Intact proviruses are enriched in the colon and associated with PD-1+TIGIT- mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy.

BACKGROUND: The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. METHODS: We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. FINDINGS: Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. INTERPRETATION: Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. FUNDING: This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

Th22 cells are efficiently recruited in the gut by CCL28 as an alternative to CCL20 but do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

Gut CD4+ T cells are incompletely restored in most HIV-1-infected individuals on antiretroviral therapy, notably Th17 cells, a key subset in mucosal homeostasis. By contrast, gut Th22 cells are usually restored at normal frequencies. Th22 cells display a CCR6+CCR10+ phenotype and could thus respond to CCL20- and CCL28-mediated chemotaxis, while Th17 cells, which express CCR6 but not CCR10, depend on CCL20. Herein, we found that CCL28 is normally expressed by duodenal enterocytes of treated HIV-1-infected individuals, while CCL20 expression is blunted. Ex vivo, we showed that Th22 cells contribute to the reduction of CCL20 production by enterocytes through an IL-22- and IL-18-dependent mechanism. Th22 cells preferentially migrate via CCL20- rather than CCL28-mediated chemotaxis when both chemokines are available in the microenvironment. However, when the CCL20/CCL28 ratio drops, as in treated HIV-1-infected individuals, Th22 cells can migrate via the CCR10-CCL28 axis, as an alternative to CCR6-CCL20. This could explain the better reconstitution of gut Th22 compared with Th17 cells on antiretroviral therapy. Lastly, we assessed the relationships between the frequencies of gut Th17 and Th22 cells and inflammatory markers related to microbial translocation, and showed that Th22 cells do not compensate for the loss of Th17 cells in treated HIV-1-infected individuals.

Hepatitis E virus replication in human intestinal cells.

OBJECTIVE: Hepatitis E virus (HEV), one of the most common agent of acute hepatitis worldwide, is mainly transmitted enterically, via contaminated water for HEV genotypes 1 (HEV1) and HEV2, or by eating raw or undercooked infected meat for HEV genotype 3 (HEV3) and HEV4. However, little is known about how the ingested HEV reaches the liver or its ability to replicate in intestinal cells. DESIGN: We developed human primary cultures of small intestine epithelial cells and intestinal explants obtained from small bowel resections. The epithelial cells were also polarised on transwells. Cells were infected with Kernow-p6 strain or clinically derived virions. RESULTS: Primary intestinal cells supported the growth of Kernow-p6 strain and HEV1 and HEV3 clinically derived virions. Polarised enterocytes infected with HEV1 and HEV3 strains released HEV particles vectorially: mostly into the apical compartment with a little basally. Iodixanol density gradient centrifugation of enterocyte-derived HEV virions gave bands at a density of 1.06-1.08 g/cm3, corresponding to that of quasi-enveloped HEV particles. Ribavirin therapy inhibited HEV excretion from the basal surface but not from the apical side of infected human enterocytes. HEV virions also infected intestinal tissue explants. Lastly, HEV RNA and antigen were detected in the intestinal crypts of a chronically infected patient. CONCLUSION: HEV can replicate in intestinal cells and reaches the liver as quasi-enveloped virions.

No selection of CXCR4-using variants in cell reservoirs of dual-mixed HIV-infected patients on suppressive maraviroc therapy.

We used ultradeep sequencing to investigate the evolution of the frequency of CXCR4-using viruses in the peripheral blood mononuclear cells of 22 patients infected with both CCR5 and CXCR4-using viruses treated with the CCR5 antagonist maraviroc for 24 weeks and a stable antiviral therapy. The mean CXCR4-using virus frequency in peripheral blood mononuclear cells was 59% before maraviroc intensification and 52% after 24 weeks of effective treatment, indicating no selection by maraviroc.

Primary resistance of CCR5-tropic HIV-1 to maraviroc cannot be predicted by the V3 sequence.

