Malaria is still a significant problem in third-world countries, what advances have been made in vaccine design and what are the challenges remaining?SUMMARYIn a phase III clinical trial RTS,S/AS01 was administered once a month for three months to African children (5-17 aged months) and infants (aged 6-12 weks) with one group receiving a fourth vaccine after 20 months (RTS, 2015). The vaccine reduced cases of malaria, although protection varied with age and deteriorated with time. Whilst the vaccine efficacy (VE) was 46% and 27% for children and infants respectively after 18 months, this efficacy declined to 28% and 18% in the 36-48 month follow up. (RTS, 2015). The decline was stalled in those who received the fourth vaccine, where a 31.5% reduction in severe malaria cases was found (RTS, 2015). The fourth vaccine appears advantageous, but further longer term studies will be required to analyse whether even further vaccines will be required if vaccine efficacy continually declines with time. One longer term phase II RTS,S/AS01 study actually found a rebound in cases of malaria 5 years after vaccination in areas with higher than average malarial parasite exposure (Olotu et al., 2016), although these children did not receive the fourth vaccination. It must be noted that bed net usage was high in both trials, so there were other protective mechanisms against malaria not merely the vaccine, however it should be noted that usage varied between study sites in the phase 3 trial (RTS, 2015). An explanation for efficacy declining over time is a reduction in IgG levels. The challenge for this vaccine design is that the specific mechanism that induces protective immunity remainsINTRODUCTION Malaria is a blood infection caused by Plasmodium parasites. Five species are known to cause the disease in humans,with P. falciparum the deadliest (Cox-Singh et al., 2008; Sarkar et al., 2010).Although a decline in cases has been observed since 2010, there were still 216 million cases and 445,000 deaths in 2016, with sub-Saharan Africa accounting for 80% of these (World Health Organisation, 2017). Whilst prevention methods such as insecticidal nets and indoor residual spraying programmes are used (West et al., 2014), eradication of the disease is unlikely unless a successful vaccine is developed.Since the first malarial vaccine trial in humans that demonstrated efficacy over 45 years ago, there is currently no licenced vaccination (Clyde et al., 1973). Drastic developments have been made however, and the “Malaria Vaccine Technology Roadmap to 2030” hopes to see a vaccine with 75% protective efficacy for either P. falciparum or P. vivax (Moorthy et al., 2013). Unfortunately, the complex, multistage lifecycle of P. falciparum makes vaccine designchallenging (Figure 1).Although there are many vaccines currently being trialled (Table 1), this review will focus primarily on vaccines targeting the pre-erythrocytic phase of P. falciparum, including the most promising current vaccine, RTS,S and a next generation design, R21. The consistent hurdles faced when designing a vaccine that meets the 75% efficacy goal will be addressed, including the ambiguity of vaccine induced protective immunity.PRE-ERYTHROCYTIC SUBUNIT VACCINATIONSWith the migration of P. falciparium sporozoites from the site of infection to the liver taking just minutes, an immediate, effective immune response is essential (Sidjanski & Vanderberg, 1997; Shin et al., 1982). Pre-erythrocytic vaccinations (PEV) aim to prevent sporozoites infecting the liver (Figure 1), to prevent blood-stage infection. As P. falciparum presents many antigens, vaccines have many possible targets, although it is difficult to design a successful vaccine as most antigens are polymorphic and exhibit clonal variation (Mwangoka et al., 2013).The current leader in the race to an approved vaccine targets the P. falciparum circumsporozoite protein (PfCSP), which is expressed on the surface of sporozoites (Figure 2). Designed to incorporate both the hepatitis B surface antigen (HBsAg) and the carboxy-terminal segment of PfCSP in a 4:1 ratio, the vaccine is known as RTS,S (Draper et al., 2018; Nielsen et al., 2018). Delivery of RTS,S requires an adjuvant as non-adjuvant RTS,S is weak at provoking an immune response (Leroux-Roels et al., 2014); when formulated with AS01 18% higher efficacy is achieved than with AS02 (Kester et al., 2009). RTS,S/AS01 has since gone on to demonstrate protective efficacy in phase III clinical trials (RTS, 2015).RTS,S/AS01 CLINICAL TRIALSIn a phase III clinical trial, RTS,S/AS01 was administered at months 0, 1 and 2 to African children (aged 5-17 months) and infants (aged 6-12 weeks), with some receiving a fourth dose in month 20 (RTS, 2015). The vaccine overall reduced cases of malaria, although protection varied with age and deteriorated with time. Whilst the vaccine efficacy (VE) was 46% and 27% for children and infants respectively after 18 months, this efficacy had declined to 28% and 18% by the 36-48 month follow up (RTS, 2015). The decline was stalled in those who received the fourth vaccine, where efficacy was 39% at follow up (RTS, 2015). Whilst this data suggests a fourth “booster” vaccine is advantageous, longer term studies will be required to assess how many doses will overall be required. With continually declining efficacy comes an increased cost of extra doses and monitoring each vaccinated child long term, which will challenge the wide scale use of RTS,S. One longer term phase II RTS,S/AS01 study actually found a rebound in malarial cases 5 years after vaccination in areas with higher than average malarial parasite exposure (Olotu et al., 2016), although these children did not receive the fourth vaccination. Bed net usage was high in both trials, however usage was inconsistent between study sites in the phase 3 trial, meaning individuals may have had differing exposures to malaria throughout the study compared with their predicted exposure level (RTS, 2015).An explanation for the waning efficacy over time is decreasing IgG levels. A challenge facing RTS,S is that the mechanism through which it induces protective immunity remains unknown, although evidence suggests it increases the concentration of anti-NANP IgGs, which positively correlates with associated protection (White et al., 2015; Kazmin et al., 2017). Other studies have shown RTS,S/AS01 induces moderate CD4+ T cell responses (Kazmin et al., 2017; Kester et al., 2009; Owusu-Agyei et al., 2009;), although challenge studies to determine this have so far been on a small scale.  