Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay
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Curated by eLife
Evaluation Summary:
This manuscript describes a fluorospot-based assay as a model for a methodical, step-wise and rigorous approach - that combines multiple reagents in a complex system - to study the cross-reactivity of antibody to polymorphic antigens using the malaria vaccine candidate, VAR2CSA, as a model. The authors apply monoclonal antibodies and the corresponding B cells to validate their multiplexed assay before testing small number of samples from malaria exposed donors in a pilot application of the assay. The data support the conclusions. This information will attract the attention of immunologists and vaccinologists, who are primarily but not exclusively, involved in research on malaria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism). However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. Using VAR2CSA-type PfEMP1—a notoriously polymorphic antigen involved in the pathogenesis of placental malaria—as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases.
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Author Response
Reviewer #1 (Public Review):
The authors attempt to optimize the FluoroSpot assay to allow for the assessment of cross-reactive antibodies targeting conserved epitopes shared by multi-allelic antigens and those specific to unique antigen variant at the B cells level. This is a critical aspect to consider when identifying targets of a broad range of cross-reactive antibody for vaccine development and the antigen VAR2CSA used in this work is one that will benefit from the method described in the manuscript.
Overall, this is a method manuscript with extensive detail of the assay validation process. The description of the assay performance steps using, first monoclonal antibodies and later hybridoma/immortalized B cells was important to understand conditions that can influence the antigen-antibody interactions in the …
Author Response
Reviewer #1 (Public Review):
The authors attempt to optimize the FluoroSpot assay to allow for the assessment of cross-reactive antibodies targeting conserved epitopes shared by multi-allelic antigens and those specific to unique antigen variant at the B cells level. This is a critical aspect to consider when identifying targets of a broad range of cross-reactive antibody for vaccine development and the antigen VAR2CSA used in this work is one that will benefit from the method described in the manuscript.
Overall, this is a method manuscript with extensive detail of the assay validation process. The description of the assay performance steps using, first monoclonal antibodies and later hybridoma/immortalized B cells was important to understand conditions that can influence the antigen-antibody interactions in the assay. This multiplex approach can assess the cross-reactivity of antibody to up four allelic variants of an antigen with the possibility to explore the affinity of antibody to a particular variant using the RSV measurements. The validation of the assay with PBMC from malaria exposed donors both men and women (that naturally acquired high titer of antibodies to VAR2CSA during pregnancy) is a strength of this work as this is in the context of polyclonal antibodies with more heterogenous antibody binding specificities.
The ability of the assay to detect cross-reactive antibodies using all four tags appear highly variable even in the context of monoclonal antibody targeting the homologous antigen labelled with all 4 tags.
We understand the concern for variability, but we think that in general the assay was very consistent. Regardless of the configuration used, we detected strikingly comparable number of spots/well, especially when the homologous antigen labelled with four tags was used (Figure 2A). Similar consistency has been previously reported when a similar assay was used to study cross-reactivity in dengue-specific antibodies.
Overall, it appears that the assessed antibody reactivity with TWIN tagged antigens was relatively low and this needs to be explained and discussed as the current multiplex method, as it is, might just be optimized for study of cross-reactive antibodies to 3 antigens.
The LED380 (used to detect and visualize the TWIN tag) indeed gave more background than the other three detection channels. We normally observed a ring of fluorescence at the edge and the middle of the wells, accompanied by lower intensity of the spots. These two characteristics are apparent in the figures and RSV plots presented in the manuscript. In an attempt to reduce these issues, we attempted to substitute the TWIN tag for a BAM tag detected with a peptide-specific antibody (data not presented). However, that approach did not improve the readout and we therefore decided to keep the TWIN-StrepTactin pair for all the experiments. Importantly, even with these issues, routine manual inspection of the wells confirmed the Apex software automatically and efficiently counted “real” spots giving us confidence on the performance of the assay. We acknowledge that exclusion of the LED380 data would lead to higher assay accuracy. However, it would result in reduced ability to assess broad antibody cross-reactivity, which was the primary objective of our study. We have added text briefly discussing this to the revised manuscript (lines 154-160).
