Multi-protein chimeric antigens, a novel combined approach for efficiently targeting and blocking the blood stage of Plasmodium falciparum

  1. Bhagyashree Deshmukh
  2. Dhruv Khatri
  3. Sanjay Kumar Kochar
  4. Chaitanya Athale
  5. Krishanpal Karmodiya

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Abstract

Plasmodium falciparum -induced malaria remains a fatal disease affecting millions of people worldwide. Mainly, the blood stage of malaria is highly pathogenic and symptomatic, rapidly damaging the host organs and occasionally leading to death. Currently, no vaccines are approved for use against the blood stage of malaria. Canonical vaccines in the past have selected the most immunodominant or essential protein to block the growth of the parasite. This strategy works efficiently for low-complexity organisms such as viruses and a few bacteria but has not shown promising results for a malaria vaccine. Plasmodium has a complex life cycle and vaccine candidates especially during blood stage are ineffective due to multiple gene families showing redundancy, immune evasion, and insufficient antibody titer. Herein, we demonstrate a novel strategy of combining multiple antigens from the blood stage of Plasmodium falciparum using only the most immunodominant peptide sequences as a way of tackling polymorphism and redundancy. We created three chimeric antigens targeting eight PfEMP1 proteins (chimeric varB) and eight merozoite surface proteins (chimeric MSP and InvP) by selecting and stitching B-cell epitopes. Our chimeric constructs show naturally circulating antibodies against individual peptides using epitope-mapping microarray as well as entire proteins in malaria-infected patients. We demonstrate that anti-varB antibodies are neutralizing in nature and significantly reduce the cytoadhesion on an organ-on-chip system with a microfluidic device mimicking physiological conditions. We have applied a Deep Learning based method to quantify the number of adhered RBCs under fluidic conditions that is used to study cytoadhesion. Furthermore, the anti-MSP and InvP antibodies show complete growth inhibition in a single cycle at a combined concentration of 0.13 mg/ml. Overall, our results show that a combination of antigenic peptides from multiple antigens can function as a next-generation vaccine and effectively block the blood stage by reducing cytoadhesion and inhibiting the parasite growth.

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    Reply to the reviewers

    #Reviewer 1 (Evidence, reproducibility and clarity):

    This manuscript by Deshmukh et al is aimed at generating chimeric antigens that can be useful for making next generation vaccines that block blood stage infection by malaria parasite. Given that there is no blood stage vaccine against malaria and available liver stage vaccine shows only limited efficacy that too only in Africa, there is dire need for having novel approaches to generate successful vaccines. In the past attempts have been made to make multivalent vaccines but have not been successful. Nevertheless, it is still a good option as single target blood-stage vaccines have failed. Authors propose to target cytoadhesion and host erythrocyte invasion. For this purpose, they have selected epitopes from PfEMP1/VarB family members, which poses a major challenge as at least 60 genes encode them and they exhibit variations which facilitate the escape from the immune system. The other two chimeras target invasion related proteins like MSPs and adhesins shed by micronemes and rhoptries, which are critical for invasion. The reported work is interesting and provides a useful approach towards developing vaccines against blood stage infection.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Comments:

    1. __ The peptides used in InvB chimera did not show good reactivity especially when compared to VarB or MSP peptides. Please discuss the possible reasons.__

    Response: Thank you for pointing out the difference in the explanation. With chimeric InvP, we see a strong response against a few peptides of SERA-5 and RH-5, while other peptides, in comparison, have lesser antibody responses. We have now included the following statement detailing this difference with possible explanations in the revised manuscript (Page 8, Line 25 to 30).

    The IgG responses to chimeric InvP were slightly different from those to chimeric varB and MSP. The intensity of IgG to peptides of SERA-5 and RH-5 was very high in comparison to the rest of the peptides used in the construct, whereas in chimeric varB and MSP, the IgG titers were comparable between the peptides. This could be a result of antigen exposure in the cohort of 19 patient samples that we used, and may change when a larger sample size is considered.

    __ It will be interesting to determine if blocking a specific VarB/PfEMP1 alters expression of other members. Based on the data provided in Fig. 4E, can a chimera be designed which only includes PfEMP1 that are represented well in HBEC-5i population?__

    Response: We agree that observing the altered expression of PfEMP1 would be an interesting phenomenon to study. The blocking of PfEMP1 using anti-chimeric varB antibodies is a transient process in our assays (just enough to quantify the cytoadhesion). It may take multiple cycles with negative selection pressure on parasites for the switching to take place. Also, it will be interesting to design chimeras based on the HBEC-5i binding PfEMP1. We can certainly plan these as prospective future experiments.

    __ Some of the invasion related proteins like RH5 and EBA175 are not present at parasite surface, instead, secreted from rhoptries and micronemes. It will be nice to perform Western blots on condition medium and see if InvP (or even MSP and VarB) antibodies recognizes the secreted version of these proteins.__

    Response: We thank the reviewer for this valuable comment and the suggestive experiment. We will perform a western blot on spent media and probe using anti-chimeric MSP and InvP antibodies to detect the proteins selected in chimeric MSP and InvP antigens.

    __ Fig. 6E- Statistics need to be provided for inhibition at 12.3 and 25ug.__

    Response: We apologize for the missed statistics. It is now included in the figure panel.

    __ Plasmodium uses multiple ligand-receptor interaction, which could depend (e.g. EBA-glycohophorins) or operate independent (e.g. RH5-basigin) of sialic acid. While there is representation from candidates from both of these families, most studies especially growth rate assays (Fig. 6E) have been carried using 3D7 strain, which does not require sialic acid. It is possible that if similar experiments were performed using sialic acid-sensitive strains, InvP and MSP antibodies may cause greater inhibition of parasite growth, which may be worth testing.__

    Response: We are grateful for the suggestion of using a sialic acid-dependent strain. Indeed, the pathway of reinvasion chosen by the parasite may determine the growth inhibition assay (GIA) outcome. We will perform the GIA assay on the Dd2 strain and 3D7 with neuraminidase treatment (Sialic acid-dependent invasion). We will also note the difference in growth inhibition potential of chimeric antibodies in sialic-acid dependent and independent pathways.

    __ The direct effect of InvP and MSP Abs should be tested directly on host erythrocyte invasion.__

    Response: We thank the reviewer for this comment. We certainly can determine the inhibitory potential of anti-chimeric MSP and InvP antibodies through invasion assays. We will include the invasion inhibition potential of these antibodies in 3D7, Dd2, with neuraminidase treatment along with GIA data.

    Reviewer #1 Significance:

    Present study proposes novel strategies for the development of anti-malarial vaccine.

