Anthony de Ronde

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.

Single Rapid Real-Time Monitored Isothermal RNA Amplification Assay for Quantification of Human Immunodeficiency Virus Type 1 Isolates from Groups M, N, and O

ABSTRACT Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 102 and 107 RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R2 = 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.

Potent antiretroviral therapy initiates normalization of hypergammaglobulinemia and a decline in HIV type 1-specific antibody responses

Next to a profound T cell immunodeficiency, HIV-1 infection induces activation and dysfunction of B cells, resulting in hypergammaglobulinemia. Whereas T cell immune reconstitution with potent antiretroviral therapy has been extensively documented, limited data are available on B cell immune reconstitution. We studied the effect of potent antiretroviral therapy on antibody titers to the viral proteins gp120 and p24 and on total IgG concentrations. Three retrospectively chosen groups were studied: a successfully treated group, untreated controls, and subjects with virological failure after several months of successful therapy. In the successfully treated group, the median total IgG concentrations normalized, whereas they remained elevated in the untreated group and rebounded after an initial decline in the therapy failure group. The HIV-1-specific antibody titers declined in the successfully treated group and followed the rebound of the HIV RNA levels in the therapy failure group. With potent antiretroviral therapy the hypergammaglobulinemia normalized whereas HIV-1-specific immune responses were weakened. The weakening of antiviral immunity with therapy may be relevant for current attempts to gain immunological control over the virus through structured treatment interruptions or therapeutic vaccinations.

Antibody response to the viral negative factor (nef) in HIV-1 infection: a correlate of levels of HIV-1 expression.

Antibody responses against the nef gene product of HIV-1 were determined in sequential sera from a longitudinally studied cohort of 194 initially asymptomatic HIV-1-seropositive individuals and 72 individuals who seroconverted for antibodies to HIV-1 structural proteins (gag/env). In the majority of men, nef-specific antibodies, once detected, persisted (67.6%). In some men, nef-specific antibodies were only transiently (6.8%), or intermittently (5.3%), detectable. No nef-specific antibodies were found in the remaining men (20.3%). Nef-specific antibodies were elicited early in infection, but rarely (2/72 men) prior to seroconversion for antibodies to HIV-1 structural proteins. An absent, transient, or intermittent nef-specific antibody response was significantly associated with the absence or disappearance of antibodies to HIV-1 core proteins, with (re)appearance and persistence of HIV-1 core antigen and with the presence of low CD4+ cell numbers, i.e. profiles previously shown to be predictive of rapid disease progression. Although more cases of AIDS and AIDS-related disease (21/86 versus 28/180) occurred in the nef-specific antibody-negative group than in the nef-specific antibody-positive group, this difference did not reach significance.

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

Infection with HIV-1 Induces a Decrease in mtDNA

Cross-sectional studies have suggested that infection with human immunodeficiency virus (HIV) type 1 could reduce the mitochondrial DNA (mtDNA) content of blood cells. We investigated mtDNA content in peripheral blood mononuclear cells (PBMCs) obtained from 36 antiretroviral therapy-naive documented HIV-1 seroconverters, before and after seroconversion. mtDNA content statistically significantly decreased 1 year after seroconversion and showed a nonsignificant decrease during the subsequent 4 years. These findings confirm that infection with HIV-1 may, itself, reduce mtDNA content, at least within PBMCs. This could have implications for the subsequent development of mitochondrial toxicities associated with the use of nucleoside analogue reverse-transcriptase inhibitors.

Subtype-Specific Sequence Variation of the HIV Type 1 Long Terminal Repeat and Primer-Binding Site

We studied sequence differences in regulatory elements of the long terminal repeat (LTR) and primer-binding site (PBS) among various human immunodeficiency virus type 1 (HIV-1) subtypes. Phylogenetic sequence analysis of a fragment of 729 base pairs (bp) covering the Gag-coding region for half of p24 and all of p17 revealed the gag subtype of all 60 viruses included in the study: A (n = 20), B (n = 12), C (n = 7), D (n = 10), E (n = 3), F (n = 4), G (n = 3), and H (n = 1). The subtype was also determined by analysis of a 689-bp fragment comprising the LTR and the PBS motif. Comparison of the LTR versus gag sequences showed a mosaic genome for seven isolates. After analysis of all sequences, we could describe subtype-specific differences in sequences encompassing the regulatory elements of the LTR and the PBS motif.

Efficient Human Immunodeficiency Virus Replication Requires a Fine-Tuned Level of Transcription

ABSTRACT Transcription represents a crucial step in the life cycle of human immunodeficiency virus (HIV) and is highly regulated. Here we show that the strength of the viral long terminal repeat (LTR) promoter is optimized for efficient replication. Artificially increasing the rate of LTR-driven transcription was strongly detrimental for viral fitness, and HIV was able to regain replication capacity by selecting for variants with a weaker LTR. Strikingly, the strength of the evolved promoter was equivalent to that of the wild-type LTR.

HIV-1 viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals

ObjectiveTo determine viral DNA load in peripheral blood mononuclear cells (PBMC) from HIV-1-infected individuals. DesignHIV-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. ResultsPBMC viral load of 19 long-term (>4 years) HIV-1-infected individuals ranged from 0.8 to 100 copies per 103 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 103 PBMC). ConclusionsATTY 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.

Suppression of virus burden by immunization with feline immunodeficiency virus Env protein

Whole inactivated virus (WIV) vaccines derived from the FLA cell line protect cats against challenge with feline immunodeficiency virus (FIV). To discover whether the protective effects of WIV could be reproduced by the isolated Env component, either WIV or immunoaffinity-purified FIV gp120 from the FL4 cell line was administered to cats. Although both vaccines induced high levels of virus neutralizing antibodies, purified Env was much less effective than WIV in protecting cats against viraemia. However, reduced virus load in PBMCs was evident in all Env-immunized cats compared to controls. Analyses of antibody responses to bacterial expression products of FIV Env, which were high in Env-immunized cats but low in animals receiving the WIV vaccine, suggested that the partially denatured, monomeric Env induces a less effective antiviral immune response than WIV. Hence, the superior immunogenicity of WIV may be due to the presentation of the native oligomeric structure of Env on virions.