Human Immunodeficiency Virus Env Research Articles

Replication-deficient recombinant adenoviruses expressing the human immunodeficiency virus Env antigen can induce both humoral and CTL immune responses in mice.

An effective vaccine against infection with human immunodeficiency virus type 1 (HIV-1) is thought likely to require both a humoral and a CTL immune response. A non-replicating adenovirus vector system has been developed that can induce both a humoral and CTL response to HIV-1 envelope in mice. It is demonstrated that the stimulatory tat/rev 5' splice-donor site sequence is required for efficient expression of HIV-1 env by this adenovirus vector system. rev can be provided bicistronically or in trans to result in good expression of env in vitro. A humoral immune response was detected after two immunizations with a bicistronic recombinant adenovirus (RAd142). The response was dose dependent, 5x10(7) p.f.u. inducing a response in some, but not all, animals and 1x10(8) p.f.u. giving a consistent antibody response. However, CTLs were induced by the lower dose of virus and after only one immunization with the higher dose. A positive CTL response was also seen consistently when the two monocistronic adenoviruses (RAd501 expressing env and RAd46 expressing rev) were given together, although two immunizations were required to give approximately the same level of response as seen with the bicistronic virus. RAd501 on its own also gave a low CTL response when two immunizations were given. It is suggested that a lower level of env expression is required to produce a CTL response than a humoral response and that this nonreplicating adenovirus vector is a good system for inducing CTL.

A human immunodeficiency virus type 1 Env-granulocyte-macrophage colony-stimulating factor fusion protein enhances the cellular immune response to Env in a vaccinia virus-based vaccine.

Vaccinia virus (VV) infection induces protective T- and B-cell responses, making recombinants based on VV good candidates for the development of effective vaccines to other viruses. VV recombinants expressing the human immunodeficiency virus (HIV) envelope protein (Env) have been generated in several laboratories and shown to induce anti-HIV cellular and humoral immune responses in vaccinated humans and in chimpanzees. To increase the immunogenicity of the Env antigen, a VV recombinant was generated that expresses a chimeric antigen consisting of the Env protein fused to an immunostimulatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The chimeric protein retained GM-CSF biological activity when expressed by this recombinant virus (VV-GM-gp120) in cells infected in vitro. Infection of BALB/c mice with VV-GM-gp120 triggered a higher HIV-specific cellular immune response, as measured by interferon-gamma production, than that induced by a VV recombinant expressing the native Env protein. Moreover, although anti-gp120 antibody titres were similar in sera from mice inoculated with either of the VV recombinants, immunization with the recombinant expressing the fusion protein elicited antibodies against a broader spectrum of Env epitopes. These results indicate that HIV Env antigen fusion to GM-CSF provides a means to improve the anti-HIV immune response.

Properties of HIV envelope expressed in the presence of SPC3, an Env-derived peptide drug under phase II clinical trials.

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.

Nonspecific interstitial pneumonitis mimicking Pneumocystis carinii pneumonia.

Bronchoalveolar lavage (BAL) and transbronchial biopsies from 351 human immunodeficiency virus (HIV)-positive patients with presumed Pneumocystis pneumonia were analyzed to determine the spectrum and frequency of interstitial lung disease mimicking Pneumocystis pneumonia. Among 67 patients without Pneumocystis, nonspecific interstitial pneumonitis (NSIP) was the most common histologic diagnosis (n = 16). Tissue sections from patients with NSIP were tested by in situ hybridization for Epstein-Barr virus, cytomegalovirus (CMV), and HIV; sections were also tested with the polymerase chain reaction (PCR) for HIV env and gag protein DNA. In patients with NSIP, Epstein-Barr virus and CMV could not be detected by in situ hybridization; HIV nucleic acid was amplifiable with PCR in 10 of 15 formalin-fixed, paraffin-embedded tissue sections. Symptoms, physical findings, and blood gas values were similar in patients with NSIP and matched controls with Pneumocystis. Patients with NSIP presented earlier in the course of HIV, with higher weight, serum albumin levels, and CD4+ T-lymphocyte counts (492 +/- 828 cells/mm3 versus 57 +/- 60 cells/mm3), and more normal lactate dehydrogenase (LDH) levels (280 +/- 113 IU/L versus 432 +/- 141 IU/L; means +/- SD). Seven to 10 d later, improvement in blood gas values was of similar magnitude for the two groups. Only one other unequivocal, treatable infection was diagnosed only with transbronchial biopsy. These results indicate that NSIP may be the most common diagnosis mimicking Pneumocystis pneumonia in acquired immune deficiency syndrome (AIDS), and that NSIP may improve during empiric therapy.

