Acquired resistance to type II collagen-induced arthritis in rhesus monkeys is reflected by a T cell low-responsiveness to the antigen.

The use of serological tests for the diagnosis of HIV infection has revealed that some non-infected persons have antibodies that react with HIV-1 gag proteins. Here, the sera of three non-infected subjects reacting with p17 and 11 non-infected subjects reacting with p24 were investigated, using an enzyme immunoassay (EIA) with six recombinant gag antigens and Western blot analysis of proteolytic peptides of two of these gag antigens. The results indicate that whereas all p17-reactive sera could react with an unique epitope, individual p24-reactive sera recognize different epitopes. Investigations by EIA also demonstrated the role of sequences located far from the epitopes in making these epitopes accessible to the antibodies or in providing them with an antigenic conformation. In addition to the 14 subjects mentioned above, another subject was shown to have antibodies reacting with the p9 (NC) gag protein. Several proteins are known as having homology with HIV-1 gag proteins. Their possible role in eliciting cross-reactive antibodies is discussed.     

Specificity of anti-P25 antibodies produced against whole HIV-1 particles or soluble forms of the protein.

Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins.     

Anti-human immunodeficiency virus type 1 antibodies of noninfected subjects are not related to autoantibodies occurring in systemic diseases.

Indeterminate Western blot (WB) (immunoblot) patterns for anti-human immunodeficiency virus type 1 (HIV-1) antibodies are often observed when testing serum samples from noninfected individuals. We investigated here the possible involvement of some frequently occurring autoantibodies (anti-SmB/B', U1snRNP [68 kDa, A, and C], Ro/SS-A [60 and 52 kDa], and Jo-1) in the generation of such indeterminate HIV-1 WB. In particular, the role of a reported sequence homology between p24 gag and the SmB/B' autoantigen was investigated. Serum samples were obtained from 50 healthy controls, 51 patients with systemic lupus erythematosus (SLE), 46 with systemic sclerosis, 6 with Sjögren's disease, 3 with mixed connective tissue disease, and 41 healthy subjects with persistent indeterminate HIV-1 WB. Reactivity to HIV-1 p24 gag was slightly but not significantly more frequent in patients with SLE than in controls (25.5% versus 14.0%; P > 0.1), whereas reactivity to HIV-1 p17 gag was significantly more frequent in the former subjects (23.5% versus 8.0%; P = 0.03). Simultaneous reactivity to p17 and p24 was observed in patients with SLE (11.8%; P = 0.014) or systemic sclerosis (8.7%; P = 0.049) but not in controls. There was no association found between the presence of any autoantibody and the occurrence of indeterminate HIV-1 WB nor between the presence of p24-reactive antibodies and anti-SmB/B'; this indicates that most p24-reactive antibodies are directed to epitopes other than the proline-rich sequences shared by p24 gag and SmB/B'.     

Detection of antibodies to trans-activator protein (p40taxI) of human T-cell lymphotropic virus type I by a synthetic peptide-based assay.

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) trans-activator protein (p40taxI) were determined in serum specimens from individuals infected with HTLV-I (n = 138) and HTLV-II (n = 19). Western blot (immunoblot) analysis using recombinant tax demonstrated the presence of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-I-associated myelopathy, 43% of those (20 of 46) with adult T-cell leukemia, and 61% of asymptomatic HTLV-I blood donors (40 of 66); only one of the HTLV-II specimens reacted with the recombinant tax protein. Synthetic peptides (Tax8(106-125), Tax22(316-335), Tax-23(331-350), and Tax-24(336-353) representing the immunodominant epitopes of¿ p40taxI detected anti-tax antibodies in 66 (48%), 50 (36%), 66 (48%), and 64 (46%) of 138 HTLV-I-positive specimens, respectively. An enzyme immunoassay using an equimolar ratio of these four peptides allowed sensitive detection of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-1-associated myelopathy, 52% of adult T-cell leukemia patients (24 of 46), and 62% of asymptomatic HTLV-1-infected donors (41 of 66). The synthetic peptide-based cocktail assay was HTLV-I specific, since none of the HTLV-II-infected specimens reacted with these peptides. Interestingly, the corresponding regions from the HTLV-II tax protein, Tax8II(106-125), and Tax-22II(312-331) did not react with either HTLV-II or HTLV-I specimens. Thus, a synthetic peptide-based assay composed of immunodominant epitopes located towards the amino terminus and the C terminus of p40taxI provides a reliable and sensitive assay for the detection of anti-tax antibodies in seroepidemiologic studies.     

