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Biomedical subjects

J Albert

Publications and source records attributed to J Albert.

At least 163 records · Page 9Linked to original sources
PubMed1991

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Amino Acid Sequence↗
PubMed1991

Few infected CD4+ T cells but a high proportion of replication-competent provirus copies in asymptomatic human immunodeficiency virus type 1 infection.

The virus load in CD4+ T cells from six asymptomatic human immunodeficiency virus type 1 (HIV-1)-infected individuals was determined by limiting-dilution analysis with a sensitive virus isolation procedure and the polymerase chain reaction (PCR). Both methods allowed detection of one HIV-1-infected cell among 10(5) uninfected cells. The number of provirus-containing CD4+ T cells was found to be 1 per 4,000 to 150,000 (median, 1 per 29,000), as determined by virus isolation and 1 per 2,500 to 26,000 (median, 1 per 12,000), as determined by PCR. Infected cells contained an average of 1 to 2 provirus copies, and a high proportion of the provirus copies (1 in 1 to 1 in 6; median, 1 in 2) were replication competent. The results suggest that only a few CD4+ T cells are likely to be lost as a direct consequence of the presence of HIV-1 in infected cells in asymptomatic individuals and that additional mechanisms may contribute to the depletion of CD4+ T cells observed in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1991

A capture enzyme immunoassay for detection of HIV-2/SIV antigen.

An antigen capture enzyme immunoassay was developed for the demonstration of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV). The assay (HIV-2 CE) has a sensitivity of approximately 250 pg/ml of HIV-2/SIV antigen. The HIV-2 CE was 4-16 times more sensitive than the Abbott HIV-1 antigen assay for detection of HIV-2/SIV antigen in cell culture supernatant. The sensitivity for detection of HIV-2/SIV antigen in serum or plasma was 98.5% in the HIV-2 CE and 95.5% in the Abbott HIV-1 antigen assay. The specificity of the HIV-2 CE was 99.2% (one false positive among 119 negative sera) whereas the Abbott assay was 100% specific. The HIV-2 CE detected antigen in supernatants from cultures of peripheral blood mononuclear cells of macaque monkeys infected with HIV-2 or SIV about 1 week before a reverse transcriptase (RT) microassay was positive and remained positive for at least 1 week after the RT assay had become negative. Three cultures from persistently infected monkeys were positive only in the HIV-2 CE, reflecting a higher sensitivity compared to the RT microassay. Thus, the HIV-2 CE was more sensitive than the Abbott HIV-1 antigen assay for detection of HIV-2/SIV antigen in culture supernatants as well as in serum/plasma. Furthermore, the HIV-2 CE showed a higher sensitivity than the RT microassay for detection of HIV-2/SIV in cell culture supernatants.

Animals↗
PubMed1990

HIV-1 infection of normal human macrophage cultures: implication for silent infection.

We have investigated the replicative capacity of 14 primary HIV-1 isolates in cultures of normal blood macrophages and PHA-stimulated peripheral blood mononuclear cells (PBMC). All viruses could infect normal macrophages as demonstrated by either reverse transcriptase (RT) activity, p24 antigen assay, or cocultivation with PBMC. One month after infection of macrophages virus could no longer be detected in the culture medium. The cells remained in this nonproductive state for another month. Virus could, however, be recovered by cocultivation with PHA-stimulated PBMC. Such macrophage-passaged virus induced pronounced cell killing with fragmentation and pyknosis and often replicated poorly in PBMC, in contrast to the original isolate. The results indicate that all primary HIV-1 isolates contain virus variants that can infect cells of the monocyte/macrophage lineage. Persistently infected, seemingly nonproducing cells, may serve as infectious reservoirs in the infected individual and spread infection to other susceptible cells over a long period of time. Moreover, the pronounced killing of PBMC by the macrophage-harbored virus may contribute to the deterioration of the immune system.

Cell Survival↗
PubMed1990

Serum levels and catabolism of 3'-azido-3'-deoxythymidine in vivo measured using a specific radioimmunoassay.

