5. Current status of malaria vaccine research

The development of effective vaccines against eukaryotic organisms is far from easy and has been particularly difficult for P. falciparum, a protozoan parasite characterized by three genomes (nuclear, mitochondrial, and apicoplastid), an adenine-thymine rich (~80%) nuclear genome [16] encoding >5000 genes, and a complex lifecycle involving several developmental stages between vertebrate and invertebrate hosts. Malaria vaccine development has been hampered by several factors during a century of effort, including the genetic diversity of P. falciparum, complexity of its biology, and difficulty obtaining long-lasting effective immunity. Interestingly, adults living in hyperendemic settings are continuously exposed to infective mosquito bites and naturally acquire immunity that controls parasitemia and reduces clinical episodes of malaria over time. Responses against some parasite proteins have been associated with this natural protection, which makes them promising vaccine targets [17].

confer protection that reduces malaria episodes, disease severity, and/or parasitemia. Additionally, immunity against VAR2CSA, a member of the P. falciparum erythrocyte membrane protein 1 family that binds to chondroitin sulfate A, may prevent placental malaria [14].

Vaccines that target the sexual stages, known as TBVs, are the focus of this chapter. TBVs do not directly protect immunized individuals but specifically block onward transmission by preventing mosquito infection. TBVs utilize antigens expressed during mosquito parasite stages (gametocytes, gametes, zygotes and ookinetes) to induce functional antibodies that attack the parasite in the mosquito and impair its viability, inhibit its development, or impede its interaction with the mosquito midgut. The effector antibody responses involved in these types of vaccines include neutralization and complement-mediated lysis. A broader concept coined as a Vaccine to Interrupt Malaria Transmission (VIMT) by the Malaria Eradication Research Agenda (MalERA) includes not only TBVs but also pre-erythrocytic and blood stage vaccines, as well as mosquito molecules involved in parasite development [15] such as Anoph-

Ideally, TBVs will elicit effective antibodies that prevent malaria parasite development in mosquitoes after uptake of blood meals. This will reduce the number of circulating infectious mosquitoes below a threshold that sustains transmission. TBVs are among the tools being encouraged for use during pre-elimination and elimination phases of malaria eradication according to malERA [15] and could be an effective alternative or adjunct to vector control. Compared to vector control interventions, TBVs are ecologically safer, cost-effective, and

Most TBV antigens are genetically conserved, which may be due to limited immune pressure. The effect of immune pressure exerted by TBV against the parasite remains unknown and will need to be monitored in future. Notably, sexual stages are critical for the generation of parasite genetic diversity and regulation of parasite virulence, hence the effects of TBVs on these phenomena also warrant monitoring. In addition, malaria parasites experience a considerable population bottleneck in the mosquito for only a handful of parasite zygotes progressing to oocysts on the mosquito midgut. Altogether, while these observations make the mosquito phase an attractive target for vaccine development, much remains to be done to achieve

The development of effective vaccines against eukaryotic organisms is far from easy and has been particularly difficult for P. falciparum, a protozoan parasite characterized by three genomes (nuclear, mitochondrial, and apicoplastid), an adenine-thymine rich (~80%) nuclear genome [16] encoding >5000 genes, and a complex lifecycle involving several developmental stages between vertebrate and invertebrate hosts. Malaria vaccine development has been hampered by several factors during a century of effort, including the genetic diversity of P. falciparum, complexity of its biology, and difficulty obtaining long-lasting effective immunity. Interestingly, adults living in hyperendemic settings are continuously exposed to infective

eles gambiae aminopeptidase 1 (AnAPN1), carboxypeptidase, and saglin.

readily enable high coverage of populations.

366 Towards Malaria Elimination - A Leap Forward

implementable and effective TBVs.

5. Current status of malaria vaccine research

Today, the most advanced malaria vaccine is RTS,S, a pre-erythrocytic stage vaccine consisting of a virus-like particle (VLP) that displays hepatitis B surface antigen alone (S) and fused with a P. falciparum circumsporozoite protein fragment containing its central repeats and T cell epitopes (RTS). RTS,S has completed Phase III clinical trial (vaccine given to thousands of people and tested for efficacy and safety) and showed an efficacy of 51.3% (95%CI, 47.5–54.9) against clinical malaria in 5- to 17-month children over 12 months after three doses of the vaccine. A fourth dose was required to sustain protection over longer periods [18]. RTS,S is currently in pilot implementation studies involving 360,000 young children, expected to be given the vaccine in Ghana, Kenya, and Malawi. Although this represents important progress given the absence of any other human vaccine against a eukaryotic pathogen, more research is needed to develop vaccines that meet the Malaria Vaccine Technology Roadmap goals of 50% efficacy against severe malaria for more than one year and ≥75% long lasting efficacy against clinical malaria. For example, alternative dosages, timing and number of doses, are being evaluated as strategies to improve RTS,S efficacy [19, 20].

