Strain-specific protective effect of the immunity induced by live malarial sporozoites under chloroquine cover

Abstract

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2-5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites' circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.

Figure 1. PSQ assay based on a SNP within the csp gene detects the strain-specific protective effect.

Toque monkeys were strain-specifically immunized with live sporozoites (by infective mosquito bites) of Pc746 and PcCeylon strains under chloroquine and challenged with a mixture of bites of PcCeylon and Pc746 live sporozoite-infected mosquitoes. Open portions of columns represent Pc746 proportions and shaded portions of columns represent PcCeylon proportions in challenge infections of (A) non-immunized, n = 4 (B) Pc746 immunized, n = 5 and (C) PcCeylon immunized monkeys, n = 4. The absolute asexual parasitaemia, as a percent of red blood cells infected, are indicated by the lines connecting filled triangles. Error bars represent the standard deviation between the 3 to 6 repeated PSQ measurements of each monkey represented in each experimental condition. The proportion of parasites of each strain was recorded for 4 to 8 consecutive days starting from and including the day on which a monkey first reached or passed 0.03% asexual parasitaemia.The day of the first such record in each monkey is designated “Day 1” in the text. The Mann-Whitney U test for two independent variables (at 95% confidence level) was used to compare the proportions of Pc746 in Pc746 immunized and PcCeylon immunized groups on each day from “Day 1” to “Day 4”. P = 0.027 for “Day 1”, “Day 3”, and “Day 4”; P = 0.014 for “Day 2”.