Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library

Abstract

Background: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (V_NAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.

Methods: In this study, new binders derived from shark V_NAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific V_NAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.

Results: The primary V_NAR domain library possessed a titre of 1.16 × 10⁶ pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.

Conclusions: Target-specific bacteriophage V_NARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as V_NAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

Fig. 1

End-titre of shark plasma against three malaria recombinant proteins over course of immunization. Shark plasma reacted against PfLDH (a), PfHRP2 (b), and PvAldolase (c). The baseline antibody response (negative control) was represented by pre-bleed (naïve sera) of wobbegong shark. The immobilized 1% BSA was used as negative antigen and reacted with serial diluted pre-bleed sera

Fig. 2

Selection and enrichment of malaria biomarker specific T7 phage after four rounds of biopanning. A plaque assay was performed to measure the size of eluted phage after each round of panning, and the size of amplified phage. The enrichment fold was calculated by dividing the eluted phage by input library size

Fig. 3

Competitive biopanning between 26 selected T7 specific clones. Twenty-six cloned selected from initial four rounds of biopanning against three different malaria biomarkers were pooled. This pool was tested competitively to select the specific clones, based on high frequency of appearance and absence of cross-reactivity. The colour bars represent the clones isolated from biopanning against different malaria antigen proteins (blue: anti-PfHRP2; red: anti-PfLDH; green: anti-PvAldolase). Yellow arrows point to four clones identified to be specific (H8 as an anti-PfHRP2 clone, P16 and P2–3 as anti-PfLDH clones, and A4–3 as an anti-PvAldolase clone)

Fig. 4

Deduced amino acid residues of four clones isolated after competitive biopanning. The PfHRP2-specific clone was represented by H8; The PfLDH-specific clones were P16 and P2–3; the PvAldolase-specific clone was A4–P4. All FR regions are shown by orange bar. The CDR regions are shown by red box. The canonical cysteine residues in FR1 and FR3 regions are highlighted in green. The non-canonical cysteine residues in CDR1 and CDR3 regions are highlighted in yellow background. The mutation sites in FR3 region of clone H8 is highlighted in pink background

Fig. 5

Analysis of deduced amino acids of V_NAR clones selected after four rounds of biopanning. a The number of cysteine residues and b represents the length of the CDR3 region encoded within the V_NAR clones

Fig. 6

Expression and purification of recombinant cytosolic H8VNAR using pET-28a vector. Proteins were separated on 15% SDS-PAGE and stained with Coomassie Blue (left panel). H8VNAR was detected by Western Blot with anti-His tag antibody and a ~ 14 kDa band clearly apparent. Lane 1 represents total protein from non IPTG-induced bacteria cells; lane 2 represents total protein from IPTG-induced bacteria cells; and lane 3 represents purified cytosolic H8VNAR protein

Fig. 7

Expression and purification of recombinant periplasmic DsbA-H8VNAR using pDSB-28Y vector. Proteins were separated on 15% SDS-PAGE and stained with Coomassie blue (left panel). DsbA-H8VNAR was detected by Western Blot with anti-His tag antibody and a ~ 14 kDa band clearly apparent. Lane 1 represents total protein from non IPTG-induced bacteria cells; lane 2 represents total protein from IPTG-induced bacteria cells; and lane 3 represents purified periplasmic DsbA-H8VNAR protein

Fig. 8

Comparison of binding efficiency of recombinant H8VNAR proteins with commercial mouse mAb C1–13 against tenfold serial diluted HRP2 proteins by ELISA. a The binding reactivity of purified cytosolic and periplasmic H8VNAR proteins was performed in a sandwich ELISA format. b The binding reactivity of positive control mAb C1–13 was performed in an indirect ELISA format. 1% BSA was used as negative antigen for rPfHRP2

Fig. 9

Comparison of binding reactivity of recombinant purified cytosolic and periplasmic H8VNAR against twofold serial diluted rPfHRP2 proteins in Dot Blot analysis. mAb C1–13 was a positive control. 1% BSA was a negative control