Attenuated whole SPZ vaccines have shown high-level sterile protection (>90%) against homologous challenge in early clinical trials [21] and thus have been heralded as a promising malaria vaccine approach. The concept of immunization using the whole SPZ was first attempted in 1910 by the French scientist Sergent using an avian model of malaria [22]. Several decades later, protective immunity was induced in mice following inoculation of X-irradiated SPZ of P. berghei [12]. In 1973, this approach was shown to be protective in humans, using Xirradiated SPZ of P. falciparum to vaccinate, followed by challenge with the non-irradiated homologous strain delivered by mosquito bites [23]. More recently, inoculation of nonattenuated fully infectious SPZ from chemo-sensitive strains along with administration of effective antimalarial drugs, known as chemoprophylaxis vaccination, was shown to induce sterilizing immunity [24]. Immunity induced by chemoprophylaxis vaccination is dosedependent and requires substantially smaller SPZ inocula compared to irradiated SPZ [25].

Finally, genetic attenuation of parasites through the deletion of liver developmental stagespecific genes by homologous recombination is also being pursued to generate whole SPZ vaccines [26]. Numerous technologies may generate genetically attenuated parasite vaccines, including flippase (Flp)/Flp recognition target, Cre/loxP recombination, zing-finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated protein 9 (CRISPR/Cas9) system [27–30]. Genetic attenuation enables generation of parasites that arrest at late liver stages, exposing a broader liver stage-specific antigen repertoire to the immune system over a longer duration. However, genetic attenuation can be incompletely effective for preventing breakthrough to blood stage parasitemia, and this needs to be monitored carefully in clinical studies. Further, the requirement for mosquitoes to deliver SPZ vaccines had been considered as an insuperable obstacle to development of a whole SPZ vaccine for mass immunization. This obstacle has been partially overcome by the production of purified, aseptic, and cryopreserved SPZ for syringe injection by Sanaria Inc. [31].

The blood stage is another important focus for malaria vaccine research, as this stage is responsible for the clinical manifestations of malaria. People living in endemic areas are repeatedly exposed to blood stage parasites and acquire protective antibodies over years that control parasitemia and prevent disease; consequently, a blood stage vaccine can be composed of antigens targeted by naturally acquired immunity that prevent clinical episodes of malaria [17, 32]. For example, immune responses to combinations of merozoite antigens were associated with 100% protection against clinical episodes of malaria in Kenyan observational cohorts [17]. Unfortunately, the efficacy of merozoite antigen vaccines in interventional trials has been poor, limited in part by antigenic diversity, which must be overcome for effective straintranscending vaccines [33, 34].

in E. coli [49, 50] or modified lichenase carrier (LiKM) produced in Nicotiana benthamiana [50]). Viral vector vaccines, such as Chad63/Modified Vaccinia Ankara, are also being assessed to

Adjuvants, such as Alhydrogel® and Montanide®, have been used for clinical trials of TBVs with reactogenicity issues observed with both; however, recent trials of Alhydrogel®-formulated TBV have demonstrated good safety and reactogenicity profiles. Recently, GSK®'s liposomal adjuvant AS01 has been considered for TBVs. AS01 incorporates the TLR4 ligand MPL and the saponin derivative QS-21, and because AS01 is used for formulating the pre-

TBV candidates that are in clinical and preclinical developments are summarized in Table 1. Human studies of Pfs25 and Pvs25 showed that priming doses of the vaccines do not induce detectable antibody levels [42, 51]. Antibody production is measurable after the first boost and then rapidly declines; additional boosts are required to retain antibody titers. However, the raised antibodies have been proven to be functional, i.e. capable of reducing oocyst formation in mosquito-feeding assays, and this activity strongly correlates to antibody titer [42, 51]. In addition to Pfs25/Pvs25 candidates, Pfs230 conjugated to EPA and adjuvanted in Alhydrogel® or AS01 has advanced to clinical trials for evaluation either alone or in coadministration with

development

Malaria Transmission-Blocking Vaccines: Present Status and Future Perspectives

http://dx.doi.org/10.5772/intechopen.77241

369

Phase 1 NCT02532049

Preclinical [55, 63]

Subunit vaccine Phase 1 NCT02942277

Subunit vaccine Phase 1 NCT02334462

Clinical trial identifier or reference number

erythrocytic vaccine RTS,S, this would simplify future efforts to combine products.

