HIV-1 Isolation Research Articles

Highly Efficient Autologous HIV-1 Isolation by Coculturing Macrophage With Enriched CD4+ T Cells From HIV-1 Patients.

We described a novel HIV autologous isolation method based in coculturing macrophages and CD4+T-cell-enriched fractions from peripheral blood collected from antiretroviral-treated (ART) HIV patients. This method allows the isolation of high viral titers of autologous viruses, over 1010HIV RNA copies/ml, and reduces the time required to produce necessary amounts for virus for use as antigens presented by monocyte-derived myeloid cells in HIV therapeutic vaccine approaches. By applying these high titer and autologous virus produced in the patient-derived cells, we intended to elicit a boost of the immunological system response in HIV therapeutic vaccines in clinical trials.

CREATURE A COLLECTION OF UP-TO-DATE HIV-1 ISOLATES INCLUDING MAJOR RUSSIAN GENETIC VARIANTS OF VIRUS

Study objectives: Fine-tuning of techniques for HIV-1 isolation from HIV patient blood samples; developing a collection of primary HIV-1 isolates; and investigating the biological features of currently circulating genetic variants of HIV-1 subtypes A and B and of their recombinant form CRF63_02A1. These objectives are important for better knowing of HIV-1 biology and for monitoring the characteristics of the spreading HIV-1 variants. Materials and methods: PBMC were isolated by whole blood centrifuging at 325 g for 30 min in 1,077 to 1,080 g/mL Ficoll gradient (MP Biomedicals, USA) followed by two washings with RPMI-1640. Donor PBMC were stimulated in RPMI-1640. The culture fluid was sampled, tested for viral p24 protein, and cryopreserved. HIV-1 reproduction was assessed by the accumulation of the viral protein in culture medium using an ELISA kit provided by Vector Best (Russia). Viral DNA was isolated from HIV patient blood plasma using RealBest DeltaMag kits and RT-PCR MasterMix kits (Vector Brest, Russia) and PCR BioMaster HS-Tag PCI-Color kits (Bilabmix, Russia). HIV-1 isolated were characterized genetically by sequencing env and pol genes using BigDyeTerminatorv31 kits (USA). Sixty-seven infectious variants of HIV-1 subtype A and B recombinants CRF63_02A1 and CRF02AG strain have been isolated and characterized.

Isolation and Biological Characterization of Non-B HIV Type 1 from Kenya

The isolation and characterization of primary strains of human immunodeficiency virus (HIV) is a vital tool for assessing properties of viruses replicating in HIV-infected subjects. HIV-1 isolation was carried out from 30 HIV-1-infected patients from a Comprehensive Care Clinic (CCC) after informed consent. Virus was successfully isolated from 9 out of the 30 samples investigated. Seven of the isolates were from drug-naive patients while two were from patients on antiretroviral drugs. The isolates were biologically phenotyped through measurement of the syncytium-inducing capacity in MT2 cells. Six of the isolates exhibited syncytia induction (SI) associated with CXCR4 coreceptor usage while three of the isolates were non-syncytia-inducing (NSI) isolates associated with CCR5 coreceptor usage. In addition, the replication capacity of the isolates was further determined in established cell line CD4(+) C8166. Indirect immunofluorescence assay was used to check the antigen expression on the cells as a supplementary test. HIV-1 isolation success was 70% (7/10) and 20% (2/20) in naive and drug-experienced patients, respectively. The majority of the viral isolates obtained (6/9) were of the SI phenotype, though SI virus strains are rare among non-B subtypes. A significant correlation between virus isolation success and viral load was established. Coreceptor use data for heavily treatment-experienced patients with limited treatment options are scanty and this is the group with perhaps the most urgent need of novel antiretroviral agents.

