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

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.

Trial registration: ClinicalTrials.gov NCT01168271.

Keywords: P. falciparum; VAR2CSA; blocking antibody; immunology; infectious disease; inflammation; malaria; microbiology; pregnancy; vaccine; variant.

Figure 1.. Flow chart of the experimental design.

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.

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

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.

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.

Percent reduction in inhibition activity after depletion of reactivities to five variants of full-length VAR2CSA ectodomain is shown.

Figure 2—figure supplement 4.. Phylogenetic analysis of variants of full-length VAR2CSA sequences.

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.

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

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

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 VAR2CSA_NF54 specific IgG.

Homologous inhibition activity of naturally acquired IgG specific to VAR2CSA_NF54 ectodomain was assessed by testing variable concentrations (100, 25, 10, 5, and 2.5 µg/mL). Data represent at least two independent experiments.

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

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.