Molecular and functional properties of human Plasmodium falciparum CSP C‐terminus antibodies

Abstract Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C‐terminal domain. Two mAbs recognized linear epitopes in the C‐terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α‐thrombospondin repeat (α‐TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α‐TSR was associated with IGHV3‐21/IGVL3‐21 or IGLV3‐1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class‐switching compared to anti‐repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C‐linker reactive mAb with cross‐reactivity to the central repeat and junction. The data provide novel insights in the human anti‐C‐linker and anti‐α‐TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

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Referee #1 (Remarks for Author): The authors set out to identify and assess the inhibitory activity of monoclonal antibodies (mAbs) specific of the C-terminal region of the P. falciparum (Pf) circumsporozoite protein (CSP), and compare it with that conferred by mAbs targeting the central repeat region of this protein. This is an important study, made particularly relevant by the endorsement of the poorly protective RTS,S vaccine and the need to design new vaccines with enhanced efficacy. The study was made possible by access to samples collected from malaria-naïve individuals who underwent immunization with Sanaria's PfSPZ vaccine, consisting of radiation-attenuated Pf sporozoites. The authors show that immunization with PfSPZ elicits the production of 73 out of 177 antibodies that recognize the C-terminus of PfCSP. These are frequently encoded by the IGHV3-21 gene and preferentially target two conformational epitopes in the alpha-TSR domain of CSP, one of the two C-terminal subregions of this protein.
However, when compared with mAb against the CSP repeat region, these C-terminal-specific mAbs showed low binding activity to Pf sporozoites and were poorly inhibitory of these parasites. In fact, only a single mAb reactive to the C-terminus of CSP, with cross-reactivity to the protein's repeat and N-terminal junction, was able to inhibit the parasite in vitro, but not in vivo.
The Introduction is thorough and well-structured, the figures are informative, and the conclusions are supported by the data. The Discussion is very well organized and very clear. Overall, I have no objections to the publication of the manuscript in its current form, except for one minor suggestion, regarding the last sentence of the Abstract and, indeed, the author's take home message summarized by that statement, which reads "The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs". In my opinion, the statement that these data "support exclusion of the PfCSP C terminus from malaria vaccine designs" overlooks the fact that this region of CSP contains T-cell epitopes that may be key to an effective protection against infection and disease. As such, I urge the authors to revise this statement or at least to discuss the importance of the C-terminal region of CSP for T-cell immunity and protective efficacy. As it is, the statement completely overlooks this aspect of immunity and, in my opinion, is misleading.

Referee #2 (Comments on Novelty/Model System for Author):
Overall, this is a very high quality study, thorough in approach and clear in outcome. The use of the rodent model to assess Mab efficacy is essential (as human trials would be extremely challenging and likely unethical without prior demonstration in a mouse anyway). As such, I believe the right experiments have been undertaken.

Referee #2 (Remarks for Author):
This is an extremely thorough study, which has surveyed the less well-investigated C terminus of the lead vaccine target CSP from the Plasmodium malaria parasite. Surveying a suite of mabs isolated from human PfSPZ challenges, the authors systematically dissect the profile and target of numerous mabs, drilling down to the important (and not to be dismissed) ultimately negative result that Mabs targetting the C terminus and on the whole non-protective. This is important data and will help refine second and third generation vaccines (e.g. RTS,S/R21 2.0) or even entirely new vaccines that wish to target CSP but avoid immunogenic but likely non-protective domains of the protein. As such, I think the study is compelling and should be published. My comments are minor and perhaps more suggestion therefore rather than a demand for more laborious experimentation/change.
1. The non-accessibility of antibodies to the C terminus on the sporozoite surface, yet their generation in vivo is (to me) intriguing. Is there anything about the PfSPZ vaccine that might bias formation of C terminal antibodies that are otherwise inaccessible? E.g. might there be degraded CSP in the cocktail that elicits a non-protective response that wouldn't otherwise exist in live sporozoites (e.g. from a mosquito bite). Said another way, is there any evidence from direct bites that C-CSP targeting antibodies are less likely? 2. In the same vein, I wondered whether the authors had considered doing labelling experiments with Pf sporozoites, rather than Pb-PfCSP sporozoites -in case their is a difference in their heterologous presentation on Pb sporozoites, or indeed if any labelling had been trialled on PfSPZ (cryo-preserved) sporozoites? It might have been nice to see some images of these labelled to partner the flow data.
3. The final section on DPN binding is interesting but I admit to having found it hard to follow (e.g. use of BSA abbreviationwhere elsewhere BSA means Bovine Serum Albumin). I just wondered whether this and Figure 5 could be simplified slightly just to make them easier for a non-structural biologist (but avid vaccinologist) to follow? 4. Figure 4 took me several reads to realise that Panel A had a separate panel -just wondered if this could be either divided into a new A/B or made distinct from Panel E below. Figure 4F mAb 1710 is listed as a test, and then in the same sentence listed as the control -this could be re-written for clarity.

