Greater and more rapid depletion of mitochondrial DNA in blood of patients treated with dual (zidovudine+didanosine or zidovudine+zalcitabine) vs. single (zidovudine) nucleoside reverse transcriptase inhibitors.

BACKGROUND: Most toxicities associated with nucleoside analogue reverse transcriptase inhibitors (NRTIs) are thought to result from mitochondrial toxicity. These toxicities include peripheral neuropathy, pancreatitis, lactic acidosis, and peripheral lipoatrophy. Unfortunately, there are no validated laboratory markers for clinically assessing, let alone predicting, the onset of mitochondrial toxicity associated with NRTI therapy. OBJECTIVES: To provide preliminary evidence of the potential clinical utility of an assay which has been developed for quantifying mitochondrial DNA (mtDNA) in clinical samples from HIV-infected patients. METHODS: A single-tube duplex real-time DNA-nucleic acid sequence-based amplification (NASBA) assay (Mitox, Primagen, Amsterdam, the Netherlands) was used to quantify mtDNA in cryopreserved peripheral blood mononuclear cells (PBMC) obtained from HIV-1-infected patients during their prior participation in a randomized placebo-controlled trial comparing zidovudine (ZDV) monotherapy with combinations of ZDV plus either dideoxycytidine (ddC) or didanosine (ddI) (the Delta trial). Patients were antiretroviral naïve prior to entering the trial. Samples obtained during the initial 48 weeks of treatment were tested. RESULTS: A significant decline of mtDNA, both in an intent-to-treat and in an as-treated analysis, was observed in patients treated with ZDV+ddC and ZDV+ddI, but not with ZDV alone, consistent with the results expected from the degree of mtDNA depletion described for each of these drugs in vitro. CONCLUSIONS: This single-tube duplex real-time DNA-NASBA assay was shown to measure mtDNA accurately in PBMC. Treatment with a combination of two NRTIs was associated with greater reductions in mtDNA than obtained for ZDV monotherapy. The relevance of these results in predicting treatment toxicity requires further evaluation.

Carrier rate of zidovudine-resistant HIV-1: the impact of failing therapy on transmission of resistant strains.

OBJECTIVE: Because maintenance of treatment success in HIV-1 infection requires viruses to remain therapy sensitive in drug-naive seropositive persons, we looked at the primary infections caused by drug-resistant HIV-1 over time. Furthermore, to study the coverage rate of therapy and therapy failure in relation to the transmission of resistant viruses a mathematical model was developed. DESIGN: The reverse transcriptase and protease genes of viruses were analysed in newly infected people in the period 1990-1998 in the Amsterdam Cohort Study on HIV infection and AIDS in homosexual men. METHODS: The mathematical model was based on the coverage of drug regimens selecting zidovudine (ZDV) resistance, the lag time in which resistance is gained or lost, the death rate of people infected with resistant virus, and the replacement of resistance-selecting regimens by more potent treatments that substantially reduce viral load and mortality. RESULTS: Of 43 individuals with a primary HIV-infection, three (7%) harboured ZDV-resistant viruses. The first of the ZDV-resistant strains was transmitted in 1995, the last two in 1996. The build-up of ZDV resistance was described by the mathematical model indicating that the equilibrium level of resistance due to treatment depends only on the treatment rate and the outflow rate of patients with resistance virus. CONCLUSIONS: Our model indicates that the frequency of viral resistance in a population is determined largely by the number of individuals on insufficient or failing therapy and is influenced only modestly by secondary transmission of ZDV-resistant strains.

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.

Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O.

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 10(2) and 10(7) 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 (R(2) = 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.

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

Epidemiology of HIV-1 and emerging problems.

Broad use of antiretroviral drugs is becoming a factor that is important to consider for understanding the HIV-1 epidemiology. Since 1993, we observe that a proportion of new infections within major risk groups in Amsterdam is caused by azidothymidine (AZT)-resistant viruses. After the introduction of combination therapy in The Netherlands in 1997, new infections with drug-resistant viruses have not been documented. Large-scale monitoring of anti-HIV-1 therapy failures revealed that antiretroviral drugs may yield previously undescribed resistant viruses, which contain a two amino acid insertion (68SS/V69) within their reverse transcriptase genes in combination with mutations at codons 67 and 215. These viruses are highly resistant to AZT, 3TC, and d4T, and moderately resistant to ddI and ddC.

Genetic analysis reveals epidemiologic patterns in the spread of human immunodeficiency virus.

