First case of new infection with zidovudine-resistant HIV-1 among prospectively studied intravenous drug users and homosexual men in Amsterdam, The Netherlands.
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Antiviral Agents↗
Biomedical subjects
A de Ronde
Publications and source records attributed to A de Ronde.
Increased number of single-LTR HIV-1 DNA junctions correlates with HIV-1 antigen expression and CD4+ cell decline in vivo.
Human immunodeficiency type 1 (HIV-1) DNA in peripheral blood cells of HIV-1 infected individuals may be present as integrated and/or unintegrated DNA. Several reports have indicated that a major proportion of HIV-1 DNA in the asymptomatic phase is linear, full-length, and unintegrated and in the symptomatic phase either circular unintegrated or integrated in the host genome. We developed a quantitative polymerase chain reaction (PCR) technique to detect single-LTR HIV-1 DNA junctions, reflecting the presence of unintegrated single-LTR circles. In vitro infection of a CD4+ T-cell line resulted first in the increase of single-LTR junctions followed by syncytium formation and a rise of p24 antigen production. The number of single-LTR HIV-1 DNA junctions was further studied in two acutely infected individuals and in 21 long-term infected individuals. The number of single-LTR junctions was significantly correlated with CD4+ cell decline, p24 antigen expression, and total HIV-1 DNA content of peripheral blood mononuclear cells (PBMC). Single-LTR HIV-1 DNA junctions were absent from PBMC containing other forms of HIV-1 DNA in four of nine non/slow progressors relative to 2 of 12 rapid progressors/AIDS patients. We conclude from our data that quantitative detection of single-LTR HIV-1 DNA junctions can be used as an early DNA marker of the transition from clinical latency to active replication in the peripheral blood.
Base Sequence↗
Vaccination against feline immunodeficiency virus using fixed infected cells.
Crandell feline kidney cells and feline thymocytes, either feline immunodeficiency virus (FIV) infected or uninfected, were fixed with paraformaldehyde and used to vaccinate cats. The cells were mixed with a 30:70 water/mineral oil emulsion containing 250 micrograms ml-1 N-acetyl-D-glucosaminyl-beta-(1-4)-N-acetyl-muramyl-L-alanyl-D-isoglutam ine. Eighteen specific pathogen-free cats were vaccinated three times with 3-week intervals and challenged 21 days after the final boost with a low dose of the homologous FIV-UT113 strain. Eight out of ten cats that had received FIV-infected cell vaccines developed significant anti-FIV antibody titres to the envelope and core antigens. Neutralizing antibodies were detectable at the moment of challenge in the sera of these animals. Within 5 weeks after challenge 15 out of 18 cats became viraemic. Three animals, two that had been vaccinated with FIV-infected thymocytes and did not develop antibody, and one that had received an uninfected thymocyte preparation, remained uninfected for 6 months. Upon rechallenge of the three animals, two again resisted infection; these cats had been immunized with the infected and the uninfected thymocyte preparations, respectively.
Animals↗
Competitive reverse transcription-polymerase chain reaction for quantitation of feline immunodeficiency virus.
A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to quantify RNA of feline immunodeficiency virus (FIV) in cats. The assay uses in vitro synthesized RNA derived from the gag region of the FIV genome as a competitive internal control. The synthesized RNA has a 22-base deletion with respect to the wild-type sequence. PCR products were quantitated by densitometric analysis of a digitalized image of the ethidium bromide stained gel. The non-radioactive method was evaluated in reconstruction experiments. RNA synthesis in FIV-infected feline thymocytes correlated well with the amount of viral p24 antigen produced. Viral RNA concentrations in the plasma of two cats experimentally infected with FIV strain UT113 were followed for 32 weeks; peak copy numbers (2.3 x 10(4) and 1.3 x 10(4) per ml, respectively) were reached 11 weeks after subcutaneous injection of ten 50% cat infectious doses. With rising antibody titers against FIV-gag and FIV-env gene products, the amount of FIV RNA in plasma decreased. Nine asymptomatic cats that had been experimentally infected 3.5 to 4.5 years earlier had copy numbers between 5.6 x 10(3) and 4.3 x 10(4) per ml. This quantitative competitive RT-PCR will be useful to study the pathogenesis of the FIV infection, to evaluate the effectiveness of vaccines and to monitor antiviral and immunomodulating drugs.
