Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria

Abstract

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Fig. 1.

Generation and maintenance of P. berghei CS₂₅₂-specific CD8 T cells. BALB/c mice were primed with 3 × 10⁵ bone marrow-derived dendritic cells coated with CS_252–260 (DC-CS). Seven days later DC-CS mice were boosted with 2 × 10⁷ LM-CS₂₅₂ (DC-CS + LM-CS) or naive mice were primed with 7 × 10⁶ LM-CS₂₅₂ (LM-CS). (A) Frequency and phenotype of splenic CD8 T cells that are CS₂₅₂ specific as determined by ICS 8 days after DC-CS prime. (B) Frequency of splenic CD8 T cells that are CS₂₅₂ specific on days 14, 41, and 96 as determined by ICS. Profiles from representative mice are shown; numbers represent mean ± SD; n = 3 per group. (C) Total number (mean ± SD, n = 3 per group) of CS₂₅₂-specific CD8 T cells in the spleen on various days after initiation of immunization. Total number (mean ± SD, n = 3 per group) of CS₂₅₂-specific CD8 T cells (D) in the spleen and (E) liver or (F) percent CS₂₅₂-specific CD8 T cells of all PBL 54 days after initiation of immunization for the indicated groups. Data are representative of three experiments.

Fig. 2.

CS₂₅₂-specific memory CD8 T cells afford protection for >3 months against a P. berghei sporozoite challenge. (A) Frequency of CS₂₅₂-specific CD8 T cells in the PBL 98 days after priming. Data (mean ± SD) are from 10 to 12 mice per group. (B) Percentage total PBL that are CS₂₅₂-specific CD8 T cells as determined by ICS in individual mice from the indicated immunization group before challenge. Filled circles indicate naïve or immune mice that developed blood-stage malaria after a challenge with 1000 P. berghei sporozoites. Numbers represent protected mice/total challenged for each group. n.d., not determined.

Fig. 3.

Long-term protection against multiple P. berghei sporozoite challenges. BALB/c mice were primed with 5 × 10⁵ splenic DC (sDC)-CS and boosted with 2 × 10⁷ LM-CS₂₅₂. (A) Total number (mean ± SD, n = 3 mice per day) of CS₂₅₂-specific CD8 T cells in the spleen on various days after the start of immunization. (B) Naïve or DC-CS + LM-CS mice were challenged with 1000 P. berghei sporozoites on day 210. (C) The percent of total PBL that are CS₂₅₂-specific CD8 T cells at day 406 after immunization was determined by ICS from individual unchallenged or previously challenged DC-CS + LM-CS immunized mice. Naïve and immune mice were challenged with 1000 P. berghei sporozoites at day 422. Filled circles indicate mice that developed blood-stage malaria. Numbers represent protected mice per total challenged for each group. n.d., not determined.

Fig. 4.

LM-CS₂₅₂-primed and LM-CS₂₅₂-boosted BALB/c mice are protected against a P. berghei sporozoite challenge. (A) In an initial experiment to evaluate conventional prime-boost responses, BALB/c mice were primed with 7 × 10⁶ LM-CS₂₅₂ and boosted with 2 × 10⁷ LM-CS₂₅₂ 53 days later (LM-CS(d53)+LM-CS). On the day of the LM-CS boost, naive BALB/c mice were primed with 7 × 10⁶ LM-CS₂₅₂ (LM-CS). (A) Total number (mean ± SD, n = 3 per group) of CS₂₅₂-specific CD8 T cells in the spleen and liver and percentage of PBL at day 61 after boost or first immunization was determined by ICS. (B) In a second experiment BALB/c mice were primed with 7 × 10⁶ LM-CS₂₅₂ and boosted with 2 × 10⁷ LM-CS₂₅₂ 44 days later (LM-CS(d44)+ LM-CS). On the day of the LM-CS₂₅₂ boost, naive BALB/c mice were primed with 7 × 10⁶ LM-CS₂₅₂ (LM-CS). Percentage of total PBL that were CS₂₅₂-specific CD8 T cells at day 75 after the last immunization was determined by ICS in individual mice. Filled circles indicate naïve or immune mice that developed blood-stage malaria after a challenge with 800 P. berghei sporozoites on day 83 after boost. Numbers represent protected mice per total challenged for each group. n.d., not determined

Fig. 5.

Numerical requirements for sterile immunity mediated by CS₂₅₂-specific CD8 T cells. BALB/c mice were primed with 2.5 × 10⁵ splenic DC-CS and boosted with titrating doses of LM-CS₂₅₂ (2 × 10⁴, 2 × 10⁵, 2 × 10⁶, or 2 × 10⁷). (A) Phenotype of PBL CS₂₅₂-specific CD8 T cells as determined by ICS at day 98. Data (mean ± SD) are from 10 mice per group except for 2 × 10⁷, in which n = 7. (B) Before sporozoite challenge the percentage of total PBL CS₂₅₂-specific CD8 T cells was determined in individual mice by ICS. Naïve and immune mice were challenged with 1000 P. berghei sporozoites. Filled circles indicate mice that developed blood-stage malaria. n.d. = not determined. (C) Cumulative results from DC-CS + LM-CS immunized mice in which the frequency of PBL CS₂₅₂-specific CD8 T cells was determined before challenge with sporozoites.