As acknowledged by the authors, the validation of this assay on PBMC from only 10 donors (7 women and 3 men) is a caveat to the conclusion and increasing this number of donors (the authors have previously excelled in B cells analyses of PfEMP1 proteins and would have PBMC readily available) will strengthen the validity of this assay.
We thank the reviewer for this comment and agree the number of donors tested is far from sufficient to provide any conclusive evidence regarding frequencies of VAR2CSA-specific and cross-reactive B cells in the context of placental malaria. However, we firmly believe that the validation of the assay – which was the objective of the study – is sufficient, especially because we included human B-cell lines isolated from donors naturally exposed to VAR2CSA-expressing parasites. Futures studies including more donors and full-length VAR2CSA antigens are certainly warranted. As the performance of assay has now been validated (this manuscript) to our satisfaction, we are indeed planning such studies.
Reviewer #2 (Public Review):
The manuscript describes the development of a laboratory-based assay as a tool designed to identify individuals who have developed broadly cross-reactive antibodies with specificity for regions that are common to multiple variants of a given protein (VAR2CSA) of Plasmodium falciparum, the parasite that causes malaria. The assay has potential application in other diseases for which the question ofacquisition of antibody-mediated immunity, either through natural exposure or through vaccination, remains unresolved.
From a purely technical/methodological viewpoint, the work described is of high quality, relying primarily on the availability of custom-designed, in-house-derived protein and antibody reagents that had, for the most part, been validated through use in earlier studies. The authors demonstrate a high degree of rigour in the assay development steps, culminating in a convincing demonstration of the ability to accurately and reproducibly quantify cross-reactive antibody types under controlled conditions using well-characterized monoclonal antibodies.
In a final step, the authors used the assay to assess the content of broadly cross-reactive antibodies in samples from a small number of malaria-exposed African men and women. Given that VAR2CSA is a parasite-derived protein that is exclusively and intimately involved in the manifestation of malaria during pregnancy, with specific localisation to the maternal placental space, the premise is that antibodies -including those with cross-reactive specificities - should be almost exclusively detectable in samples from women, either pregnant at the time of sampling or having been pregnant at least once. The assay functioned technically as expected, identifying antibodies predominantly in women rather than men, but it failed to identify broadly cross-reactive antibodies in the women's samples used, only revealing antibodies with specificity for just one of the different variants used. The latter result could have two mutually non-exclusive explanations. On the one hand, the small number of women's samples (7) screened in the assay could simply be insufficient, demanding the use of a much larger panel. On the other hand, for technical reasons the assay involves the use of only relatively restricted parts of the VAR2CSA protein, and this particular aspect may represent its primary limitation. In earlier work, the authors did identify broadly cross-reactive antibodies in samples from African women, but that work relied on the use of the whole VAR2CSA protein present in its natural state embedded in the membrane of the infected red cell, or as a complete protein produced in the laboratory. The important point being that the whole protein likely interacts with antibodies that recognize protein structures that the isolated smaller parts of the whole protein used in the assay fail to reproduce, and that the cross-reactive antibodies identified recognize these structures that are conserved across different VAR2CSAvariants. The authors recognize these potential weaknesses in their discussion of the results. It is also possible that VAR2CSA variants expressed by parasites from geographically-distinct regions (Africa, Asia, South America) are themselves distinct, and this aspect could also have affected the outcome, since the variant protein sequences used in the assay were derived from parasites originating in these different regions.
The assay could find application in the malaria research field in the specific context of assessments of antibody responses to a range of different parasite proteins that are, or have been, considered candidates for vaccine development but for which their extensive inherent allelic polymorphism has effectively negated such efforts.
