malaria vaccine aligns with one of the four key objectives of WHO's Global Malaria Programme strategy for 2024-2030: "Developing and Delivering New Tools and Innovation [1]. This objective prioritizes the evaluation and swift rollout of promising malaria interventions. This focus on new control and prevention strategies propels WHO and its partners towards achieving the ambitious goal of eradicating malaria by 2030. This includes a 90% reduction in malaria deaths and eliminating malaria in not less than 30 countries. And of course, we wouldn’t want the disease to return to countries already free from it, places like Sri Lanka, Cabo Verde, Saudi Arabia and more. That’s the aim of WHO [1]. This article presents the history of R21/Matrix-M^TM vaccinedevelopment,its efficacy, findings from clinical trials, and its limitations.

Historical Development of Malaria Vaccines

For decades, scientists have relentlessly waged a war against malaria, a top killer disease from a single parasite. This commitment has yielded both setbacks and triumphs. The journey began in the 1960s and 70s with pre-erythrocytic vaccines on the spotlight [6,7]. In the 1970s, Clyde and colleagues launched an investigative human trial of radiation-attenuated sporozoites and observed progressive evidence, even though the trial results did not meet expectations [8].

Figure 1: Life cycle of the malaria parasite and various vaccine types targeting different stages of the life cycle [23].

During the 1980s, the first malariaantigenknown as CSPwas identified inPlasmodium falciparum, leading to the development of the subunit vaccine, RTS,S in 1987 [9,10,11]. At that time, a range of vaccines that target the erythrocytic stage of the parasite (merozoites-derived antigens) also emerged [12,13], but trials efficacy was unsatisfactory [9,14,15]. In 2009 through 2019, the

subunit vaccine candidate, RTS,S, which targets the CSP antigen,demonstrated promising efficacy in clinical trials and was approved for human use by the WHOin 2015 [3,11].However, the RTS,S was imperfectand needed tobe improvedupon. This need birthed the R21/Matrix-M^TM vaccine by the University of Oxford, UK and its partners,and was approved for use in children in 2021 [4,5,16]. The key milestones in the malaria development processare highlightedin Figure 1.

Historical Background of the R21/Matrix-M^TM Vaccine

R21 is a subunit vaccine constructed by Katherine A. Collins to overcome the limitations of the initial CSP-basedRTS,Svaccine [16]. Itwasconstructedby the fusion of the N-terminus of hepatitis B virus surface antigen (HBsAg) withPlasmodium falciparum circumsporozoite protein (PfCSP), thus the name virus-like protein (VLP) [16-18]. After R21 was constructed in 2012, researchers subsequently combined it with Matrix-M^TM adjuvant to create the protein-adjuvanted vaccine R21/Matrix-M^TM [17]. The use of the Matrix-M^TM adjuvant in combination with R21 is due to its impressive historical safety and immunogenicity when used in combination with seasonal influenza vaccines in clinical trials [19,20]. The adjuvant was also well-trusted when it was usedin the development ofvaccine against SARS-CoV-2 and Ebola virus [21,22]. The original target of the R21/Matrix-M^TM vaccine isthe CSP from theP. falciparum strain NF54 [16]. The CSP, expressed on the parasite’s sporozoites, is responsible for enhancing gliding motility and hepatocellular invasion of the malaria parasite [23-25].

Katherine A. Collins and the Jenner Institute, University of Oxford, UK, in collaboration with Novavax, Inc., developed the R21/Matrix-M^TM vaccine and manufactured it on a commercial scale by the Serum Institute of India (SII) Private Ltd [2,16]. The R21/Matrix-M^TM program was led by Professor Adrian Hill, the director of the University of Oxford’s Jenner Institute at the Nuffield Department of Medicine [2]. As of May 2024, 80 million doses of the R21/Matrix-M^TM vaccine are underway in 22 African, delivered by the GAVI vaccine alliance [26]. GAVI planned to immunize 9.7 million children by the end of 2024. The vaccine was designed to induce hyperactivation of the anti-CSP antibody specific for the antigen NANP, which is a repeated amino acid sequence, asparagine-alanine-asparagine-proline, embedded within the P. falciparum circumsporozoite protein (pfCSP) [16,27], and to be administered in low doses to cut down the cost of production [28-30]. Additionally, the vaccine is targeted at children between the ages of 3 and 5, which gives credence to its potential to protect the most vulnerable individuals from malaria [1,29].

