An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies

Reviewer #2 (Public review):

The major strengths of the manuscript are in the Plasmodium falciparum genetic and phenotyping approaches. PfMSP2 knockouts are made in two different strains, which is important as it is know that invasion pathways can vary between strains, but is a level of comprehensiveness that is not always delivered in P. falciparum genetic studies. The knockout strains are characterised very thoroughly using multiple different assays and the authors should be commended for publishing a good deal of negative data, where no phenotype was detected. This is not always done but is very helpful for the field and reduces the potential for experimental redundancy, i.e. others repeating work that has already been performed but never published. The quality of the writing, referencing and figures is also generally strong.

There are certainly some areas of the manuscript that would benefit from deeper exploration, such as electron microscopy/other imaging approaches to explore whether deletion of PfMSP2 has a visible impact on merozoite surface structure, further replicates of the video microscopy assays to see whether trends in the data could reach significance (although these are very time-consuming and technically difficult assays), and follow up of some of the genes where expression is changed by PfMSP2 knockout (as the authors point out, there are no candidates that have a very obvious link to invasion suggesting that they may be compensating for PfMSP2 function, although several are expressed in schizont stages). However, there is already a substantial amount of data in the manuscript, and more detailed follow-up is reasonable to leave to future work. Overall, with the modifications made through the review process, including the addition of new controls for key experiments, the claims and conclusions are justified by the data, and the manuscript generates important new information about a highly studied Plasmodium falciparum merozoite surface protein.

Reviewer #3 (Public review):

Henshall et al. study invasion of human erythrocytes by Plasmodium falciparum merozoites and report knockout of PfMSP2, a critical merozoite surface protein with unknown function. They describe conservation of MSP2 in P. falciparum and key avian malaria parasites, unabated growth of two knockout lines (∆MSP2) produced in divergent 3D7 and Dd2 strains, no differences in expression of key invasion-associated genes, no effect on invasion kinetics (with or without protease treatment of erythrocytes), nonsignificant effects of knockout on parasite growth inhibition by antibodies directed against key invasion-associated antigens, and do find a significant effect on potentiating AMA1 invasion inhibitory antibodies. The studies are interesting and have potential for directing vaccine design targeting erythrocyte invasion, a critical step in bloodstream expansion of malaria parasites.

Major points:

(1) Much of the manuscript describes negative results and this reviewer found it arduous to get through many negative or nonsignificant results before finally getting to the significant effect on AMA1 inhibitory antibodies, not presented until Figure 6! Computational studies in Fig. 1 could be a supplementary figure. Figs. 2 and 3. demonstrate knockout in 3D7 and Dd2, respectively and could be assembled into a single figure. (Notably Fig. 2A and 3A are almost identical with use of some different primers.) Fig. 2E, 2F, 3D-H, all of Fig. 4, most of Fig. 5 are all negative or insignificant results that could also be moved to supplementary data. As MSP4, MSP5, and SUB1 are presumably included in the whole genome RNA-seq experiments shown in Fig. 4C, it makes sense to remove Fig. 4A data from the paper fully. These consolidating changes would help highlight the key finding of improved binding and block of AMA1's role in invasion.

(2) The potentiating effects on anti-AMA1 antibodies are shown with rabbit sera and purified antibodies, mouse monoclonal antibodies, and smaller i-bodies inspired by shark antibody-like receptors but not with human monoclonal antibodies (hmAbs). As naturally acquired hmAbs targeting AMA1 have been identified and characterized (PMIDs: 39632799, 40020675), would it not be important to test these antibodies in the ∆MSP2, especially as the authors emphasize the importance of their model in designing better human malaria vaccines?

(3) Fig. 7 presents quantitative fluorescence microscopy to measure anti-AMA1 binding and support a model where MSP2 serves to sterically hinder antibody access to AMA1 on individual merozoites. I understand that the negative WD33 control is useful to contrast to the positive WD34 antibody (both bind AMA1 but only WD34 exhibits parasite growth inhibitory effects), but it seems that use of smaller i-bodies rather than conventional larger mouse or ideally human monoclonal antibodies may compromise demonstration of steric hindrance by MSP2 because smaller i-bodies may be less hinder.

(4) Some explanation for why WD33 fails to inhibit growth despite targeting the same antigen as WD34 is needed. Are the epitopes known? Does one bind further from the RON2 binding pocket?

