Malaria, caused by five species of human Plasmodium, namely P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi is the most lethal human parasite infection. Of these species, infection with P. falciparum is the most prevalent and lethal, causing significant morbidity and mortality worldwide.. Rapid diagnostic tests (RDT) represent key components of current efforts to provide early diagnosis malaria and improve its control. Diagnostic biomarkers for malaria targeted in RDTs include histidine rich protein 2 (PfHRP2), a biomarker only found in P. falciparum infection; Plasmodium lactate dehydrogenase (pLDH), and fructose 1,6-biphosphate aldolase (Aldolase). These are predominantly used as candidate targets for detection infection with other Plasmodium species. Technical limitations of currently available malaria RDTs can impair the sensitivity of some tests. A possible explanation is the vulnerability of the conventional antibodies used in these kits to degrade due to harsh field conditions. Therefore, newly and more stable antigen binders represent important objectives in improving malaria RDT performance. Therefore, new and more stable antigen binders represent important objectives in improving malaria RDT performance.
Antibody phage display has been widely used as tool for the development of potent and stable monoclonal antibodies (mAbs) or fragments in diagnostic and therapeutic applications. In this technique, genes encoding the variable regions (VH and VL) of mAbs are displayed by bacteriophage, and then cloned into appropriate vectors thereby allowing the recombinant antibody fragments producion in either bacterial or mammalian expression systems. In this project, I sought to investigate new binders specific to malaria antigens from two libraries: (1) Naïve human scFv library, and (2) Immunized shark VNAR domains library.
Single chain variable fragment (scFv) are antibody fragments that consist of one heavy and one light chain V domain, typically linked to a short polypeptide linker. Owing to their small size (26–27 kDa), stability at 37 °C, and ease of expression, these molecules are becoming increasingly used for generation of high-affinity recombinant antibodies against different targets. In the first approach in this study, a naïve human scFv library (Sheets library) was used select binders targeting P. falciparum HRP2 protein. After five rounds of biopanning, 83 positive clones were identified. DNA sequence analysis showed that they belonged to two distinct groups, designated as D2 and F9.
Two representative clones were subcloned into a propriety antibody reformatting expression vector, mAbXpress for conversion to intact mAb that included a constant region at the C-terminus. The clones were subsequently transfected into the mammalian CHO cell expression system for recombinant mAb production. To verify the biological activity of these purified recombinant mAbs, functional characterization was undertaken, including binding specificity to recombinant PfHRP2 and malaria native proteins, sensitivity at different concentrations, heat-stability at various temperatures, quantification of binding affinities, and epitope mapping to PfHRP2 peptides. Binding properties and stability of these novel binders revealed both mAbs D2 and F9 are potentially working of further development.
In the second approach, I aimed to investigate alternate binders derived from shark antibodies, so called variable new antigen receptors (VNAR). VNAR are known to have excellent heat-stability, and their unique structure, entailing long CDR3 permits their penetration into cleft region of antigens. Following immunization of a wobbegong shark (Orectobolus ornatus) with three malaria biomarker proteins (PfHRP2, PfLDH and PvAldolase), and ensuring a positive antibody response was present, a single domain antibody (sdAb) library was constructed from splenocytes using a T7 phage vector system. VNAR cDNA was cloned into the library by PCR, and screened by biopanning against the target malaria biomarkers.
The results indicated that the primary library had a titre of 1.16 × 106 pfu/mL, with 55 % appearing to be the appropriate size (~450 bp). DNA sequence analysis showed that 82.5% of isolated fragments contained an in-frame sequence. The VNAR domains library constructed in this study was determined to be quality according to their diversity in CDR3 regions, isotypes of IgNAR family, and the position of canonical cysteine residues present in the conserved position of VNAR proteins.
After multiple rounds of biopanning, 26 clones were selected and verified by DNA sequence analysis. A PfHRP2-specific clone H8VNAR was selected for protein production in the E. coli pET expression system. Two types of protein expression systems were used: cytosolic and periplasmic. Further biological characterization of these proteins, including assays of the binding activities of purified H8VNAR to recombinant PfHRP2 protein indicated that it was slightly inferior to the positive control mAb C1-13.
In conclusion, the outcomes of this work may enable improving the stability of malaria RDTs and thus enhance the sensitivity and reliability of point of care diagnosis for malaria. Future approaches to improve the binding ability of these binders are discussed in the Chapter Six.