Purpose of the R21/Matrix-M^TM Vaccine

Plasmodia sporozoites move so quickly (within ≤30–60 minutes) from the bite site to the liver that the body does not generate enough immunity to overcome this process [31,32]. The purpose of the R21/matrix-M^TMvaccineismainly to outsmart this crafty maneuver by targeting sporozoites at the pre-erythrocytic stage, which ultimately stops disease and transmission [33].

Compared to the RTS,S vaccine, which has limited CSP content and only recognizes specific P. falciparum epitopes, R21/matrix-M^TM contains a higher CSP content, offering broader epitope recognition of various Plasmodium strains [22]. The Matrix-M^TM adjuvant has a high immunostimulatory ability to induce the production of T helper cells [22], leading to a high yield of anti-CSP immunoglobulin G (IgG) antibodies that can directly neutralize sporozoites in the bloodstream, preventing their progression to invade liver cells [16,22]. The adjuvant can also stimulate antigen-presenting cells (APCs) at the site where the injection is given [22]. This enhances efficient presentation of the antigen in local lymph nodes, thus intercepting the parasite at the early stage of infection [22].

Ideally, when mosquitoes bite, they introduce hundreds to thousands of sporozoites into the victim’s skin [34,35]. The mosquito species and plasmodia strain involved determine the exact number of sporozoites to be released [34,35]. Nevertheless, since these sporozoites migrate from the bite site too quickly, the ability of the R21/Matrix-M^TM vaccine to completely clear sporozoites at the pre-erythrocytic stage may not always be achieved. This allows the parasite’s life cycle to progress, eventually leading to the clinical manifestation of symptoms. Alternatively, a multistage vaccine has been proposed to close this gap [16]. One such candidate, chimpanzee adenovirus 63 (ChAd63) and modified vaccinia Ankara (MVA), encoding CSP and ME-TRAP (Multiple-Epitope Thrombospondin-Related Adhesion Protein), demonstrates remarkable antigenicity against controlled human malaria, reducing parasite levels in the liver by an impressive 79–85% [36,37].

This is especially typical for the massive production of CD8+ cytotoxic T lymphocytes (CTLs) and interferon-gamma (IFNγ), a powerful immune signaling molecule [36,37].

Components of R21/Matrix-M^TM

R21/Matrix-M^TM is an improved version of the RTS,S vaccine. The main differences between these vaccinesare intheir CSP content, HBsAg content, adjuvant, and expression host. Table 1 summarizes the key differences between them.

Table 1: Key differences between R21 and RTS,S vaccines

In RTS,S, the CSP fragment is attached to the carrier HBsAg and co-expressed in the yeastSaccharomyces cerevisiaealong with an additional free unmodified recombinant HBsAg (S) that has a fourfold molar excess (1:4) compared to that of the attached CSP protein. This results in a greater immune response against HBsAg than against the target plasmodium CSP [3,18,38,39]. In contrast, R21 contains a CSP fragment directly fused to HBsAg and co-expressed in the yeastPichiapastoris to form a hybrid particle (CSP-HBsAg) with no additional excess HBsAg. This fusion increases the concentration of CSP and is believed to enhance the immunogenic power of the CSP antigen [11,16,18,40]. In other words, R21 contains a higher concentration of the CSP antigen per dose than does RTS,S. R21 maintains an inconsequential (if at all) immune response against the HBsAg fraction. See Figure 2. However, during the preclinical investigation, adjustments were made to resolve technical issues and make the vaccine easier to purify. This involves the addition of four amino acids, EPEA (Glu-Pro-Glu-Ala) and a c-tag (R21c), which was later removed during large-scale production of the vaccine [18,41,42]. The RTS,S is combined with adjuvant Mosquirix^TM also known as AS01. This adjuvant has two immunity triggers, namely, liposome-based monophosphoryl lipid A (MPL) derived from Salmonella minnesota and QS-21, a natural saponin molecule purified from the bark of the South American treeQuillaja saponaria [40,43]. While effective in stimulating the T-helper cells, MPL has the disadvantage of safety issues because of its bacterial origin [43]. Similarly, the R21 vaccine uses theMatrix-M^TM adjuvant, a 40 nm complex containing the saponin from Quillaja extract, phospholipids and cholesterol [40,41,43], but, it does not contain the bacterial-based MPL, offering a safer profile compared to the AS01 adjuvant [41].

