Malaria remains a potentially fatal public health problem, resulting in high morbidity and mortality in tropical and subtropical regions. Recently, malaria control interventions, such as artemisinin-based combination therapy (ACT), insecticide-treated bed nets (ITNs), and other mosquito vector control strategies, have greatly reduced the incidence of malaria all over the world (O’Meara et al., 2010). However, the emergence of artemisinin derivative-resistant malaria parasites (Phyo et al., 2012; Uwimana et al., 2020) and insecticide-resistant mosquitoes (Ranson et al., 2011) has greatly hampered the effectiveness of ACTs and ITNs. Therefore, the development of a highly efficient malaria vaccine has been regarded as the most cost-effective tool for malaria control and elimination, and potentially even eradication (Crompton et al., 2010).

Malaria is caused by the infections of species in the Plasmodium genus. There are four human malaria parasites, namely, Plasmodium vivax (P. vivax), P. falciparum, P. ovale, and P. malariae. Of these, P. falciparum is the deadliest. A Plasmodium infection is initiated by sporozoite inoculation into the host skin by an infected female Anopheles mosquito. The sporozoites travel to the liver and infect a small number of hepatocytes, with a single sporozoite giving rise to tens of thousands of merozoites. Next, merozoites are released into the bloodstream when merosomes bud from an infected hepatocyte, and they invade red blood cells (RBCs), initiating blood-stage development. A merozoite can multiply up to 20-fold every 1–3 days in cycles of invasion, replication, and RBC rupture, which releases many infectious merozoites. Some of the asexual blood stages transform into sexual forms called as gametocytes, which can eventually move into the midgut of a blood-feeding mosquito and then develop into sporozoites in the salivary gland. The blood-feeding mosquito then injects these sporozoites into another human, thereby initiating a new human infection (Figure 1).

Thus, the Plasmodium life cycle involves the liver stage (pre-erythrocytic stage) and blood stage in mammals and the sexual-stage in its vector. The blood stage is the main cause of the clinical manifestations (ranging from mild to severe malaria), and a blood-stage vaccine aims to reduce mortality and morbidity in malaria patients. In contrast, the liver stage is clinically silent, and a liver-stage vaccine is promising for preventing malaria infection (Smith et al., 2012). The development of the parasite in mosquitoes is essential for malaria transmission. Therefore, a sexual-stage vaccine aims to block malaria transmission (Acquah et al., 2019; Figure 1). For each stage, two kinds of vaccines, namely subunit and whole-parasite vaccines, are being explored. As major challenges have been encountered regarding subunit malaria vaccines, whole-parasite vaccines have recently been regaining attention. Here, we discuss promising aspects regarding the development of a whole-killed blood-stage vaccine (WKV) that protects against all stages and perspectives on its optimization in the future.

In the past three decades, great efforts have been made to develop efficient subunit vaccines against the pre-erythrocytic, blood, and sexual-stages, but major challenges have been encountered. The dominant protective antigen of the pre-erythrocytic stage is the main surface protein of sporozoites, circumsporozoite protein (CSP), which plays an important role in sporozoite invasion of hepatocytes (Dame et al., 1984; Rathore et al., 2002; Kumar et al., 2006). The most advanced CSP-based subunit malaria vaccine is RTS,S/AS01. This vaccine consists of a large part of the CSP fused with hepatitis B virus surface antigen (HBV-S Ag) particle formulated with the potent liposomal adjuvant AS01 (Cohen, 1996; Casares et al., 2010). Phase 3 clinical trials showed that the vaccine’s protection efficacy was 50.4% against the first clinical episode of malaria and 45.1% against severe malaria in young children, but only 30.1% in infants (Agnandji et al., 2011, 2012). In 2015, the European Medicines Agency announced the adoption of a positive scientific opinion regarding the use of RTS,S in regions outside of the European Union, where malaria is a major problem (EMA, 2015). In 2019, the World Health Organization (WHO) recommended the large-scale pilot implementations of routine use of RTS,S in real-life settings, involving moderate-to-high malaria transmission in order to assess its protective benefits and safety (WHO, 2019). However, RTS,S/AS01 still does not meet the criteria for a licensable first-generation vaccine (50% efficacy lasting for ≥1 year; Crompton et al., 2010), as the vaccine efficacy was dropped to 28 and 18% at 3–4 years post-vaccination in children and young infants, respectively (Rts, 2015). Therefore, the main challenge of the use of RTS,S is to sustain high antibody concentrations to mediate durable protection.

