Hakim Hocini

Intracellular Neutralization of HIV Transcytosis across Tight Epithelial Barriers by Anti-HIV Envelope Protein dIgA or IgM

Human immunodeficiency virus, generated during contact between HIV-infected cells and the apical surface of an epithelial cell, can cross a tight epithelial barrier by transcytosis. We show that transcytosis of primary HIV isolates is blocked by dimeric IgA or IgM against HIV envelope proteins. Neutralization occurs intracellularly within the apical recycling endosome, and immune complexes are specifically recycled to the mucosal surface. One epitope involved in neutralization is a conserved sequence of the gp41 HIV envelope protein subunit. Finally, transcytosis also occurs across functional human mucosal tissue in a process inhibited by a serosal internalization of IgM against the HIV envelope protein. These results suggest that induction of mucosal immunity to HIV envelope proteins may impair the transcytotic route of HIV mucosal transmission.

Infectious Human Immunodeficiency Virus Can Rapidly Penetrate a Tight Human Epithelial Barrier by Transcytosis in a Process Impaired by Mucosal Immunoglobulins

Mucosal surfaces are the main natural site of entry into the body for human immunodeficiency virus (HIV). Herein, an alternative mechanism for virus spread is described. The mechanism, which involves transcytosis of endosome-internalized HIV-particles, was generated by contact of HIV-infected cells with the apical surface of an epithelial cell line. Transcytosed viruses rapidly (in 20-30 min) access the serosal side of the epithelial barrier without infecting the epithelium itself. In turn, transcytosed HIV could infect host submucosal mononucleated target cells, and thus the infection could spread. An investigation was done to determine whether mucosal antibodies could block HIV transcytosis. Both secretory IgA (S-IgA) and IgG that were purified from colostrum from HIV-seropositive women impaired HIV transcytosis, irrespective of the level of the recombinant HIV envelope anti-gp160-specific activities in an ELISA. However, specific S-IgAs were more efficient than IgG. Therefore, mucosal-specific S-IgA to HIV-1 could be relevant to reducing infectivity of HIV-1 in corporeal fluids.

Opsonization of HIV-1 by Semen Complement Enhances Infection of Human Epithelial Cells

In the present study we demonstrate that both X4- and R5-tropic HIV-1 strains are able to infect the human epithelial cell line HT-29. Infection was enhanced 2-fold when HIV was added to semen before contact with the cell cultures. The enhancing effect of semen was complement dependent, as evidenced by blockage of generation of C3a/C3adesArg in semen by heat or EDTA treatment of semen and suppression of semen-dependent enhancement with mAbs directed to complement receptor type 3 (CD11b/CD18) and soluble CD16. Infection of HT-29 cells was assessed by the release of p24 Ag in cultures and semiquantitative PCR of the HIV-1 pol gene. Inhibition of infection of HT-29 by stromal cell-derived factor 1 was decreased in the case of semen-opsonized X4- and R5-tropic virus compared with unopsonized virus. In contrast, inhibition of infection by RANTES was increased for opsonized X4-tropic HIV-1 compared with unopsonized virus. Taken together these observations indicate that activation of complement in semen may play an enhancing role in mucosal transmission of HIV-1 by facilitating infection of epithelial cells and/or enhancing infection of complement receptor-expressing target cells in the mucosa.

Secretory Anti-Human Immunodeficiency Virus (HIV) Antibodies in Colostrum and Breast Milk Are Not a Major Determinant of the Protection of Early Postnatal Transmission of HIV

The immune response to human immunodeficiency virus (HIV) type 1 was evaluated in breast milk from HIV-infected African mothers who had transmitted and those who had not transmitted HIV to their children through breast-feeding. The levels, specific activities against gp160 and 2 HIV-derived peptides from gp41 and gp120 (V3 loop), and inhibitory activity toward viral transcytosis in vitro of secretory IgA (S-IgA) and IgG purified from breast milk were investigated in 8 transmitting mothers and 18 nontransmitting mothers. S-IgA and IgG antibodies to gp160 and to peptides were found in all breast milk samples. The specific activities of S-IgA and IgG to gp160 and peptides were similar between transmitting and nontransmitting mothers. No difference of the capacity of S-IgA and IgG to block HIV transcytosis in vitro was found between the 2 groups. These results suggest that humoral mucosal immunity to HIV does not appear as a predominant factor for protection against viral transmission through breast milk.

