A major setback in malaria eradication is the emergence of drug resistance to existing antimalarials and insecticides, along with the frequent presence of asymptomatic and submicroscopic infections in endemic regions, underscoring the need for a highly effective malaria vaccine.

Ongoing efforts have been made to develop an effective vaccine for malaria control. All these efforts have provided a single recombinant vaccine RTS,S/AS01, which also has limitations in its effectiveness [31]. The natural protective immunity against malaria observed in adult individuals repeatedly exposed to the parasite in endemic regions is the basis for the malaria vaccine [32]. The major challenges accompanied by vaccine development are as follows. The extensive genetic variability/polymorphisms in many functional essential genes account for the change in the structure of proteins of a parasite, and this is the first and foremost problem in the identification of a vaccine candidate. Secondly, most of the proteins need to be functionally characterized to be targeted in vaccine studies [33]. In addition, most of the proteins are functionally redundant, so targeting a single protein would not disrupt their processes or the growth of the parasite. During infection, a parasite undergoes various morphological changes, accompanied by modifications in its proteome; therefore, it differentially provokes the effector mechanism, which may be either protective or not [34]. The differential expression of proteins at various stages thus makes it challenging to identify the actual targets that provide protective immunity against malaria [35]. Potential malaria vaccine candidates can target any of the three different stages—the hepatic/pre-erythrocytic stage, the blood stage/erythrocytic stage, or the sexual gametocyte stage (mosquito stage)—of the parasite life cycle [36].

A vaccine targeting the pre-erythrocytic stage is mainly aimed at hampering the invasion process and suppressing the sporozoites’ growth at the hepatic stage, thereby ultimately preventing the disease pathology by limiting the parasites from invading erythrocytes. The initial pre-erythrocyte vaccine trial included immunization with live attenuated sporozoites. In different Phase I trials, vaccination in humans with the irradiated sporozoites affirmed the protection against subsequent infection with P. falciparum [36,37]. In contrast, the attenuation of irradiated sporozoites is based on multiple random mutations occurring in sporozoites that ultimately lead to a blockage in liver stage development [38]. The reverse genetics approach has been used to delete the vital pre-erythrocytic stage genes, such as UIS3, UI4, and p36, causing the transgenic parasite’s sporozoites to lose the ability to mature into liver-stage merozoites [39]. The UIS3-deleted sporozoite immunized in three consecutive doses showed protection in the rodent model. Even at a high dosage of sporozoite administration, the protection remained in the immunized mice. Despite successful trials, this approach showed certain limitations, such as the cost of production and feasibility of delivery; moreover, in this case, the requirement of intravenous administration resulted in only 5% efficacy with the intradermal route [40]. In particular, GAPs (genetically attenuated parasites) have been developed, which are more stable and potent. These defined attenuated sporozoites can be produced on a large scale to immunize a large population and prevent malaria [41]. In addition, attenuation at a different pre-erythrocytic stage of development results in innumerable degrees of protection, particularly through the immune response. However, the study demonstrated that the systematic validation of the results of cryopreserved GAPs showed robust viability for more than ten years [41]. In vitro culture systems still need to be developed for the large-scale production of GAPs. The attenuation of GAPs is linked to cell-mediated protection [42]. The concept of GAPs could be translatable to the pathogenic asexual blood stage, as a low dosage also contributes to a higher level of protection. Various genes encoded on the surfaces of sporozoites and merozoites have now been identified, cloned, and expressed in various expression systems, which enable people to progress to the next level of a malaria subunit vaccine. Sporozoites are coated with CSP, which elicits strong antibody- or T-cell-mediated immunity during natural exposure or vaccination, which confers substantial protection in endemic areas. Vaccine studies of the CSP protein show low efficacy and immunogenicity [43,44,45]. Another important vaccine trial targeting the pre-erythrocytic stage, used with different formulations of various adjuvant forms, is EMTRAPE (multiepitope–thrombospondin-related protein), consisting of epitopes of B-cells and CD4+ and CD8+ T-cells from six different pre-erythrocytic proteins fused into TRAP [46]. The most advanced, successful, and only known licensed vaccine, RTS,S/AS01, also targets the pre-erythrocytic stage. RTS,S is a recombinant-protein-based vaccine designed with the NANP repeat region from the central region of the circumsporozoite protein (R) and C terminal with T-cell epitopes (T) fused with the S antigen from the hepatitis B virus (S); this fusion protein is co-expressed with HBsAg (S) in yeast formulated with the adjuvant, AS01 [47]. In Phase III clinical trials at various centers in African countries in which malaria is endemic, it has been reported with ~55% efficacy in treating children at the age group of 5–17 months. Efficacy further decreased to ~31–26% in clinical trials of 6–12-week-old infants [47,48]. Continued efforts are being made further to improve the efficacy and stability of the vaccine. RTS,S has been shown to induce a protective immune response with a high antibody titer and low T-cell response. At the same time, there were also protected volunteers found with low antibodies and higher T-cell responses. Due to the variability in the individual response, it is difficult to link the particular type of response with overall protection [49].

