An inhibition enzyme immunoassay using a human monoclonal antibody (K14) reactive with gp41 of HIV-1 for the serology of HIV-1 infections

An inhibition enzyme immunoassay (IEIA), using a human monoclonal antibody (K14) reactive with gp41 of HIV-1, was evaluated for its applicability to the serology of HIV-1 infections. Using panels of serum samples from seronegative and confirmed HIV-1-seropositive individuals, it was shown that all the HIV-1-positive samples in a panel from The Netherlands and 97% of the HIV-1-positive samples from Tanzania were identified by this IEIA. Six per cent of the IEIA-positive samples from Tanzania could not be confirmed in other assays. Testing of serial dilutions of serum samples from African individuals with confirmed HIV-1, HIV-2 or HIV(ANI70) infections in the K14 IEIA, indicated that a HIV-1-specific assay based on this principle may be developed.     

HIV-1B/C重组毒株gp160假病毒的构建及其活性检测

目的以CD4+T细胞DNA为模板,进行gp160扩增,构建艾滋病病毒Ⅰ型(HIV-1)前病毒假病毒,通过中和试验验证其具有感染活性。方法选择高效抗反转录病毒治疗(HAART)成功的病人3例,分离外周血单核细胞(PBMC),纯化CD4+T细胞,提取DNA,以其为模板扩增gp160全长基因,并克隆到pcDNA3.1(+)表达载体上,酶切验证得到阳性克隆。将此阳性克隆和pNL4-3质粒共转染,获得HIV-1假病毒。用免疫兔血清验证假病毒的感染活性。结果成功地获得了1株假病毒。用免疫后的兔血清测定半数抑制浓度(IC50)的滴度约在1∶90~1∶270之间。病毒的加入量与细胞感染率之间存在良好的线性关系,说明该假病毒感染系统可以通过细胞感染率较好地反映出感染性病毒的含量。结论获得了具有感染活性的HIV-1B/C重组型假病毒。     

Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler<Superscript>®</Superscript> Real-time PCR

Background

The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler^® real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the β-globin gene.

Methods

The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the β-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI).

Results

The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 10⁶ HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The β-globin reference gene was quantified down to a limit of 1.5 pg of DNA/μl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0,012).

Conclusion

We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs.

     

Early diagnosis of paediatric HIV-1 infection among African breast-fed children using a quantitative plasma HIV RNA assay

OBJECTIVE: To evaluate the performance of a quantitative plasma HIV-1 RNA assay for HIV infection diagnosis among African breast-fed children. METHODS: Serial plasma specimens collected in the first week, at day 45-90, 6 months and 9-12 months of age from HIV-exposed children born to HIV-1-infected women enrolled in the DITRAME ANRS 049a perinatal intervention trial (Abidjan, C?te d'Ivoire) were tested for HIV-1 plasma RNA using a branched DNA (bDNA) assay. Sensitivity and specificity of this RNA test were assessed in comparison with a qualitative DNA polymerase chain reaction (PCR) performed on the same blood samples and allowing a reliable detection of the predominant subtype A. RESULTS: Among 91 samples from 53 infected children which tested positive by DNA PCR, the sensitivity of the bDNA test was 100% [95% confidence interval (CI), 96.0-100.0] at < or = 8 days (n = 19), 6-12 weeks (n = 43), 6 months (n = 26), and 9-12 months (n = 3). The median plasma HIV-1 RNA viral load ranged from 242 000 copies/ml at < or = 8 days to more than 500 000 copies/ml at day 45-90 and at 6 months. Of 106 specimens from 106 uninfected children who were DNA PCR- negative at month 3 or 6 of age, HIV-1 RNA was undetectable in 103, yielding an overall specificity for the bDNA test of 97.2% (95% CI, 92.0-99.4). The viral load in the three remaining samples with false-positive results was low (410, 937 and 3752 copies/ml, respectively). CONCLUSIONS: The quantitative bDNA assay appears a suitable tool for early, reliable and easy diagnosis of paediatric HIV-1 infection among a population of African breast-fed children.     

HIV and HTLV infections in 1305 transfusion-dependent thalassemics in Italy. The COOLEYCARE Cooperative Group.