Mechanism of Action of R21/Matrix-M^TM

The mechanism of action of the R21/Matrix-M^TM vaccine follows the natural principle of immunologic induction and activity through multiple pathways [22,44].

Part A: Intramuscular Injection

When the R21/Matrix-M^TM vaccine is injected into muscle tissue (event 1), it initiates rapid release of local immune cells and signalling cytokines, some of which includes interferon gamma (IFN-γ), interleukin (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) [22,44,45]. These cytokines facilitate the accumulation of immune cells, including the antigen-presenting cells (APCs) like dendritic cells (DCs), at the injection site and in the draining lymph nodes (dLNs) [22,44,46]. This initial event also triggers the release of specific chemokines. For instance, chemokines like CXCL1 and CCL2 attract monocytes and granulocytes, while CXCL9 and CXCL10 recruit T cells [22,44,45].

The Matrix-M adjuvant enhances this immune response by promoting antigen uptake, transport, and efficient presentation to T-cells by the APCs in the immune-equipped dLNs (event 2-3). When the CD4+ T cells are activated, they differentiate into three special cells (event 4): Th1 cells, which orchestrate cellular immunity; Th2 cells, which stimulate antibody production; and T follicular helper (Tfh) cells, which assist in the maturation of B cells within germinal centers (event 5). Inside these germinal centers, activated B cells interact with Tfh cells and other immune cells, leading to the selection of the most effective B cells (event 6) [22]. This process results in the production of long-lasting and memory-inducing antibodies and plasma cells that neutralize the malaria parasite (event 7). The R21 was designed to induce antibody response predominantly towards the parasites CSP [16]. Additionally, memory T-cells of CD8+ types are generated, ensuring a rapid immune response during future encounters with the parasite (event 8) [22,44].

Figure 2: Schematic diagram showing the RTS,S and R21 fusion proteins. Both RTS,S and R21 include the fusion protein of HBsAg to the C-terminus and central repeats of the CSP of P. falciparum. These repeats comprise many copies of the four amino acid sequence NANP [49,50].

Part B: After Phagocytosis

The APCs engulf the antigen (event 1) and deposit it in the phagosome. Saponin components of the matrix-m adjuvant is believed to disrupt the phagosome membrane and the antigen is translocated into the lysosomes (event 2) [22]. The antigen is processed in the lysosome by lysosomal conditions (acidity and lysosomal enzymes) into small peptide fragments and is loaded onto major histocompatibility complexes [22]. The release of saponins from Matrix-M in the lysosomes increases the permeability of the lysosomal membrane (event 3). This allows intact and partially degraded antigen fragments to escape into the cytosol (event 4). Matrix-M also induces upregulation and expression of co-stimulatory molecules (CD86, CD69) on APCs (event 5) [22], improving their ability to present antigen fragments to T-cells via two pathways: fragments of antigen in the lysosome bind to MHC class II molecules to be recognized by CD4+ T lymphocytes (event 6), whereas, intact antigens that escape lysosomal degradation are further processed in the cytosol and presented by MHC class I molecules to CD8+ T lymphocytes (event 7) [22,47]. In addition, Matrix-M activates the NLRP3 inflammasome, a crucial structure responsible for releasing pro-inflammatory cytokines like IL-1β and IL-18 (event 8) [22]. This activation also stimulates the production of other cytokines, including TNF-α and IL-6, by APCs [22]. The combined release of these cytokines amplifies the immune response, creating a robust defence against the malaria parasite.

Key Observations from Clinical Trials ofR21/Matrix-M^TM

The efficacy of the R21/Matrix-M^TM malaria vaccine was assessed in several clinical trial phases, which provided key data on the vaccine’s efficacy, safety, and immunogenicity under real-world conditions and represented the data upon which the WHO relied to recommend the use of the vaccine in children who are at greater risk of malaria [17,27,29,41,42,48-50].

