Modern malaria vaccine development began with seminal studies in mice using irradiated sporozoites (). Although there is still no licensed product over 50 years later, it is important to remember the scale of the scientific and technical challenges facing those who develop vaccines against such a complex eukaryotic parasite. Moreover, steady progress is being made, especially with regard to breakthroughs in our understanding of the cellular and molecular mechanisms mediating protection in animal models and humans. The revised Malaria Vaccine Technology Roadmap to 2030 () now calls for a next-generation vaccine to achieve 75% efficacy over 2 years against P. falciparum and/or P. vivax (in an era of renewed global interest toward malaria elimination and eradication), while also retaining its original 2015 “landmark” goal of a first-generation vaccine with protective efficacy of >50% lasting more than 1 year. Achieving this next-generation vaccine goal will necessitate building on the success of current pre-erythrocytic subunit and whole sporozoite-based vaccines, as well as new strategies to add blood-stage or transmission-blocking immunity. Here we review the progress and prospects for a diverse range of approaches targeting different stages of the P. falciparum parasite’s complex life cycle ( Figure 1 ), before discussing those in development for P. vivax.

Data sources for this figure included the WHO Malaria Vaccine Rainbow Table and Clinicaltrials.gov . Vaccines for P. vivax are colored blue. The life cycle figure was adapted from

Sporozoite Subunit Vaccines

Rts, 2015 RTS,S Clinical Trials Partnership

Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Olotu et al., 2016 Olotu A.

Fegan G.

Wambua J.

Nyangweso G.

Leach A.

Lievens M.

Kaslow D.C.

Njuguna P.

Marsh K.

Bejon P. Seven-year efficacy of RTS,S/AS01 malaria vaccine among young African children. WHO, 2016 WHO

Malaria vaccine: WHO position paper-January 2016. Klein et al., 2016 Klein S.L.

Shann F.

Moss W.J.

Benn C.S.

Aaby P. RTS,S malaria vaccine and increased mortality in girls. The most extensively tested vaccine candidate for prevention of P. falciparum malaria is RTS,S/AS01; this vaccine directs immune responses against the major circumsporozoite protein (PfCSP) covering the surface of the infecting sporozoite. To accomplish this, RTS,S was designed as a virus-like particle (VLP) comprised of two components: 18 copies of the central repeat and the C-terminal domain of PfCSP fused to hepatitis B virus surface antigen (HBsAg) with extra HBsAg in a 1:4 ratio. RTS,S, formulated with the potent liposomal adjuvant system AS01 from GlaxoSmithKline, is the only vaccine that has demonstrated protective efficacy against clinical malaria in a Phase III clinical trial (), although protection is partial, wanes over time, and may be age dependent (protection was lower in infants 6–12 weeks of age than in young children 5–17 months old). In the latter, receiving three vaccinations in a 0-1-2 month schedule, the incidence of clinical malaria was reduced by 51% over the first year of follow-up post-dose three [95% CI 48%–55%]. Over 48 months of follow-up, efficacy was 26% [95% CI 21%–31%], and among children receiving a fourth dose at month 20 (18 months post-dose three), efficacy was 39% [95% CI 34%–43%]. A small Phase II study, which followed several hundred children who received the three-dose regimen over 7 years, suggests that there may also be a shifting or rebound in malaria incidence 5 years post-vaccination (). Results of a larger long-term follow-up study to the Phase III efficacy and safety trial are expected later this year. According to the World Health Organization (WHO), two safety signals (meningitis, cerebral malaria) emerged from the Phase III trial, for which the cause is unknown and they noted a confirmed risk of febrile convulsions within 7 days of vaccination in the 5–17 month age category, all of which resolved without long-term sequelae (). Following a positive opinion from European regulatory authorities in July 2015, WHO recommended large-scale pilot implementations to further evaluate the feasibility of delivering four doses, the vaccine’s potential for reducing childhood deaths, and to provide additional data on safety in the context of routine use. The pilot implementation program will include robust safety surveillance of these and other safety signals () that could not be adequately assessed in the Phase III trial due to very low mortality in the trial overall.

White et al., 2015 White M.T.

Verity R.

Griffin J.T.

Asante K.P.

Owusu-Agyei S.

Greenwood B.

Drakeley C.

Gesase S.

Lusingu J.

Ansong D.

et al. Immunogenicity of the RTS,S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Oyen et al., 2017 Oyen D.

Torres J.L.

Wille-Reece U.

Ockenhouse C.F.

Emerling D.

Glanville J.

Volkmuth W.

Flores-Garcia Y.

Zavala F.

Ward A.B.

et al. Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein. Scally et al., 2018 Scally S.W.

Murugan R.

Bosch A.

Triller G.

Costa G.

Mordmüller B.

Kremsner P.G.