Secretory Leukocyte Protease Inhibitor Inhibits Infection of Monocytes and Lymphocytes with Human Immunodeficiency Virus Type 1 but Does Not Interfere with Transcytosis of Cell- Associated Virus across Tight Epithelial Barriers

ABSTRACT In the present study, we demonstrate that recombinant human secretory leukocyte protease inhibitor (rhSLPI) inhibits infection of lymphocyte- and monocyte-derived tumor cell lines and peripheral blood lymphocytes with laboratory-adapted isolates and with the primary isolate, NDK, of free human immunodeficiency virus type 1 (HIV-1). In contrast, rhSLPI did not exhibit inhibitory activity toward transcytosis of cell-associated HIV-1 through a tight monolayer of endometrial epithelial cells. These observations indicate that the inhibitory effect of SLPI is restricted to free HIV-1 in corporal fluids.

Antibodies to C-C Chemokine Receptor 5 in Normal Human IgG Block Infection of Macrophages and Lymphocytes with Primary R5-Tropic Strains of HIV-1

In the present study, we demonstrate that normal human IgG for therapeutic use (i.v. Ig) contains natural Abs directed against the CCR5 coreceptor for HIV-1. Abs to CCR5 were isolated from i.v. Ig using an affinity matrix consisting of a synthetic peptide corresponding to the N-terminus of CCR5 coupled to Sepharose. Natural anti-CCR5 Abs inhibited the binding of RANTES to macrophages, demonstrating their interaction with the coreceptor of R5-tropic HIV-1. Affinity-purified anti-CCR5 Ig further inhibited infection of lymphocytes and monocytes/macrophages with primary and laboratory-adapted strains of HIV-1, but did not inhibit infection with X4-tropic HIV. Our results suggest that anti-CCR5 Abs from healthy immunocompetent donors may be suitable for development of novel passive immunotherapy regimens in specific clinical settings in HIV infection.

Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV

ABSTRACT The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 (HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of CT values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of CT values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.

Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells

ABSTRACT We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus.

The Phospholipid Scramblases 1 and 4 Are Cellular Receptors for the Secretory Leukocyte Protease Inhibitor and Interact with CD4 at the Plasma Membrane

Secretory leukocyte protease inhibitor (SLPI) is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1) and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention.

A novel approach for biomarker selection and the integration of repeated measures experiments from two assays

BackgroundHigh throughput ’omics’ experiments are usually designed to compare changes observed between different conditions (or interventions) and to identify biomarkers capable of characterizing each condition. We consider the complex structure of repeated measurements from different assays where different conditions are applied on the same subjects.ResultsWe propose a two-step analysis combining a multilevel approach and a multivariate approach to reveal separately the effects of conditions within subjects from the biological variation between subjects. The approach is extended to two-factor designs and to the integration of two matched data sets. It allows internal variable selection to highlight genes able to discriminate the net condition effect within subjects. A simulation study was performed to demonstrate the good performance of the multilevel multivariate approach compared to a classical multivariate method. The multilevel multivariate approach outperformed the classical multivariate approach with respect to the classification error rate and the selection of relevant genes. The approach was applied to an HIV-vaccine trial evaluating the response with gene expression and cytokine secretion. The discriminant multilevel analysis selected a relevant subset of genes while the integrative multilevel analysis highlighted clusters of genes and cytokines that were highly correlated across the samples.ConclusionsOur combined multilevel multivariate approach may help in finding signatures of vaccine effect and allows for a better understanding of immunological mechanisms activated by the intervention. The integrative analysis revealed clusters of genes, that were associated with cytokine secretion. These clusters can be seen as gene signatures to predict future cytokine response. The approach is implemented in the R package mixOmics (http://cran.r-project.org/) with associated tutorials to perform the analysisa.