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BackgroundEarly combined antiretroviral therapy (cART) limits the total HIV-DNA load in children. However, data on its impact in older children and adolescents remain scarce. This study compares HIV reservoirs in children (5–12 years) and adolescents (13–17 years) who started cART 2 years (late [L-] group).MethodsThe ANRS-EP59-CLEAC study prospectively enrolled 76 patients perinatally infected with HIV-1 who reached HIV-RNA ResultsTotal HIV-DNA levels were lower in early- versus late-treated patients (children: 2.14 vs 2.87 log copies/million PBMCs; adolescents: 2.25 vs 2.74 log; P < .0001 for both). Low reservoir was independently associated with treatment precocity, protective HLA, and low cumulative viremia since cART initiation. The 60 participants with undetectable integrated HIV-DNA started cART earlier than other patients (4 vs 54 months; P = .03). In those with sustained virological control, transitional and effector memory CD4+ T cells were less infected in the E-group than in the L-group (P = .03 and .02, respectively). Viral inducibility of reservoir cells after normalization to HIV-DNA levels was similar between groups.ConclusionsEarly cART results in a smaller blood HIV reservoir until adolescence, but all tested participants had an inducible reservoir. This deserves cautious consideration for HIV remission strategies.

Zoonotic simian foamy viruses (SFV) establish lifelong infection in their human hosts. Despite repeated transmission of SFV from nonhuman primates to humans, neither transmission between human hosts nor severe clinical manifestations have been reported. We aim to study the immune responses elicited by chronic infection with this retrovirus and previously reported that SFV-infected individuals generate potent neutralizing antibodies that block cell infection by viral particles. Here, we assessed whether human plasma antibodies block SFV cell-to-cell transmission and present the first description of cell-to-cell spreading of zoonotic gorilla SFV. We set-up a microtitration assay to quantify the ability of plasma samples from 20 Central African individuals infected with gorilla SFV and 9 uninfected controls to block cell-associated transmission of zoonotic gorilla SFV strains. We used flow-based cell cytometry and fluorescence microscopy to study envelope protein (Env) localization and the capacity of plasma antibodies to bind to infected cells. We visualized the cell-to-cell spread of SFV by real-time live imaging of a GFP-expressing prototype foamy virus (CI-PFV) strain. None of the samples neutralized cell-associated SFV infection, despite the inhibition of cell-free virus. We detected gorilla SFV Env in the perinuclear region, cytoplasmic vesicles and at the cell surface. We found that plasma antibodies bind to Env located at the surface of cells infected with primary gorilla SFV strains. Extracellular labeling of SFV proteins by human plasma samples showed patchy staining at the base of the cell and dense continuous staining at the cell apex, as well as staining in the intercellular connections that formed when previously connected cells separated from each other. In conclusion, SFV-specific antibodies from infected humans do not block cell-to-cell transmission, at leastin vitro, despite their capacity to bind to the surface of infected cells.Trial registration: Clinical trial registration:www.clinicaltrials.gov,https://clinicaltrials.gov/ct2/show/NCT03225794/.

BackgroundZoonotic simian foamy viruses (SFVs) establish persistent infections in humans, for whom the long-term consequences for health are poorly described. In this study, we aimed to characterize blood-cell phenotypes and plasma biomarkers associated with gorilla SFV infection in humans.MethodsWe used a case-control design to compare 15 Cameroonian hunters infected with gorilla SFV (cases) to 15 controls matched for age and ethnicity. A flow cytometry-based phenotypic study and quantification of plasma immune biomarkers were carried out on blood samples from all participants. Wilcoxon signed-rank tests were used to compare cases and controls.ResultsCases had a significantly higher percentage of CD8 T lymphocytes than controls (median, 17.6% vs 13.7%; P = .03) but similar levels of B, natural killer, and CD4 T lymphocytes. Cases also had a lower proportion of recent CD4 thymic emigrants (10.9% vs 18.6%, P = .05), a higher proportion of programmed death receptor 1 (PD-1) expressing memory CD4 T lymphocytes (31.7% vs 24.7%, P = .01), and higher plasma levels of the soluble CD163 scavenger receptor (0.84 vs .59 µg/mL, P = .003) than controls.ConclusionsWe show, for the first time, that chronic infection with SFV is associated with T lymphocyte differentiation and monocyte activation.

Spumaretroviruses, commonly referred to as foamy viruses, are complex retroviruses belonging to the subfamily Spumaretrovirinae, family Retroviridae, which naturally infect a variety of animals including nonhuman primates (NHPs). Additionally, cross-species transmissions of simian foamy viruses (SFVs) to humans have occurred following exposure to tissues of infected NHPs. Recent research has led to the identification of previously unknown exogenous foamy viruses, and to the discovery of endogenous spumaretrovirus sequences in a variety of host genomes. Here, we describe an updated spumaretrovirus taxonomy that has been recently accepted by the International Committee on Taxonomy of Viruses (ICTV) Executive Committee, and describe a virus nomenclature that is generally consistent with that used for other retroviruses, such as lentiviruses and deltaretroviruses. This taxonomy can be applied to distinguish different, but closely related, primate (e.g., human, ape, simian) foamy viruses as well as those from other hosts. This proposal accounts for host-virus co-speciation and cross-species transmission.