OBJECTIVES: Resistance of HIV-1 to CCR5 antagonists can occur without coreceptor switching by mutations in envelope glycoproteins that enable virus entry using the inhibitor-bound form of CCR5. We investigated whether mutations in the V3 region of HIV-1 from subjects naive to maraviroc could be associated with primary resistance to this drug. METHODS: The frequency of CCR5-tropic HIV-1 subtype B isolates harbouring putative V3 maraviroc resistance mutations was assessed among the HIV tropism database of Toulouse University Hospital, France. Phenotypic assessment of maraviroc susceptibility was performed for 14 isolates representative of the main mutation patterns and 14 controls. V3 mutations were reversed or introduced by site-directed mutagenesis. RESULTS: Ninety-three of 951 (9.8%) isolates harboured V3 mutations assumed to be associated with maraviroc resistance. Maraviroc completely blocked virus entry for all but 1 of the 14 isolates harbouring V3 mutations [IC50 8.6 nM; 95% CI (6.6-47.4)], as in the 14 control isolates [IC50 13.4 nM; 95% CI (7.7-50.3)] (P = 0.24). Primary resistance to maraviroc, with a plateau in entry inhibition, was found in one isolate (harbouring a 20F/21I genotype). Site-directed mutagenesis showed that V3 mutations are necessary but not sufficient to induce maraviroc resistance. CONCLUSIONS: The impact of V3 mutations depended on the env context in which they occurred. Simple assessment of the V3 genotype thus cannot accurately predict maraviroc resistance. Rather, phenotypic assessment of virus particles expressing the envelope glycoprotein as a whole is required. This approach revealed that primary resistance of CCR5-tropic HIV-1 subtype B isolates to maraviroc seems uncommon.

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission.

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%-90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed. Electron microscopy showed the accumulation of viral particles either into large intracellular viral-containing compartments or outside the cells, indicating that endosomal accumulation of BMP could block intracellular biogenesis of viral particles while inhibition of macropinocytosis would prevent viral particle uptake. This is the first report linking BMP metabolism with a natural steroid such as progesterone or with involvement of a phospholipase A1 activity. BMP cellular content could be used as a biomarker for efficient anti-viral drugs.

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 α4ß7 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+ß7+ 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.

Infection of macrophages and dendritic cells with primary R5-tropic human immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 antibodies purified from cervicovaginal secretions.

Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.

Inhibition of HIV-1 transmission in trans from dendritic cells to CD4+ T lymphocytes by natural antibodies to the CRD domain of DC-SIGN purified from breast milk and intravenous immunoglobulins.

The present study demonstrates that human breast milk and normal human polyclonal immunoglobulins purified from plasma [intravenous immunoglobulins (IVIg)] contain functional natural immunoglobulin A (IgA) and IgG antibodies directed against the carbohydrate recognition domain (CRD) domain of the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) molecule, which is involved in the binding of human immunodeficiency virus (HIV)-1 to dendritic cells (DCs). Antibodies to DC-SIGN CRD were affinity-purified on a matrix to which a synthetic peptide corresponding to the N-terminal CRD domain (amino-acid 342-amino-acid 371) had been coupled. The affinity-purified antibodies bound to the DC-SIGN peptide and to the native DC-SIGN molecule expressed by HeLa DC-SIGN+ cells and immature monocyte-derived dendritic cells (iMDDCs), in a specific and dose-dependent manner. At an optimal dose of 200 microg/ml, natural antibodies to DC-SIGN CRD peptide purified from breast milk and IVIg stained 25 and 20% of HeLa DC-SIGN+ cells and 32 and 12% of iMDDCs, respectively. Anti-DC-SIGN CRD peptide antibodies inhibited the attachment of virus to HeLa DC-SIGN by up to 78% and the attachment to iMDDCs by only 20%. Both breast milk- and IVIg-derived natural antibodies to the CRD peptide inhibited 60% of the transmission in trans of HIV-1(JRCSF), an R5-tropic strain, from iMDDCs to CD4+ T lymphocytes. Taken together, these observations suggest that the attachment of HIV to DCs and transmission in trans to autologous CD4+ T lymphocytes occur through two independent mechanisms. Our data support a role of natural antibodies to DC-SIGN in the modulation of postnatal HIV transmission through breast-feeding and in the natural host defence against HIV-1 in infected individuals.

Opsonization of HIV with complement enhances infection of dendritic cells and viral transfer to CD4 T cells in a CR3 and DC-SIGN-dependent manner.

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4(+) T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1(BaL) (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1(NDK) (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN(+) CR3(-) cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.

R5- and X4-HIV-1 use differentially the endometrial epithelial cells HEC-1A to ensure their own spread: implication for mechanisms of sexual transmission.

