Development of a Malaria Transmission Blocking Nanoparticle Vaccine Petr Ilyinskii 1 , Conlin P. O’Neil 1 , Takashi Kei Kishimoto 1 , Patrick E. Duffy 2 , Kazutoyo Miura 3 , Carole A. Long 3 , and Lloyd Johnston 1 . 1 Selecta Biosciences, Inc., 480 Arsenal St., Watertown, MA 02472, USA. 2 Laboratory of Malaria Immunology and Vaccinology, NIAID, National Institutes of Health, Rockville, MD, USA. 3 Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, MD, USA. We have developed a potent transmission-blocking malaria vaccine via the encapsulation of recombinant Pfs25 antigen into Selecta’s biodegradable, synthetic vaccine particle platform (SVP™) and combining it with nanoparticle formulations containing TLR7/8 or TLR9 agonists. Pfs25 is a sexual stage antigen of Plasmodium falciparum expressed on the surface of its zygote and ookinete forms. It has been long known that antibodies to Pfs25 can block the development of P. falciparum oocysts in mosquito midgut, thus Pfs25 has been extensively studied as a candidate antigen for transmission-blocking vaccines. Several formulations of SVP[Pfs25] nanoparticles were co-administered into BALB/c mice with adjuvant nanoparticles containing either CpG oligonucleotides (SVP[CpG]), or a novel polymer-resiquimod conjugate prodrug (SVP[R848]). SVP immunizations resulted in robust antibody titers over a wide range of nanoparticle-encapsulated Pfs25 doses. Titer comparison showed that SVP formulation containing just 2 ng of Pfs25 resulted in titers that were comparable to that obtained with 8 μg Pfs25 in alum, indicating a 4000-fold dose sparing effect. When tested in the standard membrane feeding assay, serum from SVP-immunized mice showed 97-99% inhibition in oocyst intensity compared to 10-70% with a standard alum formulation. These results indicate that SVP[Pfs25] formulations provide excellent potency and efficacy thus represent a promising approach to prevent malaria transmission. Transmission Blocking Approach vs. Traditional Infection Preventing Vaccine ABSTRACT Figure 1. The concept of transmission blocking malaria vaccine SVP: Application of a Modular Vaccine Platform for Induction of Long-term Humoral Immunity Figure 2. SVP technology for malaria vaccine Dose-dependent Induction of Antibodies to Pfs25 by SVP Vaccination Using R848 and CpG Adjuvants Figure 5. BALB/c mice were injected s.c. 2 or 3 times with 3-week intervals (d0, 21, 42) with either SVP (0.002-1 μg of Pfs25) or 8 μg of free Pfs25 with alum and their serum assayed for Pfs25 antibodies at d33 (A) or d54 (B). Novel SVP[R848]/SVP[CpG] Formulations: Elevated Titers and/or Ab Avidity Result in Higher SMFA Activity Figure 6. BALB/c mice were immunized with SVP on d0 and 21 and their serum assayed for Pfs25 antibodies, tested for Ab avidity and in SMFA (d33). Results for different SVP[R848] (A) and SVP[CpG] (B) formulations shown CONCLUSIONS A B ● SVP[Pfs25] nanoparticles have been formulated and tested for antibody induction in vivo ● Co-administration of SVP[Pfs25] with SVP[CpG] or SVP[R848] resulted in robust antibody titers over a wide range of SVP-encapsulated Pfs25 doses ● SVP containing 2 ng of Pfs25 resulted in titers comparable to that obtained with 8 μg Pfs25 in alum (4000-fold dose sparing) ● Serum from SVP-immunized mice showed 97-99% inhibition in oocyst formation in standard membrane feeding assay (SMFA) ● SVP[Pfs25] co-administered with novel SVP[CpG] or SVP[R848] formulations showed 100% oocyst inhibition in SMFA, which correlated either with elevated antibody titers or antibody avidity • Target sporozoite • Anti -CSPAb • CTL against CSP − Prevent