env chimeric virus technology for evaluating human immunodeficiency virus susceptibility to entry inhibitors

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.     

免疫印迹法检测HIV抗体条带常见模式分析及在随访中的价值

目的了解免疫印迹法检测人类免疫缺陷病毒(HIV)抗体阳性的常见条带模式及在随访中的价值。方法回顾分析免疫印迹法检测HIV抗体为阳性的460例患者和结果为不确定的16例患者的免疫印迹法检测结果及临床资料。结果在3个基因编码的3组HIV特异性抗体中,env基因编码的包膜蛋白抗体gp160、gp120和gp41的检出率分别为100.00%、99.35%和96.30%;pol基因编码的核酸内切酶及聚合酶抗体p66、p51和p31的检出率分别为92.61%、86.96%和91.96%;gag基因编码的核心蛋白抗体p55、p39、p24和p17的检出率分别为56.30%、13.26%、100.00%、87.17%。采用免疫印迹法检测HIV抗体,共检测到34种条带模式,最常见的条带模式为gp160/gp120/p66/p55/p51/gp41/p31/p24/p17(p39缺失,占41.74%),其次为gp160/gp120/p66/p51/gp41/p31/p24/p17(p55和p39缺失,占23.70%),全条带模式占11.30%。对16例免疫印迹法结果为不确定的患者进行随访,其中首次检测含包膜蛋白抗体条带的4例患者在随访期间HIV抗体均转为阳性。HIV感染者首诊科室共涉及到28个临床科室,其中有66.31%就诊于内科相关科室。结论 HIV感染者的免疫印迹法条带检出率存在一定的差异,患者首诊的临床科室众多,医疗机构应重视HIV检测,避免获得性免疫缺陷综合征(AIDS)被误诊和漏诊。     

Heptad-repeat-2 mutations enhance the stability of the enfuvirtide-resistant HIV-1 gp41 hairpin structure

Enfuvirtide (T20) is a peptide-based fusion inhibitor derived from the heptad repeat 2 (HR2) region of HIV-1 glycoprotein 41 (gp41). The inhibitor binds to the gp41 heptad repeat 1 (HR1) region, thereby blocking viral HR1/HR2 association. Mutations in HR1 have been reported to cause enfuvirtide resistance and reduce viral fitness. In this study, we first showed that scores obtained by a residue-specific all-atom probability discriminatory function (RAPDF) may be used as a reliable predictor of structural stability of gp41 mutants by comparing it to experimentally determined melting temperatures, and as a reliable indicator of enfuvirtide resistance by comparing it to experimentally determined fusion inhibition and viral fitness levels. We then generated an initial set of 28 theoretical structures of the HR1/HR2 hairpin complex where each structure consists of one mutation on HR1 known to cause enfuvirtide resistance and a wild-type amino acid at the corresponding HR2 residue. Mutations were then introduced in the corresponding HR2 residue of each structure where the wild-type amino acid was changed to each of the other nineteen amino acids. The enfuvirtide-resistant HR1 mutants with compensatory mutations at the corresponding HR2 residues had better RAPDF scores than those HR1 mutants with wild-type HR2. This indicates that mutations in HR2 improve structural stability of the HR1/HR2 hairpin complex and may lead to enhanced enfuvirtide resistance when present with resistant HR1 mutations. Modification of the amino acid side chains that contribute to enfuvirtide resistance using the RAPDF scores as a guide may help design of a second generation of fusion inhibitors against the enfuvirtide-resistant strains.     

Role of human immunodeficiency virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.     

