Bruno Marchou

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

Correlation between genotypic predictions based on V3 sequences and phenotypic determination of HIV-1 tropism.

Objective:Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. Design:Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. Methods:HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. Results:The 103 patients had a median CD4+ T lymphocyte count of 268 × 106 cells/l and nadirs of 98 × 106 cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. Conclusion:Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.

Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals

Depletion of CD4+ T cells from the gut occurs rapidly during acute HIV-1 infection. This has been linked to systemic inflammation and disease progression as a result of translocation of microbial products from the gut lumen into the bloodstream. Combined antiretroviral therapy (cART) substantially restores CD4+ T cell numbers in peripheral blood, but the gut compartment remains largely depleted of such cells for poorly understood reasons. Here, we show that a lack of recruitment of CD4+ T cells to the gut could be involved in the incomplete mucosal immune reconstitution of cART-treated HIV-infected individuals. We investigated the trafficking of CD4+ T cells expressing the gut-homing receptors CCR9 and integrin α4β7 and found that many of these T cells remained in the circulation rather than repopulating the mucosa of the small intestine. This is likely because expression of the CCR9 ligand CCL25 was lower in the small intestine of HIV-infected individuals. The defective gut homing of CCR9+β7+ CD4+ T cells - a population that we found included most gut-homing Th17 cells, which have a critical role in mucosal immune defense - correlated with high plasma concentrations of markers of mucosal damage, microbial translocation, and systemic T cell activation. Our results thus describe alterations in CD4+ T cell homing to the gut that could prevent efficient mucosal immune reconstitution in HIV-infected individuals despite effective cART.

R5 to X4 switch of the predominant HIV-1 population in cellular reservoirs during effective highly active antiretroviral therapy.

Summary:HIV-1 coreceptor usage plays a critical role for virus tropism and pathogenesis. A switch from CCR5 to CXCR4-using viruses can occur in the natural course of infection and correlates with subsequent disease progression. To investigate whether HIV-1 genetic evolution might lead to changes in virus coreceptor usage during highly active antiretroviral therapy (HAART), a longitudinal genotypic analysis of the virus found in cellular reservoirs was conducted in 32 patients with undetectable viral loads on HAART for 5 years. The genotype of the 3rd variable region of the env gene predicting coreceptor usage was retrospectively determined in the plasma or in peripheral blood mononuclear cells (PBMC) at baseline and then in PBMCs at months 30 and 60 of HAART. There was a switch from R5 to X4 variants in 11 of the 23 patients who harbored a majority virus population of R5 variants at baseline. X4 variants remained predominant in the 9 patients who harbored mainly X4 variants at baseline. The patients harboring predominantly X4 variants during HAART, either from baseline or after an R5 to X4 switch, tended to have lower CD4+ T-cell counts on HAART than did patients harboring continuously a majority population of R5 variants. These results suggest that potent antiretroviral therapy produces the conditions necessary for the gradual emergence of X4 variants in cellular reservoirs.

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

Background The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. Methodology/Principal Findings We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14high CD16− and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14high CD16− monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. Conclusions Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART.

Population-Based Sequencing of the V3 Region of env for Predicting the Coreceptor Usage of Human Immunodeficiency Virus Type 1 Quasispecies

ABSTRACT Genotypic population-based methods could be faster and less expensive than phenotypic recombinant assays for determining human immunodeficiency virus type 1 (HIV-1) coreceptor usage in patient samples, but their clinical use requires good genotype-phenotype correlation and concordance with clonal analyses. We have assessed these requirements by clonal analysis of the V1 to V3 env PCR products of 26 patients infected with subtype B HIV-1. We used the resulting set of molecular clones, all sequenced and characterized using a single-cycle recombinant virus phenotypic entry assay, to reevaluate genotype-phenotype correlations. Combining the previously described 11/25 and net charge rules for the V3 genotype improved the prediction of HIV-1 coreceptor usage. We also evaluated the concordance of population-based and clonal analyses for predicting the coreceptor usage of HIV-1 quasispecies. Our population-based recombinant phenotypic assay and direct sequencing of V3 were similarly sensitive for detecting the presence of minor species in the virus population, and both correlated well with clonal analysis. The improved genotype-phenotype correlation obtained by combining two simple genotypic rules and the good concordance with clonal analyses suggest that direct sequencing of V3 is a valuable alternative to population-based recombinant phenotypic assays.

