Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP

Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine

Abstract BACKGROUND: The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8(+) and CD4(+) T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. METHODOLOGY/PRINCIPAL FINDINGS: To establish the basis for a SAPN-based vaccine, B- and CD8(+) T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein ( 3c125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFN\u3b3(+), IL-2(+)) long-lived central memory CD8(+) T-cells. Furthermore, we demonstrated that these Ab or CD8(+) T-cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. CONCLUSION: The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP

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

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

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

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

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