infection − Eliminate infected hepatocytes Sporozoite surface proteins, poorly immunogenic, multiple vaccine failures • Target gametocyte or ookinetes • Anti -Pfs25 Ab − Prevent transmission Gametocyte surface proteins immunogenic ‘Altruistic’ vaccine Induction of Antibodies to Pfs25 by Several SVP[Pfs25] Formulations Figure 3. BALB/c mice were injected s.c. 3 times with 3-week intervals (d0, 21, 42) with either SVP or free Pfs25 (with or w/o alum) and their serum assayed for Pfs25 antibodies and also tested in standard membrane feeding assay (SMFA). A – Pfs25 ELISA (d54), B – SMFA (d54) T cell antigen e.g. • Disease-specific peptide epitopes • Protein antigens Adjuvant e.g. • TLR7/8 Agonist • TLR9 Agonist B cell antigen Pfs25 Targeted Synthetic Vaccine Particles (tSVP) tSVP[Pfs25] Nanoparticle polymer • Biodegradable PLGA/PLA • Particle size • Charge • Release rates R848 CpG SVP Vaccination Results in Significant Pfs25 Dose Sparing A B Figure 4. BALB/c mice were injected s.c. 3 times with 3-week intervals (d0, 21, 42) with either SVP or free Pfs25 with alum and their serum assayed for Pfs25 antibodies and tested in SMFA at d54. A – Pfs25 ELISA (d54), B – SMFA (d54) While utilization of CpG resulted in higher Ab titers than those generated with R848, these did not directly translate to improved transmission blocking in SMFA Ab Titer Correlates with SMFA if R848 used for Pfs25 vaccination F4 SVP[Pfs25] formulation selected for further development Strong Inhibition of Oocyst Formation by Serum from SVP-immunized Mice A B Targeted Synthetic Adjuvant Particles (tSAP) 2 adjuvant encapsulating formations tSVP[R848] tSVP[CpG] A B
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Development of a Malaria Transmission Blocking Nanoparticle VaccinePetr Ilyinskii1, Conlin P. O’Neil1, Takashi Kei Kishimoto1, Patrick E. Duffy2, Kazutoyo Miura3, Carole A. Long3, and Lloyd Johnston1. 1Selecta Biosciences, Inc., 480 Arsenal St., Watertown, MA 02472, USA. 2Laboratory of Malaria Immunology and Vaccinology, NIAID, National Institutes of Health, Rockville, MD, USA. 3Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, MD, USA.
We have developed a potent transmission-blocking malaria vaccine via the encapsulation of recombinant Pfs25 antigen into Selecta’s biodegradable, synthetic vaccine particle platform (SVP™) and combining it with nanoparticle formulations containing TLR7/8 or TLR9 agonists. Pfs25 is a sexual stage antigen of Plasmodium falciparum expressed on the surface of its zygote and ookinete forms. It has been long known that antibodies to Pfs25 can block the development of P. falciparum oocysts in mosquito midgut, thus Pfs25 has been extensively studied as a candidate antigen for transmission-blocking vaccines. Several formulations of SVP[Pfs25] nanoparticles were co-administered into BALB/c mice with adjuvant nanoparticles containing either CpG oligonucleotides (SVP[CpG]), or a novel polymer-resiquimod conjugate prodrug (SVP[R848]). SVP immunizations resulted in robust antibody titers over a wide range of nanoparticle-encapsulated Pfs25 doses. Titer comparison showed that SVP formulation containing just 2 ng of Pfs25 resulted in titers that were comparable to that obtained with 8 μg Pfs25 in alum, indicating a 4000-fold dose sparing effect. When tested in the standard membrane feeding assay, serum from SVP-immunized mice showed 97-99% inhibition in oocyst intensity compared to 10-70% with a standard alum formulation. These results indicate that SVP[Pfs25] formulations provide excellent potency and efficacy thus represent a promising approach to prevent malaria transmission.