Most blood-stage candidate antigens are essential invasion proteins of merozoites such as merozoite surface protein (MSP1) and MSP3, apical membrane antigen 1 (AMA1), and erythrocyte-binding antigen 175 (EBA-175). These blood stage malaria vaccine candidates seek to induce high titers of plasmodium specific antibody that inhibit erythrocyte invasion by merozoites or limit parasite replication in red blood cells. However, most of the vaccine candidates exhibit extensive polymorphism between malaria strains from different geographical regions, and immunization with these vaccines only led to partial protection against vaccine-like strains (Bull et al., 1998; Sutherland, 2007; Fowkes et al., 2010). In addition, the RBC invasion pathways of merozoite exhibit redundancy, with the blockade of one invasion pathway failing to confer protection against challenge (Ogutu et al., 2009; Sagara et al., 2009; Sirima et al., 2011). The leading vaccine candidates AMA1 did not provide significant protection against clinical malaria in vaccine trials, and an MSP3 vaccine in Burkinabe children also demonstrated short-term protection (Sirima et al., 2011; Thera et al., 2011). At present, the most promising blood-stage candidate antigen is P. falciparum reticulocyte-binding protein homolog 5 (PfRH5), which interacts with basigin (CD147) on the RBC surface during merozoite invasion (Wright et al., 2014). PfRH5 exhibits limited polymorphism, and PfRH5/basigin is essential for merozoite invasion of RBCs (Weiss et al., 2015; Volz et al., 2016). Pre-clinical studies showed that PfRH5 induced cross-strain neutralizing antibody (Douglas et al., 2011) and confers nearly complete protection against challenge with heterologous Plasmodium strains in Aotus monkeys (Douglas et al., 2015). Recent study has defined that the neutralizing and non-neutralizing epitopes of PfRH5 are both essential for red blood cell invasion by the merozoites, which will optimize the designation of PfRH5-based vaccine (Alanine et al., 2019). However, protection in monkeys required a high level of anti-PfRH5 IgG (an estimated 200 μg/ml), and there was modest or no boosting of vaccine-induced antibody by infection of the monkeys (Douglas et al., 2015). This may limit the efficacy and duration of protection conferred by a PfRH5-based subunit vaccine.

Leading candidate antigens related to the malaria sexual-stage, include P. falciparum Pfs230 and Pfs48/45, which are expressed by gametocytes, and Pfs25, which is exclusively expressed by zygotes and ookinetes. In phase 1 clinical trials, vaccines based on Pfs25 and its P. vivax ortholog Pvs25 have induced antibodies that block mosquito infection (Wu et al., 2008). However, substantial antibody levels were only achieved after four doses, and antibody levels rapidly waned after the final dose (Sagara et al., 2018). In addition, these antigens are cysteine-rich with multiple 6-cys domains and/or epidermal growth factor (EGF)-like domains, and it is difficult to prepare the properly folded recombinant protein (Barr et al., 1991).

The major issue regarding the current subunit malaria vaccines is that they always contain only a single or a small number of antigens, which do not induce broad-spectrum long-lasting immune responses. It is well known that Plasmodium encodes approximately 5,300 proteins, but only a few candidate antigens have been identified (Gardner et al., 2002; Doolan et al., 2003). Therefore, it is currently difficult to design highly efficient subunit malaria vaccines.

