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. 2022 Feb 1;11:e76264. doi: 10.7554/eLife.76264

A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates

Justin YA Doritchamou 1, Jonathan P Renn 1, Bethany Jenkins 1, Almahamoudou Mahamar 2, Alassane Dicko 2, Michal Fried 1, Patrick E Duffy 1,✉
Editors: Lars Hviid3, Betty Diamond4
PMCID: PMC8959597  PMID: 35103596

Abstract

Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti-adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is known that naturally acquired antibodies target both variant-specific and conserved epitopes. It is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA variants. Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified ~0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. In two independent antibody purification/depletion experiments with permutated order of VAR2CSA variants, IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG. These findings demonstrate that a single VAR2CSA ectodomain variant displays conserved epitopes that are targeted by neutralizing (or binding-inhibitory) antibodies shared by multiple parasite strains, including maternal isolates. This suggests that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

Research organism: P. falciparum

eLife digest

Contracting malaria during pregnancy – especially a first pregnancy – can lead to a severe, placental form of the disease that is often fatal. Red blood cells infected with the malaria parasite Plasmodium falciparum display a protein, VAR2CSA, which can recognize and bind CSA molecules present on placental cells and in placental blood spaces. This leads to the infected blood cells accumulating in the placenta and inducing harmful inflammation.

Having been exposed to the parasite in prior pregnancies generates antibodies that target VAR2CSA, stopping the infected blood cells from latching onto placental CSA or tagging them for immune destruction. Overall, this makes placental malaria less severe in following pregnancies, and suggests that vaccines could be developed based on VAR2CSA.

However, this protein has regions that can vary in structure, meaning that P. falciparaum can generate many VAR2CSA variants. Individuals exposed to the parasite naturally generate antibodies that block a wide array of variants from attaching to CSA. In contrast, first-generation vaccines based on VAR2CSA fragments have only induced variant-specific antibodies, therefore offering limited protection against infection.

As a response, Doritchamou et al. set out to find VAR2CSA structures that could be recognized by antibodies targeting an array of variants. Blood was obtained from women who had had multiple pregnancies and were immune to malaria. Their plasma was passed over five different large VAR2CSA variants in order to isolate and purify antibodies that attached to these structures.

Doritchamou et al. found that antibodies binding to individual VAR2CSA structures could also recognise a wide array of VAR2CSA variants and blocked all tested parasites from sticking to CSA. While further research is needed, these findings highlight antibodies that cross-react to diverse VAR2CSA variants and could be used to design more effective vaccines targeting placental malaria.

Introduction

Plasmodium falciparum infection in pregnant women causes placental malaria (PM) when P. falciparum-infected erythrocytes (IE) accumulate in the intervillous spaces of the placenta. PM has been linked to several adverse pregnancy outcomes (Steketee et al., 2001; Guyatt and Snow, 2001; Desai et al., 2007; Moore et al., 2017), and first-time mothers are most vulnerable (Brabin, 1983). Over successive pregnancies, PM and the related sequalae become less prevalent (Guyatt and Snow, 2001). Susceptibility to PM has been attributed to P. falciparum parasites that bind chondroitin sulphate-A (CSA) expressed by the placental syncytiotrophoblast (Fried and Duffy, 1996), and express the variant surface antigen VAR2CSA (Salanti et al., 2003; Tuikue Ndam et al., 2005). Conversely, the decrease in PM-related poor pregnancy outcomes with increasing parity is associated with the acquisition of functional antibodies to CSA-binding IE (Fried and Duffy, 1998; Ricke et al., 2000) and antibodies to VAR2CSA (Salanti et al., 2004; Ndam et al., 2015). Such functional antibodies have been characterized for two major functions: (1) blocking CSA-binding of VAR2CSA-expressing parasites and (2) opsonizing IE to promote phagocytosis (Fried and Duffy, 1998; Ricke et al., 2000; Duffy and Fried, 2003; Keen et al., 2007; Ataíde et al., 2011).

Hence, VAR2CSA represents the leading candidate for PM vaccine development. VAR2CSA is a large (∼318–478 kDa) multidomain transmembrane protein, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family encoded by var genes (Gardner et al., 2002; Hviid and Jensen, 2015). The cysteine-rich ectodomain is formed by N-terminal sequence (NTS), six and sometimes more Duffy-binding-like (DBL) domains as well as interdomain (ID) regions (Kraemer and Smith, 2006; Doritchamou et al., 2019). Recent studies showed that VAR2CSA ectodomain structure includes a stable core (NTS-DBL1X-ID1-DBL2X-ID2-DBL3X-DBL4e-ID3) flanked by a flexible arm (DBL5e -DBL6e), and the receptor interaction involves CSA threading through two channels that formed within the stable core (Ma et al., 2021; Wang et al., 2021). Multiple individual DBL domains of VAR2CSA interact with CSA in vitro (Dahlbäck et al., 2011; Clausen et al., 2012; Ma et al., 2021) and induce functional antibodies in animals (Bigey et al., 2011; Fried et al., 2013; Nielsen and Salanti, 2015; Chêne et al., 2018). Two subunit vaccine candidates (called PAMVAC and PRIMVAC) from the N-terminal region of VAR2CSA were recently tested in phase one trials (Mordmüller et al., 2019; Sirima et al., 2020). Both trial teams reported that VAR2CSA subunit vaccines were safe, immunogenic and induced functional anti-adhesion antibodies in malaria-naive and malaria-exposed women (Mordmüller et al., 2019; Sirima et al., 2020). However, functional activity was primarily against homologous parasites (same VAR2CSA sequence as vaccine) and low or absent against heterologous parasites (Sirima et al., 2020), suggesting the vaccine antigen failed to display functional epitopes shared across multiple P. falciparum variants (reviewed in Doritchamou et al., 2021).

Similarly, VAR2CSA fragments (single or double domains) fail to purify the broadly neutralizing activity of sera from PM-resistant multigravidae (Doritchamou et al., 2016). Although VAR2CSA fragments (including constructs similar to PAMVAC vaccine) purified antibodies with CSA-binding inhibitory activity against homologous parasites, broadly neutralizing activity was neither purified nor depleted after passing sera over several VAR2CSA domains and variants (Doritchamou et al., 2016). These data supports the hypothesis that functional antibodies to VAR2CSA may target conformational epitopes not displayed by any individual domain: for example, the strain-transcending human monoclonal antibody PAM1.4 is specific for an unknown conformational epitope on full-length VAR2CSA (Barfod et al., 2007).

Naturally acquired antibodies of multiparous women exhibit broadly neutralizing anti-adhesion activity (Fried and Duffy, 1998; Ricke et al., 2000; Ndam et al., 2015). However, it is unknown whether broadly binding-inhibitory antibodies target conserved epitopes on VAR2CSA or rather result from the cumulative repertoire of antibodies against variant-specific epitopes. In this study, we investigate the hypothesis that full-length VAR2CSA ectodomain may capture naturally acquired antibodies from multigravidae plasma with greater breadth of neutralizing activity than previously achieved by multiple VAR2CSA domains. We show that a single ectodomain can purify strain-transcending IgG as well as the bulk of functional activity naturally acquired by multigravid women. In parallel, the depletion of IgG reactivities to five variants of full-length VAR2CSA resulted in significantly diminished strain-transcending anti-adhesion activity. These data suggest that full-length VAR2CSA ectodomain displays functional epitopes that are absent in subunit VAR2CSA fragments and provide a basis to design improved vaccines.

Results

Depletion of IgG on five VAR2CSA ectodomains significantly reduces broad neutralizing activity

IgG specific to full-length VAR2CSA ectodomains was purified from a plasma pool prepared using samples from multigravid women participating in the previously described Immuno-epidemiology (IMEP) study (Fried et al., 2018) (see Materials and methods and Figure 1 for experimental design).

Figure 1. Flow chart of the experimental design.

Figure 1.

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VAR2CSA-specific IgG was sequentially purified from total IgG isolated in multigravidae (MG) using antigen-specific columns made with NF54, 7G8, FCR3, HB3, Malayan Camp (M. Camp) alleles of full-length VAR2CSA ectodomain. The flow-through IgG as well as eluted IgG after each VAR2CSA column were assessed by ELISA, in Flow cytometry for surface reactivity and binding inhibition assay using CSA-binding Plasmodium falciparum-infected red blood cells (RBC). Image was created with https://BioRender.com.

