Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

The nfkb1 promoter is controlled by proteins of the Ets family.

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which we refer to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. We show that EBS4 is, required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4, and a mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner.

The efficacy and safety of zidovudine with or without acyclovir in the treatment of patients with AIDS-related complex. The European-Australian Collaborative Group.

Our objective was to evaluate the efficacy and safety of zidovudine (250 mg every 6 h) alone or in combination with acyclovir (800 mg every 6 h) as treatment for AIDS-related complex (ARC). A double-blind, controlled clinical trial of 6 months therapy was conducted at teaching hospital ambulatory clinics in eight European countries and Australia; 199 patients were studied. Time to development of AIDS-defining opportunistic infections (OI) and AIDS-associated neoplasms, survival, performance status, body weight and CD4+ cell counts were measured. During the study six (9%) zidovudine recipients, five (7%) combination recipients and 12 (18%) placebo recipients developed AIDS-defining OI; the probability of developing an OI was 0.23, 0.09 and 0.08 for the placebo, zidovudine and combination recipients, respectively. Four patients in the placebo group, three in the zidovudine group and one in the combination group died during the study. Patients receiving zidovudine with or without acyclovir had moderate increases in CD4+ cell counts compared with placebo recipients and serum HIV p24 antigen level decreased significantly in all those receiving zidovudine. Fourteen (21%) patients in the zidovudine group and 16 (24%) in the combination group experienced bone-marrow suppression compared with three (5%) placebo recipients. Red-cell transfusions were administered to 6, 19 and 13% of placebo, zidovudine and combination recipients, respectively. These data confirm the efficacy of zidovudine therapy after 4 weeks' treatment in the reduction of development of OI in patients with ARC and support the use of a maintenance dose of 250 mg zidovudine 6-hourly. Given the increased development of OI in the treated groups compared with placebo during the first 4 weeks of therapy, we cannot exclude an initial adverse effect of zidovudine and recommend caution in the use of a loading dose of zidovudine. At 6 months there was no apparent difference in efficacy between the combination of zidovudine and acyclovir compared with zidovudine alone. Moreover, the addition of high-dose acyclovir resulted in a minimal increase in the risk of toxicity.

Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies.

An enzyme immunoassay (EIA) utilizing a synthetic peptide analogue of HIV gp41 (amino acids 579-599, RILAVERYLKDQQLLGIWGCS) as antigen was compared with two commercial assays (Genetic Systems, Abbott ENVACORE) for the ability to detect antibodies in the early stages of infection. Two panels, consisting of 96 sera from 15 people and 140 sera from 44 people seroconverting to HIV, were examined. In the first group the synthetic peptide assay (gp41 EIA) detected antibodies before the Genetic Systems EIA in seven out of 15 people and concurrently in the remaining eight. With the second panel the Abbott ENVACORE assay detected antibodies before the gp41 EIA in two out of 44 people while the gp41 EIA detected antibodies first in six out of 44. In the remaining 36 people antibodies were detected simultaneously by the two tests. The gp41 EIA usually detected anti-HIV antibodies before or concurrently with the two commercial assays examined suggesting that the epitope cluster represented by this peptide is recognized early in infection.

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma.

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gp120 (2-13), (55-65), gp41 (582-596) (659-670) and tatIII (71-83) were recognized by 41-67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582-596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579-599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (25%) to the tatIII peptide.

Quantitation of infectious human immunodeficiency virus using a modified plaque forming assay.

This report describes a modified plaque forming assay which has been used for quantitation of infectious human immunodeficiency virus (HIV) in cell culture supernatant fluid. The number of infectious units determined by the plaque assay correlates closely with the 50% infectious dose determined by a conventional assay in cell culture. In contrast, particle associated reverse transcriptase (RT) activity correlates poorly with the amount of infectious HIV present in culture supernatant fluids.

HTLV-I in Australia and Oceania: long-term resident or recent immigrant?

Human T-cell lymphotropic virus type I (HTLV-I) has a worldwide distribution; infection rates of up to 14% have been found in Aboriginal communities, but there is little evidence of typical HTLV-I-associated disease. The strains among Australian Aboriginals and Melanesians are more closely related to each other at the molecular level than to strains from Africa, Japan and the Caribbean basin. The clinical significance of these Oceanic strains of HTLV-I in endemically infected communities is unclear.

HTLV-I among Northern Territory aborigines.