A tightly regulated high level expression vector that utilizes a thermosensitive lac repressor: production of the human T cell receptor Vβ5.3 in Escherichia coli

A series of vectors has been constructed to express the human T cell receptor Vβ5.3 under the control of the hybrid trc promoter in Escherichia coli. Transcriptional induction of the trc promoter was achieved chemically by using isopropyl β- d-thiogalactopyranoside (IPTG) in a bacterial strain that harbors the lacI q gene, or thermally by using the mutant laclts gene that encodes a temperature-sensitive lac repressor [Bukrinsky et al. (1988) Multicopy expression vector based on temperature-regulated lac repressor: expression of human immunodeficiency virus env gene in Escherichia coli. Gene 70, 415–417]. Several of the plasmids tested also contain the E. coli heat-stable enterotoxin II (STII) signal sequence for protein secretion. In addition, the gene 10 leader sequence from bacteriophage T7 and a minicistron localized upstream of the Vβ5.3 coding sequence were tested for their potential effect on protein production. These elements increased protein yield two-fold when transcription was induced by IPTG, but had no detectable effect on protein yield when transcription was induced thermally. The highest protein yield was obtained when Vβ5.3 was expressed either from plasmid pKB containing the STII signal in strain LJ24, or from plasmid pKBi that lacks the signal sequence, in the protease deficient strain SG21173 ( lon, htpR, clp). Both plasmids contain the laclts gene, the trc promoter, the two transcription terminators of the rrnB operon, and a tetracycline selection marker. Production of Vβ5.3 using pKBi-Vβ5.3 in strain SG21173 in a 5-liter fermenter under controlled growth conditions yielded over 25 mg Vβ5.3/liter culture. Conversion of the laclts to the lacI qts gene yielded vector pKBiq-Vβ5.3 which exhibits complete repression of the trc promoter at 30°C. This stringent regulation of the thermally inducible trc promoter, the elimination of IPTG, the inclusion of the tetracycline resistance gene, and the high level of protein yield should render this expression system broadly useful for the high level production of heterologous proteins in E. coli, for both basic research and human therapeutic applications.

Induction of inflammatory cytokines in placental monocytes of gravidae infected with the human immunodeficiency virus type 1.

Placental mononuclear cells (PMC) are susceptible to infection with the human immunodeficiency virus (HIV). PMC secreted tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta), and IL-6 among other factors, which, in turn, regulate HIV replication in latently infected cells. We assessed the induction of these cytokines in PMC from HIV-infected (HIV+) and uninfected (control) gravidae following exposure to lipopolysaccharide (LPS), HIV lysate (iHIV), recombinant HIV env (GP160) and HIV gag (gag55), and synthetic HIV p17 (HGP30) antigens. In comparison to control PMC, HIV+ PMC constitutively secreted higher levels of IL-1beta and IL-6 and were refractory to stimulation by iHIV, GP160, gag55, and HGP30. Control PMC IL-1 beta levels were boosted by LPS; gag55 and HGP30 augmented IL-6 but not IL-1 beta. Both groups exhibited low basal TNF-alpha production that was augmented by LPS. HIV+ PMC exhibited higher constitutive levels of IL-1 beta, IL-6, and TNF-alpha gene transcription than control PMC. These levels could be further augmented by LPS, yet the incremental levels were lower than those obtained from PMC of uninfected women. The high basal constitutive secretion of cytokines by HIV+ PMC and their refractoriness to activation may reflect a virus-mediated dysregulation of cytokine expression culminating in compromised host defenses against secondary opportunistic infections associated with AIDS.