The affinity purification of IgG specific to five VAR2CSA ectodomain variants yielded a combined ~386.1 µg of VAR2CSA-specific IgG, representing about 0.7% of the original total IgG (Table 1). The highest yields of purified IgG were obtained on the first two variants (NF54 and 7G8) totaling 76% of the IgG purified on all five variants.

Table 1. Purification yields of purified IgG.

Order of depletion IgG Yield
- Total IgG 58,000 μg
1 Fv2-NF54 IgG 145.6 μg
2 Fv2-7G8 IgG 148.9 μg
3 Fv2-FCR3 IgG 53.4 μg
4 Fv2-HB3 IgG 14.5 μg
5 Fv2-M. Camp IgG 23.7 μg
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Variable levels of IgG reactivity to seven recombinant VAR2CSA ectodomains was measured in the pre-depletion total IgG. As expected, IgG reactivity to NF54, 7G8, FCR3, HB3, and M. Camp variants were successfully depleted from the total IgG pool, as demonstrated by the absence of reactivity in each post-depletion sample (Figure 2A, Figure 2—figure supplement 1). Of note, substantially reduced reactivity to M920 and M200101 (not used in the affinity purification assay) was seen in the post-depletion total IgG, indicating that cross-reactive IgG to these two antigens was also depleted.

Figure 2. Depletion of VAR2CSA-specific IgG and activity of Total IgG purified from Malian multigravidae.

Activity of the total IgG before (Pre-depletion) and after (Post-depletion) purification of VAR2CSA-specific IgG was assessed by (A) ELISA on different recombinant full-length VAR2CSA ectodomain, (B) by Flow cytometry, and (C) CSA-binding inhibition assay using six isolates including three recently adapted maternal isolates (M0736, M2022170, and M2001190). In panel B, histogram without IgG represents assay well with no testing IgG and similarly stained with conjugated anti-human secondary IgG. In panel C, percent inhibition activity of purified IgG was determined relative to activity obtained with control wells without any testing IgG. Data in C represent at least two independent experiments. Asterisk (*) indicates antigens used in the sequential VAR2CSA-specific IgG depletion assay.

Figure 2—source data 1. ELISA activity of total IgG before and after purification of VAR2CSA-specific IgG.
Figure 2—source data 2. CSA-binding inhibition activities before and after purification of VAR2CSA-specific IgG.

Figure 2.

Figure 2—figure supplement 1. Reactivity of the post depletion samples to VAR2CSA recombinants and CSA-binding isolates.

Figure 2—figure supplement 1.

Reactivity of initial total IgG (MG total IgG) as well as flow through (FT) IgG samples collected after each VAR2CSA-specific column were assessed by ELISA (A) on seven recombinant full-length VAR2CSA ectodomain proteins. IgG were tested at 10 µg/mL concentration (IgG conc.). (B) Ability of MG total IgG and FT IgG to bind native VAR2CSA on CSA-binding isolates was evaluated by Flow cytometry. Testing IgG is indicated in red while histogram in blue represents assay well with no testing IgG and similarly stained with conjugated anti-human secondary IgG. Median fluorescence intensity (MFI) values are indicated. For maternal isolate M2022170, no data (ND) are available for FT samples collected after NF54, 7G8, FCR3, and HB3 due to limited material.
Figure 2—figure supplement 2. VAR2CSA sequences shared by progeny of 7G8 and GB4 crossing.

Figure 2—figure supplement 2.

VAR2CSA sequences of seven progenies of 7G8 x GB4 crossing were analyzed using the Consensus Protein Pileup (CPP) tool in R package ‘DuffyNGS’ as previously described (Doritchamou et al., 2019; Renn et al., 2021). Sequence similarity among the seven variants was measured as edit distance which was determined as the number of mismatched residues of amino acid, using R function ‘adist()’. Progenies including WF12 (gray row) sharing the same VAR2CSA sequence as 7G8 are highlighted in the red box, while those with GB4 sequence are in the purple box.
Figure 2—figure supplement 3. Percent reduction in CSA-binding inhibition activity of the MG-Pool after VAR2CSA IgG purification/depletion.

Figure 2—figure supplement 3.

Percent reduction in inhibition activity after depletion of reactivities to five variants of full-length VAR2CSA ectodomain is shown.
Figure 2—figure supplement 3—source data 1. Percent reduction in CSA-binding inhibition activity of the MG-Pool after VAR2CSA IgG purification/depletion.
Figure 2—figure supplement 4. Phylogenetic analysis of variants of full-length VAR2CSA sequences.

Figure 2—figure supplement 4.

Phylogenetic trees were generated using function ‘plot.phylo()’ from the R package ‘ape’. The NTS-DBL6 truncated sequence of the M200101 variant (NTS-DBL7) was also included in the analysis.
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Similarly, reactivity to native VAR2CSA measured on the IE surface by flow cytometry was depleted in the post-depletion IgG when tested on NF54, WF12/7G8, FCR3 and Malayan Camp (M. Camp) isolates (Figure 2B, Figure 2—figure supplement 1). Of note, WF12 (here named WF12/7G8) is a progeny of 7G8 X GB4 (Hayton et al., 2008) and its genome encodes the 7G8 VAR2CSA variant (Figure 2—figure supplement 2) Further, reactivity of post-depletion total IgG to native VAR2CSA of heterologous maternal isolates (M2022170, M2001190, and M0736) was substantially reduced versus the pre-depletion total IgG.

Importantly, depletion of IgG on the five VAR2CSA variants also reduced CSA-binding inhibition activity in the total IgG, being abolished against some isolates and significantly reduced against others (Figure 2C). The inhibition activity of the original pre-depletion total IgG sample was >65% against all parasite lines including recently adapted maternal isolates, except for FCR3 whose binding was inhibited 33% (Figure 2C). After the depletion of VAR2CSA-specific IgG, the inhibition activity was substantially reduced, with the % reduction (measured as (pre-depletion % inhibition-post-depletion % inhibition)/(pre-depletion % inhibition) x 100) ranging from 45% against M2022170% to 87% against NF54 (Figure 2—figure supplement 3). CSA-binding inhibition activity was substantially reduced against the three maternal isolates (including M2022170), implying that a significant fraction of cross-inhibitory IgG to the maternal isolates were also depleted on the heterologous VAR2CSA antigens.

Highly cross-reactive IgG is purified on the first full-length VAR2CSA antigen

VAR2CSA-purified IgG exhibited cross-reactivity in ELISA to different variants including M920 and M200101 (Figure 3A), although the degree of this dropped substantially after the first purification. As expected, reactivity was largely absent when IgG was tested against VAR2CSA variants previously used in the series of purifications/depletions. Thus, NF54-purified IgG (NF54-IgG) reacted to homologous and heterologous VAR2CSA antigens at the highest level of any IgG, while IgG purified on M. Camp (the last antigen used in the purification/depletion series) showed no cross-reactivity to NF54, 7G8, FCR3, or HB3 variants. Notably, each variant purified specific reactivity to itself, albeit at lower OD values than that seen with the initial NF54 purification, which may indicate the greater abundance and/or greater affinity of cross-reactive versus variant-specific IgG acquired by multigravidae.

Figure 3. Reactivity of the purified VAR2CSA-specific IgG to different full-length VAR2CSA antigens and P. falciparum isolates.

Figure 3.

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Reactivity of VAR2CSA-specific purified IgG was assessed by ELISA (A) on seven recombinant full-length VAR2CSA ectodomain proteins including two proteins (M920 and M200101) that were not used in the purification assay. IgG were tested at 0.1 µg/mL concentration (IgG conc.); asterisk (*) indicates depleted reactivity. (B) Ability of purified VAR2CSA IgG to bind native VAR2CSA on six CSA-binding isolates was evaluated by Flow cytometry. Testing IgG is indicated in red while histogram in blue represents assay well with no testing IgG and similarly stained with conjugated anti-human secondary IgG. Median fluorescence intensity (MFI) values are indicated. Due to limited material of purified IgG on HB3 VAR2CSA (HB3-IgG), no data (ND) are available for maternal isolates (M0736, M2022170 and M2001190). Asterisk (*) indicates surface reactivity to the homologous parasite.

Figure 3—source data 1. ELISA reactivity of VAR2CSA-specific purified IgG on 7 recombinant full-length VAR2CSA ectodomain proteins.