We thank the reviewer for the kind evaluation. We fully acknowledge the need for more comprehensive studies to assess the robustness of the pilot data regarding antibody cross-reactivity after natural exposure in the present study, which was aimed to document the performance of the complicated multiplexed assay rather than to provide such evidence. As mentioned above, we are currently planning such a study. We also acknowledge the need to assess the degree of cross-reactivity to full-length antigens rather than domain-specific components of them. This is obviously particularly true for large, multi-domain antigens such as PfEMP1 (including VAR2CSA). Such an exercise is complicated by the need for appropriately tagged antigens. We are intrigued by the apparent discrepancy between the degree of antibody cross-reactivity in depletion experiments using individual DBL domains of VAR2CSA (low cross-reactivity) versus full-length VAR2CSA antigens (very substantial cross-reactivity) reported by Doritchamou et al., and are keen to apply our approach to explore that finding. Therefore, as also mentioned above, we are currently planning a study employing tagged full-length VAR2CSA allelic variants as well.
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Evaluation Summary:
This manuscript describes a fluorospot-based assay as a model for a methodical, step-wise and rigorous approach - that combines multiple reagents in a complex system - to study the cross-reactivity of antibody to polymorphic antigens using the malaria vaccine candidate, VAR2CSA, as a model. The authors apply monoclonal antibodies and the corresponding B cells to validate their multiplexed assay before testing small number of samples from malaria exposed donors in a pilot application of the assay. The data support the conclusions. This information will attract the attention of immunologists and vaccinologists, who are primarily but not exclusively, involved in research on malaria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback …
Evaluation Summary:
This manuscript describes a fluorospot-based assay as a model for a methodical, step-wise and rigorous approach - that combines multiple reagents in a complex system - to study the cross-reactivity of antibody to polymorphic antigens using the malaria vaccine candidate, VAR2CSA, as a model. The authors apply monoclonal antibodies and the corresponding B cells to validate their multiplexed assay before testing small number of samples from malaria exposed donors in a pilot application of the assay. The data support the conclusions. This information will attract the attention of immunologists and vaccinologists, who are primarily but not exclusively, involved in research on malaria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors attempt to optimize the FluoroSpot assay to allow for the assessment of cross-reactive antibodies targeting conserved epitopes shared by multi-allelic antigens and those specific to unique antigen variant at the B cells level. This is a critical aspect to consider when identifying targets of a broad range of cross-reactive antibody for vaccine development and the antigen VAR2CSA used in this work is one that will benefit from the method described in the manuscript.
Overall, this is a method manuscript with extensive detail of the assay validation process. The description of the assay performance steps using, first monoclonal antibodies and later hybridoma/immortalized B cells was important to understand conditions that can influence the antigen-antibody interactions in the assay. This multiplex …
Reviewer #1 (Public Review):
The authors attempt to optimize the FluoroSpot assay to allow for the assessment of cross-reactive antibodies targeting conserved epitopes shared by multi-allelic antigens and those specific to unique antigen variant at the B cells level. This is a critical aspect to consider when identifying targets of a broad range of cross-reactive antibody for vaccine development and the antigen VAR2CSA used in this work is one that will benefit from the method described in the manuscript.
Overall, this is a method manuscript with extensive detail of the assay validation process. The description of the assay performance steps using, first monoclonal antibodies and later hybridoma/immortalized B cells was important to understand conditions that can influence the antigen-antibody interactions in the assay. This multiplex approach can assess the cross-reactivity of antibody to up four allelic variants of an antigen with the possibility to explore the affinity of antibody to a particular variant using the RSV measurements. The validation of their assay with PBMC from malaria exposed donors both men and women (that naturally acquired high titer of antibodies to VAR2CSA during pregnancy) is a strength of this work as this is in the context of polyclonal antibodies with more heterogenous antibody binding specificities.
