What Is the most Important for Elite Control: Genetic Background of Patient, Genetic Background of Partner, both or neither? Description of Complete Natural History within a Couple of MSM.

BACKGROUND: We describe a homosexual man who strongly controlled HIV-1 for ten years despite lack of protective genetic background. METHODS: HIV-1 DNA was measured in blood and other tissues. Cell susceptibility was evaluated with various strains. HIV-1-specific (CD4 and CD8 activation markers and immune check points) and NK cells responses were assessed; KIRs haplotypes and HLA alleles were determined. FINDINGS: Two HIV-1 RNA copies/mL of plasma were detected in 2009, using an ultra-sensitive assay. HIV-DNA was detected at 1.1 and 2 copies/106 PBMCs in 2009 and 2015 respectively, at 1.2 copies/106 cells in rectal cells in 2011. WBs showed weak reactivity with antibodies to gp160, p55 and p25 from 2007 to 2014, remaining incomplete in 2017. CD4 T cells were susceptible to various strains including HIVKON, a primary isolate of his own CRF02_AG variant. CD8 T cells showed a strong poly-functional response against HIV-Gag, producing mainly IFN-γ; a robust capacity of antibody-dependant cell cytotoxicity (ADCC) was observed in NK cells. Case patient was group B KIR haplotype. Neutralizing antibodies were not detected. CD4 and CD8 blood T cells showed normal proportions without increased activation markers. Phylogenetic analyses identified the same CRF02_AG variant in his partner. The patient and his partner were heterozygous for the CCR5ΔD32 deletion and shared HLA-B*07, C*07 non-protective alleles. INTERPRETATION: This thorough description of the natural history of an individual controlling HIV-1 in various compartments for ten years despite lack of protective alleles, and of his partner, may have implications for strategies to cure HIV-1 infection.

Routinely-feasible multiple biomarkers score to predict prognosis after revascularized STEMI.

INTRODUCTION: We assessed the long-term prognostic value of an easy-to-do multiple cardiac biomarkers score after a revascularized acute myocardial infarction (MI) in order to evaluate a multimarker approach to risk stratification, based on routine biomarkers. MATERIAL AND METHODS: Blood samples from 138 patients hospitalized with acute myocardial infarction and successfully treated by primary coronary intervention (with TIMI 3 flow) were subsequently tested for creatinin level at admittance and then BNP, hsCRP, troponin I from Day 0 to day 7. The primary endpoint was a clinical evaluation comprising: new hospitalization for cardiac reasons, acute coronary events (acute coronary syndrome), and death. RESULTS: During the median follow-up period of 11.01 months [9.44-12.59], 47 events were recorded. All the following markers were able to predict events: creatinemia on admission (p=0.0057), CRP on day 3 (p, troponin I on day 1 (p<0.001), BNP (p<0.0001) and biological multimarker score (p<0.0001). Clinical events were predicted with a hazard ratio (HR) of respectively 3.30 [2.88-12.30] in BNP Q4 as compared to the three lower quartiles (Q1-3), and 3.15 [2.75-21.00] for the Multimarker approach. The multimarker score was not significantly better than BNP on day 1 alone (p=0.77), troponin on day 1 alone (p=0.43), creatininemia on admission (p=0.19) or CRPhs on day 3 alone (p=0.054). Nevertheless, the Multimarker approach leads to the selection of a smaller, hence more manageable, high-risk population (13% versus 25%). CONCLUSION: Among 138 subjects admitted for acute MI, and all successfully revascularized, a routinely multimarker approach with BNP, hsCRP, creatininemia, troponin I, is feasible. BNP is the most powerful marker, and this multimarker approach renders additional prognostic information helping to identify patients with high-risk to clinical events.

Restoration of anti-tetanus toxoid responses in patients initiating highly active antiretroviral therapy with or without a boost immunization: an INITIO substudy.

