EP4511058A1 - Compositions, dispositifs, systèmes et procédés se rapportant à la vaccination et à la protection stérile contre le paludisme - Google Patents

Compositions, dispositifs, systèmes et procédés se rapportant à la vaccination et à la protection stérile contre le paludisme

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Publication number
EP4511058A1
EP4511058A1 EP23935577.9A EP23935577A EP4511058A1 EP 4511058 A1 EP4511058 A1 EP 4511058A1 EP 23935577 A EP23935577 A EP 23935577A EP 4511058 A1 EP4511058 A1 EP 4511058A1
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Prior art keywords
reprna
mice
dose
trap
prime
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German (de)
English (en)
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Marion AVRIL
Zachary Ward MACMILLEN
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Malarvx Inc
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Malarvx Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • P. falciparum (Pf) vaccine candidates to show field efficacy are those targeting the asymptomatic pre-erythrocytic (PE) stages of infection.
  • the subunit (SU) RTS,S/AS01 vaccine is the only known licensed malaria vaccine to date, but is only modestly effective against malaria clinical disease.
  • the SU R21 vaccine candidate targets the pre-erythrocyte sporozoite (spz) circumsporozoite (CS) protein and elicit high titer antibodies that have provided high levels of protection from disease, but do not induce liver resident memory (Trm) CD8 + T cell that are potent mediators of the pre-erythrocytic immunity for long-term protection.
  • whole-organism (WO) vaccines such as radiation attenuated sporozoites (RAS) elicit both high titer antibodies and Trm, and have achieved high levels of sterilizing protection.
  • RAS radiation attenuated sporozoites
  • compositions, systems, methods, etc. for anti-malarial vaccines that are efficient over time and/or extended delivery distance (e.g., from manufacturing site to inoculation location), effective against the malaria vector, rapidly administered and/or otherwise improved over existing anti-malaria vaccines.
  • the present systems, compositions, devices, methods, etc. provide improved anti-malaria immunological responses comprising making, providing and administering vaccines comprising specific RNA molecules such as self-amplifying replicon RNA (repRNA) encoding proteins from Plasmodium such as the P. yoelii (Py) CS protein (CSP), including in some embodiments substantially target proteins encoding target antigens, for example a whole or substantially whole CSP in the repRNA.
  • repRNA self-amplifying replicon RNA
  • PrRNA self-amplifying replicon RNA
  • Pr replicon RNA
  • CDSP P. yoelii
  • substantially target proteins encoding target antigens for example a whole or substantially whole CSP in the repRNA.
  • the vaccines can comprise an advanced oil-in-water emulsion nanocarrier such as a Lipid InOrganic Nanoparticle (LIONTM) administered to a patient in conjunction with whole organism (WO) radiation attenuated sporozoites (RAS) and can be used in a ‘prime-and-trap’ heterologous vaccination strategy.
  • LIONTM Lipid InOrganic Nanoparticle
  • WO whole organism
  • RAS radiation attenuated sporozoites
  • the prime-and-trap intervals for the administration of the vaccine can comprise administration of only a single dose of the repRNA-Non- encapsulating oil-in-water emulsion nanocarriers (e.g., LIONTM) component followed by administration of as few as 3 or 2 doses, or even just a single dose, of the WO component (e.g., RAS or genetically attenuated WO) at 0 day (same day), or 1, 2, 3 , 4, 5, 10, 14, 15 days or 28 days later.
  • the repRNA-Non- encapsulating oil-in-water emulsion nanocarriers e.g., LIONTM
  • the WO component e.g., RAS or genetically attenuated WO
  • CS full-length encoded CS protein expressed by infectious spz, which protein is advantageous for motility and hepatic cell invasion.
  • CS is composed of an N-terminal region that binds heparin sulfate proteoglycans (RI), an immunodominant central repeat region of four-amino-acid (NANP) that are the target of neutralizing antibodies, and a GPI-anchored C-terminal region containing a thrombospondin- like domain (RII) and T cell epitopes.
  • RI heparin sulfate proteoglycans
  • NANP immunodominant central repeat region of four-amino-acid
  • RII thrombospondin- like domain
  • Oil-in-water emulsion nanocarrier are a particularly effective means of delivering next-generation recombinant ribonucleic acid (RNA) vaccines.
  • Suitable carriers can be nanoparticle carriers that can, for example, be solid or semi-solid nanoparticles.
  • the nanocarriers are emulsion-based delivery vehicles that utilize the framework of squalene-based adjuvants and modifies it with the addition of the cationic lipid DOTAP (l,2-dioleoyl-3-trimethylammonium-propane, chloride salt), for binding of the RNA to the nanoparticle surfaces 3 .