Study of antigenic structure of HIV-1 protein p24 using monoclonal antibodies]

During the experiments 4 murine and 3 rat hybridomas producing monoclonal antibodies (MAb) against the protein p24 of human immunodeficiency virus type 1 (HIV-1) have been obtained. Using the immunoblotting technique, it was established that all the species of MAb reacted with the same viral proteins which are derivatives of gag gene--p24 and p55. The properties of MAb have been studied in competitive binding. Their ability of binding to different fragments of the gag protein produced by the recombinant plasmids in E. coli cells have been investigated in ELISA. The analysis of the findings suggests that the HIV-1 protein p24 contains at least 3 antigenic epitopes. All species of MAb reacted with 3 different HIV-1 strains and 2 HIV-1 isolates, but failed with 2 different HIV-2 strains. The only MAb NS5E4 can be used as an immunosorbent in the antigenic capture reaction.     

New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-amino-acid sequence from a highly conserved region of the HIV-1 gp41 envelope protein. This recombinant antigen enabled the detection of antibodies to both gag and env gene products. When this assay was compared with a commercially available recombinant enzyme-linked immunoabsorbent assay (ELISA) by using four quality-control panels, the TR-FIA detected all 20 positive specimens, while the recombinant ELISA detected only 16 of them. This increased sensitivity could be demonstrated directly by the assay of dilution series of HIV-1-positive sera. The analysis of two seroconversion panels by TR-FIA and six ELISAs showed that TR-FIA allowed detection of antibody in infected individuals 16 days earlier than the other assays did. In addition to being highly sensitive, the assay was highly specific; of the 57 samples shown to be repeatedly positive by ELISA but known to be HIV-1 negative by Western immunoblot analysis, only 1 sample reacted positively in this assay. The specificity of the assay was 99.9% when 1.054 random serum specimens were tested.     

Fine Molecular Specificity of Linear and Assembled Antibody Binding Sites in HIV-1 p24

A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV-1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.     

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.     

Immune responses to six synthetic peptides of capsid protein with sera from HIV-1 infected individuals

Many B cell epitopes within p24 of human immunodeficiency virus type 1 （HIV-1） were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera （19.4%）, all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals.     

An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens

An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).     

Evidence that HIV-1 gag precursor shares antigenic sites with the major capsid protein of human cytomegalovirus.

A rabbit antiserum prepared against disrupted sucrose-banded HIV-1 virus (strain FRE-3) reacted with antigens present in nuclear inclusions, pathognomonic for human cytomegalovirus (HCMV). This cross-reactivity was observed in autopsy specimens from individuals infected with CMV, in the presence or absence of co-infection with HIV-1. A Towbin immunoassay showed that the serum reacted specifically with the HCMV major capsid protein (MCP, 153 kDa), both in the nuclear fraction of infected cells and in virions. Direct evidence that these proteins share antigenic determinants was provided by the two-way cross-reactivity of affinity-selected antibodies (i.e., anti-MCP with HIV-1 gag precursor Pr55; anti-Pr55 with MCP). All four strains of HCMV tested showed this reactivity, but the counterpart proteins of simian CMV and herpes simplex virus type 1 did not, indicating that the determinant is not common to all herpes group viruses.     

Epitope mapping of the HIV-1 gag region with monoclonal antibodies.

A new type of immunochemical mapping of the human immunodeficiency virus type 1 (HIV-1) gag region was performed. By use of native HIV-1 viral lysates or the gag recombinant p24-15 antigen, a new set of monoclonal antibodies (Mabs) to the gag region proteins was generated. Synthetic HIV-1 peptides covering the entire gag region were used to specifically localize the continuous epitopes by direct binding to the Mabs and by blocking the Mab immunoreactivity. The identified immunogenic epitopes were localized between the gag amino acids (aa) 108-127, 203-217, 208-222, 248-282, 273-302, 288-307, 308-322, 331-354 and 408-422. These continuous epitopes formed seven immunogenic regions. One strongly p17-reactive Mab appeared to react with a discontinuous epitope, the components of which were 110 aa distant in the linear sequence: aa 23-27 and 128-132. The synthetic peptides appeared to be more congruent with the Mab-reactive sites in solution than when coated to a solid phase.     

Serologic confirmation of simian T-lymphotropic virus type I infection by using immunoassays developed for human T-lymphotropic virus antibody detection.

