Samuele E. Burastero

CCR5-Reactive Antibodies in Seronegative Partners of HIV-Seropositive Individuals Down-Modulate Surface CCR5 In Vivo and Neutralize the Infectivity of R5 Strains of HIV-1 In Vitro

Exposure to HIV does not necessarily results in infection. Because primary HIV infection is associated with CCR5-tropic HIV variants (R5), CCR5-specific Abs in the sera of HIV-seronegative, HIV-exposed individuals (ESN) might be associated with protection against infection. We analyzed sera from ESN, their HIV-infected sexual partners (HIV+), and healthy controls (USN) searching for CCR5-specific Abs, studying whether incubation of PBMC with sera could prevent macrophage inflammatory protein 1β (Mip1β) (natural ligand of CCR5) binding to CCR5. Results showed that Mip1β binding to CCR5 was not modified by sera of either 40 HIV+ or 45 USN but was greatly reduced by sera of 6/48 ESN. Binding inhibition was due to Abs reactive with CCR5. The CCR5-specific Abs neutralized the infectivity of primary HIV isolates obtained from the corresponding HIV+ partners and of R5-primary HIV strains, but not that of CXCR4-tropic or amphitropic HIV strains. Immunoadsorption on CCR5-transfected, but not on CXCR4-transfected, cells removed CCR5-specific and virus-neutralizing Abs. Epitope mapping on purified CCR5-specific Abs showed that these Abs recognize a conformational epitope in the first cysteine loop of CCR5 (aa 89–102). Affinity-purified anti-CCR5-peptide neutralized the infectivity of R5 strains of HIV-1. Anti-CCR5 Abs inhibited Mip1β-induced chemotaxis of PBMC from healthy donors. PBMC from two ESN (with anti-CCR5 Abs) were CCR5-negative and could not be stimulated by Mip1β in chemotaxis assays. These results contribute to clarifying the phenomenon of immunologic resistance to HIV and may have implications for the development of a protective vaccine.

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

ABSTRACT The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.

Viral Replication-Independent Blockade of Dendritic Cell Maturation and Interleukin-12 Production by Human Herpesvirus 6

ABSTRACT Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive CD4+-T-lymphotropic betaherpesvirus that causes severe human thymocyte depletion in heterochimeric SCID-hu thy/liv mice and has been implicated as a potential cofactor in the progression of AIDS. However, the mechanisms of HHV-6-mediated immunosuppression have not yet been fully elucidated. We investigated the phenotypic and functional alterations induced by HHV-6 on peripheral blood-derived human dendritic cells (DC). The infection of DC with HHV-6 A or B was nonproductive, as revealed by calibrated real-time PCR measuring the accumulation of viral genome equivalents over time. Nevertheless, preexposure to HHV-6 markedly impaired the maturation of DC driven by gamma interferon and lipopolysaccharide, as shown by the reduced surface expression of major histocompatibility complex class I molecules, HLA-DR, CD40, and CD80. Moreover, HHV-6, but not the closely related betaherpesvirus HHV-7, dramatically suppressed the secretion of interleukin-12 (IL-12) p70 by DC, while the production of other cytokines that influence DC maturation, i.e., IL-10 and tumor necrosis factor alpha, was not significantly modified. Likewise, the secretion of the CC chemokines macrophage inflammatory protein 1β and RANTES was unaltered. Functionally, a pretreatment with HHV-6 impaired the ability of DC to stimulate allogeneic T-cell proliferation. Altogether, these data identify interference with the functional maturation of DC as a potential mechanism of HHV-6-mediated immunosuppression.

Increased expression of the CD80 accessory molecule by alveolar macrophages in asthmatic subjects and its functional involvement in allergen presentation to autologous TH2 lymphocytes.