In perinatally HIV-1-infected youths living in France, we previously reported that Gag-specific CD4 and CD8 T cell proliferation is more frequently detected in patients of black ethnicity than in those of other ethnicities. We observed that black patients had higher levels of dendritic cells (DCs) than other patients. We aimed at studying the association of DC levels with Gag-specific T cell proliferation. The ANRS-EP38-IMMIP study is an observational study of youths aged between 15 and 24 years who were perinatally infected with HIV. A single blood sample was drawn for virological and immunological assays. Data from cART-treated 53 youths with undetectable plasma HIV RNA were analyzed. Gag-specific T cell proliferation was assessed by using a CFSE-based test. Peripheral blood myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) were phenotyped by flow cytometry. Plasma markers were quantified by ELISA or multiplex assays. Logistic regression was used for univariate and multivariate analyses. Patients with Gag-specific CD4 T cell proliferative responses had significantly higher percentages and absolute counts of mDCs and pDCs in the peripheral blood than nonresponding patients. Gag-specific CD4 and CD8 T cell proliferation was associated with lower plasma sCD14 levels. Plasma levels of IFN-α, TRAIL, and chemokines involved in T cell migration to secondary lymphoid organs were not associated with T cell proliferation. Multivariate analysis confirmed the association between Gag-specific CD4 T cell proliferation and pDC levels. In conclusion, DC levels are a robust correlate of the presence of Gag-specific T cell proliferation in successfully treated youths.

International audience; Cross-species transmission of simian foamy viruses (SFVs) from nonhu-man primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently non-pathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N 96-V 110 located in the leader peptide, gp18 LP. Forty-three variant peptides with truncations, alanine substitutions, or amino acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old World monkey SFV species. The magnitude of binding to peptide N 96-V 110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodomi-nant B-cell epitope recognized by humans following zoonotic SFV infection. IMPORTANCE Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. SFVs can be transmitted to humans, in whom they establish persistent infection, like the simian lenti-and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 and human T-lymphotropic virus type 1. This is the first identification of an SFV-specific B-cell epitope recognized by human plasma samples. The immunodominant epitope lies in gp18 LP , probably at the base of the envelope trimers. The NHP species the most genetically related to humans transmitted SFV strains that induced the strongest antibody responses. Importantly, this epitope is well conserved across SFV species that infect African and Asian NHPs.

International audience; Background. A spillover of simian foamy virus (SFV) to humans, following bites from infected nonhuman primates (NHPs), is ongoing in exposed populations. These retroviruses establish persistent infections of unknown physiological consequences to the human host. Methods. We performed a case-control study to compare 24 Cameroonian hunters infected with gorilla SFV and 24 controls matched for age and ethnicity. A complete physical examination and blood test were performed for all participants. Logistic regression and Wilcoxon signed rank tests were used to compare cases and controls. Results. The cases had significantly lower levels of hemoglobin than the controls (median, 12.7 vs 14.4 g/dL; P = .01). Basophil levels were also significantly lower in cases than controls, with no differences for other leukocyte subsets. Cases had significantly higher urea, creatinine, protein, creatinine phosphokinase, and lactate dehydrogenase levels and lower bilirubin levels than controls. Cases and controls had similar frequencies of general, cutaneous, gastrointestinal, neurological, and cardiorespiratory signs. Conclusions. The first case-control study of apparently healthy SFV-infected Cameroonian hunters showed the presence of hematological abnormalities. A thorough clinical and laboratory workup is now needed to establish the medical relevance of these observations because more than half of cases had mild or moderate anemia. Clinical Trials Registration. NCT03225794.

Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8 + , CD4 + , and CD19 + lymphocyte samples and rarely in CD14 + monocyte and CD56 + NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10 5 cells in CD8 + T lymphocytes, CD4 + T lymphocytes, and CD19 + B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4 + T lymphocytes. SFV DNA levels in CD4 + T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8 + , CD4 + , and B lymphocytes are major cellular targets of SFV in the blood of infected humans. IMPORTANCE Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo . Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4 + T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.