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Henshall et al. delete the highly abundant merozoite surface protein PfMSP2 from two Plasmodium falciparum laboratory lines (3D7 and Dd2) using CRISPR-Cas9. Parasites lacking MSP2 replicate and invade red cells normally, opposing the experimental history that suggests MSP2 is essential. Unexpectedly, the knock-outs become more susceptible to several inhibitory antibodies - most strikingly those that target the apical antigen AMA1-while antibodies to other surface or secreted proteins are largely unaffected. Recombinant MSP2 added in vitro can dampen AMA1-antibody binding, supporting a "conformational masking" model. The reported data suggest that MSP2 helps shield key invasion ligands from host antibodies and may itself be a double-edged vaccine target.

Reviewer 1 did not have any comments we needed to address.

Reviewer #2 (Public review):

(1) The section describing Laverania and avian Plasmodium MSP2 comparison is a lengthy section and could be told much more concisely for clarity in delivering the key message, i.e., that conservation in distantly related Plasmodium species could indicate an important function. The identification of MSP2-like genes in avian Plasmodium species was highlighted previously in the referenced Escalante paper, so it is not entirely novel, although this paper goes into more detailed characterisation of the extent of conservation. Overall, this section takes up much more space in the manuscript than is merited by the novelty and significance of the findings.

As outlined in point (1) for Reviewer 1 (Recommendations for the authors), we have cut back through this section and focussed on the important comparisons rather than the general observation. We have also moved the elements of Table 1 to Supplementary Figures 2, 3 and 4 to streamline the manuscript. Further description of the changes is available in the Reviewer #1 (Recommendations for the authors).

(2) Characterisation of the knockout strains is generally thorough, though relatively few interactions were followed by live microscopy (Figures 3E-H). A minimum of 30 merozoites were followed in each assay (although the precise number is not specified in the figure or legend), but there are intriguing trends in the data that could potentially have become significant if n was increased.

In the Figure 3 Legend we have now indicated the number of merozoite invasions followed as per the following:

“(E-H) Key parameters of merozoite invasion were measured for both PfDd2 WT (n = 43) and PfDd2 ΔMSP2 (n = 35) parasites that had successfully invaded a RBC using live cell imaging of merozoite invasion.”

We have also removed the more general description of ‘a minimum of 30 merozoites’ from the same Figure Legend.

The number of schizont ruptures and subsequent merozoite invasions followed for each experiment is in line with previous studies that have investigated phenotypes with invasion inhibitors and gene knock-outs (e.g. Weiss et al. 2015, PLoS Pathogens). It is important to note that the data refers to merozoites that have completed invasion, and not just the number of merozoites that have been released from a schizont which is typically 2-4 times more than have invaded. This means we are comparing the kinetics of invasion across a relatively large sample size compared to other studies of inhibitory phenotypes. While it is possible that increasing the number of merozoites being filmed might lead to some statistical significance for some of the trends, we note that there is a limited growth phenotype overall in both short and long-term culture and this fits with the limited defect we are seeing. In order to better address this, as outlined in our response to point (7) for Reviewer 2 (Recommendations for the authors), we now discuss the trends seen in the data in additional detail.

(3) The comparative RNAseq data is interesting, but is not followed up to any significant degree. Multiple transcripts are up-regulated in the absence of PfMSP2, but they are largely dismissed because they are genes of unknown function, not previously linked to invasion, or lack an obvious membrane anchor. Having gone to the lengths of exploring potentially compensatory changes in gene expression, it is disappointing not to validate or explore the hits that result.

While we understand the reviewers comment, as outlined in the text we did not identify any upregulated proteins that looked like strong candidates to compensate for loss of MSP2 to explore in this manuscript. Instead, we chose to further investigate any potential loss of MSP2 phenotype that yielded the observations around improved potency of antibodies targeting some merozoite antigens with loss of MSP2. This will be explored in future studies as we try and understand the role of MSP2 in more detail and the interactions between proteins and antibodies on the merozoite surface.

(4) Given the abundance of PfMSP2 on the merozoite surface, it would have been interesting to see whether the knockout lines have any noticeable difference in surface composition, as viewed by electron microscopy, although, of course, this experiment relies on access to the appropriate facilities.

We agree with the reviewer, but this lies outside the scope of this manuscript and optimisation of the imaging platform used to gain biologically useful insights would take a considerable amount of work based on feedback from people working with these techniques.

