Amplified antigen-specific immune responses in HIV-1 infected individuals in a double blind DNA immunization and therapy interruption trial.

Immunotherapy in patients with HIV-1 infection aims to restore and broaden immunological competence, reduce viral load and thereby permit longer periods without combined antiretroviral treatment (cART). Twelve HIV-1-infected patients on cART were immunized on the skin with DNA plasmids containing genes of several HIV-1 subtypes with or without the addition of hydroxyurea (HU), or with placebo. The mean net gain of HIV-specific CD8+ T cell responses were higher and broader in the HIV DNA vaccine groups compared to non-vaccinated individuals (p<0.05). The vaccine-induced immune responses per se had no direct effect on viral replication. In all patients combined, including placebo, the viral set point after a final structured therapy interruption (STI) was lower than prior to initiation of cART (p=0.003). Nadir CD4 levels appeared to strongly influence the post-STI viral titers. After the sixth immunization or placebo, patients could stay off cART for a median time of 15 months. The study shows that HIV DNA immunization induces broader and higher magnitudes of HIV-specific immune responses compared to structured therapy interruptions alone. Although compromised by small numbers of patients, the study also demonstrates that well-monitored STI may safely function as an immunological read out of HIV vaccine efficacy.

Development and evaluation of a flow-cytometric assay of specific cell-mediated immune response in activated whole blood for the detection of cell-mediated immunity against varicella-zoster virus.

A new assay for the detection of specific cell-mediated immune (CMI) responses is described. Whole blood, diluted 1/10 in medium, was cultured in the presence or the absence of specific antigens. Results were assessed by flow cytometric analysis with or without immunophenotyping to detect proliferating lymphoblasts among cultured cells. Interferon-gamma, IL-10, and IL-5 in culture supernatants are measured by ELISAs. The assay was evaluated using samples from 37 VZV-antibody-positive children with a history of chickenpox and samples from 15 seronegative children without a history of chickenpox; it displayed a sensitivity of 95% and a specificity of 100% for the detection of varicella-zoster virus (VZV)-specific CMI. The intraassay and interassay variations of the new test were lower than with the conventional assay for CMI, detecting thymidine incorporation in peripheral blood mononuclear cells (PBMCs). Cytokines were detected in only 70% of cultures from VZV-antibody-positive subjects. The cytokine response was restricted to IFN-gamma in most cases. The Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA) is a precise and accurate yet simple and convenient test that can be readily employed for the examination of single samples as well as for large-scale studies.

Lymphocyte subset enumeration in HIV seronegative and HIV-1 seropositive adults in Dar es Salaam, Tanzania: determination of reference values in males and females and comparison of two flow cytometric methods.

The level of CD4(+) T-lymphocytes represents a useful marker with which to monitor the progression of HIV infection. Sex and geographical differences in the reference values of lymphocyte subsets have been reported. We have compared two flow cytometric methods (MultiSET and SimulSET) for the quantification of lymphocyte subsets using whole blood from 92 HIV seropositive and 241 seronegative adults, and determined the reference values of lymphocyte subsets in HIV seronegative Tanzanian subjects. In seronegative Tanzanian subjects, the percentages of CD3(+) and CD4(+) T-lymphocytes and the CD4(+):CD8(+) T-lymphocyte ratios were lower while the percentage of natural killer cells was higher compared to the levels of the corresponding parameters reported for Europeans. Seronegative Tanzanian females had significantly higher levels of CD3(+) and CD4(+) T-lymphocytes and CD4(+):CD8(+) T-lymphocyte ratios compared to seronegative males. The correlation coefficients of CD3(+), CD4(+) and CD8(+) T lymphocyte counts and percentages obtained by the two flow cytometric methods were high. The median values of the number of CD4(+) T-lymphocytes obtained by the two methods were not significantly different. In conclusion, determination of the reference values of lymphocyte subsets in HIV seronegative Tanzanian adults showed significant sex differences and differences in percentage values compared to those reported in certain other geographical areas. There was acceptable agreement in the levels of CD4(+) T-lymphocyte values obtained by the two flow cytometric methods.

Recent origin of human immunodeficiency virus type 1 variants in resting CD4+ T lymphocytes in untreated and suboptimally treated subjects.