The extreme variability of human immunodeficiency virus type 1 (HIV-1) makes it possible to conduct transmission studies on the basis of genetic analysis and to trace global and local patterns in the spread of the virus. Two such patterns are discussed in this paper. First, in many European countries (e.g., Scotland and Germany), homosexual men tend to be infected with a subtly different variant of HIV-1 than intravenous drug users. In other European countries (e.g., Norway and Sweden), a distinction is also found between the two risk groups; but based on available data, the distinction is a different one. The second pattern is a worldwide tendency for homosexual men in many different geographic regions around the world to carry HIV-1 subtype B, the variant that is most prevalent in the Americas, Europe, and Australia. In contrast, people infected via other routes (mostly heterosexual contact) in those same countries carry a mixture of other subtypes. Biologic differences between the viruses infecting different risk groups have not been found; the most likely explanation for the findings is different epidemiologic patterns. Although data are still scarce, the authors attempt to use these patterns in the reconstruction of the worldwide spread of the HIV epidemic.

Natural residues versus antiretroviral drug-selected mutations in HIV type 1 group O reverse transcriptase and protease related to virological drug failure in vivo.

HIV-1 group O viruses were first recognized as a distinct subgroup of HIV-1 with the isolation and characterization in 1990 of a virus (ANT70) from a woman (individual A) and her spouse (individual B), both from Cameroon (De Leys R, et al.: J Virol 1990;64:1207-1216). During the 5-6 years before treatment, individual A remained asymptomatic, with viral RNA levels between 2.5 and 2.8 log10 copies/ml, as measured by a newly developed group O-specific quantitative NASBA-based RNA assay. Individual B developed mild clinical symptoms, with 3.1 to 3.6 log10 copies of viral RNA per milliliter. HIV-1 sequences obtained from both individuals showed pretreatment residues in protease that confer resistance to protease inhibitors in group M viruses (10I, 36I, and 71V). Individual A showed an initial response to AZT, but shortly after addition of ddC and saquinavir, the RNA levels returned to baseline, while subsequent treatment with d4T, 3TC, and indinavir reduced the RNA level to less than 50 copies/ml for the time of follow-up. Individual B showed no response to AZT or ddC monotherapy, and a change to d4T, 3TC, and indinavir had, in contrast to individual A, only a temporary effect. While a multitude of mutations in HIV-1 group O reverse transcriptase (RT) and protease appeared that are associated with drug resistance in group M viruses, the observed T215N mutation in RT and the V15I and V22A mutations in protease have not previously been described and may represent resistance-conferring mutations specific to group O viruses. These results indicate that treatment of HIV-1 group O-infected individuals with antiretroviral drug regimens that include protease inhibitors might lead to rapid selection for resistance-conferring mutations. This probably results from preexisting protease residues contributing to reduced sensitivity of group O viruses to protease inhibitors, as is observed in vitro.

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.

Design and evaluation of a human immunodeficiency virus type 1 RNA assay using nucleic acid sequence-based amplification technology able to quantify both group M and O viruses by using the long terminal repeat as target.

Currently available human immunodeficiency virus type 1 (HIV-1) RNA quantification assays can detect most viruses of the group M subtypes, but a substantial number are missed or not quantified reliably. Viruses of HIV-1 group O cannot be detected by any commercially available assay. We developed and evaluated a quantitative assay based on nucleic acid sequence-based amplification (NASBA) technology, with primers and probes located in the conserved long terminal repeat (LTR) region of the HIV-1 genome. In 68 of 72 serum samples from individuals infected with HIV-1 subtypes A to H of group M, viruses could be detected and quantified. In serum samples from two patients infected with HIV-1 group O viruses, these viruses as well could be detected and quantified. In contrast, the currently used gag-based assay underestimated the presence of subtype A viruses and could not detect subtype G and group O viruses. The discrepancy between the results of the two assays may be explained by the number of mismatches found within and among the probe and primer regions of the subtype isolates. These data indicate that LTR-based assays, including the NASBA format chosen here, are better suited to monitoring HIV-1 therapy than are gag-based assays in an era in which multiple HIV-1 subtypes and groups are spreading worldwide.

Detection of human immunodeficiency virus type 1 nucleocapsid protein p7 in vitro and in vivo.

We developed and evaluated an immunoassay for the detection and quantification of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 using electrochemiluminescence technology. The assay had a dynamic range of 50 to 20,000 pg/ml and a lower detection limit equivalent to approximately 10(6.5) HIV-1 RNA copies/ml in culture supernatant. In vitro kinetic replication studies showed that the amount of p7 correlated strongly with the amount of p24 (R2 = 0.869; P < 0.0001) and viral RNA (R2 = 0.858; P = 0.0009). On the basis of the p7 and RNA concentrations, we calculated the median p7:RNA ratio to be approximately 1,400 p7 molecules per RNA molecule. HIV-1 p7 could be detected and quantified in culture supernatants of both group M subtype A to E viruses and group O viruses. The presence of p7 in vivo was evaluated in 81 serum samples collected from 62 HIV-1-infected individuals. Five samples were p7 positive, whereas 45 samples were HIV-1 p24 positive. Four of the five p7-positive samples were p24 positive as well. p7 could be detected only when serum HIV-1 RNA levels were greater than 10(6) copies/ml. Anti-p7 antibodies were found in six samples, and all six were p7 negative. In contrast to the in vitro results, it appeared that HIV-1 p7 could not be used as a marker for viral quantification in vivo, since more than 90% of the serum samples were p7 negative. In combination with the low prevalence of anti-p7 antibodies, this may, in turn, be advantageous: the p7 assay may be a good alternative to the p24 assay as the readout system for determination of neutralizing activity against HIV-1 in serum or other fluids containing anti-p24 antibodies.