Pfs25 (https://clinicaltrials.gov/, trials NCT02334462 and NCT02942277).

Pfs25-EPA/Alhydrogel® Subunit vaccine Phase 1 NCT01867463, 51 Pfs25 VLP-FhCMB VLP vaccine Phase 1 NCT02013687

> Viral vector & nanoparticle vaccines

Pfs25-Pfs25 Conjugate vaccine Phase 1 NCT00977899

Pfs25 & Pvs25/Montanide ISA 51 Subunit vaccine Phase 1 [43] Pvs25H/Alhydrogel® Subunit vaccine Phase 1 [42]

Plant-Produced Pfs230 LiKM Subunit vaccine Preclinical [57] Pfs48/45 Subunit vaccine Preclinical [58, 59] Pvs48/45 DNA vaccine Preclinical [60] Pvs47 DNA vaccine Preclinical [60] Pfs28 Subunit vaccine Preclinical [61, 62]

vaccine

AnAPN1 Subunit vaccine Preclinical [59, 64]

Table 1. Transmission-blocking vaccine (TBV) candidates in clinical and preclinical developments.

PfHAP2 Subunit or viral vector

Vaccine candidate Type Stage of

improve immunogenicity [49].

Pfs25M-EPA/AS01 and/or Pfs230D1M-

ChAd63 Pfs25-IMX313+/-MVA Pfs25-

Pfs230D1M-EPA/Alhydrogel® and/or Pfs25

EPA/AS01

IMX313

EPA/Alhydrogel®

#### 6. Transmission-blocking vaccine (TBV) development

The first demonstration of antibodies that prevented mosquito infection was reported in 1958 using the avian species Plasmodium gallinaceum [35]. However, it remained until 1976 for studies to show that such antibodies might recognize gamete proteins and therefore act against the parasite in the invertebrate rather than vertebrate host [36, 37]. These gamete proteins were subsequently characterized to be P230 and P48/45, and later the zygote/ookinete surface proteins P28 and P25 were shown to be TBV targets; "P" refers to Plasmodium (the antigens have homologs in all Plasmodium species to date), and the number refers to their molecular weights on SDS-PAGE [38, 39]. P28 and P25 are paralogs, most abundant on the surface of zygotes and ookinetes, glycosylphosphatidylinositol (GPI)-anchored, and involved in ookinete formation. Today, these four parasite proteins represent the leading TBV candidates. Ookinete-secreted proteins have also been identified as targets for TBVs, including chitinase 1, von Willebrand factor-A domain-related protein, thrombospondin-related anonymous protein-related protein, membrane-attack ookinete protein, secreted ookinete adhesive protein (SOAP), and celltraversal protein for ookinetes and sporozoites (CelTOS) [40, 41].

TBVs that have reached human clinical trials include only Pfs25 and its Plasmodium vivax ortholog Pvs25, and Pfs230. Early clinical trials of Pfs25 and Pvs25 yielded poor results due to either poor production of antibodies with transmission-blocking activity or to significant reactogenicity attributed to adjuvant formulations [42–44]. These challenges have been addressed by advances in vaccine expression systems, delivery platforms, and adjuvant formulations. Production of recombinant TBV antigen has been assessed in numerous systems, including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and baculovirus/insect cells, to yield better-folded proteins that are stable in solution and recreate conformational epitopes. Pfs230 and Pfs45/48 vaccines in particular are hampered by difficulty in expressing them in their appropriate conformations. To overcome this, research has focused on the expression of immunogenic fragments rather than full-length proteins [45, 46].

Several approaches to vaccine particle preparation have also been pursued to increase immunogenicity. These include conjugation to carriers (such as Pseudomonas aeruginosa exoprotein A (EPA) [47] and bacterial outer membrane protein complex (OMPC) [48]) or fusion to partners that complex to generate particles (such as C4 bp oligomerization domain (IMX313) expressed in E. coli [49, 50] or modified lichenase carrier (LiKM) produced in Nicotiana benthamiana [50]). Viral vector vaccines, such as Chad63/Modified Vaccinia Ankara, are also being assessed to improve immunogenicity [49].

