HIV type 1 subtype E-infected patients with broadened, dual (B/E) V3 loop serology have increased cross-neutralizing antibodies.

The two prevalent subtypes of HIV-1 circulating in Thailand are subtypes E and B. While the most prevalent subtype continues to be E using molecular typing assays, immunologically, a subset of subtype E-infected patients (3.4% in 1997) have binding antibodies to both the E and B V3 loops in a peptide ELISA. To assess the potential function of this dual (B/E) V3 reactivity, plasmas from patients with genetically defined HIV-1 subtype E infection and either E or B/E V3 serotypes were compared for magnitude and breadth of neutralization of seven primary and laboratory-adapted subtype B and E viruses. Dually reactive (B/E) plasmas showed significantly increased cross-neutralizing activity against subtype B viruses (p < 0.001), and increased neutralization of the panel of viruses overall (p < 0.02), as compared to monoreactive E serotype plasmas. While the total envelope binding antibody titers to both subtype B and E envelopes did not differ significantly between the E and B/E plasmas, 67% of B/E plasmas neutralized >50% of the viruses in the panel, and only 14% of E plasmas showed this broadened neutralizing activity. These data suggest that dual (B/E) V3 loop reactivity may be a marker of broader immune recognition of HIV envelope epitopes in subtype E-infected patients. V3 loop antibody, perhaps in conjunction with antibodies to additional epitopes, may play a role in neutralization of virus isolates from Thailand.

Distinguishing Plasmodium falciparum treatment failures from reinfections by restrictions fragment length polymorphism and polymerase chain reaction genotyping.

The inability to distinguish recrudescent Plasmodium falciparum infections (treatment failures) from reinfections (new infections) is an important impediment to the evaluation of antimalarial treatment regimens. Ten paired primary and recrudescent isolates collected near the Thai-Cambodian border were analyzed by restriction fragment length polymorphism (RFLP) and by polymerase chain reaction (PCR) genotyping of the genes encoding the following proteins: circumsporozite (CS) protein, erythrocyte binding antigen (EBA)-175, ring-infected erythrocyte surface antigen (RESA), merozoite surface protein-1 (MSP-1), and MSP-2. Both methods demonstrated that the fingerprint pattern of each recrudescent isolate was identical to or was contained within the pattern of the primary isolate. Each recrudescent isolate was unique when compared with the other nine primary isolates. Typing by PCR was more sensitive for the detection of multiclone infections and could be performed with small volumes of whole blood. The PCR genotyping could be a practical method for distinguishing a recrudescent from a new infection when treatment studies are conducted in areas with active malaria transmission.

Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line.

Plasmodium falciparum parasites develop in the liver before being released into the bloodstream, where they exert the potentially lethal effects characteristic of malaria. Our understanding of the hepatic phase of the life cycle is limited by the parasite's requirement for fresh human liver cells in which to mature. In this work, liver parasites completed their development within a Thai human hepatoma cell line (HHS-102), and the presence of ring-form parasites in erythrocytes overlying the liver cell culture confirmed that an entire liver cycle was completed, culminating in the production of viable blood-stage parasites. The HHS-102 cell line allows investigation of the undefined liver stage of falciparum malaria previously unavailable in the laboratory.

A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates.

BACKGROUND: Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV-1 primary isolates. METHODS: A modified neutralization assay was performed using CD8-depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tested using pooled sera or plasma from subtype B' or E infected patients. RESULTS: Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24(+) cells, depending on the virus. Less than 0.2% p24(+) cells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. CONCLUSIONS: Flow cytometric detection of intracellular HIV-1 p24 can be used as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative endpoint for HIV neutralization assays.