Human diseases of zoonotic origin are a major public health problem. Simian foamy viruses (SFVs) are complex retroviruses which are currently spilling over to humans. Replication-competent SFVs persist over the lifetime of their human hosts, without spreading to secondary hosts, suggesting the presence of efficient immune control. Accordingly, we aimed to perform an in-depth characterization of neutralizing antibodies raised by humans infected with a zoonotic SFV. We quantified the neutralizing capacity of plasma samples from 58 SFV-infected hunters against primary zoonotic gorilla and chimpanzee SFV strains, and laboratory-adapted chimpanzee SFV. The genotype of the strain infecting each hunter was identified by direct sequencing of the env gene amplified from the buffy coat with genotype-specific primers. Foamy virus vector particles (FVV) enveloped by wild-type and chimeric gorilla SFV were used to map the envelope region targeted by antibodies. Here, we showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain. Epitopes recognized by neutralizing antibodies have been conserved during the cospeciation of SFV with their nonhuman primate host. Greater neutralization breadth in plasma samples of SFV-infected humans was statistically associated with smaller SFV-related hematological changes. The neutralization patterns provide evidence for persistent expression of viral proteins and a high prevalence of coinfection. In conclusion, neutralizing antibodies raised against zoonotic SFV target immunodominant and conserved epitopes located in the receptor binding domain. These properties support their potential role in restricting the spread of SFV in the human population., Author summary Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. Simian foamy viruses (SFVs) can be transmitted to humans, in whom they establish persistent infection, as have the simian lenti- and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 (HIV-1) and human T lymphotropic virus type 1 (HTLV-1). Such cross-species transmission of SFV is ongoing in many parts of the world where humans have contact with nonhuman primates. We present the first comprehensive study of neutralizing antibodies in SFV-infected humans. We showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain that overlap with the receptor binding domain. SFV-specific antibodies target epitopes conserved over 8 million years of co-speciation with their nonhuman primate host. Greater neutralization potency in infected individuals was statistically associated with smaller SFV-related hematological changes. In conclusion, our results suggest the protective action of neutralizing antibodies against SFV infection and spread in the human population.

International audience; Background: Clinical studies support biologically independent roles of cell-free HIV particles and HIV-infected cells in disease progression. The associations between the level of infected cells and immune markers have been poorly studied, particularly in perinatally infected children. Objective: We tested the hypothesis that independent roles of cell-free virus and infected cells in HIV pathogenesis should be revealed by different associations between each of them and specific immune markers. Methods: Levels of HIV RNA and DNA, HIV-specific CD8 T lymphocytes, activated and naïve/memory T lymphocytes were determined in 44 untreated HIV-1-infected children. Pearson’s partial correlation coefficients were used to assess associations between the variables. Results: Here we provide new information, by showing a direct correlation between the percentages of CD4+HLA-DR+ lymphocytes and HIV DNA levels. Furthermore, higher HIV-specific CD8 T lymphocyte frequencies were associated with lower HIV DNA levels. In contrast, CD8+38+ lymphocytes and memory CD4 lymphocytes were correlated only to the HIV RNA level. All correlations were independent of age and CD4 depletion.Conclusion: Several immune markers were correlated to either the HIV RNA or the HIV DNA level, but never to both of them, supporting the concept that cell-free virus and infected cells play different roles in HIV-1 immunopathogenesis.

Simian foamy virus (SFV) is a ubiquitous retrovirus in nonhuman primates (NHPs) that can be transmitted to humans, mostly through severe bites. In the past few years, our laboratory has identified more than 50 hunters from central Africa infected with zoonotic SFVs. Analysis of the complete sequences of five SFVs obtained from these individuals revealed that env was the most variable gene. Furthermore, recombinant SFV strains, some of which involve sequences in the env gene, were recently identified. Here, we investigated the variability of the env genes of zoonotic SFV strains and searched for possible recombinants. We sequenced the complete env gene or its surface glycoprotein region (SU) from DNA amplified from the blood of (i) a series of 40 individuals from Cameroon or Gabon infected with a gorilla or chimpanzee foamy virus (FV) strain and (ii) 1 gorilla and 3 infected chimpanzees living in the same areas as these hunters. Phylogenetic analyses revealed the existence of two env variants among both the gorilla and chimpanzee FV strains that were present in zoonotic and NHP strains. These variants differ greatly (>30% variability) in a 753-bp-long region located in the receptor-binding domain of SU, whereas the rest of the gene is very conserved. Although the organizations of the Env protein sequences are similar, the potential glycosylation patterns differ between variants. Analysis of recombination suggests that the variants emerged through recombination between different strains, although all parental strains could not be identified. IMPORTANCE SFV infection in humans is a great example of a zoonotic retroviral infection that has not spread among human populations, in contrast to human immunodeficiency viruses (HIVs) and human T-lymphotropic viruses (HTLVs). Recombination was a major mechanism leading to the emergence of HIV. Here, we show that two SFV molecular envelope gene variants circulate among ape populations in Central Africa and that both can be transmitted to humans. These variants differ greatly in the SU region that corresponds to the part of the Env protein in contact with the environment. These variants may have emerged through recombination between SFV strains infecting different NHP species.

The ANRS-EP38-IMMIP study aimed to provide a detailed assessment of the immune status of perinatally infected youths living in France. We studied Gag-specific CD4 and CD8 T-cell proliferation and the association between the proliferation of these cells, demographic factors and HIV disease history. We included 93 youths aged between 15 and 24 years who had been perinatally infected with HIV. Sixty-nine had undergone valid CFSE-based T-cell proliferation assays. Gag-specific proliferation of CD4 and CD8 T cells was detected in 12 (16%) and 30 (38%) patients, respectively. The Gag-specific proliferation of CD4 and CD8 T cells was more frequently observed in black patients than in patients from other ethnic groups (CD4: 32% vs. 4%, P = 0.001; CD8: 55% vs. 26%, P = 0.02). Among aviremic patients, the duration of viral suppression was shorter in CD8 responders than in CD8 nonresponders (medians: 54 vs. 20 months, P = 0.04). Among viremic patients, CD8 responders had significantly lower plasma HIV RNA levels than CD8 nonresponders (2.7 vs. 3.7 log10 HIV-RNA copies/ml, P = 0.02). In multivariate analyses including sex and HIV-1 subtype as covariables, Gag-specific CD4 T-cell proliferation was associated only with ethnicity, whereas Gag-specific CD8 T-cell proliferation was associated with both ethnicity and the duration of viral suppression. Both CD4 and CD8 responders reached their nadir CD4 T-cell percentages at younger ages than their nonresponder counterparts (6 vs. 8 years, P = 0.04 for both CD4 and CD8 T-cell proliferation). However, these associations were not significant in multivariate analysis. In conclusion, after at least 15 years of HIV infection, Gag-specific T-cell proliferation was found to be more frequent in black youths than in patients of other ethnic groups, despite all the patients being born in the same country, with similar access to care.