INITIO is an open-labelled randomized trial evaluating first-line therapeutic strategies for human immunodeficiency virus-1 (HIV-1) infection. In an immunology substudy a tetanus toxoid booster (TTB) immunization was planned for 24 weeks after initiation of highly active antiretroviral therapy (HAART). All patients had received tetanus toxoid immunization in childhood. Generation of proliferative responses to tetanus toxoid was compared in two groups of patients, those receiving a protease inhibitor (PI)-sparing regimen (n = 21) and those receiving a PI-containing (n = 54) regimen. Fifty-two participants received a TTB immunization [PI-sparing (n = 15), PI-containing (n = 37)] and 23 participants did not [PI-sparing (n = 6) or PI-containing (n = 17)]. Cellular responses to tetanus antigen were monitored by lymphoproliferation at time of immunization and every 24 weeks to week 156. Proportions with a positive response (defined as stimulation index > or = 3 and Delta counts per minute > or = 3000) were compared at weeks 96 and 156. All analyses were intent-to-treat. Fifty-two participants had a TTB immunization at median 25 weeks; 23 patients did not. At weeks 96 and 156 there was no evidence of a difference in tetanus-specific responses, between those with or without TTB immunization (P = 0.2, P = 0.4). There was no difference in the proportion with response between those with PI-sparing or PI-containing regimens at both time-points (P = 0.8, P = 0.7). The proliferative response to tetanus toxoid was unaffected by initial HAART regimen. Anti-tetanus responses appear to reconstitute eventually in most patients over 156 weeks when treated successfully with HAART, irrespective of whether or not a TTB immunization has been administered.

Evaluation of the interlaboratory concordance in quantification of human immunodeficiency virus-specific T cells with a gamma interferon enzyme-linked immunospot assay.

The gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay is a reference method for the ex vivo monitoring of antigen-specific T cells and a primary tool for assessing clinical trials of human immunodeficiency virus (HIV) or cancer vaccines. Four experienced laboratories in Paris compared their results with this method by exchanging frozen blood samples from eight HIV-seronegative and eight HIV-seropositive subjects. Each laboratory measured the IFN-gamma-producing cells specific for HIV, Epstein-Barr virus, cytomegalovirus, and influenza using the same set of peptides and the same ELISPOT reader but its own ELISPOT technique. The cutoff values for positive responses (50 or 100 spot-forming cells/10(6) peripheral blood mononuclear cells over background) were consistent with the binomial statistic criterion. The global qualitative concordance, as assessed by the kappa index, ranged from 0.38 to 0.92, that is, moderate to excellent, and was better for non-HIV 9-mer peptide pools than for HIV 15-mer peptide pools. The interlaboratory coefficient of variation for the frequency of virus-specific T cells was 18.7% (data are expressed on a log scale). Clustering analysis of HIV-positive subjects showed qualitative agreement for ELISPOT results from all four laboratories. Overall, the good interlaboratory qualitative concordance of IFN-gamma ELISPOT assays with only the peptide source and ELISPOT reader in common suggests that a qualitative comparison of interlaboratory findings is feasible. Nonetheless, a single set of standard operating procedures should be used in multicenter trials to improve standardization.

Polymorphism in the proximal promoter region of the perforin gene and its impact on the course of HIV infection.

Cytotoxic T lymphocytes (CTLs) play an essential role in the control of viral replication during human immunodeficiency virus (HIV) infection. However, the efficacy of the CTL response varies between individuals. We tested the hypothesis that genetic polymorphisms in the lytic effector molecule perforin could influence the progression of HIV infection. The perforin gene was screened for single nucleotide polymorphisms (SNPs) by denaturing high-performance liquid chromatography (dHPLC). Correlations were sought between perforin genotype, perforin expression and lytic function of CD8+ T lymphocytes from HIV-positive patients. Association of perforin genotype with disease progression was investigated in 426 seroconverters enrolled in the French SEROCO cohort. AIDS-free survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test. Three SNPs were found in the proximal promoter region of the perforin gene: 63G (allelic frequency 0.029), 112G (allelic frequency 0.071) and 1012T (allelic frequency 0.070). The presence of the 1012T genotype correlated with fewer perforin+ cells among circulating CD8+ CTL. However, CTL lines from HIV(-positive) individuals heterozygous for the perforin 1012T SNP displayed normal lysis of target cells, and within the SEROCO cohort, patients heterozygous for the 1012T SNP showed normal disease progression. However, 1012T/T homozygotes showed a tendency towards slower disease progression (P = 0.08). In conclusion, polymorphism in the perforin gene is limited, and although the 1012T genotype appears to influence perforin expression, it was not conclusively associated with disease progression in HIV infection.