OBJECTIVE: To evaluate the prevalence of antibodies to HIV-1/2 and HTLV-I/II in 1305 transfusion-dependent beta-thalassemics treated in 36 centres in Italy. DESIGN: Patient serum samples were collected during 1990 and tested in Milan. METHODS: Sera were screened using an enzyme-linked immunosorbent assay (ELISA) containing viral lysate antigens from HIV-1 and HIV-2, and a particle agglutination assay for the detection of antibodies to HTLV-I and HTLV-II. Repeatedly reactive samples were examined by Western blot (WB) assays containing recombinant and viral lysate antigens. Differential diagnosis was finally made by ELISA based on synthetic peptides. RESULTS: Samples from 36 of the 1305 patients (2.76%) contained anti-HIV-1 antibodies. In four patients seroconversion occurred after the implementation of anti-HIV-1 screening in blood donors in Italy (1985). Of the 36 HIV-1-antibody-positive samples, four were HIV-2 [corrected] WB indeterminate. These four samples were negative in assays based on specific synthetic peptides, suggesting cross-reactivity. Anti-HTLV-I antibodies were found in two patients from Sicily and one from Apulia, both southern Italian regions. Anti-HTLV-II antibodies were detected in another patient from Sicily. CONCLUSIONS: Antibodies to HIV-1, HIV-2, HTLV-I and HTLV-II were detected in 2.76, 0, 0.23 and 0.08% of patients, respectively. The residual risk of HIV-1 infection through blood transfusion after the implementation of anti-HIV-1 screening in blood donors in Italy was approximately 1:50,000 blood units; this is based on an approximate number of 200,000 blood units administered to our group of patients during 1986-1990 and the occurrence of four new anti-HIV-1 seroconversions. Seroconversions to HTLV-I/II suggest that these viruses are present in Italian blood donors.     

No evidence for recombination between HIV type 1 and HIV type 2 within the envelope region in dually seropositive individuals from Senegal

To investigate the frequency of recombination between HIV-1 and HIV-2 in vivo during dual infection, we performed a retrospective analysis of blood samples from 46 dual HIV-1/HIV-2-seropositive adults for evidence of recombination. HIV viral DNA from peripheral blood mononuclear cells (PBMC) was subjected to two separate nested polymerase chain reaction (PCR) assays using opposing HIV-1 and HIV-2 primer pairs selected to flank a approximately 650-base pair region including the V3 loop of the envelope gene. In the first assay, primers were chosen to amplify recombinants with HIV-1 on the 5' end and HIV-2 on the 3' end, and in the second assay, primers were chosen to amplify recombinants with the opposite orientation. All PCR experiments were run in parallel with positive controls consisting of partial-length env fragments bearing a single central HIV-1/2 recombination site, and appropriate primer-binding sites on each end. The limit of detection for both assays was <10 copies of recombinant product per 150,000 cell equivalents of input PBMC DNA. In all 46 dually seropositive patients in this study, PCR screening of PBMC failed to detect evidence of HIV-1/HIV-2 recombinants in the C2-V5 env region. Although genetic recombination between HIV-1 and HIV-2 may occur, we conclude that any such events within env are exceedingly rare, and do not result in the outgrowth of recombinant strains.     

Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis

Serum and plasma samples were collected from blood donors who were confirmed positive for antibodies to HIV-1 in the United States, and from blood donors and individuals in West Africa and Portugal who were positive for antibodies to HIV-1, HIV-2, or both. Western blots and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) radioimmunoprecipitation assays (RIPA) utilizing native HIV-1 and HIV-2 proteins were performed on these specimens to determine the ability of these procedures to discriminate between HIV-1 and HIV-2 infections. Extensive serologic cross reactivity between HIV-1 and HIV-2 p24 was found in both populations. Antibody reactivity to the envelope protein gp120 was able to discriminate 20 of 20 (100%) U.S. specimens as HIV-1 infections. In specimens from West Africa and Portugal, Western blot and RIPA were in complete agreement on 33 of 42 samples (78.6%). Among these 33 specimens, 10 were found to be reactive for antibodies to HIV-1 only, 10 were reactive to HIV-2 only, and 13 were considered to be dually reactive, having antibodies reactive with both HIV-1 gp120 and HIV-2 gp120. Nine of the 42 specimens were discordant by Western blot and RIPA classification, being dually reactive by one procedure and reactive with only one viral gp120 by the other technique. Because of the serological cross reactivities between HIV-1 and HIV-2, in certain populations it is difficult to ascertain whether an individual is infected with HIV-1, HIV-2, a new viral type, or whether the individual is infected simultaneously with multiple viruses. More specific tests such as viral isolation or molecular probes may be necessary to distinguish between infections with these viruses in certain populations.     