    #Reviewer 2 (Evidence, reproducibility and clarity):

    The manuscript describes the vaccine potential of unstructured P. falciparum merozoite protein fragments 25 amino acid long belonging to 3 different protein families. The work is well performed, easily reproducible and clearly described.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Reviewer #2 (Significance):

    1. The use of protein fragments whose structure can be predicted by their sequence has been exploited in many studies for the development of vaccines or other biologicals. In this studies the authors selected 3 different families belonging to the red blood stage of the parasite. The table showing the sequences selected is not readable and should be clearly provided in the supplementary section.

    Response: We apologize for the readability of the sequences. The supplementary Table 1 has the proteins selected, the sequences taken, and the precise order for the stitching.

    In addition, polymorphic residues should be highlighted.

    Response: We thank the reviewer for pointing this out. We will analyze and compile the protein sequences in 3D conformation, highlighting polymorphic residues and the peptides selected in our study.

    In addition, it is not to mention why the authors used immune rabbit sera obtained by injection of the 3 poly-epitopes instead of obtaining by affinity chromatography antigen specific human antibodies from sera of individuals living in endemic regions which could provide a direct and clear answer whether a protective vaccine could be obtained.

    Response: We agree that the clear answer to the protective function of antibodies could have been answered using human antibodies. However, we did not have a sufficient volume of patient sera to perform affinity enrichment. The use of rabbits here was to ensure the generation of antigen-specific antibody responses in ample amounts. The patient sera in quantities available were used in ELISA, epitope mapping, and IP, followed by mass-spectrometry. The IP-MS clearly shows the presence of antibodies against the proteins taken in the generation of chimeric antigens (Supplementary Figure 1 D).

    #Reviewer 3 (Evidence, reproducibility and clarity):

    Multi-protein chimeric antigens... by: Deshmukh et al

    This article addresses an extremely important objective, the development of an effective prophylactic vaccine for Malaria. The disease continues to be widespread claiming the lives of hundreds of thousands of people annually, many of them children. Despite efforts towards producing Malaria vaccines, none thus far have been sufficiently protective or long term. As the authors point out vaccines can target the parasite per se, and possibly more attractive would be to focus on parasite derived antigens expressed on the surface of infected erythrocytes, hence targeting the Blood stage of the infection, which is most directly associated with Malaria pathogenesis. The authors propose a somewhat novel approach in which they have selected an array of short (25 amino acids) segments of Plasmodium derived proteins stitched together to produce 3 chimeric recombinant proteins as potential immunogens. Although a considerable amount of work is described, the results are not compelling in proving the efficacy or advantage of using chimeric antigens as worthy vaccine candidates for Malaria.

    Unfortunately, the rationale behind the experiments are not clearly defined which is a matter of concern. In addition, details of the work done and the technical aspects needs to better explained to fully understand how and why the target segments were selected and the chimeras produced. This review focuses first on scientific issues and then format and editing, both aspects demonstrate that the manuscript in its present form requires major changes for it to be of relevance to the field. This review focuses first on issues of substance and then format and editing, both aspects disqualify the publication of the manuscript in its present form.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Experiments and Results:

    1. The underlying proposal claims that chimeric antigens might be advantageous in eliciting protective antibodies. The authors produced three chimeras: var, MSP and InvP. __The var chimera contains 29 segments of PfEMP1 derived from 8 alleles. The hypothesis is that by expressing 29 different segments one will produce antibodies that can better cope with the antigenic diversity of this target. Indeed, serial monoallelic expression of anyone of the 60 PfEMP1 variants of a given P. falciparum strain has been thought to mediate immune evasion. The parasite is presumed to be able to escape immune defenses, by switching and serially expressing PfEMP1 alleles. Hence, one might assume that by introducing different segments, derived from different alleles, one will gain better protection. The authors have not really tested this idea. They have produced a single chimera and tested it without controlled comparison of performance to any single segment, or for that matter compared to alternative structural domain(s) of PfEMP. This brings me to the question of how the segments were selected and why. The authors implement IEDB-AR to identify presumably preferred B-cell epitopes. The methodology relies on a number of computational methods that predict the propensity of linear segments of proteins to have, for example, secondary structures, or be surface accessible, or relatively hydrophilic or flexible, etc. IEDB-AR is a tool to assist the identification of segments (5-25 amino acids in length), that might be associated with B-cell epitopes, or at least segments comprising linear aspects of B-cell epitopes. The input is a linear sequence of an antigen, proposing linear aspects of what could be associated with B-cell epitopes. B-cell epitopes, however, are typically conformational and discontinuous. They certainly can and do contain linear segments, but even these may require 3D conformations dictated by spatial constraints imposed by the native surrounding aspects of the natural antigen. It is hard to assume that by simply stitching 29 segments, one after the other, one can provide them with the native environment for them to assume a somewhat physiologically relevant conformation. Unfortunately, the authors have not addressed the unique characteristics of the antigen they have selected. PfEMP1, for example, is a family of antigens with discrete sub-domain structures and features (DBL and CIDR for example). It would be relevant and useful to relate the segments that they chose to the natural unique domains of the antigen and how they might best present common vs variant aspects of the antigen. There are at least 30 crystal atomic structures for PfEMP1 in complex with various physiologically relevant proteins (eg ICAM etc). The authors might have considered the 3D structure of PfEMP in their analyses and at least indicated on an atomic structure where the 29 segments lie. __

    More concerning is the fact that the expression of the chimera does not produce a crisp single protein, but rather a complex of products as illustrated in the Supplement Figure 1 B. The authors simply claim that they produce the antigen for immunization of rabbits (or one rabbit?) and they collect gel-derived band(s) of what MW?? Assuming that a 25aa segment should be about 2500-2800 daltons and so 29 such segments strung together should be about 80kDa. The gel shows bands at 124kDa, and a slew of bands shorter than 71kDa. There is no mention what the expected MW should be and there is no explanation why the protein pattern contains so many bands of different sizes and what exact bands were taken for the immunogen or why.

    Response: We thank the reviewer for this comment, as it tells us the reader's perspective on how the chimeric construct part is underexplained. We have now expanded the section on chimeric construct design, the sequences used, the functional domains they belong to in the PfEMP1 protein (Supplementary Tables 1 and 2), and the expected sizes of the proteins created. As for the B-cell epitope prediction, we have used the linear epitope prediction tool. However, we will include a 3D conformational study highlighting the placement of peptides that we have used to generate chimeric antigens.

    The sequences for chimeric constructs were synthesized commercially and confirmed using Sanger sequencing. The antigens run higher than their expected molecular weights, and we have confirmed them through western blot and mass spectrometry (Supplementary Figure 1 B and C). The chimeric varB antigen specifically shows a cleaving pattern, hence the multiple bands in western blotting (we have considered the top-most band with the highest anti-his intensity). After these confirmations, the antigens were independently injected in rabbits to generate antibodies.

    Similar considerations can be made regarding the selection of the segments for the two other chimeras, although they seem to produce a single polypeptide.

