Progress in the Development of Subunit Vaccines against Malaria

Malaria is a life-threatening disease and one of the main causes of morbidity and mortality in the human population. The disease also results in a major socio-economic burden. The rapid spread of malaria epidemics in developing countries is exacerbated by the rise in drug-resistant parasites and insecticide-resistant mosquitoes. At present, malaria research is focused mainly on the development of drugs with increased therapeutic effects against Plasmodium parasites. However, a vaccine against the disease is preferable over treatment to achieve long-term control. Trials to develop a safe and effective immunization protocol for the control of malaria have been occurring for decades, and continue on today; still, no effective vaccines are available on the market. Recently, peptide-based vaccines have become an attractive alternative approach. These vaccines utilize short protein fragments to induce immune responses against malaria parasites. Peptide-based vaccines are safer than traditional vaccines, relatively inexpensive to produce, and can be composed of multiple T- and B-cell epitopes integrated into one antigenic formulation. Various combinations, based on antigen choice, peptide epitope modification and delivery mechanism, have resulted in numerous potential malaria vaccines candidates; these are presently being studied in both preclinical and clinical trials. This review describes the current landscape of peptide-based vaccines, and addresses obstacles and opportunities in the production of malaria vaccines.


Introduction
Malaria is a major health problem worldwide and is one of the most prevalent diseases in developing countries. Globally, it is responsible for around half a million deaths annually. People living in high-poverty areas, where rainfall and temperature favor the transmission of Plasmodium parasites, are at the highest risk of contracting malaria and developing severe disease symptoms [1,2]. Human malaria is commonly caused by four different Plasmodium species: P. falciparum, P. vivax, P. malariae, and P. ovale. Severe cases of malaria in sub-Saharan Africa are typically caused by P. falciparum, while infections in southeast Asia almost always result from P. vivax and P. falciparum [3][4][5][6]. Young vaccines aim to block malaria transmission from mosquitoes to humans by preventing the malaria parasite from developing in the mosquito.
Vaccines 2020, 8, x 3 of 19 blocking vaccines aim to block malaria transmission from mosquitoes to humans by preventing the malaria parasite from developing in the mosquito. (2) The sporozoites enter into the bloodstream and head immediately for the liver, where they invade liver cells. (3)(4) The parasites reproduce in the liver to produce thousands of haploid forms, called merozoites. (5) The merozoites emerge from the liver and re-enter the bloodstream, where they infect red blood cells (RBCs). (6) They continue to reproduce asexually in the RBCs and release newly formed merozoites back into the bloodstream. Thousands of parasite-infected cells in the host's bloodstream can lead to symptoms, such as recurrent fever, chills, headache, anemia, fatigue, perspiration, anorexia, and vomiting. (7) Some merozoites develop into male and female gametocytes, which are the sexual forms of the parasite. (8)(9) When a mosquito has a blood meal from an infected person, these gametocytes are ingested in the gut of the mosquito. (10) Male and female gametes fuse together to form a diploid zygote, which develops into ookinetes that burrow in the midgut wall of the mosquito to form oocysts. (11)(12)) After a few days, oocysts burst and release sporozoites into the body cavity of the mosquito. These sporozoites then invade the salivary glands of the mosquito. The cycle continues when the mosquito bites an appropriate host, injecting the sporozoites into the animal's bloodstream [26][27][28].
Only one commercial antimalarial vaccine presently exists, RTS,S/AS01. It was approved in 2015; however, it has low efficacy (26-50%) [29]. In addition, human responses to the vaccine, both in the field and in controlled human malaria infections in non-endemic settings, are extremely variable. RTS,S includes a P. falciparum circumsporozoite (CS) protein carboxy-terminal fragment bonded to the surface antigen of hepatitis B (HBsAg). These antigens are assembled into particulate structures. The RTS,S was developed using the adjuvant AS01, which includes monophosphoryl lipid A (MPL) and saponins mixture QS21 in liposome-based formulation. RTS,S/AS01 was assessed in a phase III trial [30] and while it received a positive recommendation from the European Medicines Agency, further improvements into the vaccine's properties were recommended due to its limited efficacy and the fact that the fundamental mechanism of action is still not well-understood [31].
