Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination

  1. Katja Müller
  2. Matthew P. Gibbins
  3. Arturo Reyes-Sandoval
  4. Adrian V. S. Hill
  5. Simon J. Draper
  6. Kai Matuschewski
  7. Olivier Silvie
  8. Julius Clemence R. Hafalla

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Abstract

Vaccine discovery and development critically depends on predictive assays, which prioritise protective antigens. Immunogenicity is considered one important criterion for progression of candidate vaccines to further clinical evaluation, including phase I/II trials. Here, we tested this assumption in an infection and vaccination model for malaria pre-erythrocytic stages. We engineered Plasmodium berghei parasites that harbour a well-characterised epitope for stimulation of CD8+ T cells either as an antigen in the circumsporozoite protein (CSP), the surface coat protein of extracellular sporozoites or in the upregulated in sporozoites 4 (UIS4), a major protein associated with the parasitophorous vacuole membrane (PVM) that surrounds the intracellular exo-erythrocytic forms (EEF). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust and superior antigen-specific CD8+ T cell responses, whilst an EEF antigen evokes poor responses. However, despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine-induced, antigen-specific effector CD8+ T cells results in high levels of protection when targeting both antigens. Our study is the first demonstration that poor immunogenicity of EEF antigens does not preclude their susceptibility to antigen-specific CD8+ T cell killing. Our findings that antigen immunogenicity is an inadequate predictor of vaccine susceptibility have wide-ranging implications on antigen prioritisation for the design and testing of next-generation pre-erythrocytic malaria vaccines.

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    Reply to the reviewers

    Initial plan – Response to Reviewers’ Comments

    We thank the three reviewers for their comments, which on balance were very positive and supportive.

    The fundamental relevance and translational impact of our manuscript were reinforced by R1 and R3: The present study ”could provide important insight in the field of malaria vaccinology …” (R1), “…lead to a complete revision in how pre-erythrocytic vaccine candidates are identified and prioritized …” (R3) and “… greatly enhances our understanding of exactly how the dynamics, magnitude and quality of CD8+ T-cell responses are modulated by the timing of antigen expression …” (R3).

    R1 acknowledged our use of “using cutting edge molecular biology (techniques)” and our efforts to “… provide proof of the concept of vaccine design by evaluating if accessibility/immunogenicity of the antigen is a decisive feature on vaccine design …”. R3 emphasised that the “… manuscript is well written, concise and with a clear narrative…” and that “… conclusions drawn from the study are well supported by the data presented, and the experiments are thoroughly controlled and sufficiently replicated”.

    We have now revised the manuscript based on the reviewer’s comments and clarified valid concerns. Together, we consider the review process very helpful to further enhance the impact of our study. At this point, we have also prepared a Graphical Abstract that summarises the key findings of the manuscript.

    We address the Reviewer’s Comments below:

    Reviewer #1 (Evidence, reproducibility and clarity):

    1. General comments: … a little more of deep analysis of immune responses elicited by the transgenic parasites … how about TRM cells, what is the endogenous responses to SIINFKEL without transferring CD8 + T cells from OTI mice?

    … the potential of this study demands to be placed in the context of precedent studies that defined pre-erythrocytic stage CD8+ T cell responses. … the importance of CD8+ liver-resident memory CD8+ T cells from Health’s laboratory

    We agree with the reviewer that the field of malaria pre-erythrocytic immunology is fast-moving. This is exemplified by the more recent identification of resident memory CD8+ T cells (TRM) that patrol hepatocytes against pathogens, including malaria pre-erythrocytic stages by the Heath laboratory. Whilst the focus of our current work is on assessing the timing of expression and immunogenicity of pre-erythrocytic antigens as crucial features for vaccine design, we concur with the reviewer that the analysis of TRM is of remarkable interest to the malaria field, which we believe is currently out of the scope of the current study. Nonetheless, we have now included this in the discussion to link the findings of our study with the evolving field of TRM (lines 409-420).

    Nevertheless, we characterised CD8+ T cells using techniques that are commonplace in studying immunological response in the malaria field, while also adapting our approach to probe responses using more physiological proxies.

    Whilst we did not specifically phenotype for TRM, we measured CD8+ T cell responses in the livers of immunised mice, utilising isolation methods for intrahepatic or liver-infiltrating lymphocytes (Goossens et al., 1990, PMID: 2202764). CD8+ T cells from the livers of mice immunised by irradiated sporozoites delivered intravenously were analysed following adoptive transfer of naïve CD8+ T cells (Figure 3), as well as quantifying endogenous CD8+ T cells (Figure 4B-D). We also assessed CD8+ T cells from the livers of mice immunised with irradiated sporozoites delivered intradermally (as a proxy for the natural route of infection and parenteral vaccine administration; Figure 4E-G).

    As mentioned above, the response from endogenous CD8+ T cell, that is without adoptive transfer of naïve CD8+ T cells, are shown in Figure 4A-C (intravenous immunisation) and Figure 4D-F (intradermal immunisation).

    We apologise to the reviewer if these results were not obvious. Accordingly, we have reconfigured the figures to i) colour-coordinate intravenous from intradermal administration of sporozoites in Figure 4, and ii) to allow differentiation of pentamer staining from IFN-g production in Figure 3.

    1. … the strategy of gating on Fig2a, it is not clear if they want to track the responses from adoptively transferred CD8+ T cells to vaccine or the endogenous CD8+ T cell responses. In any case, the results is potentially interesting but need clarification.

    The purpose of adoptively transferring naïve CD8+ T cells was to augment the frequencies of naïve precursors. We used Kb-SIINFEKL pentamers to visualise the developing CD8+ T cell response, which is a combination of both OT-I and endogenous responses. As mentioned above, we have compared the kinetics of the CD8+ T response in mice administered with OT-I cells (Figure 3) or endogenous responses in the absence of OT-I cells (Figure 4).

    1. Fig 2: only the responses on the spleen are studied. In order to support the statement about the two different kinds of immunization, they should assess the responses on the liver.

    As pointed out by the reviewer, responses in the liver were not performed for Figure 2, but were assessed in Figures 3 to 5.

    1. … lack of methodology in flow cytometry analysis, a viability stain is not used, the gating is not determined by FMO … controls … For activation markers in order to assess the impact of the vaccination authors have to use gating that is already established by some of the papers they mentioned (i.e: Harty lab's studies), … the CD11a label should be CD11ahi and is not stated anywhere.

