Analysis of hepatitis G virus (HGV) RNA, antibody to HGV envelope protein, and risk factors for blood donors coinfected with HGV and hepatitis C virus.

Serologic, biochemical, and molecular analyses were used to study hepatitis G virus (HGV), antibody to the HGV envelope protein (anti-E2), risk factors, clinical significance, and the impact of HGV on coexistent hepatitis C virus (HCV). Among 329 donors with confirmed HCV infection, 12% were HGV RNA-positive and 44% were anti-E2-positive (total exposure, 56%). HGV RNA and anti-E2 were mutually exclusive except in 9 donors (1.5%); 8 of 9 subsequently lost HGV RNA but anti-E2 persisted. HGV had little impact on alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transpeptidase in donors with HGV infection alone or those coinfected with HCV. A multivariate analysis showed that intravenous drug abuse was the leading risk factor for HGV transmission, followed by blood transfusion, snorting cocaine, imprisonment, and a history of sexually transmitted diseases. In summary, HGV and HCV infections were frequently associated and shared common parenteral risk factors; HGV did not appear to cause hepatitis or to worsen the course of coexistent hepatitis C.

High-level viremia in adults and children infected with human immunodeficiency virus: relation to disease stage and CD4+ lymphocyte levels.

Sixty-eight adults and nine children infected with human immunodeficiency virus type 1 (HIV-1) were evaluated consecutively for the presence and amount of cell-free infectious virus in their plasma. Viremia was detected in 18 of 68 adults and in five of nine children; titers ranged from 10 to 100,000,000 TCID/ml plasma. Among the adults, none of 19 asymptomatic patients, 4 of 34 AIDS-related complex patients, and 14 of 15 AIDS patients had cell-free infectious virus in their plasma. None of 35 adult subjects with CD4+ lymphocyte counts greater than 400/mm3 were viremic, whereas 3 of 17 with 200-400 CD4+ lymphocytes/mm3 and 15 of 16 individuals with less than 200 CD4+ lymphocytes/mm3 were plasma viremic. In contrast to adults, each of five children infected with HIV-1 in utero or during the perinatal period were plasma viremic regardless of their CD4+ lymphocytes counts (range, 42-2227/mm3), duration of infection, or clinical stage; however, children infected by HIV-1 at older ages were less frequently plasma viremic. Therapy with zidovudine led to a 10- to 10(6)-fold decline in plasma HIV-1 TCID in all eight subjects studied before and after treatment.

Plasma viremia in human immunodeficiency virus infection.

To determine which markers of human immunodeficiency virus type 1 (HIV) replication correlate most closely with progressive disease, we compared the following: (1) the frequency of isolation of HIV from peripheral-blood mononuclear cells (PBMC), (2) the frequency of isolation of the virus from cell-free plasma (plasma viremia), (3) the presence and titer of p24 antigen in plasma, and (4) the presence and titer of antibody to p24 antigen. We studied 213 persons who were positive for HIV antibody and 71 who were negative. HIV was isolated from PBMC from 207 of the 213 antibody-positive patients (97 percent), regardless of the clinical stage of the infection. Plasma viremia, in contrast, was correlated with the clinical stage of the infection. It was detected in 11 of 48 patients (23 percent) with asymptomatic infection, 32 of 71 (45 percent) in Class IVa of the Centers for Disease Control (those with AIDS-related complex), and 75 of 92 (82 percent) in Class IVc (those with AIDS) (P less than 0.01). Plasma HIV titers ranged from 10(0) to 10(4.3) and rose from a mean of 10(1.4) in asymptomatic patients to 10(2.5) in those with AIDS (P less than 0.02). Only 45 percent of patients with plasma viremia had HIV p24 antigen in either serum or plasma, and no correlation was found between the amount of p24 antigen in plasma and the plasma HIV titers. Follow-up tests indicated that plasma viremia was associated with a more marked decline in the CD4-lymphocyte cell count and the development of symptomatic disease (P = 0.034). We conclude that plasma viremia is a more sensitive virologic marker of the clinical stage of HIV infection and viral replication than the presence of p24 antigen or antibody in plasma. Not only whole blood but cell-free plasma from HIV-infected patients should be considered potentially infectious.

