1. Introduction
Malaria infections caused by Plasmodium parasites continue to be the most important tropical disease that affects both animals and humans. Malaria was responsible for over 247 million cases and 619,000 deaths in 2021 (World malaria report 2022 , 2022).Plasmodium sporozoite infection is initiated when an infected female Anopheles mosquito probes for the blood meal and deposits sporozoites in the skin. Sporozoites migrate to the liver to invade hepatocytes and initiate a replication cycle that generates thousands of hepatic merozoites to initiate an erythrocytic cycle (Prudêncio et al. , 2006; Lindner et al. , 2012). During these replicative stages, the parasite rapidly increases its biomass, which requires many nutrients, including lipids, for growth. Lipids are the most abundant components of any living organism and are essential for cell development and division. They are not only important for membrane biogenesis but are also major signaling molecules necessary for invasive stage formation (Mazumdar and Striepen, 2007). Plasmodium parasites can either obtain lipids from the host or synthesize them de novo to maintain propagation and survival within the host (Tarun et al. , 2009; Ramakrishnan et al. , 2013). However, de novo fatty acid synthesis is only essential in liver stages and is dispensable for the asexual blood stage because these stages can survive by scavenging fatty acids from the serum (Yu et al. , 2008; Vaughan et al. , 2009).
In apicomplexan parasites, the type II fatty acid synthesis (FAS II) pathway is localized to the apicoplast and catalyzed by four key enzymes, FabB/F, FabG, FabZ, and FabI (Marrakchi et al. , 2002; Ralph et al. , 2004; van Dooren and Striepen, 2013).Plasmodium parasites are also capable of modifying de novo synthesized or scavenged fatty acids into long unsaturated fatty acids (Gratraud et al. , 2009; Ramakrishnan et al. , 2015). An endoplasmic reticulum (ER)-localized stearoyl-CoA desaturase (Scd) was identified in P. falciparum that catalyzes the formation of oleic acid from stearic acid by the insertion of a cis double bond at the Δ9 position of fatty acyl-CoAs (Shanklin et al. , 1994; Gratraudet al. , 2009). By using a Scd inhibitor, Gratraud et al. showed that methyl sterculate inhibits the synthesis of oleic acid and the development of asexual blood stage parasites (Gratraud et al. , 2009). Recently, Scd was found to be dispensable in a P. falciparum genetic screen (Zhang et al. , 2018); however, its role in mosquito and liver stages remains unknown.
The World Health Organization (WHO) recommended that the RTS,S vaccine (Zavala, 2022) might not be suitable for long-term usage because subunit vaccines occasionally do not elicit a strong or long-lasting immune response compared to whole-organism vaccines. Immunization with radiation-attenuated sporozoites (RAS) induces greater than 90% sterile protection in humans and has been the gold standard (Nussenzweiget al. , 1967). Repeated intravenous administration of RAS vaccines can achieve sterile protection in humans (Seder et al. , 2013). Immunization with live sporozoites attenuated by genetic modifications has gathered much attention, as they have been shown to produce protective immune responses equal to, or even greater than, those produced by irradiated sporozoites in rodent models (Khan et al. , 2012; Nganou-Makamdop and Sauerwein, 2013). GAPs offer several advantages over radiation-based attenuation, as they constitute a homogeneous population with a distinct genetic identity, and their attenuation is not dependent on external factors. Advances inPlasmodium genetics have enabled the generation of GAPs, which have overcome the limitations of RAS being used as a whole-organism vaccine. The obvious advantages of GAPs over RAS are that they are strategically generated by targeting genes, which are important for liver stage development, and that these KOs are clonal lines inducing a uniform immune response owing to their block at one particular stage, i.e., early to mid-liver stage (Mueller et al. , 2005b; Muelleret al. , 2005a) or late liver stage (Ishino et al. , 2009; Dankwa et al. , 2016). These studies were extended to human parasites, where the success of P. falciparum GAP was shown (Murphy et al. , 2022). The protection of these attenuated sporozoite vaccines involves antibodies elicited against sporozoite antigens that neutralize their ability to invade hepatocytes (Sederet al. , 2013). Moreover, the protection is mediated through CD8+ T-cell responses that target infected hepatocytes (Epstein et al. , 2011).
Immunization of mice with late-arresting parasites (Butler et al. , 2011) resulted in a superior immune response and protection compared with early-arresting GAP (Aly et al. , 2008). It was shown that immunization with P. falciparum sporozoites under drug coverage allows parasites to mature into the late liver stage and elicit durable protection at lower doses compared to the P. falciparumRAS sporozoites vaccine (Mordmüller et al. , 2017). These data suggest that late-arresting GAP will be a superior immunogen and an ideal whole parasite vaccine. Although the arrested parasites are a source of antigen for effective priming of the immune system, their antigenic repertoire induces cross-stage immunity only when parasites experience a block at the late liver stage. Since late liver stages exhibit a subset of antigens that are common to blood stages, identifying genes that can yield a late-arresting mutant will have a broader impact on developing an efficacious GAP vaccine. Here, by using a genetically tractable model of P. berghei , we show that Scd is expressed during the blood and liver stages and is localized to the ER. To evaluate Scd function in mosquito and liver stages, we disrupted the gene, which resulted in impaired late liver-stage development that failed to initiate blood-stage infection. Immunization with ScdKO sporozoites protects against infectious sporozoite challenges.