Ex vivo production of autologous whole inactivated HIV-1 for clinical use in therapeutic vaccines

This study provides a detailed description and characterization of the preparation of individualized lots of autologous heat inactivated HIV-1 virions used as immunogen in a clinical trial designed to test an autologous dendritic-cell-based therapeutic HIV-1 vaccine (Clinical Trial DCV-2, NCT00402142). For each participant, ex vivo isolation and expansion of primary virus were performed by co-culturing CD4-enriched PBMCs from the HIV-1-infected patient with PBMC from HIV-seronegative unrelated healthy volunteer donors. The viral supernatants were heat-inactivated and concentrated to obtain 1 mL of autologous immunogen, which was used to load autologous dendritic cells of each patient. High sequence homology was found between the inactivated virus immunogen and the HIV-1 circulating in plasma at the time of HIV-1 isolation. Immunogens contained up to 10 9 HIV-1 RNA copies/mL showed considerably reduced infectivity after heat inactivation (median of 5.6 log 10), and were free of specified adventitious agents. The production of individualized lots of immunogen based on autologous inactivated HIV-1 virus fulfilling clinical-grade good manufacturing practice proved to be feasible, consistent with predetermined specifications, and safe for use in a clinical trial designed to test autologous dendritic cell-based therapeutic HIV-1 vaccine.

V3 Sequences and Paired HIV Isolates from 52 Non-Subtype B HIV Type 1-Infected Patients

Viral isolation and V3 sequencing were performed for 52 patients with non-subtype B viruses. The HIV-1 isolation rate was 93%, and 98% of isolates were characterized as NSI. V3 sequences corresponding to NSI isolates were compared to non-subtype B sequences with corresponding SI isolates from the Los Alamos database. The two sequence groups significantly differed in number of sequences with 35 amino acids, net charge, Brigg's coefficient, loss of NGS at positions 6-8, and 11/25 genotype (p < 0.0001). Substantial discrepancies in V3 variability were also observed. Basic amino acids at positions 8, 21, 23, and 24 were more frequent in SI sequences as were uncharged amino acids at positions 5, 6, 7, 8, 12, 13, 25, and 34. When characterizing paired viral isolates and V3 sequences from patients with non-subtype B HIV-1, current V3 sequence-based criteria from subtype B appeared to discriminate well between NSI and SI sequences from non-subtype B patients.

A novel, rapid method to detect infectious HIV-1 from plasma of persons infected with HIV-1

Efficient isolation of replication-competent virus from plasma of patients infected with HIV-1 is needed to characterize important clinical parameters of virus. However, addition of plasma to in vitro cultures results in clot formation. Blood from HIV-1 infected patients was collected in the presence of three commonly used anticoagulants (ACD, heparin and EDTA) and plasma was isolated. Plasma was then used to infect HIV-1 indicator cell lines (TZM-bl and GHOST) with spinoculation in the presence or absence of additional heparin and positively charged polymers. The presence of additional heparin during inoculation significantly reduced clot formation without affecting the sensitivity of HIV-1 infection in the GHOST cell line. However, heparin reduced the frequency of HIV-1 infection of the TZM-bl cell line. Using plasma from patients with HIV RNA > 1000 copies/ml ( n = 58), the frequency of HIV-1 isolation was 92% in GHOST ( n = 51) and 54% in TZM-bl ( n = 26) cell lines. A sensitive method was developed for rapid isolation of infectious HIV-1 from plasma of patients with HIV RNA > 1000 copies/ml that includes spinoculation and the addition of heparin during infection of GHOST cells. This technique could be used for rapid evaluation of viral fitness, co-receptor usage or drug resistance without the need for viral amplification.

Discordant response to antiretroviral therapy: HIV isolation, genotypic mutations, T-cell proliferation and cytokine production.

To study virologic and immunologic factors associated with discordant treatment response in HIV-infected patients receiving highly active antiretroviral therapy (HAART). Study participants included a total of 27 patients: (a) 10 discordant patients (mean CD4+ cell count, 396.1 x 10 cells/l; mean HIV-RNA, 5.4 log copies/ml); (b) seven responder patients (mean CD4+ cell count, 997.5 x 10 cells/l); and (c) 10 failing patients (mean CD4+ cell count 66.5 x 10 cells/l; mean HIV-RNA, 5.4 log copies/ml).(10) (10) The HIV-1 isolation rate and biological phenotype, drug resistance genotypic mutations of HIV-1 strains, recall and HIV-1-specific antigen lymphocyte proliferation (LP), and interleukin (IL)-15 production were studied. Virus isolation was obtained in 30% of discordant patients, and in 100% of failing patients. A higher replication constant was reported in discordant patients. No difference in the number of drug resistance mutations and biological phenotypes of HIV-1 was found in discordant patients with respect to failing patients. Discordant patients developed positive LP responses to and HIV-1 p24. LP in response to, HIV-1 p24 and gp160 was positive in responder patients. No significant LP was found in failing patients. Increased levels of IL-15 after stimulation with lipopolysaccaride (LPS) and were found in both discordant patients and responder patients. Conversely, a strong reduction of IL-15 levels was observed in failing patients. The present results suggest that decreased virus isolation rate, restoration of both lymphocyte proliferation and IL-15 production are factors involved in the discordant antiretroviral therapy response of HIV-infected patients.