International audience; Recognition of various HIV proteins by CD8 T cells from HIV-infected children was determined by two functional assays. First, using an Elispot assay, we show that 80% of patients recognized Gag, 77% recognized Pol, 61% recognized Env, 44% recognized Nef and 29% recognized Vif. Frequencies of Gag-, Pol-, and Env-specific IFN-gamma producing CD8 T cells were higher than frequencies of Nef and Vif-specific CD8 T cells. The poor recognition of Nef by ex vivo CD8 T cells was confirmed by CTL assays performed in HAART na? children: 25% of children had positive response against Nef versus 44, 63 and 62% for Env, Gag, and Pol, respectively. Memory Gag-specific CTL were positively correlated with age, whereas Nef-specific CTL were negatively correlated with age. The poor Nef-specific CD8 T cell response in HIV-infected children contrasts with dominance of Nef-specific responses in infected adults.

International audience; Dendritic cells (DCs) are thought to play a crucial role in the pathogenesis of HIV-1 infection. DCs are believed to transport virus particles to lymph nodes before transfer to CD4(+) lymphocytes. We have investigated the role of Nef in these processes. HIV-1 replication was examined in human immature DC-lymphocyte cocultures and in DCs or lymphocytes separately. Using various R5-tropic and X4-tropic HIV-1 strains and their nef-deleted (Deltanef) counterparts, we show that Nef is required for optimal viral replication in immature DC-T cells clusters and in T lymphocytes. Nef exerts only a marginal role on viral replication in immature DCs alone as well as on virion capture by DCs, long-term intracellular accumulation and transmission of X4 strains to lymphocytes. We also show that wild-type and Deltanef virions are similarly processed for MHC-I restricted exogenous presentation by DCs. Taken together, these results help explain how HIV-1 Nef may affect viral spread and immune responses in the infected host.

HLA-A*02 tetramers complexed to human immunodeficiency virus (HIV) Gag SLYNTVATL and HIV Pol ILKEPVHGV peptides were used to characterize HLA class I-restricted CD8(+) T cells in 41 HIV-infected children. The frequencies and the phenotype of specific circulating CD8(+) T cells were determined in whole-blood samples by means of cytometric analysis. Background staining of 13 HLA-A*02-negative patients showed that the frequency of CD8(+) T cells was

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.

A CD8 + cytotoxic T-cell clone that recognized HIV p24 gag 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.

A vaccine against human immunodeficiency virus (HIV) should induce virus-specific cytotoxic T lymphocyte (CTL) activity. Immunization of uninfected volunteers with a canarypox virus express­ ing HIV envelope was carried out in a phase I trial. Two injections of canarypox expressing HIV­ l M N gp160 (months 0 and 1) were followed by two boosts of recombinant envelope protein (months 3 and 6). HIV envelope-specific CTL were detected in peripheral blood mononuclear cells stimulated with autologous HIV-l-infected blast cells. T cell lines were obtained from 18 of 20 donors: CTL were detected at least once following immunization in 7 (39%) of these 18. This activity was mediated by major histocompatibility complex class I-restricted CD3+CD8+ T cells. For two subjects, this activity was still present 2 years after the initial immunization. The CTL responses with this prime­ boost regimen are the best observed with any HIV vaccine tested in humans. Vaccination against the human immunodeficiency virus type I (HIV-I) is a key strategy for the eventual control of the AIDS pandemic. Since the discovery of the etiologic agent of AIDS, different candidate vaccines have been designed and developed, including whole killed virus, live attenuated virus, virus-like particles including pseudovirions, protein subunits based on HIV structural genes, peptides based on selected im­ munoreactive parts of the viral proteins, live recombinant viral or bacterial vectors, and DNA-based immunogens encoding one or more HIV proteins [1-3]. Live recombinant vaccinia viruses have been used successfully to induce protective immu­ nity against animal infectious diseases such as rabies [4], but because the use ofvaccinia virus for human vaccination against smallpox has been shown to induce rare but serious complica­ tions [5], other recombinant poxviruses such as avian poxvi­ ruses have been developed [6, 7]. As was the case for most live expression vectors in AIDS vaccine studies, initial efforts have focused on products based on the HIV-I envelope protein, since several epitopes of env have been described to induce neutralizing antibodies and cell-mediated immune responses, including cytotoxic T lymphocytes (CTL). In HIV-seronegative volunteers at low risk ofHIV infection, the safety and immuno

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 RNAwas undetectableintheplasmaof75%ofthesepatients.ThemedianHIV DNAloadwas 2.84(interquartilerange, 2.51–3.16) log10 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.