Sim B.K.L.

Hoffman S.L.

Levashina E.A.

et al. Rare PfCSP C-terminal antibodies induced by live sporozoite vaccination are ineffective against malaria infection. + T cell responses have been suggested to have some role in protection ( Kazmin et al., 2017 Kazmin D.

Nakaya H.I.

Lee E.K.

Johnson M.J.

van der Most R.

van den Berg R.A.

Ballou W.R.

Jongert E.

Wille-Reece U.

Ockenhouse C.

et al. Systems analysis of protective immune responses to RTS,S malaria vaccination in humans. + T cell epitopes, and a genetic analysis of the Phase III trial indicates a significant sieving effect with modestly lower efficacy for C-terminal sequence unmatched strains ( Neafsey et al., 2015 Neafsey D.E.

Juraska M.

Bedford T.

Benkeser D.

Valim C.

Griggs A.

Lievens M.

Abdulla S.

Adjei S.

Agbenyega T.

et al. Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine. Figure 2 CHMI Models for Vaccine Efficacy Testing Show full caption Payne et al., 2016 Payne R.O.

Milne K.H.

Elias S.C.

Edwards N.J.

Douglas A.D.

Brown R.E.

Silk S.E.

Biswas S.

Miura K.

Roberts R.

et al. Demonstration of the blood-stage Plasmodium falciparum controlled human malaria infection model to assess efficacy of the P. falciparum apical membrane antigen 1 vaccine, FMP2.1/AS01. Collins et al., 2018 Collins K.A.

Wang C.Y.

Adams M.

Mitchell H.

Rampton M.

Elliott S.

Reuling I.J.

Bousema T.

Sauerwein R.

Chalon S.

et al. A controlled human malaria infection model enabling evaluation of transmission-blocking interventions. Blood-stage parasitemia is monitored by qPCR with lower limit of quantification ∼20 parasites/mL blood (black dotted line), typically for a 21 day study period. Malaria-naïve volunteers are usually diagnosed and treated at ∼10,000 parasites/mL when patent by thick-film microscopy (black dashed line). Following sporozoite CHMI, sterile protection is measured or a partial vaccine effect can be assessed by analysis of the liver-to-blood inoculum (LBI) leading to a delay in time to diagnosis. For blood-stage CHMI, an inoculum of ∼1,000 parasites is administered IV on day 0, with parasites growing ∼10-fold per 48 hr (red line) (). For an effective blood-stage vaccine, a reduction in the parasite multiplication rate (PMR) would be expected. To assess transmission, CHMI is initiated by sporozoites or blood-stage inoculum (the figure depicts the latter). A low-dose drug regimen is used to treat asexual parasitemia, followed by a curative regimen if recrudescence occurs. A wave of gametocytemia then ensues, with all volunteers receiving drug treatment to clear parasites at the end of the study period (). One of the most important imperatives for future improvements to RTS,S/AS01, and all next-generation malaria vaccines, is to extend the period of protection, which will require further understanding the mechanisms of vaccine-induced efficacy. While a definitive immune mechanism remains to be determined for RTS,S/AS01, the existing data strongly suggest that high antibody concentrations against the NANP amino acid repeats are closely associated with protection, and waning of such responses is likely to be responsible for decreasing efficacy (). A direct mechanistic link between a monoclonal antibody (mAb) against the NANP epitope (isolated from a subject immunized with RTS,S []) and protection will be tested soon following passive transfer and controlled human malaria infection (CHMI) ( Figure 2 ). RTS,S also contains the C-terminal region of PfCSP; however, the role of antibody responses to this region remains unclear as a recent study showed that a number of such mAbs, obtained from a human subject immunized with sporozoites, are not protective in a mouse model (). In addition to antibody, CD4T cell responses have been suggested to have some role in protection (). In some support of this, the C terminus of PfCSP, present in RTS,S, contains two well-defined CD4T cell epitopes, and a genetic analysis of the Phase III trial indicates a significant sieving effect with modestly lower efficacy for C-terminal sequence unmatched strains (). However, a definitive immune mechanism for this effect remains to be determined, and for now the major limitation of RTS,S appears to be maintaining the high antibody levels.

Kester et al., 2009 Kester K.E.

Cummings J.F.

Ofori-Anyinam O.

Ockenhouse C.F.

Krzych U.

Moris P.

Schwenk R.

Nielsen R.A.

Debebe Z.

Pinelis E.

et al. RTS,S Vaccine Evaluation Group

Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. White et al., 2015 White M.T.

Verity R.

Griffin J.T.

Asante K.P.

Owusu-Agyei S.

Greenwood B.

Drakeley C.

Gesase S.

Lusingu J.

Ansong D.

et al. Immunogenicity of the RTS,S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Francica et al., 2017 Francica J.R.