Persistent low viral load on antiretroviral therapy is associated with T cell-mediated control of HIV replication.

BACKGROUND: It is unclear how stable low-level viral replication and CD4 cell numbers can be maintained under highly active antiretroviral therapy (HAART). This study was designed to analyse whether HIV-specific responses in stable partially controlled patients during antiretroviral therapy (ART) differ from those observed in complete HAART failure and whether they contribute to the control of viral load (VL). METHODS: Three groups of patients were selected according to plasma HIV RNA levels during 18 months of ART: persistently low VL (LoVL; HIV RNA <10,000 copies/ml; n = 28), undetectable VL (UnVL; HIV RNA <200 copies/ml; n = 29) and high VL (HiVL; HIV RNA >10,000 copies/ml; n = 14). T-cell responses were studied using lymphoproliferative and interferon (IFN)-gamma-ELISpot assays against HIV-p24, -gp160, recall antigens, and 15 pools of HIV-(Gag + RT) peptides. RESULTS: Frequencies of IFN-gamma-producing CD4 T cells against HIV-p24 were higher in LoVL than in UnVL or HiVL groups [median, 131, 47 and 23 spot-forming cells (SFC)/1 x 10 peripheral blood mononuclear cells (PBMC), respectively; P = 0.012 and P = 0.047]. Lymphoproliferative responses to HIV-p24 and recall antigens were similar in LoVL and UnVL groups but lower in HiVL (P = 0.004). Frequencies of HIV-specific CD8 T cells were higher in LoVL than in UnVL (1340 versus 410 SFC/1 x 10 PBMC; P = 0.001). They correlated negatively with VL in the LoVL and HiVL (r, -0.393, P = 0.039 and r, -0.643, P = 0.024, respectively) and positively correlated with anti-HIV CD4 cell frequencies in the LoVL group only (r, 0.420; P = 0.026). CONCLUSION: Persistently low viral replication (<10,000 copies/ml) during ART stimulates high frequencies of HIV-specific CD4 and CD8 T cells compared to full virus suppression or complete ART failure. The association of high anti-HIV activity with large numbers of HIV-specific CD8 T cells contribute to the control of viral replication.

A systematic comparison of methods to measure HIV-1 specific CD8 T cells.

Several methods are now available to evaluate the frequencies of virus-specific CD8 T cells but require a systematic comparison to help at choosing the best strategy for evaluation. First, we compared the ELISpot-IFNgamma assay, intracellular IFNgamma staining and HLA class I tetramer-binding assay to quantify the HIV-specific CD8 T cells. Second, we determined the frequency of recognition of HIV antigens and evaluated whether the mode of antigen presentation might influence the results: We compared HIV antigen presentation in the same ELISpot-IFNgamma assays by using recombinant vaccinia viruses (rVVs) encoding for HIV-LAI Gag, Pol, Env, Nef, Tat and Vif proteins, or a panel of 49 synthetic 8-11 amino acid length peptides tested either individually or pooled. Third, we compared the antigens recognized by memory CTL analysis using chromium release assay (CRA) on CTL lines and by effector CD8 cell analysis using ELISpot assay. Our results show that: (1) Flow cytometry and ELISpot assay measuring IFNgamma production give the same frequency of HIV-specific CD8 T cells; (2) tetramer-binding assay detects more HIV-specific CD8 T cells than other methods; (3) pools of optimal peptides and sum frequencies of individual optimal peptides give similar results in ELISpot assay; (4) ELISpot assays using peptides are more sensitive than those using rVV; and (5) CRA and ELISpot assay when using rVV provide a comparable profile of HIV antigen recognition by memory CTLs (CRA) and effector CTLs (ELISpot) in two thirds cases. These results have important implications for the choice of immunological methods to evaluate CD8 T cells responses to vaccines.