    Response: The antigens were confirmed using Sanger sequencing, expression using anti-his western blot, and proteins were confirmed using mass spectrometry for all three chimeric constructs (Supplementary Figure 1 B and C).

    If the point was to test a "chimera" modality as an improved vaccine, it would have been more useful to focus on one chimera and carefully characterize it and compare it to its components used separately.

    Response: The idea of chimera arises from the fact that individual proteins/components are insufficient to generate optimal responses. The proteins considered in our study have already been validated in the field (as separate components) and show that the efficacy observed was sub-optimal. Since our rationale is to include multiple proteins to tackle the redundancy and parasite virulence, we have focused on generating three chimeric constructs covering the entire blood stage of Plasmodium falciparum. Our objective is to demonstrate that a multi-protein, multi-factorial vaccine, as a proof of concept, works better in tackling malaria. We believe that in proving so, a comparison of chimera with their individual components is an unnecessary and economically unviable.

    The authors devote much effort to the fluidics system and their assay. This might warrant a paper dedicated to the methodology they have developed.

    Response: The *Plasmodium *virulence genes are extensively studied for their interactions with human endothelial receptors. Unfortunately, these studies fail to take human physiological conditions into account. We wanted to test our anti-chimeric varB antibodies in the best mimicking environment possible. Hence, the efforts were devoted to developing, standardizing, and quantifying the fluidic cytoadherence system. We thank the reviewer for their kind words of encouragement on our methodology.

    Format and Editing:

    1. The manuscript is very poorly written with multiple errors throughout. The authors use abbreviations that are not defined, eg iRBC (pg 5 line 22) or sometimes incorrectly defined, eg MSP ("merozoite-specific proteins - pg 6 line 18).

    Response: We apologize for the abbreviation error. The abbreviation for iRBC is defined in the introduction section (page no 4, Line 15); hence, it is not redefined on page 5, line 22. We have corrected merozoite-specific proteins on page 6, line 18.

    The Figures are of low resolution to the extent that they can not be read (for example Figure 3 pg 34). Figure 1 is somewhat useless and misleading. In Fig1 C - the diagram illustrates 5 hypothetical chimeras where in fact only three were produced. There really is no detail or explanation as to how the chimeras were produced.

    Response: We apologize for the low resolution of the images. We have now improved the image quality. Figure 1C represents the idea of designing the construct, not the number of chimeras we generated. We apologize for this confusion and have explicitly mentioned this in the figure panel for Figure 1C. As for the design and generation of chimeric antigens, we understand that the materials and methods section is underexplained, and we have now expanded on it with all details included.

    In the construction of the chimeras there is no mention as to whether short linkers were introduced between the segments or not. What was the expected weight of the chimera? Was the order of segments random or precise and consistent? Were the constructs sequence validated in addition to the MassSpec?

    Response: We understand that the section on the chimeric construct is underexplained for the readers, and we thank the reviewer for pointing it out. We have now expanded the section on chimeric antigen design and included the details. Chimera was tested with GSGSGS linkers and without linkers for expression. The final antigen injected in rabbits was serially attached peptides without linkers. The segments stitched were in precise order, as mentioned in Supplementary Sheet 1. The construct was commercially synthesized and sequence validated along with the anti-his western blot and mass spectrometry analysis.

    The figures of the Supplement are not numbered.

    Response: We thank the reviewer for pointing this out. The figures are now numbered.

    Note that the headings in Supplement Figure 1 B and C have overlapping text.

    Response: Thank you for pointing this out. We have now rearranged the supplementary figures 1B, and 1C.

    Most disturbing is that multiple references that are incomplete. For example: in References 15, 16, 25, 26, 27 there is no indication of the Journal.

    Response: We apologize for the mistakes in referencing. These references did not have full citations in Endnote. We have now manually checked all the references and corrected the incomplete formats of the references.

    The authors mention reference 13 [2006] in claiming that the antibodies can be protective, and then support this by referring to refs 14, 15 and 16 published in 1961, 1963 and 1962 respectively. Although, old articles can be useful, but the authors should attempt to provide current proof of such basic claims.

    Response: We thank the reviewer for pointing this out. We have now separated these two statements and not mentioned the latter as a support to the former. As for references 14, 15, and 16, these were the early studies in the field that show the protective nature of antibodies through the passive immunization process and are foundations for the idea of blood stage vaccination. Current proofs of antibodies against blood-stage antigens are included for blood-stage vaccine candidates.

    Reviewer #3 (Significance):

    The goal of the study is very important.

    The hypothesis that a chimeric presentation of select peptides could be advantageous was not rigorously tested nor well controlled in a meaningful evaluation and thus no conclusion can be made. There are no comparative analyses to test their hypothesis.

    The method for selection of epitope segments is not well justified. There is little attempt to provide rationale or description of the segments chosen and how they fit within the antigens, thus justifying segments over multiple antigens.

    The grammatical errors, lack of clarity accompanied by little attention to style and readability render the manuscript quite illegible.

    There is no excuse for so many errors in the references.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Multi-protein chimeric antigens... by: Deshmukh et al

    This article addresses an extremely important objective, the development of an effective prophylactic vaccine for Malaria. The disease continues to be widespread claiming the lives of hundreds of thousands of people annually, many of them children. Despite efforts towards producing Malaria vaccines, none thus far have been sufficiently protective or long term. As the authors point out vaccines can target the parasite per se, and possibly more attractive would be to focus on parasite derived antigens expressed on the surface of infected erythrocytes, hence targeting the Blood stage of the infection, which is most directly associated with Malaria pathogenesis. The authors propose a somewhat novel approach in which they have selected an array of short (25 amino acids) segments of Plasmodium derived proteins stitched together to produce 3 chimeric recombinant proteins as potential immunogens. Although a considerable amount of work is described, the results are not compelling in proving the efficacy or advantage of using chimeric antigens as worthy vaccine candidates for Malaria.

    Unfortunately, the rationale behind the experiments are not clearly defined which is a matter of concern. In addition, details of the work done and the technical aspects needs to better explained to fully understand how and why the target segments were selected and the chimeras produced. This review focuses first on scientific issues and then format and editing, both aspects demonstrate that the manuscript in its present form requires major changes for it to be of relevance to the field. This review focuses first on issues of substance and then format and editing, both aspects disqualify the publication of the manuscript in its present form.

    Experiments and Results:

    The underlying proposal claims that chimeric antigens might be advantageous in eliciting protective antibodies. The authors produced three chimeras: var, MSP and InvP.