57例抗HIV初筛阳性确认实验带型分析

目的 调查HIV感染初筛实验的准确率及确认抗HIV初筛试验阳性血清的感染和感染型别。方法 调查我国艾滋病高发农村，对部分初筛阳性和可疑的血清进行确认实验。结果 57例初筛为抗HIV阳性和可疑血清进行确认实验，56份为HIV—l阳性，其中1份样本出现HIV—2型反应条带；1份阴性。确认阳性标本的反应条带中，抗外膜蛋白(env)抗体阳性率最高，gpl60为100％，gpl20为94．6％，gp41为91．1％；多聚酶抗原(pol)p66为82．1％；核心抗原(gag)P24为53．6％。提示无症状携带组确认反应条带的pol和gag的阳性率显著高于艾滋病组(P＜0．01)。儿童组gag阳性率显著低于成人组(P＜0．05)。结论 HIV感染初筛实验的准确率较高，但确定感染需靠确认实验。外膜蛋白、p66和p24抗原是HIV感染的重要抗原，对确认HIV的感染具有指导意义。     

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.     

False-positive human immunodeficiency virus type 1 western blot tests in noninfected blood donors

Background: The manufacturers' criteria for a positive human immunodeficiency virus type 1 (HIV-1) Western blot (WB) test were recently revised to require reactivity to only two of the following bands: p24, gp41, and gp120/160. In a recent report, low-risk blood donors were identified in whom nonspecific reactivity to multiple env antigens in WB testing resulted in apparently false-positive WBs by these criteria. The present study was conducted to verify the existence of false-positive WBs among noninfected donors and to assess the extent of this problem. Study Design and Methods: Four donors classified as WB- positive on the basis of env-only (3 cases) or p24/env-only (1 case) patterns were investigated. Index and/or follow-up specimens were tested by polymerase chain reaction (PCR), by overlapping recombinant env antigens and synthetic peptides in enzyme immunoassays, and by deglycosylated and denatured antigen WBs. WB records from American Red Cross blood centers were reviewed to determine the frequency of env- only and p24/env-only patterns, relative to all positive WBs, from 1988 through 1993. Results: The four index-case donors denied risk and had stable WB reactivity during follow-up. HIV PCR was negative in all. Env reactivity was restricted to nonglycosylated gp41 epitopes; no gp120- specific reactivity was detected. For three of the four donors, env reactivity was mapped to a 20-amino acid N-terminal epitope of gp41. The rate of detecting WBs with these false-positive patterns increased from 0.6 percent of all positive WBs from 1988 to 1990 (4/776) to 8 percent in 1991 and 1992 (52/683), and then it declined to 6 percent in 1992 and 1993 (47/783). Env-only patterns predominated in 1991 and 1992, whereas p24/env-only patterns were more frequent following implementation of combined anti-HIV-1/HIV type 2 enzyme immunoassays in 1992. Conclusion: Low-risk blood donors can have false-positive results on WB tests. Increased detection of env-only and p24/env-only WBs appears related to the enhanced sensitivity of newer enzyme immunoassays to gp41 and p24 antibodies. Donors with these patterns should undergo follow-up testing to document the presence or absence of HIV infection.     

Genotype and phenotype patterns of human immunodeficiency virus type 1 resistance to enfuvirtide during long-term treatment

The human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral agents. In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost evolution of HIV-1. The genotype and phenotype patterns and the relative replication capacity (rRC) of enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders and 4 responders) who received the compound for more than 1 year in combination with different regimens. Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp41 N-terminal heptad repeat was observed in samples from the seven virological nonresponders but not in those from responders. In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected within 3 months. Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to enfuvirtide, with the 50% inhibitory concentrations (IC(50)s) ranging from 0.6 to 12.8 microg/ml, whereas the IC(50) for isolates with baseline sequences was 0.013 +/- 0.010 microg/ml. Interestingly, long-term monitoring of resistant variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes. A limited drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing mutations associated with resistance. Overall, the data indicate that the different genotype patterns associated with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance, and limited influence on the viral rRC.     