Animals↗
Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.
Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge.
Animals↗
A single mutation within the V3 envelope neutralization domain of feline immunodeficiency virus determines its tropism for CRFK cells.
Feline immunodeficiency virus (FIV) isolates differ in the ability to replicate in Crandell feline kidney (CRFK) cells. The difference in tropism between two variants of the Dutch isolate FIV-UT113 was studied by using molecular clones which contained the envelope genes of the variants in a background of the FIV-14 Petaluma sequence. Virus produced from clone pPET-113Th replicated in thymocytes, whereas virus from pPET-113Cr propagated in both thymocytes and CRFK cells, thereby reflecting the phenotypes of the parental variants. Exchange of envelope gene fragments showed that a 464-bp surface protein (SU)-encoding fragment encompassing the third variable region (V3) determines CRFK cell tropism. Sequence analysis of the exchanged fragments demonstrated two amino acid changes that led to an increase of the overall charge of the V3 domain: a G-->R transition at position 397 and a E-->K change at position 407. Mutational analysis of these residues revealed that the E-->K shift was responsible for the change in tropism, while the G-->R mutation improved the replication kinetics in CRFK cells. Mapping of a tropism determinant for FIV to a region which is also a major neutralization domain is reminiscent of human immunodeficiency virus type 1, in which a similar colocation was found.
Amino Acid Sequence↗
Antibody response in cats to the envelope proteins of feline immunodeficiency virus: identification of an immunodominant neutralization domain.
Overlapping fragments of the envelope protein of feline immunodeficiency virus (FIV) have been expressed in Escherichia coli. Screening of cat sera for antibodies to these fragments revealed that the immunodominant domain of the FIV envelope is localized within the transmembrane protein (amino acids 687-741) and that both the variable region 3 (SU3, aa 385-417) and the COOH-terminus (aa 599-611) of the surface protein (SU) are highly immunogenic. Of all rabbit sera raised to the envelope protein fragments only the serum directed to SU3 was neutralizing. Both FIV-infected and SU3-immunized cats elicited neutralizing antibodies to SU3. Neutralizing antibodies in sera of infected cats could be absorbed by SU3, showing that SU3 is a major neutralization domain of FIV.
Amino Acid Sequence↗
Monoclonal antibodies to immunodominant and neutralizing domains of the envelope surface protein of feline immunodeficiency virus.
Hybridomas secreting monoclonal antibodies (MAbs) specific for the surface protein (SU) of feline immunodeficiency virus were generated. Four MAbs were obtained which could be assigned to two groups based on their neutralization and competition behaviour. Using SU protein fragments expressed in Escherichia coli the antigenic site recognized by one of the MAbs (2H11) could be mapped to the c terminus. The neutralizing MAb 1E1 did not bind to any of the SU protein fragments and was directed to a conformational epitope. Binding of the MAb 1E1 to native SU protein could be blocked with a rabbit serum raised against the SU3 fragment (amino acids 361 to 445). These data indicate that at least part of the epitope is located on this SU3 domain. In competition experiments most sera of naturally infected cats were able to inhibit binding of the MAbs. This shows the conserved and immunodominant nature of the epitopes involved.
Animals↗
Host cell membrane proteins on human immunodeficiency virus type 1 after in vitro infection of H9 cells and blood mononuclear cells. An immuno-electron microscopic study.
