Endothelial activation is associated with intestinal epithelial injury, systemic inflammation and treatment regimen in children living with vertically acquired HIV-1 infection.

BACKGROUND: Premature development of cardiovascular disease in children living with HIV-1 (CLWH) may be associated with compromised gut barrier function, microbial translocation, immune activation, systemic inflammation and endothelial activation. Biomarkers of these pathways may provide insights into pathogenesis of atherosclerotic disease in CLWH. METHODS: This was a cross-sectional study of CLWH enrolled in the multicentre Early Pediatric Initiation-Canadian Child Cure Cohort (EPIC4 ) who were on antiretroviral therapy (ART) with undetectable viral load. Plasma biomarkers of intestinal epithelial injury [intestinal fatty acid binding protein-1 (IFABP)], systemic inflammation [tumour necrosis factor (TNF) and interleukin-6 (IL-6)] and endothelial activation [angiopoietin-2 (Ang2), soluble vascular endothelial growth factor-1 (sVEGFR1) and soluble endoglin (sEng)] were quantified by enzyme-linked immunosorbent assay. Correlation and factor analysis of biomarkers were used to examine associations between innate immune pathways. RESULTS: Among 90 CLWH, 16% of Ang2, 15% of sVEGFR1 and 23% of sEng levels were elevated relative to healthy historic controls. Pairwise rank correlations between the three markers of endothelial activation were statistically significant (ρ = 0.69, ρ = 0.61 and ρ = 0.65, P < 0.001 for all correlations). An endothelial activation index, derived by factor analysis of the three endothelial biomarkers, was correlated with TNF (ρ = 0.47, P < 0.001), IL-6 (ρ = 0.60, P < 0.001) and intestinal fatty acid binding protein-1 (ρ = 0.67, P < 0.001). Current or past treatment with ritonavir-boosted lopinavir (LPV/r) was associated with endothelial activation (odds ratio = 5.0, 95% CI: 1.7-17, P = 0.0020). CONCLUSIONS: Endothelial activation is prevalent in CLWH despite viral suppression with combination ART and is associated with intestinal epithelial injury, systemic inflammation and treatment with LPV/r.

Limited CD4+ T-cell renewal in early HIV-1 infection: effect of highly active antiretroviral therapy.

We show that the fraction of proliferating CD4+ lymphocytes is similar in HIV-infected subjects in the early stage of disease and in HIV-negative subjects, whereas the fraction of proliferating CD8+ lymphocytes is increased 6.8-fold in HIV-infected subjects. After initiation of antiviral therapy, there is a late increase in proliferating CD4+ T cells associated with the restoration of CD4+ T-cell counts. These results provide strong support for the idea of limited CD4+ T-cell renewal in the early stage of HIV infection and indicate that after effective suppression of virus replication, the mechanisms of CD4+ T-cell production are still functional in early HIV infection.

Comparative analysis of the T cell receptor V beta repertoire in various lymphoid tissues from HIV-infected patients: evidence for an HIV-associated superantigen.

The search to identify HIV-associated molecules with superantigenic properties has focussed on demonstration of T cell receptor V beta subset-specific perturbations in HIV-infected patients. In this report, the V beta repertoire of two HIV patients was studied in lymph nodes, where HIV-1 replication is the most intense, and compared to the repertoire found in peripheral blood. Use of this fully MHC-compatible system has allowed identification of a small, partly overlapping set of V beta determinants which appear significantly perturbed in HIV-1 infection. These results are consistent with the presence of an HIV-associated superantigen.

Isolation of drug-resistant variants of HIV-1 from patients on long-term zidovudine therapy. Canadian Zidovudine Multi-Centre Study Group.

We determined whether drug-resistant variants of HIV-1 could be isolated from the peripheral blood mononuclear cells of 20 individuals with HIV infection (Centers for Disease Control groups II and III) on long-term zidovudine (AZT) therapy. Toward this end, zidovudine (10 microM) has been included in the tissue culture medium used to isolate HIV-1. Under these circumstances, virus with a zidovudine-resistant phenotype was successfully obtained in five out of 20 cases. This property of drug resistance appeared to be stable, and did not disappear upon extended replication of such virus in the absence of drug pressure. Drug-resistant virus could also be isolated from these subjects on subsequent occasions, but was not present in samples obtained prior to therapy. Replication of these zidovudine-resistant isolates in tissue culture was inhibited by each of four other nucleoside analogues. Thus, other drugs may be useful in controlling selective zidovudine-resistant variants of HIV-1.