Similar to ELISA, flow-cytometry revealed strain-transcending reactivity to native antigen, although this decreased after the first (NF54) and again after the second (7G8) affinity columns. NF54-IgG showed the greatest breadth of reactivity (defined as the number of isolates recognized) with detectable reactivity to IE surface of all parasites including the maternal isolates (known to be polyclonal parasite samples). NF54-IgG also displayed the highest level of reactivity to all the isolates (Figure 3B). Compared to NF54-IgG, IgG subsequently purified on 7G8, FCR3, and M. Camp ectodomains demonstrated lower surface reactivity to the homologous parasite and lower cross-reactivity to the maternal isolates. Although the HB3-IgG was not tested against its homologous parasite line, this antigen-specific purified IgG displayed weaker cross-reactivity to different heterologous lab strain parasites compared to the other purified IgG. As with ELISA, reactivity by flow cytometry was lost against parasite isolates after their corresponding recombinant ectodomain had been used in the series of depletion/purifications, attesting to the complete depletion of homologous IE-surface reactivity by each homologous antigen.

A single full-length VAR2CSA variant purifies the bulk of naturally acquired binding-inhibitory IgG

Strong cross-inhibitory activity of IgG purified on VAR2CSA was observed in assays that measure inhibition of parasite binding to CSA (Figure 4—figure supplement 1). IgG binding-inhibition activity was normalized (nBIA) as a percentage of the pre-depletion total IgG, with the assumption that maximum inhibition of each parasite line is measured in IgG before depletion. NF54-IgG contained the largest fraction of the inhibitory activity, with mean nBIA values > 50% against all isolates and hovering around 100% for most (Figure 4A). This included three recently adapted heterologous maternal isolates, which showed both the lowest (M2022170) and highest (M2001190) nBIA levels respectively at 56% and 111% inhibition. Interestingly, titration of the inhibitory capacity of NF54-IgG against the homologous parasite indicated that blocking is achieved at IgG concentration as low as 10 µg/mL (Figure 4—figure supplement 2). 7G8-IgG showed lower levels of nBIA than NF54-IgG against all heterologous isolates, except against the maternal isolate M2022170 where 7G8-igG showed similar levels of nBIA to NF54-IgG; as expected 7G8-IgG (and subsequent variant-purified IgG) showed essentially no activity against NF54 parasites (Figure 4B–E). Subsequent purifications of IgG on ectodomain variants continued to yield some level of homologous activity, as seen with WF12/7G8, FCR3, and M. Camp (Figure 4C and E). Taken together, the IgG purified on VAR2CSA variants clearly demonstrated homologous inhibition activity while displaying high levels of cross-inhibition activity with initial purifications that generally waned with successive purifications. Despite significant heterologous IE surface reactivities captured on the NF54 antigen, subsequent IgG purifications on other variants captured some level blocking activity against both homologous and heterologous parasites (generally at a lower level than that of NF54-IgG).

Figure 4. CSA-binding inhibitory activity of VAR2CSA-specific purified IgG from multigravidae.

The inhibition capacity of VAR2CSA-specific purified IgG was assessed on CSA-binding isolates (A – E). Blocking activity (nBIA) of purified IgG was normalized by activity obtained with total IgG before any VAR2CSA IgG purification (Pre-depletion). Due to limited material, purified IgG on FCR3, HB3, and M. Camp was not tested on maternal isolates (M0736, M2022170, and M2001190). Black box indicates inhibition activity against homologous parasite and empty box represents activity against heterologous parasites. Data represent at least two independent experiments.

Figure 4—source data 1. Inhibition capacity of VAR2CSA-specific purified IgG against NF54.
Figure 4—source data 2. Inhibition capacity of VAR2CSA-specific purified IgG against 7G8.
Figure 4—source data 3. Inhibition capacity of VAR2CSA-specific purified IgG against FCR3.
Figure 4—source data 4. Inhibition capacity of VAR2CSA-specific purified IgG against HB3.
Figure 4—source data 5. Inhibition capacity of VAR2CSA-specific purified IgG against M. Camp.

Figure 4.

Figure 4—figure supplement 1. CSA-binding inhibitory activity of VAR2CSA-specific IgG purified from multigravidae.

Figure 4—figure supplement 1.

The inhibition capacity of VAR2CSA-specific purified IgG was assessed on CSA-binding isolates (A – E). Percent inhibition activity of purified IgG was determined relative to activity obtained with control wells without any testing IgG. Due to limited material, purified IgG on FCR3, HB3, and M. Camp was not tested on maternal isolates (M0736, M2022170, and M2001190). Black box indicates inhibition activity to the homologous parasite and empty box represents activities against heterologous parasites. Data represent at least two independent experiments.
Figure 4—figure supplement 2. Titration of homologous inhibition activity by purified VAR2CSANF54 specific IgG.

Figure 4—figure supplement 2.

Homologous inhibition activity of naturally acquired IgG specific to VAR2CSANF54 ectodomain was assessed by testing variable concentrations (100, 25, 10, 5, and 2.5 µg/mL). Data represent at least two independent experiments.
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To confirm our finding that IgG purified on the first VAR2CSA variant displays the highest level of activities against various VAR2CSA-expressing isolates, we permutated the order of antigens in a second sequential purification/depletion experiment, where IgG to M. Camp, FCR3, and NF54 variants of full-length VAR2CSA were sequentially purified (Figure 5—figure supplement 1). IgG purified on the initial variant (M. Camp) exhibited the highest cross-reactivity to heterologous variants of recombinant VAR2CSA (Figure 5A) and native antigen expressed by CSA-binding isolates (Figure 5B). In the functional assay, M. Camp-IgG strongly cross-inhibited the binding of M. Camp, FCR3 and NF54 parasites to CSA (Figure 5C, D and E). This observation is consistent with the pattern observed with IgG purified on NF54 variant of VAR2CSA in the first purification/depletion experiment.

Figure 5. Reactivity of the purified VAR2CSA-specific IgG to different full-length VAR2CSA antigens and P.

falciparum isolates in a second independent purification/depletion experiment. Reactivity of VAR2CSA-specific purified IgG was assessed by ELISA (A) on seven recombinant full-length VAR2CSA ectodomain proteins including four proteins (7G8, HB3, M920, and M200101) that were not used in this purification/depletion assay. IgG were tested at 0.1 µg/mL concentration. (B) Ability of purified VAR2CSA IgG to bind native VAR2CSA on six CSA-binding isolates was evaluated by Flow cytometry. Testing IgG is indicated in red while histogram in blue represents assay well with no testing IgG and similarly stained with conjugated anti-human secondary IgG. Median fluorescence intensity (MFI) values are indicated. Asterisk (*) indicates surface reactivity to homologous parasite. (C– E) Inhibition capacity of the VAR2CSA-specific purified IgG was assessed on three CSA-binding isolates. Black box indicates inhibition activity to the homologous parasite and empty box represent activities against heterologous parasites.

Figure 5—source data 1. ELISA reactivity of VAR2CSA-specific purified IgG.
Figure 5—source data 2. Inhibition capacity of VAR2CSA-specific purified IgG against M. Camp.
Figure 5—source data 3. Inhibition capacity of VAR2CSA-specific purified IgG against FCR3.
Figure 5—source data 4. Inhibition capacity of VAR2CSA-specific purified IgG against NF54.

Figure 5.

Figure 5—figure supplement 1. Reactivity of the post depletion samples to VAR2CSA recombinants and CSA-binding isolates.

Figure 5—figure supplement 1.

