Faculty Bibliography

Both primary isolates and laboratory strains of HIV-1 incorporate different cell-derived molecules into their envelopes depending on the host cell in which they are grown. This incorporation in not random; for example, HIV-1 has been shown to exclude CD4 from its envelope. In this study we have examined the incorporation into the envelope of HIV-1 of three coreceptors -- CXCR4,CCR5, and CCR3-- as well as CD4. For this purpose, the HIV-1 SI primary isolate BZ167 was passaged into PHA-stimulated PBMC and CEM-SS cells and the incorporation of various cell-derived molecules into the virion envelope was determined with a virus binding ELISA. As previously shown, BZ167 grown in PHA-stimulated PBMC expressed most of the adhesion molecules tested (such as LFA-1, ICAM-1, and MHC class II), while CEM-SS-grown BZ167 did not express significant levels of any of these adhesion molecules. CEM-SS cells express both CD4 and CXCR4 whereas PHA-stimulated PBMCs express CD4, CXCR4, CCR5, and CCR3. In contrast, BZ167 grown in either cell type lacked CD4 as well as the coreceptors, CXCR4, CCR5, and CCR3

Antibodies to purified, size-fractionated secreted proteins of Mycobacterium tuberculosis in sera from patients with human immunodeficiency virus (HIV) infection and active tuberculosis (HIV/TB patients), and in stored sera obtained from the same patients prior to clinical manifestation of TB, were evaluated by ELISA, and the repertoire of antigens recognized was analyzed by immunoblotting. Compared with non-HIV/TB patients, HIV/TB patients had lower levels of anti-mycobacterial antibodies, and these were directed toward a restricted set of antigens. Antibodies to an 88-kDa secreted antigen were present in the sera of 74% of HIV/TB patients during the years (1.5-6) prior to manifestation of active, clinical tuberculosis, although only 66% were positive by the time tuberculosis was diagnosed. The presence of antibodies to the 88-kDa antigen can serve as a surrogate marker for identifying HIV-infected persons with active, subclinical disease who are at a high risk of developing clinical tuberculosis

In addition to gp41 and gp120, an array of cell adhesion molecules is present on the envelope of human immunodeficiency virus type 1 (HIV-1). To examine the role of the host cell in the acquisition of these molecules by virions, both laboratory-adapted and primary isolates were sequentially passaged into different host cells. Viruses obtained from the various host cells were examined for the presence of 10 different cell-derived molecules by a virus binding enzyme-linked immunosorbent assay. Virus progeny raised in peripheral blood mononuclear cells expressed most of the adhesion molecules tested, with the level of LFA-1 being the highest. When viruses were passaged into CEM-SS or SupT1 cells, the expression of most of the adhesion molecules on the virus envelope was lost. In contrast, when viruses were passaged into MT2 cells, the virus progeny bore high levels of LFA-3, ICAM-1, and major histocompatibility complex classes I and II. These studies demonstrate for the first time the host cell dependence of the adhesion molecule profile present on the envelope of primary isolates of HIV-1. The presence of several adhesion molecules that have not previously been identified as components of the envelope of either laboratory or primary isolates is also described. In addition, we show that the adhesion molecule profile of the virions is acquired, or lost, within one passage and is maintained with subsequent passages in the same cell type

We have recently demonstrated that HIV(IIIB), when grown in different host cells, carried different adhesion molecules in its envelope. In the current study, we have examined the acquisition of adhesion molecules by primary isolates of HIV-1 under similar circumstances. Five PBMC-grown primary isolates of HIV-1 were passaged through CEM-SS, MT2, or SupT1 cells and back passaged in PBMC. The adhesion molecule profile on the progeny virions from each host cell was determined with a virus binding ELISA. The parental and back passaged primary isolates expressed high levels of LFA-1, ICAM-1, ICAM-3, and MHC class II. The profile on the envelope of the MT2-grown virions was altered in that they now bear high levels of LFA-3, ICAM-1, and MHC class I and II. In contrast, few of the adhesion molecules were detected on virions from CEM-SS or SupT1 cells. These alterations in the envelope profile of adhesion molecules were detected after a single passage and were maintained with additional passages in the same cell line. Since adhesion molecules play a critical role in virus-cell interactions, these studies suggest that the tropism of HIV-1 for various cells may be critically dependent on the host cell in which the virion was produced

