Recombinant Malaria PAN-pLDH

Plasmodium is the pathogen that causes malaria in humans. Studies have found that there are many types of Plasmodium in nature, and there are four types of Plasmodium that can parasitize humans, namely Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum. Further research on Plasmodium shows that the life cycle of Plasmodium can be divided into two stages according to the different hosts, namely the stage in the human body and the stage in the Anopheles mosquito. Studies have found that the infectious sporozoites parasitizing female Anopheles mosquitoes enter the human body with their salivary gland secretions when the Anopheles mosquito bites a person to suck blood, and quickly enter the liver through the blood circulation. In the liver cells, it takes 9-16 days for the merozoite to develop into a mature schizont. When the parasitized liver cells rupture, a large number of merozoites are released. They quickly enter the blood circulation, invade red blood cells, and begin the asexual reproduction cycle in red blood cells. After invading red blood cells, the merozoites develop into early trophozoites, i.e. ring forms, which develop into mature schizonts through trophozoites. The schizont contains several to dozens of merozoites. When the parasitized red blood cells rupture, the merozoites and metabolites are released, causing typical clinical malaria attacks. The merozoites in the blood then invade uninfected red blood cells, restarting a new round of asexual reproduction, forming clinical periodic attacks. The development cycle of Plasmodium vivax and Plasmodium ovale in red blood cells is about 48 hours. Malaria malariae is about 72 hours. The development cycle of falciparum malaria is 36-48 hours, and the development sequence is different, so the clinical attacks are also irregular. Plasmodium vivax and Plasmodium ovale have both tachysporozoites and bradysporozoites. Rapid-acting sporozoites develop quickly in liver cells and can develop into mature schizonts in just 12-20 days. Delayed-acting sporozoites develop slowly and need 6-11 months to develop into mature schizonts. Delayed-acting sporozoites, also known as hypnozoites, are the root cause of relapses of Plasmodium vivax and Plasmodium ovale. Malaria malariae and falciparum malariae do not have delayed-acting sporozoites, so there is no relapse. Some malarial parasite merozoites develop into female gametocytes and male gametocytes after 3-6 generations of proliferation in red blood cells. The survival time of gametocytes in the human body is 30 to 60 days. The mating and reproduction stage of malarial parasites in the body of Anopheles mosquito is the sexual stage. When the female Anopheles mosquito sucks blood, the gametocytes are sucked into her body and her sexual reproduction period begins. The female and male gametes develop into female and male gametes in the mosquito body, and the two combine to form a zygote, which then develops into an ookinete, which invades the intestinal wall of the Anopheles mosquito and develops into an oocyst. Each oocyst contains thousands of sporoblasts, which develop into infectious sporozoites. These sporozoites can actively migrate to the salivary glands of the Anopheles mosquito. When the Anopheles mosquito bites a human to suck blood again, the sporozoites enter the human body and continue their asexual reproduction cycle.

Figure 1. Simplified lifecycle of Plasmodium. (Sources: Fraser M, et al. 2021)

Lactate dehydrogenase (LDH) is an important functional enzyme widely distributed in microorganisms, plants and animal cells. It is one of the key enzymes in the anaerobic glycolysis pathway of intracellular carbohydrates. Studies have found that the energy source of most parasites in the body mainly comes from the anaerobic glycolysis pathway. LDH is the terminal enzyme of this metabolic pathway. It can catalyze the reversible reaction between pyruvate and lactate with the assistance of NADH and NAD+ and release energy. Therefore, once LDH is inhibited, the development of the worm will stop or even die. Studies have shown that parasite LDH is a good diagnostic molecule and a potential drug target. By studying the sequences of the LDH genes of Plasmodium vivax (P.vivax, Pv) and Plasmodium falciparum (P.falciparum, Pf), it was found that the full length of the coding region is 951 bp, encoding 316 amino acids, nucleotides and The homologies of the amino acid sequences are 75.1% and 90.2% respectively; the specific epitopes are 38 and 45 respectively; the LDH antigens of the two parasites have the highest inhibition rate against polyclonal antibodies, with Pv reaching 70.3%, while Pf is only 30.5%. In terms of diagnosis, since PLDH is an important circulating antigen that is ubiquitous in parasites and has species and genus specificity, it has important application value in the diagnosis of malaria. After the successful preparation of monoclonal antibodies for PfLDH and PvLDH, rapid detection methods such as PfLDH colloidal gold immunochromatographic test strips have been developed. In terms of drug targets, existing studies have found that PLDH and host LDH have great differences in biochemical, immunological properties and enzymology, and are considered to be potential drug targets. In view of the importance of PLDH in the energy metabolism of malarial parasites and the difference in the active site structure with host LDH, researchers have studied drugs that can inhibit the activity of PLDH and found that a variety of drugs can interact with PLDH. Research reports show that the antimalarial drug chloroquine can competitively bind to the NADH binding site on LDH, occupying a position similar to the coenzyme adenine ring, preventing the interaction between NADH and LDH, cutting off the glycolysis pathway, and thus causing the death of malarial parasites. Gossypol has a strong antimalarial effect. Its mechanism of action is to specifically inhibit the activity of PLDH and block the energy supply in the parasite, but its toxic side effects are too great and it has not yet been used as an antimalarial drug. Studies on drugs such as thioacridone, heme, and naphthalene compounds have also confirmed that these drugs have varying degrees of inhibitory effects on PLDH.

Pan-malarial pLDH
pLDH
Pan-pLDH
Pan-specific pLDH
Pan-malarial LDH
Malaria lactate dehydrogenase
Plasmodium LDH
Pan-malarial antigen