Analytical and clinical sensitivity of the Procleix Ultrio HIV-1/HCV/HBV assay in samples with a low viral load

BACKGROUND AND OBJECTIVES: The Procleix Ultrio human immunodeficiency virus type 1 (HIV-1)/hepatitis C virus (HCV)/hepatitis B virus (HBV) (Ultrio) assay simultaneously detects HIV-1 RNA, HCV RNA and HBV DNA in individual blood donations. The main objective of the study was to assess the analytical and clinical sensitivity of the multiplex and discriminatory probe assays in samples with a low viral load. MATERIAL AND METHODS: The VQC HIV RNA genotype B, HCV RNA genotype 1 and HBV DNA genotype A standard dilutions were tested in 26 repeats. The probability of detection by Ultrio was compared with previously obtained data of the Procleix Duplex HIV-1/HCV assay on the same reference panels. A selection of 121 anti-HIV-1, 138 anti-HCV and 190 HBsAg positive samples from patients receiving antiviral therapy were tested. The majority of patient samples had a viral load below the detection limit of the diagnostic nucleic acid test assays, which made them suitable to evaluate the performance of the multiplex and discriminatory assays on yield cases with a similar low viral load. RESULTS: The 95% and 50% detection end-points of the Ultrio assay along with the corresponding 95% confidence intervals are 53.7 (32.9-117.2) and 8.6 (6.2-12.1) geq/ml for HIV-1 RNA, 30.3 (19.0-62.4) and 5.2 (3.7-7.2) geq/ml for HCV RNA and 393.7 (147.9-6978) and 54.5 (22.4-143.8) geq/ml for HBV DNA. The analytical sensitivity of Ultrio expressed as a potency factor relative to previously obtained Duplex results on the same HIV-1 RNA and HCV-RNA standard dilutions was 1.09 (0.20-6.10) and 1.11 (0.21-5.89), respectively. The assay detected all 22 HIV-1 infected patients with viral load > 50 copies/ml, and 41 of 99 patients (41%) with viral load < 50 copies/ml, of which 23 (56%) were detected by the discriminatory assay. All 47 patients with HCV RNA load > 521 IU/ml and 10/91 polymerase chain reaction-negative patients with viral load < 50 IU/ml tested positive in Ultrio assay of which five were missed in the discriminatory test. The assay detected 53/55 HBV infected patients (96%) with viral load > 250 copies/ml and 108/135 patients (80%) with viral load < 250 copies/ml of which 17 (16%) were missed by the discriminatory test. CONCLUSIONS: The new Procleix Ultrio assay is as sensitive as the Procleix Duplex assay for HIV-1 and HCV detection meeting the requirements of universal guidelines. The ability of the assay to detect HBV DNA in low viral load samples could be useful for screening blood. Inevitable negative results of discriminatory probe assays caused by stochastic sample variation will reduce the chance of recognizing low viraemic blood donors detected by individual donation nucleic acid test.     

HIV type 1 subtypes among blood donors in the Mbeya region of southwest Tanzania

HIV-1 is endemic in Tanzania where three different subtypes, A, C, and D, have been identified. Information on HIV-1 genetic diversity is crucial to define requirements for an effective vaccine, in regions where HIV-1 vaccine trials are planned. To define the subtype distribution of HIV-1 in the Mbeya region of southwest Tanzania, peripheral blood mononuclear cells (PBMC) and plasma were obtained from 36 discarded HIV seropositive blood units. Multiregion hybridization assay (MHA) was performed on both PBMC DNA and plasma RNA to determine the subtype distribution. Twenty virtually full-length HIV-1 sequences were amplified from the extracted DNA, sequenced, and phylogenetically analyzed. Subtype distribution determined by all three assays was comparable. More than 50% of the samples analyzed were subtype C, followed by a high proportion of subtype C-containing intersubtype recombinants. Based on this work, subtype C appears to be the prevalent subtype in southwest Tanzania, followed by a high proportion of intersubtype recombinants.     

HIV-1 superinfections in a cohort of commercial sex workers in Burkina Faso as assessed by an autologous heteroduplex mobility procedure