Total Body Composition by DXA of 241 HIV-Negative Men and 162 HIV-Infected Men: Proposal of Reference Values for Defining Lipodystrophy

The aim of this study was to define standard values for fat mass distribution by dual-energy X-ray absorptiometry in human immunodeficiency virus (HIV)-negative men and to analyze factors associated with lipodystrophy in HIV-infected men. Total-body composition was analyzed in 241 HIV-negative men (controls) and 162 HIV-infected men. We created a fat mass ratio (FMR) as the ratio of the percentage of the trunk fat mass to the percentage of the lower limbs fat mass. We defined the FMR standard values as the mean value+/-standard deviation. We compared body mass index (BMI), fat mass percentage (%FM), lean mass (LM), bone mineral density (BMD), and FMR between the control group and HIV-infected men, by age range, according to prescription of treatment and presence of clinical lipodystrophy. The FMR standard value is equal to 1.3+/-0.2. The FMR was higher in treated HIV-infected men with or without clinical lipodystrophy. The FMR was similar for naïve HIV-infected men and controls. It was positively correlated with age, cumulative time on treatment, zidovudine, stavudine, or indinavir. BMD and fat mass were lower for treated and naïve HIV-infected men than for HIV-negative men. The FMR seems to be a valuable index for measuring fat mass distribution. We defined FMR standard values from the largest group of HIV-negative men to our knowledge. Applying FMR to HIV patients could help physicians to diagnose lipodystrophy earlier.

Shift in HIV resistance genotype after treatment interruption and short-term antiviral effect following a new salvage regimen.

ObjectiveTo investigate the changes in genotypic drug-resistance pattern, plasma HIV RNA and CD4 cell count after treatment interruption and assess the short-term antiviral effect of a new salvage regimen. DesignProspective study of 38 patients with multiple failing regimens who had completely stopped all medication for 3 months before a three to five-drug regimen was reintroduced according to clinical guidelines. MethodsPatients were tested for HIV resistance before and after treatment interruption by population-based sequencing and clonal analysis of selected patients. ResultsDiscontinuation of therapy for 3 months was associated with a median increase in HIV RNA of 0.4 log10 and a median decrease in CD4 cell count of 43 × 106/l. Sixty-one per cent of patients had a shift from the drug-resistant genotype to a predominantly wild-type genotype. The patients significantly likely to show genotype reversion were those in Centers for Disease Control groups A or B, who had been exposed to few drugs, had a low plasma HIV RNA, or a high CD4 cell count. The only independent factor predicting genotype reversion was the clinical stage. The median change in plasma HIV RNA at month 3 after treatment reintroduction was −2.3 log10 copies/ml in patients who had genotype reversion compared with −0.6 log10 copies/ml in patients without genotype reversion (P = 0.004). ConclusionSuspending treatment for 3 months after multiple failures could be a suitable strategy for optimizing salvage therapy provided it is instituted early, before the HIV disease becomes too advanced.