    In our gating strategies we relied on the population of cells with a larger FSC value (healthy cells). In previous experiments we established this population to represent the desired population by staining cells with and without Live/Dead dye. Accordingly, we have found this first gating strategy to be satisfactory and consistently excluded dead cells and cell debris.

    We apologise for not showing FMOs and have now included exemplary flow cytometry strategies in Supplementary Figure 2 and 5 to illustrate how we gated for CD8+ T cells from blood, spleen and liver. In addition to FMOs we gated for markers in our pentamer and surface stain panel and restimulation cytokine panel.

    We have focused on the enumeration of antigen-specific responses using KbSIINFEKL pentamer staining, and the measurement of the effector molecule IFN-g for the evaluation of responses to SIINFEKL. In both methodologies, responses were co-stained with CD8 and CD11a. The utilisation of the CD11a marker and identifying CD11ahi populations in models of infections were established by the Harty and Badovinac laboratories (Rai et al., 2009, PMID: 19933864). CD11ahi populations discriminate antigen-experienced but not inflammation-driven responses, particularly when analysing polyclonal populations of CD8+ T cells. We have referenced the original publication in the manuscript. Moreover, we have corrected mentions and labels of CD11a+ to CD11ahi throughout the manuscript. Thus, in addition to KbSIINFEKL pentamer and IFN-g stainings, the CD11a marker was used as a confirmatory marker for antigen-driven activation. Furthermore, we also stained the cells for canonical activation markers: CD49d, CD62L and CD44. It is notable that the numbers of CD11ahi, CD49dhi, CD62Llo, CD44hi co-stain with Kb-SIINFEKL pentamer (Figure 2 and Supplementary Figure 3), indicating that the identified cells are of effector/ effector memory phenotypes.

    1. Line 165: the statement "massive proliferative activity" is not supported by the figure, moreover there are numbers to support the statement.

    We have toned down the term from “massive” to “greater”. We have also altered the sentence to read “… immunisation with CSPSIINFEKL sporozoites led to greater expansion of Kb-SIINFEKL+ CD8+ T cells, 6x larger than that observed with UIS4SIINFEKL sporozoites…” (line 195-196), which is in agreement with that shown in Figure 2D, E.

    1. -->IFNg and other cytokines production seems too low and the stimulation assay is poorly performed because CD8 were restimulated ex-vivo only with SIINFEKL peptide in the absence of APC (antigen-presenting cells) with Brefeldin A. Also Authors omitted negative controls ( without SIINFEKL Brefeldin A) to be certain that IFNg production is du to SIINFEKL. Again we don't if they are OTI or endogenous cells.

    We have utilised stimulation and flow cytometry protocols that are widely used in the malaria pre-erythrocytic stage field (Hafalla et al., 2013, PMID: 23675294; Jagannathan et al., 2015, PMID: 25520427), as well as other fields (Hosking et al., 2014, PMID: 25015828, Nakiboneka et al., 2019, PMID: 30459072). Notably, CD8+ T cell responses to this eukaryotic pathogen have been widely published to be much lower, in contrast to those evoked by viral and bacterial pathogens (Schmidt et al., 2008, PMID: 18780790).

    As suggested, we have now included the corresponding negative controls (restimulation without peptide) in a new Supplementary Figure 6.

    1. Fig5. Are the cells from Fig5a,b SIINFEKL positive cells or only CD11a and IFNg? Are they OTI? Controls are missing to show a real IFNg production du to the ex vivo stimulation.

    The responses shown in Figure 5 were stimulated with SIINFEKL and stained for CD8+ (gated), CD11ahi, IFN-+. The mice did not receive OT-I cells, thus the data reflects the endogenous response.

    1. Fig 6. no percentages are shown in the cytometry plots, figure 6d and c seem to be inverted.

    We have now included the percentage in the flow cytometry plots. We apologise for the inversion of Figures 6C and D; this has now been corrected to match the figure legend.

    1. For the two strains, authors should show the patency in comparison whit WT parasites (currently presented as data not shown)

    We have now included the patencies of WT, CSPSIINFEKL and UIS4SIINFEKL parasites in Supplementary Figure 1f. The transgenic parasites exhibit similar patencies to WT parasites.

    1. Fig 6: how did the authors measure Sterile protection and Relative parasite load?

    We have detailed the measurements of sterile protection and relative parasite load (level) in the Methodology section. Both methodologies are standard procedures in the malaria pre-erythrocytic stage field.

    Reviewer #1 (Significance):

    The present study could provide important insight in the field of malaria vaccinology. By using cutting edge molecular biology to express the MCHI restricted epitope SIINFEKL a at different stages of the pre-erythrocytic stage of Plasmodium and used it as a surrogate marker to evaluate the CD8+T cell response to infection. The authors attempt to provide proof of the concept of vaccine design by evaluating if accessibility/immunogenicity of the antigen is a decisive feature on vaccine design. Nevertheless, the potential of this study demands to be placed in the context of precedent studies that defined pre-erythrocytic stage CD8+ T cell responses. the authors failed to fully exploit the tools that they developed (transgenic parasite) by overlooking the last studies describing the importance of CD8+ liver-resident memory CD8+ T cells from Health's laboratory or well characterized CD8 T cells responses defined by Harty's laboratory.

    If well place in the context (after revisions) this study will not only be fundamental to the malaria field but to other infectious diseases as well.

    Field of expertise: malaria immunology, vaccinology, immunomodulation, CD8+ T cell responses

    Reviewer #2 (Evidence, reproducibility and clarity):

    The manuscript b Mueller and Gibbins et al titled "Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination" compares how transgenic P. berghei parasites expressing SIINFEKL epitope from ovalbumin, as part of CSP or UIS4 present the respective epitope and how immune responses occur to each of the mutants, mostly in mice pre-treated with 2 x 10 OT-I cells expressing a SIINFEKL-specific TCR.

    their data show that when in their normal location CSP is much better than UIS4 to elicit an immune response., and that increasing UIS4 (by raising irradiated parasite numbers) does not greatly improve to reduce the difference.

    finally the authors show that mice immunized with ovalbumin can reduce liver infection of either CSPSIINFEKL or UIS4SIINFEKL sporozoite challenge infection

    the experiments presented by the authors are in my view well done and controlled, but i feel that sometimes conclusions are a bit beyond what the experimental readouts allow for.

    Reviewer #2 (Significance):

    1. In fig1 the authors show how mutants were made and that proteins with associated SIINFEKL to CSP or UIS4 localise to correct place. (could all be supplementary or Supplementary Figure 1c, d could be included in Fig1). In Fig2a is shown the gating of SIINFEKL-specific CD8+ T cells (could be supplementary).