SUMMARY 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, p15gagand 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.

Aim: To review current knowledge of CD8+ T cells in relation to their effect on the replication of HIV and on disease progression. Present knowledge: Both 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. Conclusion: These CD8+ T cells may help to maintain a low viral load in vivo, thus allowing a long asymptomatic period of infection

Virus-specific cytotoxic T lymphocytes (CTL) may be an important host defense mechanism in the control of virus replication in persons infected with human immunodeficiency virus type 1 (HIV-1). Cytotoxic T-cell lines generated by nonspecific stimulation with anti-CD3 monoclonal antibodies and interleukin 2 were used to identify regions within the HIV-1 Gag protein that are the most frequently recognized. Using autologous Epstein-Barr virus-transformed target cells infected with recombinant vaccinia viruses encoding p18gag, p24gag, and p55gag proteins of HIV-1/Lai or selected truncations of p24gag, we show that within a group of 29 infected subjects, the p24gag protein is the target of Gag-specific CTL in most donors. Using autologous Epstein-Barr virus-transformed target cells coated with different synthetic peptides spanning the Gag amino acid sequence, we found clusters of partially overlapping peptides in three conserved regions of the p24 protein (amino acids [aa] 169 to 192, aa 219 to 304, and aa 335 to 372) that are frequently recognized by CTL and presented by a variety of human leukocyte antigen class I molecules. Since there are experiments both in vitro and in vivo showing the role of CTL in the control of virus replication in HIV and simian immunodeficiency virus infections, these results may be particularly important for vaccine development.

In the USA and West Europe, nearly 80% of HIV-1-infected adults, experiencing virologic failure, harbored virus strain resistant to at least one antiretroviral drug. Limited data are available on antiretroviral drug resistance in pediatric HIV infection. The aims of this study were to analyze prevalence of HIV-1 drug resistance and to identify risk factors associated with resistance in this population. Prevalence of genotypic resistance was estimated retrospectively in treated children who experienced virologic failure (with HIV-1-RNA > 500 copies/ml) followed in Necker hospital between 2001 and 2003. Among 119 children with resistance testing, prevalence of resistance to any drug was 82.4%. Resistance ranged from 76.5% to nucleoside reverse transcriptase inhibitor (NRTI), to 48.7% to non-nucleoside reverse transcriptase inhibitor (NNRTI) and 42.9% to protease inhibitor (PI). Resistance to at least one drug of two classes and three classes (triple resistance) was 31.9 and 26.9%, respectively. Resistance was not associated with geographic origin, HIV-1 subtype, and CDC status. In multivariate analysis, resistance to any drug remained associated independently with current low viral load and high lifetime number of past PI. Triple resistance was independently associated with the high lifetime number of past PI and with gender, particularly among children aged 11 years old or more with a prevalence seven times higher in boys than in girls. In conclusion, antiretroviral resistance is common among treated HIV-1-infected children and prevalence was similar with those observed in adult population in the same year period. However, adolescent boys seem to be at greater risk.

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.

HIV-specific CD8+ T lymphocytes participate in the control of viral replication in infected patients. These responses are of low intensity in young infants and are decreased by antiretroviral therapy. In the present study, we report on a recombinant Vaccinia virus (rVV)-based Elispot assay for the detection of HIV-specific CD8+ T cells immediately after isolation of peripheral blood mononuclear cells (PBMC). The rVV-based assay was highly sensitive; 48 out of 50 children had a positive response against the rVV encoding HIV Env-Gag-Pol antigen. Interferon-gamma was produced by CD8+ T cells, and CD14+/15+ cells were the main cell subset presenting antigens expressed by rVV. We observed that the cell input per well had a critical influence on the sensitivity of the assay. Results from the ex vivo Elispot assay correlated poorly with those of the 51Cr release assay performed after expansion of PBMC in vitro; thus, both assays gave information on different subsets and/or functions of the HIV-specific T cell response.

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.

The evolution of the HIV-specific CD8+ T cell response in patients receiving potent combination therapy has been well documented in adult patients. However, no study reported whether baseline HIV-specific CD8+ T cell response is linked to treatment outcome. The aims of this study were to investigate both the impact of baseline memory cytotoxic T lymphocytes (CTL) on treatment outcome and the effect of potent therapy on memory HIV-specific CTL in HIV-1-infected pediatric patients. The study group comprised 30 children who started a first-line combination treatment including at least three drugs from two different classes and were longitudinally followed during treatment. Their memory HIV-specific responses were measured at baseline and during treatment, as well as their plasma viremia and CD4+ levels. The intensity of memory Gag-specific CTL and the breadth of the CTL response at the beginning of treatment were significantly correlated with lower plasma viral load during treatment, independently of baseline plasma viral load, CD4+ counts, and age. Children with partially controlled viral replication had enhanced Gag-specific CTL compared to their baseline value. This improvement of antiviral responses during treatment was not observed when viral replication was either fully suppressed or uncontrolled. In conclusion, our results show that higher baseline HIV-specific CTL are linked to lower viremia under combination therapy. This result adds further support to the hypothesis that cooperation between the antiviral immune response and antiviral drugs could be helpful for therapeutic management of HIV-infected patients.

