Fine mapping of an immunodominant region of the transmembrane protein of the human immunodeficiency virus (HIV-1)

Peptides of HIV sequences are significant for antibody screening systems, and because of the limited number of amino acids they have to represent immunodominant regions of the virus proteins in order to maintain sensitivity. We detected, in a region of the outside loop of the transmembrane protein gp41 of the human immunodeficiency virus HIV-1 (amino acid 586-620), two immunodominant sequences which are distinct from each other. Whereas in the first immunodominant region the sensitivity and specificity of ELISA were inadequate, a neighbouring region is well suited for use as antigen for an anti-HIV screening ELISA.     

Differentiation of human immunodeficiency virus type 1 (HIV-1) infections with HIV-2-cross-reacting antibody from mixed infections with HIV-1 and HIV-2 by serological absorption test.

The interpretation of dual seroreactivity with human immunodeficiency virus type 1 (HIV-1) and HIV-2 in blood samples is a serious problem facing AIDS researchers worldwide. Some samples of sera from HIV-1-infected patients showed a serological cross-reaction with HIV-2, causing confusion regarding the serodiagnosis. Therefore, we tried to differentiate these serum samples from those containing real mixed infections with both types of virus. Sera from patients with HIV-1 infections with HIV-2 cross-reacting antibody in Japan were distinguished from sera from patients with mixed infections with HIV-1 and HIV-2 in West Africa by our serological cross-absorption test, which proved to be highly specific and useful for serodiagnosis.     

Discrimination between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections by using synthetic peptides representing an immunodominant region of the core protein (p19) of HTLV-I and HTLV-II.

We describe enzyme immunoassays that use synthetic oligopeptides to discriminate serologically between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections. The peptides represented 20-amino acid segments between residues 111 and 130 (MA1) and residues 116 and 135 (MA2) of the p19 gag proteins of HTLV-I and HTLV-II, respectively. The assays were sensitive since 69 of 74 HTLV-positive sera were reactive to at least one of the two matrix (MA) peptides (sensitivity, 93.2%). By using the ratio of the optical density of MA1 to the optical density of MA2, which represents for every serum sample the ratio between the absorbance value obtained in the MA1 assay and the absorbance value obtained in the MA2 assay, 59 of the 69 reactive serum samples were clearly and easily typed as positive for either antibody to HTLV-I or antibody to HTLV-II. Eight of the 10 remaining reactive serum samples were analyzed further by an inhibition procedure, and their type specificities were then clearly identifiable. Therefore, the results indicate that all MA-reactive sera were serologically distinguished by our peptide assays.     

合成肽抗原抗人免疫缺陷病毒1/2型抗体酶联试剂盒的研制

根据人免疫缺陷病毒（ＨＩＶ）的基因结构和氨基酸序列，采用固相法合成了ＨＩＶ－１ｇｐ４１（ＳＰ１）、ｇｐ１２０（ＳＰ２）、ｐ２４（ＳＰ３）和ＨＩＶ－２ｇｐ３６（ＳＰ４）的４条多肽，混合包被酶标板做为固相抗原，采用间接酶联免疫吸附试验（ＥＬＩＳＡ），建立了检测抗－ＨＩＶ－１／２ＩｇＧ抗体的酶联诊断试剂盒。检测卫生部药品和生物制品检定所提供的４１份质控参比血清，其特异性、敏感性均为１００％，变异系数小于１０％。检测１８６份其它病种病人血清均为阴性，与华怡、巴斯德、金豪等公司的ＨＩＶ诊断试剂比较检测了９０份ＨＩＶ感染者和１４０份正常人血清，除与华怡试剂的阴、阳性及总符合率分别为９９２９％（１３９／１４０）、９８９０％（９０／９１）和９９５７％（２２９／２３０）外，其余均为１００％。３７℃放置４天后试剂的检测结果不受影响。     

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.     