Resting CD4(+) T lymphocytes are an important reservoir for human immunodeficiency virus type 1 (HIV-1) in treated patients with undetectable viremia. The knowledge of viral persistence in these cells is limited, however, for patients without treatment or patients for whom treatment is failing; therefore, this reservoir in such patients was characterized. Virus variants were characterized in 3 subjects who were followed-up from primary HIV-1 infection and 5 treatment-experienced subjects. No founder viral sequences and only a minority of the earlier identified drug-induced mutations were found in the resting T lymphocytes. Instead, the viral sequences were closely related to those detected simultaneously in plasma, except in 2 treatment-experienced subjects. Thus, a turnover and replenishment of this virus reservoir in peripheral blood is likely to occur in most persons with detectable viremia. However, infrequently the virus variants in plasma and resting T cells seem to be derived from independent sources.

Initiation of therapy during primary HIV type 1 infection results in a continuous decay of proviral DNA and a highly restricted viral evolution.

A latent pool of HIV-1 is established early in memory CD4+ T lymphocytes and persists during antiretroviral therapy. Also, viral replication may continue in subjects despite undetectable viremia. However, it remains unclear whether this residual replication results in any significant sequence evolution. We were therefore interested in studying the viral evolution and HIV-1 DNA dynamics in subjects with primary infection receiving or not receiving early potent antiretroviral therapy. In 16 subjects, HIV-1 DNA load was monitored from 1 to 23 days, up to 1253 days, after onset of symptoms. Extensive sequential cloning and sequence analysis of the V3 region was performed in four subjects. In the treated subjects a continuous decline in the proviral load was found, corresponding to a half-life of about 6 months. As expected in newly infected individuals the founder virus populations showed high intrasubject sequence similarity. Also, a limited increase in the viral divergence was detected during the first 6 months in three treated subjects. Thereafter, no significant sequence changes were found despite analysis of a large number of clones. Our data thus suggest that early and successful therapy in compliant subjects with primary HIV-1 infection results in a highly restricted viral evolution and a decline in the proviral load close to the decay rate of human memory T lymphocytes.

Viral dynamics in primary HIV-1 infection. Karolinska Institutet Primary HIV Infection Study Group.

OBJECTIVES: To study the natural course of viremia during primary HIV infection (PHI). METHOD: Eight patients were followed from a median of 5 days from the onset of PHI illness. Plasma HIV-1 RNA levels were measured frequently and the results were fitted to mathematical models. HIV-1 RNA levels were also monitored in nine patients given two reverse transcriptase inhibitors and a protease inhibitor after a median of 7 days from the onset of PHI illness. RESULTS: HIV-1 RNA appeared in the blood during the week preceding onset of PHI illness and increased rapidly during the first viremic phase, reaching a peak at a mean of 7 days after onset of illness. This was followed by a phase of rapidly decreasing levels of HIV-1 RNA to an average of 21 days after onset. Viral density continued to decline thereafter but at a 5- to 50-fold lower rate; a steady-state level was reached at a median of 2 months after onset of PHI. Peak viral density levels correlated significantly with levels measured between days 50 and 600. Initiation of antiretroviral treatment during PHI resulted in rapidly declining levels to below 50 copies/mL. CONCLUSIONS: This study demonstrates the kinetic phases of viremia during PHI and indicates two new contributions to the natural history of HIV-1 infection: PHI peak levels correlate with steady-state levels and HIV-1 RNA declines biphasically; an initial rapid decay is usually followed by a slow decay, which is similar to the initial changes seen with antiviral treatment.

Diagnosis of primary HIV-1 infection and duration of follow-up after HIV exposure. Karolinska Institute Primary HIV Infection Study Group.

OBJECTIVE: To determine the sensitivity of 33 currently available and seven earlier tests for the detection of HIV or HIV antibody in primary HIV-1 infection, to estimate the duration of the 'window period' and the influence of early initiated antiretroviral treatment (ART). DESIGN: A prospective cohort study of 38 patients with primary HIV-1 infection. ART was initiated at a median time of 13 (range 0-23) days after the onset of symptoms in 10 patients. MAIN OUTCOME MEASURES: The time from infection to onset of symptoms and from onset of symptoms to the appearance of HIV antibody as measured by 36 different tests, and the start and duration of viraemia, as detected by four different tests. RESULTS: The illness appeared 13-15 days after infection in 12 of 15 determinable cases, and seroconversion was detected within 1-2 weeks after the onset of illness by 27 of 30 currently available tests for HIV antibody, in contrast to the 2-7 weeks or more needed by the old tests. HIV RNA appeared during the week preceding the onset of illness and was detected in all subsequent samples, except when ART had been initiated, which also induced a delay of the antibody response. CONCLUSION: Many tests for HIV or HIV antibody can now be employed for an early confirmation of primary HIV infection (PHI). Currently available screening tests proved much more sensitive than older tests, and seroconversion was usually detected within one month after infection. Consequently, in Sweden we now recommend only 3 months of follow-up after most cases of HIV exposure.