    The var chimera contains 29 segments of PfEMP1 derived from 8 alleles. The hypothesis is that by expressing 29 different segments one will produce antibodies that can better cope with the antigenic diversity of this target. Indeed, serial monoallelic expression of anyone of the 60 PfEMP1 variants of a given P. falciparum strain has been thought to mediate immune evasion. The parasite is presumed to be able to escape immune defenses, by switching and serially expressing PfEMP1 alleles. Hence, one might assume that by introducing different segments, derived from different alleles, one will gain better protection. The authors have not really tested this idea. They have produced a single chimera and tested it without controlled comparison of performance to any single segment, or for that matter compared to alternative structural domain(s) of PfEMP. This brings me to the question of how the segments were selected and why. The authors implement IEDB-AR to identify presumably preferred B-cell epitopes. The methodology relies on a number of computational methods that predict the propensity of linear segments of proteins to have, for example, secondary structures, or be surface accessible, or relatively hydrophilic or flexible, etc. IEDB-AR is a tool to assist the identification of segments (5-25 amino acids in length), that might be associated with B-cell epitopes, or at least segments comprising linear aspects of B-cell epitopes. The input is a linear sequence of an antigen, proposing linear aspects of what could be associated with B-cell epitopes. B-cell epitopes, however, are typically conformational and discontinuous. They certainly can and do contain linear segments, but even these may require 3D conformations dictated by spatial constraints imposed by the native surrounding aspects of the natural antigen. It is hard to assume that by simply stitching 29 segments, one after the other, one can provide them with the native environment for them to assume a somewhat physiologically relevant conformation. Unfortunately, the authors have not addressed the unique characteristics of the antigen they have selected. PfEMP1, for example, is a family of antigens with discrete sub-domain structures and features (DBL and CIDR for example). It would be relevant and useful to relate the segments that they chose to the natural unique domains of the antigen and how they might best present common vs variant aspects of the antigen. There are at least 30 crystal atomic structures for PfEMP1 in complex with various physiologically relevant proteins (eg ICAM etc). The authors might have considered the 3D structure of PfEMP in their analyses and at least indicated on an atomic structure where the 29 segments lie. More concerning is the fact that the expression of the chimera does not produce a crisp single protein, but rather a complex of products as illustrated in the Supplement Figure 1 B. The authors simply claim that they produce the antigen for immunization of rabbits (or one rabbit?) and they collect gel-derived band(s) of what MW?? Assuming that a 25aa segment should be about 2500-2800 daltons and so 29 such segments strung together should be about 80kDa. The gel shows bands at 124kDa, and a slew of bands shorter than 71kDa. There is no mention what the expected MW should be and there is no explanation why the protein pattern contains so many bands of different sizes and what exact bands were taken for the immunogen or why.

    Similar considerations can be made regarding the selection of the segments for the two other chimeras, although they seem to produce a single polypeptide.

    If the point was to test a "chimera" modality as an improved vaccine, it would have been more useful to focus on one chimera and carefully characterize it and compare it to its components used separately. The authors devote much effort to the fluidics system and their assay. This might warrant a paper dedicated to the methodology they have developed.

    Format and Editing:

    The manuscript is very poorly written with multiple errors throughout. The authors use abbreviations that are not defined, eg iRBC (pg 5 line 22) or sometimes incorrectly defined, eg MSP ("merozoite-specific proteins - pg 6 line 18).

    The Figures are of low resolution to the extent that they can not be read (for example Figure 3 pg 34). Figure 1 is somewhat useless and misleading. In Fig1 C - the diagram illustrates 5 hypothetical chimeras where in fact only three were produced. There really is no detail or explanation as to how the chimeras were produced.

    In the construction of the chimeras there is no mention as to whether short linkers were introduced between the segments or not. What was the expected weight of the chimera? Was the order of segments random or precise and consistent? Were the constructs sequence validated in addition to the MassSpec?

    The figures of the Supplement are not numbered.

    Note that the headings in Supplement Figure 1 B and C have overlapping text.

    Most disturbing is that multiple references that are incomplete. For example: in References 15, 16, 25, 26, 27 there is no indication of the Journal.

    The authors mention reference 13 [2006] in claiming that the antibodies can be protective, and then support this by referring to refs 14, 15 and 16 published in 1961, 1963 and 1962 respectively. Although, old articles can be useful, but the authors should attempt to provide current proof of such basic claims.

    Significance

    The goal of the study is very important.

    The hypothesis that a chimeric presentation of select peptides could be advantageous was not rigorously tested nor well controlled in a meaningful evaluation and thus no conclusion can be made. There are no comparative analyses to test their hypothesis.

    The method for selection of epitope segments is not well justified. There is little attempt to provide rationale or description of the segments chosen and how they fit within the antigens, thus justifying segments over multiple antigens.

    The grammatical errors, lack of clarity accompanied by little attention to style and readability render the manuscript quite illegible.

    There is no excuse for so many errors in the references.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript describes the vaccine potential of unstructured P. falciparum merozoite protein fragments 25 amino acid long belonging to 3 different protein families. The work is well performed, easily reproducible and clearly described.

    Referees cross-commenting

    The polymorphic residues should be highlighted in the supplementary figure.

    Significance

    The use of protein fragments whose structure can be predicted by their sequence has been exploited in many studies for the development of vaccines or other biologicals. In this studies the authors selected 3 different families belonging to the red blood stage of the parasite. The table showing the sequences selected is not readable and should be clearly provided in the supplementary section. In addition, polymorphic residues should be highlighted. In addition, it is not to mention why the authors used immune rabbit sera obtained by injection of the 3 poly-epitopes instead of obtaining by affinity chromatography antigen specific human antibodies from sera of individuals living in endemic regions which could provide a direct and clear answer whether a protective vaccine could be obtained.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    This manuscript by Deshmukh et al is aimed at generating chimeric antigens that can be useful for making next generation vaccines that block blood stage infection by malaria parasite. Given that there is no blood stage vaccine against malaria and available liver stage vaccine shows only limited efficacy that too only in Africa, there is dire need for having novel approaches to generate successful vaccines. In the past attempts have been made to make multivalent vaccines but have not been successful. Nevertheless, it is still a good option as single target blood-stage vaccines have failed. Authors propose to target cytoadhesion and host erythrocyte invasion. For this purpose, they have selected epitopes from PfEMP1/VarB family members, which poses a major challenge as at least 60 genes encode them and they exhibit variations which facilitate the escape from the immune system. The other two chimeras target invasion related proteins like MSPs and adhesins shed by micronemes and rhoptries, which are critical for invasion. The reported work is interesting and provides a useful approach towards developing vaccines against blood stage infection.