Vertical HIV‐1 Transmission: Importance of Neutralizing Antibody Titer and Specificity

Neutralization analyses were carried out with plasma from 132 volunteer human immunodeficiency virus (HIV)-1 infected women (76% pregnant, 24% with infants suspected for HIV-1 infection) collected between 1994 and 1998, against autologous and heterologous primary- and the reference HIV-1 MN isolates. A significantly lower percentage of HIV-1 transmissions was observed after 1996, parallel to a more intense antiretroviral treatment of infected pregnant women. HIV-1 isolation was significantly more frequent from peripheral blood mononuclear cells of mothers of infected children than mothers of uninfected children (P = 0.0065). Neutralization of autologous HIV-1 isolates was comparable for HIV-1 transmitters and nontransmitters' plasma, whereas neutralization of the reference isolate HIV-1 MN was more frequent at high titers for pregnant women who did not transmit HIV to their offspring compared to pregnant women who did. Although neutralization of heterologous primary HIV-1 isolates from HIV transmitters and non transmitters by transmitter plasma occurred with similar frequency, neutralization of isolates from transmitters was much more frequent when heterologous plasma from nontransmitters were used. Macrophage-tropic heterologous HIV-1 isolates were neutralized more frequently at higher titers by plasma from nontransmitters than from transmitters. The results obtained indicate that antiretroviral treatment, lack of success of HIV-1 isolation and high titers of antibodies able to neutralize macrophage-tropic viruses appear to be of importance for protection against HIV-1 vertical transmission for the group of patients studied.

Decline in the HIV-1 isolation rate in Japan: a 12-year observation.

Since 1988, we have isolated HIV-1 from 614 HIV-1-infected persons (total sample=2,785) in Japan. During the past 12 years, we have found a decline in the HIV-1 isolation rate in Japan, with two identifiable turning points, 1991-1992 and 1996-1997. The two turning points correspond to shifts in anti-HIV-1 therapy. These findings suggest that HIV-1 in Japan is currently biologically well controlled, probably due to anti-HIV-1 therapy. On the other hand, this decline is inconsistent with the recent increase of genetic drug-resistant HIV-1 in Japan. Further studies are needed to clarify mechanisms that might explain the discrepancy.

Higher HIV-1 RNA cutoff level required in cerebrospinal fluid than in blood to predict positive HIV-1 isolation.

HIV-1 can be isolated from the vast majority of blood samples taken from HIV-1-seropositive patients not treated with antiretroviral drugs. Isolation rates from cerebrospinal fluid (CSF) samples are considerably lower, ranging between 20-70%. The objective of this study was to determine the cutoff levels for HIV-1 RNA that would yield a positive predictive value > or =90% for positive virus isolation from CSF and blood. Quantitative HIV-1 RNA PCR (Amplicor HIV monitor, version 1.0, Roche Diagnostic Systems) and virus isolation were used to examine 303 CSF samples and 278 paired blood samples from 157 HIV-1-seropositive patients. Patients on antiretroviral treatment provided 140 of the CSF samples and 131 of the blood samples. CSF samples that were positive by culture numbered 137 of 303 (45%), as compared with 216 of 278 (78%) blood samples. In the case of samples taken from patients with antiretroviral treatment, 28% were positive by culture from CSF and 63% from blood. As expected, mean HIV-1 RNA levels were higher in CSF and blood samples positive by culture than in samples negative by culture. A cutoff level of >5,000 HIV-1 RNA copies/ml was required to yield a positive predictive value for positive virus isolation from CSF samples of > or =90%, whereas the cutoff level for blood samples was just above the detection limit of the assay (>200 HIV-1 copies/ml).

Neurological disorders during HIV-1 infection correlate with viral load in cerebrospinal fluid but not with virus phenotype.