OBJECTIVE: To survey the Aboriginal community of the Northern Territory for antibodies to human T-lymphotropic virus type I (HTLV-I) and to describe the distribution of the virus. DESIGN: A sero-epidemiological study using the Serodia particle-agglutination assay, indirect immunofluorescence and western blot. Evidence of HTLV-I-related diseases was sought through clinicians, and by searching the cancer register and medical records. SERA: Samples from 1897 Aborigines, including 1569 sera received by the Royal Darwin Hospital Pathology Department for syphilis serology between March and July 1988. Most of the specimens were from public health surveys and antenatal screening. RESULTS: Ninety-four samples (5.0%) were positive by the particle-agglutination assay method but only 36 (1.9%) were positive by both particle-agglutination assay and indirect immunofluorescence. After confirmation by western blot, the seroprevalence of HTLV-I was 1.7% (95% confidence interval, 1.2-2.3%). Western blot positivity was higher in samples from the "Cattle Country" and Alice Springs regions (i.e., 4.7% and 13.9% respectively). CONCLUSION: HTLV-I is endemic among Aborigines in inland Australia. These serological findings are supported by the recognition of two cases of adult T-cell leukaemia/lymphoma in this population.

Definition of an immunodominant T cell epitope contained in the envelope gp41 sequence of HIV-1.

The majority of the immunodominant amino acid sequences of HIV-1 that have been characterized to date are coded for by hypervariable gene sequences. These variable sequences are however interspersed with sequences that are highly conserved between HIV strains. Immunogenic viral products with amino acid sequences that vary minimally between strains, and that consistently elicit both humoral and cellular immune responses, may be ideal for inclusion in a subunit vaccine. We studied HIV-seronegative and HIV-infected persons, classified as asymptomatic (AS), ARC or AIDS. Initially, we assessed the cellular immune status of each subject from results of T cell phenotype analyses, assays for serum levels of surrogate markers of disease progression, and responses to mitogens and recall antigen. In addition, we tested whether three short synthetic peptides derived from the conserved sequences of the envelope gp120 (aa 262-284) and gp41 (aa 579-601), and core p17 (aa 106-125) regions of the HTLV-IIIB isolate, could elicit B cell as well as T cell responses in HIV-infected subjects. Only the gp41-derived sequence was immunogenic at both B and T cell levels. To further characterize the gp41 epitope, we used a series of overlapping synthetic peptides derived from a conserved region of the envelope gp41 (aa 572-613). We thus identified an immunodominant 12-mer peptide sequence, gp41(8)(aa 593-604), which consistently elicited both T cell blastogenic and B cell (antibody) responses in AS HIV-seropositive individuals but not in ARC and AIDS patients. Linear regression analysis showed that in AS persons there was a strong positive correlation (P less than 0.0005) between the absolute CD8+ T cell numbers and the magnitude of blastogenic responses to the gp41(8)(aa 593-604). Furthermore, those AS subjects with T cells that proliferated in response to this gp41 analogue also had significantly greater serum levels of antibody to the same short peptide sequence than symptomatic ARC and AIDS patients. These results suggest that cellular responses to the immunodominant and highly conserved envelope sequences of HIV-1, associated with increased CD8+ T cells, may be important in the pathogenesis of HIV disease.

CD8+ T-cells from HIV-infected patients can either augment or abrogate HIV-specific lymphoproliferation.

Despite the presence of activated CD8+ T-cells that have been identified in infected individuals, these cells do not overcome natural HIV infection. To understand this better, we analyzed the CD8+ cell-dependent HIV-specific lymphoproliferation that occurs after HIV infection. Our study group of 36 individuals included 11 asymptomatic and 16 symptomatic patients (12 ARC and 4 AIDS), as well as HIV-seronegative controls. After CD8+ cell depletion of PBMC cultures, the remaining cells were tested for proliferation during culture with a well-defined and immunodominant gp41-derived HIV analog, gp41(8). After CD8+ cell depletion, three functional outcomes, which differed in accordance with the disease status of the individual, were consistently recorded, namely (i) an "abrogation effect," (ii) an "augmentation effect," or (iii) "no effect." First, removal of CD8+ cells from PBMC cultures abrogated gp41(8)-specific lymphoproliferation in gp41(8)-specific responders. Paradoxically, in other patients, including 5 symptomatics, the same inhibition of CD8+ cell function caused significant augmentation of gp41(8)-specific lymphoproliferation. These results suggest that the subpopulations of CD8+ T-cells that predominate at different stages of HIV-induced disease have different functional properties, including the ability to modulate HIV-specific cell-mediated immunity.

Altered sensitivity to antiviral drugs of herpes simplex virus isolates from a patient with the acquired immunodeficiency syndrome.

Acyclovir (ACV)-resistant herpes simplex virus type 2 (HSV-2) was isolated from a patient with acquired immunodeficiency syndrome after long-term but intermittent ACV therapy. These thymidine kinase-defective isolates were sensitive in vitro to foscarnet. While combined therapy with ACV and interferon produced only partial clinical improvement, the in vitro effect of this combination against an ACV-resistant isolate from the patient was strongly synergistic. A short course (10-12 days) of intravenous foscarnet controlled severe ulceration, and clinical improvement lasted 6 months. After recurrence and further courses of foscarnet, however, the patient responded poorly, and subsequent HSV isolates were resistant to both ACV and foscarnet and hypersensitive to aphidicolin.

