Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies

Abstract

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to naïve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.

FIG. 1.

Endpoint titrations of anti-SHIV_DH12 neutralizing activities present in purified chimpanzee IgGs. The virus-neutralizing activities in IgGs purified from a chimpanzee chronically infected with HIV-1_DH12 (top) or a virus-naïve animal (bottom) were evaluated in quadruplicate cultures of MT-4 cells following a 1-h incubation of virus (75 TCID₅₀) with threefold serial dilutions of plasma. The presence or absence of progeny virus production was measured by ³²P-reverse transcriptase assays performed on aliquots of the culture supernatant from day 14 of infection. The black spots shown in the autoradiograms indicate the presence of virion-associated reverse transcriptase activity (i.e., no virus neutralization).

FIG. 2.

Neutralization titers in plasma following the passive transfer of large amounts of purified chimpanzee IgG to naïve pig-tailed macaques. Shown are endpoint neutralization titers in pairs of monkeys receiving the maximum amounts of IgG from an HIV-1-infected (left panels) or naïve (right panels) chimpanzee. The neutralization assays were performed with samples collected immediately prior to IgG administration (day −1) and SHIV_DH12 challenge (day 0). Assays were carried out as described in the legend to Fig. 1.

FIG. 3.

PBMC-associated viral DNA loads in passively immunized pig-tailed macaques challenged with SHIV_DH12. (A to G) DNA was prepared from PBMC, collected from recipients of anti-HIV-1_DH12 IgG, and analyzed by quantitative DNA PCR over a 4-month period for the presence of SHIV_DH12. (H) Levels of PBMC-associated viral DNA in the five immunized and two naïve monkeys which became infected are shown in an expanded time scale.

FIG. 4.

Plasma viral RNA levels in passively immunized pig-tailed macaques challenged with SHIV_DH12. The number of SHIV_DH12 RNA copies in IgG recipients was determined over a 4-month period following an intravenous SHIV_DH12 challenge by quantitative RT-PCR.