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Good markers for monitoring the efficacy of antiretroviral therapy in children do not currently exist. This study examined the effect of antiretroviral therapy on human immunodeficiency virus (HIV-1) unintegrated DNA (uDNA), integrated DNA (iDNA), percent uDNA, immune complex dissociated (ICD) p24 antigenemia, and plasma viral titer. Seven children were followed at therapy initiation and at approximately 3- and 10-month intervals. HIV-1 uDNA was detected in all children prior to start of therapy (average percent uDNA, 43%). At 3 months, the percent HIV uDNA decreased in all patients to an average of 18% (p = 0.01) and at 10 months decreased to an average of 1%. In contrast, the amount of HIV iDNA was relatively constant after initiation of therapy. ICD HIV p24 antigen was detected in all patients prior to therapy (average, 538 pg/ml). Over the study period, the ICD p24 antigen level decreased in three patients and remained relatively unchanged in four patients. Plasma cultures of HIV-1 were positive in only one of the seven patients prior to therapy. Among the methods evaluated, measurement of uDNA was the only parameter which reliable decreased after initiation of nucleoside therapy.

Surrogate markers generally used for observation of patients infected with human immunodeficiency virus (HIV) and their plasma and cellular viral load were assayed in a series of 40 patients before initiation of zidovudine therapy. Plasma viremia was positive in 62.5% of patients and was statistically correlated with clinical stage, CD4+ T cell count, CD8+ T cell count, beta 2-microglobulin level, neopterin level, and immunoglobulin A level. Cellular viremia was positive in 95% of patients and was correlated with clinical stage, CD4+ T cell count, beta 2-microglobulin, neopterin levels, and disease progression during the following months. A discordance was found between p24 antigenemia, even after acid dissociation of immune complexes, and plasma viremia. In fact, p24 antigenemia was correlated with only biological markers of immune activation as beta 2-microglobulin and neopterin levels. The measurement of anti-p24 antibodies did not appear discriminative in our staging. Plasma viremia, like CD4+ T cell count, reflects the patient's status at the time of assessment. Cellular viremia could be more informative for the prediction of future clinical progression.

The calcium-binding myelomonocytic protein calprotectin (L1 protein) was quantified in serum from 51 patients with HIV infection and in 20 HIV-seronegative blood donors. Significantly elevated levels were found both in asymptomatic patients and in people with AIDS compared with controls. The calprotectin level was not related to ongoing or recent opportunistic infections. For patients with CD4+ counts above 50 x 10(6)/L, a significant negative correlation was found between serum calprotectin levels and the CD4+ counts. Serial samples from 24 patients during their first year of zidovudine (ZDV) treatment showed a further elevation of serum calprotectin during the first months of ZDV treatment, with a subsequent decline to pretreatment levels. A low calprotectin response during the first 6 months, determined as area under the curve, was associated with the occurrence of at least one AIDS-defining infection during the first year of antiviral treatment. Also, a low calprotectin maximal response during ZDV therapy was associated with short survival. Similar associations were not found for neopterin, beta 2-microglobulin, HIV p24 antigen, or CD4+ or CD8+ lymphocytes in blood. Our findings in a limited number of patients suggest that calprotectin levels may reflect immune activation and other immune mechanisms correlated with enhanced antimicrobial defense induced at least transiently by antiviral treatment.

HIV-induced cytokine dysregulation, including overproduction of the antiproliferative and cytolytic IFN alpha cytokine, represents a major component of the immune disorders characterizing AIDS. To block the overproduction of IFN alpha we designed an AIDS vaccine combination which included both an anti-HIV and/or an anti-IFN alpha immunization. The safety and immunogenicity of this multicomponent vaccine were tested in mice, Cercopithecus, two HIV noninfected individuals, and six HIV-1 seropositive immunocompromised patients enrolled in a 1-year open clinical trial. We now report the result of a 9-month short-term randomized, blind, placebo-controlled clinical trial (Phase I/II) performed in HIV-1 patients (22 individuals) to confirm safety/tolerance of the anti-IFN alpha vaccine and its immunogenicity and to evaluate whether the complex vaccine initially used could be simplified by removal of HIV component(s). Three groups of patients received inactivated IFN alpha (i-IFN alpha) associated with the immunomodulator P40 with HIV-1 antigens (groups B and C) or without (group A), and one group (D) was placebo. The clinical follow-up documented among those receiving i-IFN-alpha showed that none developed AIDS and/or required antiretroviral chemotherapy. Viral load did not increase and CD4 cell count as well as cell-mediated immunity (CMI) stabilized or even significantly increased in group A. Immunogenicity of the preparations was determined by a positive delayed-type hypersensitivity (DTH) reaction to i-IFN alpha and the presence of serum antibodies to i-IFN alpha and to HIV-1 peptides, occurring only in treated patients. As previously planned, based on these safety data, the trial has been extended for an additional year and all patients were switched to protocol A (i-IFN alpha+P40). This second period of the trial, now open and ongoing, should allow us to evaluate further the innocuity of the i-IFN alpha preparation and whether anti-IFN alpha vaccine could provide a long-lasting CD4 cell count as well as CMI stabilization.

