Mario Clerici

T Lymphocyte-Directed Gene Therapy for ADA− SCID: Initial Trial Results After 4 Years

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA− SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging.

We have tested the T helper cell (TH) potential of asymptomatic, HIV seropositive (HIV+) patients, using an in vitro assay for IL-2 production. Peripheral blood leukocytes (PBL) from 74 HIV+ patients and 70 HIV- control donors were tested for TH function when stimulated with influenza A virus (FLU), tetanus toxoid (TET), HLA alloantigens (ALLO), or PHA. Of the HIV+ patients, four different response patterns were observed: (a) patients who responded to all four stimuli (16%); (b) patients who were selectively unresponsive to FLU and TET, but responded to ALLO and PHA (54%); (c) patients who were unresponsive to FLU, TET, or ALLO, but responsive to PHA (16%); and (d) patients who failed to respond to any of these stimuli (14%). Our results indicate a time-dependent progression from a stage responsive to all four stimuli to a stage unresponsive to any of the stimuli tested, progressing in the order outlined above. The earliest TH defect is the loss of responses to FLU and TET, indicating a selective defect in CD4+ MHC self-restricted TH function. The later loss of ALLO and PHA IL-2 responses suggests more severe TH dysfunction involving both CD4+ and CD8+ T cells. None of these patterns of TH unresponsiveness in asymptomatic HIV+ individuals were correlated with CD4+ cell numbers nor with Walter Reed staging criteria. This study indicates that the in vitro TH assay used can detect multiple stages of immune dysregulation early in the course of HIV infection and raises the possibility that staging of HIV+ patients should include in vitro TH functional analyses of the type described here.

Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy

Antigen‐ and mitogen‐stimulated cytokine production by peripheral blood mononuclear cells (PBMC) of 50 pregnant women and 31 age‐ and sex‐matched non‐pregnant controls were analysed to determine whether changes in cytokine production occur during normal and pathologic human gestation. The pregnant women, consecutively enrolled during a 3‐month period, were undergoing a normal, non‐pathologic pregnancy at the time of entry into the study, and underwent ultrasound examination to ascertain the exact week of pregnancy and the vitality of the fetus. Forty of the 50 pregnancies (80%) terminated physiologically with the birth of normal babies. Spontaneous abortions were observed in 5/50 (10%) women, and five women gave birth to newborns small for gestational age (SGA). A decrease in the production of IL‐2 and interferon‐gamma (IFN‐γ) accompanied by an increase in production of IL‐4 and IL‐10, was observed in normal pregnancy, with the lowest quantities of IL‐2 and IFN‐γ and the highest quantities of IL‐4 and IL‐10 present in the third trimester of pregnancy. Statistically significant increased production of both IL‐2 and IFN‐γ and reduced production of IL‐10 characterized pathologic pregnancies and distinguished them from normal pregnancies. These preliminary data suggest that a type 2 cytokine profile may be associated with normal human pregnancy, whereas the lack of a dominant type 2 cytokine profile may be indicative of a pathologic pregnancy.

Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency virus-seropositive individuals.

Infection with HIV results in an incremental loss of T helper cell (TH) function, which can occur years before CD4 cell numbers are critically reduced and AIDS is diagnosed. All TH function is not affected, however, because B cell activation and hypergammaglobulinema are also characteristic of this period. Recently, in a murine model of AIDS an early loss in production of the CD4 cytokines IL-2 and IFN-gamma was correlated with an increase in the B cell stimulatory cytokines IL-4, IL-5, and IL-10. We therefore assessed the production of IL-4 generated by PBL from HIV-seropositive (HIV+) individuals who did not have AIDS, yet who exhibited different TH functional categories based on their IL-2 production profiles. We observed that the decreases in recall antigen-stimulated IL-2 production were accompanied by an increase in IL-4 production. The loss of recall antigen-stimulated responses in HIV+ individuals could be reversed in vitro by anti-IL-4 antibody. Our results suggest that the TH functions assessed by IL-4 production replace the normally dominant TH function of antigen-stimulated IL-2 production in the progression toward AIDS, and raise the possibility of cytokine cross-regulation in AIDS therapy.

