ISSN:0889-2229    

DOI:10.1089/aid.1991.7.1

年代:1991

数据来源: MAL

摘要:

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HTV-1 gp120 specifically at the tryptic site (GPGR ↓ AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF ↓ VT). HTV-2 gp105 is also cut by cathepsin E at a site (QIML ↓ MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this rea

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.3

年代:1991

数据来源: MAL

摘要:

A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24h.HTV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HTV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.17

年代:1991

数据来源: MAL

摘要:

CV-1 cells were infected with two recombinant vaccinia viruses carrying thegaggene with deletion of 231 bp from 3′ terminus (strain vC5) andenvgene (strain vE234L) of human immunodeficiency virus type 1 (HIV-1). Both recombinant proteins synthesized in the cells (p508gagand gp160/120env) were localized predominantly in cell membranes; however, some amount of p50 was found in cell nuclei. Thin-section immunoelectron microscopy showed accumulation of viruslike particles undistinguished from immature HIV-I virions in the culture medium of the cells infected with vC5. The similar particles containinggagandenvproteins were produced into the culture medium when the cells were coinfected with vC5 and vE234L strains. The particles contained heterogeneous cellular RNA, but no virus-specific RNA as shown by Northern blot hybridization. Immunization of the rabbits with purified viruslike particles produced virus-specific antibodies againstgagandenvproteins. The titer of antibodies was significantly higer than after immunization with cell lysate or recombinant proteins purified from the infected cells. Highly immunogenic HIV-1-like particles containinggagandenvproteins but no virus-specific RNA are good candidates for potential vaccin

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.29

年代:1991

数据来源: MAL

摘要:

Murine monoclonal antibodies (MAbs) raised against a recombinantnefprotein fragment of human immunodeficiency virus type 1 (HIV-1) strain BH10 were characterized by an epitope mapping system using overlapping decapeptides. Four different immunogenic regions were identified. Ten human HTV-1-positive sera were tested in the same epitope mapping system, seven of these were reactive with four immunogenic regions. Two of thenef-specific epitopes recognized by human sera overlapped with the epitopes defined by the murine monoclonal antibodies. The reactivity of the monoclonal antibodies with the recombinantnefprotein and with infected and uninfected cells were investigated in a variety of test systems. The results are discussed with respect to homologous regions ofnefand cellular proteins.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.37

年代:1991

数据来源: MAL

摘要:

The role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and LFA-1 in human immunodeficiency virus type 1 (HlV-l)-induced cell fusion was investigated in subclones of a T-cell leukemic cell line (CEM) with differing abilities to form syncytia. Addition of monoclonal antibodies 84H10 directed against ICAM-1 and MHM23 directed against the common β subunit of LFA-1 (CD18) resulted in greater than 50% suppression of syncytia formation in cultures of these clones infected with cell-free virus. Two subclones, 2G5-144-84 and 2G5-1, were deficient in their ability to form syncytia and expressed reduced amounts of LFA-1 compared with the parental line. The expression of ICAM-1 but not LFA-1 was upregulated on the clones following treatment with interferon-gamma (IFNγ); however, this did not overcome the delay in syncytia formation observed in these cells. The syncytia-positive subclones 1B11-39 and 17D-9 expressed high levels of LFA-1. Basal expression of ICAM-1 was upregulated on these cells by treatment with tumor necrosis factor-alpha (TNFα), which also accelerated and enhanced syncytia formation. However, anti-ICAM-1 and anti-LFA-1 (CD18) antibodies did not reverse the TNFα-induced enhancement of syncytia formation of HIV-1-infected clones 1B11-39 and 17D-9. Under conditions of low viral expression, adhesion molecules may contribute to syncytia formation if adequate levels of both receptor and ligand in the ICAM-1/LFA-1 complex are expressed. However, our data do not support the concept of an absolute requirement for those molecules in HIV-1-induced cell fusion, particularly under conditions in which large amounts of gp160 are expressed on the surface of the virus-infected c

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.45

年代:1991

数据来源: MAL

摘要:

Development of a vaccine for acquired immunodeficiency syndrome (AIDS) has proven difficult, and so alternative approaches such as idiotypic manipulation have been suggested. As applied to AIDS, this approach could involve immunizing with an anti-CD4 antibody resembling gp120, to induce anti-idiotypic antibodies which would bind to gp120. The CD4 binding site on gp120 is conserved, and so, such an immune response should protect against all variants. Induction of anti-human immunodeficiency virus (HIV) immunity has been reported using anti-Leu3a, and this result has led to testing in humans. Negative results obtained by others have been attributed to differences in immunization protocols. Because of the importance of this question, we reinvestigated the potential of anti-Leu3a to induce anti-HIV antibodies, compared with control immunizations with OKT4A (another anti-CD4 antibody) and the irrelevant Ig MOPC-21. Responses to anti-Leu3a showed induction of high-titer anti-idiotypic activity, and included combining-site-related activity. Yet sera showed no binding to gp160 above controls and no detectable neutralizing activity in a sensitive HIV plaque assay, so the anti-idiotypes induced were not internal images of CD4. We conclude that the pronounced anti-HIV responses reported with anti-Leu3a cannot be generalized, and thus that anti-Leu3a does not offer promise as an HIV vaccine. However, these results do not negate the promise of the idiotypic approach, and a vaccine for AIDS based on idiotype manipulation remains a possibility.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.55

年代:1991

数据来源: MAL

摘要:

We examined the effects of topoisomerase inhibitors on human immunodeficiency virus type 1 (HIV-1) infection of H9 cells in cell culture. Infection is blocked or substantially reduced by the topoisomerase I inhibitor camptothecin (CPT), but not by two topoisomerase II inhibitors. Significant reduction (≥ 90%) in the amount of virus released, as measured by reverse transcriptase, is obtained if the cells are treated for 1 h with 0.01-0.02 μM CPT at the time of virus infection, and expression of viral proteins is also blocked. CPT is also shown to reduce the level of infection when chronically infected cells are cocultivated with uninfected cells. These results with CPT suggest that this compound may represent a new class of drugs with antiretroviral potenti

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.65

年代:1991

数据来源: MAL

摘要:

Culture supernatants from the rabbit macrophage cell line 6083 infected with a retrovirus, human immunodeficiency virus type 1 (HIV-1), were negative for reverse transcriptase (RT) expression although the line was shown to be productively infected by all other criteria tested. Supernatants from uninfected cultures of 6083, the human monocyte line U937, and from freshly isolated peripheral human monocytes, were found to contain a monocyte-derived inhibitory factor (MDIF) which interferes with a standard assay for RT. MDIF is a heat-labile activity of approximately of 40 kD. Both substrates and products of the reverse transcriptase assay are degraded by MDIF which is not affected by reduction and alkylation of disulfide bonds. MDIF is inhibited by the addition of a particular thioated oligonucleotide (S-dG30) to the reaction mixture but this addition also inhibits RT. The optimum method to minimize MDIF interference in the RT assay is by addition of ethylene glycolbis-(β-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA); MDIF requires divalent cations for activity and has a strong preference for calcium which is preferentially chelated by EGTA. The potential presence of this inhibitory activity should be considered when using RT levels as a measure of retroviral infection

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.73

年代:1991

数据来源: MAL

摘要:

Human immunodeficiency virus type 1 (HIV-1) infection in utero was examined by isolating the virus and detecting the HIV-1 DNA sequence from different fetal tissues. The brain, thymus, lung, liver, spleen, and placenta tissues from fetuses (10-23 weeks of gestation) born to HTV-1-infected asymptomatic mothers were examined. HIV-1 was isolated from 2 of 7,1 of 7, and 1 of 7 cocultures of splenic, thymic, and trypsin-resistant cells from the liver and placenta, respectively, with peripheral blood mononuclear cells; 20-30% and 40-60% of splenic and of thymic cells were CD4+lymphoid cells and 40-80% of trypsin-resistant cells were mononuclear phagocytes. The HIV-1 DNA sequence was detected in 4 of 7,3 of 7,1 of 7,1 of 7,2 of 7, and 2 of 6 samples from the spleen, thymus, brain, lung, liver, and placenta, respectively, using the polymerase chain reaction. In one case, the intensity of the HIV-1 DNA sequence appeared to be correlated with the success of viral isolation. We indicate that fetal HIV-1 infection may frequently occur in the second trimester and the cells responsible for the infection may be CD4+lymphoid cells and mononuclear phagocytes.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.83

年代:1991

数据来源: MAL