Immunologic and Ultrastructural Characterization of HIV Pseudovirions Containing Gag and Env Precursor Proteins Engineered in Insect Cells

Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.

Cytolytic Response to HIV in Patients with HIV Disease Treated with Extracorporeal Photochemotherapy: Preliminary Study

Extracorporeal photochemotherapy (photopheresis), an immunomodulatory therapy that targets circulating T helper lymphocytes, has been applied to the management of human immunodeficiency virus (HIV) disease. Any therapy that exerts its actions on CD4+ T cells has the potential of exacerbating HIV infection. Therefore, it was necessary to observe immune function during treatment. Because cytotoxic T lymphocytes (CTL) and natural-killer cells are thought to play an important role in the response against HIV infection, we examined the effect of photopheresis on HIV cytolytic activity. The study group consisted of seven patients with late-stage HIV disease who had not received any previous treatment for HIV infection. Patients were treated exclusively with photopheresis on two consecutive days each month for 14-32 months (average, 25 months). Peripheral lymphocytes, collected at various points during treatment, were used as effectors in a 51Cr release assay. Epstein-Barr virus (EBV)-transformed autologous B cell lines transfected with recombinant vaccinia vectors that expressed the HIV env (gp120, gp41) and gag (p24) proteins were used as target cells. All seven patients demonstrated relatively constant levels of cytolysis (>10% above controls) during treatment in the context of stable CD4+ T cell counts and a stable clinical status. These results suggest that extracorporeal photochemotherapy did not impair the cytolytic response to HIV infection and may have enhanced it in some patients.

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.

Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication

We constructed a series of human immunodeficiency virus 1 (HIV-1)/simian immunodeficiency virus strain mac (SIVmac) chimeric viruses having vpr and/or nef genes of either HIV-1 or SIVmac based on a chimeric virus with LTRs, gag, pol, vif and vpx derived from SIVmac and tar, rev, vpu and env from HIV-1. All of the chimeric viruses replicated in human and macaque peripheral blood mononuclear cells (PBMCs) and in several CD4+ human cell lines, though their growth potentials were slightly different depending on whether vpr and nef were from HIV-1 or SIVmac, or were defective. The presence of nef accelerated replication in all the cells used and the replication of each chimera appeared to reflect that of the parental virus from which nef was derived. The presence of vpr had no clear effect in human and monkey PBMCs, but the replication of each chimera was influenced by the origin of vpr in H9 and A3.01 cells. NM-3rN, which carries HIV-1 vpr and SIVmac nef, was inoculated intravenously into three rhesus monkeys, three cynomolgus monkeys and two pig-tailed monkeys. From 2 to 14 weeks after inoculation, viruses were consistently re-isolated from all the monkeys and virus loads were as high as that of SIVmac reported previously. The results indicate that infection with NM-3rN is more efficient than any of our previous chimeric viruses and suggest that NM-3rN, having HIV-1 Env, will be a useful challenge virus for evaluating AIDS vaccines based on HIV-1 Env in macaque monkeys instead of chimpanzees.

Human immunodeficiency virus (HIV) nef-specific cytotoxic T lymphocytes in noninfected heterosexual contact of HIV-infected patients.

We report on the detection of HIV-specific cytotoxic T lymphocytes (CTL) among 23 regular partners of HIV-infected individuals. 15 of the 46 individuals enrolled in the study were positive for HLA-A2.1 typing. Among the 23 contacts studied, 7 were seropositive and 16 were seronegative on repeated tests. None of the 16 seronegative contacts were positive for p24 antigenemia nor were they positive by the lymphocytes coculture assay, although, in two instances HIV-1 DNA could be detected by PCR (in one case using a gag SK 38/39 primer, and in the other using a primer for the pol P3/P4 primer). These two individuals remained seronegative for 18 and 36 mo, respectively. HIV-specific cytotoxicity was performed in the 15 HLA-A2.1 subjects (7 indexes, 2 seropositive contacts, and 6 seronegative contacts) and in 4 HLA-matched HIV negative donors. CTL specific for env, gag, or nef proteins could not be detected in unstimulated bulk cultures of peripheral blood lymphocytes in any of the six seronegative contacts. However, using a limiting dilution assay we found an usually high frequency of HIV nef-specific CTL precursors (CTLp) for HIV env and gag was very similar to that observed in seronegative HLA-matched healthy donors. Because no presence of HIV could be demonstrated in these individuals, these findings argue against the possibility of a silent HIV infection and suggest that a CTL response against nef may be involved in a rapid and effective clearance of the virus after sexual exposure.