合成肽抗原抗人免疫缺陷病毒1／2型抗体酶联试剂盒…

根据人免疫缺陷病毒的基因结构和氨基酸序列，采用因相法合成了ＨＩＶ－１ｇｐ４１、ｂｐ１２０、ｐ２４和ＨＩＶ－２ｇｐ３６的４条多肽，混合包被酶标板做为固相抗原，采用间接酶联免疫吸附试验，建立了检测抗－ＨＩＶ－１／２ＩｇＧ抗体的酶联诊断试剂盒。检测卫生部药品和生物制品检定所提供的４１份质控参比血清，其特异性、敏感性均为１００％，变异系数小于１０％。检测１８６份其它病种病人血清均为阴性，与华怡、巴斯德、金     

The Honduran human immunodeficiency virus type 1 (HIV-1) epidemic is dominated by HIV-1 subtype B as determined by V3 domain sero- and genotyping.

The distribution of subtypes A through F of human immunodeficiency virus type 1 (HIV-1) in Honduras was analyzed in 120 HIV-1 positive serum samples by V3 peptide serotyping and HIV-1 cDNA sequencing. In the Honduran HIV-1 epidemic, subtype B was detected in 98 of 99 subtyped samples.     

Antibody-dependent cellular cytotoxicity (ADCC) is directed against immunodominant epitopes of the envelope proteins of human immunodeficiency virus 1 (HIV-1).

In this study, epitopes of HIV envelope proteins that are involved in ADCC were identified. Peripheral blood mononuclear cells (PBMC) were obtained from adults with asymptomatic HIV infection or early symptoms of AIDS. These PBMC, which were reported to be "armed" in vivo with HIV-specific antibodies, were used as effector cells in 51Cr release assays. Target cells consisted of CD4 lymphocytes from healthy seronegative donors, coated with the IIIB strain of HIV-1 or with one of seven synthetic peptides. Cytotoxicity was detected against CD4 lymphocytes coated with HIV-1 IIIB or with the peptides env aa 507-518, corresponding to the carboxy-terminus of gp120, and env aa 597-611, corresponding to the region of the cysteine loop of gp41. The magnitude of target cell lysis was directly related to the quantity of peptide used. In contrast, target cells coated with the peptide gag aa 129-135, corresponding to the p17/p24 cleavage region of the gag precursor, were not killed. The same immunodominant regions which were involved in ADCC were recognized in enzyme-linked immunoabsorbent assays (ELISA) by the majority of 107 sera from HIV-infected adults. We conclude that the immunodominant epitopes located at the carboxy-terminus of gp120 and the cysteine loop of gp41 serve as recognition structure for antibodies, capable of mediating ADCC against HIV-infected cells.     

Functional genomics analyses of differential macaque peripheral blood mononuclear cell infections by human immunodeficiency virus-1 and simian immunodeficiency virus

The pathogenicity of the primate lentiviruses, human, and simian immunodeficiency viruses, is host-specific. Previous studies indicated that the highly pathogenic human lentivirus HIV-1 has markedly reduced pathogenicity compared to the pathogenic simian lentivirus SIV in pigtail macaques (Macaca nemestrina). We therefore hypothesized that the pigtail macaque peripheral blood mononuclear cells (mPBMCs) would respond differently to infections of HIV-1 and pathogenic SIV. To elucidate the cellular responses to the infections of HIV-1 and SIV, we infected mPBMC with these two viruses. Like infections in vivo, HIV-1 and SIV demonstrated distinct replication kinetics in mPBMCs, with HIV-1 replicating at significantly lower levels. Similarly, gene expression profiling facilitated by macaque-specific oligonucleotide microarrays also revealed distinct expression patterns of genes between the HIV-1- and SIV-infected mPBMCs; in particular, genes associated with the antigen presentation, T cell receptor, ERK/MAPK signaling, Wnt/beta-catenin signaling, and natural killer cell signaling pathways were differentially regulated between these two viruses. Most interestingly, despite the lower levels of replication, HIV-1 triggered a more robust regulation of immune response genes early after infection; the converse was true in SIV-infected mPBMCs. Our results therefore suggest that macaques may be controlling the infection of HIV-1 at an early stage through coordinated regulation of host defense pathways.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1).