In order to direct further development and advancement in malaria vaccine strategies, it is important to consider which factors may lead to the variation in efficacy in humans. Nielsen et al. analyzed the data from six phase II trials of RTS,S [32], in which the correlation between human leukocyte antigen (HLA) allele groups and the protection mediated by RTS,S was evaluated using (2) The sporozoites enter into the bloodstream and head immediately for the liver, where they invade liver cells. (3)(4) The parasites reproduce in the liver to produce thousands of haploid forms, called merozoites. (5) The merozoites emerge from the liver and re-enter the bloodstream, where they infect red blood cells (RBCs). (6) They continue to reproduce asexually in the RBCs and release newly formed merozoites back into the bloodstream. Thousands of parasite-infected cells in the host's bloodstream can lead to symptoms, such as recurrent fever, chills, headache, anemia, fatigue, perspiration, anorexia, and vomiting. (7) Some merozoites develop into male and female gametocytes, which are the sexual forms of the parasite. (8-9) When a mosquito has a blood meal from an infected person, these gametocytes are ingested in the gut of the mosquito. (10) Male and female gametes fuse together to form a diploid zygote, which develops into ookinetes that burrow in the midgut wall of the mosquito to form oocysts. (11)(12) After a few days, oocysts burst and release sporozoites into the body cavity of the mosquito. These sporozoites then invade the salivary glands of the mosquito. The cycle continues when the mosquito bites an appropriate host, injecting the sporozoites into the animal's bloodstream [26][27][28].
Only one commercial antimalarial vaccine presently exists, RTS,S/AS01. It was approved in 2015; however, it has low efficacy (26-50%) [29]. In addition, human responses to the vaccine, both in the field and in controlled human malaria infections in non-endemic settings, are extremely variable. RTS,S includes a P. falciparum circumsporozoite (CS) protein carboxy-terminal fragment bonded to the surface antigen of hepatitis B (HBsAg). These antigens are assembled into particulate structures. The RTS,S was developed using the adjuvant AS01, which includes monophosphoryl lipid A (MPL) and saponins mixture QS21 in liposome-based formulation. RTS,S/AS01 was assessed in a phase III trial [30] and while it received a positive recommendation from the European Medicines Agency, further improvements into the vaccine's properties were recommended due to its limited efficacy and the fact that the fundamental mechanism of action is still not well-understood [31].
In order to direct further development and advancement in malaria vaccine strategies, it is important to consider which factors may lead to the variation in efficacy in humans. Nielsen et al. analyzed the data from six phase II trials of RTS,S [32], in which the correlation between human leukocyte antigen (HLA) allele groups and the protection mediated by RTS,S was evaluated using linear regression and multivariate logistic. Significant associations were found between the HLA-A/01, HLA-B/08 and HLA-DRB1/15/16 allele groups and positive protection results; whereas other allele groups, such as HLA-A/01, HLA-B/53 and HLA-DRB1/07, were associated with insufficient protection. It is worth noting that these "protective" allele groups are known to have a lower prevalence in sub-Saharan African populations compared to populations in United Kingdom or United States, where these phase II RTS,S trials took place. This finding illustrated how the HLA genotype may affect the protective efficacy of RTS,S against malaria infection [32].
Overall, the difficulty in developing effective malaria vaccines stems largely from the complexity of the malaria-causing parasites' life cycle, which includes mosquitos, human liver, and human blood stages (Figure 1), and subsequent antigenic variations of the parasite [33]. These parasites are also able to hide inside human cells to avoid being recognized by the immune system, creating further challenges [34].
Conventional vaccines use either live-attenuated/killed pathogens or recombinant protein to evoke an immune response [35,36]. The disadvantages of these vaccines include possible problems in protein expression, contamination from the expression process, difficulty in pathogen cultivation (which is especially problematic for malaria), and the risk of autoimmune and excessive inflammatory responses in humans. Therefore, peptide-based vaccine approaches have become very attractive due to: (i) the relative ease in producing peptides in large-scale with high purity; (ii) the ability for these vaccines to be freeze-dried and stored in solid form at room temperature; (iii) the possibility to target appropriate immune responses through vaccine customization; and (iv) greatly reduced risk of autoimmune and allergic responses [37]. Peptide-based vaccines are generally designed based on the minimal peptide epitope that is still able to trigger the desired immunity. Peptide-based vaccines can suffer the disadvantage of being sensitive to any mutation within the selected epitopes and too genetically constrained due to the specificity of the T-cell receptor (TCR) [38]. Further, low immunogenicity is a typical weakness of peptide-based vaccines; however, it can be minimized through the development of better adjuvant-based delivery systems.
The first parenteral peptide-based malaria vaccine, which was tested clinically in 1987, was designed based on repeat peptide sequences (NANP)3 derived from P. falciparum circumsporozoite protein (CSP) [39]. Since then, a variety of synthetic peptide vaccines have been developed for both murine (P. berghei and P. yoelii) and human (P. falciparum and P. vivax) parasites and tested for immunogenicity and efficacy. Several of these reached advanced clinical trials [40]. Additionally, the development of a database of nearly 561,924 unique B-cell and T-cell epitopes from Plasmodium proteins (Figure 2), including those from human P. falciparum and P. vivax parasites, has provided researchers with a comprehensive repository of potential peptide targets (http://www.immuneepitope.org).