The blood stage is another important focus for malaria vaccine research, as this stage is responsible for the clinical manifestations of malaria. People living in endemic areas are repeatedly exposed to blood stage parasites and acquire protective antibodies over years that control parasitemia and prevent disease; consequently, a blood stage vaccine can be composed of antigens targeted by naturally acquired immunity that prevent clinical episodes of malaria [17, 32]. For example, immune responses to combinations of merozoite antigens were associated with 100% protection against clinical episodes of malaria in Kenyan observational cohorts [17]. Unfortunately, the efficacy of merozoite antigen vaccines in interventional trials has been poor, limited in part by antigenic diversity, which must be overcome for effective strain-

The first demonstration of antibodies that prevented mosquito infection was reported in 1958 using the avian species Plasmodium gallinaceum [35]. However, it remained until 1976 for studies to show that such antibodies might recognize gamete proteins and therefore act against the parasite in the invertebrate rather than vertebrate host [36, 37]. These gamete proteins were subsequently characterized to be P230 and P48/45, and later the zygote/ookinete surface proteins P28 and P25 were shown to be TBV targets; "P" refers to Plasmodium (the antigens have homologs in all Plasmodium species to date), and the number refers to their molecular weights on SDS-PAGE [38, 39]. P28 and P25 are paralogs, most abundant on the surface of zygotes and ookinetes, glycosylphosphatidylinositol (GPI)-anchored, and involved in ookinete formation. Today, these four parasite proteins represent the leading TBV candidates. Ookinete-secreted proteins have also been identified as targets for TBVs, including chitinase 1, von Willebrand factor-A domain-related protein, thrombospondin-related anonymous protein-related protein, membrane-attack ookinete protein, secreted ookinete adhesive protein (SOAP), and cell-

TBVs that have reached human clinical trials include only Pfs25 and its Plasmodium vivax ortholog Pvs25, and Pfs230. Early clinical trials of Pfs25 and Pvs25 yielded poor results due to either poor production of antibodies with transmission-blocking activity or to significant reactogenicity attributed to adjuvant formulations [42–44]. These challenges have been addressed by advances in vaccine expression systems, delivery platforms, and adjuvant formulations. Production of recombinant TBV antigen has been assessed in numerous systems, including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and baculovirus/insect cells, to yield better-folded proteins that are stable in solution and recreate conformational epitopes. Pfs230 and Pfs45/48 vaccines in particular are hampered by difficulty in expressing them in their appropriate conformations. To overcome this, research has focused on the expression of

Several approaches to vaccine particle preparation have also been pursued to increase immunogenicity. These include conjugation to carriers (such as Pseudomonas aeruginosa exoprotein A (EPA) [47] and bacterial outer membrane protein complex (OMPC) [48]) or fusion to partners that complex to generate particles (such as C4 bp oligomerization domain (IMX313) expressed

transcending vaccines [33, 34].

368 Towards Malaria Elimination - A Leap Forward

6. Transmission-blocking vaccine (TBV) development

traversal protein for ookinetes and sporozoites (CelTOS) [40, 41].

immunogenic fragments rather than full-length proteins [45, 46].

Adjuvants, such as Alhydrogel® and Montanide®, have been used for clinical trials of TBVs with reactogenicity issues observed with both; however, recent trials of Alhydrogel®-formulated TBV have demonstrated good safety and reactogenicity profiles. Recently, GSK®'s liposomal adjuvant AS01 has been considered for TBVs. AS01 incorporates the TLR4 ligand MPL and the saponin derivative QS-21, and because AS01 is used for formulating the preerythrocytic vaccine RTS,S, this would simplify future efforts to combine products.

TBV candidates that are in clinical and preclinical developments are summarized in Table 1. Human studies of Pfs25 and Pvs25 showed that priming doses of the vaccines do not induce detectable antibody levels [42, 51]. Antibody production is measurable after the first boost and then rapidly declines; additional boosts are required to retain antibody titers. However, the raised antibodies have been proven to be functional, i.e. capable of reducing oocyst formation in mosquito-feeding assays, and this activity strongly correlates to antibody titer [42, 51]. In addition to Pfs25/Pvs25 candidates, Pfs230 conjugated to EPA and adjuvanted in Alhydrogel® or AS01 has advanced to clinical trials for evaluation either alone or in coadministration with Pfs25 (https://clinicaltrials.gov/, trials NCT02334462 and NCT02942277).


Table 1. Transmission-blocking vaccine (TBV) candidates in clinical and preclinical developments.
