Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.

Peptide serology for analysis of the inter- and intra-individual variation in the HIV-1 V3 domain.

OBJECTIVE: To investigate whether the specificity of antibody responses to the gp120 V3 domain in HIV-1-infected individuals is related to the variability of this region. METHODS: Sera from a cohort of 22 HIV-1-infected Ugandans were tested against peptides derived from each individual's autologous proviral V3 apex sequence. Autologous peptide reactivity was compared with reactivity to peptides derived from two Ugandan consensus sequences and previously isolated US/European and African viruses. Peptides from individuals with heterogeneous V3 apex sequences, representing different HIV-1 variants, were obtained and tested against the corresponding sera. RESULTS: A notable cross-reactivity to different V3 apex peptides was observed. However, in the majority of sera, antibody reactivity to the autologous peptides was found to exceed reactivity to any of the other peptides tested. V3 proviral sequences from the Ugandan cohort studied have been shown to be closely related to the HIV-1MN isolate and thus, their sera gave better reactivity to V3MN and related peptides than to peptides representing other African HIV-1 isolates. In individuals with heterogeneous V3 proviral sequences, we could distinguish divergent antibody responses to the genomic variants differing by single amino acids. CONCLUSION: Analysis of seroreactivity to peptides might constitute a relevant tool for investigating the variability of the HIV-1 gp120 V3 domain within infected populations and single individuals.

Ugandan HIV-1 V3 loop sequences closely related to the U.S./European consensus.

The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-1) envelope protein is an important determinant for virus neutralization and cell tropism. V3 loop sequences from uncultured lymphocytes obtained in 1990 from 22 Ugandan HIV-1-infected patients could, with the exception of two patients' sequences, be divided into two groups (A and B) on the basis the V3 loop size and sequence. The V3 loop consensus sequences from both groups showed a high degree of homology to a U.S./European consensus, a characteristic also reflected by the results of peptide serology. In the case of group B the difference in sequence was only five amino acids. In contrast, the V3-flanking regions for both groups showed greater homology to an earlier (1986/1987) Ugandan consensus. The discovery of these two new Ugandan V3 loop genotypes, which are closely related to the U.S./European consensus, has implications for the understanding of the evolution of HIV-1 and for the future design of a vaccine for use in Africa.

Revival of the Ugandan Blood Transfusion System 1989: an example of international cooperation.

10 yr of civil war in Uganda had destroyed the Blood Transfusion Service when the government came to power in 1986. AIDS had become recognized as a problem of severe proportion. In 1987, the E.E.C. pledged to rehabilitate the central blood bank. This paper describes the first year of operation from December 1988. Over 5000 units of blood, largely from volunteer donors, were delivered to 19 hospitals. The overall incidence of HIV-1 seropositivity was 14.6% and Hepatitis B surface antigen was 5.5%. The cost was 21.5 ECU (US $25) for each unit of HIV negative, H.B.s.Ag. negative, blood.

Autoantibodies to intermediate filaments in sera of patients with Schistosoma mansoni infection.

Autoantibodies to the intermediate filament proteins vimentin and keratin were studied in sera of 50 Caribbean patients with Schistosoma mansoni infection and 50 control subjects. Autoantibodies were detected by indirect immunofluorescence on HEp-2 cells pretreated with colchicine. The incidence of anti-vimentin antibodies in patients' sera was 94% for IgM, 12% for IgG, and 4% for IgA; in the control subjects incidence was 52%, 0%, and 4%, respectively. Anti-keratin antibodies were found in 82%, 4%, and 4% of patients' sera and 42%, 0%, and 2% in controls, respectively. The difference between the geometric means of titres for patients (1:150) and controls (1:26) was highly significant (P less than 0.001). The possible role and genesis of autoantibodies to intermediate filaments is discussed.

Anti-intermediate filament antibodies, antikeratin antibody, and antiperinuclear factor in rheumatoid arthritis and infectious mononucleosis.