Pre-Erythrocytic-Stage Vaccines
The pre-erythrocytic parasite stage is the most advantageous target for vaccine development against malaria because the inhibition of the liver stage of parasite growth blocks merozoites from entering the bloodstream. As such, symptomatic malaria can be prevented (Table 1).  ND: no data. LSA-1: DNA sequence coding for a P. falciparum liver-stage-specific antigen composed of repeats of 17 amino-acids.

Circumsporozoite Protein
Studies in transgenic mice that were tolerant to CSP T-cell epitopes confirmed that CSP is the immunodominant target of protective immune responses elicited by irradiated sporozoites [63]. Neutralizing antibodies against sporozoites targeted the species-specific central repeat region of CSP, and the antibodies recognizing the anti-repeat region bound to the surface of the sporozoites, inhibited motility, and blocked invasion of the host's hepatocytes [64].
RTS,S (licensed by GlaxoSmithKline Biologicals) is the only approved malaria vaccine on the market. It is based on the CSP of P. falciparum 3D7 clone. The vaccine antigen is comprised of two peptides; RTS and S, which are recombinantly co-expressed in Saccharomyces cerevisiae (Table 1) [48,65]. RTS corresponds to amino acids 207-395 of CSP. It is conjugated to S, which is the N-terminus peptide of HBsAg, which consists of 226 amino acids. The RTS,S vaccine specifically targets the pre-erythrocytic stage of P. falciparum. It confers a moderate to low level of protection against infection by P. falciparum sporozoites in humans [47,66].
An improved version of this vaccine, RTS,S/AS02, was developed based on a new adjuvant (AS02, an oil in water-based formulation containing the immunostimulants MPL, a nontoxic derivative of lipopolysaccharide (LPS), and QS21). RTS,S/AS02 achieved elevated antibody titers and enhanced cell-mediated immune responses in both malaria-naive and malaria-experienced individuals [48]. A separate phase III clinical trial of RTS,S/AS01 (NCT00866619) was conducted in Africa with 6537 infants (age 6-12 weeks) and 8923 children (age 5-17 months). In this study, parasite clearance occurred in 46% of vaccinated children and 27% of infants [30]. Therefore, further improvements are needed to potentiate the efficacy of RTS,S. Although RTS,S/AS01 has been approved for active immunization against malaria in children between the ages of 6 weeks to 17 months by the European Medicines Agency, WHO has not recommended the implementation of RTS,S/AS01 in the Expanded Program on Immunization (EPI) [29].
Alternative versions of RTS,S equivalent vaccines have also been examined for P. vivax circumsporozoite (CSV) protein and a truncated region containing repeat sequences from both VK210 and VK247 subtypes of the parasite expressed in E. coli [60]. CSV-S,S included CSV-S, a fusion protein between VMP001 and S (HBsAg peptide) expressed in S. cerevisiae. CSV-S,S/AS01 induced higher levels of vaccine-specific antibodies than VMP001/AS01 in rhesus monkeys [60]. However, no challenge study was performed.