    Comments:

    1. The peptides used in InvB chimera did not show good reactivity especially when compared to VarB or MSP peptides. Please discuss the possible reasons.
    2. It will be interesting to determine if blocking a specific VarB/PfEMP1 alters expression of other members. Based on the data provided in Fig. 4E, can a chimera be designed which only includes PfEMP1 that are represented well in HBEC-5i population?
    3. Some of the invasion related proteins like RH5 and EBA175 are not present at parasite surface, instead, secreted from rhoptries and micronemes. It will be nice to perform Western blots on condition medium and see if InvP (or even MSP and VarB) antibodies recognizes the secreted version of these proteins.
    4. Fig. 6E- Statistics need to be provided for inhibition at 12.3 and 25ug.
    5. Plasmodium uses multiple ligand-receptor interaction, which could depend (e.g. EBA-glycohophorins) or operate independent (e.g. RH5-basigin) of sialic acid. While there is representation from candidates from both of these families, most studies especially growth rate assays (Fig. 6E) have been carried using 3D7 strain, which does not require sialic acid. It is possible that if similar experiments were performed using sialic acid-sensitive strains, InvP and MSP antibodies may cause greater inhibition of parasite growth, which may be worth testing.
    6. The direct effect of InvP and MSP Abs should be tested directly on host erythrocyte invasion.

    Significance

    Present study proposes novel strategies for the development of anti-malarial vaccine.

  5. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    #Reviewer 1 (Evidence, reproducibility and clarity):

    This manuscript by Deshmukh et al is aimed at generating chimeric antigens that can be useful for making next generation vaccines that block blood stage infection by malaria parasite. Given that there is no blood stage vaccine against malaria and available liver stage vaccine shows only limited efficacy that too only in Africa, there is dire need for having novel approaches to generate successful vaccines. In the past attempts have been made to make multivalent vaccines but have not been successful. Nevertheless, it is still a good option as single target blood-stage vaccines have failed. Authors propose to target cytoadhesion and host erythrocyte invasion. For this purpose, they have selected epitopes from PfEMP1/VarB family members, which poses a major challenge as at least 60 genes encode them and they exhibit variations which facilitate the escape from the immune system. The other two chimeras target invasion related proteins like MSPs and adhesins shed by micronemes and rhoptries, which are critical for invasion. The reported work is interesting and provides a useful approach towards developing vaccines against blood stage infection.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Comments:

    1. __ The peptides used in InvB chimera did not show good reactivity especially when compared to VarB or MSP peptides. Please discuss the possible reasons.__

    Response: Thank you for pointing out the difference in the explanation. With chimeric InvP, we see a strong response against a few peptides of SERA-5 and RH-5, while other peptides, in comparison, have lesser antibody responses. We have now included the following statement detailing this difference with possible explanations in the revised manuscript (Page 8, Line 25 to 30).

    The IgG responses to chimeric InvP were slightly different from those to chimeric varB and MSP. The intensity of IgG to peptides of SERA-5 and RH-5 was very high in comparison to the rest of the peptides used in the construct, whereas in chimeric varB and MSP, the IgG titers were comparable between the peptides. This could be a result of antigen exposure in the cohort of 19 patient samples that we used, and may change when a larger sample size is considered.

    __ It will be interesting to determine if blocking a specific VarB/PfEMP1 alters expression of other members. Based on the data provided in Fig. 4E, can a chimera be designed which only includes PfEMP1 that are represented well in HBEC-5i population?__

    Response: We agree that observing the altered expression of PfEMP1 would be an interesting phenomenon to study. The blocking of PfEMP1 using anti-chimeric varB antibodies is a transient process in our assays (just enough to quantify the cytoadhesion). It may take multiple cycles with negative selection pressure on parasites for the switching to take place. Also, it will be interesting to design chimeras based on the HBEC-5i binding PfEMP1. We can certainly plan these as prospective future experiments.

    __ Some of the invasion related proteins like RH5 and EBA175 are not present at parasite surface, instead, secreted from rhoptries and micronemes. It will be nice to perform Western blots on condition medium and see if InvP (or even MSP and VarB) antibodies recognizes the secreted version of these proteins.__

    Response: We thank the reviewer for this valuable comment and the suggestive experiment. We will perform a western blot on spent media and probe using anti-chimeric MSP and InvP antibodies to detect the proteins selected in chimeric MSP and InvP antigens.

    __ Fig. 6E- Statistics need to be provided for inhibition at 12.3 and 25ug.__

    Response: We apologize for the missed statistics. It is now included in the figure panel.

    __ Plasmodium uses multiple ligand-receptor interaction, which could depend (e.g. EBA-glycohophorins) or operate independent (e.g. RH5-basigin) of sialic acid. While there is representation from candidates from both of these families, most studies especially growth rate assays (Fig. 6E) have been carried using 3D7 strain, which does not require sialic acid. It is possible that if similar experiments were performed using sialic acid-sensitive strains, InvP and MSP antibodies may cause greater inhibition of parasite growth, which may be worth testing.__

    Response: We are grateful for the suggestion of using a sialic acid-dependent strain. Indeed, the pathway of reinvasion chosen by the parasite may determine the growth inhibition assay (GIA) outcome. We will perform the GIA assay on the Dd2 strain and 3D7 with neuraminidase treatment (Sialic acid-dependent invasion). We will also note the difference in growth inhibition potential of chimeric antibodies in sialic-acid dependent and independent pathways.

    __ The direct effect of InvP and MSP Abs should be tested directly on host erythrocyte invasion.__

    Response: We thank the reviewer for this comment. We certainly can determine the inhibitory potential of anti-chimeric MSP and InvP antibodies through invasion assays. We will include the invasion inhibition potential of these antibodies in 3D7, Dd2, with neuraminidase treatment along with GIA data.

    Reviewer #1 Significance:

    Present study proposes novel strategies for the development of anti-malarial vaccine.

    #Reviewer 2 (Evidence, reproducibility and clarity):

    The manuscript describes the vaccine potential of unstructured P. falciparum merozoite protein fragments 25 amino acid long belonging to 3 different protein families. The work is well performed, easily reproducible and clearly described.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Reviewer #2 (Significance):

    1. The use of protein fragments whose structure can be predicted by their sequence has been exploited in many studies for the development of vaccines or other biologicals. In this studies the authors selected 3 different families belonging to the red blood stage of the parasite. The table showing the sequences selected is not readable and should be clearly provided in the supplementary section.

    Response: We apologize for the readability of the sequences. The supplementary Table 1 has the proteins selected, the sequences taken, and the precise order for the stitching.

    In addition, polymorphic residues should be highlighted.

    Response: We thank the reviewer for pointing this out. We will analyze and compile the protein sequences in 3D conformation, highlighting polymorphic residues and the peptides selected in our study.

    In addition, it is not to mention why the authors used immune rabbit sera obtained by injection of the 3 poly-epitopes instead of obtaining by affinity chromatography antigen specific human antibodies from sera of individuals living in endemic regions which could provide a direct and clear answer whether a protective vaccine could be obtained.

