Blocking-ELISA for detection of specific antibodies to the glycoproteins of the human immunodeficiency virus type 1 (HIV-1)

A blocking ELISA was developed to confirm the specificity of screening tests for anti-HIV-1 antibodies. A murine monoclonal antibody (McAb) raised against recombinant gp160 was used in combination with a commercial technique (ELA-VIA-1). After determining the optimal experimental conditions, the assay was applied to 92 samples presenting different reactivities by Western blot (WB) analysis. All the sera containing antibodies to gp160/gp120 (53) were positive in our assay. The six patients who sero converted showed a low positivity by ELAVIA-1 (optical density near the cutoff value) reacted by blocking-ELAVIA-1 with an McAb binding inhibition greater than 85%. By contrast, negative samples (29) and specimens that exhibited reactivity only against gag-proteins (10) were not detected (McAb binding inhibition smaller than 15%). This sensitive and specific blocking-ELAVIA-1 represents a convenient alternative to WB as a confirmatory test. The technique is time-saving and inexpensive and can easily be integrated with a screening test for diagnostic or epidemiologic studies on HIV-1 infection.     

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).     

An ultrastructural observation of negatively-stained human immunodeficiency virus type 1 (HIV-1) cores

Cores of human immunodeficiency virus type 1 (HIV-1) were observed using negative staining for electron microscopy. On the surfaces of cores isolated from viral particles with a Nonidet P40 and glutaraldehyde mixture, mortar-like units were detected in the wide and narrow margins. In some of the cores which were burst with distilled water, the units were linked together revealing a beaded structure.     

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.     

前列腺素A1体外对人免疫缺陷病毒1型的复制作用

在感染人免疫缺陷病毒１型的人Ｔ细胞ＣＤ４的ＣＥＭｓ细胞中，应用核酸杂交法检测病毒ＲＮＡ水平，观察前列腺素Ａ１对人免疫缺陷病毒１型复制作用的影响。结果表明，在低感染多重性下，用前列腺素Ａ１处理组，病毒ＲＮＡ水平在病毒感染７２～９６小时明显低于对照组，感染２４小时后给予前列腺素Ａ１，仍可抑制病毒ＲＮＡ。连续用前列腺素处理，其抑制作用达９天。     

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: effects on virion morphogenesis and RNA maturation

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag–Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC₉₀), most of the Gag and Gag–Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.     

Perinatal infection by human immunodeficiency virus type 1 (HIV-1): relationship between proviral copy number in vivo, viral properties in vitro, and clinical outcome.

Human immunodeficiency virus type 1 (HIV-1) isolates from 25 perinatally HIV-1 infected children were classified according to their capacity to replicate in vitro as rapid (R), intermediate (S/R) and slow (S) variants. R-type viruses replicated on peripheral blood mononuclear cells (PBMCs) and grew better in T-lymphoid cells, even though 9 out of 12 isolates also maintained tropism for monocytoid cells. The S/R-type isolates replicated efficiently after several days of culture, while the S-type viruses displayed only a low and transient replication activity; however, both S/R- and S-type isolates exerted viral transactivation activity in an indicator monocytoid cell line. Replication patterns in vitro were significantly associated in vivo with the number of HIV-1 copies in PBMCs as determined by polymerase chain reaction: in children with R-type isolates, the number of HIV-1 proviral DNA molecules/10(5) PBMCs ranged from 62 to 571, and in children with S/R and S isolates the range was 5-43. Seven children had severe symptomatic HIV-1 infection, and in all an R-type virus was identified; 18 children had no or only mild symptoms, and among these, S-, S/R-, and R-type isolates were found in 5, 8, and 5 cases, respectively. Besides demonstrating HIV-1 variability in perinatal infection, these findings suggest that R-type virus might be a prerequisite for disease progression.     

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

A cell line (H2-5) producing defective doughnut-shaped particles of human immunodeficiency virus type 1 (HIV-1) was found to contain proviral DNA with a large deletion of 2558 bases, corresponding to the 3 half ofpol gene, the entirevif andvpr genes, and the 5 terminal of thetat gene.     

Characterization of human immunodeficiency virus type 1 Pr160 gag-pol mutants with truncations downstream of the protease domain

We have constructed a series of HIV-1 Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain. Effects of the truncation mutations on virus particle production and Gag particle processing were analyzed. Analysis indicated that removal of the integrase (IN) domain had no major effect on the efficiency of particle processing, but resulted in a marked reduction in virus particle budding. Deletion of both the IN and RNase H domains, however, restored the production of virus particles to wild-type level. The proteolytic processing of virus particle was significantly impaired when the p51RT domain was truncated. All of the truncated Gag-pol proteins could be incorporated into virus particles and demonstrated an immunofluorescence staining pattern similar to that of the wild type (wt). Our data are consistent with the proposal that signals for directing the Gag-pol transport and particle incorporation are determined by its N-terminal Gag domain. Truncated Gag-pol retaining an intact p51RT was able to complement a PR-defective mutant to produce infectious pseudotyped virions, with a virus titer 20-70% of that of wt. Pseudotyped virions produced by the Gag-pol lacking an intact p51RT were noninfectious or poorly infectious. This suggests that an intact p51RT domain is required for the Gag-pol to mediate production of mature infectious virus particles in trans.     