OBJECTIVE: To describe and evaluate a simple procedure to identify HIV-1 co- or super-infections based on a heteroduplex mobility assay (HMA). METHODS: To identify heteroduplexes corresponding to divergent viral populations in a the same individual, HMA was applied to single DNA samples from each subject in a prospective cohort of commercial sex workers (CSW) in Bobo-Dioulasso, Burkina Faso. After denaturation and renaturation of env DNA amplicons, hybridized DNA was separated by electrophoresis through polyacrylamide gel. HIV-1 co-infections were suspected by slow migration of heteroduplex, at a level comparable to that of mixed reference strains. Following electrophoresis, DNA in bands representing heteroduplex was extracted and cloned in a plasmid vector; identification of phylogenetically distinct clones was confirmed by sequencing divergent clones identified through a second HMA step of a pair of two mixed clones. RESULTS: Among 147 HIV-1 infected CSW, four had an autologous HMA profile comparable to low mobility of hybridized DNA from mixed reference strains representing most frequent HIV-1 clades and circulating recombinant forms (CRF) prevalent in Burkina Faso. In two of them, two phylogenetically distinct HIV-1 populations were coexisting. The first woman presented with a CRF02-AG and CRF06-cpx co-infection, and the second with a CRF02-AG and a divergent virus co-infection not significantly related to any other known subtypes. In both women, retrospective analyses of stored samples by the same HMA procedure showed acquisition of a second virus concomitent with an increasing plasma HIV RNA. CONCLUSIONS: Autologous HMA procedure followed by acrylamide extraction of heteroduplexes allowed identifying HIV-1 co- and super-infections in a large cohort study. HIV-1 super-infection is not an uncommon phenomenon.     

HIV-1 DNA is detected in bone marrow populations containing CD4+ T cells but is not found in purified CD34+ hematopoietic progenitor cells in most patients on antiretroviral therapy

Identifying cellular reservoirs of human immunodeficiency virus type 1 (HIV-1) in patients on antiretroviral therapy (ART) is critical to finding a cure for HIV-1. In addition to resting CD4(+) T cells, CD34(+) hematopoietic progenitor cells have been proposed as another reservoir. We obtained bone marrow aspirates from 11 patients on ART who had undetectable plasma HIV-1 RNA. HIV-1 DNA was detected in CD4(+) T cells from peripheral blood in all patients and from bone marrow cellular fractions containing T cells in most patients. We did not find HIV-1 DNA in highly purified CD34(+) populations using either a sensitive real-time polymerase chain reaction assay or a coculture assay for replication-competent HIV-1.     

Study of HIV type 1 gag/env variability in The Gambia, using a multiplex DNA polymerase chain reaction

A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants.     

Analysis of length variation in the V1-V2 region of env in nonsubtype B HIV type 1 from Uganda

We optimized an assay for analysis of length variation in the V1-V2 region of HIV-1 env in plasma samples from Uganda. V1-V2 env length variation was analyzed in 31 plasma samples containing subtype A, C, D, or A/D recombinant HIV-1. DNA corresponding to the V1-V2 region was amplified by nested PCR. One of the primers in the second step of the PCR was fluorescently labeled. Successful amplification was confirmed by agarose gel electrophoresis. V1-V2 length variation of PCR products was analyzed with an ABI PRISM 3100 genetic analyzer and GeneScan software. A diversity score was generated for each sample on the basis of the degree of fragment length variation. The V1-V2 region was successfully amplified from 30 of 31 samples. Fragment length analysis was successful for all of those 30 samples. The diversity score and lengths of V1-V2 fragments were unique for each sample. This assay can be used for analysis of V1-V2 length variation in subtypes commonly found in Uganda. This assay may be helpful for studies examining the impact of env length diversity on HIV-1 transmission and pathogenesis in regions where these subtypes are prevalent.     

Absence of infectious human immunodeficiency virus type 1 in "natural" eccrine sweat.

Although human immunodeficiency virus type 1 (HIV-1) has been found in numerous body fluids, there are no reports of attempts to demonstrate this virus in eccrine sweat, a fluid frequently encountered during person-to-person interactions. "Natural" eccrine sweat samples and blood from 50 HIV-1-seropositive patients and 2 HIV-1-seronegative controls were cultured for HIV-1 by a cocultivation method. Polymerase chain reaction for HIV-1 RNA and proviral DNA was done on 40 sweat samples (39 patients, 1 control). HIV-1 was isolated from peripheral blood mononuclear cells of 39 (78%) of 50 patients but from none of 52 sweat samples. No HIV-1 viral DNA or RNA was detected in the 40 sweat samples tested. With present methodology, infectious HIV-1 cannot be demonstrated in "natural" eccrine sweat samples from HIV-infected patients.     