Primary virus-induced lymphomas evade T cell immunity by failure to express viral antigens

T lymphoma induction by the mink cell focus-inducing murine leukemia virus MCF 1233 in C57BL/10 and C57BL/6 mice is influenced by a strongly Th-dependent, H-2I-A-restricted antiviral immune response (25). We compared the MHC class I as well as viral env and gag antigenic cell surface profiles of frequent T lymphomas of H-2I-A nonresponder-type mice to that of rare T lymphomas of H-2I-A responder-type mice. Membrane immunofluorescence studies, with a panel of anti-env mAbs (reactive with the highly conserved gp70f epitope, the p15Ec epitope, and the gp70-p15E complex), a polyclonal anti-p30 serum, and anti-H-2 class I mAbs, showed that all 17 nonresponder tumors tested expressed high levels of both env and gag viral proteins, and 15 of these 17 nonresponder tumors expressed high levels of H-2 class I K and D antigens. In contrast, 10 of 11 responder lymphomas lacked env and/or gag determinants. The only responder lymphoma with both strong env and gag expression failed to express H-2K and -D antigens. Preferential loss of env or gag expression did not correlate with H-2 class I allelic specificities. Both responder and nonresponder T lymphoma DNA contained multiple, predominantly MCF-like, newly acquired proviral integrations. Differences in viral antigen cell surface expression were confirmed at cytoplasmic and RNA levels. The amounts of 8.2- and 3.2-kb viral RNA were greatly reduced in two responder lymphomas when compared with four nonresponder lymphomas. In both responder lymphomas, aberrantly sized viral RNA species were found. Upon in vivo passage of these responder lymphomas in either immunocompetent or T cell-deficient nu/nu mice, it was found that various molecular mechanisms may underlie the lack of viral antigen expression at the cell surface of these lymphomas. One lymphoma re-expressed viral antigens when transplanted with nu/nu mice, whereas the other remained stably gag negative. The combined findings indicate that an H-2I-A-regulated antiviral immune response not only strongly reduces T lymphoma incidence, but also forces T lymphomas that still arise to poorly express viral antigens, thus explaining their escape from immunosurveillance.     

Amplified env and gag products on AKR cells. Origin from different murine leukemia virus genomes

Thymocytes of AKR mice express two species of gp70, the envelope glycoprotein of murine leukemia virus (MuLV), encoded by the env gene. One is denoted Ec+ gp70 in reference to the type-antigen Ec and association with ecotropic virus. The other, Ec- gp70, resembles gp70 found also on thymocytes of mouse strains that are not overt producers of MuLV, and has no evident relation to ecotropic virus. Expression of Ec- gp70 type, but not of Ec+ gp70 type, is amplified with age on AKR thymocytes. In contrast, viral core polyproteins, encoded by the gag gene and simultaneously amplified with age, appear to be related to ecotropic virus. These observations imply selective amplification of products of env and gag genes from two sorts of provirus, a phenomenon which may be connected to the dual genetic origin of recombinant mink- cell-focus inducing viruses in AKR mice.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

Resistance to enfuvirtide, the first HIV fusion inhibitor

Fusion inhibitors are a new class of antiretroviral drugs (ARVs) for the treatment of human immunodeficiency virus infection. Enfuvirtide is the first in this class to reach market approval. Fusion inhibitors block the last step in the three-step viral entry process consisting of attachment, co-receptor binding and fusion, thereby preventing viral capsid entry into the host cell. Enfuvirtide has a unique mechanism of action and high viral target specificity, and in clinical trials has been shown to exhibit both high efficacy and low toxicity. Enfuvirtide is a peptide mimetic of an essential region within viral envelope glycoprotein gp41 that functions by blocking gp41 structural rearrangements at a transitional pre-fusion conformation. Although different clinical isolates show variation in susceptibility to enfuvirtide, primary resistance has not been observed, and thus enfuvirtide-naive isolates remain clinically sensitive. Acquired resistance centres round a 10 amino acid motif between residues 36 and 45 in gp41 that forms part of the binding site of enfuvirtide. The 10 amino acid motif is critical for viral fusion, and enfuvirtide-resistant mutants show poor replicative capacity compared with wild type. Reversion to a wild-type, drug-sensitive state has been reported following enfuvirtide withdrawal.     

Conferral of an antiviral state to CD4+ cells by a zipper motif envelope mutant of the human immunodeficiency virus type 1 transmembrane protein gp41.