In
Referee #3 (Comments on Novelty/Model System for Author): This is really nice work and on the whole is done well. These are not the first human antibodies against CSP purified and tested, including others made against similar regions. The focus on the C-terminal domain and antibodies against this region is an interesting addition to the field and could help design better vaccines going forward. The finding of non functional (non protective) antibodies at this region is unfortunate, but this information should be used to build a better vaccine construct.

Referee #3 (Remarks for Author):
Oludada et al describe purification and characterisation of a series of human derived monoclonal antibodies that target the Cterminus of the malaria sporozoite circumsporozoite protein (C-CSP). The manuscript is well written and the data nicely presented, I quite enjoyed reading about this study. The manuscript establishes a series of antibodies with similar origins, specificity and functional properties and shows that antibodies to this region of CSP have limited efficacy in blocking sporozoite host-cell entry. Although the inability to find a blocking antibody targeting this domain is unfortunate for the overall impact of the study, the fact that this paper helps to rule out much of the c-terminal domain as a target is of benefit for future vaccine design. The authors have done a very nice job of putting this paper together and I found little to criticise. To what degree the C-CSP-reactive serum antibodies recognize C-CSP specifically or cross-react with the repeat and junction has not been determined. Therefore, it is unclear whether protection is associated with C-CSP-specific or cross-reactive antibodies.
The summary suggests here that high titres of vaccine induced C-CSP antibodies must have some role in protection, perhaps through targeting domains other than some of the ones focussed on here. However, could not the antibody titres received during vaccination be simply a marker of antibody production (i.e. antibodies are made against the region in high titres because it is part of the vaccine), with the anti-NANP antibodies doing the bulk of the protective work and the C-CSP antibodies themselves do little functional protection? The data presented in this study demonstrate convincingly that a lot of antibodies to the C-CSP epitope have limited/no functional activity. While its possible that cell-mediated responses may confer protection via C-CSP targeting antibodies, this goes against the theory postulated here that antibodies targeting this domain don't work because they can't access it. In effect, the anti-C-CSP antibodies titres might be significant only as markers of exposure and antibody production towards the vaccine and have no role in protection. Is this possible and worth considering in the discussion?

RESPONSE TO REVIEWERS:
Referee #1 (Comments on Novelty/Model System for Author): This is an important and novel study, made particularly relevant and potentially impactful by the recent endorsement of the poorly protective RTS,S vaccine and the need to design new vaccines with enhanced efficacy. The study was made possible by access to samples collected from malaria-naïve individuals who underwent immunization with Sanaria's PfSPZ vaccine, consisting of radiation-attenuated Pf sporozoites, which constitutes a most appropriate model system to conduct this study.
We thank the reviewer for the positive feedback.

Referee #1 (Remarks for Author):
The authors set out to identify and assess the inhibitory activity of monoclonal antibodies (mAbs) specific of the C-terminal region of the P. falciparum (Pf) circumsporozoite protein (CSP), and compare it with that conferred by mAbs targeting the central repeat region of this protein. This is an important study, made particularly relevant by the endorsement of the poorly protective RTS,S vaccine and the need to design new vaccines with enhanced efficacy. The study was made possible by access to samples collected from malaria-naïve individuals who underwent immunization with Sanaria's PfSPZ vaccine, consisting of radiation-attenuated Pf sporozoites. The authors show that immunization with PfSPZ elicits the production of 73 out of 177 antibodies that recognize the C-terminus of PfCSP. These are frequently encoded by the IGHV3-21 gene and preferentially target two conformational epitopes in the alpha-TSR domain of CSP, one of the two C-terminal subregions of this protein. However, when compared with mAb against the CSP repeat region, these Cterminal-specific mAbs showed low binding activity to Pf sporozoites and were poorly inhibitory of these parasites. In fact, only a single mAb reactive to the C-terminus of CSP, with cross-reactivity to the protein's repeat and N-terminal junction, was able to inhibit the parasite in vitro, but not in vivo. The Introduction is thorough and well-structured, the figures are informative, and the conclusions are supported by the data. The Discussion is very well organized and very clear. Overall, I have no objections to the publication of the manuscript in its current form, except for one minor suggestion, regarding the last sentence of the Abstract and, indeed, the author's take home message summarized by that statement, which reads "The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs". In my opinion, the statement that these data "support exclusion of the PfCSP C terminus from malaria vaccine designs" overlooks the fact that this region of CSP contains T-cell epitopes that may be key to an effective protection against infection and disease. As such, I urge the authors to revise this statement or at least to discuss the importance of the C-terminal region of CSP for T-cell immunity and protective efficacy. As it is, the statement completely overlooks this aspect of immunity and, in my opinion, is misleading.
We would like to thank the reviewer for raising this important point, which we now address as follows in the discussion: "Future studies will need to determine whether efforts to design a second generation PfCSP vaccine might benefit from suppressing or even abrogating the humoral response against C-CSP, e.g. by boosting the anti-repeat and junction response. However, exclusion of the complete domain, especially of the highly immunodominant α-TSR, would eliminate the main T helper cell epitopes with likely strong negative effects on the quality and strength of the humoral response against the potent repeat and junction epitopes. Inclusion of linear peptide epitopes rather than the complete C-CSP may be sufficient to provide efficient T cell help without inducing non-protective humoral responses (Wahl et al., 2022). Alternatively, non-PfCSP T cell epitopes could substitute for the loss of T cell help and promote affinity maturation of the PfCSP-specific response. "