Feline immunodeficiency virus subunit vaccines that induce virus neutralising antibodies but no protection against challenge infection.

Three experimental vaccines against feline immunodeficiency virus (FIV), all based on viral antigens presented via immune stimulating complexes (iscoms), were tested for their capacity to induce protection in cats from FIV infection. The respective vaccines consisted of FIV propagated in Crandell feline kidney (CrFK) cells (FIV-iscoms); FIV-iscoms spiked with recombinant vaccinia virus expressed FIV envelope glycoprotein incorporated into iscoms (FIV-iscoms + vGR657x15-iscoms) and vGR657x15-iscoms spiked with recombinant FIV Gag protein incorporated into iscoms (vGR657x15-iscoms + FIV-Gag-iscoms). Simian immunodeficiency virus envelope glycoprotein incorporated into iscoms, iscoms prepared with uninfected CrFK cells, and PBS served as controls. All cats vaccinated with vGR657x15-iscoms combined with FIV-iscoms or FIV-Gag-iscoms developed Env-specific plasma antibody responses. These antibodies neutralised FIV infection in CrFK cells, but failed to neutralise FIV infection in primary feline thymocytes. FIV-iscoms induced poor Env-specific responses and only one out of six cats developed antibodies that neutralised FIV in the CrFK cell based assay. Four weeks after challenge all cats proved to be infected, showing that none of the vaccine preparations provided protection. In contrast, 2 weeks after infection, virus infected peripheral blood mononuclear cells were only observed in cats vaccinated with FIV-iscoms + vGR657x15-iscoms or CrFK-iscoms and to a lesser extent in cats vaccinated with FIV-iscoms and vGR657x15-iscoms + FIV-Gag-iscoms, but not in cats vaccinated with SIV-iscoms or PBS. The differences found in cell associated virus loads amongst the respective groups are discussed in the light of antibody mediated enhancement of infectivity and protective effects provided by Gag-specific T cell responses.

Salmonella typhimurium aroA recombinants and immune-stimulating complexes as vaccine candidates for feline immunodeficiency virus.

Two experimental feline immunodeficiency virus (FIV) vaccines were tested, either alone or in combination, in four groups of cats (A-D). One vaccine (SL3261-FIV) was composed of live attenuated Salmonella typhimurium aroA (SL3261) strains expressing the capsid (Gag) and part of the envelope (Env) proteins of FIV. The other was composed of FIV Gag and Env proteins incorporated into immune-stimulating complexes (iscom-FIV). Cats of group A were immunized four times with SL3261-FIV. Cats of group B were immunized twice with SL3261-FIV and then twice with iscom-FIV. Cats of group C were immunized twice with SL3261 expressing the B subunit of cholera toxin (SL3261-CtxB) and then twice with iscom-FIV. Cats of group D, which served as negative controls, were immunized twice with SL3261-CtxB and then twice with iscom into which the Gag and Env proteins of simian immunodeficiency virus (SIV) had been incorporated (iscom-SIV). Two weeks after the last immunization, all cats were challenged with FIV. At this time, cats immunized with iscom-FIV (groups B and C) showed strong plasma antibody responses to Gag and Env, whilst these responses were weak or undetectable in the cats immunized four times with SL3261-FIV (group A). Seven weeks after FIV challenge, Env-specific antibody responses had increased considerably in cats of all groups except group A. The mean virus loads in the cats of this group proved to be lower than those of the other groups at all time points, indicating partial protection.

A single amino acid substitution in the transmembrane envelope glycoprotein of feline immunodeficiency virus alters cellular tropism.

The cellular tropism of the feline immunodeficiency virus (FIV) is affected by changes in variable region 3 (V3) of the surface (SU) envelope glycoprotein (Verschoor, E. J., et al., J. Virol. 69:4752-4757, 1995). By using high-dose DNA transfection, an FIV molecular clone with a non-CRFK-tropic V3 acquired the ability to replicate in CRFK cells. A single point mutation from a methionine to a threonine in the ectodomain of its transmembrane (TM) envelope glycoprotein was responsible for this change in viral tropism. This substitution is located in the putative SU interactive region, between the fusion peptide and the membrane-spanning region. Our results show that this region of the TM envelope glycoprotein constitutes an additional determinant for cell tropism.