Mouse monoclonal antibodies with high human immunodeficiency virus type 1 (HIV-1) neutralizing titers were used for passive immunotherapy of eleven late-state HIV-infected patients. In five patients the serum level of the core protein p24 decreased, while in five cases it remained unchanged. The level of viral RNA in plasma as measured by quantitative polymerase chain reaction (PCR) decreased in four cases, was stable in another four, and increased in three cases. An anti-mouse (HAMA) response developed in eight patients and anti-idiotypic antibodies appeared in six. Immune complexes that formed in patient sera during the treatment were shown to contain mostly envelope glycoprotein gp120 which decreased in nine of the eleven treated patients toward the end of treatment. Antibodies inhibiting gp120 binding to CD4 became detectable or increased in six patients during immunotherapy. Serology of the HIV-1 V3 region was studied for both the HIV-1 IIIB and MN strains with no or very small changes in titer or avidity after treatment. No change in neutralizing titers to strain HTLVIIIB was observed in serum samples collected before and after treatment was terminated. In nine of the eleven patients stimulation of the T lymphocytes to proliferate in vitro when activated by phytohemagglutinin (PHA) was shown to be increased compared to before treatment. Increased T-cell proliferation was also noted with several antigens such as HIV-1 recombinant antigens, cytomegalovirus (CMV), tetanus toxoid (TT), and purified protein derivate of mycobacterium tuberculosis (PPD). These findings indicate a decreased total gp120 content in serum, permitting better T-cell activation.

It has been reported that HIV-1 p24 antigen (p24 Ag) detection is improved after dissociation of immune complexes using acid treatment (ICD assay). In order to evaluate the clinical significance of p24 Ag detected by the standard assay and by the ICD assay in pediatric patients, we related these measurements to clinical status, level of p24 antibody, and percentage of CD4+ lymphocytes. Fifty-nine plasma specimens from 20 symptomatic HIV-1-infected children, collected prospectively over a 1-year period, were tested for these markers. Plasma was collected at the beginning of zidovudine therapy and approximately 7 and 12 months thereafter. Compared with the standard assay, the ICD assay showed a higher number of samples positive for p24 Ag (78% versus 34%) and an increase in the levels of p24 Ag (median value of 129 versus 24 pg/ml). The anti-p24 antibody level was inversely correlated with the p24 Ag level measured by either assay. Four children negative for p24 Ag by both assays had a stable clinical course. In contrast, 50% of the children negative by the standard assay but positive for ICD p24 Ag and 75% of the children positive by both assays had progression of disease. No patients were positive by the standard assay but negative by the ICD assay. Children whose plasma tested positive by both assays had lower percentages of lymphocytes that were CD4+ by comparison with children who were negative by both assays; children whose plasma tested positive only by the ICD assay formed an intermediate group.(ABSTRACT TRUNCATED AT 250 WORDS)

To ascertain whether immune complex dissociation (ICD) improves the value of p24 antigen as a prognostic marker for progression of HIV infection, 53 patients were followed over a 3-year period, including at least one visit per year. All had CD4+ counts at entry > 400/mm3; progressors (n = 18) were defined as having CD4+ counts < 200/mm3 and nonprogressors (n = 35) as having CD4+ counts still > 400/mm3 at the end of follow-up. Serum specimens were collected at each annual visit and assayed for p24 antigen with and without ICD treatment. At entry, the percentage of progressors positive for ICD p24 antigen was significantly higher than the percentage of positive nonprogressors (39% versus 3%, p < 0.01). The sensitivity of p24 antigen over all visits in terms of predicting the progression increased from 61% before ICD to 83% after. The specificity of p24 antigen in terms of predicting progression decreased from 97% before ICD to 89% after. The relative risk of progression in individuals positive for p24 antigen was 6.7 before ICD and increased after ICD to 12.7. When evaluating the respective prognostic value of the p24 antigen and of the ICD p24 antigen, only ICD p24 was significant (RR 10.2, 95% CI 2.2-46.9). ICD p24 antigen appears to be a marker of progression that may be detected earlier than p24 antigen without ICD.