    We deem the depiction of CSP and UIS4 in Figure 1B and C to be important for the concept and impact of the study. We would like to adhere to common practice in immunology studies and keep one representative flow cytometry gating strategy in the main paper (in Figure 2B), to illustrate an example of our analysis methods going forward.

    1. In Fig 2b the authors show that the highest CD8T cell specific for SIINFEKL is on the first day analysed (d4) and I would like to see how day 2 and 3 would look like.
      specially because proliferative differences don't seem massive to me, CFSE should decrease with each cell division and reach different fluorescence values if replication numbers differ. however here the CFSE fluorescence signal is similar on d5 fig 2c, indicating a similar number of replicative rounds, but probably a different starting numbers of cells that would replicate. Or that CFS labelling was too low to allow distinguishing the number of replicative rounds occuring in that time. so when the authors conclude that proliferative activity was 6x larger than that observed with UIS4SIINFEKL sporozoites, i think they would have to show before that numbers of cells prior to replication was the same

    This is a good suggestion, but unfortunately, we did not perform CFSE experiments on days 2 and 3. We agree that the resulting CD8+ T cell responses to both parasites seem to have similar replication rounds (number of cell division), yet the frequencies of those recruited to the immune response are much more elevated in the CSPSIINFEKL as compared UIS4SIINFEKL parasites (5.05 vs 0.84, respectively – as shown in Figure 2D). A better representation is shown in Figure 2E, which is gated on KbSIINFEKL+, CD11ahi, CD8+ T cells.

    For our study, we have used published and standard CFSE labelling protocols (Lundie et al., 2008, PMID: 18799734).

    In light of Reviewer 1 and 2 both commenting on our use of terminology regarding proliferation, we altered and corrected the text in the manuscript to address that there is a 6x increase (5.05 vs.0.84) in recruitment of SIINFEKL-specific CD8+ T cells rather than proliferation (line 195-196). The same number of cell divisions were undergone, however the level of expansion was greatly increased when mice were immunised with CSPSIINFEKL.

    1. I think it would be nice to show when is infection stopped in these two groups os mice, but looking at EEF in the liver if the two groups of mice.

    We believe that the reviewer is referring to the outcomes of the protection experiments. We utilised a widely used quantitative PCR method to quantify the EEF in the liver after challenge of vaccinated mice. We agree with the reviewer that it will be interesting to determine whether the kinetics of killing by vaccine-induced CD8+ T cells of CSPSIINFEKL and UIS4SIINFEKL parasites are different. While presently out of the scope, we would have to establish ex vivo quantitative imaging and hope to advance on this in the future.

    14 … could show that an adenovirus carrying UIS4 (and CSP) would result in the same as observed here with the ovalbumin one).**

    The vaccine efficacy of an Adenovirus vaccine expressing CSP has been established (Rodrigues et al., 1997, PMID: 9013969; Bruña-Romero et al., 2001, PMID: 11553779; Gilbert et al., 2002, PMID: 11803063. Since there are no known ‘immunodominant’ CD8+ T cell epitopes in UIS4 such a vaccine construct is likely to only serve as negative control.. We and others have previously systematically screened for CD8+ T cell epitopes in the pre-erythrocytic stages, including from UIS4, of Pb, but experimental testing yielded only few peptides, with none from UIS4.

    15 … discuss the advantages and problems of the two SPZ and PVM locations, assuming that indeed an adenovirus carrying UIS4/CSP would also result in similar protection upon challenge, regarding potential boost from natural Infection, and how variable/conserved each of the proteins are and what could be expected in field trials ion the falciparum counterpart.

    We have now included these points in the discussion. Thus far, the consensus in the field is that T cell responses to pre-erythrocytic stage antigens are low in endemic areas (Heide et al., 2019, PMID: 30949162), and there is a striking paucity of data on the impact of boosting (primary infection vs. multiple infections) in the field (Doolan et al., 1993, PMID: 7680226); Khusmith et al., 1999, PMID: 10774643). Previous work in rodent models has demonstrated that boosting of T cell responses to liver stage antigens is poor (Murphy et al., 2013, PMID: 23530242), and this was also documented for CSP (Hafalla et al., 2003, PMID: 12847268). With the very low responses to UIS4SIINFEKL, we reasoned whether they could be enhanced by increased dose of immunisation. However, Figure 5 rejected this hypothesis.

    It is noteworthy that we selected CSP and UIS4 as the best characterized representatives of sporozoite and EEF vacuolar antigens, respectively. Following up on the reviewer’s comments, it would be interesting to contrast the allelic diversity of sporozoite and EEF antigens, since this information will be important for vaccine design.

    Reviewer #3 (Evidence, reproducibility and clarity):

    1. The authors assume a reader familiarity with the use of ovalbumin, the SIINFEKL epitope, the transgenic T-cell receptor OT-1 mice and adoptive transfer experiments to assay immunogenicity. These concepts are not comprehensively introduced in the introduction, and the relationship between these tools are not delineated sufficiently to allow the non-expert reader to follow the logic and methodology of the experiments right from the start. Background information given in Results (Line 139-144, 204-208) and Discussion section, (Line 288-293) could with advantage be synthesized into one paragraph and presented in the introduction to bring all readers onboard from the start.

    We thank the reviewer for this important point and have now addressed this in the introduction which reads as follows:

    “To control for epitope specificity, we generated Pb transgenic parasites that incorporate the MHC class I H-2-Kb epitope SIINFEKL, from ovalbumin, in either the CSP or UIS4 protein. The resulting transgenic parasites develop normally as wild-type (WT) Pb in the mosquito vector and mammalian host. However, SIINFEKL would be expressed at the same time and space as its respective Plasmodium protein, enabling the CD8+ T cell response against these proteins to be tracked in an epitope-specific physiological manner. In line with previous studies (8,15), to augment low numbers of CD8+ T cell in the naïve response, cells from OT-I mice, which express SIINFEKL-specific TCRs on their CD8+ T cells, were initially adoptively transferred to mice prior to them receiving sporozoite immunisations” (lines 117-127).

    1. Results Page 11 Line 255-260, Figure legend Fig. 6 page 27 Line 258-665 The punchline of the paper is that the despite differences in immunogenicity between γ-irradiated CSP SIINFEKL or UIS4 SIINFEKL sporozoites, both CSP SIINFEKL or UIS4 SIINFEKL are targets of protective CD8+ T-cell responses resulting in sterile immunity in a challenge following vaccination with full-length ovalbumin in OT-I cell recipient mice. This section is a cornerstone for the conclusions of the paper and would benefit from being better supported by its explanatory text and presentation of data.