Expression of a functional p75 interleukin-2 receptor on lung lymphocytes from patients with human immunodeficiency virus type 1 (HIV-1) infection

Lung involvement in patients affected by HIV-1 infection is characterized by an alveolitis sustained by the accumulation of CD8+ T lymphocytes. To investigate whetherin situ T cell growth plays a relevant role in the pooling of CD8+ lymphocytes, we have analyzed the activity of two lymphokines involved in the mechanisms of T cell proliferation, i.e., interleukin-2 (IL-2) and interleukin-4. To this aim, following appropriate triggering and blocking, the expression and the functional role of IL-2 receptors (IL-2R) (both p55 and p75 chains) and IL-4 receptors have been analyzed on T lymphocytes obtained from the bronchoalveolar lavage (BAL) of 16 HIV-1+ patients. Molecular and phenotypic studies we performed demonstrated that CD8+ lymphocytes from the BAL of HIV-1 + patients strongly expressed the p75 chain of IL-2 receptor, while neither p55 mRNA nor its surface membrane product (Tac antigen) was detectable; in addition, there was no expression of IL-4 receptors. IL-2 stimulation was able to induce T cell growth in a dose-dependent manner, whereas IL-4 did not. Finally, using mAbs which specifically block the p55 or p75 IL-2R, we showed that both subunits of IL-2R were involved in the proliferative activity of lung lymphocytes. The results obtained in the present study directly demonstrate that BAL T lymphocytes of HIV-1 + patients express a fully functional IL-2 receptor apparatus, pointing to the role for this lymphokine in maintaining the alveolitis taking place in the lungs of AIDS patients.     

Characterization of human T-cell lines harboring defective human immunodeficiency virus type 1

Defective HIV-producing T-cell lines were subcloned from MT-4/HIV_HTLV-IIIB MOLT-4/HIV_HTLV-IIIB, and H9/HIV_HTLV-IIIB cell lines chronically infected with HIV. The NY-M10 cell line derived from MOLT-4/HIV_HTLV-IIIB and the NY-H6 cell line derived from H9/HIV_HTLV-IIIB produce defective HIV, which lacks the ability to infect human T-cell lines. NY-M10 cells retain the capacity to form multinucleated giant cells in cocultivation with HIV-uninfected CD4-positive cells. However, NY-H6 cells failed to fuse with CD4-positive cells. Electron microscopic analysis indicated that the defective HIV produced from NY-M10, like those reported previously, lacked the structure of the nucleocapsid, and the virion released from NY-H6 was indistinguishable from those of authentic HIV particles. Southern and Northern blotting analyses of NY-M10 and NY-H6 cleared that the genome of those defective viruses was not significantly deleted, suggesting minor mutation(s) should take place on the viral genome.     

国产HIV-1p24抗原检测试剂盒的评价

目的探讨国产的HIV-1 p24抗原检测试剂盒进行药物筛选研究的可行性。方法对国产试剂盒的使用性能与Biomerieux公司商品化的Vironostika试剂盒进行比较,评估国产试剂盒的敏感性、重复性及检验效能。结果国产试剂盒具有较高的敏感性和重复性,在体外药效学筛选实验中国产试剂盒的阳性检出率及药物评价结果与Vironostika试剂盒差异无统计学意义(P>0.05)。结论国产试剂盒具有较好的使用特性,在药物筛选中可以用国产试剂盒替代Vironostika试剂盒。     

Analysis of intracellular human immunodeficiency virus (HIV)-1 drug resistance mutations in multi-failed HIV-1-infected patients treated with a salvage regimen: 72-week follow-up

The human immunodeficiency virus (HIV) mutational archive of proviral DNA was monitored during a 72-week follow-up in 20 multidrug-experienced HIV-1-infected patients treated with a darunavir/ritonavir-based salvage therapy. At the beginning of the study, all patients harboured a number of intracellular drug resistance-associated mutations (RAMs) in peripheral blood mononuclear cells. In some patients, a significant fluctuation in the number of RAMs was observed during the observation period. However, all patients, notwithstanding the presence or the fluctuation of intracellular RAMs, showed a persistently undetectable viraemia. The data suggest that the archived resistant viral variants change during suppressive therapy, but that the variants are unable to re-emerge and to affect virological response.     

Evaluation of the new VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA

BackgroundThe VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of HIV-1 RNA has recently been introduced.ObjectivesIn this study, the performance of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0.Study designAccuracy, linearity, interassay and intra-assay variations were determined, and a total of 196 routine clinical samples including a high number of HIV-1 subtype non-B samples were investigated.ResultsWhen accuracy of the new kit was tested, all of the quantifiable results were found to be within ?0.5 log₁₀ unit of the expected panel results. Determination of linearity resulted in a quasilinear curve up to the initial concentration of 3.4 × 10⁵ copies/mL. The interassay variation ranged from 12 to 20%, and the intra-assay variation ranged from 8 to 16%. When clinical samples were tested by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, the results for 95% of all samples with positive results by both tests were found to be within ±1.0 log₁₀ unit. The viral loads for all samples measured by the Siemens and Roche assays showed a high correlation (R² = 0.94); quantitative results obtained by the Siemens assay were usually found to be lower than those obtained by the Roche assay.ConclusionsThe new VERSANT HIV-1 RNA 1.0 Assay (kPCR) proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.