To verify the compartmentalization of HIV-1 within the central nervous system (CNS) and to define whether viral phenotype of HIV-1 isolates from cerebrospinal fluid (CSF) samples and CSF viral load correlate with the presence and type of neurological disorders. A total of 33 HIV-1-infected patients with and without neurological disorders were included in the study. HIV-1 isolation from paired CSF and peripheral blood mononuclear cell (PBMC) samples was attempted by a standard cocultivation technique; the biological phenotype of HIV-1 isolates was assessed by the MT-2 cell assay. CSF and plasma HIV-RNA levels were measured by a quantitative reverse transcripase-polymerase chain reaction. The rate of HIV-1 isolation from CSF and PBMC was 66% (22 isolates) and 85% (28 isolates), respectively. Seventeen out of 22 (77%) CSF HIV-1 isolates were characterized as non-syncytium-inducing, and 15 out of 28 (68%) isolates from PBMC were typed as syncytium-inducing (SI). The presence of SI isolates in CSF was limited to patients with HIV-1-, cytomegalovirus- or JC virus-related disorders and was often associated with high levels of HIV-1 RNA in the CSF. Our results demonstrate a correlation between high levels of HIV RNA in CSF and the presence of neurological disorders thus indicating a possible role for HIV-1 RNA in the CSF as a biological marker of neurological disease. The finding of viruses with a different phenotype in paired CSF and PBMC indicates that HIV-1 may evolve differently in the brain and in the blood. This suggests compartmentalization of HIV-1 within the CNS.

Nosocomial transmission of Mycobacterium bovis resistant to 11 drugs in people with advanced HIV-1 infection

Since 1990, several nosocomial outbreaks of multidrug-resistant (MDR) tuberculosis have occurred, none of which have involved Mycobacterium bovis. We describe an epidemic of nosocomial and primary MDR M bovis tuberculosis from December, 1993, to February, 1995, among HIV-1-infected patients in a district of Madrid. We undertook genetic characterisation of the M bovis strain and investigated its presence in a tuberculosis epidemic in a Madrid hospital in a case-controlled study. We assessed 19 cases diagnosed with MDR tuberculosis due to M bovis during the study period. For the control group, we randomly selected 33 patients with HIV-1 infection and isolation of a strain of M tuberculosis susceptible to isoniazid, rifampicin, or both, who were treated in Ramón y Cajal Hospital. Infection-control policies and practices were implemented. We detected 19 cases in HIV-1-infected patients of primary MDR tuberculosis produced by M bovis resistant to 11 antituberculosis drugs. We found phenotypic and genotypic similarities in the strains of M bovis. In the case group, the index case and two other cases had had previous contact with another hospital that had had an MDR tuberculosis outbreak. All patients died after a mean of 44 days (range 2-116), despite multidrug treatment with first-line and second-line antituberculosis drugs. The cases with M bovis MDR tuberculosis were significantly more likely than controls to have been admitted to a hospital ward at the same time as patients already infected with MDR tuberculosis during the 10 months before their diagnosis (adjusted odds ratio 94.6 [95% CI 9.4-956.3], p < 0.0001). Advanced HIV-1 immunosuppression was associated with the development of MDR tuberculosis. Implementation of control measures stopped the epidemic. An M bovis primary MDR tuberculosis epidemic that cannot be treated effectively and with high mortality has emerged in Europe and has been transmitted between hospitals.

HIV-1 isolation from plasma specimens. HEC/FIOCRUZ AIDS Clinical Research Group.

HEC/FIOCRUZ AIDS Clinical Research Group (INI): B Grinszteijn, MCG Galhardo, MRC Guimaraes, S Cavalcante, VC Rolla.

A nationwide effort to sistematically monitor HIV-1 diversity in Brazil: preliminary results. Brazilian Network for the HIV-1 Isolation and Characterization.