Role of T‐Cell Derived Cytokines in the Downregulation of Immune Responses in Parasitic and Retroviral Infection

Parasitic infection is frequently accompanied by a downregulation in host cell-mediated immunity. Recent studies suggest that this modulation of helper T cells and effector cell function can at least in part be attributed to the action of a set of inhibitory cytokines produced by T lymphocytes as well as by a number of other cell types. The best characterized of these inhibitory lymphokines are IL-4, IL-10 and TGF-beta. Interestingly, both IL-4 and IL-10 are produced by the Th2 but not the Th1 subset of CD4+ helper cells. The former subset dominates in many situations of chronic or exacerbated parasitic infection and is thought to suppress Th1 function as a consequence of the cross-regulatory activity of these two cytokines. The latter hypothesis is supported by recent experiments demonstrating that mAb-mediated neutralization of IL-10 reverses suppressed IFN-gamma responses and/or disease susceptibility in mice with parasitic infections. In vivo neutralization of TGF-beta has also been reported to increase host resistance to parasite challenge. In addition to suppressing T-cell differentiation, function or proliferation, IL-4, IL-10 and TGF-beta each inhibit the ability of IFN-gamma to activate macrophages for killing of both intracellular and extracellular parasites. Moreover, the three cytokines are able to synergize with each other in downregulating these parasiticidal effects. Interestingly, each of the cytokines inhibits the production of reactive nitrogen oxides, an effector mechanism previously demonstrated to play a major role in parasite killing by activated macrophages. In the case of IL-10, this suppression of nitrogen oxide production appears to result from an inhibition of TNF-alpha synthesis leading to defective macrophage stimulation. While distant from parasites in their biology and phylogeny, some retroviruses also appear to induce an over-production in downregulatory cytokines which is closely associated with the onset of immunodeficiency. Thus, in an animal model involving infection of mice with LP-BM5 MuLV and in human HIV infection, Th2 (IL-10 and/or IL-4) cytokine synthesis is increased while Th1 (IFN-gamma and/or IL-2) cytokine production is suppressed. These observations suggest that cytokine-mediated cross-regulation may play a role in the pathogenesis of acquired immune deficiency disease, contributing both to the progression of retroviral infection and the increase in susceptibility to opportunistic infections and malignancy. Observations of similar cytokine cross-regulatory activities in organisms as diverse as helminths, protozoa and retroviruses predict that comparable mechanisms may operate in a wide variety of infectious diseases.

Role of interleukin-10 in T helper cell dysfunction in asymptomatic individuals infected with the human immunodeficiency virus.

The loss of T helper cell (TH) function in asymptomatic HIV type 1-infected individuals occurs before the decline in CD4+ T cells. At least part of the loss in TH function results from changes in immunoregulatory cytokine profiles. To investigate the role of IL-10 in such dysregulation, we tested whether: (a) expression of IL-10-specific mRNA would be upregulated in PBMC from asymptomatic, HIV-infected (HIV+) individuals; (b) PBMC from these same individuals would produce increased levels of IL-10 when stimulated in vitro with phytohemagglutinin; and (c) defective antigen-specific TH function could be restored by anti-IL-10 antibody. We observed that IL-10-specific mRNA was marginally upregulated, and increased levels of IL-10 were produced by PBMC from HIV+ individuals compared with PBMC from uninfected individuals. Those individuals whose TH function was more severely compromised produced higher levels of IL-10. Additionally, defective antigen-specific TH function in vitro could be reversed by anti-IL-10 antibody, including the response to HIV envelope synthetic peptides. Furthermore, the antigen-specific TH responses of HIV-uninfected PBMC could be reduced with IL-10, a process reversed by anti-IL-10. These results confirm that the early loss of TH function in HIV+ individuals is due at least in part to cytokine-induced immune dysregulation, and support the hypothesis of a switch from a predominant type 1 state to a predominant type 2 condition in HIV infection.

Redistribution of body fat in HIV-infected women undergoing combined antiretroviral therapy.