The selection of antigens of Mycobacterium tuberculosis for most studies of humoral responses in tuberculosis patients has been restricted to molecules that were either immunodominant in immunized animals or amenable to biochemical purification rather than those that were reactive with the human immune system. Delineation of antigens that elicit humoral responses during the natural course of disease progression in humans has been hindered by the presence of cross-reactive antibodies to conserved regions on ubiquitous prokaryotic antigens in sera from healthy individuals and tuberculosis patients. The levels of cross-reactive antibodies in the sera were reduced by preadsorption with Escherichia coli lysates, prior to studying their reactivity against a large panel of M. tuberculosis antigens to which the human immune system may be exposed during natural infection and disease. Thus, reactivity against pools of secreted, cellular, and cell wall-associated antigens of M. tuberculosis was assessed by an enzyme-linked immunosorbent assay (ELISA). Initial results suggested that the secreted protein preparation contained antigens most frequently recognized by the humoral responses of pulmonary tuberculosis patients. The culture filtrate proteins were subsequently size fractionated by preparative polyacrylamide gel electrophoresis, characterized by reaction with murine monoclonal antibodies to known antigens of M. tuberculosis by an ELISA, and assessed for reactivity with tuberculous and nontuberculous sera. Results show that a secreted antigen of 88 kDa elicits a strong antibody response in a high percentage of patients with pulmonary tuberculosis. This and other antigens identified on the basis of their reactivity with patient sera may prove useful for developing serodiagnosis for tuberculosis

The ability of antibodies to the V3 region and the CD4-binding domain (CD4bd) of human immunodeficiency virus type 1 (HIV-1) to act in synergy to neutralize HIV has been demonstrated previously. However, synergy between antibodies to other HIV-1 epitopes has not been studied. We have used 21 combinations of human monoclonal antibodies (MAbs) directed against different epitopes of the gp120 and gp41 proteins of HIV-1 to evaluate their ability to act in synergy to neutralize HIV-1. Combinations of anti-V3 and anti-CD4bd antibodies, anti-V3 and anti-gp120 C-terminus antibodies, anti-CD4bd and anti-C-terminus antibodies, anti-V3 and anti-gp41 antibodies, and anti-CD4bd and anti-gp41 antibodies were tested. Our results show that some, but not all anti-V3 antibodies can act in synergy with anti-CD4bd antibodies. In addition, for the first time, antibodies to the C-terminus region have been found to act in synergy with the anti-CD4bd antibodies. Various anti-CD4bd MAbs also act in synergy when used together. The use of such cocktails of human MAbs for passive immunization against HIV-1 may prove to be important for therapy in postexposure settings and for prevention of maternal-fetal transmission of the virus. The results also provide information on the types of antibodies that should be elicited by an effective vaccine

Identification of molecules of M. tuberculosis which are expressed and immunogenic in the context of a dysfunctional immune system may provide insights into the pathogenesis of reactivation of latent tuberculosis. Use of such antigens (and antibodies to them) for the design of serodiagnostic tests may yield specific and sensitive assays that could be used for screening populations at high risk of reactivation. Antibodies to antigens of M. tuberculosis have been studied in the sera of HIV-seropositive individuals with confirmed clinical pulmonary TB. Sera were obtained from these individuals (a) several months prior to clinical manifestations of TB (b) at the time of clinical diagnosis of TB and (c) post-diagnosis. Unfractionated total cellular proteins from four strains of M. tuberculosis obtained from HIV-TB patients, and grown after minimal passaging in the laboratory, were used as antigen. The sera were depleted of antibodies to cross-reactive, ubiquitous bacterial antigens prior to being tested in an ELISA employing the pooled M. tuberculosis antigen preparation. Results show that antibodies to M. tuberculosis were detectable several months prior to clinical TB in 7/10 individuals. Only 4/10 patients had anti-M. tuberculosis antibodies at the time of clinical diagnosis of tuberculosis, and the proportion increased (9/15) after initiation of anti-tuberculosis therapy. The identity of the M. tuberculosis antigens identified by these patients is being analyzed by SDS-PAGE and immunoblotting

Studies have shown that combinations of anti-V3 region and anti-CD4 binding domain (CD4bd) monoclonal antibodies are synergistic for neutralization of HIV-1. We have used a panel of human monoclonal antibodies (humAb) directed against various immunogenic regions on the HIV envelope, to perform a systematic analysis of the epitopes that must be targeted simultaneously to achieve enhanced neutralization. The panel included 2 humAbs to the gp120-V3 region, 3 to the gp120-CD4bd, 2 to the gp120-C-terminal region, 1 each to gp41 cluster I (aa571-641) and II (aa644-663), and 1 (2F5 from H. Kattinger) to a gp41 region adjacent to cluster II (aa662-667). The assay used was the modified syncytium formation microassay (Nara et al., Laal et al.) that measures cell free virus neutralization. The results show that a) not all anti-V3 and anti-CD4bd humAb pairs show synergy; b) anti-CD4bd humAbs can act in synergy both with anti-V3 and anti-C-terminus humAbs; c) anti-V3 humAb 447-D is able to act in synergy with 5 different anti-CD4bd humAbs; d)none of the anti-gp41 humAbs is able to act in synergy with either anti-V3 or anti-CD4bd humAbs. These results have important implications for rational design of regimens for active and passive immunization against HIV