In contrast to the narrow antigen spectrum related to subunit vaccines, whole-parasite vaccines maximize the spectrum of antigens, greatly enhancing protective immunity, and they have attracted more attention in recent years (Tarun et al., 2008; Ting et al., 2008; Moorthy and Ballou, 2009; Aly et al., 2010; Demarta-Gatsi et al., 2016; Mordmuller et al., 2017; Table 1). Attenuated sporozoites are the most promising vaccines against the pre-erythrocytic stage. It has been reported that either irradiation-, genetic-, or chemo-attenuated sporozoites (also called as chemoprophylaxis with sporozoites, CPS) could induce sterilizing protective immunity against homologous sporozoite challenge in both mice and humans (Nussenzweig et al., 1967; Mueller et al., 2005; Roestenberg et al., 2009; Epstein et al., 2011; Bijker et al., 2013; Seder et al., 2013). An irradiation-attenuated sporozoite vaccine could also induce a strain-transcending T cell response and durable protection against heterologous controlled human malaria infection (CHMI; Lyke et al., 2017). PfSPZ vaccine was confirmed to be extremely well tolerated and showed the significant protection in Mali adults against P falciparum infection in phase 1 trial (Sissoko et al., 2017). Furthermore, in the recent study by Roestenberg et al. (2020) in phase 1/2a trial, genetically attenuated malaria vaccine PfSPZ-GA1 also showed the modest protective immunity to mosquito-bite challenge. However, safety remains a great concern because the breakthrough of genetically attenuated sporozoites has been reported (Vaughan et al., 2010; Spring et al., 2013). Meanwhile, sporozoites can only be developed in mosquitoes, and sourcing and delivering aseptic, purified, and cryopreserved sporozoites limit the broad application of sporozoite vaccines (Prinz et al., 2018).

Compared to the sourcing of sporozoites, the culture of the Plasmodium falciparum blood stage is a well-established process, so whole-parasite blood-stage vaccines can easily be manufactured. Therefore, efforts have been made to develop genetically or chemically attenuated whole-parasite blood-stage vaccines. Attenuated P. yoelii (Plasmodium yoelii) with purine nucleoside phosphorylase (PNP), nucleoside transporter 1 (NT1), or histamine-releasing factor (HRF) deficiency confer complete sterile protection against both homologous and heterologous blood-stage challenge (Ting et al., 2008; Aly et al., 2010; Demarta-Gatsi et al., 2016). Regarding chemical attenuation, the blood stage can be chemically attenuated both in vivo and in vitro. In vivo, this has been achieved by creating a persistent subpatent blood-stage infection using a low dose of malaria parasite followed by treatment with anti-malaria drugs, which induces sterile protection against both high-dose homologous and heterologous blood-stage challenge (Pombo et al., 2002; Elliott et al., 2005). In vitro, several approaches have been used to chemically attenuate the blood stage, such as treatment with the parasite DNA-binding drugs centanamycin (CM; Good et al., 2013) and tafuramycin-A (TF-A; Stanisic et al., 2018), or the delayed death-causing drugs doxycycline or azithromycin (Low et al., 2019). The vaccination of in vitro chemically attenuated blood stage has also been reported to induce protective immunity against both homologous and heterologous blood-stage challenges (Good et al., 2013; Raja et al., 2016; Low et al., 2019). However, safety concerns regarding the breakthrough of genetically or chemically attenuated blood stages remains unavoidable. In contrast, a WKV, which is an inactive vaccine prepared by several cycles of freezinge/thawing of pRBCs (mostly containing schizonts), is theoretically much safer than other forms of whole-parasite blood-stage vaccine. Furthermore, our previous study and other research have demonstrated that the WKV induced strong protective immunity against both homologous and heterologous blood-stage challenge (Liu et al., 2013; Lu et al., 2017).

Most importantly, the protection brought about by genetically or chemically attenuated blood-stage vaccines involved both antibodies and CD4⁺ T cell responses (Good et al., 2013; Raja et al., 2016; Low et al., 2019). However, WKV with CpG as the adjuvant is dependent on CD4⁺ T cell responses, which might target the antigens that are conserved across strains (Pinzon-Charry et al., 2010). Thus, whole-parasite blood-stage vaccines, such as WKV, induce strong CD4⁺ T cell responses against universal epitopes, allowing them to confer species-transcending protection, unlike most subunit malaria vaccines against the polymorphic B cell epitopes.