Serum specimens from diverse species of Old World monkeys, categorized as seropositive (n = 97) or seronegative (n = 23) for human T-lymphotropic virus (HTLV) infection, were tested by using recombinant env-spiked Western immunoblot assays and synthetic peptide assays for simultaneous detection and discrimination of simian T-lymphotropic virus (STLV) infection. Of the 97 seropositive specimens, 93 reacted with the recombinant transmembrane (r21env) protein and 90 reacted with a recombinant, MTA-1, derived from the central region of the external glycoprotein of HTLV-I (rgp46env), thus yielding test sensitivities of 96 and 93%, respectively. While 1 of the 23 negative monkey specimens reacted with r21env, none reacted with rgp46env, for overall specificities of 96 and 100%, respectively. Analysis of synthetic peptide-based immunoassays demonstrated that while 85 of 97 (88%) seropositive specimens reacted with HTLV-I-specific epitope (p19gag), none of the specimens reacted with HTLV-II-specific epitope (gp52env). These results show that recombinant envelope-spiked Western blots provide a simple means for serologic confirmation of STLV-I infection and that type-specific synthetic peptides can be used to confirm the virus type in seropositive monkey specimens.     

Fine specificity of IgG subclass response to group antigens in HIV-1-infected patients

There is an association between the clinical stage of HIV-1 infection and the presence of antibodies against viral gag proteins (p17 and p24). The IgG subclass (G1 and G3) pattern against these antigens was analysed in stable patients and HIV patients progressing to AIDS. Antibodies were analysed with whole viral or peptide ELISA (using sequentially overlapping peptides) and Western Blots. IgG1 was found to be the dominant anti-HIV-1 IgG subclass and IgG1 antibodies declined in progressing patients against all HIV antigens evaluated in Western blot, including p17, p24, p31, gp41, p64, gp120 and gp160. In contrast IgG3 antibodies, which were found to be predominantly directed against gag proteins, and which could be detected in almost all patients, remained in the circulation during disease progression. By peptide assays distinct immunogenic regions were found in p17 in contrast to more evenly distributed epitopes in p24. A decreased divergence of antibody reactivity to both p17 and p24 peptides in the group of patients who developed AIDS was seen. No reaction to any single gag epitope related to disease progression. The difference between IgG1 and IgG3 anti-gag antibodies in relation to clearance during disease progression may depend on different properties of immune complexes formed by these two IgG subclasses.     

An analysis of blood sera by the immunoblot method using natural and recombinant antigens of the human immunodeficiency virus

A recombinant antigen produced in CV-I cells infected with vaccinia virus vC5 carrying the HIV-I gag gene was used to test sera. This antigen (rp50) reacted with 95 serum specimens shown to have anticore antibodies by immunoblot based on natural HIV antigens. Six sera from blood donors positive in ELISA contained antibodies to p17, p24, or p55 by natural antigen-based blot. All these sera did not react with rp50. These patients did not belong to any known risk groups and showed no dynamics in the immunoblot pattern. We consider the reaction of their serum samples as false positive. We believe that the recombinant antigen rp50 may be used for verification of positive ELISA results.     

Evaluation of the Gen-Probe Aptima HIV-1 RNA qualitative assay as an alternative to Western blot analysis for confirmation of HIV infection

The Gen-Probe Aptima HIV-1 RNA qualitative assay was evaluated as an alternative to Western blot analysis for the confirmation of HIV infection using serum samples that were repeatedly reactive for HIV antibodies. The Aptima HIV assay readily discriminated between HIV-1-infected and -uninfected individuals and effectively reduced the number of indeterminate results relative to Western blot analysis.     

Molecular epidemiology of HIV-1 strains in the Moscow Region

The Moscow Region is one of the HIV-1-affected subjects of the Russian Federation; there were 34613 HIV-1-infected subjects as of October 31, 2009. To characterize the molecular epidemiology of HIV-1 in the Moscow Region, the investigators obtained and studied HIV-1 variants from 61 infected subjects of the region, who were major risk groups: intravenous drug users (IDUs) and hetero- and homosexually infected persons. Genetic analysis of HIV-1 variants was carried out by sequencing the gag genes (729 nucleotides in length, including full-length protein p17 and partial p24) andlor env (270 nucleotides in length, V3 region) with further phylogenetic analysis. The findings demonstrated that HIV-1 subtype A variants are dominant in the Moscow Region and detectable in 93.5% of IDUs and 100% of heterosexually infected persons. Phylogenetically (and accordingly epidemiologically) unrelated HIV-1 subtype B strains were revealed in 4 patients, including 2 IDUs.