Patrick H. M. Boers

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes

Recent studies indicate that the time required for virus‐infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV‐1‐specific CTL, we measured virus production in cultures containing CD4+ T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev‐specific CTL prevented at least 2 log10 more HIV‐1 production, in 10 days, than similar numbers of RT‐specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL‐target cell interactions during successive infection cycles. The results show that substantially higher CTL‐mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT‐specific CTL were at least as effective as the Rev‐specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Antiviral resistance of biologic HIV-2 clones obtained from individuals on nucleoside reverse transcriptase inhibitor therapy

Objective: 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.

Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure.

ABSTRACT Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.

HIV-2-infected individuals with undetectable plasma viremia carry replication-competent virus in peripheral blood lymphocytes.

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 (rs = −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.

Antibody-Mediated Enhancement of Human Immunodeficiency Virus Type 1 Infectivity Is Determined by the Structure of gp120 and Depends on Modulation of the gp120-CCR5 Interaction

ABSTRACT 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.

Impact of natural sequence variation in the V2 region of the envelope protein of human immunodeficiency virus type 1 on syncytium induction: a mutational analysis

Several studies have demonstrated a functional role for the V1-V2 region of the human immunodeficiency virus type 1 (HIV-1) envelope surface glycoprotein gp120 in the membrane fusion processes underlying viral entry and syncytium induction. In a study with chimeric primary envelope genes, we have previously demonstrated that the exchange of V2 regions was sufficient to transfer syncytium-inducing capacity to a non-syncytium-inducing envelope protein. The exchanged V2 regions, comprising a number of variable amino acids, conferred changes to both the predicted secondary structure and to the net positive charge of the V2 loops. In a syncytium-forming assay based on transient envelope protein expression in CD4+ SupT1 cells, we have extended this observation by mutating the variable positions of the V2 region to determine the relative contribution of individual amino acids to syncytium formation. It can be shown that simultaneous mutation of multiple amino acids is needed to interfere with the V2 region-determined syncytium-inducing phenotype. Single amino acid changes either influencing charge of predicted secondary structure of the V2 loop proved to be insufficient to abolish V2 region-controlled syncytium formation. This robust V2 organization may allow the virus to accumulate mutations, while retaining its biological phenotype.

Decline of Simian Immunodeficiency Virus (SIV)-Specific Cytotoxic T Lymphocytes in the Peripheral Blood of Long-Term Nonprogressing Macaques Infected with SIVmac32H-J5

The evolution of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte precursors (CTLps) and their relationship with virus replication were studied in SIV-infected macaques. After primary viremia, 3 of 8 macaques lost culturable virus and polymerase chain reaction-detectable provirus in peripheral blood. Although proviral DNA persisted in the spleen and lymph nodes, virus loads were below or barely above detection levels. Throughout the study, the 3 macaques remained asymptomatic, with stable CD4+ cell counts. These findings were associated with the detection of CTLps directed against both structural and regulatory SIV proteins. The response peaked during the first 7 months of infection but waned subsequently. CTLps increased after rechallenge of 1 macaque, suggesting that limited antigenic stimulation contributed to their disappearance from circulation. Transient viremia with increasing CTLp frequencies and antibody titers also suggested at least partial susceptibility to reinfection. These findings bear implications for vaccination strategies aimed at inducing protective CTLs against lentiviruses.

Characterization of recombinant influenza A virus as a vector for HIV-1 p17Gag

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.

CCR5-Restricted HIV Type 2 Variants from Long-Term Aviremic Individuals Are Less Sensitive to Inhibition by β-Chemokines Than Low Pathogenic HIV Type 1 Variants

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-1alpha than HIV-1 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.

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef

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.