Investigation of human spleen dendritic cell phenotype and distribution reveals evidence of in vivo activation in a subset of organ donors.

Although the mouse spleen dendritic cell (DC) is perhaps the most intensively studied DC type, little has been published concerning its human equivalent. In this report, rare event flow cytometry and in situ immunofluorescence were used to study the surface phenotype and distribution of HLA-DR(+) CD3(-)14(-)16(-)19(-) human spleen DC. Spleens from organ donors with different clinical histories were used. Most (81% +/- 9%; n = 14) spleen DCs expressed high levels of the integrin CD11c. CD11c(+) DCs were distributed in 3 distinct regions-the peri-arteriolar T-cell zones, the B-cell zones, and the marginal zone, where they formed a ring of cells surrounding the white pulp, just inside a ring of CD14(+) red pulp macrophages, apparently more regularly organized than the previously described marginating DC population in the mouse spleen. The T-cell zones contained CD86(+) DCs, among which a subpopulation expressed CD83. These mature/activated CD86(+) DCs represented a minority (12% +/- 8%) of total spleen DCs in most organ donors: most spleen DCs are immature. In 3 of 18 (17%) donors, however, most (54%-81%) of spleen DCs were CD86(+), suggesting that in vivo DC activation had occurred. In one donor, a radical shift in DC distribution from the marginal zone to the T-cell zones was also observed. This activation of spleen DCs in vivo was reminiscent of the effects of experimental microbial product injection in mice, and it seemed to correlate with bacterial infection or multiple trauma. (Blood. 2001;97:3470-3477)

HIV-specific effector cytotoxic T lymphocytes and HIV-producing cells colocalize in white pulps and germinal centers from infected patients.

Human immunodeficiency virus (HIV) infection is characterized by the massive infiltration of secondary lymphoid organs with activated CD8(+) T lymphocytes. While converging data indicated that these cells were HIV-specific cytotoxic T lymphocytes (CTLs) responsible for HIV spread limitation, direct evidence was lacking. Here, the presence of HIV-specific effector CTLs was demonstrated directly ex vivo in 15 of 24 microdissected splenic white pulps from an untreated patient and in 1 of 24 tonsil germinal centers from a second patient with incomplete viral suppression following bitherapy. These patients had plasma HIV RNA loads of 5900 and 820 copies per milliliter. The frequencies of HIV-1 DNA(+) cells in their lymphoid organs were more than 1 in 50 and 1 in 175, respectively. Spliced viral messenger RNA (a marker for ongoing viral replication) was present in most immunocompetent structures tested. Conversely, CTL activity was not found in spleens from 2 patients under highly active antiretroviral therapy, with undetectable plasma viral load. These patients had much lower spleen DNA(+) cell frequencies (1 in 2700 and 1 in 3800) and no white pulps containing spliced RNA. CTL effector activity as well as spliced viral messenger RNA were both concentrated in the white pulps and germinal centers. This colocalization indicates that viral replication in immunocompetent structures of secondary lymphoid organs triggers anti-HIV effector CTLs to these particular locations, providing clues to target therapeutic intervention.

Transient mobilization of human immunodeficiency virus (HIV)-specific CD4 T-helper cells fails to control virus rebounds during intermittent antiretroviral therapy in chronic HIV type 1 infection.

Immune control of human immunodeficiency virus (HIV) is not restored by highly active antiretroviral therapies (HAART) during chronic infection. We examined the capacity of repeated structured therapeutic interruptions (STI) to restore HIV-specific CD4 and CD8 T-cell responses that controlled virus production. Eleven STI (median duration, 7 days; ranges, 4 to 24 days) were performed in three chronically HIV-infected patients with CD4 counts above 400/mm(3) and less than 200 HIV RNA copies/ml after 18 to 21 months of HAART; treatment resumed after 1 week or when virus became detectable. HIV-specific T-cell responses were analyzed by proliferation, gamma interferon (IFN-gamma) production, and enzyme-linked immunospot assays. Seven virus rebounds were observed (median, 4,712 HIV-1 RNA copies/ml) with a median of 7 days during which CD4 and CD8 counts did not significantly change. After treatment resumed, the viral load returned below 200 copies/ml within 3 weeks. Significant CD4 T-cell proliferation and IFN-gamma production against HIV p24 appeared simultaneously with or even before the virus rebounds in all patients. These CD4 responses lasted for less than 3 weeks and disappeared before therapeutic control of the virus had occurred. Increases in the numbers of HIV-specific CD8 T cells were delayed compared to changes in HIV-specific CD4 T-cell responses. No delay or increase in virus doubling time was observed after repeated STI. Iterative reexposure to HIV during short STI in chronically infected patients only transiently mobilized HIV-specific CD4 T1-helper cells, which might be rapidly altered by virus replication. Such kinetics might explain the failure at delaying subsequent virus rebounds and raises concerns about strategies based on STI to restore durable HIV-specific T-cell responses in chronic HIV infection.