To determine whether human immunodeficiency virus (HIV) viral load has short term stability, eight clinically stable subjects infected with HIV and having CD4 counts ranging between 10-600/mm3, had blood samples taken at 0800 and 1700 on 3 consecutive days and then weekly at 0800 for 1 month (8-10 observations/subject). Plasma HIV RNA, peripheral blood mononuclear cell (PBMC) proviral DNA, serum p24 antigen levels, and mononuclear cell subsets were measured at each time point. Mean plasma HIV RNA, PBMC HIV DNA, and p24 antigen [both regular and immune complex dissociated (ICD)] levels did not change significantly between mornings and afternoons or on successive days or weeks. CD4+, CD8+, and CD56+ number demonstrated a diurnal variation in those subjects with > 200 CD4 cells/mm3. We conclude that HIV viral load demonstrates short-term stability in clinically stable subjects. This stability has important implications for monitoring HIV disease progression or antiretroviral therapy.

We conducted a double-blind, randomized phase II study to evaluate the safety and activity of combination therapy with N-butyl-deoxynojirimycin (SC-48334) (an alpha-glucosidase I inhibitor) and zidovudine versus zidovudine alone. Patients with 200 to 500 CD4 cells/mm3 who tolerated < or = 12 weeks of prior zidovudine therapy received SC-48334 (1000 mg every 8 h) and zidovudine (100 mg every 8 h) or zidovudine and placebo. Sixty patients received combination therapy and 58, zidovudine and placebo. Twenty-three patients (38%) and 15 (26%), in the combination and zidovudine groups, respectively, discontinued therapy (p = 0.15). The mean SC-48334 steady-state trough level (4.04 +/- 0.99 micrograms/ml) was below the in vitro inhibitory concentration for human immunodeficiency virus (HIV). The mean increase in CD4 cells at week 4 was 73.8 cells/mm3 and 52.4 cells/mm3 for the combination and zidovudine groups, respectively (p > 0.36). For patients with prior zidovudine therapy, the mean change in CD4 cells in the combination and zidovudine groups was 63.7 cells/mm3 and 4.9 cells/mm3 at week 8 and 6.8 cells/mm3 and -45.1 cells/mm3 at week 16, respectively. The number of patients with suppression of HIV p24 antigenemia in the combination and zidovudine groups was six (40%) and two (11%) at week 4 (p = 0.10) and five (45%) and two (14%) at week 24 (p = 0.08), respectively. Diarrhea, flatulence, abdominal pain, and weight loss were common for combination recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

In 1990 the results of a placebo-controlled study conducted within the AIDS Clinical Trials Group (ACTG 019) showed that in the short term, zidovudine was effective in slowing progression to advanced disease in HIV-infected asymptomatic patients with low CD4 cell counts. More recently, the preliminary results of the Concorde Trial suggested that while the data at 1 year agreed with those of ACTG 019, no sustained clinical benefit was detectable at 3 years for early versus deferred therapy. Therefore, the length of the clinical usefulness of zidovudine in this population is still to be determined. We evaluated the 2-year outcome of zidovudine therapy in asymptomatic patients through the prospective follow-up of a cohort of 936 subjects with low CD4+ (< 500/mm3) counts who strictly satisfied, at enrollment, the inclusion and exclusion criteria of the ACTG 019 trial. The clinical end point of the analysis was the development of AIDS. The majority (72.2%) of the individuals in the cohort acquired HIV infection through intravenous drug use; 26.6% were women. The median baseline CD4 cell count was 308/mm3. At 55 weeks of mean follow-up, the progression rate to AIDS (3.2 events per hundred person-years) appears to be comparable to that already reported at the same mean follow-up time for the ACTG 019 zidovudine-treated asymptomatic patients. After 124 weeks of mean follow-up, the overall rate of progression to AIDS was 5.2 per hundred person-years.(ABSTRACT TRUNCATED AT 250 WORDS)

The usefulness of traditional methods of HIV plasma titration has been limited by poor detection capacity in the asymptomatic phase of HIV disease. We analyzed plasma samples from asymptomatic seropositive or early symptomatic patients, comparing the classic plasma culture method with the following techniques: phorbol 12-myristate 13-acetate treatment of target cells, centrifugal inoculation of the virus, heat treatment of cultures, addition of monocyte/macrophages to cultures, and polyethylene glycol (PEG) treatment of the plasma. Only PEG treatment significantly increased the percentage of HIV isolation. The increase of HIV isolation after PEG treatment is more evident in patients with higher CD4+ cell counts and those without detectable levels of p24 antigen. In the p24-negative samples, HIV was isolated in 17 of 24 (71%) with PEG treatment versus nine of 24 (37%) with the classic method (p < 0.01). A number of discordant samples were found using the classic and PEG methods. Combining the positive results obtained with either technique, we obtained an overall HIV detection rate of 76%. The increased sensitivity of the combination of PEG and classic methods may allow a wider use of plasma viremia as part of the virological evaluation of anti-HIV drug efficacy in asymptomatic patients.

