Keiji Mitamura

Quantitative analysis of human immunodeficiency virus type-1 DNA in asymptomatic carriers using the polymerase chain reaction

A method for detecting human immunodeficiency virus type 1 (HIV-1) provirus DNA in lymphocytes with improved sensitivity and reproducibility was developed using the polymerase chain reaction (PCR). Amplified HIV-1 DNA was hybridized with a 32P-labeled probe and quantitated with a beta-scanner after electrophoresis. A linear relationship was obtained between the common logarithms of the counts detected and the number of HIV-1 DNA copies applied to the PCR. Detectability was from 3 copies/10(5) lymphocytes, and linearity was maintained from 10 to 10(3) copies. HIV-1 DNA was detected in all 9 asymptomatic carriers tested (18 to 2,857 copies/10(5) CD4+ T lymphocytes). The viral burden was inversely related to the CD4+ lymphocyte count, suggesting that quantitation of provirus levels may serve as a predictor of progress in early HIV infection.

Inhibition of human immunodeficiency virus type-1-induced syncytium formation and cytopathicity by complestatin

Complestatin, an anti-complement agent, was shown to be a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) infection in vitro. It inhibited HIV-1-induced cytopathicity and HIV-1 antigen expression in MT-4 cells; the 50% effective doses for these effects were 2.2 and 1.5 micrograms/ml, respectively. No toxicity for MT-4 cells was observed at concentrations up to 400 micrograms/ml. In addition, the agent inhibited the focus formation in HT4-6C cells (CD4-positive HeLa cells); the concentration for 50% focus reduction was 0.9 microgram/ml. HIV-1-induced cell fusion in cocultures of MOLT-4 cells and MOLT-4/HTLV-IIIB were also blocked by complestatin (the concentration for 50% cell fusion inhibition, 0.9 microgram/ml). Complestatin had no ability to inhibit HIV-1 reverse transcriptase activity. When MT-4 cells were pretreated with complestatin for 2 hrs prior to the exposure to HIV-1, the HIV-1-induced cytopathicity was markedly inhibited, while pretreatment of HIV-1 with the agent did not affect the infection. These results suggest that complestatin primarily interacts with cells and inhibits viral adsorption to the cell surface as well as adsorption of infected cells to adjacent cells.

Quantitative estimation of human immunodeficiency virus type-1 provirus in CD4+ T lymphocytes using the polymerase chain reaction

The proviral burden of peripheral blood lymphocytes in 29 patients infected with human immunodeficiency virus type-1 (HIV-1) was estimated using a polymerase chain reaction method which we recently refined. The mean numbers of HIV-1 provirus in eight patients with AIDS and AIDS-related complex treated with zidovudine (AZT), in 10 asymptomatic patients treated with AZT, and in 11 asymptomatic untreated patients were 892, 436, and 406 copies in 1 x 10(5) CD4- T lymphocytes, respectively. These results demonstrate that patients may have more HIV-1 provirus copies in an advanced than early stage of disease, and that the fraction of the infected lymphocytes is much higher than previously thought, especially in asymptomatic patients. There was no difference in the viral burden in CD4+ T lymphocytes regardless of whether AZT had been administered or not. These findings validate the method used here to quantitate HIV-1 provirus DNA and confirm that AZT is not effective in reducing the amount of provirus DNA in lymphocytes.

Immune Response of Mice Infected with Recombinant Vaccinia Viruses Carrying the HIV gag Gene

We examined mouse immune response to 4 kinds of recombinant vaccinia viruses carrying the HIV gag gene, including vac‐gag/pol, which produces HIV‐like particles with processed gag proteins; vac‐gag, which also produces HIV‐like particles but with unprocessed gag protein; and vac‐gag‐pol‐fuse and vac‐es‐gag/pol, neither of which produces such particles but releases reverse transcriptase and gag protein, respectively, from infected cells. Although infection of mice with recombinant vaccinia viruses induced production of the anti‐p24 antibody in all mice, vac‐gag/pol and vac‐es‐pol induced higher production than the other two recombinants. Increase in [3H]thymidine uptake by splenic lymphocytes following p24 antigen stimulation was most evident in mice infected with vac‐gag/pol. Thus, the highest immune reaction, both humoral and cellular, was elicited by vac‐gag/pol, indicating that among those tested, this recombinant vaccinia virus is the best candidate for a vaccine that induces anti‐HIV gag immunity.