Reactivity of pre-depletion total IgG (T-IgG) as well as flow through (FT) IgG samples collected after each VAR2CSA-specific column was assessed by ELISA (A) on seven recombinant full-length VAR2CSA ectodomain proteins. IgG were tested at 10 µg/mL concentration. IE-surface reactivity of IgG evaluated by Flow cytometry is shown in panel (B). Testing IgG is indicated in red while histogram in blue represents assay well with no testing IgG and similarly stained with conjugated anti-human secondary IgG. Median fluorescence intensity (MFI) values are provided.
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Discussion

VAR2CSA is the leading vaccine candidate to protect malaria-exposed pregnant women against PM and related adverse outcomes. Two vaccines based on VAR2CSA fragments have been recently tested in clinical trials (Mordmüller et al., 2019; Sirima et al., 2020), showing good safety and immunogenicity but a general inability to induce broadly neutralizing antibodies, such as those described in sera of PM-resistant multigravid women (Fried and Duffy, 1998; Ricke et al., 2000; Duffy and Fried, 2003; Ndam et al., 2015). The limitations of VAR2CSA domain-based subunit vaccines have also been established from the studies of naturally acquired antibodies in PM-resistant multigravidae, where single domains or domain combinations from different variants failed to deplete heterologous binding-inhibition activity (Doritchamou et al., 2016). Among the probable explanations, the restricted array of protective epitopes displayed by VAR2CSA subunit vaccines, as well as the contribution of antibodies targeting other variants or antigens (including non-VAR2CSA proteins), are of major interest. It is therefore likely that larger fragments of VAR2CSA would offer a broader spectrum of functional epitopes including conformational epitopes and those present in other fragments missing from the subunits tested. Supporting this hypothesis, the present data demonstrate that strain-transcending naturally acquired anti-adhesion antibodies can be purified on a single recombinant VAR2CSA ectodomain, providing additional evidence that define VAR2CSA as the major target of functional antibodies.

This conclusion is well-supported by previous studies showing that naturally acquired anti-adhesion antibodies in PM target VAR2CSA (Bigey et al., 2011; Doritchamou et al., 2016), even though other invariant proteins are highly expressed by placental parasites (Francis et al., 2007; Fried et al., 2007; Tuikue Ndam et al., 2008; Bertin et al., 2013). Anti-adhesion activity of antibodies targeting these invariant proteins has not been reported thus far, and whether these proteins play a role in parasite binding to CSA is still unclear. Our data demonstrate that the removal of IgG specific to five variants of VAR2CSA ectodomain significantly reduced/abolished IE-surface reactivity and the CSA-binding inhibition activity of total IgG from multigravidae. This observation suggests that antibodies to surface-expressed non-VAR2CSA proteins might have limited or no blocking activity against CSA-binding parasites, although a synergistic effect of these antibodies with anti-VAR2CSA cannot be excluded.

Naturally acquired antibodies of malaria-exposed multigravid women recognize placenta-binding parasites from diverse geographical origins (Fried and Duffy, 1998; Ricke et al., 2000). One leading hypothesis is that pregnant women acquire antibodies to both cross-reactive and variant-specific epitopes over successive pregnancies and exposure to diverse placenta-binding parasites. We show here that the purification of IgG to VAR2CSA recombinants resulted in loss of IE surface reactivity against the three homologous parasites, and significantly reduced heterologous cross-reactivity of total IgG to maternal isolates. Consequently, the VAR2CSA-purified IgG exhibited strong cross reactivity to all isolates tested in this study, with IgG purified on the first variant demonstrating the highest level of cross-reactivity to diverse CSA-binding isolates. Of note, the isolates under study included WF12/7G8 that expresses the South American VAR2CSA 7G8 allele, in addition to isolates from South East Asia (M Camp), East Africa (M0736), and West Africa (NF54, M2022170, M2001190), thus representing geographically diverse origins. Overall, it may be difficult to estimate the fraction of variant-specific or cross-reactive antibodies contributed by each variant to the measured activities, knowing that each sequence of VAR2CSA could have multiple epitopes that are variably shared with some or all variants (Benavente et al., 2018; Otto et al., 2019). However, highly cross-reactive antibodies can be purified from a single recombinant VAR2CSA ectodomain, suggesting that immunization with a single variant may prove highly effective.

The full-length VAR2CSA ectodomain-based vaccine approach has been previously investigated in preclinical studies in which antisera raised in rodent models had strong homologous functional activity (Khunrae et al., 2010). However, these antibodies generated in rodents did not cross-inhibit different CSA-binding isolates, while exhibiting a broadly strain-transcending surface reactivity to these isolates (Avril et al., 2011). In contrast to this observation, our data demonstrate that naturally acquired IgG specific to a single variant and purified from multigravidae sera cross-inhibited multiple isolates. Notably, most of the binding-inhibition activity to CSA-binding parasites was depleted on the first variant and the resulting IgG purified on NF54 (in the first experiment) or M. Camp (in the second experiment) demonstrated strain-transcending blocking activity to all isolates tested in this study. Antibody response to VAR2CSA may differ between species or may differ based on exposure to vaccine antigen or to natural infection. For example, recently published data demonstrated that naturally acquired PfEMP1-specific antibodies are strongly afucosylated, while immunization with a subunit (VAR2CSA) vaccine results in fully fucosylated specific IgG (Larsen et al., 2021). Future studies that investigate differences in the host immune response to VAR2CSA in human versus other species will also elucidate any discrepancies between the functional activity of human versus rodent antibody.

Our data support the idea that one VAR2CSA variant might induce antibody with broad functional activity, acknowledging the challenges in developing a full-length VAR2CSA-based vaccine (reviewed in Doritchamou et al., 2021), including the possibility that non-functional epitopes may interfere with the optimal functional antibody response. It is also possible that the flanking flexible arm composed of DBL5ɛ–6ɛ or DBL5ɛ–7ɛ (in some cases) may play a role in masking functional epitopes on the core (NTS-ID3) structure of VAR2CSA (Doritchamou et al., 2019; Ma et al., 2021). Although recombinant expression of full-length VAR2CSA may not be feasible for manufacturing, novel technologies such as mRNA vaccine platform (reviewed in Pardi et al., 2018) should be explored in future studies. To conclude, we report that naturally acquired antibody purified on VAR2CSA ectodomains exhibited strong strain-transcending surface reactivity and blocking activity to CSA-binding isolates. Interestingly, IgG purified against a single variant captured the bulk of strain-transcending IE reactivity and CSA-binding inhibition activity from total IgG of PM-resistant multigravidae. Therefore, the full-length VAR2CSA ectodomain appears to display both variant-specific and shared epitopes targeted by functional antibodies and as such, would be a credible alternative to the VAR2CSA subunit approach in PM vaccine development. Moreover, cross-reactive antibodies could result from both conserved linear epitopes and conformational (tertiary structure related) epitopes, and the human monoclonal antibody PAM1.4 (Barfod et al., 2007) provides strong evidence of the latter. Although such antibodies may contribute to the broadly neutralizing activity of VAR2CSA-specific IgG described in this study, quantifying their relative contribution will inform future PM vaccine development.

Materials and methods

Key resources table.

Reagent type (species) or resource Designation Source or reference Identifiers Additional information
Antibody Goat anti-Human IgG Fc - Affinity Pure, HRP Conjugate (goat polyclonal) ImmunoReagents, Inc GtxHu-004-DHRPX 1:3000 dilution
Antibody Goat anti-Human IgG Fc Secondary Antibody, PE, eBioscience (goat polyclonal) Invitrogen, Carlsbad, CA 12-4998-82 1:800 dilution
Biological samples (Human) Plasma Malaria Research and Training Centre, Bamako, Mali, Fried et al., 2018
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant NF54 Patrick E Duffy, Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant 7G8 Patrick E Duffy, 
Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant FCR3 Patrick E Duffy, Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant HB3 Patrick E Duffy, Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant M920 Patrick E Duffy, Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant M. Camp Patrick E Duffy, Renn et al., 2021
Protein, recombinant protein Full-length VAR2CSA ectodomain, variant M200101 Patrick E Duffy, Renn et al., 2021
Strain, strain background (Plasmodium falciparum) NF54 Patrick E Duffy, Doritchamou et al., 2019
Strain, strain background (Plasmodium falciparum) FCR3 Patrick E Duffy, Doritchamou et al., 2016
Strain, strain background (Plasmodium falciparum) M.Camp Patrick E Duffy
Strain, strain background (Plasmodium falciparum) M0736 Patrick E Duffy, Doritchamou et al., 2016
Strain, strain background (Plasmodium falciparum) M2022170 Patrick E Duffy
Strain, strain background (Plasmodium falciparum) M2001190 Patrick E Duffy
Strain, strain background (Plasmodium falciparum) WF12 Thomas E Wellems, NIH, Hayton et al., 2008
Commercial assay or kit NHS-Activated Sepharose 4 Fast Flow GE Healthcare Life Sciences 17090601
Chemical compound, drug SYBR Green I Nucleic Acid Gel Stain - 10,000 X LifeTechnologies S7563 Used at 0.1 X
Software, algorithm Graphpad PRISM 9.0 GraphPad Software, Inc.
Software, algorithm FlowJo 10 Tree Star, Inc.
Software, algorithm R package ‘ape’ http://ape-package.ird.fr/
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Plasma pool, recombinant full-length VAR2CSA and P. falciparum cultures

For this study, a plasma pool was prepared using samples from multigravid women participating in the previously described Immuno-epidemiology (IMEP) study (Fried et al., 2018). Briefly, pregnant women aged 15–45 years without clinical evidence of chronic or debilitating illness were enrolled from 2010 to 2013 into a longitudinal cohort study of mother-infant pairs conducted in Ouélessébougou, Mali. All participants provided signed informed consent after receiving a study explanation form and oral explanation from a study clinician in their native language. The study protocol and study procedures were approved by the institutional review board of the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health (ClinicalTrials.gov ID NCT01168271), and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali. Aliquots of the plasma pools used in the IgG purification/depletion experiments are fractions of two larger stocks prepared by pooling up to 500 µl of plasma from two batches of 119 and 98 randomly selected multigravid women.