Zak D.E.

Linde C.

Siena E.

Johnson C.

Juraska M.

Yates N.L.

Gunn B.

De Gregorio E.

Flynn B.J.

et al. Innate transcriptional effects by adjuvants on the magnitude, quality, and durability of HIV envelope responses in NHPs. It therefore seems critical for future PfCSP-based subunit vaccines to take into account the parameters of vaccine-induced antibody concentration decay. RTS,S is administered with the adjuvant AS01—the leading formulation for induction of high antibody concentrations in humans. Peak polyclonal anti-NANP serum IgG responses after the final immunization averaged ∼150 μg/mL in malaria-naive adults (), and in the Phase III trial antibody levels declined sharply with initial half-life of ∼40 days followed by a period of slower decline of ∼600 days (). The requirement for such high and sustained antibody concentrations to mediate durable protection poses a substantial challenge. The impact of novel adjuvants and vaccine delivery platforms may help to provide a solution in the future, especially if they can be demonstrated to skew immune responses toward improved induction of long-lived plasma cells. Antibody decay parameters for an HIV envelope protein delivered with eight different clinical adjuvants have been studied in non-human primates (NHP) (), and the data demonstrate some differences in how adjuvants can influence durability. Nevertheless, improving upon the magnitude of antibody responses by adjuvants other than AS01 may be difficult to achieve. Consequently, an alternative strategy to achieve sustained protection with lower antibody concentrations is by improving the potency or breadth of the polyclonal antibody (pAb) response. The induction of pAb functional at lower concentrations should result in improved vaccine efficacy during a longer segment of the IgG decay curve. Altered vaccine regimens or identification of new neutralizing epitopes on PfCSP could guide such an approach, given that RTS,S does not contain the N-terminal region and portions of the repeat region. Similarly, a more detailed understanding of the contribution made by antibody Fc-mediated effector functions as well as binding parameters, such as affinity, to sporozoite blockade will be important when seeking to design vaccines that induce optimal pAb potency.

Regules et al., 2016 Regules J.A.

Cicatelli S.B.

Bennett J.W.

Paolino K.M.

Twomey P.S.

Moon J.E.

Kathcart A.K.

Hauns K.D.

Komisar J.L.

Qabar A.N.

et al. Fractional third and fourth dose of RTS,S/AS01 malaria candidate vaccine: a phase 2a controlled human malaria parasite infection and immunogenicity study. Interestingly, the most recent advance in RTS,S/AS01 vaccine development has been made through a modification of dose and schedule: 10/16 (62%) volunteers given the full dose at the standard 0-1-2 month regimen were protected against CHMI 3 weeks after the last immunization; in contrast, 26/30 (86%) volunteers were protected when the third vaccine administration occurred 6 months after the second with the dose reduced to one-fifth of the original dose (“fractional dose,” Fx). Immunological analysis to determine the mechanism for these findings showed that the titers of anti-NANP antibody were similar between the two groups but the Fx regimen may affect antibody avidity, somatic hypermutation, and isotype switching (). While it remains unclear whether the prolonged interval and/or the reduced dose of the final vaccine are mediating these effects, the data raise interesting questions about how this simple alteration controls antibody quality. However, it is important to note that while short-term protection was strikingly improved with the Fx regimen, only 3/7 subjects were protected following secondary CHMI 8 months later. These data suggest that durability of protection will need to be further assessed in future clinical trials and likely improved and, importantly, it remains to be determined whether this altered schedule will lead to improved protection in the field.

Collins et al., 2017 Collins K.A.

Snaith R.

Cottingham M.G.

Gilbert S.C.

Hill A.V.S. Enhancing protective immunity to malaria with a highly immunogenic virus-like particle vaccine. Genito et al., 2017 Genito C.J.

Beck Z.

Phares T.W.

Kalle F.

Limbach K.J.

Stefaniak M.E.

Patterson N.B.

Bergmann-Leitner E.S.

Waters N.C.

Matyas G.R.

et al. Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013. Espinosa et al., 2015 Espinosa D.A.

Gutierrez G.M.

Rojas-López M.

Noe A.R.

Shi L.

Tse S.W.

Sinnis P.

Zavala F. Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. Important new information will also emerge from clinical testing of next-generation vaccine designs. The R21 vaccine is composed of a single subunit (equivalent to “RTS” alone without the 4-fold excess HBsAg) (). The display of a higher proportion of PfCSP and less HBsAg per VLP may lead to improved anti-NANP IgG responses in comparison to RTS,S. Clinical testing is underway using Matrix-M or AS01 adjuvants. In an alternative approach, full-length PfCSP containing the N-terminal non-repeat region has been manufactured for clinical testing (), providing the potential to induce antibodies against additional epitopes (not present in RTS,S) that show anti-parasitic function in mice ().