We showed in a transient coexpression study that a single proline substitution for any of the five conserved leucine or isoleucine residues located in the envelope (Env) transmembrane protein gp41 zipper motif of the human immunodeficiency virus type 1 dominantly interferes with wild-type Env-mediated viral infectivity. In the present study, we intended to explore the feasibility of developing a genetic anti-HIV strategy targeting the zipper motif. Stable HeLa-CD4-LTR-beta-gal clones that harbored silent copies of Tat-regulated expression cassettes encoding the zipper motif Env mutants were first generated. Expression of any of the five Env mutants in transfectants interfered with exogenously expressed homologous HXB2 Env-mediated cytopathic effects. Mutant transfectants 566, 573, and 580 were further examined. Viral transmission mediated by the laboratory-adapted T cell-tropic HXB2 and NL4-3 viruses was greatly reduced in these transfectants compared with that observed in the env-defective control deltaKS and wt env transfectants. Moreover, viral replication mediated by the NL4-3 virus and a macrophage-tropic ADA-GG virus was delayed or reduced in human T cells harboring the mutant 566 or 580 env construct as opposed to those observed in cells harboring the control deltaKS or mutant 573 env construct. The wt and mutant Env proteins formed a hetero-oligomer when they were coexpressed. These results demonstrate that zipper motif Env mutants 566 and 580 confer an anti-HIV state to the host CD4+ cells, which indicates that dominant inhibitory mutants targeting the gp41 zipper motif might function as genetic anti-HIV agents to combat HIV-1 infection.     

Antibody reactivity to deletion mutants of the HIV-1 SF2 envelope

In human immunodeficiency virus type 1 (HIV-1) infected individuals, the antibody response to the external envelope (gp120) is associated with in vitro neutralization. To further characterize the anti-gp120 response, we examined the IgG reactivity of 75 HIV-1-seroconverted and 200 HIV-1-seropositive individuals to deletion mutants of gp120 in an enzyme immunoassay. We used yeast-derived, non-glycosylated recombinant HIV-1 SF2 gp120 equivalent and-variants deleted in variable regions. We observed two distinctive response patterns: IgG non-responders (SF2-V3-restricted responders) and IgG responders to conserved regions of gp120. This divergence in response pattern occurred soon after gag/env HIV-1 antibody seroconversion and persisted in time within an individual. In addition, the SF2-V3-restricted responders had a higher frequency of HIV-1 core antigen positivity and HIV-1 core antibody negativity than the non-restricted responders. These results suggest that specific and persistent host antibody response patterns to gp120 develop early in HIV-1 infection and that these patterns are associated with differences in HIV-1 expression.     

Identification of tryptic peptides unique to a 110,000-molecular weight polyprotein encoded by the T-8 isolate of murine leukemia virus

Mink cells nonproductively-infected with the weakly-transforming T-8 isolate of murine leukemia virus (MuLV) express a 110,000 mol wt polyprotein designated T-8 P110. By immunoprecipitation analysis, T-8 P110 is shown to contain AKR-MuLV amino terminal gag gene-specific components (p15, p12) but to lack p30, p10, gp70, and p15(E) antigenic determinants. These observations are further substantiated by tryptic peptide analysis indicating T-8 P110 to share approximately six lysine-containing tryptic peptides with AKR-MuLV Pr65gag, and none with AKr-MuLV Pr82env. Furthermore, of seven methionine-containing T-8 P110 tryptic peptides, at least four can be conclusively shown not to be present in either AKr-MuLV Pr180gag/pol or Pr82env. A clonal mink cell line nonproductively infected by T-8, and expressing high levels of P110, although not morphologically transformed, is shown to lack elevated levels of tyrosine-specific protein kinase activity and reduction of epidermal growth factor binding sites characteristic of cells transformed by many other RNA-transforming viruses. These findings argue either that the T-8 viral genome contains acquired cellular sequences encoding a portion of P110, or that T-8 P110 represents an inphase deletion of AKR-MuLV Pr180gag/pol with extensive posttranlational modification and that an as yet unidentified protein is responsible for T-8 associated transformation.