Recombinant full-length VAR2CSA ectodomains of seven genetically diverse P. falciparum parasite strains (NF54, 7G8, FCR3, HB3, M. Camp, M920, and M200101) (Figure 2—figure supplement 4) were expressed in a mammalian expression system as in a previous study (Renn et al., 2021). Three CSA-binding maternal isolates (M2022170, M2001190, and M0736) expressing VAR2CSA as well as four lab-strains (NF54, FCR3, M. Camp and WF12/7G8, a progeny of 7G8 X GB4 (Hayton et al., 2008) that encodes the 7G8 VAR2CSA variant) selected to bind CSA (Supplementary file 1) were maintained in culture for the functional assays.

Purification of IgG specific to full-length VAR2CSA ectodomains

Total IgG was initially purified from 4 mL of the multigravidae plasma pool using Protein G Sepharose 4 Fast Flow resin (GE Healthcare Life Sciences) according to the manufacturer’s instructions. For VAR2CSA-specific IgG purification and depletion, an antigen-specific affinity column was prepared as previously described (Doritchamou et al., 2016) for each VAR2CSA variant. Briefly, each ectodomain variant was chemically cross-linked to N-hydroxysuccinimide (NHS)-activated Sepharose beads (GE Healthcare Life Sciences) according to the manufacturer’s instructions. IgG specific to five VAR2CSA variants (NF54, 7G8, FCR3, HB3, and MC) were sequentially removed from the total IgG by passage through the affinity column and by applying the flow-through sample from one column to the next column until all specificities were depleted (Figure 1: Flow chart). IgG purification was performed repeatedly on each VAR2CSA antigen to ensure complete depletion of the specific IgG. The purified IgG were eluted with IgG Elution buffer, pH 2.8 (Invitrogen, Carlsbad, CA), neutralized with 2 M Tris buffer pH 9.0, and dialyzed into PBS pH 7.4. Variable number of passages were needed for full depletion of NF54 (x 3), 7G8 (x 3), FCR3 (x 2), HB3 (x 1) and M. Camp (x 1) specific IgG. The different aliquots of purified IgG were quantified using a Nanodrop (ND2000, Thermo Fisher Scientific, Waltham, MA).

Reactivity of the naturally acquired IgG to VAR2CSA recombinants and VAR2CSA-expressing isolates

Total IgG (before and after each variant-specific depletion) or VAR2CSA-specific IgG purified on each the five ectodomain variants was assessed by ELISA as previously described (Doritchamou et al., 2019). Briefly, recombinant VAR2CSA proteins were coated at 1 μg/mL and incubated overnight at 4 °C. Plates were blocked at room temperature (RT) for 2 hr (hr) and 100 µL of total IgG at 10 µg/mL and VAR2CSA-specific IgG at 0.1 µg/mL were added in duplicate wells for 1 hr at RT. The plates were washed and 100 µL of 1:3000 diluted HRP-conjugated anti-human IgG antibody (ImmunoReagents, Inc) added to each well for 1 hr incubation at RT followed by a final wash. One hundred microliters TMB (SeraCare) was added to each well for 10 min of incubation in dark at RT and the reaction was stopped by adding an equal volume of Stop Solution (SeraCare). Optical density (OD) values at 450 nm were acquired using the MultiskanFC (Thermo Fisher) plate reader.

IgG reactivity to native VAR2CSA expressed on IE surface was assessed by flow cytometry using 6 isolates of P. falciparum as previously described (Doritchamou et al., 2016). Briefly, enriched mature trophozoite/schizont stages of IE were incubated with total IgG (at 100 µg/mL) or the antigen-specific IgG (at 1 µg/mL), washed, and bound IgG labeled with PE-conjugated anti-human IgG (Invitrogen, Carlsbad, CA) in a buffer containing 0.1% SYBR (LifeTechnologies). IgG-labeled IE were quantified using an LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 10 (Tree Star, Inc). The median fluorescence intensity (MFI) was determined and background intensity from a well containing IE without immune IgG was subtracted from the MFI value detected for each test IgG.

Inhibition of IE binding to CSA

The CSA-binding inhibition activity of the purified IgG was evaluated in a static binding inhibition assay using immobilized CSPG receptor as previously described (Doritchamou et al., 2016). Briefly, the proteoglycan decorin (Sigma) at 2 µg/mL in 1 X PBS was coated as 15 µL spots on a 100 × 15 mm Petri dish (Falcon 351029) by overnight incubation at 4 °C in a humid chamber. The spots were blocked with 3% BSA in 1 X PBS at 37 °C for 30 min. Before binding assay, enriched mature trophozoite/schizont stages of IE from different P. falciparum strains were adjusted to 20% parasite density at 0.5% hematocrit and incubated with total IgG (at 1, 2, and 4 mg/mL) or VAR2CSA-specific IgG (at 0.1 mg/mL) for 30 min at 37 °C. IE suspended in IgG solution were then allowed to bind duplicate receptor spots for 30 min at RT. Unbound IE were washed away and bound IE were fixed, stained and quantified by microscopy. The percentage of inhibition was calculated relative to the wells containing IE without IgG.

Acknowledgements

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The authors thank J Patrick Gorres for editing the manuscript and Robert Morrison for bioinformatic analysis. We are grateful to women in Ouélessébougou, Mali for participation in the IMEP study. We also thank Thomas E Wellems for generously gifting us the WF12 parasite clone.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Patrick E Duffy, Email: patrick.duffy@nih.gov.

Lars Hviid, University of Copenhagen, Denmark.

Betty Diamond, The Feinstein Institute for Medical Research, United States.

Funding Information

This paper was supported by the following grants:

  • Division of Intramural Research, National Institute of Allergy and Infectious Diseases to Justin YA Doritchamou, Jonathan P Renn, Bethany Jenkins, Michal Fried, Patrick E Duffy.

  • National Institutes of Health to Justin YA Doritchamou, Jonathan P Renn, Bethany Jenkins, Michal Fried, Patrick E Duffy.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Conceptualization, Data curation, Formal analysis, Investigation, Supervision, Validation, Visualization, Writing - original draft.

Conceptualization, Investigation, Writing – review and editing.

Formal analysis, Investigation, Writing – review and editing.

Writing – review and editing, Conducted the clinical study in Mali and provided samples used in the study.

Writing – review and editing, Conducted the clinical study in Mali and provided samples used in the study.

Funding acquisition, Supervision, Writing – review and editing.

Conceptualization, Funding acquisition, Supervision, Writing – review and editing.

Ethics

Human subjects: For this study, a plasma pool was prepared using samples from multigravid women participating in the previously described Immuno-epidemiology (IMEP) study (Fried et al., 2018). Briefly, pregnant women aged 15-45 years without clinical evidence of chronic or debilitating illness were enrolled from 2010-2013 into a longitudinal cohort study of mother-infant pairs conducted in Ouélessébougou, Mali. All participants provided signed informed consent after receiving a study explanation form and oral explanation from a study clinician in their native language. The study protocol and study procedures were approved by the institutional review board of the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health (ClinicalTrials.gov ID NCT01168271), and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali.

Additional files

Supplementary file 1. CSA-binding level of the isolates.
elife-76264-supp1.docx (12.8KB, docx)
Transparent reporting form

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; source data files have been provided for all figures.