OBJECTIVES To investigate the prevalence, metabolic features and risk factors of a particular pattern of fat redistribution (FR), characterized by a progressive enlargement of breast and abdominal girth associated with a wasting of the lower limbs, observed in HIV-infected women treated with combined antiretroviral (ARV) therapy. DESIGN Cross-sectional study. SETTING Outpatients attending the Institute of Infectious Diseases, University of Milan, Milan, Italy. PATIENTS AND METHODS HIV-infected women treated with two or more ARV drugs, observed between December 1997 and February 1998. FR was confirmed by means of a physical examination and dual-energy X-ray absorptiometry (DEXA). The metabolic and endocrinological measurements in patients with FR were compared with those in FR-free women. RESULTS FR was observed in 32 out of 306 women (10.5%). DEXA revealed more trunk fat (P < 0.01) and less leg fat (P < 0.001) in the patients with FR than in the matched controls. There were no significant differences in laboratory test results between the two groups. All of the FR patients were taking lamivudine-containing regimens; 20 of them were also taking a protease inhibitor (PI). The association of FR with lamivudine-including regimens was statistically significant (P = 0.017). Among the patients taking lamivudine, the risk associated with treatments including PI was 1.8 (95% CI 0.8-3.8, P = 0.12). A total duration of ARV therapy of more than 1000 days was associated with a greater risk of developing FR (OR 10.8; 95% CI 1.4-80.5; P = 0.0207). Stepwise logistic regression analyses indicated that prolonged ARV therapy and a viral load of more than 10000 copies per ml at the beginning of the last ARV regimen were the only variables that significantly and independently correlated with the risk of FR. CONCLUSIONS The observed body modifications are caused by a redistribution of body fat without fat loss that is apparently not associated with hyperlipidemia, altered glucose metabolism or other endocrinological disorders. The development of FR in patients receiving only reverse transcriptase (RT) inhibitors suggests the presence of a PI-independent mechanism that deserves further investigation.

HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers.

OBJECTIVES Most HIV-1 transmission is sexual; therefore, immune responses in the genital mucosa may be important in mediating protection against HIV infection. This study examined HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan female sex workers. METHODS HIV-1-specific immune responses were compared in HIV-1-resistant and HIV-1-infected sex workers, and in lower risk uninfected women. Cervical and vaginal samples from each group were tested for HIV-1-specific IgA and IgG by enzyme immunoassay. Systemic T-helper lymphocyte cell responses to HIV-1 envelope peptide epitopes were assayed using an interleukin 2 bioassay. HIV-1 risk-taking behaviours were assessed using standardized questionnaires. RESULTS HIV-1-specific IgA was present in the genital tract of 16 out of 21 (76%) HIV-1-resistant sex workers, five out of 19 (26%) infected women, and three out of 28 (11%) lower risk women (P < 0.0001). Among lower risk women, the presence of HIV-1-specific IgA was associated with HIV-1 risk-taking behaviour. Systemic T-helper lymphocyte responses to HIV-1 envelope peptides were present in 11 out of 20 (55%) HIV-1-resistant women, four out of 18 (22%) infected women, and one out of 25 (4%) lower risk women (P < 0.001). T-helper lymphocyte responses did not correlate with the presence or titre of virus-specific mucosal IgA in any study group. CONCLUSIONS HIV-1-specific IgA is present in the genital tract of most HIV-1-resistant Kenyan sex workers, and of a minority of lower risk uninfected women, where it is associated with risk-taking behaviour. These data suggest a role for mucosal HIV-1-specific IgA responses in HIV-1 resistance, independent of host cellular responses.

ENV-specific cytotoxic T lymphocyte responses in HIV seronegative health care workers occupationally exposed to HIV-contaminated body fluids.

Identification of the components of protective immunity are crucial for the development of effective prophylactic and therapeutic vaccine strategies. Analysis of HIV-specific responses in exposed but uninfected individuals might thus provide a unique resource to elucidate the components and correlates of protective immunity to HIV. In the present study we analyzed HIV-specific cytotoxic and helper T lymphocyte responses in health care workers (HCW) exposed to body fluids from HIV-positive individuals. HCW exposed to blood from HIV-negative individuals as well as healthy donors served as controls. Cytotoxic T lymphocyte (CTL) responses to HIV envelope (env) peptides were detected in 7/20 (35%) HCW exposed to HIV-positive blood and in none of the 20 health care workers exposed to uninfected blood or the seven healthy blood donors studied. HIV-specific CTL responses were detected only after in vitro stimulation, and were MHC class I restricted. No MHC class I restriction elements were uniformly identified among the different responders. 21/28 (75%) HCW exposed to contaminated blood responded to env as measured by IL-2 production to the peptides, in contrast to only 9/38 (24%) HCW exposed to HIV seronegative blood and 3/35 (9%) healthy blood donors. All the HIV exposed individuals were seronegative on repeated ELISA tests, and no evidence of infection was obtained by PCR analysis. These findings indicate that a single exposure to HIV can induce CTL immunity to HIV antigens, in the absence of other evidence of infection.

Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.