Broad spectrum of in vivo fitness of human immunodeficiency virus type 1 subpopulations differing at reverse transcriptase codons 41 and 215.

Viral populations in a human immunodeficiency virus type 1 (HIV-1)-infected individual behave as a quasispecies with a rated distribution of fitness variants. Fitness distributions in naturally occurring viral populations have been difficult to study due to the lack of markers for individual virus clones and complicating inter- and intrahost factors like the presence of multiple cell types with distinct tropisms, differences in route of transmission, and intervening immunity. Here, we quantitated the relative fitness in vivo of three subpopulations of HIV-1 marked by mutations at codons 41 and 215 of reverse transcriptase (RT) directly related to zidovudine resistance in an untreated individual who was infected by a zidovudine-resistant strain transmitted from a donor on therapy. The transmission event did not have a substantial impact on the distribution of mutants within the dominant virus population replicating to high levels in the recipient. The evolution of the RT gene was monitored for 20 months. All 102 clones obtained from the donor and the recipient at the different time points contained the M41L mutation, which is associated with a fourfold reduction in zidovudine sensitivity. The leucine at position 41 was stable, although it was encoded by TTG and CTG triplets that fluctuated in abundance partially due to founder effects of clones with nonsilent mutations at codon 215. Of the three subpopulations in the patient, distinguished by a tyrosine (TAC), aspartic acid (GAC), or serine (TCC) at the 215 position of RT, the relative fitness of the GAC variant was calculated to be 10 to 25% higher than the initial TAC variant, and the relative fitness of the TCC variant was 1% higher than that of the GAC variant. Similar to other RNA viruses, lentivirus populations like HIV-1 in patients with a high virus load apparently consist of a broader spectrum of fitness variants than the 1 to 2% fitness difference sufficient for significant replicative advantage.

Human immunodeficiency virus fitness in vivo: calculations based on a single zidovudine resistance mutation at codon 215 of reverse transcriptase.

We monitored a subject newly infected with a zidovudine-resistant human immunodeficiency virus type 1 strain and found that in the absence of drug, the viral population with the resistance-conferring tyrosine (TAC) codon 215 of reverse transcriptase was gradually replaced. By using standard formulas to model the effects of selection at a single locus in an asexual haploid population, the relative fitness gain of the viral population with a single mutation at codon 215 creating a serine (TCC) was calculated. We concluded that a viral population with a serine at reverse transcriptase codon 215 conferring zidovudine sensitivity was between 0.4 and 2.3% more fit.

[Quantification of feline immunodeficiency virus (FIV) RNA in the plasma of infected cats]. / Quantifizierung feliner Immunschwächevirus (FIV)-RNS im Plasma infizierter Katzen.

A competitive reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify RNA of feline immunodeficiency virus (FIV) in cats. The assay uses in vitro synthesized RNA 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 digitized image of the ethidium bromide stained gel. 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. Cats treated for six weeks with 9-(2-phosphonylmethoxy-propyl)-2,6-diaminopurine [PMPDAP] (20 mg/kg body weight s.c. three times a week) showed a significant decrease of RNA copy numbers in plasma. 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.

[Zidovudine-resistant HIV strains in intravenous drug users and homosexual men in Amsterdam]. / Zidovudine-resistente HIV-stammen bij intraveneuze-drugsgebruikers en homoseksuele mannen in Amsterdam.

OBJECTIVE: To investigate the spread of HIV strains resistant to antiviral drugs prescribed in the Netherlands. DESIGN: Descriptive. SETTING: Amsterdam. METHOD: In several cohorts of homosexual men and intravenous drug users being followed in Amsterdam, in cases of newly acquired HIV infections in the period 1992-1995 HIV RNA was isolated from serum. The nucleic acid sequence encoding the first 250 amino acids of the HIV reverse transcriptase was determined in order to detect mutations conferring resistance to reverse transcriptase inhibitors. RESULTS: Among participants of the Amsterdam cohort studies, 12 new HIV infections of homosexual men and 23 of IV drug users were observed. In the group of homosexual men the first infection by a zidovudine-resistant HIV was observed in 1995. In the group of IV drug users the first infection by a zidovudine-resistant strain was noticed in 1993 with two more infections in 1995. The mutations regarded positions 41, 67, 70 and 215 of the HIV reverse transcriptase. No HIV strains resistant to didanosine, deoxycytidine, lamuvidine or nevirapine were found in untreated persons with an acute infection. CONCLUSION: As zidovudine is a vital part of the latest and most efficacious combination therapies of HIV infection, testing for zidovudine-resistance prior to treatment is recommended.