International audience; In the pediatric human immunodeficiency virus type-1 (HIV-1) infection, the presence of cytotoxic T lymphocytes (CTL) is associated with a slow progression to AIDS. The secretion of cytokines by CTLs may be critical in the control of viral infection. We used the combination of cell surface and intracellular staining to study the functionality of tetramer binding CD8+ T cells recognizing two HIV-1 immunodominant epitopes, in peripheral blood mononuclear cells from HIV-1-infected children. A fraction of tetramer positive CD8+ T cells produce cytokines (IFN-gamma, TNF-alpha) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlation between the plasma viral load and the percentage of CD8+ Tetramer Gag+ T cells secreting IFN-gamma. This is the first report in the context of pediatric HIV-1 infection showing that only a fraction of HIV-1-specific CD8+ T cells have the capacity to produce cytokines and chemokines implicated in their antiviral functions.

Dendritic cells (DCs) facilitate HIV-1 spread in the host by capturing virions and transferring them to permissive lymphocytes in lymphoid organs. Lectins such as DC-specific ICAM-grabbing non-integrin (DC-SIGN) are involved in HIV-1 uptake by DCs, through high-affinity binding to viral envelope glycoproteins. We examined the role of DC-SIGN on the fate of incoming virions and on major histocompatibility complex class I (MHC-I)–restricted HIV-1 antigen presentation. We show that DC-SIGN expression in B-cell lines dramatically enhances viral internalization. In these cells, and also in primary DCs, most of the captured virions are rapidly degraded, likely in a lysosomal compartment. In addition, a fraction of incoming viral material is processed by the proteasome, leading to activation of anti–HIV-specific cytotoxic T lymphocytes (CTLs) by DC-SIGN–expressing cells. In DCs, DC-SIGN is not the only receptor involved, and redundant pathways of virus capture leading to antigen presentation likely coexist. Altogether, our results highlight new aspects of DC-SIGN interactions with HIV-1. The lectin does not significantly protect captured virions against degradation and promotes MHC-I exogenous presentation of HIV-1 antigens.

Human immunodeficiency virus (HIV)-specific CD8+-T-cell responses are critical in restricting viral replication and altering the course of HIV infection (38, 48). Ex vivo cytotoxic T lymphocytes (CTL) are much less frequent in vertically infected children than in adults (9, 10, 41, 42, 46, 60). In contrast, after in vitro culture, memory CTL can be readily detected in HIV-infected children, with magnitude, breadth, and specificity similar to those observed in adults (9, 10, 41, 46). These memory CTL can be detected during the first weeks or months of life (10, 41, 56, 75), and the presence of HIV-specific CTL is associated with slower evolution toward disease (10). Complete suppression of viral replication following combined therapy is less frequent in children than in adults (40, 51, 52), and the kinetics of viral decrease is slower (49). However, children have a more active thymus function than adults (19, 44), which allows better lymphocyte regeneration following combined therapy (13, 69). In children treated with combined therapy before 3 months of age, and with undetectable viral load, neither HIV-specific antibodies nor HIV-specific CD4 or CD8 cells were detected (43). This contrasts with the beneficial effect of early treatment on preservation of HIV-specific T-cell immunity in adults (8, 50). New immunotherapeutic interventions are being developed for adults (8, 50). These treatments are of great interest for children due to observance problems and because complete viral suppression is more difficult to obtain in children than in adults. Therefore, it is crucial to characterize the dynamics of HIV-specific T cells in pediatric HIV infection. On encountering viral antigen, naive CD8+ T cells proliferate and differentiate into effector cells able to lyse infected cells and to secrete cytokines. As virus is cleared, most activated effector cells undergo apoptosis, but some survive and enter the memory pool that persists for long periods (76). Most memory cells are in a resting state, unable to secrete cytokine or to lyse infected cells, until reactivation on reexposure to viral antigen (61). During persistent infection, continuous stimulation of T cells may lead to dysfunction, anergy, or clonal exhaustion. In the absence of CD4+ T cells, exhaustion and anergization of CD8+ T cells is more rapid (78). The complex interplay between the antigen load, virus-specific CD8+-T-cell dynamics and function, and the immune status of the infected host has been illustrated by a number of studies of HIV-infected adults. In untreated chronically HIV-infected patients, CTL numbers are inversely correlated with the plasma viral load (5, 24, 25, 34, 54, 58). On the other hand, reduction of HIV replication by potent antiretroviral therapy is associated with a decline in HIV-specific CD8+ T cells (11, 55). Evidence that a fraction of HIV-specific CD8+ T cells are not functional was recently obtained (22, 31). Furthermore, loss of gamma-interferon (IFN-γ)-producing cells with persistence of tetramer binding cells was observed in subjects progressing to AIDS (32). CTL derived from the memory pool after in vitro expansion are easily detected by the standard 51Cr release assay in most HIV-infected children (9). In contrast, ex vivo-activated effector cytolytic cells are infrequently detected immediately after isolation (9). Cytokine synthesis following short-term ex vivo stimulation with the antigen can be measured using assays that are more sensitive than the chromium release assay and could be used to quantify the effector subset of HIV-specific CD8+ T cells (3, 18). Cytokine production can be measured at the single-cell level using the enzyme-linked immunospot (ELISPOT) technique, allowing direct calculation of T-cell frequencies (15). This technique is very sensitive and has been found to reliably detect CD8+ T cells in various human diseases (35, 63, 64, 74), including HIV infection (16, 36). The aim of the present work was to evaluate the use of the ELISPOT assay for the ex vivo study of HIV-specific CD8+ T cells from HIV-infected children. We chose to focus our initial effort on two immunodominant HLA-A∗0201-restricted HIV epitopes that are frequently recognized by infected children, as we showed previously using tetramers (66). In this cross-sectional study, HLA-A∗0201-positive HIV-infected children were systematically tested for the presence of HIV-specific CD8+ T cells using the ELISPOT assay. The frequencies of HIV-specific IFN-γ-producing CD8+ T cells were compared to the frequencies of HIV-specific CD8+ T cells measured by tetramer labeling, and relationships with biological parameters of HIV infection were investigated. The results from the ELISPOT and the tetramer assays were well correlated, but a comparison of the results from both assays suggests that a significant fraction of CD8+ T cells were unable to produce IFN-γ. Most importantly, the frequencies of ex vivo-activated HIV-specific CD8+-T-cell-mediated IFN-γ production were positively correlated with plasma HIV RNA, showing that this subset of antiviral CD8+ T cells is dependent upon continuous antigenic stimulation.

