The vaccine candidate Liver Stage Antigen 3 is exported during Plasmodium falciparum infection and required for liver-stage development

Reviewer #1 (Public review):

Summary:

The extent to which P. falciparum liver stage parasites export proteins into the host cell is unclear. Most blood-stage exported proteins tested in liver stages were not exported. An exception is LISP2, which is exported in P. berghei but not P. falciparum liver stages. While the machinery for export is present in liver stages, efforts to demonstrate export have so far been mostly unsuccessful. Parasite proteins exported during the liver stage could be presented by MHC and thereby become the target of immune control, an incentive to study liver stage export and identify proteins exported during this stage. However, particularly for P. falciparum, it is very difficult to study liver stages.

This work studies LSA3 in P. falciparum blood and liver stages. The authors show that this protein is exported into the host cell in blood stages, but in liver stages, no or only very little export was detected. A disruption of LSA3 reduced liver stage load in a humanized mouse model, indicating this protein contributes to efficient development of the parasites in the liver.

The paper also studies the localization of LSA3 in blood stages and uses a known inhibitor to show that it is processed by plasmepsin 5, a protease important for protein trafficking. The work also shows that LSA3 is not needed for passage through the mosquito.

Strengths:

The main strength of this work is the use of the humanized mouse model to study liver stages of P. falciparum, which is technically challenging and requires specialized facilities. The biochemical analysis of LSA3 localization and processing by plasmepsin 5 is thorough and mostly overcame adverse issues such as a cross-reactive antibody and the negative influence of the GFP-tag on LSA3 trafficking. The mosquito stage analysis is also notable, as these kinds of studies are difficult with P. falciparum. However, there was no evidence for a function of LSA3 in mosquito stages.

Weaknesses:

The cross-reactivity of the antibody, together with the co-infection strategy, prevents reliable assessment of LSA3 localization in liver stages. Despite this, it seems LSA3 is not exported in liver stages, and the paper does not bring us closer to the original goal of finding an exported liver stage protein.

While the localization analysis in blood stages is well done and thorough, the advance is somewhat limited. LSA3 may be in structures like J dots, but this hypothesis was not tested. Although parasites with a disrupted LSA3 were generated, the function of this protein was not explored. Given that a previous publication found some inhibitory effect of LSA3 antibodies on blood stage growth, a comparison of the growth of the LSA3 disruption clones with the parent would have been very welcome and easy to do. At this point, LSA3 is one more of many proteins exported in blood stages for which the function remains unclear.

It might be possible to refine some of the conclusions. The impact on liver stage development is interesting, but which phase of the liver stage is affected, and the phenotype remains largely unknown. The co-infection (WT together with LSA3 mutant) has the advantage of a direct comparison of the mutant with the control in the same liver, but complicates phenotypic analysis if the LSA3 antibody is also cross-reactive in liver stages. This issue adds a question mark to the shown localization and precludes phenotypic comparisons. The authors write that they do not know if the cross-reactive protein is expressed at that stage. But this should be immediately evident from the mixed WT/mutant infection. If all cells are positive for LSA3, there is a cross-reaction. If about half of the cells are negative, there isn't. In the latter case, the localization shown in the paper is indeed LSA3, and morphological differences between WT and LSA3 disruption could be assessed without additional experiments.

Significance:

The conclusion from the paper that "our study presents just the second PEXEL protein so far identified as important for normal P. falciparum liver-stage development and confirms the hypothesized potential of exported proteins as malaria vaccine candidates" is partially misleading. Neither LISP2 nor LSA3 seems to be exported in P. falciparum liver stages, and we can't confirm the potential of vaccines with proteins exported in this stage. LSA3 is still important and may still be the target of the immune response, but based on this work, probably not due to export in liver stages.

Reviewer #2 (Public review):

Summary:

Immunogenic Plasmodium falciparum proteins that could be targeted to prevent parasite development in the liver are of significant interest for novel anti-malarial vaccine development. In this study, McConville et al evaluate the trafficking and functional importance of LSA3, a protein expressed in the blood and liver stages and previously shown to provide protection in immunized chimpanzees. LSA3 contains a PEXEL motif, but the authors have previously shown that this protein does not appear to be exported beyond the PVM in the liver stage (McConville et al, PNAS 2024). However, LSA3 trafficking and functional importance have not been comprehensively evaluated across stages. In the present study, the authors find that blood-stage LSA3 undergoes PEXEL processing, and a portion of the protein is exported into the erythrocyte, where it localizes to punctate structures distinct from Maurer's clefts. Using a knockout mutant, LSA3 is shown to be dispensable for blood and mosquito stages but important to liver-stage development. Collectively, these results validate LSA3 as a liver-stage target and place it among several other PEXEL proteins that display differential trafficking beyond the PVM in the erythrocyte but not the hepatocyte.

Strengths:

(1) The authors present a thorough analysis of LSA3 trafficking in the blood stage. PEXEL processing by Plasmepsin 5 is clearly demonstrated through a combination of mini LSA3-GFP reporters and Plasmepsin 5 inhibitors. Importantly, an LSA3 knockout mutant is used to show that the LSA3-C anti-sera also react with additional, unidentified parasite proteins in the blood stage. Nonetheless, comparison between the WT and KO parasites clearly indicates that a portion of LSA3 is exported into the erythrocyte, which is further supported by protease-protection assays with fractionated iRBCs. This contrasts with the liver stage, where LSA3 does not appear to traffic beyond the PVM, similar to what has been observed for other PEXEL proteins in the rodent malaria model.

(2)This study provides the first direct analysis of LSA3 function by reverse genetics, showing this protein is important for liver stage development in chimeric human liver mice. Several PEXEL proteins in P. berghei have been shown to be exported into the host cell in the blood stage, but do not appear to cross the PVM in the liver stage. These observations reinforce that even without detectable export into the hepatocyte, PEXEL proteins play critical roles during liver stage development.

Weaknesses:

(1) A previous study reported that anti-LSA3 antibodies inhibit blood-stage growth, suggesting a role for LSA3 during erythrocyte infection. While the authors carefully evaluate the LSA3 mutant in mosquito and liver stages, the impact on blood stage fitness is not tested. While the knockout shows LSA3 is not essential in the blood stage, its importance during erythrocyte infection remains unclear.

(2) The authors previously reported that anti-LSA3-C signal in the liver stage localizes within the parasite and at the parasite periphery but is not exported into the hepatocyte. In the present study, it is shown that anti-LSA3-C reacts with other parasite proteins beyond LSA3 in the blood stage, and this may also occur in the liver stage. However, since liver-stage IFAs were only performed on samples co-infected with both WT and ∆LSA3 parasites, non-specific anti-LSA3-C reactivity at this stage could not be determined, and the localization of LSA3 in the liver stage remains somewhat unclear.

Reviewer #3 (Public review):

Summary:

This manuscript provides a comprehensive characterization of the Plasmodium falciparum protein LSA3, combining biochemical, genetic, and in vivo approaches. The authors convincingly demonstrate that LSA3 is expressed during liver stage infection and that disruption of the gene leads to a modest but reproducible reduction in liver stage parasite load in humanized mice.

Strengths:

Their biochemical and cell biological analysis of blood stages provides strong evidence that LSA3 is exported to the infected erythrocyte, and the detailed analysis of its PEXEL motif processing is well executed.

Weaknesses:

The study suggests LSA3 as one of only two known P. falciparum PEXEL proteins contributing to this stage, although there is no evidence for the export beyond the vacuolar membrane. Several key conclusions, particularly regarding antibody specificity, localization in liver stage parasites, and the interpretation of the phenotypic data, are not fully supported by the current experiments.