Human immunodeficiency virus type 1 (HIV-1)-infected H9 and blood mononuclear cells (MNCs) were studied by immunogold electron microscopy for the presence of HIV-1 gag p24 protein, env gp41 and gp120 proteins, and host cell molecules CD4, CD11a, CD25, CD54, CD63, HLA class I and HLA-DR. Uninfected H9 cells and MNC membranes labelled for CD4, HLA class I and class II, and, at low density, CD11a and CD54; lysosomal structures in the cytoplasm labelled for CD63. The infected cell surface showed immunolabelling for HIV-1 proteins, as did budding particle-like structures. Immunogold labelling of the cell membrane for CD4 was almost non-existent. The level of immunolabelling for CD11a and CD54 on infected cells was greater than that on uninfected cells; this is presumably related to a state of activation during virus synthesis. Budding particle-like structures and free virions in the intercellular space were immunogold-labelled for all host cell markers investigated. This was confirmed by double immunogold labelling using combinations of HIV-1 gag p24 labelling and labelling for the respective host cell molecule. We conclude that virions generated in HIV-1-infected cells concentrate host-derived molecules on their envelope. Also molecules with a prime function in cellular adhesion concentrate on the virion.
Antigens, CD↗
Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis.
The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleavage occurred in Sf9 cells expressing the envelope precursor, but further processing was not observed. Crude lysates of insect cells infected with the wild-type baculovirus or with the recombinant viruses were used to develop an enzyme-linked immunosorbent assay for the detection of feline immunodeficiency virus-specific antibodies in cat sera. The assay showed a higher sensitivity and specificity than immunofluorescence and Western blotting (immunoblotting).
Animals↗
Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes.
HIV-1 NEF genes were isolated directly from peripheral blood lymphocyte DNA of two HIV-1-infected individuals and cloned into an HXB-2-infectious molecular clone. The effect of NEF on virus production in T-cell lines and primary human lymphocytes was studied. Naturally occurring NEF accelerates virus production in primary human lymphocytes, but not in T-cell lines.
Amino Acid Sequence↗
Modulation of the human papillomavirus type 16 induced transformation and transcription by deletion of loci on the short arm of human chromosome 11 can be mimicked by SV40 small t.
The human papillomavirus (HPV) type 16 enhancer-promoter has been shown to be active in human fibroblasts with a deletion on the short arm of one chromosome 11 (karyotype 46,del(11)(p11.11p15.1)) but is virtually inactive in diploid human fibroblasts (Smits, Smits, Jebbink, and ter Schegget, 1990b, Virology, 176, 158-165). In diploid human embryonic fibroblasts, activation of the HPV16 enhancer-promoter could be achieved by expression of the SV40 small t. By cotransfecting SV40 small t cDNA together with HPV16 DNA into diploid cells, it was possible to increase the transforming activity of HPV16 by 10- 15-fold. Furthermore, SV40 small t was essential for the SV40 large T-induced morphological transformation of human diploid fibroblasts, whereas SV40 small t was dispensable for transformation of del-11 cells. We propose that, as a result of the deletion of loci on the short arm of chromosome 11 in del-11 cells, functions are expressed that mimic those of SV40 small t in transformation and trans-activation.
Antigens, Polyomavirus Transforming↗
HIV-1 viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals.
OBJECTIVE: To determine viral DNA load in peripheral blood mononuclear cells (PBMC) from HIV-1-infected individuals. DESIGN: HIV-1 copy numbers were determined using a quantitative polymerase chain reaction (PCR), the PCR-aided template titration assay (PATTY). PATTY utilizes an internal plasmid control DNA, which is amplified within the same tube and using the same primers as the PBMC target DNA. HIV-1 copy numbers were confirmed by limiting-dilution PCR analysis. RESULTS: PBMC viral load of 19 long-term (greater than 4 years) HIV-1-infected individuals ranged from 0.8 to 100 copies per 10(3) PBMC. Significantly higher copy numbers were found among p24-antigen-positive than among p24-antigen-negative individuals. In addition, the PBMC viral load of two HIV-1-infected individuals was monitored during the first 3 months after acute infection. For both patients, the HIV-1 copy numbers were shown to peak at the time of HIV-1-antibody seroconversion and decline subsequently (range, 0.6-10 copies per 10(3) PBMC). CONCLUSIONS: PATTY is a useful method for assessing the HIV-1 copy numbers in PBMC DNA. Viral DNA load peaks shortly after infection and reaches an individual specific level that is probably stable within a few months of infection. Viral DNA load in PBMC varies widely among long-term HIV-1-infected individuals.