Immunogenicity of mutations induced by nucleoside reverse transcriptase inhibitors for human immunodeficiency virus type 1-specific cytotoxic T cells.

The impact of drug resistance mutations induced by nucleoside reverse transcriptase (RT) inhibitors (NRTI) on cytotoxic T-lymphocyte (CTL) recognition of human immunodeficiency virus type 1 strain LAI (HIV-1(LAI)) RT was addressed in 35 treated or untreated patients. Two HIV-1(LAI) RT regions encompassing mutation M41L, L74V, M184V, and T215Y/F were recognized in 75 and 83% mutated and in 33 and 42% unmutated samples, respectively. A total of 41 new CTL epitopes overlapping these mutations were predicted. Mutations enhanced HLA-binding scores of 17 epitopes, decreased scores of 5, and had no effect in 19. Four predicted epitopes containing mutations 41, 74, and 184 were tested and recognized by CD8 cells from mutated or unmutated samples, with frequencies up to 270 gamma interferon spot-forming cells per 10(6) peripheral blood mononuclear cells. Therefore, RT mutations induced by NRTI can increase the immunogenicity of RT for CTL and might allow a better immune control of resistant viruses in vivo, suggesting that specific immune therapy might help prevent these mutations.

Dynamics of HIV-specific CD8+ T lymphocytes with changes in viral load.The RESTIM and COMET Study Groups.

The influence of HIV burden variations on the frequencies of Ag-specific CD8+ T cell responses was evaluated before and during highly active antiretroviral therapy by analyzing the number, diversity, and function of these cells. The frequencies of HLA-A2-restricted CD8+ PBL binding HLA-A2/HIV-epitope tetramers or producing IFN-gamma were below 1%. A panel of 16 CTL epitopes covering 15 HLA class I molecules in 14 patients allowed us to test 3.8 epitopes/patient and to detect 2.2 +/- 1.8 HIV epitope-specific CD8+ subsets per patient with a median frequency of 0.24% (0.11-4. 79%). During the first month of treatment, viral load rapidly decreased and frequencies of HIV-specific CD8 PBL tripled, eight new HIV specificities appeared of 11 undetectable at entry, while CMV-specific CD8+ PBL also appeared. With efficient HIV load control, all HIV specificities decayed involving a reduction of the CD8+CD27+CD11ahigh HIV-specific effector subset. Virus rebounds triggered by scheduled drug interruptions or transient therapeutic failures induced four patterns of epitope-specific CD8+ lymphocyte dynamics, i.e., peaks or disappearance of preexisting specificities, emergence of new specificities, or lack of changes. The HIV load rebounds mobilized both effector/memory HIV- and CMV-specific CD8+ lymphocytes. Therefore, frequencies of virus-specific CD8 T cells appear to be positively correlated to HIV production in most cases during highly active antiretroviral therapy, but an inverse correlation can also be observed with rapid virus changes that might involve redistribution, sequestration, or expansion of these Ag-specific CD8 T cells. Future strategies of therapeutic interruptions should take into account these various HIV-specific cell dynamics during HIV rebounds.

Up-regulation of adhesion and MHC molecules on splenic monocyte/macrophages in adult haemophagocytic syndrome.