Breast milk specimens from human immunodeficiency virus type 1 (HIV-1)-seropositive and HIV-1-seronegative women were examined for the presence of HIV-1 p24 antigen by the antigen capture method and for viral DNA using the polymerase chain reaction. HIV-1 DNA was present in 70% of milk specimens collected from 47 HIV-seropositive women 0-4 days after delivery and in approximately 50% of specimens collected 6 and 12 months postpartum. p24 antigen, present in 24% of milk specimens collected from 37 seropositive women within the first 4 days postpartum, was not detected in any of the subsequent specimens. The presence of HIV-1 DNA or p24 antigen in milk was not significantly associated with maternal CD4 lymphocyte count, beta 2-microglobulin level, or fulfillment of the AIDS clinical case definition. Although the correlation of either HIV-1 proviral DNA or p24 antigen with the presence of infectious virus is not known, these data indicate the need for additional studies examining the role of breastfeeding in maternal-infant transmission of HIV-1.

Measles-mumps-rubella vaccine (MMR) is recommended for human immunodeficiency virus-infected (HIV+) adults. Data concerning MMR vaccination of HIV+ patients are limited to children. We evaluated 39 HIV+ (97% with > 200 CD4+ lymphocytes) and 17 non-HIV+ control adults receiving measles-rubella vaccine (MR). Clinical adverse events did not differ between groups. Prevaccination, three HIV+ and two control vaccinees were measles seronegative; no HIV+ and one control vaccinee seroconverted. No initially measles-seropositive vaccinee had a significant antibody elevation. Four HIV+ and three control vaccinees were rubella seronegative prevaccination; three HIV+ and two control vaccinees seroconverted. Among those initially rubella seropositive, two HIV+ and one control vaccinee had significant antibody elevations. There were no significant percentage CD4+ or CD8+ lymphocyte changes between groups. Three HIV+ vaccinees were p24 antigen positive pre- and postvaccination. Although MR vaccination appears safe in HIV+ adults, questions remain about the response of measles and rubella antibody-negative HIV+ adults and those with < 200 CD4+ lymphocytes.

The use of full-dose intensive regimens of chemotherapy in patients with HIV-associated lymphoma has often resulted in severe toxicity, treatment delay, and reduced subsequent dosing. We conducted a Phase I trial to evaluate the toxicity of the combination of m-BACOD (methotrexate, Bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone) with granulocyte-macrophage colony stimulating factor (GMCSF) in these patients. A total of 17 patients were entered and treated at three dose levels of m-BACOD in combination with a fixed dose of GMCSF. Eight patients received standard dose m-BACOD plus GMCSF without experiencing dose-limiting hematologic toxicity, although significant nonhematologic toxicity was seen. We conclude that it is feasible to treat patients with HIV-associated lymphoma using standard dose m-BACOD plus GMCSF, but further study is needed to determine whether full-dose regimens improve survival when compared with reduced dose regimens in these individuals.

Differences in avidity between HIV-1 antibodies transmitted passively and antibodies synthesized by children born to HIV-1-positive mothers can be measured using a commercially available competitive enzyme immunoassay kit. The avidity determination method is based on the competition between an anti-HIV-1-peroxidase-labeled antibody at a stable and known concentration and the anti-HIV-1 antibodies (IgA, IgG, IgM) present in the child's serum at various and increasing dilutions. The shift in the competition/dilution curves between serum samples taken at the third and the sixth month of the child's life showed either the loss or the synthesis of anti-HIV-1 antibodies. The antibody avidity determination combined with a test detecting free or complexed p24 antigen is a workable and inexpensive serological method for the follow-up of children born to seropositive mothers. Combining these two complementary methods, HIV-1 infection has been established at 6 months of age in 13 of 13 infants, and positive results were confirmed by coculture and by PCR. An HIV-1 infection was excluded at 6 months of age in 17 of 17 infants, results otherwise confirmed by virological and clinical follow-up. These new and convenient approaches to the diagnosis of vertically acquired HIV-1 could be used worldwide, including in developing countries.

