Sequences, formation and structural analysis of SAPN.

<p>(A) The amino acid sequences of the monomers used to make the SAPN in this study. Black: flanking regions (His-tag, thrombin cleavage site, proteosomal cleavage sites, linkers). Green: coiled-coil pentamer domain (Trp-zipper); Blue: coiled-coil trimer domain; Red: predicted B-cell epitopes of <i>P. falciparum</i> or <i>P. vivax</i> CSP repeat region; Yellow: predicted human HLA restricted CD8⁺ T-cell epitopes <i>P. falciparum</i> CSP; Magenta: universal CD4 T-helper epitope (PADRE) as a part of the trimer domain. (B) SAPNs are formed by the oligomerization of 3- and 5-stranded coiled-coiled domains within a single polypeptide monomer. Shown is the <i>in silico</i> prediction of the SAPN with icosahedral symmetry. Colors are representative of the sequences as described in (A). (C) Individual nanoparticles are visualized using transmission electron microscopy. The bar represents 100 nm. (D) The size distribution of the nanoparticles in solution is monitored using dynamic light scattering.</p

Amino acid sequences of the three predicted <i>P. falciparum</i> CSP CD8⁺ T-cell epitopes (K, M and Y) based on binding to major human HLA haplotypes.

<p>Shown are comparisons to the known sequence in the 3D7 strain of <i>P. falciparum</i> CSP (used for human volunteer challenge trials) and to the Wellcome Strain of <i>P. falciparum</i> CSP sequence expressed in the Tg-<i>Pb/Pf</i>CSP sporozoites (used in these mouse studies). Underlined residues in Wellcome strain highlight the differences from the 3D7 strain.</p

Analysis of purified monomers.

<p>(A) Coomassie Blue stained SDS-PAGE gel of monomer proteins. Lane 1: Molecular weight marker proteins; Lane 2: <i>Pf</i>CSP monomer; Lane 3: <i>Pv</i>CSP monomer; Lane 4: <i>Pf</i>CSP-KMY monomer; Lane 5: <i>Pv</i>CSP-KMY monomer.</p

SAPN vaccination induces protective cellular immune responses in mice.

<p>SAPN based vaccines present CD8⁺ T-cell epitopes to stimulate a protective cellular immune response. (A) C57BL/6 mice immunized with a SAPN containing only <i>P. falciparum</i> CSP B-cell epitopes (<i>Pf</i>CSP-SAPN) or a SAPN containing both <i>P. falciparum</i> CSP B- and T-cell epitopes (<i>Pf</i>CSP-KMY-SAPN). n = 10; Error bars show means ± s.d. of three separate experiments. (B) Either sera or total splenocytes were transferred from immunized mice to naïve mice which were challenged 24 h post-transfer. n = 10; data shown is one of two experiments. (C) In order to determine if CD8⁺ T-cells were involved in protection we immunized wild-type C57BL/6 mice (WT) and MHC Class I knockout (MHC1 KO) mice with SAPN containing <i>Pf</i>CSP specific CD8⁺ epitopes. Mice were challenged with 5,000 Tg-<i>Pb/PfCSP</i> sporozoites. n = 10. *P<0.01; ***P<0.0001.</p

Consistency of protection against lethal challenge with malaria parasites in mice immunized with <i>Pf</i>CSP-SAPNs.

<p><i>Pf</i>CSP-SAPN vaccines were administrated at wk 0, 2 and 4 in three independent experiments Two wks post 3^rd dose of <i>Pf</i>CSP-SAPN, C57BL/6 mice were challenged with 5,000 and Balb/C with 10,000, Tg-<i>Pb/Pf</i>CSP sporozoites. Shown here are three separate experiments with C57BL/6 mice and two experiments with Balb/C mice. n = 10. Infectivity sham control mice were given PBS. Mice were considered protected if they had no detectable parasitemia by day 15 following challenge. nd = not determined.</p

Sera or Cell Transfer Studies.

<p>(<b>A</b>) Pooled sera isolated from mice immunized with <i>Pf</i>CSP-SAPN or <i>Pf</i>CSP-KMY-SAPN but not sera from <i>Pv</i>CSP-SAPN or <i>Pv</i>CSP-KMY-SAPN immunized mice transferred to naïve mice conferred protection from challenge with Tg-<i>Pb/PfCSP</i> sporozoites. (<b>B</b>) In a separate experiment sera or washed splenocytes from mice were transferred. Whereas sera from <i>Pf</i>CSP-SAPN or <i>Pf</i>CSP-KMY-SAPN transferred protection sera from <i>Pv</i>CSP-SAPN or <i>Pv</i>CSP-KMY-SAPN immunized mice did not. On the contrary, total splenocytes from <i>Pf</i>CSP-KMY-SAPN or <i>Pv</i>CSP-KMY-SAPN transferred protection while splenocytes from <i>Pf</i>CSP-SAPN or <i>Pv</i>CSP-SAPN did not. (<b>C</b>) Two wks post final immunization with <i>Pf</i>CSP-KMY-SAPN 1.33×10⁶ enriched CD8⁺ T-cells were adoptively transferred to naïve animals which were then challenged 72 hrs post transfer. *P<0.05. Mice were challenged with 5,000 Tg-<i>Pb/PfCSP</i> sporozoites. n = 10.</p

Antibody responses and protective efficacy induced by SAPN vaccinations in Mice.