The RTS,S vaccine development approach was first proposed in 1987 by the Walter Reed Army Institute of Research (WRAIR) and the GlaxoSmithKline (GSK) group. The first pilot study was conducted in 2019 in a malaria-endemic region [22]. The RTS,S vaccine is able to target a sequence of four amino acids present on the surface of the P. falciparum sporozoite. It is stated that the RTS,S/AS01 vaccine is a combination of repeated T-epitopes (RTS) derived from PfCSP (Plasmodium falciparum circum-sporozoite protein) with the S-antigen derived from HBSAg (hepatitis B surface antigen) and the AS01 adjuvant, which is based on liposomes obtained by combining two immune-stimulants, MPL and QS-21, as an adjuvant. RTS,S/AS01 is a recombinant antigen expressed in yeast Saccharomyces cerevisiae [22,47]. The CS protein was modified to include the entire C-terminal region of the protein, which contains specific T-cell epitopes required for T-cell response induction. The RTS,S vaccine contains 25% fusion protein RTS and 75% wild-type HBsAg(S) antigen to maximize antigenicity and is only effective against P. falciparum disease. Adjuvant systems AS02 and AS01 are used to administer the fusion protein RTSs [50,51]. AS02 is a water-in-squalene emulsion containing monophosphoryl lipid A and saponin from Quillaja Saponaria bark. The AS01 formulation contains monophosphoryl lipid A and Quillaja Saponaria as an immunostimulant component, and the oil-in-water emulsion components are replaced with liposomes. The final vaccine for administration is obtained by reconstituting a lyophilized preparation of RTS,S antigen with AS01 and injecting it intramuscularly [51]. This vaccine is also known as Mosquirix TM. In Ghanaian children, trials were carried out using the RTS,S antigen with the AS01 and AS02 adjuvant systems; the RTS,S antigen with AS01 showed a greater response. Thus, the AS01 adjuvant system was selected for further Phase 3 trials. The Phase 3 randomized controlled trials were conducted involving 15,459 infants (6–12 weeks) and young children (5–17 months) across 11 centers in 7 sub-Saharan African countries: Burkina Faso, Gabon, Ghana, Kenya, Malawi, Mozambique, and Tanzania [22,50]. The study started in March 2009 and continued until January 2011, with 18 months of follow-up study. Vaccine efficacy for all episodes in this population was 55.1%. Vaccine efficacy for severe malaria was found to be 47.3% for children aged 5–17 months old and 34.8% for children aged 6 weeks to 17 months old [52,53]. These initial results confirm the validity of the efficacy rates observed in Phase 2 trials. The vaccine is well tolerated in infants and children. Preliminary results from the Phase 3 study have reported few cases of meningitis and seizures after increasing to seven days of vaccination. Future analyses will determine whether the vaccine response is sustainable over a 32-month follow-up period [22,50,52]. The RTS,S vaccine study was well designed, but it had some limitations: the field study covered only African infants and children, despite the fact that malaria affects a wide range of populations, including those in Asia and South America. Furthermore, the follow-up periods in these studies were short; it is unclear for how long the vaccine retains its efficacy, making it difficult to predict the time intervals required for booster dose administration. In October 2021, the WHO approved the RTS,S/ASO1 vaccine for use in pilot and large-scale studies in children in malaria-endemic areas such as sub-Saharan Africa and other areas with high