Effect of zidovudine on serum human immunodeficiency virus core antigen levels. Results from a placebo-controlled trial.

We assessed the effect of antiviral therapy on serum human immunodeficiency virus core antigen (HIV-Ag) levels in patients enrolled in the phase II trial on zidovudine for acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. Human immunodeficiency virus core antigen was detected in 45% of subjects at entry (59% with AIDS and 37% of patients with AIDS-related complex). Median HIV-Ag levels in zidovudine-treated subjects fell from 111 pg/mL at entry to 46 pg/mL at four weeks, while levels in placebo recipients did not change significantly. Decline in HIV-Ag in zidovudine recipients was sustained through 16 weeks of treatment and was significantly different from the placebo group. Anti-p24 antibody levels did not change in either group. We conclude that in patients with HIV-antigenemia changes in HIV-Ag level are an important marker of anti-retroviral activity.

False-positive results of screening for antibodies to human immunodeficiency virus in chronic hemodialysis patients.

Eighty-three chronic hemodialysis patients were tested for human immunodeficiency virus (HIV) infection. Testing included screening enzyme immunoassay (EIA) for HIV antibodies, competitive EIA for envelope and core antibodies, EIA for HIV antigen, and lymphocyte culture. Five (6%) of the patients had positive screening EIA at low reactivity. Four of these five had antibodies to H-9 cellular antigens. Comparison of the five seropositive patients to matched controls showed no significant differences in number of lymphocytes or helper/suppressor ratio. Six months later, the five patients had negative screening EIA results using a kit with a manufacturing change approved by the Food and Drug Administration that provided improved specificity. In addition, their Western blot analysis was negative. We conclude that (1) false-positive screening EIA results are more common in chronic hemodialysis patients than other populations; (2) evaluation of chronic hemodialysis patients for HIV infection requires confirmatory tests; and (3) newer EIA screening kits appear to have improved specificity.

Treatment of the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex with a regimen of 3'-azido-2',3'-dideoxythymidine (azidothymidine or zidovudine) and acyclovir. A pilot study.

On the basis of observation that acyclovir potentiates the in-vitro antiviral activity of 3-azido-2',3'-dideoxythymidine (also known as azidothymidine or zidovudine) against human immunodeficiency virus (HIV), we administered a regimen of azidothymidine and acyclovir to eight patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. An oral regimen of 100 mg of azidothymidine and 800 mg of acyclovir every 4 hours was in general well tolerated, with the principal toxicity being megaloblastic erythroid changes. The pharmacokinetics of the two drugs were independent of each other. Six patients received the drug combination for at least 10 weeks; all had increased numbers of T4+ lymphocytes (P = 0.028), and two of three assessable patients had reversal of anergy. Two patients tested positive for serum HIV p24 antigen at entry, but became negative with treatment. Data for this small group suggest that this drug combination can be tolerated in patients with severe HIV infections; this study can be used as a basis for larger studies of this drug combination.

HIV-1 inhibition by azidothymidine in a concurrently randomized placebo-controlled trail.