A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry.

A flow cytometric assay for the combined measurement of cell-mediated cytotoxicity and conjugate formation has been developed. Cytolysis is detected by propidium iodide uptake. Target cells, effector cells and conjugates between targets and effectors are separated by post-culture immunophenotyping and their scatter profiles. Pre-assay staining of cells is thus not required. Each cluster of cells can be further examined at the single-cell level by simultaneously performed additional immunophenotyping. Two applications were established: the assessment of NK cell activity against K562 cells and the evaluation of LAK cell cytotoxicity against both K562 and Daudi cells. A comparison with the standard 51Cr release assay for the detection of NK cytotoxicity showed that the two assays were strongly correlated, but the sensitivity of the flow cytometric assay was significantly higher.

Recruitment of monocyte derived dendritic cells ex vivo from SIV infected and non-infected cynomolgus monkeys.

This study shows that characteristic dendritic, antigen presenting cells, can be generated from adherent peripheral blood mononuclear cells (PBMC)/monocytes of uninfected and SIVsm-infected cynomolgus monkeys after stimulation in vitro with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4. The recruitment of monocyte derived dendritic cells (MDDC) was usually possible irrespective of the level of immunodeficiency (CD4-level) and viremia. The cynomolgus MDDC closely resembled their human counterpart (immature MDDC) with regard to capacity to upregulate CD1a, CD40, CD86 and human leukocyte antigen (HLA)-DR and develop dendrites and veiled processes. Such MDDC also increased their capacity for antigen uptake (dextran endocytoses/macropinocytosis) and for induction of T-cell proliferation in mixed leukocyte reaction (MLR) assays. However, although no clear difference with regard to phenotype and morphology was seen between MDDC from SIV-infected and uninfected monkeys, a reduction in MLR responsiveness in MDDC from SIV infected monkeys was consistently detected within each experiment.

Reappearance of founder virus sequence in human immunodeficiency virus type 1-infected patients.

Different patterns of temporal evolution in human immunodeficiency virus type 1 V3 and p17 regions are described for eight patients studied during the first years following primary infection. In samples from three patients, a rapid replacement of the major sequence occurred but the original sequence reappeared later simultaneously with clinical deterioration and increased plasma viral load.

Long-term prognosis following zidovudine monotherapy in primary human immunodeficiency virus type 1 infection.

Eighty-five subjects with symptomatic primary (P) human immunodeficiency virus (HIV) type 1 infection were analyzed in a retrospective cohort study to investigate the long-term clinical benefit of antiretroviral treatment during PHIV infection. Zidovudine treatment was initiated (PHIV treatment group) in 21 persons a median of 9 days after onset of PHIV symptoms and continued for a median of 55 days (range, 21-99). Sixty-four subjects did not receive early antiretroviral treatment (PHIV nontreatment group). After follow-up for 3-10 years, 33 subjects had developed AIDS and 22 subjects had died of AIDS. The median times for progression to AIDS and death were 6.4 and 9.1 years, respectively. Progression rates did not differ between the PHIV treatment and nontreatment groups. Zidovudine treatment initiated during PHIV infection did not improve long-term outcome after symptomatic PHIV infection.

Low relative frequencies of CD26(+) CD4(+) cells in long-term nonprogressing human immunodeficiency virus type 1-infected subjects.

A broad antibody panel was used for immunophenotyping of human immunodeficiency virus type 1 (HIV-1)-infected patients who were long-term nonprogressors (LTNP). The LTNP were compared with patients in the early phase of infection and patients who had progressed to advanced immunodeficiency. Changes in CD8(+) subset distribution were observed mainly at acquisition of HIV-1 infection, whereas CD4(+) subset changes appeared during progression of HIV-1 infection. The decreasing levels of CD4(+) cells were characterized by an increasing frequency of cells expressing the activation markers HLA-Dr and CD45RO but not the CD28 surface antigen. The LTNP exhibited significant changes compared to HIV-negative patients in almost all markers. Compared to patients in the early phase of infection, the only difference was a relatively lower frequency of CD4(+) cells expressing CD26 among the LTNP. The results show that HIV-1-infected persons who have no signs of immunodeficiency despite many years of infection have an immunophenotypic pattern that is substantially different from that of noninfected persons. Despite the long duration of infection, the LTNP exhibit a pattern similar to that of newly infected persons, with the exception of lower expression of CD26 on CD4(+) cells.