AIDS Serodiagnosis↗
Analysis of human immunodeficiency virus type 1 LTR-LTR junctions in peripheral blood mononuclear cells of infected individuals.
Circularized DNA species containing two long terminal repeat circle junctions were analysed in peripheral blood mononuclear cells of human immunodeficiency virus type 1 (HIV-1)-infected individuals. The circle junction fragments found could be classified into four groups: fragments containing a normal circle junction, fragments with deletions at the circle junction, fragments containing the primer binding site inserted at the circle junction, and fragments containing insertions at the circle junction derived from other regions of the HIV-1 genome.
Base Sequence↗
Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity.
Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.
Amino Acid Sequence↗
Immunodominance and antigenic variation of the principal neutralization domain of HIV-1.
The principal neutralization domain (PND) of HIV-1 is located in the third variable region (V3) of the envelope glycoprotein gp 120. Cross-reactivity of experimental and natural sera with recombinant proteins containing the V3 region of four HIV-1 variants showed that a group of viruses (among which HIV-1 MN) had antigenically similar V3 regions. The V3 regions of HIV-1 IIIB and HIV-1 RF were antigenically distinct. Antibodies raised to V3 domains of two isolates from the "MN group" neutralized HIV-1 MN (and not HIV-1 IIIB), thus confirming the antigenic similarity of V3 of these isolates to that of HIV-1 MN. Human antibodies to the V3 region were shown to be mainly directed to the central area in V3, where the neutralization domain is. In addition, antibody reactivities in sera of 397 Dutch and 39 Tanzanian HIV-1-infected individuals show that the V3 neutralization domain is highly antigenic, and that viruses from the MN group predominate in The Netherlands and to a lesser extent in Tanzania. Thus, if the protective value of antibodies to the PND can be established, then PND (poly)peptides derived from the MN group may be important components of a subunit vaccine against HIV-1.
Amino Acid Sequence↗
Contribution of antibody response to recombinant HIV-1 gene-encoded products nef, rev, tat, and protease in predicting development of AIDS in HIV-1-infected individuals.
The relation between antibody-response profiles to Escherichia coli-produced HIV-1 nef, rev, tat, and protease proteins and the risk of developing AIDS was studied using stored serum samples taken sequentially from a cohort of 195 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. The AIDS attack rates at 39 months follow-up were significantly higher in the men with negative versus positive antibody profiles to nef, tat, and protease, respectively. [Difference (D) between attack rates = 11.279, 5.884, and 8.322, respectively]. No significant difference was found between men with negative versus positive antibody profiles to rev. The above differences between AIDS attack rates were clearly lower than those reported from the same cohort for men who were serum HIV-1 antigen positive versus negative, and for men with low versus normal CD4+ lymphocyte counts, but with respect to nef antibody-response profiles, resembled the difference reported between anti-HIV-1 core antibody-negative versus antibody-positive men. In the subgroup of men without any of the markers previously found to be predictive of progression to AIDS in the cohort (persistent HIV-1 p24 antigenemia, low anti-HIV-1 anti-core antibody reactivity, and low CD4+ cell counts), antibody profiles to nef, rev, tat, and protease did not contribute to the prediction of outcome of infection. When used in combination with persistent HIV-1 p24 antigenemia and low CD4+ cell counts, negative antibody profiles to nef and protease, respectively, were equally sensitive and specific in predicting progression to AIDS, as was low anti-HIV-1 anti-core antibody reactivity.
Acquired Immunodeficiency Syndrome↗
Predominance of HIV-1 serotype distinct from LAV-1/HTLV-IIIB.
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Antibodies, Viral↗