Thevaccine effectively elicited immune responses specific for NANP repeats on the pfCSP for up to 12 months in children. However, the immunity lowered at 24 months [17,27,29]. Younger age groups had the highest 12-month vaccine efficacy of 79% and 80% [17,29]. This is correlated with better vaccine effectiveness at a dose ratio of 10 µg/50 µg (R21/Matrix-M^TM) administered 3 times at intervals of 4 weeks, followed by a booster shot 12 months after the third dose [17,27,29]. However, the immune induction power depends on the quality of the CSP contained in each vaccine [17]. The safety profile of the vaccine was satisfactory, with only mild reactions such as fever and pain at the injection site. Post-vaccination fevers vary from mild to none across regions and within groups included in the studies. Severe adverse effects (AEs) were mild and resolved within 48 hours [41]. These symptoms are less common with the R21/Matrix-M^TM vaccine than with the RTS,S/AS01 vaccine [3,32,51]. This combination of efficacy and safety is unique and positions the R21/Matrix-M^TM vaccine as a smartintervention for malaria prevention in children living in malaria-endemic regions.

Limitations of the R21/Matrix-M^TM vaccine

First, the ability of the R21/Matrix-M^TM malaria vaccine to completely clear Plasmodium sporozoites in the early stages of infection is limited. This is because of its long-term window for producing higher titers of antibodies. Even though the window period is not explicitly stated in clinical trials, higher titers of malaria-specific anti-NANP antibodies were observed 28 days after the third vaccination, indicating the vaccine’s delayed impact, during which the plasmodium sporozoites must have advanced [17,27,29,41]. Second, the efficacy of the R21/Matrix-M^TM vaccine was sustained for up to two years after the booster was administered [17], suggesting continued monitoring of the vaccine to establish the extent of its protection beyond this timeframe [4]. Third, R21/Matrix-M^TM vaccination should be further evaluated for potential changes in age-related effectiveness. For example, younger children (5–17 months old) had greater NANP-specific antibody titers than older children (18–36 months old), showing that age may influence the immune response and effectiveness [29]. Furthermore, participants’ voluntary withdrawals across clinical trials can explain the potential reluctance of taking the vaccine or incomplete adherence to the required doses, as already observed by Datoo and colleagues among participants in Burkina Faso [27]. Finally, the R21/Matrix-M^TM is limitedby its restricted applicability in children aged 3 to 5 years. No malaria vaccine for use in the adult population hasbeen licensed.

Conclusion

The R21/Matrix-M^TM vaccine is a revolutionary intervention against malaria. It has the ability to elicit an immune response toward the NANP central repeat of P. falciparum and intercept the Plasmodium sporozoites in the pre-erythrocytic stage of the malaria life cycle. This is a wonder the world has been expecting. The vaccine is only allowed to be used in children between the ages of 3 and 5. No definite evidence of the vaccine’s efficacy in adult. We strongly encourage continued search for malaria vaccines for the adult population to overcome inequality and form a more formidable force for the eradication of malaria globally. Furthermore, since the WHO announced the approval of the R21/Matrix-M^TM vaccine in 2021, the world seems to be less excited about this novel intervention. Could the enduring familiarity with malaria and availability of other existing interventions be the reason for this? As wider rollout is set to begin, should this be a concern? Does this factors have the potential to interfere with the R21/Matrix-M^TM uptake and/or adherence to the required doses? But the good news is that, after decades of fervent research marked by repeated setbacks, and now an efficacy of 77%, the R21/Matrix-M^TM is simply a wonder. There’s need for public awareness about this vaccine as soon as possible in order to minimize likely underutilization in targeted regions.

Abbreviations

APCs: Antigen-presenting cells

ChAd63: Chimpanzee adenovirus 63

CSP: Circumsporozoite protein

PfCSP: Plasmodium falciparum circumsporozoite protein

Tfh: T follicular helper

VLP: Virus-like protein

Supporting information: None

Ethical Considerations: Not applicable

Acknowledgments: None

Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author contribution statement: All authors (Initials of authors) contributed equally and attest they meet the ICMJE criteria for authorship and gave final approval for submission.

Data availability statement: Data included in article/supp. material/referenced in article.

Additional information: No additional information is available for this paper.

Declaration of competing interest: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Clinical Trial: Not applicable

Consent for publication: Note applicable

References