Giuseppe Scala

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A–F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

TNF-α-Induced Secretion of C-C Chemokines Modulates C-C Chemokine Receptor 5 Expression on Peripheral Blood Lymphocytes

Peripheral blood lymphocytes express CCR5, a chemokine receptor for immune cell migration and calcium signaling that serves as an important coreceptor for the HIV. After in vitro stimulation, CCR5 expression is dramatically increased on mature T lymphocytes, especially on the CD45RO+ memory subset. In this study, we report that TNF-α delays the surface expression of CCR5 on PBLs after activation and diminishes CCR5 irrespective of its initial level. Functional loss of CCR5 is reflected in a decreased capability of the treated cells to migrate and signal calcium after MIP-1β stimulation. The effect is mediated via the p80 type II TNF receptor (TNFR2), which induces NF-κB among other factors, leading to an enhanced secretion of the chemokines macrophage-inflammatory protein-1α, macrophage-inflammatory protein-1β, and RANTES. Expression of these chemokines directly down-regulates CCR5. These findings reveal a new regulatory mechanism utilized by activated peripheral T cells to modulate their chemotaxis and potentially other functions mediated by CCR5, including the infection of T lymphocytes by macrophage-tropic HIV strains.

Production of multiple cytokines by clones of human large granular lymphocytes.

SummaryLGL in addition to mediating natural killer (NK) activity, can secrete a variety of lymphokines, depending on the stimulus used: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon α and γ (IFN), and B-cell growth factor (BCGF). To define more directly whether cells with NK activity can also secrete one or more cytokines, we obtained clones by limiting dilution assays from highly purified preparations of human LGL and cultured them in IL-2-containing medium for several weeks. All the clones tested spontaneously produced detectable levels of IFN-γ and 35 of 40 clones (87%) produced higher levels when stimulated with PHA. A smaller proportion (16%) of clones (9 of 54) secreted IL-1 after stimulation with LPS, while 34% of the clones (17 of 49) produced IL-2 in response to PHA stimulation. Cytokine production was associated with both cytotoxic and noncytotoxic clones and did not correlate with their surface phenotype, as has been observed for fresh LGL. The ability to produce IL-1 or IL-2 was not usually found within the same clones following PHA and LPS stimulation, respectively; however two clones produced both IL-1 and IL-2 when stimulated in different experiments, but not at the same time. In addition, two of nine cloned LGL simultaneously produced IFNγ and IL-1. These results indicate that LGL-derived clones have the ability to produce multiple cytokines, suggesting that the LGL population may play an important immunoregulatory role and may also be capable of self-regulation of cytolytic activity.

Potent and stable attenuation of live-HIV-1 by gain of a proteolysis-resistant inhibitor of NF-kappaB (IkappaB-alphaS32/36A) and the implications for vaccine development.

Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-κB inhibitor (IκB-αS32/36A) in the nef region. HIV-1 expressing IκB-αS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IκB-αS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivopassaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IκB-αS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.

Effects of recombinant interferon-γ on HLA-DR antigen shedding by human peripheral blood adherent mononuclear cells

Two reproducible and sensitive assays have been developed for the detection of cell‐free HLA‐DR antigen, an antibody‐mediated complement‐dependent cytotoxicity inhibition assay (CIA) and a competition enzyme‐linked immunosorbent assay (CELISA). One unit of cell‐free HLA‐DR antigen has been quantitated to be the equivalent of 27.5 ng pure DR antigen/ml. Human peripheral blood adherent mononuclear cells (PBAMC), as well as DR+ monocytes and B lymphoblastoid cell lines but not T lymphocytes, were observed to shed DR‐bearing vesicles in vitro. Human recombinant IFN‐γ induces the expression of Ia/DR antigen by PBAMC by increasing both the number of cells expressing DR antigen and the density per cell. After incubation with IFN‐γ, PBAMC also shed significantly more DR antigen. The degree of DR expression and shedding is dependent on the dose of IFN‐γ. The shedding of DR antigen occurred concomitantly with the inductive phase of cell membrane antigen expression, and very little DR antigen appeared in culture supernatants subsequent to the removal of IFN‐γ. The possible physiologic significance of shed DR antigen is discussed.