New mechanisms of viral persistence in primary human immunodeficiency virus (HIV) infection.

Viruses, including the Human Immunodeficiency Virus (HIV), have evolved multiple strategies to overcome host immune defenses, allowing them to persist in the host. Molecular and cellular approaches were simultaneously used to provide sensitive and unbiased delineation of the diversity and dynamics of the immune response, and to study the relative compartimentalization of HIV-specific CTL clones in patients undergoing primary HIV infection. This approach revealed that some HIV-specific CTL clones can be deleted in presence of high levels of antigen, a phenomenon analogous to high-dose tolerance or clonal exhaustion described in murine models of persistent viral infections. Also, HIV-specific CTL clones were found to accumulate preferentially in peripheral blood as compared to lymph nodes, even though the large majority of viral replication during primary HIV infection takes place within lymph nodes. These two mechanisms may decrease the effectiveness of the host cell-mediated immune responses, and favor the establishment of virus persistence during primary HIV infection.

The moving target: mechanisms of HIV persistence during primary infection.

The vigorous host immune responses detected during primary HIV infection fail to prevent the establishment of chronic infection because HIV has evolved multiple strategies to make these responses ineffective. Here, Hugo Soudeyns and Giuseppe Pantaleo discuss the mechanisms that allow HIV to persist in the host.

Identification of a novel glucocorticoid response element within the genome of the human immunodeficiency virus type 1.

We have shown that (HIV-1) replication can be regulated by interaction between glucocorticoid hormones and the viral genome; treatment of acutely infected lymphoid and monocytoid cell lines with cortisol and dexamethasone increased HIV-1 production in culture. The magnitude of this response correlated with glucocorticoid receptor (GR) and GR message in responder and non-responder cell lines. Furthermore, treatment of each of two HIV-infected cell lines with glucocorticoids led to enhancement of HIV-1 gene expression. We have identified a novel intragenic glucocorticoid response element (GRE) within the genome of HIV-1 at position +5002 in the vif open reading frame, as well as a second potential GRE, previously identified by other researchers at position -257 in the HIV-1 negative regulatory element (LTR-NRE). Our data indicate that only the motif at position +5002 represents a true GRE that confers glucocorticoid inducibility to a MMTV-luciferase reporter gene construct. The GRE located at -257 lacked significant functional activity in its native configuration in that it could bind GR but did not transactivate reporter gene constructs. However, this sequence was able to impart glucocorticoid inducibility when inverted or dimerized. These results suggest that as in the case of other retroviruses, HIV-1 has evolved to interface with the GR signal transduction pathway to gain replicative advantage in target cells.

The T cell receptor V beta repertoire in HIV-1 infection and disease.

Viral superantigens (SAg) were shown in mice to induce anergy and deletion of T cells bearing specific T cell receptor V beta subsets, these perturbations being mainly restricted to CD4+ T cells. In accordance with this model, a putative HIV-associated SAg could contribute to the pathogenesis of HIV-1 infection and AIDS. To reveal the presence of this putative molecule, three study protocols were designed that relied on the fact that similarity of the expressed V beta repertoire of a given pair of individuals is proportional to the relative likeness of their MHC background: (1) by using a quantitative PCR technique that allows simultaneous typing of 24 V beta families, the V beta repertoires of HIV-discordant monozygotic twins were compared; (2) the V beta repertoire found in lymph nodes of HIV-infected subjects was contrasted to that found in peripheral blood of the same individuals; (3) the V beta repertoire of a cohort of HIV-infected mothers was compared with that of their HIV-infected and uninfected children. Results from these approaches revealed that significant perturbations of the TCR V beta repertoire were taking place in HIV-infected subjects, and that these alterations were restricted to T cells expressing specific V beta s. These results are consistent with the presence of an HIV-associated SAg in HIV-1 infection.

