Protective efficacy and safety of liver stage attenuated malaria parasites

Abstract

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Figure 1. Generation and characterization of P. berghei ANKA uis3(–) parasites.

(A) Schematic of the uis3 replacement strategy using the 5′ and 3′UTRs of uis3 to integrate a resistance cassette by double crossover into the P. berghei strain ANKA genome. Locations of primers used for PCR in (B) are indicated. (B) Diagnostic PCR to investigate generation of uis3(–) parasites. WT: wild type, KO: uis3(–) parasite line. Numbers below the gel indicate expected amplicon sizes. (C) Liver stages in HepG2 cells formed by WT and uis3(–) sporozoites 65 hours post infection labeled with antibodies against EXP-1 and MSP1; Hoechst reveals host and parasite DNA. Images on the right show the merged images. Scale bar: 10 μm.

Figure 2. Generation and characterization of ANKA LISP2(–) and N-LISP2 parasites.

(A) LISP2 locus and differently modified gene loci as generated in previous studies (i, ii) and in the current study (iii, iv). The region encoding the N-terminus is colored in orange. Boxes not drawn to scale. (B) Schematic of the LISP2 replacement strategy using the 5′ and 3′UTRs of LISP2 to integrate a resistance cassette by double crossover into the genome of the P. berghei strain ANKA. Note that this resistance cassette (yellow) included a negative selection marker to enable recycling of the cassette. The locations of primers used for PCR in (C) are indicated. (C) Diagnostic PCR to investigate generation of LISP2(–) parasites. Numbers below the gel indicate expected amplicon sizes. (D) Liver stages in HepG2 cells formed by ANKA WT and LISP2(–) sporozoites 65 hours post infection labeled with antibodies against EXP-1 and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. (E) Schematic of the strategy used to integrate the N-terminus of LISP2 into the LISP2(–) parasite. The locations of primers used for PCR in (F) are indicated. The N-terminal fragment contained a start and stop codon. (F) Diagnostic PCR to investigate generation of N-LISP2 parasites. Numbers below the gel indicate expected amplicon sizes. KO: LISP2(–) parasites, N-L: N-LISP2 parasites. (G) Meyer-Kaplan plot to indicate the number of blood stage positive mice after injection of sporozoites i.v. and by bite. 4 groups of 4 mice each were infected; of these 15 became blood stage patent.

Figure 3. Characterization of a ANKA LISP2(–)/uis3(–) double knockout parasite line and comparison with single knockout lines.

(A) Liver stages in HepG2 cells formed by WT and LISP2(–)/uis3(–) sporozoites 65 hours post infection as revealed by labelling with antibodies against EXP-1, and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. (B) Percentage of breakthrough infections (black) and infected mice per 1 million sporozoites injected (white) of the different parasite lines, see also Appendix Table SII. (C) Number of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2(–), uis3(–) and LISP2(–)/uis3(–) sporozoites. All data normalized to the mean of WT duplicates in each individual experiment. Raw data are shown in Supplementary Fig. S4. (D) Sizes of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2(–), uis3(–) and LISP2(–)/uis3(–) sporozoites. (E,F) Relative liver load of two mice 40 and 56 hours post infection (hpi) of LISP2(–), uis3(–) and LISP2(–)/uis3(–) sporozoite infected C57BL/6 mice. 18S rRNA abundance was normalized to the average value of 2 PbANKA infected mice for each time point. Note that at 56 hpi some WT parasites have already emerged from the liver, hence the lower relative levels.