    Firstly, there is a mix up, between panel 6c and 6d, where 6c shows "% Sterile protection" and 6d shows "Parasite load in the liver", while it says the opposite in main text and figure legend.

    We apologise for this error, which has now been corrected. We also added more explanatory text in the results section to avoid reader’s missing the punchline and impact of our study, which reads as follows: “Strikingly, contrary to the differential CD8+ T cell responses induced by CSP and UIS4, there was no statistical difference in the protection observed when vaccinated mice were challenged with either CSPSIINFEKL or UIS4SIINFEKL sporozoites. Consistent with these findings, both groups of vaccinated mice challenged with either CSPSIINFEKL or UIS4SIINFEKL sporozoites exhibited sterile protection of comparable levels…” (lines 305-310).

    1. Secondly, while it is clear that qPCR is used to measure liver parasite load at 24 hours after challenge. It is not immediately clear from neither main text nor figure legend that sterile immunity is measured by microscopy on blood films. The use of the term "sterile immunity" naturally implies this to the initiated reader, but it should be spelled-out that this was the case and that it was monitored from day 3-14 following challenge, which is outlined only in the methods section. Rewriting and restructuring this section to make this clearer would greatly help guide the reader through the results. Currently it reads at first pass as if qPCR on liver samples harvested at 42 hours was used to generate the data in both 6C and 6D.

    We agree that this important point should be consistently described and have now added the necessary clarification in the results (line 310-311) and figure legend (line 799-800) to indicate that we used microscopy to assess blood smears for parasitaemia.

    19: Thirdly, protective efficacy here is given as a percentage of those mice that become protected, presumably remaining negative by day 14. Authors should provide the actual blood stage parasitaemia in graph or table format in Figure 6 or as a supplemental figure to show that sterile immunity is obtained and maintained until day 14, and that in the control groups patency develops as normal. This will also give clearer insight into how many mice developed patency in the control groups and at what point break-through was observed.

    We have now included prepatency in our manuscript to illustrate if and when non-vaccinated and vaccinated animals became parasitaemic. Mice were monitored up to day 14, after which they were deemed sterilely protected. This is found in the new Supplementary Table 2.

    1. In a similar vein, qualitative and / or quantitative presentation of microscopy data of EEFs (as presented in Figure 1C) would strengthen conclusions drawn from the qPCR parasite liver load data.

    We have included a graph detailing quantitative data of EEF counts as Supplementary Figure 1E.

    1. Fourthly, the authors should also comment on why there is such a great variation in the number of mice used in the different studied groups, it says maximum of n=11 mice per group but one group only has as n=3 mice and another n=4, and make a convincing argument this does not affect the conclusions drawn and statistical analysis undertaken.

    In this experiment (Figure 6D), we placed particular emphasis on the quantification of the liver load in AdOVA-immunized mice challenged with UIS4SIINFEKL sporozoites. We included cumulative data from multiple challenge experiments. The other groups of mice serve as controls and consistently displayed high parasite loads in non-immunized or WT sporozoite-challenged controls and very low parasite loads in CSPSIINFEKL sporozoite-challenged mice, respectively.

    1. Finally, the authors characterise CD8+ T-cell responses in absence of preceding OT-I adoptive transfer but do not report on whether the ovalbumin-immunization was tried on mice without preceding OT-I cell transplant. Was this tried? If not authors should discuss whether this is likely to be successful or not for readers to understand if both sporozoite and EEF presented antigens are likely to induce sterile immunity in a natural setting without artificial enrichment for epitope specific T-cells.

    We thank the reviewer for highlighting this point. We did not vaccinate mice in the absence of OT-I cells. Previous work with Py and _Pb_CSP-based adenovirus vaccines yielded only up to 40% sterile immunity, despite up to 97% reduction in parasite load in the liver after challenge with viable sporozoites (Rodrigues et al, 1997, PMID: 9013969; Rodrigues et al., 1998, PMID: 9795385). Thus, we augmented the numbers of naïve antigen-specific CD8+ T cell precursors by adoptively transferring OT-I prior to vaccinating with recombinant adenovirus. This methodology was chosen in order to attain optimal levels of vaccine-induced effector CD8+ T cells producing IFN-g in a single vaccination, and to obtain reliable frequencies comparable to those achieved by prime-boost vaccinations with recombinant adeno- followed vaccinia viruses, or with peptide-loaded dendritic cells followed by recombinant Listeria. Previous work by colleagues and ourselves have shown that in order to achieve sterile protection in both the Py- and Pb-Balb/c model, vaccine-induced CSP-specific CD8+ T cells must exceed a threshold of >1% of all CD8+ T cells in peripheral blood (Bruña-Romero et al., 2001, PMID: 11553779; González-Aseguinolaza et al., 2003, PMID: 14557672; Schmidt et al, 2011, 21460205). Moreover, B10 backgrounds (including C57BL/6) further increases the threshold necessary for sterile protection through a CD8+ T cell-extrinsic mechanism. In our current study, the mean frequencies of antigen-specific CD8+ T cells induced following adenovirus vaccination was 7.5% (Figure 6C), which translated to 80% sterile protection (combined data from CSSIINFEKL and UIS4SIINFEKL groups).

    In the current manuscript, we believe that we have successfully provided proof-of-concept evidence to assess the timing of expression and immunogenicity of pre-erythrocytic antigens as crucial parameters for vaccine design. Nonetheless, we have added a comment in the discussion on our chosen approach to test for vaccine efficacy, and on the importance of achieving relatively high levels of CD8+ T cells to enable high vaccine efficacy:

    “Regardless of their differing immunogenicities in the context of parasitic infection, we further demonstrated that both sporozoite and EEF antigens are effectively targeted by antigen-specific effector CD8+ T cells, which were generated by vaccination using priming and boosting with recombinant viruses expressing the epitope. This method of prime-boost using recombinant viruses has been consistently shown to induce high numbers of antigen-specific CD8+ T cells (39-43) necessary for protection(20). Importantly, mice harbouring similarly high levels of vaccine-induced, antigen-specific CD8+ T cells were comparably protected when challenged with either CSPSIINFEKL or UIS4SIINFEKL” (lines 371-379).

    1. Line 723, 725 clarify if data is from independent biological repeats, i.e. different infected mice fed to different pots of mosquitoes, in which case the data is sufficiently replicated.

