Randomized Phase I: Safety, Immunogenicity and Mucosal Antiviral Activity in Young Healthy Women Vaccinated with HIV-1 Gp41 P1 Peptide on Virosomes.

UNLABELLED: Mucosal antibodies harboring various antiviral activities may best protect mucosal surfaces against early HIV-1 entry at mucosal sites and they should be ideally induced by prophylactic HIV-1 vaccines for optimal prevention of sexually transmitted HIV-1. A phase I, double-blind, randomized, placebo-controlled trial was conducted in twenty-four healthy HIV-uninfected young women. The study objectives were to assess the safety, tolerability and immunogenicity of virosomes harboring surface HIV-1 gp41-derived P1 lipidated peptides (MYM-V101). Participants received placebo or MYM-V101 vaccine at 10 µg/dose or 50 µg/dose intramuscularly at week 0 and 8, and intranasally at week 16 and 24. MYM-V101 was safe and well-tolerated at both doses administered by the intramuscular and intranasal routes, with the majority of subjects remaining free of local and general symptoms. P1-specific serum IgGs and IgAs were induced in all high dose recipients after the first injection. After the last vaccination, vaginal and rectal P1-specific IgGs could be detected in all high dose recipients. Approximately 63% and 43% of the low and high dose recipients were respectively tested positive for vaginal P1-IgAs, while 29% of the subjects from the high dose group tested positive for rectal IgAs. Serum samples had total specific IgG and IgA antibody concentrations ≥ 0.4 µg/mL, while mucosal samples were usually below 0.01 µg/mL. Vaginal secretions from MYM-V101 vaccinated subjects were inhibiting HIV-1 transcytosis but had no detectable neutralizing activity. P1-specific Th1 responses could not be detected on PBMC. This study demonstrates the excellent safety and tolerability of MYM-V101, eliciting systemic and mucosal antibodies in the majority of subjects. Vaccine-induced mucosal anti-gp41 antibodies toward conserved gp41 motifs were harboring HIV-1 transcytosis inhibition activity and may contribute to reduce sexually-transmitted HIV-1. TRIAL REGISTRATION: ClinicalTrials.gov NCT01084343.

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

A Role for cis Interaction between the Inhibitory Ly49A receptor and MHC class I for natural killer cell education.

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

Stable masking by H-2Dd cis ligand limits Ly49A relocalization to the site of NK cell/target cell contact.

Ly49A is an inhibitory receptor, which counteracts natural killer (NK) cell activation on the engagement with H-2D(d) (D(d)) MHC class I molecules (MHC-I) on target cells. In addition to binding D(d) on apposed membranes, Ly49A interacts with D(d) ligand expressed in the plane of the NK cells' membrane. Indeed, multivalent, soluble MHC-I ligand binds inefficiently to Ly49A unless the NK cells' D(d) complexes are destroyed. However, it is not known whether masked Ly49A remains constitutively associated with cis D(d) also during target cell interaction. Alternatively, it is possible that Ly49A has to be unmasked to significantly interact with its ligand on target cells. These two scenarios suggest distinct roles of Ly49A/D(d) cis interaction for NK cell function. Here, we show that Ly49A contributes to target cell adhesion and efficiently accumulates at synapses with D(d)-expressing target cells when NK cells themselves lack D(d). When NK cells express D(d), Ly49A no longer contributes to adhesion, and ligand-driven recruitment to the cellular contact site is strongly reduced. The destruction of D(d) complexes on NK cells, which unmasks Ly49A, is necessary and sufficient to restore Ly49A adhesive function and recruitment to the synapse. Thus, cis D(d) continuously sequesters a considerable fraction of Ly49A receptors, preventing efficient Ly49A recruitment to the synapse with D(d)+ target cells. The reduced number of Ly49A receptors that can functionally interact with D(d) on target cells explains the modest inhibitory capacity of Ly49A in D(d) NK cells. This property renders Ly49A NK cells more sensitive to react to diseased host cells.

Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alpha-defensin production.

Although human CD56(+)CD3(-) natural killer (NK) cells participate in immune responses against microorganisms, their capacity to directly recognize and be activated by pathogens remains unclear. These cells encode members of the Toll-like receptor (TLR) family, involved in innate cell activation on recognition of pathogen-associated molecular patterns (PAMPs). We therefore evaluated whether the 2 bacterial protein PAMPs, the outer membrane protein A from Klebsiella pneumoniae (KpOmpA) and flagellin, which signal through TLR2 and TLR5, respectively, may directly stimulate human NK cells. These proteins induce interferon-gamma (IFN-gamma) production by NK cells and synergize with interleukin-2 (IL-2) and proinflammatory cytokines in PAMP-induced activation. Similar results were obtained using CD56(+)CD3(+) (NKR-expressing) T cells. NK cells from TLR2(-/-) mice fail to respond to KpOmpA, demonstrating TLR involvement in this effect. Defensins are antimicrobial peptides expressed mainly by epithelial cells and neutrophils that disrupt the bacterial membrane, leading to pathogen death. We show that NK cells and NKR-expressing T cells constitutively express alpha-defensins and that KpOmpA and flagellin rapidly induce their release. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to pathogen components through TLRs and evidence defensins as a novel and direct cytotoxic pathway involved in NK cell-mediated protection against microorganisms.

Receptor/ligand avidity determines the capacity of Ly49 inhibitory receptors to interfere with T-cell receptor-mediated activation.

The specificity and the relative affinity of many Ly49 receptors for major histocompatibility complex class I ligands have been studied in detail in various adhesion and binding assays. However, how the level of cell surface expression of a given Ly49 receptor and its ligand affinity influence the strength of the inhibition signal is not well documented. To address this issue, we developed a series of human Jurkat T-cell transfectants expressing the whole range of Ly49A and Ly49C levels found in vivo on natural killer and T cells and evaluated their capacity to alter superantigen-induced NF-AT activation and interleukin-2 production. We show that the strength of the inhibition induced by Ly49A/H-2Dd interaction correlates with Ly49A density up to a certain level after which increasing expression does not further inhibit significantly the T-cell receptor-induced activation. This system also represents a valuable tool for the determination of the relative strength of the inhibitory signals of Ly49 receptors following their interactions with different ligands. Even at high levels of expression there was no evidence that engagement of Ly49A with H-2b class I molecules provided an inhibitory signal. Moreover, we showed that functional inhibitory interactions of Ly49C with H-2b class I molecules were only the result of H-2Kb and that H-2d represent lower affinity ligands for Ly49C than H-2b. Therefore, depending on the relative affinity of Ly49 receptors for their ligands, the modulation of their expression level will be determinant for the functional outcome of activated T cells.