BACKGROUND Alveolar macrophages (AMs) are more efficient antigen-presenting cells in allergic individuals than in nonatopic subjects. OBJECTIVE We studied whether this difference may be correlated to increased expression of membrane costimulatory molecules, such as the B7 molecules (CD80 and CD86). METHODS Eleven subjects with allergic asthma sensitized to Dermatophagoides pteronyssinus and 5 healthy nonatopic volunteers underwent bronchoalveolar lavage, and the costimulatory molecule expression on AMs was evaluated. Peripheral blood T cells, either freshly isolated or as established D pteronyssinus -specific cell lines, were cultured with autologous monocytes or AMs as antigen-presenting cells. In vitro allergen-induced proliferation and cytokine production were evaluated in the presence of B7-blocking reagents. RESULTS Allergic individuals had a significantly higher proportion of AMs expressing the CD80 molecule than control subjects (28.5% +/- 14.8% vs 1.4% +/- 1.2%; P <.001), whereas no difference was observed in CD86 expression (2.0% +/- 2.3% vs 1.1% +/- 0.6; P >.1). In a large proportion of the asthmatic subjects we studied, AMs were presenting soluble antigens (tetanus toxoid and streptolysin-O) to freshly isolated T cells more efficiently than AMs from nonatopic control subjects. Finally, both T-cell proliferation and cytokine production of D pteronyssinus- specific established T-cell lines were inhibited by a CD80-blocking antibody in a dose-dependent manner. CONCLUSION Costimulation by means of CD80 expressed by AMs is probably involved in the amplification of the allergen-specific T-lymphocyte response in the airways of asthmatic subjects.

Anti-Cell Antibodies in Exposed Seronegative Individuals with HIV Type 1-Neutralizing Activity

Despite repeated exposures to HIV-1, some individuals remain seronegative. This study reports that sera from a fraction of exposed seronegative (ESN) subjects showed HIV-neutralizing activity; 5 of 17 ESN sera and none of 17 controls neutralized two different HIV-1 primary isolates (range of neutralizing titers: 1/20 to 1/60). The neutralizing activity was associated with the IgG fraction of 4 of 4 neutralizing ESN sera. Moreover, in 11 of 17 and 9 of 17 ESN sera (but none of the control sera) we found antibodies against HLA class I and CD4, respectively. One of the ESN sera (EU22) neutralized efficiently the primary virus derived from the seropositive partner and showed a good broadly cross-reactive neutralization. Immunoadsorption of two IgG fractions from EU19 and EU22 on peripheral blood mononuclear cells (PBMC) removed virus-neutralizing antibodies. The correlations between the ESN status and neutralizing activity (p<0.05), anti-HLA antibodies (p<0.0002), and anti-CD4 antibodies (p<0.001) were statistically significant. However, there was no statistically significant correlation between neutralizing activity and either anti-HLA or anti-CD4 antibodies. It can therefore be said that exposure to HIV-1 without seroconversion is, in some individuals, associated with HIV-neutralizing antibodies (not directed against viral antigens) and/or with anti-cell autoantibodies, which are possibly specific for cellular antigens involved in the infection/entry process.

Frequency of allergen-specific T lymphocytes in blood and bronchial response to allergen in asthma☆

BACKGROUND This study was designed to investigate whether the bronchial response to the sensitizing allergen in asthma is correlated with the frequency of allergen-specific T lymphocytes. METHODS Twenty-three asthmatic patients sensitized to Dermatophagoides pteryonyssinus who had never received hyposensitizing therapy and 11 healthy control subjects were studied. Allergen-specific T lymphocytes were enumerated in peripheral blood with limiting dilution cultures. Bronchial challenge with methacholine was performed in all subjects; patients with asthma also underwent an allergen bronchial challenge. Correlations between allergen-specific T cell frequencies and nonspecific bronchial hyperresponsiveness to methacholine as independent variables and early and late bronchial responsiveness to allergen challenge as dependent variables were investigated by means of stepwise-multiple regression analysis. RESULTS We found that the frequency of allergen-specific T lymphocytes was higher than in control subjects in both patients with asthma with (p < 0.001) and those without (p < 0.05) late-phase asthmatic response to allergen. Moreover, the provocative does of allergen necessary to produce an early 15% fall of forced expiratory volume in 1 second could be predicted in part (59%) by an equation that incorporates methacholine sensitivity and allergen-specific T cell frequency. CONCLUSIONS We conclude that allergen-specific T lymphocytes, which have an established influence on immunoglobulin E production, play an additional role in the induction of the bronchospastic response to inhaled allergen.

HIV-specific antibodies but not t-cell responses are associated with protection in seronegative partners of HIV-1-infected individuals in Cambodia.