Biological comparison of wild-type and zidovudine-resistant isolates of human immunodeficiency virus type 1 from the same subjects: susceptibility and resistance to other drugs.

We used a viral endpoint dilution assay to show changes in the proportion of zidovudine (azidothymidine; AZT)-resistant viruses within a heterogeneous mixture of human immunodeficiency virus type 1 (HIV-1) quasispecies isolated from patients on long-term AZT therapy. Several HIV-1 isolates, which could replicate in 10 microM AZT, were susceptible to both 2',3'-dideoxycytidine and a novel cytosine analog BCH-189, in which a sulfur atom replaces the 3' carbon of the pentose ring. In certain instances, cross-resistance was seen with 3'-didehydro-2',3'-dideoxythymidine. Although most strains of AZT-resistant HIV-1 displayed reduced susceptibility to 3'-azido-2',3'-dideoxyuridine, two strains were identified for which this was not the case.

Major expansion of CD8+ T cells with a predominant V beta usage during the primary immune response to HIV.

A SIGNIFICANT proportion (up to 70%) of individuals experience an acute clinical syndrome of varying severity associated with primary infection with the human immunodeficiency virus (HIV). We report here studies on six individuals who showed an acute HIV syndrome which generally resolved within four weeks, concomitant with a dramatic downregulation of viraemia. To characterize the T-cell-mediated primary immune response to HIV, we used combined semiquantitative polymerase chain reaction assay and cytofluorometry to analyse the T-cell antigen receptor repertoire in sequential peripheral blood mononuclear cells from the patients. We found major oligoclonal expansions in a restricted set of variable-domain beta-chain (V beta) families. Cells expressing the expanded V beta s predominantly expressed the CD8 T-cell differentiation antigen and mediated HIV-specific cytotoxicity. Major oligoclonal expansions of these CD8+ T lymphocytes may represent an important component of the primary immune response to viral infections and may help to clarify both the immunopathogenic and the protective mechanisms of HIV infection.

Analysis of the T-cell receptor beta-chain variable-region (V beta) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific V beta segments in CD4+ T cells of the virus-positive twins.

We analyzed the T-cell receptor (TCR) V beta repertoire in human immunodeficiency virus type 1 (HIV-1)-infected individuals at different stages of disease. To circumvent the effect of HLA and other loci on the expressed TCR repertoire, we compared the TCR repertoire in nine pairs of monozygotic twins who were discordant for HIV infection. A semiquantitative polymerase chain reaction (PCR) assay and flow cytometry enabled us to show distinct differences in the V beta repertoire in the HIV-positive twin compared with the HIV-negative twin. By combining PCR and cytofluorometry, these differences were restricted to a specific set of TCR V beta segments, with members of the V beta 13 family perturbed in six out of seven cases and those of the V beta 21 family perturbed in four out of seven cases studied. Most of the other V beta families remained unchanged. Our results provide direct evidence for a skewed TCR repertoire in HIV infection.

Anti-human immunodeficiency virus type 1 activity and in vitro toxicity of 2'-deoxy-3'-thiacytidine (BCH-189), a novel heterocyclic nucleoside analog.

We describe a novel nucleoside analog, 2'-deoxy-3'-thiacytidine (BCH-189), in which the 3' carbon of the ribose ring of 2'-deoxycytidine has been replaced by a sulfur atom. In MT-4 T cells, this compound had significant time- and dose-dependent antiviral activity against five different strains of human immunodeficiency virus type 1 (HIV-1) (mean 50% inhibitory dose, 0.73 microM); known 3'-azido-3'-deoxythymidine (AZT)-resistant HIV-1 variants did not exhibit cross-resistance to it. BCH-189 also suppressed HIV-1 replication in the U937 monocytoid cell line as well as in primary cultures of human peripheral blood mononuclear cells; in these latter systems, suppression was fuller and longer lasting than that induced by AZT. Moreover, BCH-189 was less toxic than AZT in cell culture. BCH-189 may be a promising drug for the treatment of HIV-1-associated disease.

