Th1/Th2 cytokine responses following HIV-1 immunization in seronegative volunteers. The AIDS Vaccine Evaluation Group

Abstract

The Th1/Th2 profile that follows human vaccination may profoundly influence the subsequent course of disease after infection. However, the ability to detect IL-4 has been limited outside trials of live vaccination. By using methods in which memory effector cells are allowed to antigenically expand by short term culture, followed by low-dose mitogenic stimulation, we have been able to follow the Th1/Th2 profile in HIV-1 volunteers enrolled in two phase I studies of HIV immunogens (a recombinant gp120 and a multivalent, octomeric V3 loop peptide). Antigen-specific interferon-gamma (IFN-gamma) could be detected in primary stimulation, but IL-4 was observed only after antigenic expansion and restimulation. In both of these studies the responses after initial immunizations were dominated by IFN-gamma, with IL-4 appearing only after multiple rounds of immunization, and IL-4 was temporally related to antibody production. Concomitant with the IL-4 production, the amount of supernatant IFN-gamma declined. Antigen-specific IL-10 was not detected in either study. Such techniques, which have been shown to correlate with outcomes in immunotherapy, may prove useful as future surrogates of human vaccine response.

Fig. 1

A representative experiment in a normal volunteer in which peripheral blood mononuclear cells (PBMC) were stimulated with the antigen as described in the text. Supernatant harvest at 4–7 days in the absence of IL-2 failed to detect IL-4 in almost all cases, whereas expansion of the cells with IL-2 for 10 days followed by restimulation led to easily measurable levels of IL-4.

Fig. 2

Peripheral blood mononuclear cells (PBMC) in culture were surface stained as described at day 0 or prior to phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) restimulation. The populations were gated on the FL3 (CyC) channel by either CD4 (a) or CD8 (b), and the percent of other markers was determined by setting gates using an isotype control. The figure represents PBMC from three individuals stimulated with media alone and two antigens (either Candida, influenza, or tetanus).

Fig. 3

The cytokine values were measured as described in supernatants harvested at 4 days (▪) or at the end of the 10-day restimulation period (□). IFN-γ is shown as ng/ml, whereas IL-4 is shown as pg/ml. When cell numbers were limited, antigens were used in the following order: V3 loop, media control, Candida, phytohaemagglutinin (PHA). The error bar represents the s.e.m. (a) Cells from vaccine recipients preimmunization stimulated with media alone (n = 8), the V3 loop (n = 9), and Candida antigen (n = 5). (b) The cytokine values 2 weeks after the second immunization (n = 9, 10 and 3, respectively). (c) The cytokine values 2 weeks after the third immunization (n = 6, 7, and 4, respectively).

Fig. 4

Supernatant amounts of IL-10 (± s.e.m.) produced by effector cells after restimulation with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) at 10 days in five (preimmunization), six (post-second immunization), and five (post-third immunization) volunteers who received the V3 peptide immunogen. No IL-10 was detectable in the primary culture supernatant harvested at day 4.

Fig. 5

The logarithmic scale lymphocyte proliferative indices (± s.d.) are shown to Candida, tetanus, and gp120 (10 μg/ml) in four placebo recipients (a) and a variable number of vaccinees (b) (depending on cell viability). The graph shows that excellent proliferation was achieved 2 weeks after the second immunization (visit 6), and increased thereafter at the post-third immunization at 6 months (visit 11) and the 2 weeks after post-fourth immunization (visit 19) at 12–18 months. CHO, Chinese hamster ovary.

Fig. 6

IFN-γ (a) and IL-4 levels (b) in supernatants of cultures (± s.e.m.) stimulated with Candida, the gp120 protein, or phytohaemagglutinin (PHA) harvested at 5 days in primary culture, or 24 h after restimulation at 10 days.