Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of thePlasmodium–infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specificPlasmodiumsp is that most of the existing methods for malaria diagnosis focus on detectingP. falciparum. Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiatePlasmodiumspecies in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances.

Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice. We expressed in Pichia pastoris two chimeric proteins by merging the three central repeat regions of different CSP alleles (VK210, VK247, and P. vivax-like). The first construct (yPvCSP-All_FL) contained the fused repeat regions flanked by N- and C-terminal regions. The second construct (yPvCSP-All_CT) contained the fused repeat regions and the C-terminal domain, plus RI region. Mice were vaccinated with three doses of yPvCSP in adjuvants Poly (I:C) or Montanide ISA720. We also used replication-defective adenovirus vectors expressing CSP of human serotype 5 (AdHu5) and chimpanzee serotype 68 (AdC68) for priming mice which were subsequently boosted twice with yPvCSP proteins in Poly (I:C) adjuvant. Regardless of the regime used, immunized mice generated high IgG titres specific to all CSP alleles. After challenge with P. berghei ANKA transgenic parasites expressing Pb/PvVK210 or Pb/PvVK247 sporozoites, significant time delays for parasitemia were observed in all vaccinated mice. These vaccine formulations should be clinically tried for their potential as protective universal vaccine against P. vivax malaria.

Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP), we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP). In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like), as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminus of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C). Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210). Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multi-allele vaccine against P. vivax malaria.

Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP—All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSP_R) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSP_CT). To elicit stronger humoral and cellular responses, Pichia pastoris yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSP_CT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.