Initiation of antiretroviral therapy during primary HIV-1 infection induces rapid stabilization of the T-cell receptor beta chain repertoire and reduces the level of T-cell oligoclonality.

Major T-cell receptor beta chain variable region (TCRBV) repertoire perturbations are temporally associated with the down-regulation of viremia during primary human immunodeficiency virus (HIV) infection and with oligoclonal expansion and clonal exhaustion of HIV-specific cytotoxic T lymphocytes (CTLs). To determine whether initiation of antiretroviral therapy (ART) or highly active antiretroviral therapy (HAART) during primary infection influences the dynamics of T-cell-mediated immune responses, the TCRBV repertoire was analyzed by semiquantitative polymerase chain reaction in serial blood samples obtained from 11 untreated and 11 ART-treated patients. Repertoire variations were evaluated longitudinally. Stabilization of the TCRBV repertoire was more consistently observed in treated as compared with untreated patients. Furthermore, the extent and the rapidity of stabilization were significantly different in treated versus untreated patients. TCRBV repertoire stabilization was positively correlated with the slope of HIV viremia in the treated group, suggesting an association between repertoire stabilization and virologic response to treatment. To test whether stabilization was associated with variations in the clonal complexity of T-cell populations, T-cell receptor (TCR) heteroduplex mobility shift assays (HMAs) were performed on sequential samples from 4 HAART-treated subjects. Densitometric analysis of HMA profiles showed a reduction in the number of TCR clonotypes in most TCRBV families and a significant decrease in the total number of clonotypes following 7 months of HAART. Furthermore, a biphasic decline in HIV-specific but not heterologous CTL clones was observed. This indicates that ART leads to a global reduction of CD8(+) T-cell oligoclonality and significantly modulates the mobilization of HIV-specific CTL during primary infection. (Blood. 2000;95:1743-1751)

Transient T cell receptor beta-chain variable region-specific expansions of CD4+ and CD8+ T cells during the early phase of pediatric human immunodeficiency virus infection: characterization of expanded cell populations by T cell receptor phenotyping.

T cell receptor (TCR) repertoire perturbations are commonly detected in CD8+ T cells during adult primary human immunodeficiency virus (HIV) infection and have been associated with HIV-specific cytotoxic T cell responses. By use of flow cytometry, transient high-level TCR beta-chain variable region-specific expansions of CD4+ and CD8+ T cells were observed more frequently in HIV-infected children than in children exposed to HIV who remained uninfected. TCR beta-chain diversity analysis and diversity-specific polymerase chain reaction were used to study the clonality of expanded CD4+ and CD8+ subsets. In CD8+ T cells, structural features of the complement-determining regions 3 were altered during the course of the expansion, and persistent TCR clonotypes were observed, consistent with antigen-driven selection. In contrast, TCR beta-chain variable region-specific expansions without clonotypic overrepresentation or persistence were observed in CD4+ T cells, possibly related to HIV-specific helper T cell responses or to the progressive destruction of the CD4+ cell compartment.

Selective pressure exerted by immunodominant HIV-1-specific cytotoxic T lymphocyte responses during primary infection drives genetic variation restricted to the cognate epitope.

HIV-specific cytotoxic T lymphocytes (CTL) play a central role in the control of HIV-1 replication during primary infection. It has been hypothesized that the appearance of CTL escape mutants represents an important mechanism by which HIV-1 escapes the host cell-mediated immune response. However, evidences for a direct relationship between CTL responses and emergence of CTL escape mutants are still limited. Here we report detailed longitudinal analysis of DNA sequence variation performed over the entire HIV-1 envelope in two subjects during primary HIV infection. Estimates of the frequencies of synonymous (ds) and non-synonymous (dN) nucleotide substitutions were used to identify regions of the HIV-1 envelope which were subjected to significant levels of selective pressure. These regions were shown to comprise defined epitopes recognized by CTL. Furthermore, dN mutation fixed within these epitopes effectively abolished recognition by the host CTL response. These results provide compelling evidence that the CTL epitope mutations directly resulted from the selective pressure exerted by the virus-specific cytotoxic response.

Evidence for rapid disappearance of initially expanded HIV-specific CD8+ T cell clones during primary HIV infection.

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.