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Decision letter

Editor: Lars Hviid1
Reviewed by: Lars Hviid2, Stephen J Rogerson3

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for resubmitting the paper entitled "A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates" for further consideration by eLife. Your revised article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. We are sorry to say that we have decided that this submission will not be considered further for publication by eLife.

While all reviewers found your work of interesting to the community, they also agreed that the findings reported are too preliminary to fully justify the conclusions drawn. In particular, the lack of permutated depletion experiments was considering a critical shortcoming, and that substantial additional experimentation will be necessary to alleviate this concern and the other concerns raised and detailed below.

Reviewer #1:

This is a follow-up of a similar study by the same authors (Doritchamou, 2016). While they previously used recombinant single- and double-domain constructs of VAR2CSA to affinity-purify IgG from placental malaria-exposed multigravidae, they now employ full-length VAR2CSA ectodomain constructs. In contrast to the findings from their earlier study, the authors now report that affinity purification of IgG on full-length VAR2CSA yielded IgG that contained broadly neutralizing (adhesion-blocking) activity. The study reports what appears to be essentially a single sequential experiment that is consistent with the authors' interpretation, but in my opinion falls somewhat short of fully justifying it.

Reactivity and neutralization data of experiments all variants and single variant-depleted and single variant-purified IgG would strengthen the study markedly. Furthermore, it would be interesting to see results from experiments where the order of the sequential depletion was permutated. Finally, the authors should present absolute adhesion and adhesion inhibition data, as normalized data can be quite misleading (e.g., they can produce impressive adhesion inhibition data for a parasite line that shows almost no adhesiveness initially).

Specific comments:

– L. 15 ("…it is still unclear… epitopes"): The authors themselves have previously reported data supporting the existence of broadly cross-reactive IgG acquired in response to natural infection (Barfod et al. 2010).

– L. 25-8: I find this conclusion too categorical, considered the limited evidence (the above-mentioned "single" experiment and the fact that only a few maternal isolates – from the same study site and temporally related? – were tested).

– L. 41-2: Expression of VAR2CSA was not formally demonstrated in Fried 1998 or Ricke 2000 as both studies predate the discovery of VAR2CSA.

– L. 46-8 could deserve referencing – either original studies or a recent review (e.g., Hviid and Jensen 2015).

– L. 51-4: Suggest adding Wang et al. Nat Commun 2021.

– L. 54-8: Nine references seems a bit excessive here.

– L. 153-5 ("Although… NF54-IgG"): Why did the authors not test for IgG reactivity against native 7G8-VAR2CSA and HB3-VAR2CSA on the surface of IEs?

– L. 155ff ("As with ELISA… antigen"): How does this sentence fit with the fact that reactivity against 7G8-IEs was not tested?

Reviewer #2:

This work builds on both work from this team suggesting that antibodies to individual VAR2CSA domains do not show broad cross reactive neutralising activity, and on two phase 1 trials of vaccines based on the DBL2 domain of VAR2CSA and flanking sequences, which showed limited heterologous protection in in vitro studies. This work has led to the idea that it might be necessary to include a cocktail of key variants to produce a successful vaccine. The current authors instead investigated whether antibodies purified using the whole protein ectodomain were markers of potential heterologous protection, and find that this is the case.

Generally the experiments presented and the interpretations of the findings appear logical and consistent. The figures show that depletion of antibodies using one VAR2CSA variant decreases antibody reactivity to other variants by ELISA and that the more the plasma is depleted, the greater the loss of antibody reactivity. Broadly similar data are shown for the ability of plasma to inhibit adhesion of infected erythrocytes (IEs) to CSA, and for ability to recognise the IE surface using flow cytometry. The validation of their ELISA findings with live parasites is a strength of this study.

The results are consistent with the idea that immunity to a single VAR2CSA variant might be associated with a fairly broad range of cross-reactive antibody to different VAR2CSA isolates that might broadly protect against placental malaria. It is not presently clear whether production of such an antigen at scale for use in vaccines is a realistic possibility, and this challenge might be mentioned in the discussion. It is also not clear whether immunisation with a single variant would result cross reactive immunity (recognition of an antigen by antibodies being different to an antigens ability to elicit antibodies). The results contrast with the animal immunisation studies mentioned in the discussion; however, the authors do not clearly discuss why this may be the case.

Specific suggestions

Line 84-87: should this be "that are absent in 'individual' vaccine fragments"? As this study does not confirm that all vaccine fragments together do not contain epitopes.

Lines 105 and 113-114: The data presented in figures 2 are different to those in supplementary figure 1. Therefore, the results from Figures in the main file and supplement should be described separately.

Line 263-7: We are missing details of how the ectodomains were expressed. There is a reference to Renn et al., JEM 20210848P which may be a submitted manuscript. This information is important in understanding how the protein expression was evaluated for conformation and folding, as it appears tertiary or higher-level structural considerations are important for understanding the difference between this study and previous efforts using single domains.

Discussion: Could the authors comment on whether it is that they have used a more complete protein or whether it is the tertiary structure which results in the cross-reactive antibodies? Would the use of for example 6 fragments (making up the whole VAR2CSA) give a similar result?

Figure 2 A: what do the brackets and asterisk refer to?

Figure 2 C: There is a comment about "histogram without IgG" – what does this refer to? Does it refer to B rather than C?

Figure 2 D may be redundant as it can be derived from figure 2C.

Figure 3 X axis should not be VAR2CSA, it is antibody reactivity to IEs.

Line 179- should this be 'heterologous parasites' not homologous as it is clear that other purifications did indeed capture IgG that could block activity against heterologous parasite strains (though not at the same level as that of the first depletion).

Line 202: what is an 'invariant protein'?

Methods:

Line 279-281 More detail of the elution buffer is also needed as low pH can cause changes to antibody conformation that might alter protein binding.

Lines 290,298-9 please provide details of the reagents used including detection antibodies and SYBR.

How many times did plasma need to be passed through column before activity was deemed fully depleted?

Reviewer #3:

1) It has been shown in a previous work (in particular by the first author of this work), that despite exposure to several field P. falciparum isolates, multigravid women could nevertheless be susceptible to placental infections by certain rare parasite variants (resembling in their var2csa sequence to strain WRO80). The authors should have discussed their findings in relation to these very relevant observations from this previous study?

2) Including laboratory strains 7G8 and HB3 in the cellular testings (surface reactivity and binding inhibition) would have helped to substantiate the authors' conclusions. The same applies to parasite isolates M920 and M200101 from which the FL var2csa recombinants have been produced.

3) The order of filtration of the MG IgG pool on the antigens during depletion / purification steps seems decisive. If this order had been different would the authors have expected the same activity pattern of the purified antibodies? It would have been interesting to reproduce this experiment by reversing this order. Carrying out this experiment in my opinion would have further strengthened the study and better reflect the title chosen by the authors. One can wonder whether all the VAR2CSA variants used in this study would be functionally comparable in this operation, if not the title of this study would be better suited to IgG purified on the VAR2CSA variant of NF54, as the depletion / purification activity on this protein (first in the series) presents the least bias compared to the others used afterwards.

4) Despite these comments which could only further help improve this work if addressed, the content is already of definite interest and may be published or with some few modifications.

5) Adding a figure (even as a supplementary) that shows the dispersion or phylogenic relation between the 7 VAR2CSA sequences used in this work would have been a good added value, as this will inform about their relationship with the main variants (3D7, FCR3, 7G8, WRO80) and help to further interpret the results.

6) The total IgGs purified from the MG pool show a strong reactivity against the 7 recombinants VAR2CSA used as well as on the native protein of the cultured parasites. This surface reactivity of the pre-depletion IgG pool is surprisingly less on the strain NF54 (Figure 2B). In Figure 3 the affinity-purified IgGs on the NF54 antigen (the first in the chain of depletion/purification) also show strong reactivity against the 7 recombinants used as well as on the native protein of all the 6 parasites cultured, including NF54. The authors should explain or discuss this weak recognition of the pre-depletion pool on the NF54 parasite which contrasts with the inhibitory properties observed (Figure 2C)?