Microparticle peptide-based malaria vaccines were developed by Powell et al. using layer-by-layer (LBL) fabrication of polypeptide films on solid CaCO 3 cores [50]. These vaccines were comprised of tri-epitope CS peptide T1BT*, including the B-cell epitope of the CS repeat region B and the highly conserved T-cell epitope (T1), as well as the universal epitope T* that is detected by multiple molecules of HLA class II [67]. Parasite-neutralizing antibodies and malaria-specific T-cell responses, including cytotoxic effector T-cells, were produced upon immunization of mice. When the challenge was performed with live sporozoites from infected mosquitoes, protection from infection was found to be associated with levels of neutralizing antibodies. However, some immunized mice produced low or undetectable neutralizing antibodies levels, yet were still protected. Furthermore, mice immunized with only T-cell epitopes carrying microparticles developed parasitemia, indicating that cellular immunity alone is not sufficient for preventing infection. The immune response against this vaccine was potentiated with the incorporation of the TLR2 agonist Pam 3 Cys [50,68,69].
Mitchell et al. used the skin scarification technique to deliver malaria repeat peptide containing a protective B-cell epitope of P. falciparum CS formulated with TLR-7/8 and -9 agonists [51]. The vaccine elicited high levels of neutralizing antibodies, triggered protective cell-mediated immunity in mice, and resulted in mice resistant to disease when challenged through bites by infected mosquitoes. In this study transgenic P. berghei sporozoites expressing P. falciparum CS repeats were used for infection of mosquitoes.
Wilson et al. evaluated Poly I:C and Montanide as adjuvants and polystyrene nanoparticles as a carrier for the delivery of the immunodominant CD8 T-cell epitope, KI (SYIPSAEKI), of P. berghei CSP (named pb9 or KI) in the murine model. The systems induced the production of IFN-γ and cellular immunity [70]. The highest CD8 T-cell responses were induced against KI vaccine adjuvanted with Montanide or covalently conjugated to polystyrene nanoparticles (40-50 nm), rather than adjuvanted with Poly I:C or delivered as a mixture with polystyrene nanoparticles.
Several other epitopes derived from CS protein were examined; however, immune responses were poor, non-selective, or the vaccines were not protective [71][72][73].

Other Liver-Stage Peptide Specific Sequences
Liver-stage antigen 1 (LSA-1) is expressed during the liver stage of malaria in the parasitophorous vacuole [74]. It consists of 17 amino acid repeat units (ALKEKLQ-X-QQSDLEEQR, wherein X is Glu or Gly) and plays a vital role in late liver-stage schizogony [74][75][76]. LSA-1 naturally evokes antibody responses, especially those of the IgG1 subclass, in humans exposed to P. falciparum infection [57]. Recombinant LSA-1, either alone or in combination with AS01/AS02, elicited high antibody titers in humans and induced CD4 + cells to produce IFN-γ and IL-2, but did not protect against P. falciparum infection [77]. However, liver-stage antigen 3 (LSA-3) was identified following immunization with irradiated sporozoites in humans [42,78]. Immunization with LSA-3 in chimpanzees induced full protection against successive heterologous challenges with a large numbers of P. falciparum sporozoites [58]. Efficacy of the vaccine candidate has not yet been demonstrated in humans [79].