  • DOTAP l,2-dioleoyl-3-trimethylammonium-propane, chloride salt
  • An example of a suitable nanocarrier is a Lipid InOrganic Nanoparticle (LIONTM).
  • the priming dose can be administered before and on the same day as the trapping dose, or the priming dose can be administered first followed by the trapping dose(s) from 12 - 120 hours later, from 121 hours to 28 days later or more than 28 days later,
  • FIG. 1 Preliminary study in mice.
  • Figure 1-1 shows RepRNA-PyCS/LION nanocarrier formulation prior to injection.
  • FIGs 3-1 A through 3- IB Accelerated vaccine induces sterile i:lrotection.
  • A) Mice were primed with 5 pg repRNA-PyCS, 5-day (green) or 28-day (purple) prior to trapping dose of WO Py RAS SPZ. Two doses (Prime-Boost) of repRNA-PyCS (orange, 5pg, 28-day) and prime-trap control (trap only repRNA-PfCS+RAS) (blue, 5pg, 28- day) were used as controls.
  • Primary Boost Prime-trap control
  • FIGs 4-1A through 4-1B Immunogenicity of repRNA-LION-CS dual antigen vaccine.
  • FIGs 6-1A through 6-1C Preliminary data on 5-day and 0-day vaccine durability.
  • B) CS+ tetramer liver resident COS+ T cells were assessed by flow cytometry 4 weeks post-trap.
  • Figure 1-2A to 1-2B LION/repRNA-CS vaccine design. After RNA transcription and capping, repRNA-PyCS or -PfCS or -PvCS was transfected into BHK cells and 24 to 48 hours later, the transfected cells lysate (R, reduced, NR, non-reduced) or null transfection used as control, were analyzed by Western blot, using the rabbit polyclonal antibodies for immunodetection.
  • Supplemental Figures 1-2A to 1-2C Supplemental Figures 1-2A to 1-2C. CS replicon and protein sequences.
  • SP signal peptide
  • RI signal peptide
  • RII the region II
  • VEE Venezuelan equine encephalitis virus
  • FIGs 2-2A to 2-2C Immunogenicity and efficacy of a prime-boost vaccine with repRNA encoding either PyCS (orange circle), PfCS (opened circle) or PvCS (black circle) formulated with LION.
  • PyCS and -PfCS antibody responses were determined by repeat region peptide PyCS or PfCS titration ELISA, respectively.
  • FIGS 3-2A to 3-2C Immunogenicity of an accelerated prime-and-trap immunization regimens in Balb/cJ mice.
  • Control cohorts are prime-boost repRNA- PyCS (orange data) or trap cohort (repRNA-PfCS +RAS, dark blue data).
  • FIG. 4-2A To 4-2C 5-day prime-and-trap and trap alone (RAS) regimens of immunization induce higher-frequency liver Trm cells than 14-day prime-and-trap regimen.
  • Control cohorts are prime-boost repRNA-PyCS (orange data) or trap cohort (repRNA-PfCS +RAS, black data).
  • FIGS 5-2A to 5-2E Efficacy of an accelerated prime-and-trap immunization regimens in Balb/cJ mice.
  • FIGS 6-2A to 6-2D Figures 6-2A to 6-2D.
  • Prime-and-trap vaccine improves protection against stringent challenge in Balb/cJ mice.
  • Control cohort is a 5ug 5-day prime-boost repRNA- PyCS (orange data) or a Trap cohort (repRNA-PfCS +RAS, dark blue data). Two months later mice were challenged intravenously with 10,000 live spz isolated from infected mosquitos.
  • FIG. 7-2A to 7-2E Prime-and-trap vaccine in C57BL/6 mice.
  • Prime-and-trap vaccine composed of a 5ug 5-day regimen of prime with repRNA-PyCS (green data) followed by trap dose of 25,000.
  • Control cohort is a 5ug 5-day prime-Boost repRNA-PyCS (orange data) or a trap cohort (repRNA-PvCS +RAS, purple data) or double trap (RAS +RAS, dark blue data), and a cohort of naive mice.
  • mice One- month post-trap (day 35), sera was collected. Two months post-trap, mice were challenged intravenously with 5,000 live spz isolated from infected mosquitos. B) Posttrap sera were harvested (day 35) and CS specific IgGs titer were analyzed by ELISA. C) Patency curves (>1% parasitemia) of mice and protection post-challenge per cohort. Number of mice per cohort indicated above bar graph. D) Parasitemia post-challenge of all cohorts. Emphasized is the parasitemia peak at day 12, where each dot represents a mouse, and the bar is the mean of the cohort.