7) Authors should describe how the plasma pool was prepared. It would be helpful to clarify how many multigravid women have been used as donors? If they were selected it would be necessary to clarify on which criteria and if not why.

eLife. 2022 Feb 1;11:e76264. doi: 10.7554/eLife.76264.sa2

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

This is a follow-up of a similar study by the same authors (Doritchamou, 2016). While they previously used recombinant single- and double-domain constructs of VAR2CSA to affinity-purify IgG from placental malaria-exposed multigravidae, they now employ full-length VAR2CSA ectodomain constructs. In contrast to the findings from their earlier study, the authors now report that affinity purification of IgG on full-length VAR2CSA yielded IgG that contained broadly neutralizing (adhesion-blocking) activity. The study reports what appears to be essentially a single sequential experiment that is consistent with the authors' interpretation, but in my opinion falls somewhat short of fully justifying it.

In an additional set of experiments, we have now reproduced our findings in studies in which we changed the order of VAR2CSA antigens used for the sequential purification/depletion of IgG. Our data confirm that antibody purified on the first antigen exhibits broadly neutralizing activity, and this is not dependent on the VAR2CSA variant used for initial purification.

Reactivity and neutralization data of experiments all variants and single variant-depleted and single variant-purified IgG would strengthen the study markedly. Furthermore, it would be interesting to see results from experiments where the order of the sequential depletion was permutated. Finally, the authors should present absolute adhesion and adhesion inhibition data, as normalized data can be quite misleading (e.g., they can produce impressive adhesion inhibition data for a parasite line that shows almost no adhesiveness initially).

We thank the reviewer for these suggestions. Unfortunately, our lab does not have both 7G8 and HB3 isolates. However, in this revised manuscript, we have included data generated on WF12, a progeny of 7G8 X GB4 whose genome encodes the 7G8 VAR2CSA allele (Hayton et al., Cell Host Microbe 2008). We have provided detailed information on WF12 in Supplemental Figure S2. We have also permutated the order of antigens in a second sequential depletion experiment using full-length VAR2CSA (FV2) representing Malayan Camp (MCamp), with FCR3 and NF54 variants (in that order) for purification/depletion. In our additional data, we show that IgG purified on FV2 MCamp had the highest cross-reactivity and cross-inhibitory activity against recombinant FV2 variants as well as against native antigen expressed by MCamp, FCR3 and NF54 parasites (Figure 5). This finding is consistent with the pattern observed with IgG purified on NF54 variant in the first sequential depletion experiment in our original manuscript. We have provided average CSA binding level for the isolates used in the BIA experiments as Table S1.

– L. 25-8: I find this conclusion too categorical, considered the limited evidence (the above-mentioned "single" experiment and the fact that only a few maternal isolates – from the same study site and temporally related? – were tested).

As noted above, we have now generated new data, using a different order of FV2 variants for antibody purification/depletion on the multigravidae plasma pool, and depleting initially with a west African variant (NF54) in the first set of experiments and in the second set of experiments depleting initially with southeast Asian variant (M Camp). We have incorporated an additional parasite (that expresses the South American VAR2CSA 7G8 allele), in addition to the isolates from SE Asia (M Camp), east Africa (M0736) and west Africa (NF54, M2022170, M2001190) reported in the original manuscript. We believe that the additional data from the second sequential depletion experiment and the expanded set of parasites support our conclusions.

– L. 41-2: Expression of VAR2CSA was not formally demonstrated in Fried 1998 or Ricke 2000 as both studies predate the discovery of VAR2CSA.

These two references referred to antibodies that bind to placental IE. The sentence has been modified for clarity (lines 43-44).

– L. 46-8 could deserve referencing – either original studies or a recent review (e.g., Hviid and Jensen 2015).

As suggested, references have been added (lines 50-51).

– L. 51-4: Suggest adding Wang et al. Nat Commun 2021.

The suggested reference has been added (line 57).

– L. 153-5 ("Although… NF54-IgG"): Why did the authors not test for IgG reactivity against native 7G8-VAR2CSA and HB3-VAR2CSA on the surface of IEs?

As indicated above, our lab does not have both 7G8 and HB3 isolates. We have now tested the purified IgG on WF12, a progeny of 7G8 X GB4 that expresses the same native VAR2CSA as South American 7G8 parasite.

Reviewer #2:

This work builds on both work from this team suggesting that antibodies to individual VAR2CSA domains do not show broad cross reactive neutralising activity, and on two phase 1 trials of vaccines based on the DBL2 domain of VAR2CSA and flanking sequences, which showed limited heterologous protection in in vitro studies. This work has led to the idea that it might be necessary to include a cocktail of key variants to produce a successful vaccine. The current authors instead investigated whether antibodies purified using the whole protein ectodomain were markers of potential heterologous protection, and find that this is the case.

Generally the experiments presented and the interpretations of the findings appear logical and consistent. The figures show that depletion of antibodies using one VAR2CSA variant decreases antibody reactivity to other variants by ELISA and that the more the plasma is depleted, the greater the loss of antibody reactivity. Broadly similar data are shown for the ability of plasma to inhibit adhesion of infected erythrocytes (IEs) to CSA, and for ability to recognise the IE surface using flow cytometry. The validation of their ELISA findings with live parasites is a strength of this study.

The results are consistent with the idea that immunity to a single VAR2CSA variant might be associated with a fairly broad range of cross-reactive antibody to different VAR2CSA isolates that might broadly protect against placental malaria. It is not presently clear whether production of such an antigen at scale for use in vaccines is a realistic possibility, and this challenge might be mentioned in the discussion. It is also not clear whether immunisation with a single variant would result cross reactive immunity (recognition of an antigen by antibodies being different to an antigens ability to elicit antibodies). The results contrast with the animal immunisation studies mentioned in the discussion; however, the authors do not clearly discuss why this may be the case.

We thank the reviewer for these comments, and we have made revisions and clarifications to our manuscript to address them. We share the reviewer’s concern about the realistic possibility to produce such a large antigen at scale for use in vaccines. However, we believe the mRNA vaccine platform (among other advanced technologies) could be a feasible approach to explore and have discussed this in the revised manuscript (Lines 272-275). We also recognized the limitation of antibodies induced against FV2 in animals to display broadly neutralizing activity, similar to what is seen in naturally acquired immunity. It is possible that differences in host responses and context of the antigen may alter quality of immunity. For example, recently published data demonstrated that naturally acquired PfEMP1-specific antibodies are strongly afucosylated, while immunization with a subunit (VAR2CSA) vaccine results in fully fucosylated specific IgG (Larsen et al., 2021). This study provides new understanding of key differences in antibody features between natural- and vaccine-induced immunity. The Discussion section has been amended with this new observation in the revised manuscript (lines 258-264).

Lines 105 and 113-114: The data presented in figures 2 are different to those in supplementary figure 1. Therefore, the results from Figures in the main file and supplement should be described separately.

Figure 2 and Supplementary Figure S1 are both used to demonstrate depletion of reactivity to VAR2CSA antigens and parasites from total IgG. However, Supplementary Figure S1 shows reactivity after each individual antigen-specific IgG purification/depletion, while Figure 2 only displays the difference between Pre-depletion IgG versus IgG post-depletion by all variants used.

Line 263-7 We are missing details of how the ectodomains were expressed. There is a reference to Renn et al., JEM 20210848P which may be a submitted manuscript. This information is important in understanding how the protein expression was evaluated for conformation and folding, as it appears tertiary or higher-level structural considerations are important for understanding the difference between this study and previous efforts using single domains.

That is an interesting point, and the reference has been updated in this revised version of the manuscript. We cite our new publication (Renn et al., Comms Bio 2021, DOI : 10.1038/s42003-021-02787-7) that provides a detailed description of the expression and functional characterization of full length VAR2CSA ectodomain variants (lines 301-306).

Discussion: Could the authors comment on whether it is that they have used a more complete protein or whether it is the tertiary structure which results in the cross-reactive antibodies? Would the use of for example 6 fragments (making up the whole VAR2CSA) give a similar result?

At the current state of understanding, it appears that cross-reactive antibodies could result from both conserved linear epitopes and conformational (tertiary structure related) epitopes. The strain-transcending human monoclonal antibody PAM1.4 provides strong evidence of a strain-transcending antibody targeting conformational epitopes on full-length VAR2CSA (Barfod et al., 2007). Although such antibodies may be contributing to the broadly neutralizing activities of PM IgG described in this study, quantifying their level of contribution will certainly provide crucial data to inform PM vaccine development. Similarly, whether the use of the 6 individual domains (making up the entirety of the VAR2CSA ectodomain) will give a similar result remains to be explored. The Discussion has been amended accordingly (lines 281-286)

Line 202: what is an 'invariant protein'?

