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.