Fidelity of HIV-1 Reverse Transcriptase Copying a Hypervariable Region of the HIV-1 env Gene

The unusually high mutation frequency exhibited by the human immunodeficiency virus (HIV) is a major impediment to developing effective vaccines against the virus and to designing nucleotide analogs that inhibit viral replication. To investigate the molecular basis of HIV hypermutability, we established cell-free assays to measure the fidelity of HIV-1 reverse transcriptase (RT) in copying either DNA or both RNA and DNA templates that contain the hypervariable region 1 of the HIV-1 env gene (V-1). The fidelity of DNA synthesis was measured by repetitively copying the envelope gene (V-1) DNA by HIV-1 RT, followed by cloning and sequencing these newly synthesized DNA products. We found that the error rate of HIV RT copying either RNA or DNA of the env V-1 region is about one misincorporation per 5 kb polymerized. This rate is similar to that found with the M13mp2 forward mutation assay using the lacZα gene as a template. This similarity suggests that the HIV env hypervariable sequence is not inherently hypermutable. The high error rate of HIV RT suggests that misincorporation by this enzyme is a major source of mutations throughout the viral genome and a determinant for rapid viral evolution. The spectrum of mutations produced by HIV RT in vitro partially correlates with the spectrum of HIV mutations observed in AIDS patients. The differences between these spectra highlight the contribution of phenotypic selection during HIV-1 infection. The overall uniformity of misincorporation of HIV-1 RT further suggests an alternative anti-HIV strategy based on increasing viral mutagenesis by nucleotide analogs.

Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron.

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regulation of HIV env expression is critically dependent on whether the env region is defined as an intron. This strengthens the hypothesis that Rev interacts with components of the splicing machinery to release splicing factors and enable export of the mRNA before splicing occurs.

Induction of Human Immunodeficiency Virus-Specific T Cell Responses in Rhesus Monkeys by Synthetic Peptides from gp160

We have previously identified several synthetic peptides from conserved regions of the human immunodeficiency virus (HIV) envelope protein gp160 that have the capacity to induce broadly reactive T cell responses against gp160 in mice of several major histocompatibility complex (MHC) haplotypes. In the present investigation three rhesus monkeys were immunized with a mixture of eight synthetic peptides that are capable of inducing T cell activity in mice. Peripheral blood mononuclear cells (PBMCs) from these monkeys were monitored every 2 weeks for a period of 34 weeks for proliferative responses against individual peptides and recombinant gp160. Peripheral blood mononuclear cells from all three rhesus monkeys showed good proliferative responses with peptides 104 (aa 45-55), 111 (aa 118-130), and 63 (aa 519-543), whereas weak responses were observed with peptides 113 (aa 204-216) and 116 (aa 240-252). Two of the three rhesus monkey-derived PBMC preparations also showed good proliferative responses with peptide 61 (aa 586-598). Significant responses were not observed with peptides 105 (aa 48-61) and R15K (aa 315-329) in any of the monkeys immunized. However, PBMCs from all three monkeys showed significantly high proliferative responses with recombinant gp160, the HIV-1 envelope protein precursor. These results demonstrate that mixtures of synthetic peptides from HIV env gene product can prime gp160-specific T cell responses in rhesus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein.

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2-terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428-440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)-synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.

The galactosyl ceramide/sulfatide receptor binding region of HIV-1 gp120 maps to amino acids 206-275.

