties of four primary human immunodeficiency virus type 2 (HIV-2) isolates and two primary HIV-2 biological clones were studied in an in vivo human-to-mouse chimeric model. The cell-associated viral load and the ability to reduce the severity of the induced graft-versus-host disease symptoms, the CD4/CD8 ratio and the level of repopulation of the mouse tissues by the graft, were determined. All HIV-2 strains, irrespective of their in vitro biological phenotype, replicated to high titres and significantly reduced graft-versus-host disease symptoms as well as the CD4/CD8 ratios. Reduction of graft repopulation caused by infection with the respective HIV-2 strains showed that the in vitro replication rate, syncytium-inducing capacity and ability to infect human macrophages did influence the in vivo pathogenic potential whereas broadening of coreceptor usage did not.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.