Two independent measures of human immunodeficiency virus type 1 (HIV-1) infection, virus isolation, and serum levels of p24 antigen were evaluated in a double-blind randomized clinical trial of the safety and efficacy of a nucleoside analogue, 3'-azido-3'-deoxythymidine (AZT) versus placebo in a single center. Pretreatment studies from 38 AIDS and AIDS-related complex (ARC) patients were comparably positive for virus isolation from their lymphocytes; all patients were qualitatively virus positive. Before AZT treatment, there was significantly decreased virus recovery in patients with higher numbers of CD4-positive lymphocytes. Within 1 month of AZT therapy, the time in culture required to register virus positivity was increased markedly in the AZT-treated group, and over the following several months progressive diminution in virus recovery was noted. Similar changes were not seen in patients concurrently receiving placebo treatment. Before treatment, 16 of 20 and 12 of 16 patients in the AZT and placebo groups, respectively, were p24 antigen positive. Marked reduction in serum p24 levels were noted in 11 of 16 (69%) of the p24 antigen-positive AZT-treated patients compared to 3 of 12 (25%) of the p24 antigen-positive placebo-treated patients (p = 0.02). There was a marked virologic response in 14 of 20 (70%) of the AZT-treated patients compared to 4 of 18 (22%) placebo-treated patients (p = 0.004). A higher frequency of positive clinical and immunological effects also were noted in the AZT-treated patients relative to placebo-treated patients (p = 0.02 and p = 0.06, respectively).

Long-term evaluation of HIV antigen and antibodies to p24 and gp41 in patients with hemophilia. Potential clinical importance.

To investigate the relation between human immunodeficiency virus (HIV) antigenemia and clinical manifestations of HIV infections, we studied 96 patients with hemophilia who were positive for HIV antibody, for a median of 34 months. Every 4 to 10 months a clinical and laboratory examination was performed and serum samples were tested for three HIV markers: HIV antigen, antibody to p24, and antibody to gp41. Twenty-two subjects (23 percent) were found to be positive for HIV antigen: 8 were positive upon entry and remained so (Group 1), and 14 became positive during the study, 4 to 26 months after HIV antibody appeared (seroconversion), 13 of whom remained positive for HIV antigen (Group 2). Most subjects positive for HIV antigen had low or undetectable titers of antibody to p24, whereas the antibody titer to gp41 remained high. In Group 2, patients with low p24 antibody titers had further decreases in their titers before or at the time HIV antigen appeared. Once present, HIV antigen persisted and tended to increase in concentration. In contrast to Group 3 (negative for HIV antigen, low anti-p24 titer) and 4 (negative for HIV antigen, high anti-p24 titer), the groups positive for HIV antigen had significantly higher incidences of acquired immunodeficiency syndrome (P = 0.05), immunodeficiency-related infections (P less than 0.001), and immune thrombocytopenia (P = 0.001), and had more severe disease as measured by the Walter Reed staging system (P less than 0.001). In this study, HIV antigen appeared to be a better predictive marker of HIV-related complications than the absolute T4+ count. These results suggest that HIV antigenemia indicates a poor clinical prognosis.

Long latency precedes overt seroconversion in sexually transmitted human-immunodeficiency-virus infection.

Signs of latent HIV infection were sought in stored serum samples collected before overt seroconversion, confirmed by enzyme-linked immunosorbent assay (ELISA), from 9 subjects with human-immunodeficiency-virus (HIV) infection, in serum from 25 seronegative sexual partners of HIV-seropositive men and from 23 other seronegative, homosexually active men. Free HIV antigen and/or low-titre antibodies to recombinant structural (core, env) or non-structural (3' orf, sor, tat) proteins were seen 6-14 months before seroconversion in all 9 subjects who seroconverted. Antibodies against core proteins detected by western blot were usually the first sign of latent HIV infection. 5 of the 25 ELISA-negative exposed partners have shown HIV antigenaemia and antibodies against core proteins for 16-34 months. By in-situ hybridisation, HIV-specific RNA was detected in peripheral-blood non-lymphoid mononuclear cells in some of the latently infected partners. All subjects with latent HIV infection had normal numbers of T4 lymphocytes but half of them lost their in-vitro proliferative T-cell response to a recall antigen (purified protein derivative of tuberculin). Early HIV infection, characterised by a low-level and restricted antibody response towards HIV core and regulatory proteins, seems mainly to affect antigen-presenting cells.