Erythrocytic-Stage Vaccines
Malaria parasites attack red blood cells (RBCs) during their merozoite stage. RBCs cannot stimulate T-cell responses, as they do not express MHC class I molecules. Thus, parasite infection at this stage can generate only humoral immune responses. Two types of antibodies are produced against merozoites at the blood stage: (a) antibodies that recognize antigens from merozoite surface proteins and (b) antibodies that recognize parasite antigen expressed on infected RBCs, which initiates Antibody-Dependent Cell-Mediated Cytotoxicity [80]. The blood stage vaccines are designed based on these antigens (Table 2); among which MSP-1, MSP-2 and MSP-3 are the most commonly employed [81].

MSP and Glutamate-Rich Protein
MSP-1 is expressed from the schizogony stage of malaria parasites and is involved in RBC invasion by parasites in the merozoite stage [88]. MSP-1 has highly conserved regions recognized by B-and T-cells in both human and murine models. To identify potent vaccine candidates, two synthesized multiple epitope peptides (MEP), P1 and P2, containing B-and T-cell epitopes from two N-terminal conserved regions of blood-stage antigen MSP1 and RESA (ring-infected erythrocyte surface antigen) of P. falciparum, respectively, were tested in mice [89]. Both P1 and P2 were able to stimulate high levels of antibody titers without the need for any carrier proteins. Upon challenge with a lethal dose of P. yoelii nigeriensis blood-stage parasites, only P1 adjuvanted with alum provided protection in BALB/c mice. In contrast, P1 adjuvanted with theoretically much stronger adjuvant, complete Freund's adjuvant (CFA), conveyed partial protection.
MSP-2 is a 45 to 52 kDa parasite integral membrane protein located on the merozoite surface. It has highly conserved N-and C-terminal regions that are recognized by human and murine immune cells. B-cell epitope (SNTFINNA) from the N-terminal conserved region of MSP-2 conjugated to the entire N-terminal peptide sequence (KNESKY-SNTFINNA-YNMSIRRSM), but not peptides separately, was found to be immunogenic in both BALB/c and C57BL/6 mice [90]. BALB/c mice immunized with the conjugate adjuvanted with CFA were able to resist challenge with P. yoelii 265BY strains, while C57BL/6 mice did not generate protective immunity.
Protective effects of human antibodies against MSP-3 and glutamate-rich protein (GLURP) have been suggested by a number of immuno-epidemiological studies. This followed demonstration that the levels of two antigen-specific cytophilic antibodies (IgGl and IgG3) were significantly associated with a reduced incidence of malaria [82][83][84]. The malaria vaccine candidate GMZ2 was designed as a hybrid polypeptide to include the N-terminal region of GLURP linked with the C-terminal region of MSP-3 [85]. GMZ2 adjuvanted with aluminum hydroxide induced high levels of IgG antibodies.
Both malaria-naive adults and malaria-exposed preschool children produced vaccine-specific antibodies with broad inhibitory activity against geographically diverse P. falciparum isolates [85]. A recent phase II trial of GMZ2/aluminum hydroxide vaccine with a cohort of 1849 children demonstrated that GMZ2 was well-tolerated and induced high antibody titers, which unfortunately corresponded with poor protection against malaria (14%) [91][92][93].

SPf66
SPf66 was one of the first synthetic peptide-based vaccines developed against malaria. It is a synthetic 45 amino acid peptide based on four P. falciparum proteins, including three asexual blood-stage protein fragments (83, 55 and 35 kDa), linked by the repeat region of circumsporozoite protein. The vaccine construct is primarily intended to target blood-stage malaria parasites. Initially, two studies were conducted to evaluate the efficacy of SP66 in humans [94,95]. The first study evaluated the immune response induced by SPf66 in humans from the Colombian Pacific coast. The Spf66 vaccine construct was able to induce higher antibody titers and protection against parasites after the third boost. The second study was conducted with 9957 children aged less than 1 year old to evaluate vaccine safety. Clinical observation was carried out 30 min and 48 h after each immunization and no adverse reactions were recorded in 97% of the cases. When tested in Colombian volunteers who were semi-immune, the vaccine demonstrated 38-60% protective efficacy against P. falciparum [96]. However, subsequent studies with participants from Tanzania [97], Gambia [98] and Thailand [99] found that Spf66 failed to provide protection against P. falciparum malaria. Following these results, justification could no longer be made for further trials of SPf66 using the same formulation [41,86].

