Genetically attenuated Plasmodium berghei liver stages induce sterile protracted protection that is mediated by major histocompatibility complex Class I-dependent interferon-gamma-producing CD8+ T cells

Abstract

At present, radiation-attenuated plasmodia sporozoites ( gamma -spz) is the only vaccine that induces sterile and lasting protection in malaria-naive humans and laboratory rodents. However, gamma -spz are not without risks. For example, the heterogeneity of the gamma -spz could explain occasional breakthrough infections. To avoid this possibility, we constructed a double-knockout P. berghei parasite by removing 2 genes, UIS3 and UIS4, that are up-regulated in infective spz. We evaluated the double-knockout Pbuis3(-)/4(-) parasites for protective efficacy and the contribution of CD8(+) T cells to protection. Pbuis3(-)/4(-) spz induced sterile and protracted protection in C57BL/6 mice. Protection was linked to CD8(+) T cells, given that mice deficient in beta (2)m were not protected. Pbuis3(-)/4(-) spz-immune CD8(+) T cells consisted of effector/memory phenotypes and produced interferon- gamma . On the basis of these observations, we propose that the development of genetically attenuated P. falciparum parasites is warranted for tests in clinical trials as a pre-erythrocytic stage vaccine candidate.

Fig. 1. Pbγ-spz and Pbuis3(−)/4(−) spz induce similar populations of phenotypically distinct subsets of CD8+ T cells

(A) Hepatic mononuclear cells (HMC) were isolated from livers of 3 individual naïve or 3 immune mice 6 days after the indicated immunization and analyzed by flow cytometry. Lymphocytes labeled with anti-CD8 mAbs were gated on a forward-side scatter plot, and gates were applied to identify CD8+ T cells. CD8+ T cell subsets were revealed using anti-CD45RB and anti-CD44 mAbs and percentages of subsets are shown as a dot plot from a representative mouse. The experiments were performed 3 times, with 3 mice/group and cells from individual mice were assayed. The data in the text are shown as the mean ± SD of responses observed in 9 individual mice.

Fig. 2. CD8+ T_EM cells are maintained after challenge and re-challenge of Pbγ-spz and Pbuis3(−)/4(−) spz-immunized mice

(A) HMC were isolated from livers of individual mice at the indicated time points after immunization and challenge and analyzed as described in Fig. 1. The percentages of CD8+ T cell subsets are shown as a dot plot from a representative mouse. The experiments were performed 3 times, with 3 mice/group and cells from individual mice were assayed. The data in the text are shown as the mean ± SD of responses observed in 9 individual mice. (B) Mononuclear cells were isolated from peripheral blood of naïve mice or immune mice 6 days after the 2^nd boost with Pbγ-spz or Pbuis3(−)/4(−) spz, and analyzed as in (A). Results are from 1 of 3 representative experiments.

Fig. 2. CD8+ T_EM cells are maintained after challenge and re-challenge of Pbγ-spz and Pbuis3(−)/4(−) spz-immunized mice

(A) HMC were isolated from livers of individual mice at the indicated time points after immunization and challenge and analyzed as described in Fig. 1. The percentages of CD8+ T cell subsets are shown as a dot plot from a representative mouse. The experiments were performed 3 times, with 3 mice/group and cells from individual mice were assayed. The data in the text are shown as the mean ± SD of responses observed in 9 individual mice. (B) Mononuclear cells were isolated from peripheral blood of naïve mice or immune mice 6 days after the 2^nd boost with Pbγ-spz or Pbuis3(−)/4(−) spz, and analyzed as in (A). Results are from 1 of 3 representative experiments.

Fig. 3. Genetically-attenuated P. berghei fail to induce protective immunity in β2m−/− mice

WT (n=5) and β2m−/− mice (n=19) were immunized with 75K, 20K and 20K Pbuis3(−)/4(−) spz given one week apart. One week after the final immunization, mice were challenged with 10K infectious P. berghei spz. Naïve WT (n=5) were used as infectivity controls. Pbuis3(−)/4(−) spz-immune WT mice were re-challenged with 10K infectious P. berghei spz 6 months after the first challenge. Parasitemia was monitored by microscopy of Giemsa stained blood smears every other day, starting 2 days after challenge. (A) The percentage of mice remaining parasite-free at the indicated time points. (B) Results indicate the average % +/− SEM of parasitized red blood cells per mouse. Naïve WT control mice are indicated by a filled square and a solid line; immunized WT mice are indicated by a diamond and a broken line; immunized β₂m −/− mice are indicated by a filled circle and a dashed line.

Fig. 3. Genetically-attenuated P. berghei fail to induce protective immunity in β2m−/− mice

WT (n=5) and β2m−/− mice (n=19) were immunized with 75K, 20K and 20K Pbuis3(−)/4(−) spz given one week apart. One week after the final immunization, mice were challenged with 10K infectious P. berghei spz. Naïve WT (n=5) were used as infectivity controls. Pbuis3(−)/4(−) spz-immune WT mice were re-challenged with 10K infectious P. berghei spz 6 months after the first challenge. Parasitemia was monitored by microscopy of Giemsa stained blood smears every other day, starting 2 days after challenge. (A) The percentage of mice remaining parasite-free at the indicated time points. (B) Results indicate the average % +/− SEM of parasitized red blood cells per mouse. Naïve WT control mice are indicated by a filled square and a solid line; immunized WT mice are indicated by a diamond and a broken line; immunized β₂m −/− mice are indicated by a filled circle and a dashed line.

Fig. 4. Similar levels of anti-P. berghei CS protein antibodies in β2m−/− and WT mice

β2m−/− (n = 5) and WT (n = 5) mice were immunized with 75K, 20K and 20K Pbuis3(−)/4(−) spz given one week apart. Sera were obtained 6 days following tertiary immunization and assayed for anti-P. berghei CS protein antibodies by ELISA. Sera from naïve mice served as negative controls. Filled bar = WT mice; open bar = β₂m −/− mice

Fig. 5. Higher frequency of IFNγ-producing CD8+ T cells in the liver of Pbuis3(−)/4(−) spz-immunized vs Pbγ-spz-immunized mice

HMC were isolated 6 days after both prime and prime-boost immunizations and at 24hr, 72hr, 144hr and 216hr after challenge from livers of Pbγ-spz or Pbuis3(−)/4(−) spz immunized mice. IFN-γ secreting CD8+ T cells were identified by fluorescent labeling using an IFN-γ secretion assay (see Materials and Methods). The percentage of IFNγ-secreting T cells in the gated CD3+CD8+ T cell populations were determined by flow cytometry and are indicated in the upper quadrants. Dots plots are representative of 3 mice per group.