    The mosquito infectivity is from 14 different mosquito feedings and the sporozoite numbers per mosquito were calculated from 18 (UIS4SIINFEKL and WT) and 21 (CSPSIINFEKL n=21) independent infections.

    1. Page 6 Line 132 Please show blood-stage infection data / growth rates for CSP SINFEKL and UIS4 SINFEKL compared to WT as supplemental figure, if available.

    We have now included prepatency data for the two transgenic parasites (Supplementary Figure 1f).

    1. Figure 1. If quantitative data is available for EEF, as indicated by the mean numbers with SD given within the microscopy pictures it would be nice to see these plotted. Does the reduced EEF numbers for CSP SINFEKL compared to UIS 4SINFEKL and WT mean anything? If not perhaps worth stating this in figure legend, or consider different presentation. Distracting when looking at the figure.

    We have generated a graph depicting the numbers of EEFs developing in vitro from sporozoite of Huh7 cells from two independent experiments. This is now found in Supplementary Figure 1E.

    1. Figure 2. Would benefit from a panel with a simple schematic that shows the overall experimental design with irradiation of sporozoites, OT-1 transfer, administration of parasites and sampling with the timings for each event clearly marked out.

    We thank Reviewer 3 for this suggestion and have now included timelines of our experimental design for Figure 2, as well Figures 3-6.

    1. Figure Panel 2b would benefit from the in-figure legend stating CSP SINFEKL + OT-1, UIS4 SINFEKL + OT-1, WT + OT-1 and OT-1 only. Similar to as in Figure 3.**

    We thank the reviewer for this suggestion. Since OT-1 transfer was done in all groups of mice, and, hence, is not a distinctive feature, we have instead included a timeline on top of the graph with clear colour coding showing administration of OT-I cells prior to sporozoite immunisation (Figure 2A). We believe this is sufficient to guide the reader through the figure. Similarly, we reduced the labelling in Figure 3, and instead added a timeline as a reference for the experimental design **(Figure 3A)

    1. Figure 3 and Figure 4. Label within figure more clearly what is being measured, i.e. what is the difference between panels a,b,c vs. d,e,f (e.g. intravenously v.s. intradermal administrations), gets confusing since Figure 3 and Figure 4 are very similar within the figures (a,b,c vs. d,e,f) and between the figures.

    We thank the reviewer for this suggestion and have now added colour coding to Figures 3 and 4 and an experimental schematic to guide the reader through the data. We have included segregation lines above the flow cytometry plots to further guide the reader, i.e. Figure 3B-D denotes Kb-SIINFEKL pentamer data, while Figure 3E-G denotes IFN-g production following restimulation. Further, in Figure 4 segregation lines have been added and labelled to allow easy discernibility of panels 4B-D (intravenous immunisation of sporozoites) vis-a-vis panels 4E-G (intradermal immunisation of sporozoites).

    1. Figure 3, 4, The Panel indicating letters (a, b, c, d...) become smaller as figures get bigger and become hard to read for Figure 3 and Figure 4.

    This has now been adjusted, as suggested.

    1. Line 158 - Throughout manuscript, when it says administration of WT, CSP SINFEKL and / or UIS4 SINFEKL sporozoites it would be good to always have it preceded by irradiated when referring to irradiated sporozoites, e.g. Line 158 and only use sporozoites on its own when referring to live sporozoites (or even better spell out also when using live sporozoites e.g. Line 255).

    We have followed the advice of the reviewer including "g-radiation attenuated” or “live” as appropriate (lines 188, 202-203, 216, 236, 248-249, 260-261, 280 and 299).

    1. The authors could measure the total amounts of IFN-ɣ being secreted in the tissues after immunization to both antigens and investigate if the level of other IFN-ɣ secreting cells might compensate for the weak response of CD8+ T cells, particularly against UIS4. If completed it has the potential to help the authors to in more detail understand the mechanism and contributing factors to the successful CD8+ T-cell targeting of UIS4. As antigen protection is dependent not only cellular response but also on antibody responses induced against the antigens, authors should analyze by ELISA IgG and IgM responses induced against the two antigens.

    We thank the reviewer for raising interest on possible future directions for our study. We have specifically engineered SIINFEKL to be a part of either CSP or UIS4 and utilised an OVA-expressing adenovirus to focus on CD8+ T cell responses. However, we agree with the great idea from the reviewer that justifies further work in dissecting the multifaceted mechanisms underlying CD8+ T cell-mediated protection to malaria pre-erythrocytic stages, as well as future combinations to assess contributions of antibody responses.

    1. Page 6 Line 121-123 he authors reason that addition of the SIINFEKL epitope to the immediate C-terminus of the UIS4 protein might confer enhanced antigen presentation through increase MHC-I antigen presentation. Supplementary experiments particularly a MHC I stabilization assay might help confirm this. Complementary experiments looking at MHC-II antigen presentation to APC would also be very relevant.

    We have appended the SIINFEKL to the C-terminus of the UIS4, based on earlier studies in Toxoplasma gondii that the potency of an immunodominant epitope was associated with its C-terminal location, allowing for enhanced presentation by infected cells. Whilst this information is not defined for UIS4, studies on the basic biology of pre-erythrocytic stages have demonstrated for several ETRAMPs (UIS4 is a member of the ETRAMP protein family) that the C-terminus faces the host-cell cytoplasm, which might enhance exposure to the MHC I machinery. Our findings showing that vaccine-induced effector CD8+ T cell responses eliminate both transgenic parasites, argues against potential defects in antigen processing and presentation of SIINFEKL in both systems.

    Again, we agree that future studies aiming at dissecting the molecular mechanisms of MHC-I antigen presentation in infected host cells and cross-presentation via MHC-II are warranted. Another long-standing goal of the community is to elude Plasmodium peptides from MHC molecules, similar to the pioneer work by Rammensee and co-workers. One potential, albeit challenging, research direction could be to focus on rare EEF-derived peptides, since they might proof to be excellent and hitherto neglected subunit vaccine candidates, as exemplified in the present proof-of-concept study.

    1. The authors have described the effect of both antigens in the response of CD8+ T cells and the generation of memory. A more detailed characterization of the differential phenotypes of memory in CD4 and CD8 T cells in the spleen and liver following immunostimulatory therapy would increase the relevance of the data presented.

    We entirely agree that a more detailed characterisation of the different phenotypes of not only memory CD8+, including TRM, but also CD4+ T cell responses, is warranted. Whilst these suggestions clearly inspire further work using the transgenic parasites of this study by colleagues and ourselves, we believe that these are out of the scope of the current study.

