Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver

Abstract

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites.

Fig 1. Experimental design and P. knowlesi parasitemia during CPS immunization.

(A) Fifteen healthy naive rhesus macaques were administered 3 sequences of bites from 15–20 P. knowlesi-infected Anopheles dirus mosquitoes (immunized group; n = 8) or uninfected mosquitoes (control group; n = 7) on days 0, 28 and 56. Each immunization was followed by 14 days of daily oral chloroquine (CQ) treatment at a dose of 10 mg/kg/day in order to kill blood parasites at trophozoite stage. Seven animals (4 immunized /3 controls) were sacrificed between days 105–113 for evaluation of pre-challenge immune responses, while eight animals (4 immunized /4 controls) were sacrificed between days 153–162 (days 39–48 post challenge). (B) Transient blood-stage P. knowlesi parasitemia in seven of eight animals in the experimental group during CPS immunization.

Fig 2. Residual CQ levels and malaria patency post-challenge.

(A) Plasma CQ levels dropped below the lower limit of quantification by day 7 following administration in animals in the immunized group (n = 4) and control group (n = 4). (B) Blood-stage P. knowlesi parasitemia measured by light microscopy following challenge from the bites of 5 infected mosquitoes at day 114. Animals who developed malaria patency were treated with artesunate and quinine dihydrochloride once parasitemia reached 0.2–2%.

Fig 3. Kinetics of serum antibody responses.

Measurement of antibody responses was conducted from serum of control animals (n = 7) and immunized animals (n = 8). Serum antibody responses to (A) P. knowlesi sporozoites measured by IFA, (B) CSP measured by ELISA, (C) AMA1 measured by ELISA and (D) CelTOS measured by ELISA. Each data point represents the GMT± SE (*p < 0.05, Bonferroni correction).

Fig 4. Kinetics of peripheral blood T cell responses.

Peripheral blood T cell cytokine responses by ICS assay for (A) CD4⁺ and (B) CD8⁺ T cells against P. knowlesi sporozoites were measured in control animals (n = 7) and immunized animals (n = 8). Data shown are means ± SE of IFN-γ producing cells in the CD4⁺ or CD8⁺ T cell population (*p < 0.05, Bonferroni correction).

Fig 5. Anatomical distribution of sporozoite-specific memory CD4⁺ plus CD8⁺ T cells.

The frequencies of P. knowlesi sporozoite-specific IFN-γ producing T cells isolated from tissue compartments from euthanized animals at pre-challenge in A) control animals (n = 3) and B) immunized animals (n = 4), and 39–46 days post challenge in a second cohort of C) control animals (n = 4) and D) immunized animals (n = 4). Data shown are means ± SE.

Fig 6. Memory phenotype and chemokine receptor expression of P. knowlesi sporozoite-specific T cells.

Expression of central memory (CD45RA^- CCR7⁺), effector memory (CD45RA^- CCR7^-) and terminal effector (CD45RA⁺ CCR7^-) T cell markers on sporozoite-specific memory T cells isolated from different compartments (A). IFN-γ producing CD4⁺ and IFN-γ producing CD8⁺ T cells were gated and then analyzed for the expression of CCR7 and CD45RA. Data are representative of two animals (R704 and R827). CXCR3, CCR5 and CXCR6 expression by P. knowlesi sporozoite-specific memory T cells isolated from different compartments (B). IFN-γ producing CD4⁺ and IFN-γ producing CD8⁺ T cells were gated and then analyzed for the expression of CXCR3, CCR5 and CXCR6. Data are representative of three animals (R704, R827 and R919).

Fig 7. Phenotypic T_RM cell marker expression on P. knowlesi sporozoite-specific memory T cells.

Percentage of P. knowlesi sporozoite-specific IFN-γ producing (A) CD8⁺ and (B) CD4⁺ T memory T cells that expressed CD103. IFN-γ response in P. knowlesi sporozoite-specific (C) CD8⁺ and (D) CD4⁺ memory T cells following depletion of CD69-positive cells. Mean ± SE of 3 animals (R704, R827 and R919) are shown.