The ability of the assay to detect cross-reactive antibodies using all four tags appear highly variable even in the context of monoclonal antibody targeting the homologous antigen labelled with all 4 tags. Overall, it appears that the assessed antibody reactivity with TWIN tagged antigens was relatively low and this needs to be explained and discussed as the current multiplex method, as it is, might just be optimized for study of cross-reactive antibodies to 3 antigens. As acknowledged by the authors, the validation of this assay on PBMC from only 10 donors (7 women and 3 men) is a caveat to the conclusion and increasing this number of donors (the authors have previously excelled in B cells analyses of PfEMP1 proteins and would have PBMC readily available) will strengthen the validity of this assay.
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Reviewer #2 (Public Review):
The manuscript describes the development of a laboratory-based assay as a tool designed to identify individuals who have developed broadly cross-reactive antibodies with specificity for regions that are common to multiple variants of a given protein (VAR2CSA) of Plasmodium falciparum, the parasite that causes malaria. The assay has potential application in other diseases for which the question of acquisition of antibody-mediated immunity, either through natural exposure or through vaccination, remains unresolved.
From a purely technical/methodological viewpoint, the work described is of high quality, relying primarily on the availability of custom-designed, in-house-derived protein and antibody reagents that had, for the most part, been validated through use in earlier studies. The authors demonstrate a high …
Reviewer #2 (Public Review):
The manuscript describes the development of a laboratory-based assay as a tool designed to identify individuals who have developed broadly cross-reactive antibodies with specificity for regions that are common to multiple variants of a given protein (VAR2CSA) of Plasmodium falciparum, the parasite that causes malaria. The assay has potential application in other diseases for which the question of acquisition of antibody-mediated immunity, either through natural exposure or through vaccination, remains unresolved.
From a purely technical/methodological viewpoint, the work described is of high quality, relying primarily on the availability of custom-designed, in-house-derived protein and antibody reagents that had, for the most part, been validated through use in earlier studies. The authors demonstrate a high degree of rigour in the assay development steps, culminating in a convincing demonstration of the ability to accurately and reproducibly quantify cross-reactive antibody types under controlled conditions using well-characterized monoclonal antibodies.
In a final step, the authors used the assay to assess the content of broadly cross-reactive antibodies in samples from a small number of malaria-exposed African men and women. Given that VAR2CSA is a parasite-derived protein that is exclusively and intimately involved in the manifestation of malaria during pregnancy, with specific localisation to the maternal placental space, the premise is that antibodies - including those with cross-reactive specificities - should be almost exclusively detectable in samples from women, either pregnant at the time of sampling or having been pregnant at least once. The assay functioned technically as expected, identifying antibodies predominantly in women rather than men, but it failed to identify broadly cross-reactive antibodies in the women's samples used, only revealing antibodies with specificity for just one of the different variants used. The latter result could have two mutually non-exclusive explanations. On the one hand, the small number of women's samples (7) screened in the assay could simply be insufficient, demanding the use of a much larger panel. On the other hand, for technical reasons the assay involves the use of only relatively restricted parts of the VAR2CSA protein, and this particular aspect may represent its primary limitation. In earlier work, the authors did identify broadly cross-reactive antibodies in samples from African women, but that work relied on the use of the whole VAR2CSA protein present in its natural state embedded in the membrane of the infected red cell, or as a complete protein produced in the laboratory. The important point being that the whole protein likely interacts with antibodies that recognize protein structures that the isolated smaller parts of the whole protein used in the assay fail to reproduce, and that the cross-reactive antibodies identified recognize these structures that are conserved across different VAR2CSA variants. The authors recognize these potential weaknesses in their discussion of the results. It is also possible that VAR2CSA variants expressed by parasites from geographically-distinct regions (Africa, Asia, South America) are themselves distinct, and this aspect could also have affected the outcome, since the variant protein sequences used in the assay were derived from parasites originating in these different regions.
The assay could find application in the malaria research field in the specific context of assessments of antibody responses to a range of different parasite proteins that are, or have been, considered candidates for vaccine development but for which their extensive inherent allelic polymorphism has effectively negated such efforts.
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