A previous study showed that, during the first year of life, the presence of cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus (HIV)–infected children is associated with a lack of rapid progression to acquired immunodeficiency syndrome. The goal of the study was to address the role of CTLs in children who survived after age 5 years. Memory HIV-specific CTLs directed against Env, Gag, Nef, and Pol proteins were measured in a group of 47 highly active antiretroviral therapy–naive HIV-infected children. Both Gag- and Polspecific CTLs were positively correlated with CD4 + T cell counts. Gag-, Nef-, and Pol-specific CTLs were inversely correlated with virus load. The inverse correlation between virus load and Gag-specific CTLs was independent of CD4 + T cell counts. In conclusion, this study showed the beneficial role of HIV-specific CTLs in children who survived after age 5 years. The role of virus-specific cytotoxic T lymphocytes (CTLs) in the control of human immunodeficiency virus (HIV)andsimian immunodeficiency virus (SIV) replication has been established by a great number of studies. The elimination of CD8 T cells in rhesus macaques results in a dramatic increase in SIV load in both acute and chronic infection [1‐4]. The vaccination of monkeys capable of inducing SIV-specific cellular immune responses results in improved control of viral replication and longer survival after challenge [5]. In HIV-infected adults,acute viremia resolves with the appearance of CTLs, which precedes the detectable production of any neutralizing antibodies [6‐10]. The selection of virus mutants in vivo that are no longer recognized by CTLs was observed in both acute and late HIV infection, because virus levels increased [11‐13]. Long-term nonprogression is associated with the maintenance of high levels of both effector [14] and memory CTLs [15, 16]. Finally, the presence of effector CTLs against Gag [17], memory CTLs

Pathogens attempt to evade immune recognition by expressing mutated antigens. The present study shows that two mechanisms happen in vivo during the course of HIV infection to limit the escape of antigenic variants from cytotoxic T lymphocyte (CTL) recognition: recognition of several epitope variants by the same TCR and generation of several CTL populations specific for a single epitope but recognizing different variant sequences. We have studied two CTL populations directed towards the HIV-p24 gag amino acids 176‐184 QASQEVKNW epitope, presented by HLA-B5301. Both CTL populations were derived from a long-term asymptomatic HIV-infected child and they express different TCR. Each of the two CTL recognizes five of the 10 naturally occurring variants. These variants are distinct for both CTL and thus a total of eight variants are recognized. Thus, polyclonality of CTL specific for the same epitope but differing in variant sequences recognized may improve the control of variant viruses’ replication in vivo. In addition to cross-recognition of several variant epitopes, promiscuous recognition of exogenous peptides complexed to allogeneic HLA-B molecules occurs, showing that the TCR can tolerate amino acid changes on both the peptide and the MHC molecule. This flexibility of the TCR is probably of great importance for control of viruses with high genetic variability, such as HIV.

International audience; 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.

The activities of HIV-specific cytotoxic T lymphocytes (CTLs) were evaluated in 10 HIV-infected children, born to infected mothers who did not receive AZT during pregnancy. CTL activities were present as early as 4 months of age. The five children that progressed to AIDS before 1 year of age had reduced in vivo and in vitro CTL activities, when compared with children who remained AIDS free after 1 year of age. The latter children had weak in vivo activated CTL responses but strong memory CTLs. No relation was found between viral load, lymphocyte populations, and CTL responses between birth and 6 months of age. Between 7 and 12 months old, children with broader in vitro activated CTLs had higher absolute numbers of CD4+ and CD8+ T lymphocytes and lower plasma viral load. These data support a beneficial role of CTLs in pediatric HIV infection.