Apical Membrane Antigen (AMA-1)
AMA-1 from P. falciparum is one of the most promising target antigens for the development of erythrocytic-stage malaria vaccines (Figure 2) [53]. The entire region of this protein can be divided into three domains; the majority of antibodies recognize strain-specific epitopes in domain I. AMA-1-derived peptide comprising residues 446 to 490 was conjugated to the influenza virosome and tested in a murine model. All mice immunized with this virosome produced high antibody (IgG) levels, which were able to inhibit P. falciparum growth in vitro (95%).
In another study, virosomal formulations of peptide-phosphatidylethanolamine peptidomimetic conjugates, including the NANP repeat region of P. falciparum CSP (Pf CSP, UK-39) and P. falciparum AMA-1 (Pf AMA1, AMA49-C1), were administered to immunize healthy malaria-naive adults [55]. UK-39-and AMA49-C1-loaded virosomes induced a long-lasting parasite growth-inhibiting antibody response in humans. The antibodies from immunized volunteers inhibited sporozoite migration and the invasion of hepatocytes in vitro, suggesting the potential of influenza virosomes as a human-compatible antigen delivery platform for the development of subunit vaccines [54]. To investigate the efficacy of virosome-formulated P. falciparum AMA-A-and CSP-derived peptides as a malaria vaccine, ten Tanzanian adults and 40 children aged between 5 and 9 years old living in a malaria-endemic region were immunized with one or two doses of the vaccine on days 0 and 90. After vaccination, the incidence rate of clinical malaria episodes in children was reduced by 50% compared with control children. Only two adults showed malaria episodes. While it appears that this virosomal formulation has higher efficacy in adults, a larger-scale clinical study is required to test this observation.

Clinical Trials
Over 20 malaria vaccines have been recently evaluated in clinical trials (Table 3). These were mostly designed to protect humans against P. falciparum infection. Only one, ChAd63/MVA PvDBP, was against P. vivax, and it has only reached phase I trials. More than dozens of malaria parasite proteins (or their fragments) have been selected as antigens. For example, SE36 is a malaria vaccine candidate bearing the N-terminal 47 kDa domain of P. falciparum serine repeat antigen 5 (Pf SERA5) [100]. SE36 inhibited parasite growth in vitro; however, there was a negative correlation between parasite density and antibody level [100,101]. In phase Ib trials, BK-SE36 vaccine induced 72% protective efficacy after 130-365 days post-second vaccination for Ugandan children aged 6-20 months who suffered from symptomatic malaria [102]. A leading alternative or potentially complementary strategy in fighting malaria is heterologous prime-boost immunization with sequential administration of chimpanzee adenovirus serotype 63 (ChAd63) and modified Ankara (MVA) vaccines. Both encode the malaria antigenic sequence called ME-TRAP. Venkatraman et al. conducted a controlled phase I study to evaluate the immunogenicity and safety of a ChAd63 vaccine candidate containing Matrix-M as an adjuvant [103,104]. Matrix-M is a promising Quillaja saponins-based vaccine adjuvant with demonstrated acceptable safety [105] and the ability to improve both cellular and humoral immune responses of vaccines [104,106,107]. In the abovementioned study, 22 healthy individuals were intramuscularly immunized with either (a) ChAd63-MVA ME-TRAP alone, or adjuvanted with (b) 25 µg or (c) 50 µg of Matrix-M. All groups received boost vaccination 8 weeks later. The group that received 50 µg of Matrix M had a significant increase in cytokine production at day 63. In all three groups, IgG production related to TRAP was highest 28 days after the boost vaccination. The vaccine was clinically safe and well-tolerated, with most local and systemic adverse reactions being mild in nature. Matrix-M did not increase local adverse effects; however, systemic adverse reactions were more frequently reported by volunteers receiving adjuvanted vaccine compared to the control group [103].

