**4. Vaccine containing the three allelic variants of the** *plasmodium vivax* **circumsporozoite antigen**

Immunologically, the course of infection by *Plasmodium* depends on the production of pro- and anti-inflammatory cytokines. In cases where an inflammatory pattern is prevalent, the disease tends to be more severe. Nonetheless, the upregulation of anti-inflammatory cytokine appears to occur after the increase in inflammatory cytokines, due to a regulatory mechanism, to prevent the exacerbation of inflammatory response and its deleterious effects [76, 77]. Moreover, *P. vivax* sporozoites are covered with CSP, a highly immunogenic protein, recognized mainly by B lymphocytes.

The good-responder immune profile against CS repeats of VK247 in individuals carrying the typically Amerindian HLA specificity DRB1\*16 and the non-responder profile against CS repeats of VK210 in individuals carrying HLA DRB1\*07 was previously determined [31]. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In other Brazilian Amazon areas (Maranhão and Pará state), the CSP polymorphism is associated with the increase of nonregulated inflammatory immune responses, which in turn may be associated with the outcome of infection [59]. In addition, Individuals with the rs16944 CC genotype in the *IL1β* gene have higher antibody levels to the CSP of *P. vivax* of VK247 and *P. vivax*-like variants [29].

The development of a vaccine with satisfactory efficacy for malaria would be an important strategy for the control of the disease, mainly because it provides a tool for the prevention of this parasitosis, with relevant aspects of the cost–benefit relation and would bring a solution that overlays the adaptive strategies of parasites and vectors. Despite the general obstacles that need to be overcome in the development of vaccines against parasitic diseases, researchers who develop vaccines against *P. vivax* face other adversities. One of them is the fact that these protozoa have the formation of hypnozoites, which can cause relapses months and even years after the primary infection [78]. In endemic areas of malaria transmission, individuals with repeated exposure to the parasite tend to develop clinical immunity to both P. falciparum and *P. vivax* malaria.

Since the central portion of CSP is highly immunogenic and the induction of a protective response in animals immunized with sporozoites and in humans has been observed in experimental models, CSPs are being investigated as candidates for a human vaccine against malaria. However, despite decades of research, a highly effective vaccine still remains elusive. To date, only one vaccine formulation against *P. falciparum* has been licensed, RTS, S, manufactured by GlaxoSmithKline (GSK), which showed limited protective efficacy in young children (approximately 36%). RTS, S is a recombinant vaccine produced in *Saccharomyces cerevisiae*, which comprises the C-terminal portion of PfCSP and repeat regions fused to the surface antigen (S) of hepatitis B virus. This vaccine may contribute substantially to the control of malaria when used in combination with other control measures, especially in high-risk areas [79].

This vaccine is based on CSP, the major surface antigen present in sporozoites that is critical in liver-stage development during the pre-erythrocytic life cycle [29]. CSP has a central immunodominant region of tandem repeats flanked by two highly conserved regions that encode the amino terminal and carboxy terminal regions. The most advanced vaccine candidate (phase II clinical trial) to prevent malaria by the *P. vivax* also targets CSP. Nonetheless, differently from *P. falciparum*, the PvCSP exhibits diversity in its central repetitive domain, defining the variants VK210, VK247, and *P. vivax*-like. The *P. vivax* CSP vaccine was also combined into multivalent formulations or chimeric synthetic molecules. Peptides based on the N-terminal, central repeat, and C-terminal regions of PvCSP were immunogenic in individual administrations of experimental models (mice and monkeys) as well as in healthy human volunteers [80].

Another major difficulty that must be overcome is the absence of *P. vivax* infection in rodents, and in this way, preclinical evaluations on the vaccine protective efficacy are mainly restricted to nonhuman primates. In addition to the ethical issues, these