malaria transmission [52]. One of the main goals of this RTS,S/AS01 vaccine is to eliminate and provide 100% protection from malaria; consequently, the RTS,S/AS01 vaccine is scheduled to enter Phase 4 clinical trials in various malaria-endemic regions to be evaluated. Clinical study results from the RTS,S/AS01E vaccine study were published in 2013, and 447 infants and children aged 5 to 17 months were enrolled in Kilifi, Kenya. All of these children received three doses of the vaccine (NCT00872963) [22,52,54]. The results showed that the effectiveness of the RTS,S/AS01E vaccine decreased by approximately 44% (95% CI; 16–62) to 0 between the first and fourth subsequent years. However, in 2015, the second clinical trial was conducted and the results of a Phase 3 vaccine trial involving 15,459 children aged 5 to 17 months at 11 sites in 7 sub-Saharan African countries were reported (NCT00866619) [53]. With a 20-month booster dose, the results of the RTS,S/AS01 vaccine showed that targets were only partially met and the vaccine provided partial protection against clinical malaria with a median follow-up of 48 months. However, even after a booster vaccination of this vaccine, there was no change in the children (age group 6–12 weeks) regarding protection against severe malaria [53]. In addition, further studies are ongoing to evaluate the effectiveness of the vaccine in children with four vaccinations. The RTS,S/AS01 vaccine was found to provide better protection against malaria caused by Plasmodium species matching the protein used in the vaccine [22,53]. In the following year, 2016, two expert panels, the Strategic Advisory Group of Experts on Immunization (SAGE) and the Malaria Policy Advisory Committee (MPAC), decided not to recommend the RTS,S/AS01 vaccine for general use, pending the results of further clinical studies [22]. However, in July 2015, after the EMA (European Medicines Agency) had received good results and good scientific support for the RTS,S/AS01 malaria vaccine, the WHO recommended several pilot studies on the use of the RTS,S/AS01 vaccine in children aged 5 months in different malaria-endemic locations [49,55]. The fourth vaccine dose was only given between 15 and 18 months after the first three doses, at least 1 month apart [31,49]. The results of clinical trials and mathematical modeling showed that the RTS,S/AS01 vaccine, when used in conjunction with other antimalarial therapies, enhances protection against malaria infection and also showed that the RTS,S/AS01 vaccine has the potential to significantly improve public health [55]. In addition, the study showed that infants from the age of 5 months benefit the most from four doses of the vaccine. Future investigation of these preliminary results in real-world conditions is needed to determine the vaccine’s safety, the mortality implications, and the feasibility of administering four doses in an immunization/vaccination program on a broad basis in different African countries and severely malaria-affected regions [22,55]. In contrast to infection with malaria parasites that have an allelic mismatch, additional research is needed to assess the potential of RTS,S/AS01 vaccines towards different malaria parasites with a PfCSP allele that links with the vaccine target. The only malaria vaccine with ongoing large-scale post-licensing pilot implementation initiatives is currently RTS,S/AS01. Thousands of newborns and children received the RTS,S/AS01 vaccine this year in most malaria transmission countries, such as Ghana, Malawi, and Kenya [22,56].