    Response: We agree that the clear answer to the protective function of antibodies could have been answered using human antibodies. However, we did not have a sufficient volume of patient sera to perform affinity enrichment. The use of rabbits here was to ensure the generation of antigen-specific antibody responses in ample amounts. The patient sera in quantities available were used in ELISA, epitope mapping, and IP, followed by mass-spectrometry. The IP-MS clearly shows the presence of antibodies against the proteins taken in the generation of chimeric antigens (Supplementary Figure 1 D).

    #Reviewer 3 (Evidence, reproducibility and clarity):

    Multi-protein chimeric antigens... by: Deshmukh et al

    This article addresses an extremely important objective, the development of an effective prophylactic vaccine for Malaria. The disease continues to be widespread claiming the lives of hundreds of thousands of people annually, many of them children. Despite efforts towards producing Malaria vaccines, none thus far have been sufficiently protective or long term. As the authors point out vaccines can target the parasite per se, and possibly more attractive would be to focus on parasite derived antigens expressed on the surface of infected erythrocytes, hence targeting the Blood stage of the infection, which is most directly associated with Malaria pathogenesis. The authors propose a somewhat novel approach in which they have selected an array of short (25 amino acids) segments of Plasmodium derived proteins stitched together to produce 3 chimeric recombinant proteins as potential immunogens. Although a considerable amount of work is described, the results are not compelling in proving the efficacy or advantage of using chimeric antigens as worthy vaccine candidates for Malaria.

    Unfortunately, the rationale behind the experiments are not clearly defined which is a matter of concern. In addition, details of the work done and the technical aspects needs to better explained to fully understand how and why the target segments were selected and the chimeras produced. This review focuses first on scientific issues and then format and editing, both aspects demonstrate that the manuscript in its present form requires major changes for it to be of relevance to the field. This review focuses first on issues of substance and then format and editing, both aspects disqualify the publication of the manuscript in its present form.

    We appreciate the time and effort given by our reviewer in thoroughly reading the manuscript. We are thankful for all the comments and suggestions for better shaping the article.

    Experiments and Results:

    1. The underlying proposal claims that chimeric antigens might be advantageous in eliciting protective antibodies. The authors produced three chimeras: var, MSP and InvP. __The var chimera contains 29 segments of PfEMP1 derived from 8 alleles. The hypothesis is that by expressing 29 different segments one will produce antibodies that can better cope with the antigenic diversity of this target. Indeed, serial monoallelic expression of anyone of the 60 PfEMP1 variants of a given P. falciparum strain has been thought to mediate immune evasion. The parasite is presumed to be able to escape immune defenses, by switching and serially expressing PfEMP1 alleles. Hence, one might assume that by introducing different segments, derived from different alleles, one will gain better protection. The authors have not really tested this idea. They have produced a single chimera and tested it without controlled comparison of performance to any single segment, or for that matter compared to alternative structural domain(s) of PfEMP. This brings me to the question of how the segments were selected and why. The authors implement IEDB-AR to identify presumably preferred B-cell epitopes. The methodology relies on a number of computational methods that predict the propensity of linear segments of proteins to have, for example, secondary structures, or be surface accessible, or relatively hydrophilic or flexible, etc. IEDB-AR is a tool to assist the identification of segments (5-25 amino acids in length), that might be associated with B-cell epitopes, or at least segments comprising linear aspects of B-cell epitopes. The input is a linear sequence of an antigen, proposing linear aspects of what could be associated with B-cell epitopes. B-cell epitopes, however, are typically conformational and discontinuous. They certainly can and do contain linear segments, but even these may require 3D conformations dictated by spatial constraints imposed by the native surrounding aspects of the natural antigen. It is hard to assume that by simply stitching 29 segments, one after the other, one can provide them with the native environment for them to assume a somewhat physiologically relevant conformation. Unfortunately, the authors have not addressed the unique characteristics of the antigen they have selected. PfEMP1, for example, is a family of antigens with discrete sub-domain structures and features (DBL and CIDR for example). It would be relevant and useful to relate the segments that they chose to the natural unique domains of the antigen and how they might best present common vs variant aspects of the antigen. There are at least 30 crystal atomic structures for PfEMP1 in complex with various physiologically relevant proteins (eg ICAM etc). The authors might have considered the 3D structure of PfEMP in their analyses and at least indicated on an atomic structure where the 29 segments lie. __

    More concerning is the fact that the expression of the chimera does not produce a crisp single protein, but rather a complex of products as illustrated in the Supplement Figure 1 B. The authors simply claim that they produce the antigen for immunization of rabbits (or one rabbit?) and they collect gel-derived band(s) of what MW?? Assuming that a 25aa segment should be about 2500-2800 daltons and so 29 such segments strung together should be about 80kDa. The gel shows bands at 124kDa, and a slew of bands shorter than 71kDa. There is no mention what the expected MW should be and there is no explanation why the protein pattern contains so many bands of different sizes and what exact bands were taken for the immunogen or why.

    Response: We thank the reviewer for this comment, as it tells us the reader’s perspective on how the chimeric construct part is underexplained. We have now expanded the section on chimeric construct design, the sequences used, the functional domains they belong to in the PfEMP1 protein (Supplementary Tables 1 and 2), and the expected sizes of the proteins created. As for the B-cell epitope prediction, we have used the linear epitope prediction tool. However, we will include a 3D conformational study highlighting the placement of peptides that we have used to generate chimeric antigens.

    The sequences for chimeric constructs were synthesized commercially and confirmed using Sanger sequencing. The antigens run higher than their expected molecular weights, and we have confirmed them through western blot and mass spectrometry (Supplementary Figure 1 B and C). The chimeric varB antigen specifically shows a cleaving pattern, hence the multiple bands in western blotting (we have considered the top-most band with the highest anti-his intensity). After these confirmations, the antigens were independently injected in rabbits to generate antibodies.

    Similar considerations can be made regarding the selection of the segments for the two other chimeras, although they seem to produce a single polypeptide.

    Response: The antigens were confirmed using Sanger sequencing, expression using anti-his western blot, and proteins were confirmed using mass spectrometry for all three chimeric constructs (Supplementary Figure 1 B and C).

    If the point was to test a "chimera" modality as an improved vaccine, it would have been more useful to focus on one chimera and carefully characterize it and compare it to its components used separately.

    Response: The idea of chimera arises from the fact that individual proteins/components are insufficient to generate optimal responses. The proteins considered in our study have already been validated in the field (as separate components) and show that the efficacy observed was sub-optimal. Since our rationale is to include multiple proteins to tackle the redundancy and parasite virulence, we have focused on generating three chimeric constructs covering the entire blood stage of Plasmodium falciparum. Our objective is to demonstrate that a multi-protein, multi-factorial vaccine, as a proof of concept, works better in tackling malaria. We believe that in proving so, a comparison of chimera with their individual components is an unnecessary and economically unviable.

    The authors devote much effort to the fluidics system and their assay. This might warrant a paper dedicated to the methodology they have developed.