(5) One of the key findings is that deletion of PfMSP2 increases inhibition by some antibodies/nanobodies (some anti-CSS2, some anti-AMA1) but not others (anti-EBA/RH, anti-EBA175, anti-Rh5, anti-TRAMP, some anti-CSS2, some anti-AMA1). The data supporting these changes in inhibition are solid, but the selectivity of the effect (only a few antibodies, and generally those targeting later stages in invasion) is not really discussed in any detail. Do the authors have a hypothesis for this selectivity? The authors make attempts to explore the mechanisms for this antibody-masking (Figure 7), but the data is less solid. Surface Plasmon Resonance was non-conclusive, while an ELISA approach co-incubating MSP2 and anti-AMA1 antibodies to wells coated with AMA1 lacks appropriate controls (eg, including other merozoite proteins in similar experiments).

As outlined in our response to point (7) for Reviewer 2 (Recommendations for the authors), we have repeated the ELISA based assessment of recombinant MSP2s impact on anti-AMA1 antibody binding. In addition, we have included two comparator control proteins, the intrinsically disordered MSP4 of P. falciparum and the globular domain of the neural cell adhesion molecule (NCAM, CD56, 16 kDa), and found these proteins did not impact binding of anti-AMA1 antibodies. This strengthens the data that links the presence of MSP2 to reduced activity of anti-AMA1 antibodies.

As covered in our response to point (7) for Reviewer 2 (Recommendations for the authors) we provide additional discussion of this phenotype. We note that the list of inhibitory antibodies tested is not exhaustive, and additional antibodies may be identified where loss of MSP2 could improve potency. So although we see a consistent effect with a relatively small number of antibody targets, this does not rule out additional examples that may act earlier in invasion (for example, we noticed a small, but not statistically significant, trend for mildly inhibitory antibodies targeting MSP1-19 as well) and this makes speculating on why these two initial antibody targets at this time problematic.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) If feasible, perform ex vivo assays to demonstrate that the masking effect operates with physiologically relevant antibodies.

For this manuscript, we focussed on characterising the MSP2 knock-out parasites using the best reagents available. We remain interested in understanding whether these lines can be used to investigate the activity of functional antibodies from malaria exposed human serum and this will be the subject of future studies.

Reviewer #2 (Recommendations for the authors):

(1) As noted in the Public Review, the section describing MSP2 orthologues in other Laverania and avian Plasmodium species is overly long and not the most novel section of the manuscript. It could be really radically trimmed back.

We have taken this suggestion for the reviewer on board and have significantly cut back on our descriptions of the basic similarity properties of the conserved N and C-terminal regions as well as the description of the central variable region. Effectively, we have cut back the number of words through this section from 864 across 3 paragraphs to 478 across 2 paragraphs. While we have chosen to greatly economise our description of the N and C-terminal conserved regions, we have maintained much of the description of the similarities and differences in the central variable region as we believe the observation that this variant region still maintaining repeats, though they differ in size, number and amino acid composition, across such evolutionary distances is of interest.

Taking the reviewers comment on board, we have also removed Table 1 from the manuscript (shows amino acid sequence properties of these regions) and instead have inserted the tables relevant for each alignment in Supplementary Figures 2, 3 and 4 as appropriate. This will streamline the main manuscript and better align amino acid property and alignment data in the one Figure. We thank the reviewer for this feedback and believe that this has helped focus the text on the most important observations.

(2) Figure 2C - As MSP2 has stage-specific expression, it could be informative to incorporate an antibody targeting another gene with a similar stage-specific expression pattern, such as AMA,1 into the blot. This would confirm that both protein samples were collected at a similar point during blood stage development.

We have modified Figure 2C to include both the original comparison using PfAldolase as the loading control and also the merozoite expressed PfGAP45 as a loading/stage specific control as per the Figure.

(3) Figure 2D - Magenta and red are hard to distinguish in the merge channel. Is it possible to pseudocolour one of these channels a different colour? Also, it would be simpler to keep PfMSP2 a consistent colour in both rows.

Thank you for this suggestion and we agree that the comparison could be made clearer. For this figure, we have coloured DAPI to label the nuclei (Cyan), and antibodies targeting PfMSP2 (Magenta), PfAMA1 and PfMSP1-19 (Yellow). This is also reflected in the merged image. The Figure legend now reads:

“(D) Distribution of key merozoite surface proteins in the presence or absence of PfMSP2 was visualised by immunofluorescence. PfMSP2 (magenta), the nucleus stained by DAPI (cyan) and PfAMA1 (yellow, top two rows) or PfMSP1-19 (yellow, bottom two rows), and the coloured merge of the preceding panels. Scale bar = 0.7 µm. Representative images shown from a minimum of 10 schizonts imaged per condition.”

(4) Figure 2F - Static growth relative to shaking growth is plotted in this panel; perhaps this could be more clearly described in the legend or mentioned in the text that there was not a significant alteration in growth in static or shaking conditions.