The R21/MM malaria vaccine has elicited researchers’ interest due to the high potency response seen in 2022. It is known that this vaccine is a combination of the R21 virus-like protein based on the PfCSP fused to the N-terminus of HBsAg and combined with the Matrix-M adjuvant [57]. This vaccine is manufactured by Oxford University and the R21/MM vaccine contains Matrix-M, a patented saponin-based adjuvant manufactured by Novavax. The results of an exploratory Phase 2b research study on the human safety and the effectiveness of this malaria vaccine, R21/MM, were published in May 2021 (NCT03896724) [57]. Convincing data from Phase 2b randomized vaccine trials have been obtained in various malaria-endemic areas, such as West Africa, Burkina Faso, and other regions where high seasonal malaria transmission is reported. A study was conducted by two organizations, namely, the Institut de Recherche en Sciences de la Sant (IRSS) and the Nanoro Clinical Research Unit (CRUN). Additionally, this Phase 2b trial included 450 infants and children aged between 5 and 17 months. In this Phase 2b clinical trial, the organization chose to administer three doses of the vaccine at different doses, and children received either a low dose, such as 5μg R21 with 25μg MM, or a high dose of 5μg R21 with 50μg MM, and a Rabies vaccine was used as a control group [57]. Finally, the effects and efficacy of this vaccine were observed after six months, and they found 74% (95% CI; 63–82) efficacy in the low-dose R21/MM adjuvant group and 77% (95% CI; 67–84) efficacy in the high-dose R21/MM adjuvant group [57]. Moreover, no major adverse effects have been reported with R21/MM immunization [22]. However, it was observed that the children who received three doses of the R21/MM vaccine, 28 days apart, had significant antimalarial antibody titers found at the higher adjuvant dose [57]. Moreover, 77% of newborns and children showed good vaccination effectiveness. In addition, a booster dose of the vaccine given one year after the initial three doses of this vaccine as part of an R21/MM immunization regimen can elevate antibody titers to values close to the highest antibody titers [22,57]. Therefore, with regard to the efficacy and safety profile of this human R21/MM vaccine, as well as the ability to maintain immunity in children with annual R21/MM vaccine boosters, optimism about the possibility of eradicating and treating the malaria infection has been renewed [58]. Notably, due to its high R21/MM vaccine efficacy/potency of 77%, this malaria vaccine was the first to reach the 75% efficacy target set by the WHO [54]. The Phase 2b study was extended for a further two years to evaluate the effectiveness of additional booster doses of the malaria vaccine R21/MM by the funding organization [22]. In early 2023, the results of an ongoing clinical study (NCT05252845) to evaluate the immunogenicity and safety of this malaria R21/MM vaccine in adult humans in Thailand will be published [59]. In addition, Oxford University investigators are testing the protection and immunogenicity of this human R21/MM vaccine in adult Thai individuals with the aim of determining whether the concomitant use of different antimalarial drugs has an impact on the immunogenicity of the R21/MM vaccine [59]. Furthermore, antimalarial drugs such as piperaquine and a single low-dose primaquine treatment will be evaluated for their pharmacokinetics and absorption when co-administered with the R21/MM vaccine [22,59]. It is hoped that adults will experience the high potency and immunogenicity of malaria vaccines seen in infants and children. In addition, in early 2023, researchers anticipate the completion/receipt of Phase 3 testing results, which are ongoing at scale to verify the safety and efficacy of the vaccine. In addition, in this Phase 3 trial, 4800 infants and children between the ages of 5 and 36 months will be enrolled in four different African countries, namely, Mali, Tanzania, Burkina Faso, and Kenya [60]. Therefore, should the results of these ongoing clinical trials be positive, regulatory approval of this human R21/MM malaria vaccine in 2023 would represent a significant improvement in the safety and health of the world and potentially save the lives of millions of people.