    Response: The *Plasmodium *virulence genes are extensively studied for their interactions with human endothelial receptors. Unfortunately, these studies fail to take human physiological conditions into account. We wanted to test our anti-chimeric varB antibodies in the best mimicking environment possible. Hence, the efforts were devoted to developing, standardizing, and quantifying the fluidic cytoadherence system. We thank the reviewer for their kind words of encouragement on our methodology.

    Format and Editing:

    1. The manuscript is very poorly written with multiple errors throughout. The authors use abbreviations that are not defined, eg iRBC (pg 5 line 22) or sometimes incorrectly defined, eg MSP ("merozoite-specific proteins - pg 6 line 18).

    Response: We apologize for the abbreviation error. The abbreviation for iRBC is defined in the introduction section (page no 4, Line 15); hence, it is not redefined on page 5, line 22. We have corrected merozoite-specific proteins on page 6, line 18.

    The Figures are of low resolution to the extent that they can not be read (for example Figure 3 pg 34). Figure 1 is somewhat useless and misleading. In Fig1 C - the diagram illustrates 5 hypothetical chimeras where in fact only three were produced. There really is no detail or explanation as to how the chimeras were produced.

    Response: We apologize for the low resolution of the images. We have now improved the image quality. Figure 1C represents the idea of designing the construct, not the number of chimeras we generated. We apologize for this confusion and have explicitly mentioned this in the figure panel for Figure 1C. As for the design and generation of chimeric antigens, we understand that the materials and methods section is underexplained, and we have now expanded on it with all details included.

    In the construction of the chimeras there is no mention as to whether short linkers were introduced between the segments or not. What was the expected weight of the chimera? Was the order of segments random or precise and consistent? Were the constructs sequence validated in addition to the MassSpec?

    Response: We understand that the section on the chimeric construct is underexplained for the readers, and we thank the reviewer for pointing it out. We have now expanded the section on chimeric antigen design and included the details. Chimera was tested with GSGSGS linkers and without linkers for expression. The final antigen injected in rabbits was serially attached peptides without linkers. The segments stitched were in precise order, as mentioned in Supplementary Sheet 1. The construct was commercially synthesized and sequence validated along with the anti-his western blot and mass spectrometry analysis.

    The figures of the Supplement are not numbered.

    Response: We thank the reviewer for pointing this out. The figures are now numbered.

    Note that the headings in Supplement Figure 1 B and C have overlapping text.

    Response: Thank you for pointing this out. We have now rearranged the supplementary figures 1B, and 1C.

    Most disturbing is that multiple references that are incomplete. For example: in References 15, 16, 25, 26, 27 there is no indication of the Journal.

    Response: We apologize for the mistakes in referencing. These references did not have full citations in Endnote. We have now manually checked all the references and corrected the incomplete formats of the references.

    The authors mention reference 13 [2006] in claiming that the antibodies can be protective, and then support this by referring to refs 14, 15 and 16 published in 1961, 1963 and 1962 respectively. Although, old articles can be useful, but the authors should attempt to provide current proof of such basic claims.

    Response: We thank the reviewer for pointing this out. We have now separated these two statements and not mentioned the latter as a support to the former. As for references 14, 15, and 16, these were the early studies in the field that show the protective nature of antibodies through the passive immunization process and are foundations for the idea of blood stage vaccination. Current proofs of antibodies against blood-stage antigens are included for blood-stage vaccine candidates.

    Reviewer #3 (Significance):

    The goal of the study is very important.

    The hypothesis that a chimeric presentation of select peptides could be advantageous was not rigorously tested nor well controlled in a meaningful evaluation and thus no conclusion can be made. There are no comparative analyses to test their hypothesis.

    The method for selection of epitope segments is not well justified. There is little attempt to provide rationale or description of the segments chosen and how they fit within the antigens, thus justifying segments over multiple antigens.

    The grammatical errors, lack of clarity accompanied by little attention to style and readability render the manuscript quite illegible.

    There is no excuse for so many errors in the references.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Multi-protein chimeric antigens... by: Deshmukh et al

    This article addresses an extremely important objective, the development of an effective prophylactic vaccine for Malaria. The disease continues to be widespread claiming the lives of hundreds of thousands of people annually, many of them children. Despite efforts towards producing Malaria vaccines, none thus far have been sufficiently protective or long term. As the authors point out vaccines can target the parasite per se, and possibly more attractive would be to focus on parasite derived antigens expressed on the surface of infected erythrocytes, hence targeting the Blood stage of the infection, which is most directly associated with Malaria pathogenesis. The authors propose a somewhat novel approach in which they have selected an array of short (25 amino acids) segments of Plasmodium derived proteins stitched together to produce 3 chimeric recombinant proteins as potential immunogens. Although a considerable amount of work is described, the results are not compelling in proving the efficacy or advantage of using chimeric antigens as worthy vaccine candidates for Malaria.

    Unfortunately, the rationale behind the experiments are not clearly defined which is a matter of concern. In addition, details of the work done and the technical aspects needs to better explained to fully understand how and why the target segments were selected and the chimeras produced. This review focuses first on scientific issues and then format and editing, both aspects demonstrate that the manuscript in its present form requires major changes for it to be of relevance to the field. This review focuses first on issues of substance and then format and editing, both aspects disqualify the publication of the manuscript in its present form.

    Experiments and Results:

    The underlying proposal claims that chimeric antigens might be advantageous in eliciting protective antibodies. The authors produced three chimeras: var, MSP and InvP.

