Immune responses to Plasmodium falciparum-merozoite surface protein 1 (MSP1) antigen, II. Induction of parasite-specific immunoglobulin G in unsensitized human B cells after in vitro T-cell priming with MSP119

Abstract

A baculovirus recombinant antigen corresponding to the C-terminal 19 000 MW fragment of Plasmodium falciparum merozoite surface protein 1 (MSP119), has been used to prime T cells from individuals with no previous exposure to malaria, to provide help for the induction of a parasite specific antibody response in vitro. Although MSP119 alone could induce a small but detectable T-cell response, which included interleukin-4 (IL-4) secretion, this response was significantly increased by the presence of IL-2. In addition, IL-4 was shown to synergize with IL-2 for the induction of antigen-specific T-cell responses. If interferon-gamma (IFN-gamma), IL-12, or neutralizing anti-IL-4 antibody was present at the time of priming, the T-cell responses were abolished. Parasite-specific immunoglobulin G (IgG) could be detected after secondary restimulation with MSP119, IL-10 and anti-CD40 monoclonal antibody in cultures containing MSP119 primed T cells, autologous B cells, IL-2 and IL-4. No antibody was secreted in the absence of primed T cells in this B-cell culture assay. These data show that recombinant MSP119, a leading malaria vaccine candidate, can prime non-immune human lymphocytes under defined in vitro experimental conditions, which include regulatory cytokines and/or other costimulatory molecules. This is a complementary approach for exploring immunogenic mechanisms of potential vaccine candidates such as P. falciparum antigens in humans.

Figure 1

T-cell responses after in vitro priming with MSP1₁₉ in the presence of various cytokines. Purified, naive T cells were exposed to weekly stimulation with antigen in the presence of various cytokines. After 28 days, blastic cells were recovered from individual cultures and counted by conventional microscopy. The geometric means±SD of six individual experiments are shown. Asterisks indicate statistical significance (P < 0·05).

Figure 2

CD4⁺ T cells can be primed in vitro by MSP1₁₉ in the presence of IL-2+IL-4. Purified CD4⁺ T cells and unseparated (CD4⁺ and CD8⁺) T cells were exposed to weekly stimulation with antigen in the presence of IL-2+IL-4, as described in Materials and Methods. After 28 days, blastic cells were recovered from three individual cultures and counted.

Figure 3

Optimization of T–B cell coculture conditions for maximum parasite specific IgG production after restimulation with MSP1₁₉. The T–B cocultures were carried out for 10 days, in the presence of IL-10 plus anti-CD40 mAb. Data are expressed as individual mean OD ratios (from duplicates) measured in different culture conditions in two individual experiments. The threshold for positivity is an OD ratio >1·5. Baseline values for actual ODs were ≈0·06. (a) T cells primed under conditions defined in the text were cocultured with autologous B cells at different T:B ratios. (b) T cells were cocultured with B cells for varying times following stimulation and/or restimulation with antigen as described in the text. The T:B cell ratio used was 1:10.

Figure 4

T cells primed with MSP1₁₉ in the presence of IL-2+IL-4 provide help for parasite-specific IgG production in vitro. Purified, naive T cells were expanded with MSP1₁₉ in the presence of IL-2 (a,d), IL-2+IL-4 (b,e) or IL-2+IL-4+ neutralizing anti-IFN-γ Ab (c,f). The T–B coculture was performed for 10 days, under various culture conditions, either in the presence of IL-4 (left histograms), IL-2+IL-10 (centre histograms) or IL-10+anti-CD40 mAb (left histograms). (a–c) Total IgG production; (d–f) Parasite-specific IgG production. Data are expressed as geometric means±SD of OD ratios (from duplicates) in six (a,b,d,e) or 12 (c,f) individual experiments. The threshold for positivity is an OD ratio >1·5 (dotted line). The asterisk indicates statistical significance (P < 0·05). Baseline values for actual ODs were ≈0·09 for total IgG and 0·06 for parasite-specific IgG.

Figure 5

Relative affinity of specific IgG antibodies produced in vitro after restimulation with P. falciparum MSP1₁₉ recombinant protein. Culture supernatants of either PBMC cultures or blood T- and B-cell cocultures were tested for the presence of IgG specific to the MSP1₁₉ recombinant protein used for stimulation (open symbols) or to a crude antigenic preparation enriched for P. falciparum merozoite proteins (closed symbols). Twofold dilutions of each culture supernatant were tested (1/2 to 1/16). The dashed line indicates the threshold for positivity (OD ratio ≥1·5). (a) Specific Ab reactivity in cultures of PBMC from P. falciparum-immune individuals. The means±SD of five individual cultures in which Abs were detected are shown. (b) Specific Ab reactivity in T- and B-cell cocultures. The values obtained from three independent cultures ae designated with different symbols. Coculture supernatants diluted 1/16 were not tested against MSP1₁₉.