The World Health Organization (WHO) Global Program on AIDS (GPA) organized the WHO Network for HIV-1 Isolation and Characterization to monitor HIV-1 variability. Brazil is one of the HIV vaccine trial sites selected by WHO-GPA. HIV-1 subtypes B F and C have thus far been found in the country. A study involving 235 Brazilian isolates found subtype B to prevail in 88.5% of cases subtype F in 8.9% and subtype C in 1.7%. 2 samples (0.9%) were variants resulting from a recombination between subtypes B and F. Further studies have found that Brazilian HIV-1 strains have genetic and antigenic differences compared to North American/European prototype strains potentially affecting the success of immunoprophylactic programs based upon HIV-1 vaccine candidates currently proposed for testing in Brazil. A Brazilian Network for HIV-1 Isolation and Characterization (BNHIC) was thus established in March 1993 as part of the National Program of HIV/AIDS Vaccine Development and Evaluation. The BNHIC was organized upon a 3-tier basis including primary site central reference laboratory and secondary laboratories. The authors discuss efforts made to achieve network goals in Brazil.

HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay

We have previously demonstrated that detection of syncytium-inducing (SI) HIV-1 in asymptomatic seropositive individuals is associated with rapid progression to AIDS. In the present study, we sought to develop and evaluate an HIV-1 phenotyping assay for the screening of large numbers of individuals. Efficiency of HIV-1 isolation from patient peripheral blood mononuclear cells (PBMC) was studied with donor PBMC or seven different CD4+ T-cell lines as target cells. The biological phenotype of sequential isolates from 20 long-term asymptomatic HIV-1-seropositive individuals was determined by two different assays. Non-SI isolates, efficiently recovered by cocultivation with donor PBMC, were never isolated with T-cell lines as target cells. Direct cocultivation with MT-2 cells, but not with six other CD4+ T-cells, resulted in the efficient recovery of SI isolates. HIV-1 MT-2 tropism and SI capacity were shown to be coupled properties at the clonal level. SI isolates emerged in 10 out of 20 longitudinally-studied individuals. In these long-term infected individuals, appearance of SI isolates was associated with progression to AIDS. Direct cocultivation of patient PBMC with the MT-2 cell line is a sensitive, specific and convenient method to detect SI isolates. The availability of an assay suitable for the screening of large groups allows further study of the value of HIV-1 biological phenotyping as a prognostic marker.

Prognostic value of HIV-1 isolation from peripheral blood mononuclear cells: A study in a group of apulian hemophiliacs

Prognostic value of HIV-1 isolation from peripheral blood mononuclear cells: A study in a group of apulian hemophiliacs

Cell cultures from small amounts of heparinized whole blood enhance HIV-1 isolation rate

Cell cultures from small amounts of heparinized whole blood enhance HIV-1 isolation rate

Suppression of HIV-1 Reverse Transcriptase Activity by Mycoplasma Contamination of Cell Cultures

The detection of HIV-1 in human peripheral blood lymphocytes is routinely carried out by cocultivation of test cells with normal peripheral blood mononuclear cells (PBMC). The presence of virus is evidenced by cytologic observation of syncytia or by detecting viral reverse transcriptase (RT) and/or p24 antigen in the culture supernatant fluid. Syncytia formation is almost always associated with the presence of virus as measured by RT, although many RT-positive cultures do not form syncytia. As part of a large screening program, we identified three cultures that showed syncytia but were RT negative. The basis for these discrepant observations was contamination of cultures with mycoplasma that interfered with the RT assay and thereby obscured virus detection. Treatment of cultures with BM-cycline removed mycoplasma contamination and restored RT activity. The present findings indicate the need for caution in the interpretation of negative RT results during HIV-1 isolation and especially in cultures that show evidence of syncytia formation.

Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with HIV-1

Neopterin, a biochemical marker for the activation of cell-mediated immune reactions, was determined in serum and cerebrospinal fluid (CSF) from patients infected with HIV-1. A significant correlation was found between serum and CSF neopterin concentrations, although concentrations of neopterin in serum were more closely correlated with the clinical severity of HIV-1 infection than those in CSF. However, higher CSF levels were observed in patients with neurologic/psychiatric symptoms than in unaffected patients. Also, quotients of CSF neopterin versus serum neopterin concentrations were increased, indicating intrathecal production of neopterin. Positive HIV-1 isolation from peripheral blood mononuclear cells (PBMC) was associated with higher neopterin concentrations in serum, when compared with negative HIV-1 isolation. Neopterin in CSF appears to be a suitable biochemical marker in patients with HIV-1 infection for detecting overt neurologic/psychiatric disturbances. The data suggest that in HIV-1 infected patients, cell-mediated immune reactions might be activated intrathecally and might contribute to neuropsychiatric disease.