11th Mar 2023 1st Authors' Response to Reviewers
Referee #2 (Comments on Novelty/Model System for Author): Overall, this is a very high-quality study, thorough in approach and clear in outcome. The use of the rodent model to assess Mab efficacy is essential (as human trials would be extremely challenging and likely unethical without prior demonstration in a mouse anyway). As such, I believe the right experiments have been undertaken.
We thank the reviewer for the positive feedback.
Referee #2 (Remarks for Author): This is an extremely thorough study, which has surveyed the less well-investigated C terminus of the lead vaccine target CSP from the Plasmodium malaria parasite. Surveying a suite of mabs isolated from human PfSPZ challenges, the authors systematically dissect the profile and target of numerous mabs, drilling down to the important (and not to be dismissed) ultimately negative result that Mabs targetting the C terminus and on the whole nonprotective. This is important data and will help refine second and third generation vaccines (e.g. RTS,S/R21 2.0) or even entirely new vaccines that wish to target CSP but avoid immunogenic but likely non-protective domains of the protein. As such, I think the study is compelling and should be published. My comments are minor and perhaps more suggestion therefore rather than a demand for more laborious experimentation/change.
1. The non-accessibility of antibodies to the C terminus on the sporozoite surface, yet their generation in vivo is (to me) intriguing. Is there anything about the PfSPZ vaccine that might bias formation of C terminal antibodies that are otherwise inaccessible? E.g. might there be degraded CSP in the cocktail that elicits a non-protective response that wouldn't otherwise exist in live sporozoites (e.g. from a mosquito bite). Said another way, is there any evidence from direct bites that C-CSP targeting antibodies are less likely?
The reviewer raises a very interesting point. To the best of our knowledge, it is unclear whether the PfSPZ antigens are presented differently to the immune system than the antigens of mosquito-transmitted sporozoites. The overall weak response induced by natural parasites and strong differences in the number of transmitted parasites in the field vs. PfSPZ vaccination and different routes of parasite injection (s.c. for mosquito-bites vs. i.v. for PfSPZ vaccination) make direct quantitative comparisons difficult.
2. In the same vein, I wondered whether the authors had considered doing labelling experiments with Pf sporozoites, rather than Pb-PfCSP sporozoites -in case their is a difference in their heterologous presentation on Pb sporozoites, or indeed if any labelling had been trialled on PfSPZ (cryo-preserved) sporozoites? It might have been nice to see some images of these labelled to partner the flow data.
We used transgenic Pb-PfCSP parasites for quantification of antibody binding capacity to live sporozoites because they express mCherry, a red fluorescence marker that allows us to accurately gate single sporozoites for flow cytometry analyses. We have used live Pf and Pb-PfCSP sporozoites in IFA analyses and did not detect differences in the staining pattern, however, this method has low resolution for detection of fine differences related to heterologous expression. The reviewer raised an interesting question about potential changes in antibody binding to irradiated or cryo-preserved parasites. However, to the best of our knowledge, this question has not yet been quantitatively assessed, likely due to the lack of a Pf fluorescence reporter line compatible with A. gambiae mosquitoes.
3. The final section on DPN binding is interesting but I admit to having found it hard to follow (e.g. use of BSA abbreviation -where elsewhere BSA means Bovine Serum Albumin). I just wondered whether this and Figure 5 could be simplified slightly just to make them easier for a non-structural biologist (but avid vaccinologist) to follow?
We appreciate the complexity of the structural data, and have included some additional context with the results. No changes have been made to the figures, however, additional guidance on interpreting the figures has been provided in the results, and the important takehome messages from the structural data are emphasized in the discussion.
4. Figure 4 took me several reads to realise that Panel A had a separate panel -just wondered if this could be either divided into a new A/B or made distinct from Panel E below.
We agree with the reviewer that Panel A was difficult to read and have modified Fig. 4 accordingly. For clarity, we now show the data for each antibody separately and indicate the respective affinity in Panel A. The raw data panel has been moved to extended view figure 4. Figure 4F mAb 1710 is listed as a test, and then in the same sentence listed as the control -this could be re-written for clarity.