Challenges and Prospects
Both cellular and humoral immune pathways play significant roles in defending against malaria. It is important to note that scientists are still not able to articulate clear correlates of protection [108]. Therefore, the major impact of the various immune mechanisms remains to be defined, and as such all factors of the immune response are still under consideration in the design of vaccination strategies against this complex parasite disease [109].
Three decades following the first clinical trials on malaria vaccine, we are still yet to develop an effective strategy for prevention of the disease. However, a large number of peptide antigens have been identified. Pre-erythrocytic vaccines based on these peptides have delivered protection to some extent, or have at least led to reduced parasite growth [73]. Yet, research and development of malaria vaccines for liver and blood stages of the parasites face enormous challenges, as many vaccine candidates are poorly immunogenic and have shown toxicity in mice. Plasmodium can evade immune responses via a few reported mechanisms [110], therefore, non-natural (absent in infected individuals) immune responses stimulated by vaccine could overwhelm Plasmodium defense systems [111,112].
RTS,S is the first malaria vaccine able to develop significant immunity against malaria parasites. Early success with RTS,S led to reduced priority for, and less funding being directed at vaccines targeting blood-stage malaria, delaying vaccine development for infection by parasites at this stage. However, once RTS,S was found to have poor immunogenicity in women and children and increased pediatric morbidity, research interest in blood-stage malaria vaccines significantly intensified [30,113,114].
Another challenge faced in malaria vaccine development is that the common human malaria-causing parasite, P. falciparum, is not a rodent pathogen. The most common mouse models of malaria employ the rodent-specific parasite species P. berghei, P. yoelii, and P. chabaudi. These species generate unique pathologies and immune responses. While they are still employed to model various manifestations of human disease, the immune response patterns observed in these models are not fully transferable to humans [73].
For future efforts, many of the major problems in vaccine development can be minimized through the following: (a) using new adjuvants to improve immunogenicity; (b) discovery of new potent classical and cryptic epitopes [115] from a variety of P. falciparum proteins [116][117][118][119]; (c) modification to, and development of new, vaccine delivery systems, for example, nanoparticulate liposomal forms [112]; and (d) the use of multi-antigenic vaccines, including multiple epitopes from both blood and liver stages of malaria [57,119].

Conclusions
Malaria disease prevalence has a significant impact on society, resulting in high demand for vaccines against the disease. This is especially true for developing countries, where malaria is most detrimental. Low immunogenicity, adverse effects and storage limitations are some of the major challenges for malaria vaccine development. One of the most common targets for malaria vaccine development is sporozoite surface protein CSP. However, this might prove to be a "dead end" target, as a variety of vaccines containing CSP have failed to produce the desired protection.
Many vaccine candidates are currently in clinical trials. For example, ChAd63-MVA completed phase I clinical trials, showing high efficacy in the induction of cellular immunity against malaria with no evidence of dose-limiting toxicity. This vaccine is currently in phase II clinical trials. Peptide vaccines, especially those bearing strong immune stimulants, have been able to improve antibody and cellular responses in mice, but often at the expense of increased adverse effects. Therefore, extensive research has been devoted to the development of new adjuvants and new antigenic targets to minimize side-effects and increase the efficacy and immunogenicity of peptide-based malaria vaccines. In developing malaria vaccines and identifying new Plasmodium antigens, several methods are used, such as array technology, immunoinformatics, PCR deep sequencing technology and whole-proteome screening. Many antigens/proteins are yet to be utilized for vaccine development; screening for their antigenicity should be performed.

Conflicts of Interest:
The authors declare no conflict of interest.