    Reviewer #3 (Significance):

    This paper would be of interest to malaria parasite biologists, immunologists and vaccinologists alike. The significance of this paper is three-fold. Firstly, the authors demonstrate contrasting immunogenic profiles between a sporozoite and EEF presented antigen. They comprehensively characterize respective CD8+ T-cells responses, with the sporozoite expressed antigen displaying enhanced immunogenicity compared to the the EEF expressed antigen. Secondly, the authors demonstrate that despite these stark differences in immunogenicity, both the sporozoite and EEF expressed antigens are effective targets of epitope specific CD8+ T-cell responses capable of eliciting sterile immunity. This has the important implication that low immunogenicity as defined by conventional immunological assay fails to capture all antigens that are capable of inducing sterile immunity, and thus could be prioritized as vaccine targets, but instead risks leading investigators down a path where so to speak "the baby is thrown out with the bath water". Thirdly, this work shows for the first time that EEF expressed antigens are potential vaccine targets, and thus effectively expands the pool of available pre-erythrocytic vaccine targets for the research community to explore.

    The data presented here should thereby lead to a complete revision in how pre-eryhtrocytic vaccine candidates are identified and prioritized. In terms of basic biology, the fact that CD8+ T-cells are critical in mediating immunity is been well established, however this paper greatly enhances our understanding of exactly how the dynamics, magnitude and quality of CD8+ T-cell responses are modulated by the timing of antigen expression.

    Keywords for main reviewer expertise: Malaria, Plasmodium berghei, genetic manipulation, host-parasite interactions

    Keywords for ECR co-reviewer expertise: Immunity, host-pathogen interactions.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The study by Müller et al. uses the rodent malaria parasite Plasmodium berghei, which gives access to the mouse in vivo infection model. The authors investigate the initiation and development of CD8+ T cell responses during parasite liver-stage infection when the peptide antigen SIINFEK is presented either early or late during liver-stage infection by fusing SIINFEK to the Circumsporozoite Protein (CSP SIINFEK) or the Up-regulated in Infective Sporozoites 4 protein (UIS4 SIINFEK). CSP is expressed already in the motile sporozoite that invades the liver while UIS4, is expressed only in the later exoerthrocytic forms developing in the infected hepatocytes.

    Using the SIINFEK peptide the authors can control for epitope differences between CSP and UIS4 and it allows them to use the OT-I transgenic mouse line that produces high numbers of CD8+ T-cells that specifically recognizes MHC class I presented ovalbumin (OT-I cells), which can be used in adoptive transfer experiments. Using this approach the authors provide detailed kinetic and phenotypic analysis of CD8+ T-cell responses to CSP SIINFEK and UIS4 SIINFEK antigen-specific response in vivo and ex vivo, chiefly using FACS. The authors found that despite having different timing of expression and immunogenicity both CSP SIINFEK and UIS4 SIINFEK induce similar levels of CD8+ mediated targeting, resulting in a high degree of sterile immunity in a vaccination challenge experiment.

    Major comments

    Overall the manuscript is well written, concise and with a clear narrative. The conclusions drawn from the study are well supported by the data presented, and the experiments are thoroughly controlled and sufficiently replicated. The manuscript is suited for publication but presentation of key concepts and data could be made clearer and impact enhanced if the authors address some of the following commentary.

    1. Introduction -The authors assume a reader familiarity with the use of ovalbumin, the SIINFEKL epitope, the transgenic T-cell receptor OT-1 mice and adoptive transfer experiments to assay immunogenicity. These concepts are not comprehensively introduced in the introduction, and the relationship between these tools are not delineated sufficiently to allow the non-expert reader to follow the logic and methodology of the experiments right from the start. Background information given in Results (Line 139-144, 204-208) and Discussion section, (Line 288-293) could with advantage be synthesized into one paragraph and presented in the introduction to bring all readers onboard from the start.
    2. Results Page 11 Line 255-260, Figure legend Fig. 6 page 27 Line 258-665 The punchline of the paper is that the despite differences in immunogenicity between γ-irradiated CSP SIINFEKL or UIS4 SIINFEKL sporozoites, both CSP SIINFEKL or UIS4 SIINFEKL are targets of protective CD8+ T-cell responses resulting in sterile immunity in a challenge following vaccination with full-length ovalbumin in OT-I cell recipient mice. This section is a cornerstone for the conclusions of the paper and would benefit from being better supported by its explanatory text and presentation of data.

    Firstly, there is a mix up, between panel 6C and 6D, where 6C shows "% Sterile protection" and 6D shows "Parasite load in the liver", while it says the opposite in main text and figure legend.

    Secondly, while it is clear that qPCR is used to measure liver parasite load at 24 hours after challenge. It is not immediately clear from neither main text nor figure legend that sterile immunity is measured by microscopy on blood films. The use of the term "sterile immunity" naturally implies this to the initiated reader, but it should be spelled-out that this was the case and that it was monitored from day 3-14 following challenge, which is outlined only in the methods section. Rewriting and restructuring this section to make this clearer would greatly help guide the reader through the results. Currently it reads at first pass as if qPCR on liver samples harvested at 42 hours was used to generate the data in both 6C and 6D.

    Thirdly, protective efficacy here is given as a percentage of those mice that become protected, presumably remaining negative by day 14. Authors should provide the actual blood stage parasitaemia in graph or table format in Figure 6 or as a supplemental figure to show that sterile immunity is obtained and maintained until day 14, and that in the control groups patency develops as normal. This will also give clearer insight into how many mice developed patency in the control groups and at what point break-through was observed.

    In a similar vein, qualitative and / or quantitative presentation of microscopy data of EEFs (as presented in Figure 1C) would strengthen conclusions drawn from the qPCR parasite liver load data.

    Fourthly, the authors should also comment on why there is such a great variation in the number of mice used in the different studied groups, it says maximum of n=11 mice per group but one group only has as n=3 mice and another n=4, and make a convincing argument this does not affect the conclusions drawn and statistical analysis undertaken.

    Finally, the authors characterise CD8+ T-cell responses in absence of preceding OT-I adoptive transfer but do not report on whether the ovalbumin-immunization was tried on mice without preceding OT-I cell transplant. Was this tried? If not authors should discuss whether this is likely to be successful or not for readers to understand if both sporozoite and EEF presented antigens are likely to induce sterile immunity in a natural setting without artificial enrichment for epitope specific T-cells.

    1. Line 723, 725 clarify if data is from independent biological repeats, i.e. different infected mice fed to different pots of mosquitoes, in which case the data is sufficiently replicated.

