Kinetics of lymphocyte proliferation during primary immune response in macaques infected with pathogenic simian immunodeficiency virus SIVmac251: preliminary report of the effect of early antiviral therapy

Abstract

The aim of this study was to evaluate the kinetics of lymphocyte proliferation during primary infection of macaques with pathogenic simian immunodeficiency virus (SIV) and to study the impact of short-term postexposure highly active antiretroviral therapy (HAART) prophylaxis. Twelve macaques were infected by intravenous route with SIVmac251 and given treatment for 28 days starting 4 h postexposure. Group 1 received a placebo, and groups 2 and 3 received combinations of zidovudine (AZT), lamivudine (3TC), and indinavir. Macaques in group 2 received AZT (4.5 mg/kg of body weight), 3TC (2.5 mg/kg), and indinavir (20 mg/kg) twice per day by the oral route whereas macaques in group 3 were given AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice per day, to improve the pharmacokinetic action of these drugs, and a higher dose of indinavir (60 mg/kg). The kinetics of lymphocyte proliferation were analyzed by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake ex vivo and by fluorescence-activated cell sorting analysis. HAART did not protect against SIV infection but did strongly impact on virus loads: viremia was delayed and lowered during antiviral therapy in group 2, with better control after treatment was stopped, and in group 3, viremia was maintained at lower levels during treatment, with virus even undetectable in the blood of some macaques, but there was no evidence of improved control of the virus after treatment. We provide direct evidence that dividing NK cells are detected earlier than dividing T cells in the blood (mostly in CD45RA(-) T cells), mirroring plasma viremia. Dividing CD8(+) T cells were detected earlier than dividing CD4(+) T cells, and the highest percentages of proliferating T cells coincided with the first evidence of partial control of peak viremia and with an increase in the percentage of circulating gamma interferon-positive CD8(+) T cells. The level of cell proliferation in the blood during SIV primary infection was clearly associated with viral replication levels because the inhibition of viral replication by postexposure HAART strongly reduced lymphocyte proliferation. The results and conclusions in this study are based on experiments in a small numbers of animals and are thus preliminary.

FIG. 1.

Longitudinal analysis of plasma SIV RNA load (A), proliferation of blood lymphocyte subpopulations (B to E), and lymphocyte blood counts (F to I) in four control macaques inoculated with 50 AID₅₀ of pathogenic SIVmac251 (group 1). The results of ex vivo BrdU incorporation are given for each individual. Lines indicate the median values. Animals received a placebo from 4 h after virus inoculation to day 28 p.i., which is indicated by a vertical dashed line. In graph A, the limit of quantitative measurement of plasma virus load is indicated by a horizontal line at the level of 40 copies/ml. Negative values are plotted on the x axis.

FIG.2.

(A) Evolution of proliferating (BrdU⁺) cell percentages in blood lymphocyte subpopulations and evolution of plasma viral load of one representative SIV-infected macaque of group 1 (Z860). The limit of quantitative measurement of plasma virus load is indicated by a horizontal line at the level of 40 copies/ml. Negative values are plotted on the x axis. (B) The electronic gates and representative dot plots used for the determination of proliferating NK cells and CD4⁺ and CD8⁺ T cells are shown. Percentages of BrdU positive cells are indicated in each panel.

FIG. 3.

Longitudinal analysis of the proliferation of naive and memory CD4⁺ and CD8⁺ T lymphocytes after SIV infection of group 1 macaques. CD45RA was used to designate CD3⁺ CD8⁺ and CD3⁺ CD8⁻ as having a naive (CD45RA⁺) or memory (CD45RA⁻) phenotype. Curves represent median values. Open squares represent CD45RA⁻ cells, and closed triangles represent CD45RA⁺ cells. For better visualization, the results obtained for CD45RA⁺ cells are also shown alone with a magnified scale (small graphs).

FIG. 4.

(A) Lymphocyte proliferation and cell-associated virus loads in the lymph nodes of group 1 control macaques inoculated with a pathogenic SIVmac251. LNMC, lymph node MNCs. (B) Comparison of the percentages of proliferating T and B cells in the blood (14 and 16 days p.i.) and in the lymph nodes (15 days p.i.). (C) Percentages of proliferating cells in CD45RA⁺ (naive) or CD45RA⁻ (memory) T cells in the lymph nodes on day 15 after SIV infection. Median values ± standard deviations of the medians are represented.

FIG. 5.

Effect of HAART on plasma viral load (top panels) and on proliferation of NK cells and CD4⁺ and CD8⁺ T lymphocytes (middle and bottom panels) in macaques treated with HAART after SIV exposure. Group 2 macaques (left panels) were treated twice per day orally with a combination of AZT (4.5 mg/kg), 3TC (2.5 mg/kg), and indinavir (20 mg/kg); group 3 macaques (right panels) were treated twice per day subcutaneously with a combination of AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) and orally with indinavir (60 mg/kg). Treatment was started 4 h postexposure and was stopped on day 28 p.i., which is indicated on each panel by a vertical dashed line. Curves show medians of four macaques per group. In the top panels, the median of plasma viral loads of group 1 control monkeys which were treated with a placebo is superimposed (dashed line), and the limit of quantitative measurement of plasma virus load is indicated by a horizontal line at the level of 40 copies/ml. Negative values are plotted on the x axis.

FIG. 6.

Intracellular IFN-γ expression in blood CD8⁺ T cells over time. PBMC were labeled ex vivo. CD8⁺ T cells were defined on the basis of membrane expression of both CD3 and CD8. Curves represent median values from four macaques per group. Group 1, placebo-treated macaques; groups 2 and 3, macaques treated postexposure with different combinations of AZT, 3TC, and indinavir.