Characterization of the viral population during primary HIV-1 infection.

OBJECTIVE: To study viral heterogeneity at a very early phase of primary HIV-1 infection. DESIGN: Samples were drawn very early during primary HIV-1 infection. A virus population-based approach was used to study the viral heterogeneity in the C2-V3 and p17 regions. METHODS: Plasma samples (n = 33) were obtained before or shortly after onset of acute symptoms in 15 patients. In all subjects, the first sample was drawn within 10 days after onset of symptoms. Peripheral blood mononuclear cells (PBMC) were available in two patients. The number of polymorphic sites in the C2-V3 (15 patients) and p17 regions (eight patients) were determined by direct sequencing. RESULTS: The sequence heterogeneity was restricted in most patients, although only two out of 15 patients had a completely homogeneous C2-V3 sequence. However, pronounced individual differences were seen. Rapid sequence changes occurred during the first month in two patients. In one patient, the major DNA species at day 12 later became the major species in plasma. CONCLUSIONS: The viral population is seldom completely homogeneous during primary HIV-1 infection, although the heterogeneity is restricted in most, but not all, patients. These individual differences do not seem to be due to sex or viral subtype. Rapid changes of the virus population may occur during primary HIV-1 infection. The DNA species detected in PBMC do not only represent earlier viral quasispecies but are also a potential source of future viral RNA species.

Evaluation of 14 commercial HIV-1/HIV-2 antibody assays using serum panels of different geographical origin and clinical stage including a unique seroconversion panel.

The performance of 14 commercially available HIV-1/2 antibody assays were compared using well-characterized serum panels containing in total 1500 1800 sera. The panels included consecutive HIV-negative blood donor sera from Sweden, unselected blood donor and patient sera from Tanzania and unselected sera from outpatient clinics in Guinea-Bissau. Furthermore selected HIV-1 antibody positive sera from Sweden and Tanzania and HIV-2 antibody positive sera from Guinea-Bissau were included in the panels. The HIV-1 antibody positive sera were from individuals at various stages of HIV infection, from primary infection, to asymptomatic phase and late stage disease. 12 of the 14 assays identified correctly all HIV-1 and HIV-2 antibody positive sera. One Tanzanian HIV-1 antibody positive sample with complete banding pattern on Western blot was not detected by two of the ELISAs employing synthetic peptides. There were small differences in sensitivity between the assays when used for analysis of seroconversion panels. The most sensitive assay, Abbott IMx HIV-1/HIV-2 III Plus detected antibodies in all nine samples collected from four individuals during the first week after onset of symptoms of primary HIV-1 infection. Most of the assays became reactive during the second week after onset of symptoms and the least sensitive assays were reactive from the third week. The assays showed a high specificity ranging from 99.2 to 100% when used for analysis of Swedish blood donor sera, while most of the assays showed a significantly lower specificity, 91.9-99.6%, when used for testing African specimens.

Effects of hormone replacement on growth hormone and prolactin exercise responses in postmenopausal women.

Exercise elevates growth hormone (GH) and prolactin (PRL) blood concentrations in premenopausal women. Postmenopausal women taking hormone replacement therapy (HRT) maintain higher estrogen levels that could affect GH and PRL. The purpose of the study was to determine the effects of HRT on GH and PRL responses to treadmill exercise. Seventeen healthy women who were postmenopausal (naturally or surgically) [8 on HRT; 9 not on HRT (NHRT)], completed 30 min of treadmill exercise at 79.16 +/- 1.2% maximal O2 consumption (HRT group) and 80.19 +/- 0.91% maximal O2 consumption (NHRT) group). Blood samples were collected from an intravenous catheter during an exercise session and during a control session without exercise. GH and PRL concentrations were significantly higher in the exercise trial than in the nonexercise trial, whereas resting concentrations were similar for both trials. GH and PRL peaked at 10.8 +/- 1.60 and 12.67 +/- 2.58 ng/ml, respectively, for HRT subjects and at 4.90 +/- 1.18 and 9.04 +/- 2.17 ng/ml, respectively, for NHRT subjects. GH concentrations in the exercise trial were significantly higher for HRT than for NHRT subjects. This is the first study to demonstrate that HRT enhances treadmill-exercise-induced GH release and that similar PRL responses to treadmill exercise occur in postmenopausal women regardless of HRT status.