    Minor comments:

    1. Page 6 Line 132 Please show blood-stage infection data / growth rates for CSP SINFEKL and UIS4 SINFEKL compared to WT as supplemental figure, if available.
    2. Figure 1. If quantitative data is available for EEF, as indicated by the mean numbers with SD given within the microscopy pictures it would be nice to see these plotted. Does the reduced EEF numbers for CSP SINFEKL compared to UIS 4SINFEKL and WT mean anything? If not perhaps worth stating this in figure legend, or consider different presentation. Distracting when looking at the figure.
    3. Figure 2. Would benefit from a panel with a simple schematic that shows the overall experimental design with irradiation of sporozoites, OT-1 transfer, administration of parasites and sampling with the timings for each event clearly marked out.
    4. Figure Panel 2b would benefit from the in-figure legend stating CSP SINFEKL + OT-1, UIS4 SINFEKL + OT-1, WT + OT-1 and OT-1 only. Similar to as in Figure 3.
    5. Figure 3 and Figure 4. Label within figure more clearly what is being measured, i.e. what is the difference between panels a,b,c vs. d,e,f (e.g. intravenously v.s. intradermal administrations), gets confusing since Figure 3 and Figure 4 are very similar within the figures (a,b,c vs. d,e,f) and between the figures.
    6. Figure 3, 4, The Panel indicating letters (a, b, c, d...) become smaller as figures get bigger and become hard to read for Figure 3 and Figure 4.
    7. Line 158 - Throughout manuscript, when it says administration of WT, CSP SINFEKL and / or UIS4 SINFEKL sporozoites it would be good to always have it preceded by irradiated when referring to irradiated sporozoites, e.g. Line 158 and only use sporozoites on its own when referring to live sporozoites (or even better spell out also when using live sporozoites e.g. Line 255).
    8. The authors could measure the total amounts of IFN-ɣ being secreted in the tissues after immunization to both antigens and investigate if the level of other IFN-ɣ secreting cells might compensate for the weak response of CD8+ T cells, particularly against UIS4. If completed it has the potential to help the authors to in more detail understand the mechanism and contributing factors to the successful CD8+ T-cell targeting of UIS4.

    Suggested extra experiments:

    1. As antigen protection is dependent not only cellular response but also on antibody responses induced against the antigens, authors should analyze by ELISA IgG and IgM responses induced against the two antigens
    2. Page 6 Line 121-123 he authors reason that addition of the SIINFEKL epitope to the immediate C-terminus of the UIS4 protein might confer enhanced antigen presentation through increase MHC-I antigen presentation. Supplementary experiments particularly a MHC I stabilization assay might help confirm this. Complementary experiments looking at MHC-II antigen presentation to APC would also be very relevant.
    3. The authors have described the effect of both antigens in the response of CD8+ T cells and the generation of memory. A more detailed characterization of the differential phenotypes of memory in CD4 and CD8 T cells in the spleen and liver following immunostimulatory therapy would increase the relevance of the data presented.

    Significance

    This paper would be of interest to malaria parasite biologists, immunologists and vaccinologists alike. The significance of this paper is three-fold. Firstly, the authors demonstrate contrasting immunogenic profiles between a sporozoite and EEF presented antigen. They comprehensively characterize respective CD8+ T-cells responses, with the sporozoite expressed antigen displaying enhanced immunogenicity compared to the the EEF expressed antigen. Secondly, the authors demonstrate that despite these stark differences in immunogenicity, both the sporozoite and EEF expressed antigens are effective targets of epitope specific CD8+ T-cell responses capable of eliciting sterile immunity. This has the important implication that low immunogenicity as defined by conventional immunological assay fails to capture all antigens that are capable of inducing sterile immunity, and thus could be prioritized as vaccine targets, but instead risks leading investigators down a path where so to speak "the baby is thrown out with the bath water". Thirdly, this work shows for the first time that EEF expressed antigens are potential vaccine targets, and thus effectively expands the pool of available pre-erythrocytic vaccine targets for the research community to explore.

    The data presented here should thereby lead to a complete revision in how pre-eryhtrocytic vaccine candidates are identified and prioritized. In terms of basic biology, the fact that CD8+ T-cells are critical in mediating immunity is been well established, however this paper greatly enhances our understanding of exactly how the dynamics, magnitude and quality of CD8+ T-cell responses are modulated by the timing of antigen expression.

    Keywords for main reviewer expertise: Malaria, Plasmodium berghei, genetic manipulation, host-parasite interactions

    Keywords for ECR co-reviewer expertise: Immunity, host-pathogen interactions.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript b Mueller and Gibbins et al titled "Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination" compares how transgenic P. berghei parasites expressing SIINFEKL epitope from ovalbumin, as part of CSP or UIS4 present the respective epitope and how immune responses occur to each of the mutants, mostly in mice pre-treated with 2 x 10 OT-I cells expressing a SIINFEKL-specific TCR.

    their data show that when in their normal location CSP is much better than UIS4 to elicit an immune response., and that increasing UIS4 (by raising irradiated parasite numbers) does not greatly improve to reduce the difference.

    finally the authors show that mice immunized with ovalbumin can reduce liver infection of either CSPSIINFEKL or UIS4SIINFEKL sporozoite challenge infection

    the experiments presented by the authors are in my view well done and controlled, but i feel that sometimes conclusions are a bit beyond what the experimental readouts allow for.

    Significance

    In fig1 the authors show how mutants were made and that proteins with associated SIINFEKL to CSP or UIS4 localise to correct place. (could all be supplementary or Supplementary Figure 1c, d could be included in Fig1).

    In Fig2a is shown the gating of SIINFEKL-specific CD8+ T cells (could be supplementary). In Fig 2b the authors show that the highest CD8T cell specific for SIINFEKL is on the first day analysed (d4) and I would like o see how day 2 and 3 would look like. specially because proliferative differences don't seem massive to me, CFSE should decrease with each cell division and reach different fluorescence values if replication numbers differ. however here the CFSE fluorescence signal is similar on d5 fig 2c, indicating a similar number of replicative rounds, but probably a different starting numbers of cells that would replicate. Or that CFS labelling was too low to allow distinguishing the number of replicative rounds occuring in that time.

    so when the authors conclude that proliferative activity was 6x larger than that observed with UIS4SIINFEKL sporozoites, i think they would have to show before that numbers of cells prior to replication was the same

    Figs 3 and 4 show that response to CSP is stronger than response to UIS4, and in the spleen larger than in the liver and that this was true for mice adoptively transferred with OT-I cells prior to intravenously immunisation or without that, and that with the transfer responses were much higher.