Sera from patients with rheumatoid arthritis (RA), patients with infectious mononucleosis (IM), and blood donors were tested by indirect immunofluorescence for the presence of antikeratin antibody (AKA), antibody to cytoskeletal intermediate filaments of prekeratin or vimentin type (AIFA) and antiperinuclear factor (APF). In 81.9% of the RA sera and 92.5% of the IM sera AIFA of IgM class was found at titres up to and in some cases exceeding 1/160. In blood donors the incidence of AIFA was 26%, at titres not exceeding 1/20. AKA and APF, always of IgG class, were found in 54.2% and 73.6% of rheumatoid sera. A weak correlation was found in RA between the incidence of AIFA and APF. AKA was not present in either IM or blood donor sera, and APF was found in only 2.5% and 3.2% of IM or blood donors respectively.

Incidence of anti-intermediate filament antibody in serum samples of students with suspected glandular fever.

Serum samples from 40 students with suspected infectious mononucleosis were tested for the presence of antibodies to intermediate filaments (AIFA) of the cytoskeleton. Twenty had antibodies to the Epstein-Barr virus capsid antigen before their illness, and during it their sera remained negative by the Paul-Bunnell test. The other 20 patients did not have antibodies to the Epstein-Barr virus capsid antigen before their illness and seroconverted during the illness. These patients (true infectious mononucleosis group) developed positive Paul-Bunnell tests. Sera from normal subjects (blood donors) were also tested for AIFA. AIFA was present in titres greater than 1/10 in 80% of the infectious mononucleosis group (mean titre 1/40-1/80), 10% of the Paul-Bunnell negative glandular fever group, and 8.5% of the normal blood donors.

Stimulation of autoantibody production in normal blood lymphocytes by malaria culture supernatants.

Supernatants from Plasmodium falciparum cultures containing soluble parasite material were mitogenic for normal human peripheral blood mononuclear cells (MNC) in vitro. This was evidenced by blast transformation and significant incorporation of 3H-thymidine and confirms earlier reports of the mitogenic potential of malaria parasites. Lymphocyte activation by these malaria derived products was polyclonal as demonstrated by increased secretion of IgA, IgG and IgM by the stimulated cells. Using rat tissues and Hep-2 cells as substrates, autoantibody activity was found in the IgM fraction of the secreted immunoglobulin. Speckled anti-nuclear (ANA) antibody, anti-globulins (rheumatoid factor) and anti-intermediate filament antibodies were produced by the stimulated lymphocytes. No significant immunoglobulin secretion with autoantibody specificity was found in control cultures in which normal MNC were incubated with supernatants from non-parasitized red cell cultures. The data supports the suggestion that polyclonal lymphocyte activation by parasite derived products occurs in vivo and, in addition, provides an explanation for the presence of autoantibodies in the serum of malaria patients.

Plasmodium falciparum products enhance human lymphocyte transformation by Epstein-Barr virus.

Supernatants obtained from the in vitro culture of Plasmodium falciparum infected erythrocytes induced prolonged lymphocyte survival in culture for more than 8 weeks in six cultures and permanent cell lines were established in four of these. The cells in the latter showed lymphoblastoid features similar to those seen in parallel cultures to which transforming Epstein-Barr (EB) virus instead of P. falciparum derived substances had been added. Cells from the same donors stimulated with other mitogens (pokeweed mitogen, Salmonella paratyphi culture supernatants) ceased to proliferate and died after 3-4 weeks. A 195 Kd polypeptide obtained from P. falciparum parasites also exhibited the potential to transform normal lymphocytes. Characterization of the cell lines indicated a B lymphocyte origin and the presence of EB virus in these lines suggests the possibility that P. falciparum products may activate latent EB virus genomes. These observations appear relevant to both the choice of P. falciparum derived antigens as vaccines, and to the interaction of EB virus and malaria in the aetiology of African Burkitt's lymphoma (BL).