Haemophagocytic syndrome (HPS) has been associated with the abnormal activation of mono/macrophages and increased cytokine production. However, neither the phenotype of haemophagocytic monomacrophages nor the cellular origin of cytokine production have been described. We studied splenic monomacrophages and lymphocytes from five patients with HPS (two HIV- and three HIV+) and from controls without HPS (three normal HIV and two pathological HIV+). Using flow-cytometry, we observed a marked increase in the expression of MHC class I and II, M-CSF-receptor and adhesion molecules LFA-1, LFA-3, ICAM-1 (P<0.05) on HPS+ splenic monomacrophages compared to HPS-, which was independent of their HIV status. A high percentage of CD8+ lymphocytes from 4/5 HPS+ patients produced TNF alpha and IFNgamma, but no IL-6 upon in-vitro activation. In a fifth patient CD4+ but not CD8+ lymphocytes produced these cytokines. Although other cytokines might be involved in the pathophysiology of HPS as suggested by the high expression of M-CSF-receptor, these results suggest that TNF alpha and IFNgamma secretion by T cells might play a role in the up-regulation of adhesion and MHC molecules on monomacrophages from HPS.

Cytotoxic T-cell responses to HIV-1 reverse transcriptase, integrase and protease.

OBJECTIVES: To determine immunodominant regions and new epitopes for cytotoxic T cells (CTL) directed against the HIV-1 pol products reverse transcriptase (RT), integrase and protease in a large cohort of patients at different stages of disease. DESIGN AND METHODS: Cross-sectional analysis of 98 patients from the French IMMUNOCO cohort (CD4 counts: 125-1050 x 10(6) cells/l), monitored for CTL recognition of HIV-1 pol products using recombinant vaccinia virus constructs and synthetic peptides. RESULTS: Memory CTL responses against HIV-1 pol products were detected in 78% of all patients whatever the stage of disease. RT was more immunogenic (81%, 30 out of 37 patients) than integrase and protease (51% and 24%, respectively). CTL recognition of RT was more frequent against Pol amino acids 310-460 (61%, 11 out of 18 patients) than against the other three portions (Pol 168-310, Pol 450-600, Pol 590-728) in patients with CD4 counts > 400 x 10(6)/l, whereas in patients at advanced stages no prominent differences were observed. Two new clusters of antigenic regions were found in the NH2 segment: three epitopes between amino-acids Pol 200 and 217 and four epitopes between amino-acids Pol 346 and 387, using five different HLA-restricting elements. A new cluster of three conserved epitopes was found in the COOH segment of RT. CONCLUSIONS: This study shows that memory CTL responses against HIV-1 RT, integrase and protease are detectable in most patients at different stages of disease. The capacity of CTL to recognize simultaneously clusters of epitopes may become important for the immune control to reinforce antiretroviral drug efficiency.

CTLs from lymphoid organs recognize an optimal HLA-A2-restricted and HLA-B52-restricted nonapeptide and several epitopes in the C-terminal region of HIV-1 Nef.

In a previous analysis of HIV-1-specific CTLs in lymphoid organs from HIV-seropositive patients, we reported high frequencies of in vivo differentiated CTLs directed against two immunodominant regions in the central and in the C-terminal part of the HIV-1 Nef protein. The present study analyzes the epitopes recognized by CTLs in the carboxyl terminus of Nef (amino acids 182-205). In addition to several epitopes that are recognized in association with different HLA molecules (A1, A2, A25(10), B35, B52), we defined an optimal nonapeptide (190-198). This nonapeptide was recognized by CTLs down to nanomolar concentrations in the context of at least two HLA molecules, HLA-B52 and HLA-A2, including three HLA-A2 subtypes: HLA-A2.1, -A2.2, and -A2.4. We also determined the relative frequencies of effector CTLs directed against peptide 190-198 to be as high as 10(-4), as opposed to lower frequencies ranging between 5 x 10(-5) and 5 x 10(-6) observed for the other peptides recognized in the same region, thus confirming the optimal presentation of this nonapeptide in vivo. Molecular modeling of the interactions between HLA-A2.1 and Nef peptide 190-198 suggests the formation of a stable complex and allowed us to study sequence motifs that are important for the binding of the HIV-1 peptide in the pockets of the HLA-A2.1 molecule.