The CCR5 gene encodes one of the major human immunodeficiency virus type 1 (HIV-1) coreceptors. A 32-bp deletion in this gene (delta ccr5) is associated with relative resistance to disease progression in heterozygous HIV-1-infected persons. The effect of this mutation on virologic and immunologic parameters was determined in a cohort of 45 perinatally HIV-1-infected children prospectively followed after 5 years of age. At a median age of 8.3 years, heterozygous children had significantly lower virus load than homozygous children (median, 3.3 vs. 4.1 log copies/mL, P < .009) and higher percentages of CD4 T cells (median, 26% vs. 17%, P < .07). However, there was no discernible influence of the CCR5 genotype on the percentages of CD8 T cells (P < .27) or on HIV-specific cytotoxic T lymphocyte activities (P < .65). There was a trend for lower rates of progression to AIDS (CDC stage C) in heterozygous children. These data confirm a major role for the CCR5 coreceptor in HIV-1 pathogenesis in children.

Cell-mediated cytotoxic (CMC) responses were measured in a group of human immunodeficiency virus type 1 (HIV-1)-seropositive donors against target cells expressing the envelope protein of either the HIV-1 strain Lai or strain MN. In primary CMC assays using freshly isolated peripheral blood mononuclear cells, seropositive individuals more commonly had CMC responses against HIV-1Lai than HIV-1MN. Moreover, each of the responders to HIV-1MN envelope also had primary CMC responses against HIV-1Lai envelope. Cytotoxic T cells generated by nonspecific in vitro stimulation recognized both strains at a similar frequency. These results may indicate the existence of multiple effector populations or that the cellular immune response is directed at regions of the envelope outside the V3 loop. By contrast, serologic studies done on similar populations have shown that most persons have antibodies capable of recognizing the V3 loop of HIV-1MN but rarely that of HIV-1Lai.

In the classic model of antigen processing and presentation, viral antigens must be synthesized within the cytoplasm of infected cells to be processed and presented to CD8+, MHC class I-restricted cytotoxic T lymphocytes (CTLs). We have examined the utility of a retroviral vector (pNeoNef) expressing the human immunodeficiency virus type (HIV-1)Lai Nef protein for the development of target cells to study HIV-specific CTLs. Autologous Epstein-Barr-transformed B cell lines (EBV-B cells) transduced with pNeoNef were efficiently lysed by CTL lines from donors capable of lysing EBV-B cells infected with a recombinant vaccinia virus (rVV) expressing Nef. Also, the transduced cells were efficient stimulator cells for the generation of Nef-specific CTL lines. The CTL lines thus generated recognized the same epitopes as CTL lines from the same donor generated by nonspecific stimulation. The use of similar cell lines transduced with retroviral vectors expressing HIV proteins may be useful in the study of CTLs in HIV-infected donors and in the study of the ability of candidate vaccines, including rVV, to induce HIV-specific CTLs. As antigen-presenting cells, the cell lines may be useful in the generation of antigen-specific CTL lines.

CD8+ T cells may play a beneficial role in human immunodeficiency virus (HIV)-infected patients by two mechanisms: HIV-specific cytotoxic activity and secretion of a soluble mediator(s) that inhibits HIV replication in vitro. Here we characterized both activities mediated by an HIV p24 gag -specific cytotoxic T lymphocyte (CTL) CD8+ clone derived from an HIV-infected patient. When the CTL clone was mixed with HIV-infected autologous CD4+ T cells, viral replication was suppressed. This viral inhibition was observed in heterologous CD4+ T cells and when CD8+ and CD4+ populations were separated by a semipermeable membrane, demonstrating the involvement of a diffusible factor(s). The lysis of autologous HIV-infected T cells was also detected. However, HIV suppression was more efficient when CD4+ and CD8+ T cells shared major histocompatibility complex alleles and were in direct contact. Thus, one and the same CD8+ T cell population can mediate both lysis of HIV-infected targets and nonlytic suppression of HIV replication. These results underline the multiple roles of CD8+ T lymphocytes in the suppression of HIV-infected cells.

International audience; Human T-cell Lymphotropic Virus type 1 (HTLV-1) is a human retrovirus that infects at least 5–10 million people worldwide, and is the etiological agent of a lymphoproliferative malignancy; Adult T-cell Leukemia/Lymphoma (ATLL); and a chronic neuromyelopathy, HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), as well as other inflammatory diseases such as infective dermatitis and uveitis. Besides sexual intercourse and intravenous transmission, HTLV-1 can also be transmitted from infected mother to child during prolonged breastfeeding. Some characteristics that are linked to mother-to-child transmission (MTCT) of HTLV-1, such as the role of proviral load, antibody titer of the infected mother, and duration of breastfeeding, have been elucidated; however, most of the mechanisms underlying HTLV-1 transmission during breast feeding remain largely unknown, such as the sites of infection and cellular targets as well as the role of milk factors. The present review focuses on the latest findings and current opinions and perspectives on MTCT of HTLV-1.