The thymidine analogue 3'-azido-3'-deoxythymidine is an effective inhibitor of HIV replication in vitro and is used in the treatment of acquired immunodeficiency syndrome. We report here upon a rapid sensitive radioimmunoassay for the detection of azidothymidine in serum or plasma. The assay is simple to perform and levels as low as 1 ng azidothymidine/ml can be detected. The assay is specific for azidothymidine and shows almost no cross-reaction with closely related nucleoside analogues or with naturally occurring nucleosides. Using this radioimmunoassay we were able to measure the azidothymidine levels in the serum of monkeys and acquired immunodeficiency syndrome patients treated with AZT. Individual variation in the peak serum level and clearance rate of azidothymidine were seen, which emphasizes the need to tailor the dose to the individual.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Naturally occurring HIV-1 isolates with differences in replicative capacity are distinguished by in situ hybridization of infected cells.

Replication of human immunodeficiency virus type 1 (HIV-1) isolates in peripheral blood mononuclear cells (PBMC) has been studied by in situ hybridization using the riboprobe BH10-R3 from HTLV-IIIB. Two series of isolates were tested: (a) 20 isolates from individuals with varying severity of HIV-1 infection and (b) sequential isolates from 5 subjects showing signs of clinical progression over a 45 month observation period. The results show that HIV-1 isolates with distinct replicative capacity can be distinguished by the intensity of radioactive labeling over single infected cells after in situ hybridization. Sequential isolates from patients with clinically progressive HIV-1 infection show a gradual increase in replicative capacity over time. In PBMC cultures infected with such sequential isolates, intensity of radioactive label over single infected cells increases and is strongest with isolates obtained at the time of low CD4 counts in blood. The results suggest that the restriction of virus replication that operates in the early stages of HIV-1 infection is gradually lost with progression of the disease.

HIV Infections↗
PubMed1990

Rapid development of isolate-specific neutralizing antibodies after primary HIV-1 infection and consequent emergence of virus variants which resist neutralization by autologous sera.

The kinetics of appearance and specificity of HIV-1 neutralizing antibodies was studied in four individuals. HIV-1 was isolated during symptomatic primary HIV-1 infection and repeatedly thereafter, and tested against autologous sera collected in parallel. Our patients developed isolate-specific low-titer neutralizing antibodies within 2-4 weeks, and the titers to the first isolates increased with time. We documented the emergence of virus variants with reduced sensitivity to neutralization by autologous, but not heterologous, sera in three patients. These virus variants were not, however, resistant to neutralization per se, since they were readily neutralized by the positive control serum. Our patients did not develop antibodies capable of neutralizing the new virus variants during the observation period. This suggests either a failure of the immune system to respond to the new virus variants or a mechanism by which the virus evades detection by the immune system. The emergence of neutralization-resistant virus variants was not directly correlated with disease progression since two patients have remained asymptomatic after the emergence of such virus variants. It is, however, likely that the emergence of virus variants which the patient fails to neutralize in the long run contributes to disease progression.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Replicative capacity of HIV-2, like HIV-1, correlates with severity of immunodeficiency.

We have obtained 15 HIV-2 isolates from the peripheral blood mononuclear cells (PBMCs) of 24 HIV-2-infected west African people. The frequency of virus isolation correlated with the severity of HIV-2 infection; only three isolates were obtained from 11 asymptomatic individuals, whereas virus was isolated from nearly all (12 of 13) individuals with symptoms. The HIV-2 isolates showed distinct replicative and cytopathic characteristics and, similarly to HIV-1 isolates, could be divided into two major groups: rapid/high and slow/low. Rapid/high isolates, i.e. isolates with the ability to replicate in tumour cell lines, were obtained from individuals with symptomatic HIV-2 infection and CD4+ lymphocyte counts less than 360/microliters blood; these isolates induced syncytia in PBMC cultures. HIV-2 isolates unable to replicate continuously in tumour cell lines (slow/low isolates) induced small syncytia, cell death, or no cytopathic effect at all. All HIV-2 isolates obtained from asymptomatic individuals showed a slow/low replication pattern.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection.