Antibody mediated and cell mediated immune responses to the envelope glycoproteins gp120 and gp41 of the human immunodeficiency virus (HIV-1) are considered important for protection against infection and for attenuation of disease symptoms after infection. Virus neutralizing antibodies are mostly subtype specific and primarily directed against epitopes on a hypervariable loop from the V3 region of HIV-1 gp120. Such epitopes are recognized by helper and cytotoxic T-cells suggesting that all protective immune responses to HIV-1 are predominantly subtype specific. The extraordinary primary sequence variability of gp120 indicates that a combination of subtype specific components will be required to design a broadly effective protective immunogen against HIV-1. Peptides from hypervariable loops of the V3 region of 21 distinct HIV-1 isolates (clones) were synthesized and used to raise rabbit antisera. The antisera contained high levels of antibodies recognizing the homologous peptides and the parent gp120 sequence. The serological cross-reactivity between the distinct peptides was evaluated and related to amino acid divergence. The corresponding relationship approximated a linear regression with a correlation coefficient r = 0.718. The 21 peptides were combined into a single immunogen which elicited broadly reactive antibodies recognizing all 21 peptides as well as gp120 from the only isolate tested, HIV-1 IIIB. The results suggest the possibility of developing broadly protective HIV-1 immunogens by combining judiciously selected subtype specific peptides derived from envelope glycoproteins of divergent virus isolates.     

Biological mechanisms of vertical human immunodeficiency virus (HIV-1) transmission

In the absence of interventions, 30-45% of exposed infants acquire human immunodeficiency virus type 1 (HIV-1) through mother-to-child transmission. It remains unclear why some infants become infected while others do not, despite significant exposure to HIV-1 in utero, during delivery and while breastfeeding. Here we discuss the correlates of vertical transmission with an emphasis on factors that increase maternal HIV-1 levels, either systemically or locally in genital secretions and breast milk. Immune responses may influence maternal viral load, and data suggest that maternal neutralising antibodies reduce infection rates. In addition, infants may be capable of mounting HIV-specific cellular immune responses. We propose that both humoral and cellular responses are necessary to reduce infection because cell-free as well as cell-associated virus appears to play a role in vertical transmission. These distinct forms of the virus may be targeted most effectively by different components of the immune system. We also discuss the use of antiretrovirals to reduce transmission, focusing on the mechanisms of action of regimens currently used in developing country settings. We conclude that prevention relies not only on reducing maternal HIV-1 levels within blood, genital tract and breast milk, but also on pre- and/or post-exposure prophylaxis to the infant. However, HIV-1 has the capacity to mutate under drug pressure and rapidly acquires mutations conferring antiretroviral resistance. This review concludes with data on persistence of low-level resistance after delivery as well as recent guidelines for maternal and infant regimens designed to limit resistance.     

Biologic heterogeneity of human immunodeficiency virus type 2 (HIV-2) strains

Seven HIV-2 isolates recovered from peripheral blood mononuclear cells (PBMC) of patients from the Ivory Coast have been biologically characterized. All seven strains replicated well in primary human lymphocyte and macrophage cultures and in established human T cell lines. They showed differences in infectivity and replicating ability in primary PBMC cultures from chimpanzees, rhesus macaques, and baboons. Moreover, variations in levels of virus replication in PBMC from 13 seronegative donors were observed. Four strains (UC2, UC3, UC7, and UC8) were highly cytopathic and caused extensive surface CD4 antigen depletion in acutely infected PBMC and SupT1 cells. Two strains (UC1 and UC6) showed minimal or no cytopathology, no CD4 down-modulation, and much lower levels of virus protein expression in SupT1 cells. These findings reflect the heterogeneity of HIV-2 strains and suggest that these biological properties could influence pathogenesis in the host.     

Relationship between human immunodeficiency virus (HIV-1) infection and chronic periodontitis

Introduction: Current studies show that, even in the era of antiretroviral therapies, HIV-1 infection is associated with more severe and frequent refractory chronic periodontitis.

Areas covered: This review, based on a systematic analysis of the literature, intends to provide an update on factors that may be involved in the pathogenesis of periodontal disease in HIV-1-infected patients, including local immunosuppression, oral microbial factors, systemic inflammation, salivary markers, and the role of gingival tissue as a possible reservoir of HIV-1.