    Fig 5 show that increasing # of irradiated UIS4SIINFEKL 8x does not bring levels of response to anywhere close than the observed against 1x CSPSIINFEKL.

    and fig 6 show that if a response is obtained (in the case with an adenovirus expressing ovalbumin which will generate e a response recognising SIINFEKL) both CSPSIINFEKL or UIS4SIINFEKL infection challenge can be blocked and protective immunity equally achieved.

    I think it would be nice to show when is infection stopped in these two groups os mice, but looking at EEF in the liver if the two groups of mice.

    Also the authors could show that an adenovirus carrying UIS4 (and CSP) would result in the same as observed here with the ovalbumin one).

    I also think the authors should discuss the advantages and problems of the two SPZ and PVM locations, assuming that indeed an adenovirus carrying UIS4/CSP would also result in similar protection upon challenge, regarding potential boost from natural Infection, and how variable/conserved each of the proteins are and what could be expected in field trials ion the falciparum counterpart.

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    Referee #1

    Evidence, reproducibility and clarity

    In this study by Müller et al, the authors study if immunogenicity is an adequate predictor for vaccine development in malaria and more precisely against malaria pre-erythrocytic stage. For that the used two different strains of the murine parasite Plasmodium berghei They based their study on the use of the MCH I restricted epitope SIINFEKL to follow CD8 T cell responses. For that, they integrated the sequence SIINFEKL sequence into the protein CSP expressed by the infective form sporozoite and at the end of the sequence of the protein UIS4 expressed exclusively by the exo-erythrocytic forms (EEF) of the parasite. They compared then the CD8+ T cell responses elicited by each strain of the parasite and came to the conclusion that whilst antigen origin results in very different immunogenicity responses both sporozoite and EEF expressed antigens elicit antigen-specific effector CD8+ T cell responses with a high level of protection.

    Major comments:

    Whilst rational of parasite strain design is adequate and well-performed and the concept of low immunogenicity novel potentially interesting, there are several methodological flaws that make the conclusions somewhat speculative and need to be addressed to really support the conclusions. Given the fact that authors are top-level scientists in the malaria vaccinology field, I am confident that they can address the following comments that will help to improve the manuscript and its impact;

    • General comment: this reviewer was expecting a little more of deep analysis of immune responses elicited by the transgenic parasites that authors developed and not only a superficial analysis, how about TRM cells, what is the endogenous responses to SIINFKEL without transferring CD8 + T cells from OTI mice? This should be addressed

    • Fig 2-3: Authors compared the CD8+ T cell responses elicited by the two different strains of P.berghei. In order to evaluated if the two strains allowed to track anti-SIINFEKL, they immunized mice with both irradiated parasite strains or their control WT. To track these responses mice were adoptively transferred with CD8+ T cells from OT-I mice and immunized with irradiated parasites. They track responses by using a SIINFEKL tetramer expressing CD8+ T cells in the blood and the marker for antigen-experienced T cells CD11a. The problem here is that with the strategy of gating on Fig2a, it is not clear if they want to track the responses from adoptively transferred CD8+ T cells to vaccine or the endogenous CD8+ T cell responses. In any case, the results is potentially interesting but need clarification.

      • Fig 2: only the responses on the spleen are studied. In order to support the statement about the two different kinds of immunization, they should assess the responses on the liver.
      • There is also a lack of methodology in flow cytometry analysis, a viability stain is not used, the gating is not determined by FMO (fluorescence minus one) controls and seems aleatory. For activation markers in order to assess the impact of the vaccination authors have to use gating that is already established by some of the papers they mentioned (i.e: Harty lab's studies), it is difficult to evaluate the responses if we don't know how many of the CD11a/Cd49d cells are Memory effector or effector (CD62L and CD44 markers). Moreover, the CD11a label should be CD11ahi and is not stated anywhere.

    Line 165: the statement "massive proliferative activity" is not supported by the figure, moreover there are numbers to support the statement. - IFNg and other cytokines production seems too low and the stimulation assay is poorly performed because CD8 were restimulated ex-vivo only with SIINFEKL peptide in the absence of APC (antigen-presenting cells) with Brefeldin A. Also Authors omitted negative controls ( without SIINFEKL Brefeldin A) to be certain that IFNg production is du to SIINFEKL. Again we don't if they are OTI or endogenous cells.

    • Fig5. Are the cells from Fig5a,b SIINFEKL positive cells or only CD11a and IFNg? Are they OTI? Controls are missing to show a real IFNg production du to the ex vivo stimulation.
    • Fig 6. no percentages are shown in the cytometry plots, figure 6d and c seem to be inverted. An interesting observation is that the level of protection against both strains of parasites is the same when vaccinated mice with AdOVA are challenged. The authors make the interpretation that immunogenicity does not predict effector responses. This is one of the central conclusions of the paper. The authors only show level of protection but don't characterize the phenotype of CD8+ T cells in the liver of vaccinated and challenged mice. Can cells from Fig6a be find in the liver? Are they liver TRm (resident memory CD8+ T cells), know to be an important class of cells for protection against malaria.

    Minor comments:

    • For the two strains, authors should show the patency in comparison whit WT parasites (currently presented as data not shown)
    • Gating strategy for markers is missing, FMO as well
    • Fig 6: how did the authors measure Sterile protection and Relative parasite load?

    Significance

    The present study could provide important insight in the field of malaria vaccinology. By using cutting edge molecular biology to express the MCHI restricted epitope SIINFEKL a at different stages of the pre-erythrocytic stage of Plasmodium and used it as a surrogate marker to evaluate the CD8+T cell response to infection. The authors attempt to provide proof of the concept of vaccine design by evaluating if accessibility/immunogenicity of the antigen is a decisive feature on vaccine design. Nevertheless, the potential of this study demands to be placed in the context of precedent studies that defined pre-erythrocytic stage CD8+ T cell responses. the authors failed to fully exploit the tools that they developed (transgenic parasite) by overlooking the last studies describing the importance of CD8+ liver-resident memory CD8+ T cells from Health's laboratory or well characterized CD8 T cells responses defined by Harty's laboratory.

    If well place in the context (after revisions) this study will not only be fundamental to the malaria field but to other infectious diseases as well.

    Field of expertise: malaria immunology, vaccinology, immunomodulation, CD8+ T cell responses

  6. Version published to 10.1101/2020.09.03.281238 on bioRxiv