Simian immunodeficiency virus (SIV) infection in cynomolgus macaques leads to severe immunodeficiency with a fatal outcome. In contrast, HIV-2 infects these primates without apparently causing any immunological abnormalities. In this study three cynomolgus monkeys were experimentally infected with HIV-2 strain SBL-K135 and 168 days later challenged with 10-100 animal infectious doses of the closely related SIV strain SM to study protective immunity. At the time of SIV challenge the HIV-2-infected monkeys had neutralizing antibodies against HIV-2, but virus could no longer be recovered from their peripheral blood mononuclear cells (PBMCs) and no clinical symptoms or decrease in CD4+ lymphocytes were observed. Follow-up for 9 months after challenge with SIV showed that the HIV-2-infected monkeys were protected against SIV-induced immunodeficiency (no decrease of CD4+ lymphocytes) and lymphadenopathy. However, they were not resistant to SIV infection since virus could be recovered from their PBMCs and they developed anamnestic antibody responses. Four naive control monkeys which were inoculated with the same dose of SIV became persistently infected and developed a decrease of the absolute numbers of CD4+ cells and showed a marked lymphadenopathy. Two out of four control animals died 58-265 days postinfection with an immunosuppressive disease. Immunohistochemical examination showed abundant viral antigen in lymph-node biopsies from the SIV-infected control monkeys but absence of SIV or HIV-2 antigens in the biopsies from the three HIV-2-preinfected and SIV-superinfected monkeys. The present study demonstrates possibilities for induction of immunity against immunodeficiency induced by a primate lentivirus, a concept with application also to HIV infection and AIDS in man.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.

Base Sequence↗
PubMed1989

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations.

A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.

Amino Acid Sequence↗
PubMed1989

IgG subclass responses to a transmembrane protein (gp41) peptide in HIV infection.

The IgG subclass distribution to the E34/E32 peptides, derived from the HIV-1 glycoprotein 41 transmembrane protein, was analyzed in ELISA. Sera from individuals at different stages of the disease were assayed. A restricted subclass response of mainly IgG1 and IgG2 was found. The subclass response was of a different type than the one observed to HIV whole Ag and to a synthetic peptide from the C'-terminal part of the HIV-1 p24 core protein. An increased subclass restriction was observed in progressed stages of the disease.

Amino Acid Sequence↗
PubMed1989

Human immunodeficiency virus type 1 p24 production and antigenic variation in tissue culture of isolates with various growth characteristics.

Two monoclonal antibodies (Mabs) reacting with different epitopes of the human immunodeficiency virus type 1 core protein p24 (HIV p24) were used either singly or in combination as tracers in enzyme-linked immunosorbent assays. The culture supernatant of 215 samples of peripheral blood mononuclear cells from 112 patients were measured for HIV p24 and reverse transcriptase (RT) activity during cultivation. One hundred forty-one cultures were positive for HIV p24 and 122 for RT after 32 days of cultivation. After 8-9 days, HIV p24 was detected in 50.4% and RT in 27.8% of the cultures later judged as HIV positive. Two patients seemed to have substrains of HIV-1 not reactive with one of the Mabs.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid.

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.

AIDS Dementia Complex↗
PubMed1989

Molecular and biological characterization of a replication competent human immunodeficiency type 2 (HIV-2) proviral clone.

We obtained complete genomic clones of human immunodeficiency virus type 2 (HIV-2) from the DNA of the neoplastic human cell line HUT 78 freshly infected with a HIV-2 isolate, strain SBL6669. The recombinant phage DNA was transfected into the lymphocytes of CD4-positive HUT 78 cell line to test the replication competence of the proviral DNA. One genomic clone, designated HIV-2SBL/ISY, yielded retroviral particles after a few weeks of culture of the transfected cells. The HIV-2SBL/ISY clone contained a complete provirus and cellular flanking sequence. We obtained the DNA sequence of the provirus and compared it with the published sequence of two other HIV-2 isolates. The degree of variability among HIV-2 isolates is comparable to that observed among African HIV-1 isolates sequenced to date. Immunologically, HIV-2SBL/ISY is similar to the parental virus (HIV-2SBL6669) but differs in the envelope transmembrane protein that is truncated (gp32-34) in the parental virus and not in HIV-2SBL/ISY (gp41). Both the parental and the cloned viruses are infectious and cytopathic for some human T-cell lines, induce syncytia, and infect a human macrophage cell line (U937) in vitro. The availability of a biologically active HIV-2 clone provides the means to study the role and interaction of HIV-2 genes in vitro as well as to assess the functional similarities among HIV-1 and HIV-2 genes. Since HIV-2SBL/ISY cloned virus infects fresh peripheral blood T cells from Rhesus macaques in vitro and infects the same animal in vivo, its use in animals may represent a model for functional study of viral genes in vivo as well as for development of experimental approaches to prevent and cure retroviral infection in humans.

Blotting, Southern↗