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. 2015 May;83(5):1799-808.
doi: 10.1128/IAI.02980-14. Epub 2015 Feb 17.

Alga-produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated With a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium Falciparum Infection of Mosquitoes

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Alga-produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated With a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium Falciparum Infection of Mosquitoes

Kailash P Patra et al. Infect Immun. .
Free PMC article

Abstract

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene-oil-in-water emulsion, and GLA plus squalene-oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene-oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene-oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.

Figures

FIG 1
FIG 1
Analysis of purified alga-produced Pfs25. (A) FLAG tag affinity-purified alga-produced rPfs25 was resolved by SDS-PAGE and stained with InstantBlue. (B) Western immunoblot of FLAG tag affinity-purified alga-produced rPfs25 probed with the anti-Pfs25 MAb 4B7.
FIG 2
FIG 2
Anti-Pfs25 IgG titers in mice vaccinated with alga-produced Pfs25 antigen in combination with human-compatible adjuvants. Ten mice per group were vaccinated with or without affinity-purified alga-produced Pfs25 antigens mixed with AL007 (2 mg/ml alum), AL019 (0.5 mg/ml alum plus GLA), EM081 (squalene–oil-in-water emulsion), and EM082 (squalene–oil-in-water emulsion plus GLA). Each group of mice was vaccinated with the initial dose of 20 μg alga-rPfs25 antigen plus adjuvant, followed by 3 injections of 10 μg of antigen at 3-week intervals. The vaccinated mice were bled 2 weeks after the final immunization, sera were pooled, and anti-Pfs25 IgG titers were determined by ELISA. Titers for rPfs25 plus adjuvants are reported after subtracting signals obtained using serum from each respective adjuvant-alone group. Pfs25 monoclonal antibody secreting hybridoma (4B7) was obtained from MR4 and cultured in vitro, and the MAb was purified and used as a positive control in this assay at a stock solution concentration of 1 mg/ml. Pooled prebleed mouse sera were used as negative control. ELISA plates were coated with alga-Pfs25 antigens (100 ng/well) in sodium bicarbonate buffer, pH 9.6, and serial serum dilutions (1/500 to 1/64,000) were used to determine titers.
FIG 3
FIG 3
Comparison of anti-Pfs25 IgG levels among four different adjuvant groups against P. falciparum parasite lysate. Plasmodium falciparum mixed gamete/zygote stage lysate-coated ELISA plate (100 ng/well) and the pooled serum (10 mice) samples (1/500 dilutions) were used to measure anti-Pfs25 IgG levels among the four adjuvant plus alga-Pfs25-vaccinated animals AL007 (2 mg/ml alum), AL019 (0.5 mg/ml alum plus GLA), EM081 (squalene–oil-in-water emulsion), and EM082 (squalene–oil-in-water emulsion plus GLA). Titers for rPfs25 plus adjuvants are reported after subtracting signals obtained using serum from the adjuvant-alone group. Pfs25 MAb obtained from MR4 (concentration, 1 mg/ml) was used as a positive control; a sample from pooled sera of prevaccinated mice (prebleed) was used as a negative control. Statistical analysis was carried out using Student's t test between various adjuvant groups, and statistical significance is indicated by horizontal lines above the values.
FIG 4
FIG 4
Western immunoblot analysis of P. falciparum mixed gamete/zygote lysate probed with anti-alga Pfs25 serum raised with four different adjuvants. Reduced (+DTT) and nonreduced (−DTT) mixed gamete/zygote stage lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and cut into individual strips. The blots were probed individually with postvaccinated sera from the four vaccine groups (AL007, AL019, EM081, and EM082), prebleed mouse sera (negative control), and anti-Pfs25 monoclonal antibodies (4B7) as a positive control under the same conditions to determine the comparative binding efficiency levels of anti-Pfs25 antibodies induced by different adjuvants.
FIG 5
FIG 5
Pfs25-specific IgG subtypes of antibodies along with four different adjuvant groups vaccinated with alga-produced Pfs25. ELISA plates were coated with alga-produced, affinity-purified rPfs25 recombinant protein as capture antigen (100 ng/well) in bicarbonate buffer and incubated with serum samples (1/500 dilution) from different adjuvant (with rPfs25 antigen) and control (adjuvant alone) groups in duplicate wells. Groups were AL007 (2 mg/ml alum), AL019 (0.5 mg/ml alum plus GLA), EM081 (squalene–oil-in-water emulsion), and EM082 (squalene–oil-in-water emulsion plus GLA). Pooled serum samples from pre- and postimmunization sera were used for the IgG subtyping ELISA. Statistical analysis was carried out using Student's t test between various adjuvant groups, and the values mentioned for significant differences (P value < 0.05) are marked with horizontal lines above them.
FIG 6
FIG 6
Comparison of relative avidity indexes of anti-Pfs25 antibodies against recombinant alga-Pfs25 antigen from four vaccination groups. Avidity of the anti-Pfs25 IgG was measured by continued binding to alga-Pfs25 antigen in the presence of 8 M urea compared to no urea as a control. Serum samples from alga-Pfs25 antigens vaccinated in combination with AL007 (2 mg/ml alum), AL019 (0.5 mg/ml alum plus GLA), EM081 (squalene–oil-in-water emulsion), and EM082 (squalene–oil-in-water emulsion plus GLA). The avidity index was calculated as the ratio of average optical density at 450 nm (OD450) for urea-treated samples to the average OD450 for PBS-Tween 20-treated samples. Statistical analysis was carried out using Student's t test to compare various adjuvant groups, and the values mentioned for significant differences between groups (P value < 0.05) are marked with a horizontal line above them. Vaccination with EM082 (oil-in-water plus GLA) showed a significantly higher avidity index than vaccination with alum (AL007) and than vaccination with oil-in-water emulsion without GLA (EM081).
FIG 7
FIG 7
Immunofluorescence microscopy images show the binding of anti-alga Pfs25 antiserum to native parasite protein expressed in the in vitro-cultured gametocyte (GC), female gamete (GM), and zygote (ZY) stages of P. falciparum. BF, Nomarski (differential interference contrast) bright-field images; DAPI, nucleus stained blue; FITC (fluorescein isothiocyanate), Pfs25 protein stained green; Merge, merged images to show both the nucleus and the Pfs25 protein localization in P. falciparum parasite stages. Serum samples from rPfs25 antigens in the four vaccine groups were used as primary antibodies for probing the specimens.
FIG 8
FIG 8
Adjuvant effect on transmission-blocking activity of anti-Pfs25 antibodies. Standard membrane feeding assays were carried out with pooled sera from four groups of mice vaccinated with alga-produced Pfs25 in combination with the four adjuvants (AL007, AL019, EM081, and EM082). Preimmune sera were used as negative controls. Oocyst counts reflect the successful development of P. falciparum inside the Anopheles stephensi mosquitoes. Two separate membrane feeds were done using serum from each group, and oocyst counts were pooled for statistical analysis. The table shows P. falciparum infectivity and oocyst counts in the different groups (AL007, AL019, EM081, and EM082). Statistical analysis (Mann-Whitney test and Fisher's exact test) was carried out using GraphPad Prism 5. Sig, significant.

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