The mechanism of viral transmission across the mucosal barrier is poorly understood. Using the endometrial epithelium-derived cell line HEC-1A, we found that the cells are capable of sequestering large numbers of HIV-1 particles but are refractory to cell-free viral infection. The removal of heparan sulfate moieties of cell-surface proteoglycans (HSPG) from the apical pole of HEC-1A accounted for at least 60% of both R5- and X4-HIV-1 attachment, showing their important implication in viral attachment. HEC-1A cells also have the capacity to endocytose a weak proportion of the attached virus and pass it along to underlying cells. Fucose, N-acetylglucosamine and mannosylated-residues inhibited the transcytosis of some virus isolates, suggesting that mannose receptors can be implicated on the both R5- and X4-HIV-1 transcytosis. The inhibition of HIV transcytosis by blocking CCR5 mAb suggests the implication of specific interaction between the viral gp120 and sulfated moiety of syndecans during the transcytosis of mostly R5- and X4-HIV-1. At the basolateral pole of HEC-1A, HSPG sequestered X4- and not R5-HIV-1, highlighting the important role of HEC-1A as an X4 virus reservoir. The cell-free virus particles that have transcytosed could infect activated T cells but with a weaker efficiency than virus that had not transcytosed. The specific stimulation of HEC-1A by R5-HIV-1 increased the release of monocytes/chemokines-attracting chemokines (IL-8 and GR0) and proinflammatory cytokines (TNF-beta and IL-1alpha) that enhanced the production of virus by activated T cells. This study suggests that R5 and X4 viruses can differentially use epithelial cells to ensure their own spread.

IFN-gamma-activated monocytes weakly produce HIV-1 but induce the recruitment of HIV-sensitive T cells and enhance the viral production by these recruited T cells.

The ability of macrophages to adapt to changing cytokine environments results in the dominance of a particular functional phenotype of macrophages, which would play a significant role in HIV pathogenesis. In comparison with untreated macrophages (M0), we examined the role of macrophages derived from IFN-gamma-activated monocytes (M1) in the HIV spread. We show that M0 and M1 bind with the same efficiency HIV-1 with a predominant role of C-type lectins in the R5-HIV attachment and of the heparan sulfate proteoglycans in the X4-HIV attachment. Despite similar levels of R5- and X4-HIV DNA, M1 replicates and weakly transmits the virus to activated T cells by releasing CXCR4- and CCR5-interacting chemokines. The blockade of dendritic cell-specific ICAM-3-grabbing nonintegrin expressed on M1 by mAb does not interfere with the viral transfer. Uninfected M1 recruits HIV-sensitive T cells efficiently and releases soluble factors, enhancing the viral production by these recruited cells. This study highlights the role of IFN-gamma to induce a population of macrophages that archive HIV-1 within a latent stage and cause the persistence of the virus by favoring the recruitment of T cells or enhancing the viral replication in infected CD4(+) T cells.

Differential modulation of human lactoferrin activity against both R5 and X4-HIV-1 adsorption on epithelial cells and dendritic cells by natural antibodies.

Human lactoferrin (Lf) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to Lf. In the present study, we showed that Lf limited specifically adsorption of R5- and X4-HIV-1-free particles on endometrial epithelial HEC-1A cells, by inhibiting virus adsorption on heparan-sulfated proteoglycans. But, Lf did not interfere with both R5 and X4-HIV transcytosis. We showed also the efficacy of Lf in preventing R5 and X4-HIV capture by dendritic cells. Conversely, we demonstrated that Lf-reacting natural Abs (NAbs) present within i.v. Ig-enhanced HIV attachment on dendritic cells by forming HIV-Lf-NAbs. HIV particles were able to directly interact with Lf following its interaction with NAbs. We also found Lf-reacting natural Abs within cervicovaginal secretions, suggesting the existence of Lf-NAbs complexes in women genital tract in vivo. In conclusion, this study highlights Lf as a potent microbicides and reports new function for NAbs within the genital compartment that may compartment that may abolish the inhibitory activity of microbicide compounds. Thus, we proposed a model in which Lf would appear as a double-edged sword that could have beneficial or detrimental effects depending on both cellular and molecular environments. This study highlights the use of Lf derivates as microbicide candidates to limit such interferences.

Natural antibodies to CCR5 from breast milk block infection of macrophages and dendritic cells with primary R5-tropic HIV-1.

In the present study, we demonstrate that breast milk of 66% and 83% of HIV-seronegative and seropositive women, respectively, contains natural Abs of the secretory IgA and IgG isotypes directed against the CCR5 coreceptor for R5-tropic strains of HIV-1. Abs to CCR5 were affinity purified on a matrix to which a synthetic peptide corresponding to the second extracellular loop of CCR5 had been coupled. The purified Abs bound to the CCR5 peptide in a dose-dependent fashion and to both native CCR5 expressed by Chinese hamster ovary cells transfected with CCR5 gene, macrophages, and immature dendritic cells. Although the avidity differed, the amount of anti-CCR5 Abs did not significantly differ between breast milk of HIV-seropositive and -seronegative women. Purified anti-CCR5 Abs inhibited up to 75% infection of macrophages and dendritic cells with HIV(BaL) and HIV(JR-CSF). Our observations provide evidence for a role of natural Abs to CCR5 in breast milk in controlling transmissibility of HIV through breastfeeding.