REVISED COPY Plasmodium vivax in Pichia pastoris yeast · 40 expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble 41 protein in the methylotrophic yeast Pichia
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REVISED COPY 1
Invasion-inhibitory antibodies elicited by immunization with 2
Mauricio M. Rodriguesg, Bruce Russellf,h and Irene S. Soaresa# 9
10 aDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências 11
Farmacêuticas, Universidade de São Paulo, 05508-900, São Paulo, SP, Brazil; 12 bInstituto Butantan, São Paulo, SP, Brazil; 13 cShoklo Malaria Research Unit (SMRU), Mae Sot, Tak Province, Thailand; 14 dMahidol-Oxford-University Research Unit, Bangkok, Thailand; 15 eCentre for Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United 16
Kingdom; 17 fSingapore Immunology Network, Biopolis, Agency for Science Technology and 18
Research, Singapore; 19 gCTCMOL, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade 20
Federal de São Paulo-Escola Paulista de Medicina, Rua Mirassol, 207, São Paulo 21
04044-010, SP, Brazil; 22 hDepartment of Microbiology, Yong Loo Lin School of Medicine, 23
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Figure 1. The recombinant protein was expressed in P. pastoris with a hexa-histidine tag and purified from the supernatant by affinity chromatography, followed by anion exchange chromatography, as described in the Methods section. (A) SDS-PAGE analysis of purified recombinant PvAMA-1 stained with Coomassie blue (1 mg of protein per lane)protein per lane). (B) Immunoblotting analysis of purified recombinant PvAMA-1 using an anti-histidine tag antibody.(C) RP-HPLC profile of purified recombinant PvAMA-1. Purified PvAMA-1 produced under cGLPwas analyzed by RP-HPLC using a C4 column, as described in the Methods section. The plot represents the mean residue ellipticity (m.r.e.) of the recombinant protein.(D) Circular dichroism spectrum of recombinant PvAMA-1. The spectrum was recorded from 200(D) Circular dichroism spectrum of recombinant PvAMA 1. The spectrum was recorded from 200 to 260 nm using a JASCO-J720 spectropolarimeter. The plot represents the mean residue ellipticity (m.r.e.) of the recombinant protein.
Figure 2. Immunoblotting analysis of recombinant PvAMA-1 using monoclonal antibodies and a comparative evaluation of PvAMA-1 expressed in E. coli or P. pastoris for recognition by human sera. (A) Recombinant PvAMA-1 was subjected to 12% SDS-PAGE performed under reducing (R) and non-reducing (NR) conditions. Immunoblotting was performed using the indicated monoclonal antibodies. (B) S f 208 i di id l ith t t P i l i f d i f(B) Sera from 208 individuals with patent P. vivax malaria from endemic areas of Brazil were tested against recombinant PvAMA-1 produced in E. coli or P. pastoris. The values are the OD492 measurements of each protein. The tendency line and the value of the Spearman correlation coefficient (r) are represented.
Figure 3. Serum IgG titers of mice immunized with recombinant PvAMA-1and/or plasmid DNA containing the pvama-1 gene.Mice were immunized 4 times with 100 μg of DNA (D) i.m. or 10 μg ofμg ( ) μgrecombinant protein (P) s.c. with a 20-day interval. The protein wasadministered without adjuvant or emulsified in CFA/IFA. Five days prior toimmunization with DNA, some groups received cardiotoxin (D*). Theanimals from control groups were immunized with empty DNA vector (Dctrl)or CFA/IFA diluted in PBS. The results are expressed as the mean titers inlog10 ± SEM. [n.s= non significant, p>0.05; *p<0.05; *p<0.01; ***p<0.001].
Figure 4. Serum IgG titers of mice immunized with recombinant PvAMA-1using formulations containing different adjuvants.g g j(A) Groups of BALB/c mice were immunized s.c. with 10 μg of the PvAMA-1protein in the presence of different adjuvants, as described in the Methodssection. These adjuvants were tested alone or in combination (Alum + MPLAand Alum + FliC). The results are expressed as the mean titers in log10 ± SEM.[n.s.= non significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001].(B) Comparative serum IgG subclass profile after mouse immunization withPvAMA-1 using different adjuvants. The results are expressed as theIgG1/IgG2a ratio of titer ± SEM.(C) Immunogenicity of the PvAMA-1 protein produced under conditions ofGood Laboratory Practice (cGLP). The results are expressed as the means ±SEM of antibody titer (log10). [n.s.= non significant, p>0.05; *p<0.05; **p<0.01;***p<0.001].
Figure 5. Indirect immunofluorescence analysis using sera from BALB/cg y gmice immunized with the cGLP PvAMA-1 protein in formulationscontaining different adjuvants. The second row of panels show amagnified region of the corresponding panels above. These zoomed inimages highlight the apical staining pattern of the anti-PvAMA-1 + MPLA.Microscope slides containing fixed P. vivax obtained from patients fromThailand were incubated with sera from mice immunized with PvAMA-1 inthe presence of MPLA or Quil A (diluted 1:100).
Figure 6. Invasion-inhibitory activity of sera from mice immunized withPvAMA-1 (cGLP) in the presence of Quil A (1:100) against merozoites fromfour Thai isolates of P. vivax (Mean Inhibition=25.15% +/- SD 15.77). A flowcytometry method using a field deployable flow cytometer (BD Accuri® C6Flow Cytometer) was used to quantify the amount of invasion inhibitiony ) q yrelative to the treatment free control (1:100 prebleed mouse sera) (FL1-A=Sybr Green and FL2-A = Dihydroethidium). Each assay was compared to apositive control (anti DARC (2C3 region)) (Mean Inhibition=85.81% +/- SD3.72). Surrounding the central graph we provide FACs scatter plots of theinhibition assays. The FACs plot on the left side of the Y-axis shows a gatecontaining 2.42% rings (post invasion) in the untreated control (1:100prebleed mouse sera) compared to 1.37% and 0.26% in the AMA-1 treatmentand DARC positive control, respectively.