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.

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.

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.

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.

Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals.

Antibodies to E. coli-produced HIV-1 vpr and vpu were determined by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 196 initially symptom-free men who were positive for such antibodies at study entry. First detection of vpr- and vpu-specific antibodies always was within 12 months of seroconversion for antibodies to structural proteins. In the combined cohort of 268 men, vpr- and vpu-specific antibodies were found persistently in 26 and 43% of men, respectively. Vpr- and vpu-specific antibodies were transiently detected in 3 and 7%, respectively, and intermittently detected in 18 and 13% of men, respectively. No association was found between the patterns of vpr- or vpu-specific antibody response and clinical outcome. In subjects with different patterns of vpr- and vpu-specific antibody response, no clear temporal relationship existed between the appearance or disappearance of antibodies and the onset of HIV-1-related disease.

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.

Low antigenicity of HIV-1 rev: rev-specific antibody response of limited value as correlate of rev gene expression and disease progression.

An enzyme immunoassay based on an E. coli-produced HIV-1 rev gene product was used to detect rev-specific antibodies in longitudinally collected serum samples from 196 initially symptom-free men who were seropositive for antibodies to HIV-1 structural proteins and 72 men who seroconverted for such antibodies. In 61% of men no rev-specific antibodies were detected at all, 30% had persistently detectable rev-specific antibodies, and in 9% rev-specific antibodies were only transiently or intermittently detected. When a persistent rev-specific antibody response occurred in subjects who seroconverted to structural proteins, it was always, with one exception, found within 12 months of seroconversion. The rev-specific antibodies were also studied in a transectional sample of sera from the men who remained symptom-free and from those who developed AIDS-related conditions or AIDS, as well as in sera from 31 other men with AIDS-related conditions and in sera from 6 of these men at the time they developed AIDS. The rev-specific antibodies were found in 34% of symptom-free men, in 28% of patients with AIDS-related conditions, and in 16% of patients with AIDS. The low incidence of rev-specific antibodies early after infection may be due to low antigenicity of rev. The lower prevalence of rev-specific antibodies in sera from patients with AIDS, compared with patients with AIDS-related conditions and symptom-free HIV-1-infected individuals, may be explained by a progressive HIV-1-induced immunodeficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum reactivity to HIV-1 accessory gene products distinguishes East African from West African HIV strains as infecting agent.

PIP: The existence of dual infections with human immunodeficiency virus (HIV)-1 and 2 in West African countries has been controversial, although the current consensus is that dual infection is not the cause of the extensive cross-reactivity observed between these 2 viruses. To evaluate the role of antibody reactivity to the HIV-1 accessory gene products in type-specific HIV serology, proteins encoded for nef, tat, rev, vpr, and vpu were developed and used as an antigen. 5 of the 7 exclusively HIV-2 reactive sera were not reactive to the HIV-1 accessory gene products. Moreover, the 2 sera that showed reactivity to the HIV-1 envelope were the only ones reactive to HIV-1 accessory gene products. These findings indicate that type 2 viruses may be as diverse as type 1 viruses. A subsequent analysis of sera from 24 West Africans revealed reactivity with a simian immunodeficiency virus (SIV) peptide but not with an HIV-1 peptide previously shown to be discriminatory in a direct binding assay between HIV-1 and HIV-2. Compared to 29 control sera from East Africans, the West Africa sera had significantly lower reactivity to antibodies specific to nef, tat, and rev; there was not reactivity to vpr and vpu. 38% of the West African sera compared with 93% of the East African sera showed reactivity to HIV-1 accessory gene products. It is concluded that, while reactivity to the HIV-1 accessory gene products vpr and vpu indicate HIV-1 infection, reactivity to the other accessory gene products cannot be used to identify virus type given the documented cross-reactivity to HIV-1 accessory gene products of antibodies elicited by HIV-2 strains.^ieng

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.

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.

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.

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.

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.

[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.

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.

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.