The binding domain of Plasmodium vivax Duffy binding protein (PvDBP-II) is a promising blood-stage vaccine candidate for vivax malaria. For the development of a successful vivax malaria vaccine based on DBP-II, the antigenic diversity and also naturally occurring functional antibodies to different PvDBP-II variant types in the various populations must be determined. However, similar to other blood-stage antigens, allelic variation within the PvDBP-II is a fundamental challenge for the development of a broadly efficient vaccine. The present study was performed to define whether the polymorphisms in PvDBP-II influence the nature of functional inhibitory activity of naturally acquired or induced anti-DBP-II antibodies in mice. In this investigation, five genetically distinct variants of PvDBP-II were transiently expressed on the COS-7 cell surface. Erythrocyte-binding inhibition assay (EBIA) was performed using human sera infected with corresponding and non-corresponding P. vivax variants as well as by the use of mice sera immunized with different expressed recombinant PvDBP-IIs. EBIA results showed that the inhibitory percentage varied between 50 and 63 % by using sera from infected individuals, and in case of mouse antisera, inhibition was in the range of 76-86 %. Interestingly, no significant difference was detected in red blood cell binding inhibition when different PvDBP-II variants on the COS-7 cell surfaces were incubated with heterologous and homologous sera infected with PvDBP-II variants. This suggests that the detected polymorphisms in all five forms of PvDBP-II may not affect functional activity of anti-DBP-II antibodies. In conclusion, our results revealed that there are functional cross-reactive antibody responses to heterologous PvDBP-II variants that might provide a broader inhibitory response against all, or at least the majority of strains compared to single allele of this protein that should be considered in development of PvDBP-II-based vaccine.

Naloxone (NLX) has the ability to shift the immune response to a Th1 profile. Therefore, the adjuvant efficacy of NLX with recombinant P. vivax apical membrane antigen-1(rPvAMA-1) in BALB/c mice was evaluated. Mice were immunized subcutaneously with purified rPvAMA-1 formulated with NLX (doses of 5 mg/kg body weight) alone or in combination with IFA. A significant increase in anti-PvAMA-1 IgG antibody after the second boost (mean OD = 2·08 and 2·17, in groups received, rPvAMA-1/NLX and rPvAMA-1/NLX/IFA, respectively) was detected. IgG1 and IgG2b were the predominant isotypes in all immunized mouse groups. In immunized mice with rPvAMA-1/NLX (mean: 1036 pg/mL) and with rPvAMA-1/NLX/IFA (mean: 1024 pg/mL), IFN-γ was elicited in response to rPvAMA-1 after the second boost. No detectable IL-4 secretion was determined in all tested groups. In conclusion, the administration of NLX alone or NLX/IFA with rPvAMA-1 in BALB/c mice, which induced mixed Th1/Th2 immune responses, was comparable with that of the same recombinant antigen with CFA/IFA adjuvant. The results indicate that NLX alone may possibly not be considered as a potent Th1 adjuvant in PvAMA-1-based vaccine. However, in order to modulate immune responses from mixed Th1/Th2 to strong and protective Th1 response, further study is warranted on combination of NLX with other adjuvants such as CpG motifs or MPL in proper vaccine formulation. Additionally, dose-response study is necessary to determine the effect of different doses of antigen combined with NLX (at various doses) in Balb/c mice.

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