Maintaining high IgG levels therefore appears to be the aim to improve efficacy, which could be achieved with novel adjuvants or potentially by altering dose quantity and time between doses. Regules et al., (2016) found 86% of volunteers were protected when given doses at months 0, 1 and 7, with the third dose at 20% of the original. Manipulating dose timings and concentrations may improve vaccine efficacy, however this study highlights a problem often seen in malarial vaccination development, using volunteers not from areas of high malarial prevalence. Western travellers represent a small proportion of the malarial vaccine target population, yet research is predominantly carried out on them, wh.Although the RTS,S vaccine will begin pilot implementation in 2019, it will not completely eradicate malaria. A 39% peak efficacy at phase 3 is lower than that of common vaccines, and the “Malaria Vaccine Technology Roadmap to 2030” goal (Mahmoudi and Keshavarz, 2017; Moorthy et al., 2013). Determining the vaccine immunity mechanism should be a focus, so RTS,S design can be manipulated to achieve sustained high IgG concentrations. Further larger scale studies investigating dosing intervals and quantities should be carried out in Africa, to build upon early promising results found by Regules et al., (2016).R21: A NEXT GENERATION VACCINEA next generation vaccine R21 gets its design basis from RTS,S, although was developed to be more immunogenic. Where RTS,S was composed of truncated CSP and HBsAg in a 1:4 ratio (Figure 2), R21 particles are formed using a 1:1 ratio. The novel design incorporates a higher proportion of PfCSP, thought to enhance both antigen recognition by B cell receptors and antibody production. In mouse models using R21 very high NANP-specific IgG levels were found at doses as small as 0.5 ?g (Collins et al., 2017). Investigations proved high NANP-specific IgG titres can be achieved when R21 is given in 3 doses 8 weeks apart, which will be optimal when combined with viral vectors (Collins et al., 2017). Further, almost complete sterile immunity was found with a Matrix M adjuvant after infection with P. berghei (a rodent malaria parasite which expresses PfCSP), higher than other adjuvants, including MF59. Interestingly however, the titres of NANP-specific IgG did not differ significantly with either adjuvant, suggesting that high levels of IgG are not solely responsible for protective efficacy (Collins et al., 2017), which is a focus of the RTS,S vaccine discussed earlier. Phase 1b clinical trials for R21 will be interesting to see if the promising efficacy found in mice can be replicated in humans (ClinicalTrials.gov, 2018). If found to be the case, this RTS,S adapted, higher proportion PfCSP vaccine offers a potential highly efficacious malaria vaccine.A NEW APPROACH: COMBINING VACCINES Combining vaccine types to target multiple stages of the malaria lifecycle (Figure 1) has been proposed and preliminarily tested to combat the low efficacy commonly observed with PEVs (Sherrard-Smith et al., 2018). Characteristically, as mentioned previously, PEV use and development is challenged because only partial malarial protection is achieved despite high antibody titres (Collins et al., 2017). This could be because the probability of at least one sporozoite escaping immune detection and reaching the liver is high, which can then go on to replicate and cause blood-stage infection (White et al., 2013). Transmission blocking vaccines (TBVs), have been shown to reduce the sporozoite density in mosquito saliva (Bompard et al., 2017) and reduce transmission to mosquitoes (Hoffman et al., 2015); by using TBVs in conjunction with PEVs, it was hypothesised that a reduction in the number of parasites infecting a person would allow PEVs to work more effectively.Sherrard-Smith et al., (2018) demonstrated the TBV and PEV combination by passing P. berghei between mice through the direct feeding of Anopheles mosquitoes, and found a combined efficacy of 90.8%, greater than estimated. Further, the lower the parasite load, the higher the PEV efficacy.  This shows combining relatively efficacious vaccines may be an option to drawing out the declining reduction in efficacy experienced with PEV alone, although this may be dependent on vaccine type, with Rampling et al., (2016) combined RTS,S and a liver stage sununit vaccination and finding no improved protection.but longer term studies are needed to assess the rate of IgG decline. Although this offers promise, it is a preliminary study in mice, and the effects are yet to be replicated in humans, but will no doubt be an avenue explored in future.CONCLUDING REMARKSThis review has focussed on pre-erythrocytic vaccines against P. falciparum, however it is clear this has a wider scope in developing a successful vaccine for Malaria. With 4 other species causing malaria, each with complex multistage lifecycles, the big challenge is finding an efficacious vaccine against the highly diverse strains. 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