Heterogeneous effects of exogenous IL-2 on HIV-specific cell-mediated immunity (CMI).

A characteristic feature associated with HIV-1 infection of the human host is a chronic decline in circulating CD4+ T helper/inducer cell numbers. Impaired cell-mediated immune functions usually occur in parallel with the decline in CD4+ T cells. Activated CD4+ T helper cells are a major source of endogenous IL-2 which is required for the immunoregulation of both antigen-specific B cells and CD8+ T cells. HIV-specific T cell proliferative responses are said to be weak and inconsistent, even during the asymptomatic phase of disease. We thus wished to determine how exogenous IL-2 affected HIV-specific T cell proliferation at different stages of the disease. Our cohort of 81 included both asymptomatic and symptomatic HIV-infected patients as well as uninfected normal donors. Proliferative responses of peripheral blood mononuclear cells (PBMC) that were elicited during culture with an immunodominant gp41-derived synthetic peptide, gp41[8], and which were known to be CD8+ cell-associated in asymptomatics only, were used to analyse the effects of exogenous IL-2. IL-2 had three main effects on HIV-specific proliferation, namely (i) an additive effect, (ii) a synergistic effect, and (iii) an induced effect. More specifically, low dose exogenous IL-2 frequently augmented lymphoproliferation in both asymptomatic and symptomatic gp41[8] responders. In most symptomatics, however, who were predominantly gp41[8] non-responders, exogenous IL-2 induced lymphoproliferation. Flow cytometric analyses using dual immunofluorescence were used to analyse the T cell subset distribution of proliferating PBMC cultures. During culture with gp41[8], both CD4+ and CD8+ T cell numbers increased. However, after the addition of exogenous IL-2 to gp41[8]-containing cultures, CD8+ cell-associated lymphoproliferative responses were preferentially augmented. These results suggest that in symptomatics there is an inadequate supply of endogenous IL-2 to help maintain the strong and effective CD8+ cell-associated anti-viral immunity, and an exogenous supply of IL-2 may be required.

Spotted fever in East Gippsland, Victoria: a previously unrecognised focus of rickettsial infection.

A new focus of spotted fever group rickettsial infection has been recognised in East Gippsland, Victoria. Seven cases have been identified among Melbourne residents after they holidayed in the area. The infections were confirmed serologically. The precise identity of the Rickettsia has not been determined.

Antibodies to gp41 and nef in otherwise HIV-negative homosexual man with Kaposi's sarcoma.

A homosexual man with histologically confirmed Kaposi's sarcoma remained seronegative for HIV-1, HIV-2, and HTLV-1 on conventional tests over a 4-year period. HIV cultures were also negative on thirteen separate occasions. However, serum antibodies to synthetic peptide analogues of the gp41 and nef regions of HIV-1 were consistently detected on an enzyme immunoassay. Tests with the polymerase chain reaction with primers directed to the gag and env regions were negative. The antigens to which the antibodies were produced might have come from a defective HIV mutant, another retrovirus, or a hitherto unknown "agent of Kaposi's sarcoma" with similar antigenic epitopes.

Patterns of migration indicate sexual transmission of HTLV-I infection in non-pregnant women in Papua New Guinea.

The prevalence of infection with human T-cell leukaemia virus (HTLV-I) was studied in Madang Province on the north coast of Papua New Guinea. Serum specimens collected from non-pregnant women in 17 villages were tested for anti-HTLV-I by gelatin particle agglutination screening and confirmed by immunofluorescence and Western blotting. Overall, 13.9% of subjects were antibody-positive, with the prevalence of antibodies varying from less than 10% to 30% in villages situated less than 10 km apart. Two groups of migrant women were identified, and in both a parity-related increase in antibody prevalence which occurred only after marriage suggested that the predominant mode of transmission in migrant women was sexual. There was no parity-associated increase in anti-HTLV-I in indigenous women, and in contrast to migrant women, nulliparous indigenous women had a high prevalence of antibody (16.8% vs. 0%; p = 0.005). Vertical transmission cannot be excluded in indigenous women. No correlation was detected between the prevalence of anti-HTLV-I and a variety of indices of malarial infection.

New guidelines for management and prevention of meningococcal disease in Australia. Meningococcal Disease Working Party of the National Health and Medical Research Council.

The incidence of invasive meningococcal disease in Australia has increased over the past decade, and in April 1997 the National Health and Medical Research Council published guidelines for management of patients with meningococcal disease and their contacts. These guidelines emphasise the need for immediate intravenous antibiotic treatment of patients with suspected meningococcal disease, before transfer to hospital or lumbar puncture. When possible, blood for culture should be collected before antibiotic therapy, if this does not delay treatment.