Evaluation of the Impact of Codon Optimization and N-Linked Glycosylation on Functional Immunogenicity of Pfs25 DNA Vaccines Delivered by In Vivo Electroporation in Preclinical Studies in Mice

Abstract

bPlasmodium falciparum sexual stage surface antigen Pfs25 is a well-established candidate for malaria transmission-blocking vaccine development. Immunization with DNA vaccines encoding Pfs25 has been shown to elicit potent antibody responses in mice and nonhuman primates. Studies aimed at further optimization have revealed improved immunogenicity through the application of in vivo electroporation and by using a heterologous prime-boost approach. The goal of the studies reported here was to systematically evaluate the impact of codon optimization, in vivo electroporation, and N-linked glycosylation on the immunogenicity of Pfs25 encoded by DNA vaccines. The results from this study demonstrate that while codon optimization and in vivo electroporation greatly improved functional immunogenicity of Pfs25 DNA vaccines, the presence or absence of N-linked glycosylation did not significantly impact vaccine efficacy. These findings suggest that N-glycosylation of Pfs25 encoded by DNA vaccines is not detrimental to overall transmission-blocking efficacy. Malaria caused by Plasmodium species is still endemic in 97 countries, and WHO estimated that 3.3 billion people are at risk of disease, with ϳ584,000 deaths reported in 2013 (1). Plasmodium falciparum is responsible for the most morbidity and mortality and is thus the major focus of current vaccine development efforts (2). The malaria eradication research agenda (malERA) initiative of 2011 underscored the need for a multipronged approach for malaria control and elimination that includes vaccines targeting infection (3) and transmission along with various control measures currently in use such as indoor residual spraying and insecticide-treated mosquito nets (4).Malaria transmission-blocking vaccines (TBVs) target the sexual life cycle stages of the parasite that develop in the mosquito vector with the goal of interrupting transmission and further spread of the infection (5). The primary mode of action of TBVs is via induction of antibodies that target surface antigens expressed in the sexual stages of the parasite. P. falciparum TBV candidates include prefertilization antigens Pfs230 and Pfs48/45 and postfertilization antigens Pfs25 and Pfs28 (6, 7). So far, vaccine approaches based on recombinant protein-adjuvant formulations have met with limited success due to the complex conformational nature of these antigens, often resulting in improperly folded, unstable, and aggregated proteins (6). DNA vaccines encoding specific P. falciparum TBV target antigens offer alternatives to traditional platforms as seen in murine (8) and nonhuman primate (9) models. Additional benefits for use of DNA vaccines include ease of design and sequence modification, stability, and transportability (10). Studies in mice with Pfs25 DNA plasmids showed high TBV activity with Ͼ95% oocyst reduction in the mosquitoes (8). Similar studies in rhesus macaques, however, revealed only modest immunogenicity even after four immunization doses and required heterologous boosting with recombinant protein for ...

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation

“…Plasmid DNA (<30 EU/mg) was produced and supplied at 2.5 mg/mL (Aldevron, Fargo, ND). For combination immunization, VR1020 encoding SYN Pfs25 (minus N-terminal signal and C-terminal anchor sequences and codon optimized for optimal expression in mammalian cells) was employed [30]. Combination immunization were conducted by mixing SYN Pfs48/45 and SYN Pfs25 DNA together as described below.…”

“…Total IgG was purified using protein A-Sepharose beads from pooled sera at terminal bleeds after 3 DNA immunization (50 ug DNA; WT, SYN and MUT1 and MUT2 DNA groups) as described and stored as sterile solutions in PBS [30]. IgG from no-EP immunization groups (WT, SYN, MUT1 and MUT2) were tested at 1 mg/mL and 0.5 mg/mL and from EP immunized groups at 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL.…”

“…The percent inhibition of oocyst development and mosquito infectivity differences were analyzed as described [30]. Statistical analysis was performed using the GraphPad Prism software package.…”

“…With respect to malaria TBV, considerable progress has been made with Pfs25 based DNA vaccines, evaluated in mice and nonhuman primates [25–30]. While the initial studies reported suboptimal immunogenicity of Pfs25 DNA vaccine in various animals [26], significant advances were made with the use of in vivo electroporation (EP) delivery [28, 29].…”

Comparative functional potency of DNA vaccines encoding Plasmodium falciparum transmission blocking target antigens Pfs48/45 and Pfs25 administered alone or in combination by in vivo electroporation in rhesus macaques

“…DNA vaccines encoding Pfs25 and Pvs25 (a P. vivax ortholog) have revealed highly potent immunogenicity, especially when administered using in vivo electroporation in mice and nonhuman primates [11–16]. We have recently also reported on induction of transmission-blocking antibodies in mice by DNA vaccine encoding Pfs48/45 [17].…”