Detection of HIV antigen and specific antibodies to HIV core and envelope proteins in sera of patients with HIV infection.

The sera of well-characterized populations were examined for three markers of human immunodeficiency virus (HIV) infection; HIV antigen (HIV Ag), and antibodies to HIV envelope (gp41) and core (p24) proteins. Of 563 serum samples tested, 251 were from HIV-infected patients diagnosed as having AIDS manifested by opportunistic infections (AIDS-OI), AIDS-associated Kaposi's sarcoma (AIDS-KS), or AIDS-related complex (ARC). One hundred seventy-six specimens tested were from asymptomatic high-risk individuals, and 136 were from heterosexual control subjects or patients with non-AIDS-related disease. None of the 136 control individuals tested had HIV Ag or HIV antibodies to either p24 or gp41. Of the 427 HIV-seropositive individuals, 99% to 100% were positive for gp41 antibodies to HIV. In contrast, the seroprevalence of p24 antibodies to HIV varied from 23% to 83% and appeared to be inversely associated with the severity of the patients' clinical symptoms. When specimens were analyzed for the presence of HIV Ag, in seropositive individuals the prevalence rate for this marker was lowest (1.4%) in asymptomatic individuals and highest (50%) in the AIDS-OI diagnosed group. Also, 240 cases with AIDS-KS, AIDS-OI, and ARC and the group of asymptomatic high-risk individuals were analyzed for T helper/T lymphocytes (T4) cell number and T4/T8 ratio; only one (2.0%) HIV Ag-positive case showed a T4 cell number greater than 400 and a normal T4/T8 ratio. These studies appear to demonstrate a direct correlation between the presence of HIV Ag and the severity of clinical complications of HIV infection.

Herpes simplex virus binding and entry modulate cell surface protein mobility.

Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.

Changes in lectin receptor lateral mobilities accompany lymphocyte stimulation.

Changes in lateral mobilities of rabbit lymphocyte membrane components in response to succinyl concanavalin A (S Con A) have been studied by fluorescence photobleaching recovery (FPR). During hrs 0 to 3 after exposure to S Con A, lectin receptor mobilities on both T and B cells fall about 2-fold. Reduced mobility of T cell lectin receptors persists until hr 18. From hr 18 to 24 rapid recovery of original mobility occurs if and only if lectin is present. In contrast, nonresponding B cells recover original receptor mobility gradually over hr 4 to 48. Metabolic inhibitors added at hr 3 restore original receptor mobilities, but cytoskeletal disruptors have this effect on T cells only. From hr 0 to 15, washing lectin from the cell surface is decreasingly effective in restoring T cell receptor mobility. After hr 15, mobility cannot be enhance by lectin removal. Parallel DNA synthesis studies show that, for T cell stimulation, lectin must be present on the cell surface during hr 0 to 3 and 18 to 24. These are the periods when FPR measurements show lectin receptor mobilities being restricted and released, respectively. Stimulation of B cells by anti-Ig shows several interesting features. First, stimulation by intact anti-Ig fails to reduce the mobilities of Con A receptors in a manner similar to that produced by S Con A. Second, S Con A does reduce mobility of surface Ig. Thus, Con A receptors would appear to exert a unique anchorage modulation of mobilities of other membrane molecules.

Fluorescence photobleaching recovery measurement of protein absolute diffusion constants.

The technique of fluorescence photobleaching recovery [Axelrod et al., Biophys. J. 16 (1976) 1055] has been applied to the measurement of absolute diffusion constants of a number of fluoiescein isothiocyanate-labeled proteins. Measured diffusion constants agree to within +/- 7% of published values for the underivatized proteins. The method has sufficient sensitivity to reveal the concentration dependence at neutral pH of the diffusion constant of alpha-chymotrypsin. The rapidity with which the labelling and measurements can be performed and the small amount of material required suggest the technique may be useful in rapid characterization of small protein samples. Some developments in optical and electronic systems and in data processing for this technique are discussed.