Zika virus in semen and spermatozoa

The current Zika virus epidemic is a major challenge for the medical and scientific communities for at least two reasons: the severe clinical situation associated with Zika virus infection (neurological complications and adverse fetal outcomes) and the unexpected sexual viral transmission from men to women. These recent findings have shifted the paradigm of arbovirus–host interactions, modifying standard epidemiology and clinical patterns. High infectious Zika virus loads have been detected in semen, but data for viral persistence after symptomatic infections are scarce and even nonexistent for asympto matic ones, with the remaining key issue: how long does semen contain infectious Zika virus? As long as this question is unanswered, the adaptation of preventive measures such as the use of condoms and the abstinence of semen donation will be hampered. Here, we report the longitudinal follow up of Zika virus RNA in the semen of a 32-year-old man returning from French Guyana. At admission, the patient had moderate fever, maculopapular rash, myalgia, and arthralgia and was diagnosed for Zika virus infection upon detection of viral RNA in plasma and urine 2 days after onset of symptoms. The molecular diagnosis for dengue and chikungunya viruses was negative (in-house RT-PCR). The patient was seronegative for HIV, had a normal immunological status, and recovered in few days. Semen (11 samples), blood (ten), and urine (five) were prospectively collected for 141 days after symptom onset. Zika virus RNA was detected on each semen sample and was still positive after 141 days, with viral load decreasing from 8·6 log copies per mL to 3·5 log copies per mL. It was detected until day 37 in both blood (2·5 log copies per mL) and urine (3·7 log copies per mL; fi gure). Such a prolonged RNA Zika virus excretion is quite different from other fl aviviruses, which are rapidly cleared by the immune response and for which the detection of viral nucleic acids is typically limited to a short window after symptom onset. In addition to the present case, we investigated five other symptomatic men for the presence of Zika virus RNA in semen; RNA was still detected 69 days and 115 days after the symptom onset in two patients, but not detected at day 20 in the other three individuals (data not shown). These data suggest that the length of Zika virus excretion varies, probably depending on viral and host characteristics, but long-lasting excretion might be frequent among adults who experienced a symptomatic infection. Such long-lasting excretion of genomic material was recently described in semen from Ebola survivors. The viral persistence in semen is of major concern and could be related to a viral tropism for male sexual cells. Accordingly, we report the immunohistochemical detection of Zika virus in the head of spermatozoa obtained from the fi rst patient (fi gure; appendix). The proportion of infected spermatozoa was estimated at 3·52% (SD 0·71), as assessed by counting Zikavirus-positive sperma tozoa on three smears by fl uorescence micro scopy. The use of both confocal and stimulated emission depletion microscopy (appendix), allowed the unambiguous demonstration that Zika virus antigens are indeed present inside spermatozoa. Additional immuno stainings of sperm from a healthy non-infected control donor for Zika virus antigens or with a mouse IgG2a isotype control confi rmed the specifi city of the staining (data not shown). Spermatozoa do not express the candidate Zika virus entry receptor Axl. Because Sertoli cells highly express Axl and have essential roles in spermatogenesis, we hypothesise that they might be involved in Zika virus transmission to spermatozoa. Appropriately designed studies are needed to discover the modes of spermatozoa infection and length of risk of sexual transmission. Answers to these questions will allow public health authorities to recom mend urgently needed universal safe practices.

Mutations conferring resistance to zidovudine diminish the antiviral effect of stavudine plus didanosine

This study evaluated the influence of zidovudine (ZDV) resistance mutations on the antiviral effect of the combination of stavudine (D4T) plus didanosine (ddI) in patients treated previously with ZDV plus zalcitabine (ddC). Twenty patients who had been treated with ZDV plus ddC for a median duration of 11 months (range, 7–42 months) were switched to D4T (40 mg twice a day [BID]) + ddI (200 mg BID) in an open pilot study lasting 6 months. The CDC classes were A (n = 10) and B (n = 10). The median baseline CD4 count was 285/mm3 and the median baseline plasma virus RNA (Amplicor HIV Monitor RT‐PCR assay) was 4.6 log copies/ml. Population‐based sequence analysis detected mutations associated with resistance to reverse transcriptase (RT) inhibitors in the RT domain of virus RNA from baseline plasma samples in 13/20 (65%) patients. Twelve patients had mutations associated with zidovudine resistance (3 T215Y ‐ M41L ‐ L210W; 3 T215Y ‐ M41L; 2 T215Y ‐ L210W; 3 T215Y; 1 K70R) and 1 patient had a multi‐dideoxynucleoside resistance mutation (QI5IM). Patients with a resistance mutation had a significantly lower RNA suppression after 3 and 6 months (median RNA reduction −0.5 log and −0.1 log) than the remaining patients (−1.6 log and −2 log). Fifty percent of patients with wild‐type viruses had undetectable plasma RNA after 24 weeks of D4T plus ddI therapy, whereas all those with mutated viruses had HIV RNA concentration > 3 log copies/ml at week 24 (P < .05). Our finding may have implications when deciding on a second line therapy with three or four drugs that includes two new nucleoside analogues. Cross‐resistance between nucleoside analogues deserves maximal attention to ensure optimal antiretroviral therapy and design algorithms for antiretroviral management based on genotypic antiretroviral resistance testing. J. Med. Virol. 59:507–511, 1999.