In
For clarity, we modified the legend as follows: "Capacity of the passively-transferred indicated antibodies (100 μg) to protect mice (n=10 for mAb 317 (Oyen et al., 2017), n=8 for mAb 1961, and n=9 for mAb 1710(Scally et al., 2018) from parasitemia after the bite of three PbPfCSP(mCherry)-infected mosquitoes." Referee #3 (Comments on Novelty/Model System for Author): This is really nice work and on the whole is done well. These are not the first human antibodies against CSP purified and tested, including others made against similar regions. The focus on the C-terminal domain and antibodies against this region is an interesting addition to the field and could help design better vaccines going forward. The finding of non functional (non protective) antibodies at this region is unfortunate, but this information should be used to build a better vaccine construct.
Referee #3 (Remarks for Author): Oludada et al describe purification and characterisation of a series of human derived monoclonal antibodies that target the C-terminus of the malaria sporozoite circumsporozoite protein (C-CSP). The manuscript is well written and the data nicely presented, I quite enjoyed reading about this study. The manuscript establishes a series of antibodies with similar origins, specificity and functional properties and shows that antibodies to this region of CSP have limited efficacy in blocking sporozoite host-cell entry. Although the inability to find a blocking antibody targeting this domain is unfortunate for the overall impact of the study, the fact that this paper helps to rule out much of the c-terminal domain as a target is of benefit for future vaccine design. The authors have done a very nice job of putting this paper together and I found little to criticise.
We thank the reviewer for the positive feedback.
Major comments -Discussion: The authors state that. In response to RTS,S/AS01 immunization, anti-NANP and anti-C-terminus antibody titers correlate with protection (Chaudhury et al, 2021;Chaudhury et al, 2016;Dobano et al, 16 2019). To what degree the C-CSP-reactive serum antibodies recognize C-CSP specifically or cross-react with the repeat and junction has not been determined. Therefore, it is unclear whether protection is associated with C-CSP-specific or cross-reactive antibodies. The summary suggests here that high titres of vaccine induced C-CSP antibodies must have some role in protection, perhaps through targeting domains other than some of the ones focussed on here. However, could not the antibody titres received during vaccination be simply a marker of antibody production (i.e. antibodies are made against the region in high titres because it is part of the vaccine), with the anti-NANP antibodies doing the bulk of the protective work and the C-CSP antibodies themselves do little functional protection? The data presented in this study demonstrate convincingly that a lot of antibodies to the C-CSP epitope have limited/no functional activity. While its possible that cell-mediated responses may confer protection via C-CSP targeting antibodies, this goes against the theory postulated here that antibodies targeting this domain don't work because they can't access it. In effect, the anti-C-CSP antibodies titres might be significant only as markers of exposure and antibody production towards the vaccine and have no role in protection. Is this possible and worth considering in the discussion?
The reviewer raises a very valid point. We would like to clarify that we fully agree that most of the protective capacity comes from anti-repeat antibodies and that C-CSP specific antibodies alone would not show any parasite inhibitory effect. Indeed, the C-CSP antibodies might simply be a marker of exposure and of the overall strength of the anti-parasite response. To clarify this point, we have modified the discussion as follows: "In response to RTS,S/AS01 immunization, anti-NANP and anti-C-terminus antibody titers correlate with protection (Chaudhury et al, 2021;Chaudhury et al, 2016;Dobano et al, 2019). To what degree the C-CSP-reactive serum antibodies recognize C-CSP specifically or cross-react with the repeat and junction has not been determined. Therefore, it is unclear whether protection is associated with C-CSP-specific or cross-reactive antibodies or whether C-CSP antibodies are simply a marker of the overall strength of the anti-parasite response whereas protection is mediated by anti-repeat antibodies. " We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.
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