  • FIGS 8-2A to 8-2G Immunogenicity and efficacy of a same-day prime-and- trap vaccine in Balb/cJ mice.
  • Prime-and-trap vaccine composed of a 5ug 5-day regimen (green data) or 5ug same-day interval (pink data) of prime with repRNA-PyCS followed by trap dose of 25,000.
  • Control cohort is 5ug same- day interval of a trap cohort (repRNA-PfCS +RAS, dark blue data).
  • repRNA-PfCS +RAS dark blue data.
  • present systems, compositions, devices, methods, etc. provide significantly improved, and even same-day administration, anti-malaria vaccines and immunization processes.
  • Such methods, etc. include making, providing and administering vaccines comprising specific RNA molecules such as self-replicating RNA (repRNA) encoding proteins from Plasmodium such as the P. yoelii (Py) CS protein (CSP), including in some embodiments target proteins substantially encoding target antigens, for example a whole or substantially whole CSP in the repRNA.
  • repRNA self-replicating RNA
  • the vaccines can comprise an advanced oil- in-water emulsion nanocarrier such as a Lipid InOrganic Nanoparticle (LIONTM) administered to a patient in conjunction with whole organism (WO) radiation attenuated sporozoites (RAS) and can be used in a ‘prime-and-trap’ heterologous vaccination strategy.
  • LIONTM Lipid InOrganic Nanoparticle
  • WO whole organism
  • RAS radiation attenuated sporozoites
  • the prime-and-trap intervals for the administration of the vaccine can comprise administration of only a single dose of the repRNA-Non-encapsulating cationic nanocarriers (e.g., LIONTM) component followed by administration of as few as 3 or 2 doses, or even just a single dose, of the WO component (e.g., RAS or genetically attenuated WO) at 0 day (same day), or 1, 2, 3 , 4, 5, 10, 14, 15 days or 28 days later.
  • the repRNA-Non-encapsulating cationic nanocarriers e.g., LIONTM
  • the WO component e.g., RAS or genetically attenuated WO
  • LION RNA nanoparticles are immunogenic in animals. When introduced into cells, repRNA initiates biosynthesis of antigen-encoding mRNA, raising and prolonging antigen expression and thereby enhancing humoral and cellular immune responses 4 . Moreover, repRNA elicits more robust immune responses after a single dose than conventional mRNA formulations, offering an attractive approach for emerging infectious diseases, such as Dengue 5 , Zika 3 , and SARS-CoV-2 4 . However, the in vivo instability of RNA and the requirement for transport through lipid bilayers have stimulated development of novel vehicles for intracellular RNA delivery.
  • repRNA-CoV2S a stable and highly immunogenic vaccine candidate comprising repRNA formulated with a Lipid InOrganic Nanoparticle (LIONTM) to enhance vaccine stability, delivery, and immunogenicity 4 (Fig.l).
  • LIONTM Lipid InOrganic Nanoparticle
  • the LION-repRNA-CoV2S construct elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes, indicative of a Type 1 T-helper response, in mice 4 .
  • a single-dose administration in NHP elicited antibody responses that potently neutralized SARS-CoV-2 4 .
  • LION nanoparticles represent a stable and easily manufacturable strategy for malaria vaccines:
  • the vaccines herein do not need to be encapsulated into lipid nanoparticles under a regulated manufacturing process; formulating variant-specific vaccines with LION is more flexible and can be rapidly customized.
  • the lipid component can be produced and stockpiled separately and then combined with the target- specific RNA as desired.
  • the LION formulation can be stored long-term at 4°C, rendering it practical for use in low- and-middle income countries (LMICs).
  • LION nanoparticles are highly immunogenic in malaria models: Applicant has employed the nanostructured LION particle to deliver repRNA to prime immune responses to the P. yoelii CS antigen (repRNA-PyCS) (Fig. 2A). Vaccine immunogenicity was evaluated by serum ELISAs (Fig. 2B) and splenocyte IFNy ELISPOTs (Fig. 2C). Anti-PyCS IgG antibody levels were substantially higher than after PyRAS immunization alone and were boosted by either a second dose of repRNA-PyCS or a dose of PyRAS (Fig. 2B). Boosting by PyRAS confirmed the fidelity of PyCS epitope presentation by repRNA-LION-expressing cells.