Effects of estrogen replacement therapy on dehydroepiandrosterone, dehydroepiandrosterone sulfate, and cortisol responses to exercise in postmenopausal women.

OBJECTIVE: To determine the effects of hormone replacement therapy (HRT) on dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and cortisol (F) responses to treadmill exercise. DESIGN: Controlled clinical study. SETTING: Female volunteers in an academic research environment. PATIENT(S): Sixteen healthy, postmenopausal women (7 were receiving HRT, 9 were not). INTERVENTION(S): Blood samples were taken from an intravenous catheter before, during, and after 30 minutes of treadmill exercise following an overnight fast. A second session was conducted one month later for the same subjects using the same blood sampling protocol without exercise. MAIN OUTCOME MEASURE(S): Serum DHEA, DHEAS, and F concentrations. RESULT(S): The HRT and untreated DHEA area under the curve (AUC) for the exercise trials was significantly greater than that for the control trials. The untreated, but not the HRT, DHEAS AUC for the exercise trials was significantly greater than that for the control trials. The HRT and untreated F AUC for the exercise trials was significantly greater than that for the control trials. The AUC for the HRT exercise trials was significantly higher than the untreated exercise trials for DHEA and F, but not DHEAS. CONCLUSION(S): Data suggest that treadmill exercise elevates DHEA, DHEAS, and F levels in postmenopausal women and that HRT enhances the DHEA and F responses.

Longer survival after HIV infection for injecting drug users than for homosexual men: implications for immunology.

BACKGROUND: Comparisons of progression in HIV-1 infection between injecting drug users (IDU) and homosexual men have been inconclusive due to the short follow-up periods, often with less well-defined starting points and endpoints. In addition, comparisons of survival after injection have been to some extent obscured by higher non-AIDS mortality in IDU. METHOD: In a retrospective cohort study, homo-/bisexual men and IDU were followed, with dates of seroconversion defined within +/- 1 year by a previously negative HIV antibody test. Endpoints were CD4 cell count below 200 x 10(6)/l, AIDS and death from AIDS. RESULTS: Sixty-three homo-/bisexual men and 125 IDU fulfilled the entry criteria, with no significant differences in age at or date for seroconversion. Mean follow-up times were 6.7 and 7.0 years, respectively. The homo-/bisexual group had a significantly accelerated progression rate to all three endpoints: time to CD4 cell count below 200 x 10(6)/l (P = 0.002), to AIDS (P = 0.0003), and to death from AIDS (P < 0.0001). Adjusting for age and sex only made marginal alterations. Ten years after infection, 54% of homosexual men had developed an AIDS-defining condition and 51% had died from AIDS, whereas the corresponding precentages in the IDU group were 26 and 15, respectively. There was, however, no difference in overall mortality due to almost constant, non HIV-related, yearly mortality of some 4% in IDU. CONCLUSIONS: In our cohort there was a highly significant difference in disease progression and death from AIDS between homo-/bisexual men and IDU. This difference was proposed to be due to the transmission route determining the initial immune response and suggested that this route may have played a more important role than virus variability of the subsequent prognosis.

A new method for measuring lymphoproliferation at the single-cell level in whole blood cultures by flow cytometry.

A simple and reproducible method is described for the measurement of proliferative responses to mitogens and antigens. Whole blood (WB) cultures are used and separation procedures are not necessary. Proliferative responses are monitored as development of lymphoblasts determined by flow cytometry (FC). Lymphocytes and lymphoblasts were shown to be exclusively selectable from mixed cell populations of WB by their light scatter profile on FC analysis. More than 90% of the lymphoblasts identified by FC were replicating DNA as determined by incorporation of the thymidine analogue 2-bromo-5-deoxyuridine. The WB/FC technique can be combined with triple immunofluorescence which was illustrated by determining subsets of lymphoblasts developing following stimulation with various mitogens and antigens. The new method was compared with the conventional method (peripheral blood mononuclear cell culture and proliferation measured by detection of DNA synthesis). The reproducibility of the methods were not different. The antigen-induced responses recorded by both methods correlated, but with significantly higher stimulation indices obtained by WB/FC. The latter procedure was also technically easier to perform and less time-consuming.