Transmission Blocking Approach vs. Traditional Infection Preventing Vaccine
ABSTRACT
Figure 1. The concept of transmission blocking malaria vaccine
SVP: Application of a Modular Vaccine Platform for Induction of Long-term Humoral Immunity
Figure 2. SVP technology for malaria vaccine
Dose-dependent Induction of Antibodies to Pfs25 by SVP Vaccination Using R848 and CpG Adjuvants
Figure 5. BALB/c mice were injected s.c. 2 or 3 times with 3-week intervals (d0, 21, 42) with either SVP (0.002-1 μg of Pfs25) or 8 μg of free Pfs25 with alum and their serum assayed for Pfs25 antibodies at d33 (A) or d54 (B).
Novel SVP[R848]/SVP[CpG] Formulations: Elevated Titers and/or Ab Avidity Result in Higher SMFA Activity
Figure 6. BALB/c mice were immunized with SVP on d0 and 21 and their serum assayed for Pfs25 antibodies, tested for Ab avidity and in SMFA (d33). Results for different SVP[R848] (A) and SVP[CpG] (B) formulations shown
CONCLUSIONS
A
B
● SVP[Pfs25] nanoparticles have been formulated and tested for antibody induction in vivo
● Co-administration of SVP[Pfs25] with SVP[CpG] or SVP[R848] resulted in robust antibody titers over a wide range of SVP-encapsulated Pfs25 doses
● SVP containing 2 ng of Pfs25 resulted in titers comparable to that obtained with 8 μg Pfs25 in alum (4000-fold dose sparing)
● Serum from SVP-immunized mice showed 97-99% inhibition in oocyst formation in standard membrane feeding assay (SMFA)
● SVP[Pfs25] co-administered with novel SVP[CpG] or SVP[R848] formulations showed 100% oocyst inhibition in SMFA, which correlated either with elevated antibody titers or antibody avidity
• Target sporozoite• Anti -CSPAb• CTL against CSP
− Prevent infection− Eliminate infected
hepatocytesSporozoite surface proteins, poorly immunogenic, multiple vaccine failures
• Target gametocyte or ookinetes
• Anti -Pfs25 Ab−Prevent
transmissionGametocyte surface proteins immunogenic‘Altruistic’ vaccine
Induction of Antibodies to Pfs25 by Several SVP[Pfs25] Formulations
Figure 3. BALB/c mice were injected s.c. 3 times with 3-week intervals (d0, 21, 42) with either SVP or free Pfs25 (with or w/o alum) and their serum assayed for Pfs25 antibodies and also tested in standard membrane feeding assay (SMFA). A – Pfs25 ELISA (d54), B – SMFA (d54)
T cell antigene.g. • Disease-specific
peptide epitopes• Protein antigens
Adjuvante.g. • TLR7/8 Agonist• TLR9 Agonist
B cell antigen
Pfs25
Targeted Synthetic Vaccine Particles (tSVP)
tSVP[Pfs25]
Nanoparticle polymer• Biodegradable PLGA/PLA• Particle size• Charge• Release rates
R848
CpG
SVP Vaccination Results in Significant Pfs25 Dose Sparing
A
B
Figure 4. BALB/c mice were injected s.c. 3 times with 3-week intervals (d0, 21, 42) with either SVP or free Pfs25 with alum and their serum assayed for Pfs25 antibodies and tested in SMFA at d54. A – Pfs25 ELISA (d54), B – SMFA (d54)
While utilization of CpG resulted in higher Ab titers than those generated with R848, these did not directly translate to improved transmission blocking in SMFA
Ab Titer Correlates with SMFA if R848 used for Pfs25 vaccination
F4 SVP[Pfs25] formulation selected for further development
Strong Inhibition of Oocyst Formation by Serum from SVP-immunized Mice
A
B
Targeted Synthetic Adjuvant Particles (tSAP)2 adjuvant encapsulating formations
tSVP[R848]
tSVP[CpG]
A
B
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