Florence Buseyne

MHC-I–restricted presentation of HIV-1 virion antigens without viral replication

Dendritic cells and macrophages can process extracellular antigens for presentation by MHC-I molecules. This exogenous pathway may have a crucial role in the activation of CD8+ cytotoxic T lymphocytes during human viral infections. We show here that HIV-1 epitopes derived from incoming virions are presented through the exogenous MHC-I pathway in primary human dendritic cells, and to a lower extent in macrophages, leading to cytotoxic T-lymphocyte activation in the absence of viral protein synthesis. Exogenous antigen presentation required adequate virus-receptor interactions and fusion of viral and cellular membranes. These results provide new insights into how anti-HIV cytotoxic T lymphocytes can be activated and have implications for anti-HIV vaccine design.

Distinct Trafficking Pathways Mediate Nef-Induced and Clathrin-Dependent Major Histocompatibility Complex Class I Down-Regulation

ABSTRACT The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.

Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load

ABSTRACT HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106 peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.

Cytotoxic T Lymphocytes in Human Immunodeficiency Virus Infection: Regulator Genes

Like all retroviruses, the human immunodeficiency virus (HIV) provirus contains two long terminal repeat elements (LTR) along with the three structural genes—gag, pol, and env—that are essential for virus replication. In addition to these genetics elements, HIV contains at least six additional genes: tat, rev, nef, vif, vpr, and vpu, whose functions impart both positive and negative effects on the HIV life cycle (for review see Cullen 1991; Greene 1991; Haseltine 1991; Steffie and Wong Staal 1991; Feinberg and Greene 1992).

HIV-specific CD8+ T-cell immune responses and viral replication

AimTo review current knowledge of CD8+ T cells in relation to their effect on the replication of HIV and on disease progression. Present knowledgeBoth CD8+ cytotoxic T lymphocytes capable of killing cells expressing HIV antigens and CD8+ lymphocytes that suppress HIV replication in vitro are detectable in response to HIV infection. ConclusionThese CD8+ T cells may help to maintain a low viral load in vivo, thus allowing a long asymptomatic period of infection.

Relationships Between HIV Disease History and Blood HIV-1 DNA Load in Perinatally Infected Adolescents and Young Adults: The ANRS-EP38-IMMIP Study

BACKGROUND Our aim was to study the impact of lifelong human immunodeficiency virus (HIV) disease history on the current immune and virological status of perinatally infected patients reaching adulthood. We evaluated blood cell-associated HIV DNA load as an indicator of cell-associated HIV reservoirs and an independent predictor of disease progression. METHODS The ANRS-EP38-IMMIP Study included 93 patients aged 15-24 years who were infected with HIV during the perinatal period. HIV DNA load was quantified by real-time polymerase chain reaction. RESULTS Eighty-five percent of patients were receiving highly active antiretroviral therapy (HAART), and HIV RNA was undetectable in the plasma of 75% of these patients. The median HIV DNA load was 2.84 (interquartile range, 2.51-3.16) log(10) copies per 10(6) peripheral blood mononuclear cells. In patients with viral suppression, HIV DNA load was independently associated with cumulative HIV RNA viremia over the last 5 years. HIV DNA load was negatively correlated with CD4 cell count in patients with active replication but not in those with undetectable HIV RNA. CONCLUSIONS In perinatally infected youths who are successfully treated, sustained viral suppression is associated with a low HIV DNA load. The absence of association between current HIV DNA load and CD4 cell counts suggests that the unique physiological characteristics of pediatric infection persist after adolescence. CLINICAL TRIALS REGISTRATION NCT01055873.

Multispecific and heterogeneous recognition of the gag protein by cytotoxic T lymphocytes (CTL) from HIV-infected patients: factors other than the MHC control the epitopic specificities

The HIV gag polyprotein is a major target for recognition by CTL in infected humans. Using recombinant vaccinia viruses (rVV) expressing truncations of the p24gag, and the p18gag pl5gagand HIV‐2 p56gag proteins, the characterization of epitope regions recognized by in vitro‐stimulated peripheral blood mononuclear ceils (PBMC) from 18 infected patients has been studied. The gag‐specific response of most individuals is polyclonal and multispecific, and inter‐individual variations between target epitope regions were frequently observed, despite shared MHC alleles. As CTL may play an important role in the control of HIV replication in infected hosts, these results have important implications for designing vaccine strategies.

The Frequency of HIV-Specific Interferon-γ–Producing CD8 T Cells Is Associated with Both Age and Level of Antigenic Stimulation in HIV-1–Infected Children

Ex vivo interferon (IFN)- gamma -producing CD8 T cells specific for human immunodeficiency virus (HIV) Env, Gag, and Pol antigens were measured in the peripheral blood of 55 children not receiving highly active antiretroviral therapy (HAART) and 70 children receiving HAART. In children not receiving HAART, the frequency of HIV-specific IFN- gamma -producing CD8 T cells was positively correlated with age and was not associated with plasma viral load or CD4 T cell levels. In children receiving HAART, the frequency of HIV-specific IFN- gamma -producing CD8 T cells was directly correlated with plasma viral load, and its association with age remained significant. In conclusion, the frequency of HIV-specific IFN- gamma -producing CD8 T cells in children is primarily determined by both age and plasma viral load.

Characterization of an HIV-1 p24gag epitope recognized by a CD8+ cytotoxic T-cell clone

A CD8+ cytotoxic T-cell clone that recognized HIV p24gag was isolated from an infected individual. The minimal epitope was localized to amino acids 308-316 (QASQEVKNW). Using allogeneic target cells, we found that lysis was restricted by the HLA-Cw0401 molecule. We observed that C1R cells, that express the HLA-Cw0401 allele are able to present the peptide to the cytotoxic clone, but with reduced efficiency. Other B-cell lines, that have been genotyped as HLA-Cw0401+ were unable to present the peptide to the clone, suggesting the existence of other variants of HLA-Cw0401 or a loss of cell surface expression of this molecule.

Cytotoxic T lymphocytes generation capacity in early life with particular reference to HIV.

Most of our knowledge concerning the presence of virus specific cytotoxic T lymphocytes (CTL) in early life has been provided by studies of CTL activities against human immunodeficiency virus type 1 (HIV-1) in infected infants born to HIV-infected mothers. HIV-specific cytolytic responses were found to be similar in perinatally infected children compared with adults, with respect to the nature of effector cells, protein recognized and the ability to control viral replication. CTL responses measured immediately after PBMC isolation (ex vivo activated CTL) were observed predominantly in children with no or mild symptoms, and the presence of in vitro activated CTL was found to be associated with the absence of severe symptoms during the first year of life and survival over 5 years.