Qualitative T-helper responses to multiple viral antigens correlate with vaccine-induced immunity to simian/human immunodeficiency virus infection

Abstract

Evidence is accumulating that CD4(+) T-helper (Th) responses play a critical role in facilitating effector responses which are capable of controlling and even preventing human immunodeficiency virus (HIV) infection. The present work was undertaken to determine whether immunization with multiple antigens influenced individual Th responses and increased protection relative to a single antigen. Rhesus macaques were primed with DNA and boosted (immune-stimulating complex-formulated protein) with a combination of regulatory and structural antigens (Tat-Env-Gag) or with Tat alone. Immunization with combined antigens reduced the magnitude of the responses to Tat compared to the single-antigen immunization. Interestingly, the Th immune responses to the individual antigens were noticeably different. To determine whether the qualitative differences in vaccine-induced Th responses correlated with vaccine efficacy, animals were challenged intravenously with simian/human immunodeficiency virus (strain SHIV(89.6p)) 2 months following the final immunization. Animals that developed combined Th1- and Th2-like responses to Gag and Th2 dominant Env-specific responses were protected from disease progression. Interestingly, one animal that was completely protected from infection had the strongest IFN-gamma and interleukin-2 (IL-2) responses prior to challenge, in addition to very strong IL-4 responses to Gag and Env. In contrast, animals with only a marked vaccine-induced Tat-specific Th2 response (no IFN-gamma) were not protected from infection or disease. These data support the rationale that effective HIV vaccine-induced immunity requires a combination of potent Th1- and Th2-like responses best directed to multiple antigens.

FIG. 1.

Immunization-and-challenge schedule. Animals were immunized i.m. at weeks 0, 2, and 6 with DNA constructs and boosted at weeks 10, 14, and 24 with protein formulated with ISCOM matrix. At weeks 0, 2, and 6, animals from group A received HIV-1_IIIB tat-, SHIV-1_89.6p env-, and SIV_mac239 gag-expressing DNA plasmids. At weeks 10, 14, and 24, the same animals were boosted with HIV-1_IIIB Tat, HIV-1_89.6 Env gp140, and SIV_mac239 Gag proteins formulated with ISCOM matrix, while animals from group B only received HIV-1_IIIB tat-expressing DNA plasmids and HIV-1_IIIB Tat protein formulated with ISCOM matrix. Control animals received empty vectors and ISCOM matrix. All animals were subsequently challenged (i.v.) at week 32 (8 weeks after the last immunization) with 50 MID₅₀ of SHIV_89.6p (1 ml of a 1:1,000 dilution of the virus stock). S.U., subunit.

FIG. 2.

Protein-specific immune responses of immunized macaques. Tat-, Env-, and Gag-specific immune responses of Tat-Env-Gag-immunized animals (group A [EEG, WJ8, R300, and Ri178]) are displayed on the left site of the dashed line. Tat-specific immune responses of Tat-immunized animals (group B [R113, R357, 94059, and 95053]) are presented on the right site of the dashed line. From top to bottom, the number of antigen-specific IFN-γ-, IL-2-, and IL-4-secreting cells; antigen-specific lymphoproliferation (LP); and antibody (Ab) titers are depicted. The Env- and Gag-specific cell-mediated immune responses of Tat-immunized group B animals were negative and are not shown. At the bottom, the first horizontal line represents the time points of DNA immunization (weeks 0, 2, and 6), the second horizontal line represents the times of protein boosts (weeks 10, 14, and 24), and the arrow indicates the time of challenge (week 32).

FIG. 3.

Peptide-specific IFN-γ responses. The number of antigen-specific IFN-γ-secreting cells generated in immunized animals, indicative of potential CD8⁺ CTL responses, as measured by ELIspot assay, 2 weeks after the last immunization. Peptide-specific (20-mers, 10-aa overlap) IFN-γ production to pools of peptides (2 μg of peptide per ml) from SIV_mac251 Gag, SHIV_89.6p Env, and HIV-1_LAI Tat of each individual monkey per million PBMC is represented.

FIG. 4.

Viral RNA loads and absolute number of CD4⁺ T cells in peripheral blood. Viral RNA loads (RNA equivalents per milliliter of plasma; left panels) and absolute numbers of CD4⁺ T cells (per microliter of blood; right panels) are given for animals immunized with Tat-Env-Gag (top), Tat (middle), and vector controls (bottom). At week 32, animals were exposed to 50 MID₅₀ of SHIV_89.6p (arrowhead).