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.

Antibodies recognizing conformational epitopes in Pfs48/45, an antigen expressed on the surface of Plasmodium falciparum gametes and zygotes, have firmly established Pfs48/45 as a promising transmission blocking vaccine (TBV) candidate. However, it has been difficult to reproducibly express Pfs48/45 in a variety of recombinant expression systems. The goal of our studies was to evaluate functional immunogenicity of Pfs48/45 using DNA vaccine format in rhesus macaques. An additional goal was to ensure that when used in combination with another malarial antigen, specific immunity to both antigens was not compromised. For testing combination vaccines, we employed Pfs25 DNA plasmids that have previously undergone evaluations in rodents and nonhuman primates. Pfs25 is expressed on the surface of parasites after fertilization and is also a lead TBV candidate. DNA plasmids based on codon-optimized sequences of Pfs48/45 and Pfs25 were administered by in vivo electroporation, followed by a final recombinant protein boost. Our studies demonstrate that Pfs48/45 encoded by DNA plasmids is capable of inducing potent transmission blocking antibody responses, and such transmission blocking immune potency of Pfs48/45 was not compromised when tested in combination with Pfs25, These findings provide the evidence in favor of further studies on Pfs48/45 and Pfs25, either alone or in combination with other known malaria vaccine candidates for developing effective vaccines capable of interrupting malaria transmission.

Datta D ，Bansal GP ，Grasperge B ，Martin DS ，Philipp M ，Gerloff D ，Ellefsen B ，Hannaman D ，Kumar N ... -  《-》

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

Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes.

Datta D ，Bansal GP ，Gerloff DL ，Ellefsen B ，Hannaman D ，Kumar N ... -  《-》

Evaluation of combination vaccines targeting transmission of Plasmodium falciparum and P. vivax.

Transmission-blocking vaccines interrupting malaria transmission within mosquitoes represent an ideal public health tool to eliminate malaria at the population level. Plasmodium falciparum and P. vivax account for more than 90% of the global malaria burden, co-endemic in many regions of the world. P25 and P48/45 are two leading candidates for both species and have shown promising transmission-blocking activity in preclinical and clinical studies. However, neither of these target antigens as individual vaccines has induced complete transmission inhibition in mosquitoes. In this study, we assessed immunogenicity of combination vaccines based on P25 and P48/45 using a DNA vaccine platform to broaden vaccine specificity against P. falciparum and P. vivax. Individual DNA vaccines encoding Pvs25, Pfs25, Pvs48/45 and Pfs48/45, as well as various combinations including (Pvs25 + Pvs48/45), (Pfs25 + Pfs48/45), (Pvs25 + Pfs25), and (Pvs48/45 + Pfs48/45), were evaluated in mice using in vivo electroporation. Potent antibody responses were induced in mice immunized with individual and combination DNA vaccines, and specific antibody responses were not compromised when combinations of DNA vaccines were evaluated against individual DNA vaccines. The anti-Pvs25 IgG from individual and combination groups revealed concentration-dependent transmission-reducing activity (TRA) in direct membrane feeding assays (DMFA) using blood from P. vivax-infected donors in Brazil and independently in ex vivo MFA using Pvs25-transgenic P. berghei. Similarly, anti-Pfs25 and anti-Pfs48/45 IgGs from mice immunized with Pfs25 and Pfs48/45 DNA vaccines individually and in various combinations revealed antibody dose-dependent TRA in standard membrane feeding assays (SMFA) using culture-derived P. falciparum gametocytes. However, antibodies induced by immunization with Pvs48/45 DNA vaccines were ineffective in DMFA and require further vaccine construct optimization, considering the possibility of induction of both transmission-blocking and transmission-enhancing antibodies revealed by competition ELISA. These studies provide a rationale for combining multiple antigens to simultaneously target transmission of malaria caused by P. falciparum and P. vivax.

Cao Y ，Hayashi CTH ，Araujo MDS ，Tripathi AK ，Andrade AO ，Medeiros JF ，Vinetz J ，Kumar N ... -  《-》

Functional evaluation of malaria Pfs25 DNA vaccine by in vivo electroporation in olive baboons.

Plasmodium falciparum Pfs25 antigen, expressed on the surface of zygotes and ookinetes, is one of the leading targets for the development of a malaria transmission-blocking vaccine (TBV). Our laboratory has been evaluating DNA plasmid based Pfs25 vaccine in mice and non-human primates. Previously, we established that in vivo electroporation (EP) delivery is an effective method to improve the immunogenicity of DNA vaccine encoding Pfs25 in mice. In order to optimize the in vivo EP procedure and test for its efficacy in more clinically relevant larger animal models, we employed in vivo EP to evaluate the immune response and protective efficacy of Pfs25 encoding DNA vaccine in nonhuman primates (olive baboons, Papio anubis). The results showed that at a dose of 2.5mg DNA vaccine, antibody responses were significantly enhanced with EP as compared to without EP resulting in effective transmission blocking efficiency. Similar immunogenicity enhancing effect of EP was also observed with lower doses (0.5mg and 1mg) of DNA plasmids. Further, final boosting with a single dose of recombinant Pfs25 protein resulted in dramatically enhanced antibody titers and significantly increased functional transmission blocking efficiency. Our study suggests priming with DNA vaccine via EP along with protein boost regimen as an effective method to elicit potent immunogenicity of malaria DNA vaccines in nonhuman primates and provides the basis for further evaluation in human volunteers.

Kumar R ，Nyakundi R ，Kariuki T ，Ozwara H ，Nyamongo O ，Mlambo G ，Ellefsen B ，Hannaman D ，Kumar N ... -  《-》

Induction of Plasmodium falciparum transmission-blocking antibodies in nonhuman primates by a combination of DNA and protein immunizations.

Coban C ，Philipp MT ，Purcell JE ，Keister DB ，Okulate M ，Martin DS ，Kumar N ... -  《-》