Rapid and specific diagnosis of HIV-1 and HIV-2 infections: an evaluation of testing strategies

To identify cost-effective testing strategies for HIV-1 and HIV-2 infections, we evaluated different combinations of tests on serum specimens from 1134 consecutive patients attending tuberculosis treatment centers in Abidjan, C?te d'lvoire. Virus-specific whole-virus enzyme-linked immunosorbent assay (WVE), Western blot (WB) and synthetic peptide enzyme-linked immunosorbent assay (SPE) were used in sequential fashion to determine the true prevalence of infection; 27% were reactive to HIV-1, 5% to HIV-2, and 10% to both viruses. Of 239 specimens positive on WB for both HIV-1 and HIV-2, SPE diagnosed 38% as HIV-1-reactive and 16% as HIV-2-reactive, while 46% remained reactive to both viruses. Using WVE or one of two rapid (5-10 min) mixed (HIV-1 and HIV-2) antigen tests (RMATs) as a screening test, followed by SPE as a supplemental test, gave results with sensitivity of 97.3-99.2%, specificity of 99.5-99.7%, and positive predictive value for diagnosing HIV infection of 99.4-99.6%, with important savings in time and reagent costs. SPE allows more specific distinction between HIV-1 and HIV-2 infections than WB, and could replace it as a supplemental test in many settings. WB may be required for specimens reactive on screening tests but negative on SPE, until sensitivity of the SPE is further evaluated. A mixed antigen screening test followed by SPE seems to be an efficient testing strategy for diagnosing HIV-1 and HIV-2 infections.     

Cellular HIV-1 DNA load predicts HIV-RNA rebound and the outcome of highly active antiretroviral therapy

OBJECTIVE: To assess whether cellular HIV-1 DNA prior to highly active antiretroviral therapy (HAART) initiation predicts its outcome. DESIGN AND METHODS: Patients included all 51 hemophiliacs of the Greek component of the Multicenter Hemophilia Cohort Study who had initiated HAART and for whom cryopreserved lymphocyte samples before HAART initiation were available. Cellular HIV-1 DNA quantification was performed by a molecular beacon-based real-time PCR assay in multiple samples per patient with a median (interquartile range) follow-up of 76 (45-102) weeks. RESULTS: The median (range) baseline HIV-1 DNA load was 297 (< 10 to 3468) copies per 1 x 10(6) peripheral blood mononuclear cells. Baseline HIV-1 DNA load did not predict initial virological response (VR). None of the patients with initial VR and baseline HIV-1 DNA load at or below the median experienced a subsequent virological rebound, while the cumulative probability of virological rebound by week 104 was 55% among those with HIV-1 DNA load greater than the median (P < 0.008). Cellular HIV-1 DNA load was the only parameter associated with sustained virological response as shown by univariate or multivariate analyses [adjusted odds ratio (95% confidence interval) 0.197 (0.048-0.801) per 1 log10 increase in DNA copies, P = 0.023]. CONCLUSION: Low cellular HIV-1 DNA load is a marker of sustained virological response in patients with initial VR and it can reliably predict the long-term success of HAART.     

Quantitation of HIV viral burden by PCR in HIV seropositive Navy personnel representing Walter Reed stages 1 to 6.

To quantitate the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells (PBMC) of 78 infected individuals, we have developed a polymerase chain reaction (PCR) assay that is both quantitative and sensitive. Quantitation was based on incorporation of a 32P end-labelled primer (SK39) in the PCR reaction and on comparison after electrophoresis with known amounts of HIV DNA. A linear relationship was obtained between the natural logarithms of the radioactive counts detected and the number of HIV-1 DNA copies (10-1000 copies) from the standard DNA. HIV copy numbers from patient samples were then extrapolated from the standard curves. This sensitive and reproducible method was compared with virus isolation which is a semiquantitative evaluation of viral burden. HIV DNA levels correlated with virus isolation, i.e., high viral burden (100-1000 HIV copies) were found in most samples from which virus was isolated after only 7 days in culture; low viral burden (less than 100 HIV copies) was observed in samples from which virus was isolated after 14 to 21 days in culture. These estimates of viral burden were then compared with the clinical stage of the individuals.     

HIV-2 infection in an American

HIV-2 is endemic in West Africa but rare elsewhere. In the USA there have been 18 reported cases of HIV-2 infection; most identified people have been West Africans. We recently diagnosed the first case of HIV-2 infection in a native-born US citizen, a woman whose serum was found to be reactive to anti-HIV-1 enzyme immunoassay (EIA) when she attempted to donate blood in 1986. Although both HIV-1- and HIV-2-specific EIAs were reactive, the anti-HIV-2 Western blot (WB) was positive, while the anti-HIV-1 WB was positive or indeterminate on different occasions. Synthetic peptide testing was reactive for HIV-2 but not HIV-1. HIV-2 DNA was detected using the polymerase chain reaction procedure. Although she had travelled to West Africa, it is unclear how she became infected with HIV-2.