Expert commentary: The therapeutic revolution of ART made HIV-1 infection a chronic controllable disease, reduced HIV-1 mortality rate, restored at least partially the immune response and dramatically increased life expectancy of HIV-1-infected patients. Despite all these positive aspects, chronic periodontitis assumes an important role in the HIV-1 infection status for activating systemic inflammation favoring viral replication and influencing HIV-1 status, and also acting as a possible reservoir of HIV-1. All these issues still need to be clarified and validated, but have important clinical implications that certainly will benefit the diagnosis and management of chronic periodontitis in HIV-1-infected patients, and also contributes to HIV-1 eradication.     

Confronting the hypervariability of an immunodominant epitope eliciting virus neutralizing antibodies from the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1)--II. Synthetic peptides linked to HIV-1 carrier proteins gag and nef.

Combining of subtype specific peptides from the hypervariable loop of the envelope glycoprotein gp120 of divergent HIV-1 isolates may help in designing a broadly protective immunogen against HIV-1 infection. To enhance the immunogenicity of such a polyvalent antigen, in the absence of oil-containing adjuvants, it is necessary to link the peptides to a protein carrier. It is preferable to use as carriers those proteins from HIV-1 itself which may contribute to eliciting protective immunity. The structural and non-structural proteins, gag P18 and nef, respectively, which can be prepared in high yields by recombinant DNA techniques in Escherichia coli, were selected for this purpose. The corresponding peptide-protein conjugates, each containing 21 distinct peptides, were prepared using the cross-linking reagents N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS). Conjugates prepared by the second method elicited approximately 10-100 times higher levels of antibodies recognizing the homologous peptides and the HIV-1 envelope glycoproteins. The sulfo-MBS conjugation procedure preserved the antigenicity of both gag P18 and nef and the respective conjugates elicited an immune response to these proteins. Despite the low immunization dose of single peptides (10 micrograms) present in the mixture of peptides collectively linked to the carriers, antibody responses to most of the individual peptides were high (dilution endpoints 1: greater than 16,000, 1: greater than 80,000 for the nef and gag P18 conjugates, respectively). Conjugates consisting of a multitude of HIV-1 envelope-derived peptides in combination with gag P18 and nef carriers are expected to elicit broadly protective immunity against distinct HIV-1 subtypes.     

Human immunodeficiency virus type 1 (HIV-1) and human hematopoietic progenitor cells

Summary Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.     

Detection of human immunodeficiency virus type 1 (HIV-1) antibody by western blotting and HIV-1 DNA by PCR in patients with AIDS.

The human immunodeficiency virus type 1 (HIV-1) Western blotting (immunoblotting) band patterns and the sensitivity of an HIV-1 DNA PCR assay were determined by testing the blood of patients with AIDS. Plasma and cell pellets processed from the peripheral blood of 199 patients with absolute CD4 cell counts of less than 200 cells per mm3 were tested by a licensed enzyme immunoassay (EIA; Abbott HIV-1) and Western blot assay (Cambridge-Biotech) for HIV-1 antibody. The Roache HIV-1 AMPLICOR DNA PCR assay was used to test cell pellets from 125 of the 199 patients for HIV-1 gag DNA sequences. All plasma samples from these 199 sequential patients were reactive for HIV-1 antibody by EIA and were positive by Western blot assay using the criteria recommended by the Centers for Disease Control and Prevention. The majority of samples (192 of 199; 96.5%) displayed at least six of nine bands characteristic of the virus by Western blotting, with the lowest number of bands characteristic of the virus displayed by any sample being three. However, 39 and 48% of all patients exhibited no bands to p17 and p55 antigens, respectively, whereas 0 to 7.5% of all patients exhibited no bands to the other antigens. HIV-1 gag DNA sequences were detected in 117 (93.6%) of 125 cell pellets processed from the peripheral blood of these same patients. All eight patients initially negative by PCR tested positive when a second pellet which had been produced from the same blood sample was tested. Despite a decrease in antibody reactivity to HIV Gag and Pol proteins, patients with advanced HIV-1 infection remained positive for HIV-1 antibody by EIA and Western blot testing. Confirmation by the HIV-1 Western blot assay still appears to be the more sensitive assay for the diagnosis of HIV-1 infection in those individuals with advanced HIV-1 infection in the United States.