Genetic diversity of Plasmodium knowlesi invasion-related proteins

In the present study, several potential P. knowlesi pre-erythrocytic and erythrocytic stage malaria vaccine candidates (PkRON2, PkTRAMP, PkSPATR, PkCSP, PkDBPαII, and PkNBPXa) will be studied for their genetic diversity and natural selection pressure.

The effects of genetic polymorphisms in these proteins on stimulation and regulation levels of immune responses in hosts (animal and human) will then be assessed.

Knowlesi-positive blood samples will be collected from patients in hospitals and field samplings. The targeted genes will be amplified by PCR. Sequencing analyses will be performed and the genetic diversity of each gene will be studied. Suitable haplotypes of each gene will be selected and cloned. Recombinant proteins of these haplotypes will be expressed and purified using a bacterial expression system. The purified recombinant proteins of the haplotypes will be used for animal immunization to study their effects on cytokine profile and antibody regulation. Raised rabbit antibodies will be purified, and human antibodies against each haplotype will also be affinity purified from infected patient sera. The protective level of these antibodies against malarial infection will be evaluated using merozoite invasion inhibition assay. The differences found between groups will be compared.

The findings in this present study can generate essential information on the function and immunogenicity of the selected proteins, and provide insights on the effect of antigen genetic polymorphisms in malarial vaccine design.

To investigate the genetic diversity and selection pressure of targeted genes PkRON2, PkTRAMP, PkSPATR, PkCSP, PkDBPαII, and PkNBPXa:

• Primers design and amplification of targeted genes

The oligonucleotide primers for targeted genes will be designed based on Plasmodium knowlesi strain H sequences obtained from GenBank (NCBI). PCR will be carried out using high fidelity DNA polymerase to obtain proofreading activity for accurate and effective amplification of long DNA amplicons.

• Nucleotide sequence analyses

Nucleotide sequencing on PCR amplicons will be performed by commercial laboratory (MyTACG Bioscience Enterprise, Malaysia) using respective primers. Nucleotide peaks for each sequence chromatogram will be examined to confirm for ambiguity, noise and double peaks. Sequences will be aligned using online-available alignment software CLUSTAL-Omega (http://www.ebi.ac.uk/Tools/msa/clustalo).

• Phylogenetic and sequence polymorphism analyses

MEGA 6 software will be used to construct a phylogenetic tree using the neighbour-joining method (Tamura et al. 2013). Bootstrap replicates of 1000 will be performed to evaluate the robustness of the tree. 

To evaluate the effect of genetic polymorphisms of targeted genes in host immune responses using animal model:
a) Selection of haplotypes in targeted genes
b) Construction of recombinant plasmids 
c) Expression and purification of recombinant proteins
d) Animal immunization
e) Measurement of cytokine levels in mice
f) Antibody characterization
g) Purification of antibodies from rabbit serum
h) Evaluation of protective level of raised antibodies using merozoite invasion inhibition assay

Plasmodium knowlesi A1H1 strain will be cultured in O+ human erythrocytes and RPMI-1640 complete medium supplemented with 10% horse serum at 37°C in a humidified atmosphere of 90% N2, 5% CO2 and 5% O2 (Moon et al. 2013). Daily monitoring will be performed. When parasites in culture are majority in late stages, synchronization will be performed by using histodenz to obtain schizont stage parasites. The synchronized parasites will be plated into 96-wells plate with 2% parasitemia and 3% haematocrit (180μL/well), and incubated with 20 μL of serial diluted raised antibodies (50 μg/mL to 3 mg/mL) at 37°C with 90% N2, 5% CO2 and 5% O2 for 5-10 hours (until all the schizonts in negative control well has developed into ring stage). Parasites in each well will be harvested and evaluated by microscopy examination. Thin blood smear will be prepared from each well and stained with Giemsa.
Ring stage parasites will be counted using microscope. Merozoite invasion inhibition efficiencies of each antibody concentration will be determined. Assays will be performed in triplicates for each antibody concentration. Data will be analysed using statistical software and significant difference between groups will be determined at p ≤ 0.05 significance level.

To evaluate the effect of genetic polymorphisms of targeted genes in humoral responses of human host:
a) Affinity purification of antibodies from patient serum
b) Evaluation of protective level of human antibodies using merozoite invasion inhibition assay

To develop an enhanced multi-host mathematical model to evaluate the dynamics of P. knowlesi in Malaysia:
A multi-host model for P. knowlesi will be modified by incorporating its transmission between human, macaque and mosquito. Using parameters, such as infectious period and mosquito biting rate, derived from literature, the extent to which infection is sustained by different host populations will be projected. The potential for sustained transmission within human-mosquito in the absence of the macaque population will also be assessed. Other spatial and geographical factors such as deforestation will be incorporated into the enhanced model empirically, aiding the authority in mitigating the widespread of P. knowlesi.

All these relevant data will be collected from other projects in the program.

Genetics and Genomics
Measurement of Transmission
Modeling

Jan 2019 — Dec 2022

Malaysia

A multi-pronged approach in combating knowlesi malaria

Plasmodium knowlesi