As suggested, we have clarified the result in the Figure legend text as follows:

“(E-F) Growth of Pf3D7 WT compared to Pf3D7 ΔMSP2 P. falciparum parasites, measured as fold increase in parasitaemia, over one (48 hrs) or two (96 hrs) cycles in either standard (still- (E)) or shaking (F) conditions, with no measurable difference between parasite growth rates seen between standard or shaking conditions.”

Please also describe the shaking conditions used (i.e., speed, culture size, and vessel) in the methods.

We have updated the methods to provide information on the growth conditions used in the standard versus shaking growth assays:

“The initial parasitemia of cultures was determined by flow cytometry and then measured again after the 50 mL cultures in 96 well plates were maintained under standard (still) or shaking (50 rpm) conditions for 48 hrs or 96 hrs of growth.”

(5) Figure 3G - Annotate legend for strength of deformation to describe what 1,2, or 3 refers to.

We have added the following to the Figure legend of Figure 3G:

“Deformation scores are as defined by Weiss et al (Weiss et al., 2015), with 1 = weak deformation of the RBC membrane at the point of contact, 2 = strong deformation leading to the RBC membrane extending up the sides of the merozoite and changes in RBC membrane curvature beyond the point of contact and 3 = extreme deformation indicated by the merozoite being deeply embedded in the RBC membrane and strong deformation of the RBC well beyond the point of contact.”

There is a small visible shift in the deformation event scores. Is this also not significant? Even if deformation is not significantly longer, could this small effect alter the exposure of epitopes on other proteins for antibody targeting?

We did test the deformation event scores and the differences were non-significant. We have considered this possibility raised by the reviewer, but we are cautious in over interpreting the possibility that these trends might contribute to the increased potency of certain antibodies in the absence of additional data. We note that, although deformation may happen over a slightly longer timescale and show more aggressive deformations with PfMSP2 knock-out, this also seems to translate into a weak trend for faster overall entry for those merozoites that go on to invade. Therefore, although deformation may be longer and stronger, antibodies may have less time to block invasion overall. We are not confident that we can interpret around what might be happening at the molecular scale here based on this data and have chosen not to discuss this possibility in the manuscript. However, we have added the following to the results to better explain the phenotype the phenotype we observed.

“This analysis showed that, although there was a trend for PfDd2 ΔMSP2 knock-out parasites to have a higher mean time to attach to the RBC, as well as for the length and strength of RBC deformation, these trends did not reach significance. For those merozoites that did invade the RBC, on average it took less time for PfDd2 ΔMSP2 knock-out parasites to invade then PfDd2 WT, but this again did not reach significance (Figure 3 E-H). Together these data show PfMSP2 is not essential for blood-stage replication in vitro in two P. falciparum laboratory isolates from different geographical regions and knock-out of PfMSP2 does not seem to significantly impact parasite growth or merozoite invasion in vitro.”

(6) Figure 4C - Legend refers to black lines, but on the figure, they are red? Is the horizontal red line in the correct place, or should some of the dots below it be black rather than blue if they fall outside the adjusted p-value significance cut-off? Were 4 schizont harvests performed in total, or 4 for each cell line?

We thank the reviewer for pointing this out and we have now changed the text to say red lines. We have also provided more information in the Figure legend to more clearly define what data is represented. In short, 4 harvests were performed for each cell line (8 in total across the 2 cell lines) and the data represents the distribution from one of these harvests. The blue shaded genes are those that, on average, across the 4 Pf3D7 WT and Pf3D7 ΔMSP2 paired harvests show up or down-regulated expression. This is why some of the blue shaded genes lie near or below the cut-off values represented by the red line. The Figure legend text has now been modified as follows.

“(C) Log2(fold change) for differentially expressed genes, including multigene families, between the transcriptome of Pf3D7 WT and Pf3D7 ΔMSP2 schizonts. Plot represents the results for one of four independent schizont RNA harvests for Pf3D7 WT and Pf3D7 ΔMSP2 parasites and red lines differentiate genes with a log2 (fold change) > 0.5 and < -0.5 with adjusted p-value < 0.01. Genes shaded blue represent those genes that were found to have an average log2 (fold change) > 0.5 (dark blue) or < -0.5 (light blue) across the four replicate samples compared. Significance determined as below p< 0.05 after correction for multiple testing.”

(7) Figure 7D - ELISA results don't show a convincing concentration-dependent inhibition, and repeating with another recombinant protein is essential before inferring that the effect is specific to PfMSP2

We have repeated the ELISA experiment using recombinant PfMSP2 to reduce variability across the assay and again found a dose dependent reduction of anti-PfAMA1 binding with increasing concentrations of recombinant PfMSP2. It should be noted that this is a completely new set of experiments that recapitulate the original findings. See updated Figure 7D.