It is the blood stage that is amenable to all disease pathologies during malaria, and natural immunity to malaria is also observed due to many blood-stage antigens. Experiments with the passive transfer of antibodies were able to reduce the disease symptoms and parasitemia in children with malaria infection. The vaccine development strategies propose an approach that can elicit an immune response in children that would also protect adults from disease manifestation [61]. Considering the above, it is speculated that RTS,S might induce immunity to the blood stage by rendering the extended exposure of circulating blood-stage parasites at a lower density [62,63]. Most of the blood-stage vaccine studies focus on the invasion of merozoites, during which the parasites are transiently exposed to the host immune system. The surface proteins of the merozoites are in direct contact with the immune cells, and these proteins are crucial for the parasite’s growth as well; therefore, these proteins are considered candidates in developing a blood-stage vaccine [64,65]. SPf66 was the first blood-stage asexual malaria vaccine to be developed. It contains a multi-stage peptide vaccine mixed with alum as an adjuvant, but further development of this vaccine has been halted because numerous Phase III studies have shown that the efficacy of this vaccine is very low [66]. Various vaccine candidates are merozoite surface proteins found to produce an effective immune response in preclinical trials [67]. Some of the well-studied vaccine candidates under clinical trials are apical membrane antigen 1 (AMA1), serine repeat antigen 5 (SERA5), ring-infected erythrocyte surface antigen (RESA), erythrocyte-binding antigen 175 (EBA175), merozoite surface protein (MSP)-1, MSP-2, MSP-3, erythrocyte-binding antigen 175, and glutamate-rich protein (GLURP), which are all exposed to the surfaces of the blood-stage antigens [67,68,69]. The technological challenges of generating Plasmodium proteins in a recombinant form that is biochemically active is one of the main problems arising from the use of recombinant proteins for the production of blood-stage vaccines. The functional redundancy between the merozoite ligands involved in erythrocyte invasion is a significant obstacle to the creation of a highly effective blood-stage malaria vaccine. Merozoites are thought to have an assemblage of overlapping ligands expressed on their surfaces, giving the parasite access to multiple invasion pathways. In order to effectively prevent a wide range of invasion routes, it is believed that effective blood-stage vaccination should target different merozoite antigens simultaneously [69]. The large allelic variation observed in many Plasmodium strains is a significant obstacle to the creation of an effective blood-stage malaria vaccine, in addition to the parasite’s ability to gain access to multiple invasion routes. The two foremost blood-stage vaccine candidates, AMA1 and MSP142, were not protective in African adolescents, partly because of antigenic differences. Despite sufficient antibody titers, cross-protection against different malaria strains was insufficient [70,71]. However, it was recently discovered that RH5 and basigin interact in a way that is generally required for erythrocyte invasion, raising new expectations for the development of a very effective blood-stage vaccination [72]. Low levels of anti-basigin monoclonal antibodies can completely prevent erythrocyte invasion; this effect has been observed in numerous laboratory-adapted P. falciparum lines and field isolates [72,73]. Conversely, in nine separate P. falciparum strains harboring all five of the most common RH5 polymorphisms, polyclonal antibodies produced against the 3D7 variant of RH5 were able to prevent erythrocyte invasion [74]. In addition, RH5 delivered via a virus can elicit antibodies that prevent a variety of parasite genetic variations from entering erythrocytes. Therefore, it is expected that the discovery of the interaction between RH5 and Basigin will lead to the creation of effective therapeutic approaches targeting the blood stages of the parasite. The first Phase Ib results of the VV-RH5 immunization and the RH5.1 protein vaccine in African infants are therefore eagerly awaited by researchers (ClinicalTrials.gov: NCT03435874 and NCT04318002) [75]. These results will be crucial in determining whether the CHMI overestimated the effectiveness of RH5-based vaccines in adults in the UK due to the significantly weaker antibody response. To further increase the effectiveness of RH5-based vaccines, the other recently identified antigens, CyRPA and RIPR, which combine with RH5 to form a heterotrimeric protein complex, could be included. Similar to RH5, these antigens are extremely conserved, necessary, and capable of inducing significant levels of anti-growth antibodies following animal vaccination [72,76,77]. Finally, it is crucial to consider logical adaptations to the structure and mode of delivery of the RH5 immunogen. Two methods to achieve large increases in the quantity and/or quality of vaccine-induced polyclonal anti-RH5 IgG include the increased delivery of innovative RH5 immunogen arrays on virus-like particles and the analysis of vaccine-induced human anti-RH5-mAbs to inform structure-based vaccine design [75,76,77,78].