It is thought that a subunit vaccine developed to induce a response against a certain stage of the malaria parasite induces stage-specific, but not cross-stage, immunity. In contrast, whole-parasite vaccines against the pre-erythrocytic stage, such as chemically and genetically attenuated sporozoites, have been shown to induce cross-stage protection against the blood stage (Nahrendorf et al., 2015b; Sack et al., 2015). Although the protection induced by chemically attenuated blood-stage P. yoelii parasites was stage specific as immunized mice were not protected against intravenous or mosquito bite sporozoite challenges (Raja et al., 2016), our study showed that the WKV not only conferred the cross-stage immunity against sporozoite challenge but also blocked parasite development in mosquitoes (Figure 2; Lu et al., 2017; Zhu et al., 2017). Therefore, the WKV not only prevents malarial infection and reduces mortality and morbidity among malaria patients but also blocks malaria transmission. Compared to the genetically or chemically attenuated blood-stage vaccines, the WKV is more acceptable and easily manufactured and shows promise regarding controlling and even eradicating malaria worldwide.

The main limitation of the WKV is the high dose required per vaccine due to its low immunogenicity. The immunization dose of WKV typically needs to be at least 1.5 × 10⁷ parasites or parasite equivalents per vaccine dose (Liu et al., 2013). Therefore, it is a significant challenge due to the difficulties in vaccine manufacture, which involves the human red blood cells. The use of human blood products may also have the possibility to contaminate the vaccine with infectious adventitious agents in the manufacturing process. In the final WKV formulation, an appropriate human-compatible adjuvant is required to induce protection with the lowest dose of parasite. Up to now, all the whole-parasite blood-stage vaccines are at a very early stage of clinical evaluation, and there is no WKV enters clinical trial for the assessment of safety and immunogenicity (Stanisic et al., 2018). Hence, the clinical evaluation of WKV remains a significant challenge. Additionally, the safety concerns regarding the WKV should also be considered, as the parasite is grown in human RBCs, and immunization with pRBCs may induce anti-RBC antibodies and possibly lead to autoimmunity following immunization (Good, 2011). Although no adverse events have been reported, the immunogenicity of the current WKV still needs to be improved. One way to potentially do this is to gain further understanding into the underlying mechanisms of the WKV against each of the three stages, and another is to develop human-compatible adjuvants.

Although the burden of malaria has been greatly reduced by control measures in the past decades, a highly efficient and safe vaccine is a high priority to ultimately control and even eradicate malaria around the world. Plasmodium is a complicated organism, encoding more than 5,000 proteins, but only a few protective antigens have been identified. Therefore, the current subunit vaccine strategy has encountered major challenges, as these vaccines can only induce narrow immune responses, and the future development of malaria subunit vaccine is not optimistic. Although multi-stage or multi-antigen vaccines are theoretically promising to control malaria, it is dependent on the identification of protective antigens. In contrast, the immune responses induced by whole-parasite vaccines are broad, sparking a renewed interest in these vaccines. Owing to the difficulties in sourcing and delivering cryopreserved sporozoites, whole-parasite blood-stage vaccine manufacturing is more feasible, as the culture of P. falciparum blood stage is a well-established process. Furthermore, the Plasmodium life cycle is very complicated, with three developmental stages, each of which has different antigens. An ideal vaccine would confer cross-stage immunity against all three stages, and WKV, an inactive whole-parasite vaccine, is safe and can induce cross-protection against both the liver and sexual-stages.

However, the immunogenicity of WKV is low and needs to be improved. To optimize WKV, two major elements should be identified: (1) malaria parasite components that positively regulate the host immune responses and (2) the mechanisms and parasite components that suppress or evade the host immune responses, which would affect the magnitude and duration of the protective immune response induced by WKV. With this knowledge, negative regulatory components could be removed and positively components could be strengthened from the parasite by genetic manipulation to greatly improve the immunogenicity and protective efficacy of the WKV.

WX and TL: conceptualization, writing-review and editing, and funding acquisition. JC, SC, XL, and FZ: writing-original draft. All authors contributed to the article and approved the submitted version.