Our recent studies have indicated that galactosyl ceramide (GalCer) or sulfatide (sul) may serve as an alternate receptor for human immunodeficiency virus (HIV) in neural cells. In this paper, we describe the mapping of GalCer/sul binding region of HIV env glycoprotein gp120. Deglycosylated gp120 binds to GalCer, suggesting that the amino acids of glycoprotein gp120 and not the carbohydrates are responsible for the observed binding. Specific regions of gp120 responsible for the binding were analyzed by using varying-length truncations of gp120 expressed in Escherichia coli and vaccinia virus. Purified recombinant gp120 containing amino acids 200-295 of gp120 bind to GalCer/sul, whereas recombinant env proteins that deleted this region did not bind. These recombinant proteins also bind to SK-N-MC-derived neuroblastoma cells, the binding of which is inhibited by anti-GalCer. In addition, 125I-labeled gp120 binding to GalCer is inhibited by these proteins. Studies using lysates containing truncated gp120 expressed in vaccinia virus also gave similar results. By eliminating the overlapping regions that do not bind, we conclude that the amino acids responsible for GalCer/sul binding reside between amino acids 206 and 275. The significance of this mapping is discussed in relation to the neurotropism of HIV.

Idiotypic expression of anti-gp120 antibodies in unrelated human immunodeficiency virus-infected individuals.

Although it is recognized that human immunodeficiency virus (HIV) env genes exhibit a high degree of variability, little is known about the molecular heterogeneity of gp120-specific antibodies in infected individuals. As a first step to approach this issue, we investigated the idiotypic relatedness of anti-gp120 antibodies present in the serum of HIV-infected individuals. Idiotypic determinants (idiotopes) are fingerprints of the variable region of the antibody molecule and, as such, they represent unique probes with which to explore the diversity of the immune response. We isolated IgG anti-gp120 antibodies from the serum of a seropositive asymptomatic individual by affinity chromatography. The purified antibodies were shown to bind gp120 and gp160 by ELISA, Western blotting and radio-immunoprecipitation. They also recognized HIV-infected human T cells as detected by immunofluorescence. Anti-idiotypic reagents were generated against this gp120 idiotype, and one of them was used to study anti-gp120 idiotypic diversity in a panel of 65 sera drawn from AIDS and AIDS-related complex patients, and from HIV seropositive asymptomatic individuals. Sixty normal human sera were used as negative controls. We found no evidence for common idiotopes on anti-gp120 antibodies of unrelated individuals. In contrast, we also noticed that the idiotypic profile expressed sequentially at two different intervals in a persistently infected individual showed little variation. Finally, when the diversity of murine anti-gp120 antibodies with a monoclonal anti-idiotype was analysed, no evidence of cross-reactive idiotopes in the murine system was found.

Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura

Twenty-one human immunodeficiency virus (HIV)-positive patients, including 11 acquired immunodeficiency syndrome (AIDS)-free patients with immune thrombocytopenic purpura (ITP), were studied to determine whether the megakaryocytic/platelet lineage was infected by HIV. Because purification of platelets did not reach a level sufficient for unequivocal results by the polymerase chain reaction, in situ hybridization was thus performed. Purified marrow megakaryocytes (MK) from 10 HIV-infected ITP patients were studied using a 35S HIV riboprobe, antisense of an HIV ENV sequence. HIV transcripts were clearly detected in MK from five of these 10 patients, although heterogeneity among MK was observed. In three of these five cases, small amounts of HIV glycoproteins were detected in MK by means of immunofluorescence. In addition anti-HIV antibodies could be eluted from platelets of all patients. In contrast, HIV transcripts were not detected in MK derived from colony-forming units-MK (CFU-MK) cultured in suspension, suggesting either that MK are infected by HIV during terminal differentiation or that HIV-infected CFU-MK are unable to differentiate in vitro. In conclusion, this study suggests that HIV infection of MK may be implicated in the pathogenesis of thrombocytopenia of HIV-positive patients.

Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.

Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENV gp) that were recognized by antibodies. Two epitopes of HIV ENV gp, ENVP 466 (amino acids 466–481) and ENVP 497 (amino acids 497–509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV 497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENV gp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.