Vaccines that block transmission are not intended to provide direct protection; rather, the aim is to inhibit the formation of gametocytes, which would ultimately lower the transmission rate and thereby reduce the rate of infection [80]. The transmission-blocking vaccines interfere with the sexual stage of the parasite. Earlier experiments on malaria models have reported malaria transmission-blocking immunity. When turkeys or chickens are vaccinated with extracellular male or female gametes or gametocytes of the malaria parasite, the animals develop immunity that may eventually protect them from further infection with the same parasite by infected mosquitoes. Based on this concept, mosquitoes are allowed to feed on the blood of rodents and humans, containing antibodies against the gametocytes, ookinete, or zygote [80]. These antibodies will attack the respective stage of antigens and block the development of the parasite to further stages. Many vaccine trials for antigens have been initiated. However, the most promising antigens are Pfs-48/45 and Pfs-230 of P. falciparum’s and their orthologs in P. vivax, expressed on both macrogametes and microgametes [81]. Pfs-48/45 and Pfs-230 antigens have a 6-cysteine structure, represented in the human host [81]. These proteins contain polymorphic sequences and have heavily conserved domains. Antigens Pfs-25 and Pfs-28 are expressed in zygotes and on the matured ookinetes of the malaria parasite [82]. These proteins are abundant in cysteines such as the Pfs-12 family of proteins, but the arrangement of cysteines is different, having four complete 6-cysteines or four partial cysteine EGF-like domains [83].

Therefore, the results from human clinical trials of two transmission-blocking antigen (TBV) vaccine candidates, Pfs230 (pre-fertilization antigen) and Pfs25 (post-fertilization antigen), were obtainable [84]. According to related studies, it was found that human immunity against sex-stage malaria parasites can act in the mosquito. On the other hand, NIAID researchers coupled both Pfs25 and Pfs230 to the immunogenic carrier protein exoprotein (EPA) and administered adjuvants to overcome the antigens’ inherently low immunogenicity [84]. Furthermore, human clinical studies of Pfs25-EPA performed with Alhydrogel demonstrated the safety and immunogenicity of the antigen in humans. In addition, functional antibodies can be induced by the antigen in laboratory tests to prevent parasite transmission to mosquitoes. Serum activity also showed a correlation with antibody titers [85]. However, the majority of vaccine recipients required four doses to produce functional serum activity, and this activity was transient as the anti-Pfs25 antibody levels fell rapidly after each dose [86,87]. Remarkably, the titers against the carrier protein EPA decreased more slowly than the titers against Pfs25 IgG. Ongoing studies are associating and merging the vaccine antigens Pfs25 and Pfs230 [84]. Standard membrane feeding assays (SMFAs) and the direct feeding of mosquitoes to vaccine recipients are both used to measure vaccine efficacy. Since Pfs230 vaccination is well recognized for the complement to be reliant on, the SMFA requires a new complement. GSK’s AS01 adjuvant is being used in an ongoing experiment with both the Pfs230 and Pfs25 conjugate human vaccines, as pre-clinical studies indicate that this formulation could dramatically increase antibody titers and consequently lead to efficient serum activity following immunization [87,88,89,90]. Overall, the research group has completed the first clinical trial and demonstrated its safety and immunogenicity in an alum-adjuvanted formulation of the Pfs25 vaccine. Thus, the serum from these subjects had a macerating but suboptimal transmission-blocking effect, on the order of a 50% reduction in the infectivity of P. falciparum to mosquitoes compared to controls [88,89,90].