Journal: Infection and Immunity

Article Title: Evaluation of the Antigenic Diversity of Placenta-Binding Plasmodium falciparum Variants and the Antibody Repertoire among Pregnant Women ▿ †

doi: 10.1128/IAI.01365-09

Figure Lengend Snippet: Polymorphic loops formed by variable sequence blocks of DBL3 were recognized by vaccine-induced and acquired antibodies. (A) Rabbit antiserum raised against the DBL1 or DBL3 domain of IT4 VAR2CSA was tested by ELISA for reactivity against synthetic peptides, which represent polymorphic loops of the DBL3 domain (IT4). Antibodies generated by DNA vaccination (aDBL3 DNA) recognized only loop 5, whereas antibodies generated by immunization with recombinant protein (aDBL3 Pp) reacted with IT4 DBL3 loops 5 and 3. Negative control sera against the VAR2CSA DBL1 domain were generated by DNA vaccination (aDBL1 DNA) or with recombinant protein (aDBL1 Pp) and did not react with either DBL3 loop. Subsequently, human sera from malaria-exposed Malawian (n = 72) and PNG (n = 56) women were tested for antibodies to IT4 DBL3 loop 5 peptide by an ELISA. The peptide was recognized by PG women, MG women, and several sympatric men from both countries, but not by Melbourne controls. Results for a representative selection of sera from Malawi (B) are shown. All women included were in the top quartile of responders to CS2 in FACS-based assays (see Table ​Table1).1). In panel B samples 27, 71, 98, and 161 were samples from MG women, samples 66 and 145 were samples from PG women, and sample c10 was a sample from a Melbourne control donor. All samples were run in duplicate; means and standard deviations are shown. FB, final bleed; PI, preimmunization serum; OD (405), optical density at 405 nm. (C and D) Identical or nearly identical variable block 5 (C) and variable block 3 (D) sequences could be identified for isolates from diverse geographic origins. Each group contains sequences from isolates from different geographic regions, and the sequences were grouped using sequences obtained from one of the placenta-binding isolates used in antibody studies (3D7-CSA, CS2/IT4, HCS3, XIE-CSA, HB3-CSA, Pf2004-CSA, and Pf2006-CSA). Sequence alignments were constructed on the basis of identity in the region corresponding to synthetic peptides used in immunoassays (A and B). Blue shading indicates the predicted loop (additional residues are also shown), and red shading indicates an amino acid change compared to the majority of the sequences. The overall sequence alignment is indicated by bars; red indicates a 100% match, while orange, green, and blue indicate increasing numbers of sequence differences. The origins of the parasites are as follows: CYK34.1, CYK37, and CYK42.1, Senegal; D10, PNG; MC, Malaysia; Mplc21-1, Malawi; PC49, Peru; T2C6, Thailand; XHA clones, PNG; XIE par (non-CSA-selected XIE clones), PNG; XJA clones, PNG.

Article Snippet: IT4 DBL3 vb3 (IIEKGTPQQKDKIGGVGS), vb4 (biotin-SGSGAITKINKKNNNSIFNGD), vb5 (TINGKNEKKCINSKSGQGDKIQGA, linear or biotin or BSA conjugated), and Dd2 DBL3 vb5 (INKDMEQNKCINSKNNNEKEIQGD) were synthesized by Mimotopes (Clayton, Victoria, Australia).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Generated, Recombinant, Negative Control, Selection, Control, Blocking Assay, Binding Assay, Construct, Clone Assay

Journal: Infection and Immunity

Article Title: Evaluation of the Antigenic Diversity of Placenta-Binding Plasmodium falciparum Variants and the Antibody Repertoire among Pregnant Women ▿ †

doi: 10.1128/IAI.01365-09

Figure Lengend Snippet: Globally diverse isolates share DBL3 variable block sequences. Analysis of VAR2CSA DBL3 variable block 3 and 5 amino acid sequences demonstrated that isolates having diverse geographic origins shared the same polymorphic loop sequences. A diagram of the DBL3 domain is shown at the top. The positions and lengths of variable blocks 1 to 5 in the DBL3 sequences are indicated. The locations of primers used for PCR amplification are indicated by arrowheads. The filled arrowheads indicate primers used to amplify PNG sequences in this study, and the open arrowheads indicate the locations of primers used previously to amplify sequences from Senegal isolates (1). The amino acid position of the primers is counted from the first amino acid in the IT4 VAR2CSA sequence (accession no. AAQ73926). The segmentation structures for variable blocks 3 and 5 in representative PNG sequences are shown below the diagram of the DBL3 domain (a full alignment of all sequences is shown in Fig. S1 in the supplemental material). Variable block 3 is composed of three segments, segments a, b, and c, and variable block 5 is composed of two segments, segments a and b. The locations of variable block peptides used for serological analysis are indicated under the alignments. PNG sequences that were identical to the IT4 VAR2CSA peptide sequences are indicated by filled circles.

Article Snippet: IT4 DBL3 vb3 (IIEKGTPQQKDKIGGVGS), vb4 (biotin-SGSGAITKINKKNNNSIFNGD), vb5 (TINGKNEKKCINSKSGQGDKIQGA, linear or biotin or BSA conjugated), and Dd2 DBL3 vb5 (INKDMEQNKCINSKNNNEKEIQGD) were synthesized by Mimotopes (Clayton, Victoria, Australia).

Techniques: Blocking Assay, Amplification, Sequencing

Journal: Infection and Immunity

Article Title: Evaluation of the Antigenic Diversity of Placenta-Binding Plasmodium falciparum Variants and the Antibody Repertoire among Pregnant Women ▿ †

doi: 10.1128/IAI.01365-09

Figure Lengend Snippet: Overlap and differences in distribution of polymorphic segments in variable blocks 3 and 5. The major segment types found in variable blocks 3 and 5 in the VAR2CSA DBL3 domain were quantified. A total of 124 DBL3 sequences were compared (16 sequences from Southeast Asia, 54 sequences from PNG, 43 sequences from Senegal, 8 sequences from other African countries, and 3 sequences from Central and South America [C/S America], as well as one sequence of unknown origin (3D7). (A) Major segment types of vb3 segment a. (B) Major segment types of vb3 segment b. (C) Major segment types of vb3 segment c. (D) Major segment types of vb5 segment a. (E) Major segment types of vb5 segment b. When data for PNG (n = 54) and Senegal (n = 43) were compared, there were statistically significant differences in the distribution of sequences in variable block 3 and 5 (P < 0.0001 for differences in the proportions of vb3 segment a and vb3 segment b sequences; P = 0.045 for differences in the proportions of vb3 segment c sequences; P = 0.061 and P = 0.023 for differences in the proportions of vb5 segment a and vb5 segment b sequences, respectively [Fisher's exact test]).

Article Snippet: IT4 DBL3 vb3 (IIEKGTPQQKDKIGGVGS), vb4 (biotin-SGSGAITKINKKNNNSIFNGD), vb5 (TINGKNEKKCINSKSGQGDKIQGA, linear or biotin or BSA conjugated), and Dd2 DBL3 vb5 (INKDMEQNKCINSKNNNEKEIQGD) were synthesized by Mimotopes (Clayton, Victoria, Australia).

Techniques: Sequencing, Blocking Assay