Summary: To study biological factors related to protection against HIV-1 infection in Cambodia, we recruited 48 partners of HIV-1-infected patients who remained uninfected (exposed uninfected individuals, EUs) despite unprotected sexual intercourse for more than 1 year and 49 unexposed controls (UCs). HIV-1-specific antibodies (IgA anti-gp41 and IgG anti-CD4-gp120 complex), T-cell responses, and cellular factors that may be involved in protection (peripheral blood mononuclear cell [PBMC] resistance to HIV-1 infection and &bgr;-chemokine production) were evaluated. Anti-HIV-1 antibodies were higher in EUs than those in UCs (P = 0.01 and P = 0.04 for anti-gp41 and anti-CD4-gp120, respectively). We observed a decreased susceptibility to a primary Cambodian isolate, HIV-1KH019, in EU PBMCs as compared with UC PBMCs (P = 0.03). A weak T-cell response to one pool of HIV-1 Gag peptides was found by ELISpot in 1 of 19 EUs. Whereas T-cell specific immunity was not associated to protection, our results suggest that HIV-specific humoral immunity and reduced cell susceptibility to infection may contribute to protection against HIV-1 infection in Cambodian EUs.

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte‐derived DCs to the NO donor (z)‐1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino] diazen‐1‐ium‐1,2 diolate (DETA‐NO) during their maturation process induced by treatment with tumor necrosis factor α or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA‐NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo‐antigens, or cross‐linking of the CD3–T cell receptor complex. This effect persists after removal of DETA‐NO, depends on the generation of cyclic guanosine 5′‐monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)‐12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL‐12 release was observed after DC exposure to DETA‐NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.

Recruitment of circulating allergen-specific T lymphocytes to the lung on allergen challenge in asthma.

BACKGROUND In allergic subjects with asthma, the migration of CD4+ T cells to the lungs in the hours after allergen exposure may contribute to allergic inflammation in the target organ. OBJECTIVE We studied allergen-specific T cells from the peripheral blood and lungs of allergic subjects with asthma at baseline and after allergen challenge. METHODS In each patient, blood samples were taken 10 minutes before and 24 hours after the inhalation of a major sensitizing allergen. In vitro proliferation of peripheral blood CD4+ T cells specific for the same allergen used in the in vivo challenge was assessed. In one patient two Dermatophagoides pteronyssinus-specific T-cell clones (TCCs) were derived from peripheral blood, and their T-cell receptors were sequenced to determine their clonotypic determinants on the beta chains. The T-cell receptor determinants of the allergen-specific TCCs were sought in blood and bronchoalveolar lavage samples taken from this patient. RESULTS We found that allergen inhalation is followed by a decrement in the specific proliferation of peripheral CD4+ T cells to the same allergen used for bronchial provocation. In one patient the clonotypic determinants of two allergen-specific TCCs diminished in the peripheral blood, whereas they were simultaneously expanded in the lower respiratory tract. CONCLUSION Our data suggest that allergen-specific T cells are recruited from the peripheral blood to the bronchial lumen after allergen challenge.

Depletion of circulating allergen-specific TH2 T lymphocytes after allergen exposure in asthma

BACKGROUND In allergic asthma, CD4+ T lymphocytes are a fundamental component of local chronic inflammation. Their cytokine profile is oriented toward a TH2 phenotype, characterized by production of IL-4, IL-5, IL-10, and IL-13. Egress of T cells from blood to airways after allergen challenge has been described. OBJECTIVE We have studied a cohort of six patients with asthma who had multiple allergies to investigate how exposure to allergen affects the proliferation of peripheral CD4+ T lymphocytes with different allergen specificities and lymphokine profiles. METHODS For each patient, CD4+ T-cell lines were generated by in vitro stimulation with sensitizing and with nonsensitizing allergens, and IL-4 and interferon-gamma production by these lines was assessed. Proliferation of peripheral blood CD4+ T lymphocytes in response to the same allergens was measured before and 24 hours after inhalation challenge with a sensitizing allergen. RESULTS We found that each single sensitizing allergen can deplete peripheral blood of TH2-type CD4+ T lymphocytes specific for all sensitizing allergens, but not of TH1-type CD4+ T lymphocytes. CONCLUSIONS Our results suggest the existence of mechanisms capable of sorting disease-associated antigen specificities together with defined lymphokine patterns into T lymphocytes that can migrate to target organs, in allergic asthma.