We agree with the reviewer that the experiment and interpretation of the data would be strengthened by comparing any potential inhibitory impact on anti-PfAMA1 binding to a different recombinant protein. Therefore, we have completed identical experiments using the similarly intrinsically disordered PfMSP4 recombinant protein (40 kDa) and the highly structured 16 kDa immunoglobulin domain of human neural cell adhesion molecule (NCAM). We find that there is no dose dependent loss of anti-PfMAMA1 binding to recombinant PfAMA1 with addition of PfMSP4 or NCAM immunoglobulin domain recombinant protein. These controls are contained in Supplementary Figure 6, the relevant text is provided below.

‘In contrast, increasing concentrations of the intrinsically disordered MSP4 from P. falciparum 3D7 (40 kDa) and the highly structured immunoglobulin domain of neural cell adhesion molecule (NCAM, CD56, 16 kDa) recombinant proteins did not impact on binding of anti-PfAMA1 antibodies to recombinant AMA1 (Supplementary Figure 6).’

(8) Again, as noted in the public review, the target-specificity of the inhibition-masking effect is perhaps the most surprising aspect of the data - this could do with much more thorough discussion. Why only these proteins, both of which function late in invasion?

Overall, we tested several growth inhibitory and non-inhibitory antibodies shown to bind specifically to individual or some combination of nine P. falciparum merozoite surface and secreted proteins. However, we do not consider this to be an exhaustive list of potentially invasion inhibitory antibodies by any means. We mostly did not observe any non-inhibitory antibodies becoming significantly more growth inhibitory to PfMSP2 KO lines, indicating that these antibodies were not impacted by loss of PfMSP2 or had no functional inhibitory effect in these assays.

What we do demonstrate here is that we see a consistent impact with different rabbit, mouse monoclonal and i-body growth inhibitory antibodies targeting PfAMA1, indicating that it is not a spurious result from a single antibody or antibody type. We also find a second example, with nanobodies targeting the PfPCRCR complex protein PfCSS potentiated with loss of PfMSP2. This opens up the possibility that other growth inhibitory antibodies to the antigens tested here, or growth inhibitory antibodies targeting other antigens involved in merozoite invasion, may also become more potent with MSP2KO. Although both PfAMA1 and PfCSS function late in invasion, it is too early to say whether this is a functional trend or an observation that is related to the panel of antibodies tested. Therefore, further testing using lines developed in this study could yield additional examples of antibodies that become more inhibitory with MSP2 KO and provide additional information on the potential impact that MSP2 may have on their vaccine potential. In order to address this, we have added the following text to the discussion:

“Here we show consistent potency improvement with PfMSP2 knock-out for growth inhibitory rabbit, mouse monoclonal and i-body antibodies targeting PfAMA1, as well as demonstrate improved activity for and Fc-tagged nanobody targeting PfCSS, indicating that these are not outlier results from a single antibody or antibody type. However, increased antibody potency was not shared across all antibodies tested, possibly because the specific function or localisation of a target protein, the region that an antibody binds to or the functional activity (or lack thereof) of an antibody may all play a role in determining whether loss of PfMSP2 can potentiate growth inhibitory activity. Further investigation using the parasite lines developed in this study and a wider panel of antibodies that target different stages of the merozoite invasion process could shed more light on this potentially novel mechanism of vaccine derived antibody efficacy.”

(9) Typos/minor editorial points:

L111 – conserved

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L235-237 - check the wording in this sentence for clarity

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Figure 3E - 'attachment' on axis

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L350 - mentions eight 'proteins' having expression increase, instead 'transcripts' should be referred to when describing RNAseq data, as transcript levels may not correspond directly with protein levels. Also, be careful when referring to transcript or protein throughout this paragraph.

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Figure 4A - instead of 'transcription during schizonts', better to say 'schizont transcript abundance'

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L514 - 'detectable binding to PfAMA1'

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L589 - Is it a mouse Fc region or a human Fc region that is added? The human Fc region is mentioned in the results.

In the growth inhibition assays anti-AMA1 WD34 i-body with a human FC region was used and in the ELISA assays anti-AMA1 WD34 i-body with a mouse FC region (to enable detection of AMA1 binding use the same secondary anti-body for both the WD34 i-body and the 4G2 mouse monoclonal antibody) was used. The text has been been checked and modified accordingly to clearly say this.

Supplementary figure 3 - 'repeats'

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