To promote progress in the research and development of early diagnostic tests for HIV in young children an international workshop was held on January 17-18, 1992, in Siena, Italy. Experts on pediatric AIDS, diagnostic retrovirology, and immunodiagnosis discussed and summarized the state-of-the-art and made recommendations for general application of several tests and further evaluation and continued research for other candidate tests. From the discussions it was clear that the field has advanced beyond the time when it was necessary to wait until the infant reached 18 months of age before attempting the diagnosis with conventional serologic tests for HIV. About half of infected infants can now be identified at birth, approximately 90% by 3 months of age, and almost all by 6 months of age using HIV culture and polymerase chain reaction assays. IgA antibody tests and p24 antigen tests have also proved useful, although they are not as sensitive in newborn infants. The fact that HIV can be detected in only one-half of infected infants at the time of birth points to the need for further research on the timing of transmission and the natural history of perinatally acquired HIV infection to understand the limitations of current early diagnostic tests and to develop new approaches to overcome these problems.

Quantitative culture of human immunodeficiency virus (HIV) was performed on 121 plasma samples from 76 HIV-infected individuals to determine the sensitivity of the assay at different stages of disease and to measure the effect of antiviral therapy on plasma viremia. Plasma virus was detected in 49 of 76 (64%) of patients, primarily those with AIDS and AIDS-related complex (36 of 38) versus asymptomatic subjects (13 of 38) (p less than 0.001, chi 2). Similarly, plasma cultures were more often positive in patients with less than 250 CD4+ T cells per microliter (38 of 40) than in those with greater than 250 CD4+ T cells per microliter (11 of 36) (p less than 0.001, chi 2). Plasma virus cultures were also more likely to be positive in patients with detectable serum p24 antigen (24 of 26) than in those without detectable p24 antigen (25 of 50) (p = 0.0023, chi 2). An effect of zidovudine (ZDV) treatment on plasma viremia was seen in a comparison of treated and untreated patients with less than 250 CD4+ T cells per microliter. Geometric mean titers of plasma viremia from 16 patients treated with ZDV for more than 3 months were significantly lower than titers from 24 untreated patients (10(1.3) versus 10(2.1), p less than 0.05, Student's t test. A comparison of pre- and posttherapy titers in 33 patients receiving antiviral treatment showed that plasma virus was not detectable at either time in 17 patients; there was a fall in plasma virus titer in 12; and titers were unchanged or increased in 4. In patients with advanced disease, plasma viremia is a potential marker of antiviral drug activity.

Experimental conditions affecting the successful propagation of HIV-1 from the plasma of seropositive individuals were examined. It was determined that whole blood samples collected with lithium heparin as the anticoagulant, immediate plasma separation, and immediate culturing were best suited for obtaining viable virus from plasma. Virus was isolated by infecting fresh phytohemagglutinin-stimulated normal donor peripheral blood mononuclear cells (PBMCs) with plasma followed by weekly cocultivation with new target cells. The plasma virus isolation rate was the greatest and HIV-1 titers were the highest for those individuals with less than 200 CD4+ cells/mm3 and decreased as the level of CD4+ cells approached normal values. We were able to obtain positive cultures from 29.5% of those patients with CD4+ counts greater than 500/mm3. HIV-1 titers in plasma also correlated with high serum p24 antigen levels when serum was treated with glycine to dissociate antigen-antibody complexes.

The p24 antigen is only detectable in a minority of asymptomatic HIV infected individuals, which is in part felt to be secondary to immune complex formation with p24 antibody. We sought to determine if the recovery of p24 antigen could be enhanced by an acidification procedure in an attempt to dissociate p24 antibody-antigen complexes. We tested 291 HIV antibody positive serum samples in duplicate, comparing the standard Coulter p24 Antigen Assay with the same assay which was modified by pretreating samples for 60 min with 1.5 M glycine (pH 1.85) and then neutralizing the sample with 1.5 M Tris (pH 9.0). Using the assay cutoff (approximately 7.8 pg/ml), the standard assayed sera and the acid pretreated sera were positive in 65/291 (22.3%) and 167/291 (57.4%) of the samples, respectively. This difference between procedures was statistically significant (p < 0.0001) by McNemar chi 2. There was also a statistically significant difference between Walter Reed stages for p24 antigen quantification by ANOVA (p < 0.0001). We have demonstrated that the detection of p24 antigen may be significantly enhanced by acid pretreatment of sera which dissociates immune complexed antigen. We have also shown significant differences exist between Walter Reed stages for quantitative p24 antigen levels. Enhanced detection of p24 antigen may be important prognostically and may allow for the monitoring of antiretroviral therapy.