    The var chimera contains 29 segments of PfEMP1 derived from 8 alleles. The hypothesis is that by expressing 29 different segments one will produce antibodies that can better cope with the antigenic diversity of this target. Indeed, serial monoallelic expression of anyone of the 60 PfEMP1 variants of a given P. falciparum strain has been thought to mediate immune evasion. The parasite is presumed to be able to escape immune defenses, by switching and serially expressing PfEMP1 alleles. Hence, one might assume that by introducing different segments, derived from different alleles, one will gain better protection. The authors have not really tested this idea. They have produced a single chimera and tested it without controlled comparison of performance to any single segment, or for that matter compared to alternative structural domain(s) of PfEMP. This brings me to the question of how the segments were selected and why. The authors implement IEDB-AR to identify presumably preferred B-cell epitopes. The methodology relies on a number of computational methods that predict the propensity of linear segments of proteins to have, for example, secondary structures, or be surface accessible, or relatively hydrophilic or flexible, etc. IEDB-AR is a tool to assist the identification of segments (5-25 amino acids in length), that might be associated with B-cell epitopes, or at least segments comprising linear aspects of B-cell epitopes. The input is a linear sequence of an antigen, proposing linear aspects of what could be associated with B-cell epitopes. B-cell epitopes, however, are typically conformational and discontinuous. They certainly can and do contain linear segments, but even these may require 3D conformations dictated by spatial constraints imposed by the native surrounding aspects of the natural antigen. It is hard to assume that by simply stitching 29 segments, one after the other, one can provide them with the native environment for them to assume a somewhat physiologically relevant conformation. Unfortunately, the authors have not addressed the unique characteristics of the antigen they have selected. PfEMP1, for example, is a family of antigens with discrete sub-domain structures and features (DBL and CIDR for example). It would be relevant and useful to relate the segments that they chose to the natural unique domains of the antigen and how they might best present common vs variant aspects of the antigen. There are at least 30 crystal atomic structures for PfEMP1 in complex with various physiologically relevant proteins (eg ICAM etc). The authors might have considered the 3D structure of PfEMP in their analyses and at least indicated on an atomic structure where the 29 segments lie. More concerning is the fact that the expression of the chimera does not produce a crisp single protein, but rather a complex of products as illustrated in the Supplement Figure 1 B. The authors simply claim that they produce the antigen for immunization of rabbits (or one rabbit?) and they collect gel-derived band(s) of what MW?? Assuming that a 25aa segment should be about 2500-2800 daltons and so 29 such segments strung together should be about 80kDa. The gel shows bands at 124kDa, and a slew of bands shorter than 71kDa. There is no mention what the expected MW should be and there is no explanation why the protein pattern contains so many bands of different sizes and what exact bands were taken for the immunogen or why.

    Similar considerations can be made regarding the selection of the segments for the two other chimeras, although they seem to produce a single polypeptide.

    If the point was to test a "chimera" modality as an improved vaccine, it would have been more useful to focus on one chimera and carefully characterize it and compare it to its components used separately. The authors devote much effort to the fluidics system and their assay. This might warrant a paper dedicated to the methodology they have developed.

    Format and Editing:

    The manuscript is very poorly written with multiple errors throughout. The authors use abbreviations that are not defined, eg iRBC (pg 5 line 22) or sometimes incorrectly defined, eg MSP ("merozoite-specific proteins - pg 6 line 18).

    The Figures are of low resolution to the extent that they can not be read (for example Figure 3 pg 34). Figure 1 is somewhat useless and misleading. In Fig1 C - the diagram illustrates 5 hypothetical chimeras where in fact only three were produced. There really is no detail or explanation as to how the chimeras were produced.

    In the construction of the chimeras there is no mention as to whether short linkers were introduced between the segments or not. What was the expected weight of the chimera? Was the order of segments random or precise and consistent? Were the constructs sequence validated in addition to the MassSpec?

    The figures of the Supplement are not numbered.

    Note that the headings in Supplement Figure 1 B and C have overlapping text.

    Most disturbing is that multiple references that are incomplete. For example: in References 15, 16, 25, 26, 27 there is no indication of the Journal.

    The authors mention reference 13 [2006] in claiming that the antibodies can be protective, and then support this by referring to refs 14, 15 and 16 published in 1961, 1963 and 1962 respectively. Although, old articles can be useful, but the authors should attempt to provide current proof of such basic claims.

    Significance

    The goal of the study is very important.

    The hypothesis that a chimeric presentation of select peptides could be advantageous was not rigorously tested nor well controlled in a meaningful evaluation and thus no conclusion can be made. There are no comparative analyses to test their hypothesis.

    The method for selection of epitope segments is not well justified. There is little attempt to provide rationale or description of the segments chosen and how they fit within the antigens, thus justifying segments over multiple antigens.

    The grammatical errors, lack of clarity accompanied by little attention to style and readability render the manuscript quite illegible.

    There is no excuse for so many errors in the references.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript describes the vaccine potential of unstructured P. falciparum merozoite protein fragments 25 amino acid long belonging to 3 different protein families. The work is well performed, easily reproducible and clearly described.

    Referees cross-commenting

    The polymorphic residues should be highlighted in the supplementary figure.

    Significance

    The use of protein fragments whose structure can be predicted by their sequence has been exploited in many studies for the development of vaccines or other biologicals. In this studies the authors selected 3 different families belonging to the red blood stage of the parasite. The table showing the sequences selected is not readable and should be clearly provided in the supplementary section. In addition, polymorphic residues should be highlighted. In addition, it is not to mention why the authors used immune rabbit sera obtained by injection of the 3 poly-epitopes instead of obtaining by affinity chromatography antigen specific human antibodies from sera of individuals living in endemic regions which could provide a direct and clear answer whether a protective vaccine could be obtained.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    This manuscript by Deshmukh et al is aimed at generating chimeric antigens that can be useful for making next generation vaccines that block blood stage infection by malaria parasite. Given that there is no blood stage vaccine against malaria and available liver stage vaccine shows only limited efficacy that too only in Africa, there is dire need for having novel approaches to generate successful vaccines. In the past attempts have been made to make multivalent vaccines but have not been successful. Nevertheless, it is still a good option as single target blood-stage vaccines have failed. Authors propose to target cytoadhesion and host erythrocyte invasion. For this purpose, they have selected epitopes from PfEMP1/VarB family members, which poses a major challenge as at least 60 genes encode them and they exhibit variations which facilitate the escape from the immune system. The other two chimeras target invasion related proteins like MSPs and adhesins shed by micronemes and rhoptries, which are critical for invasion. The reported work is interesting and provides a useful approach towards developing vaccines against blood stage infection.

    Comments:

    1. The peptides used in InvB chimera did not show good reactivity especially when compared to VarB or MSP peptides. Please discuss the possible reasons.
    2. It will be interesting to determine if blocking a specific VarB/PfEMP1 alters expression of other members. Based on the data provided in Fig. 4E, can a chimera be designed which only includes PfEMP1 that are represented well in HBEC-5i population?
    3. Some of the invasion related proteins like RH5 and EBA175 are not present at parasite surface, instead, secreted from rhoptries and micronemes. It will be nice to perform Western blots on condition medium and see if InvP (or even MSP and VarB) antibodies recognizes the secreted version of these proteins.
    4. Fig. 6E- Statistics need to be provided for inhibition at 12.3 and 25ug.
    5. Plasmodium uses multiple ligand-receptor interaction, which could depend (e.g. EBA-glycohophorins) or operate independent (e.g. RH5-basigin) of sialic acid. While there is representation from candidates from both of these families, most studies especially growth rate assays (Fig. 6E) have been carried using 3D7 strain, which does not require sialic acid. It is possible that if similar experiments were performed using sialic acid-sensitive strains, InvP and MSP antibodies may cause greater inhibition of parasite growth, which may be worth testing.
    6. The direct effect of InvP and MSP Abs should be tested directly on host erythrocyte invasion.

    Significance

    Present study proposes novel strategies for the development of anti-malarial vaccine.

  9. Version published to 10.1101/2023.11.22.568251v1 on bioRxiv