<p>C57BL/6 and Balb/C mice make high titer antibodies when immunized with the <i>Pf</i>CSP SAPN. (A) Mice were given vaccine intraperitoneally (i.p.) or intramuscularly (i.m.) at wk 0, 2 and 4. Titers were determined 2 wks after each dose. n = 10 per group per experiment; data are representative of one of three independent experiments with similar results. (B) Two wks post 3^rd dose of <i>Pf</i>CSP-SAPN, C57BL/6 mice were challenged with 5,000 Tg-<i>Pb/Pf</i>CSP, Balb/C with 10,000, Tg-<i>Pb/Pf</i>CSP sporozoites. Shown are three separate experiments with C57BL/6 mice and two experiments with Balb/C mice. n = 10. Infectivity sham control mice were given PBS. Mice were considered protected if they had no detectable parasitemia by day 15 following challenge. (C) Titer of anti-<i>Pf</i>CSP repeat antibody of the serum from individual mice in each SAPN vaccinated group two days before challenge. The line represents the mean titers of the individual mice in that group. n = 6 or 7 depending if serum was available. Those SAPN immunized mice that were protected against malaria are represented by (•); those that developed parasitemia and died by (▴). (D) Protection following challenge. At the predetermined time point, from wk 6 to wk 52 of the study, C57BL/6 mice in a select immunized group (n = 6 or 7) and a time matched PBS-sham vaccinated group were challenged with 5,000 sporozoites. Mice receiving <i>Pf</i>CSP-SAPN immunization (black bars) and matched sham PBS vaccinated mice (white bars). ***P<0.0001.</p

Anti-SAPN antibody titer, avidity index and protection in mice immunized with <i>Pf</i>CSP-SAPNs.

<p>Analysis of sera from individual mice immunized with <i>Pf</i>CSP-SAPNs for anti-SAPN specific antibody titer, IgG concentration and avidity index vis-à-vis the protection status of each mouse. All sera used in this analysis were taken 48 wks post-3^rd immunization (wk 52 of study), one day before mice were challenged. P = protected from challenge dose of transgenic sporozoites. NP = not protected from challenge dose of transgenic sporozoites.</p

Schematic representation of redesigning the scaffold for SAPN-based <i>P. falciparum</i> CSP vaccine.

<p>The cartilage oligomerization matrix protein (COMP) was replaced with a <i>de novo</i> designed sequence (Trp-zipper) that, like COMP, forms a pentameric coiled-coil domain. Sequences coding for the universal CD4 T-cell helper epitope, the pan-allelic DR epitope (PADRE) were incorporated into the trimeric coiled-coil domain without disrupting the stoichiometry needed to oligomerize. The <i>P. berghei</i> circumsporozoite surface protein repeat (<i>Pb</i>CSP) epitopes were replaced with <i>Pf</i>CSP repeat epitopes to form <i>Pf</i>CSP-SAPN. Three <i>Pf</i>CSP CD8+ T cell epitopes were then engineered into the pentameric coiled coil domain to form <i>Pf</i>CSP-KMY-SAPN.</p

CD8⁺ T-lymphocyte population profiles.

<p><i>Pf</i>CSP-KMY-SAPN immunized mice show induction of long-lived Ag-specific memory cells and residency in both secondary lymph and non-lymphoid organs (A–D). Two to five wks post final immunization total lymphocytes from designated organs were cultured, stimulated with peptides, stained and characterized by flow cytometry. Data are presented as a percentage of the CD8+ T-cell population normalized to peptide stimulated saline control minus media alone. Data was segregated based on phenotype and location for (A) naïve, (B) effector memory, (C) central memory, and (D) long-term central memory cell populations and characterized as Naïve, T_EM, T_CM or T_LCM based on expression levels of CD44, CD62L, IL-2 and IFNγ. The FACS gating strategy is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048304#pone.0048304.s001" target="_blank">Figure S1</a>. Error bars represent the mean ± s.d. observed in 4 separate experiments, two mice each, over the course of 5 wks post vaccination. LN = lymph node; SPL = spleen.</p