Here, an “invariant protein” refers to a highly conserved protein across different variants of P. falciparum encoded by a single-copy gene. For example, we have a new publication that describes such an invariant protein on the surface of CSA-binding IE (Keitany et al., J Infect Dis. doi: 10.1093/infdis/jiab550).

Reviewer #3:

1) It has been shown in a previous work (in particular by the first author of this work), that despite exposure to several field P. falciparum isolates, multigravid women could nevertheless be susceptible to placental infections by certain rare parasite variants (resembling in their var2csa sequence to strain WRO80). The authors should have discussed their findings in relation to these very relevant observations from this previous study?

We thank the reviewer for this comment. We have indicated in this manuscript and Renn et al., 2021 that the VAR2CSA sequence of M920 has a similar ID1 motif as WR80. Our data here show that IgG purified on the first FV2 variant significantly cross-reacted with the M920 variant of VAR2CSA. Unfortunately, the M920 isolate was not available to us to generate any surface reactivity and binding inhibition data. At this point, we believe that our interpretation should be limited to the observed ELISA cross-reactivity to a WR80-like VAR2CSA.

2) Including laboratory strains 7G8 and HB3 in the cellular testings (surface reactivity and binding inhibition) would have helped to substantiate the authors' conclusions. The same applies to parasite isolates M920 and M200101 from which the FL var2csa recombinants have been produced.

We do not have 7G8 and HB3 strains but have included data generated on WF12 (a progeny of 7G8 X GB4 that expresses the same VAR2CSA sequences as 7G8 (Hayton et al., Cell Host Microbe 2008)) in this revised version of the manuscript. We believe that data generated from maternal isolates M2022170 (Mali), M2001190 (Mali) and M0736 (Tanzania, for which no VAR2CSA) protein was produced, have provided reasonably similar findings (substantial reduction of reactivities and inhibition after depletion of IgG specific to multiple variants of VAR2CSA) as those from M920 (parasite not available) and M200101 (fails to maintain expression of VAR2CSA in culture) would have provided.

3) The order of filtration of the MG IgG pool on the antigens during depletion / purification steps seems decisive. If this order had been different would the authors have expected the same activity pattern of the purified antibodies? It would have been interesting to reproduce this experiment by reversing this order. Carrying out this experiment in my opinion would have further strengthened the study and better reflect the title chosen by the authors. One can wonder whether all the VAR2CSA variants used in this study would be functionally comparable in this operation, if not the title of this study would be better suited to IgG purified on the VAR2CSA variant of NF54, as the depletion / purification activity on this protein (first in the series) presents the least bias compared to the others used afterwards.

We thank the reviewer for this suggestion and have carried on generating similar data from a second series of sequential FV2 IgG purifications on MCamp, FCR3 and NF54 variants, in that order. This time, IgG first purified on MCamp (SE Asia variant) displayed the highest level of cross-reactivities and cross-inhibition activity, similar to what was seen in our original experiment with IgG purified first on NF54 (west African variant). These new data support the idea that different full-length VAR2CSA variants used as a first antigen to deplete/purify IgG from multigravidae will produce comparable functional activities as seen with NF54 and MCamp.

5) Adding a figure (even as a supplementary) that shows the dispersion or phylogenic relation between the 7 VAR2CSA sequences used in this work would have been a good added value, as this will inform about their relationship with the main variants (3D7, FCR3, 7G8, WRO80) and help to further interpret the results.

A larger phylogenic analysis of VAR2CSA sequences including the 7 variants that were expressed is reported in our newly published paper that described the expression of these FV2 recombinants (Renn et al., 2021). We have also provided a phylogenetic tree with all the 7 variants of FV2 used in this study as Supplemental Figure S7. The NTS-DBL6 version of M200101 (NTS-DBL7) VAR2CSA sequence has been also included here as reference.

6) The total IgGs purified from the MG pool show a strong reactivity against the 7 recombinants VAR2CSA used as well as on the native protein of the cultured parasites. This surface reactivity of the pre-depletion IgG pool is surprisingly less on the strain NF54 (Figure 2B). In Figure 3 the affinity-purified IgGs on the NF54 antigen (the first in the chain of depletion/purification) also show strong reactivity against the 7 recombinants used as well as on the native protein of all the 6 parasites cultured, including NF54. The authors should explain or discuss this weak recognition of the pre-depletion pool on the NF54 parasite which contrasts with the inhibitory properties observed (Figure 2C)?

The lower reactivity of the pre-depletion total IgG (used at 100ug/mL) compared to the antigen-specific IgG (used at 1ug/mL) could be explained by the low concentration of VAR2CSA-specific IgG in the total IgG. Indeed, based on the antibody yield in Table 1, NF54-specific IgG might represent 1/400 fraction of the total IgG. In a tentative extrapolation of the Flow data, a level of surface reactivity similar to that of NF54-specific IgG might be obtained if pre-depletion total IgG was used at 400ug/mL.

7) Authors should describe how the plasma pool was prepared. It would be helpful to clarify how many multigravid women have been used as donors? If they were selected it would be necessary to clarify on which criteria and if not why.

The plasma pools used in this study are fractions of two larger stocks prepared by pooling up to 500ul of plasma from 119 (first purification/depletion experiment) and 98 (second purification/depletion experiment) randomly selected multigravid women. We have now provided this detail in the revised manuscript.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 2—source data 1. ELISA activity of total IgG before and after purification of VAR2CSA-specific IgG.
    elife-76264-fig2-data1.xlsx (8.9KB, xlsx)
    Figure 2—source data 2. CSA-binding inhibition activities before and after purification of VAR2CSA-specific IgG.
    elife-76264-fig2-data2.xlsx (9.2KB, xlsx)
    Figure 2—figure supplement 3—source data 1. Percent reduction in CSA-binding inhibition activity of the MG-Pool after VAR2CSA IgG purification/depletion.
    elife-76264-fig2-figsupp3-data1.xlsx (8.9KB, xlsx)
    Figure 3—source data 1. ELISA reactivity of VAR2CSA-specific purified IgG on 7 recombinant full-length VAR2CSA ectodomain proteins.
    elife-76264-fig3-data1.xlsx (9.2KB, xlsx)
    Figure 4—source data 1. Inhibition capacity of VAR2CSA-specific purified IgG against NF54.
    elife-76264-fig4-data1.xlsx (8.8KB, xlsx)
    Figure 4—source data 2. Inhibition capacity of VAR2CSA-specific purified IgG against 7G8.
    elife-76264-fig4-data2.xlsx (8.8KB, xlsx)
    Figure 4—source data 3. Inhibition capacity of VAR2CSA-specific purified IgG against FCR3.
    elife-76264-fig4-data3.xlsx (9.2KB, xlsx)
    Figure 4—source data 4. Inhibition capacity of VAR2CSA-specific purified IgG against HB3.
    elife-76264-fig4-data4.xlsx (8.7KB, xlsx)
    Figure 4—source data 5. Inhibition capacity of VAR2CSA-specific purified IgG against M. Camp.
    elife-76264-fig4-data5.xlsx (8.6KB, xlsx)
    Figure 5—source data 1. ELISA reactivity of VAR2CSA-specific purified IgG.
    elife-76264-fig5-data1.xlsx (9.2KB, xlsx)
    Figure 5—source data 2. Inhibition capacity of VAR2CSA-specific purified IgG against M. Camp.
    elife-76264-fig5-data2.xlsx (8.9KB, xlsx)
    Figure 5—source data 3. Inhibition capacity of VAR2CSA-specific purified IgG against FCR3.
    elife-76264-fig5-data3.xlsx (8.9KB, xlsx)
    Figure 5—source data 4. Inhibition capacity of VAR2CSA-specific purified IgG against NF54.
    elife-76264-fig5-data4.xlsx (8.9KB, xlsx)
    Supplementary file 1. CSA-binding level of the isolates.
    elife-76264-supp1.docx (12.8KB, docx)
    Transparent reporting form
    elife-76264-transrepform1.docx (246.3KB, docx)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting file; source data files have been provided for all figures.

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