  • repRNA-PyCS When a single dose of repRNA-PyCS was benchmarked against gene gun PyCS (the basis for priming in the first-generation prime-and-trap vaccine 1,2 ), the LION repRNA-PyCS vaccine elicited >10-fold more IFNy-producing T cells than gene gun vaccination (Fig. 2C). However, two doses of repRNA-PyCS alone do not provide sterile protection against Py wild-type SPZ challenge in mice (Fig. 2D-E). These repRNA-PyCS data support the robust and consistent immunogenicity of this platform and its ability to synergize with liver-targeted trapping vaccines to provide high levels of protection.
  • LION nanoparticles accelerate the protective prime-and-trap vaccination schedule: Applicant next attempted prime-and-trap vaccination, using the SU repRNA- PyCS to prime and WO PyRAS to trap. Applicant tested the prime-and-trap approach for immunogenicity and efficacy with several control groups, including two doses of repRNA-PyCS, trap-only (having an irrelevant repRNA-PfCS control followed by PyRAS), a single dose of PyRAS alone, and naive animals (Fig. 3 A). Applicant compared different prime-and-trap regimens on accelerated schedules (1-day and 5-day) vs. the standard 28-day schedule.
  • Accelerated Prime-Trap immunization against liver-stage malaria can be optimized. For example, following priming with repRNA-PyCS, mice or other patients including humans will be boosted with a trapping dose of PyRAS at various i nlerval s and challenged one month after trapping. Other groups will be subjected to immunogenicity endpoints using ELISA, ELISpot, and/or flow cytometry to assess the mechanisms of protection. The results indicate that the efficacy of the best schedule of immunization for our prime-and-trap strategy will be improved.
  • the anti-PyCS and control anti-PfCS antibody responses elicited by the immunization regimens herein can be optimized. Cellular immune responses to immunization based on the regimens herein can be optimized. .
  • RNA self-amplifying (replicon) RNA (repRNA) as formulated herein (Fig.1-1) initiates biosynthesis of antigen-encoding mRNA in the host, raising and prolonging antigen expression and enhancing humoral and cellular immune responses 4 .
  • repRNA self-amplifying (replicon) RNA
  • Fig.1-1 self-amplifying (replicon) RNA
  • Fig.1-1 initiates biosynthesis of antigen-encoding mRNA in the host, raising and prolonging antigen expression and enhancing humoral and cellular immune responses 4 .
  • repRNA elicits more robust immune responses after a single dose than conventional mRNA formulations, offering an attractive approach for emerging infectious diseases, such as Dengue 5 , Zika 3 , and SARS-CoV-2 4 .
  • the use of LION eases manufacturing of malaria vaccines.
  • the LION oil-in-water emulsion nanocarrier was used to deliver repRNA to prime immune responses to the P. yoelii (Py) CS antigen (repRNA-PyCS) (Fig. 1, 2-1 A). Immunogenicity was evaluated by serum ELISAs (Fig. 2-1B) and splenocyte IFNy ELISPOTs (Fig. 2-1C). Anti-PyCS IgG antibody levels were substantially higher than after PyRAS immunization alone and were boosted by either a second dose of repRNA- PyCS or a trap dose of PyRAS (Fig. 2-1B). Boosting by PyRAS confirmed the fidelity of PyCS epitope presentation by repRNA-LION-expressing cells.
  • LION nanocarriers accelerated the protective prime-and-trap (P&T) vaccination schedule.
  • Applicant compared different P&T regimens on an accelerated schedule (5- day) vs. the standard 28-day schedule (Fig. 3-1A). Consistent with findings described above, none of the mice that received two doses of control repRNA-PyCS were protected, and mice treated with PyRAS (a “trap-only” vaccine) were only 40% protected (Fig. 3- 1B). Yet, all immunization schedules of the P&T regimen produced sterile (100%) protection against WT PySPZ challenge in mice (Fig. 3-1B).
  • LION nanocarriers induced a broad antibody response against two antigens.
  • a two-antigen mixture repRNA-PyCS and -PfCS
  • Applicant achieved broad humoral immune responses (Fig. 4-1 A, 4-1B) with a comparable magnitude of response from the mixed vaccine and single antigens alone (Fig. 4- IB), indicating that under these conditions, any immune interference present is insignificant.
  • VEE Venezuelan equine encephalitis
  • TC-83 strain 6 Applicants incorporated the coding sequences of the CS full length protein from Plasmodium yoelii into the alphavirus expression vector to create a repRNA malaria vaccine.
  • the coding sequences of the CS full length protein from P. falciparum and P. vivax were incorporated into the same expression vector as controls (Supplemental Figure 1-2A).
  • the repRNA-PyCS or -PvCS or - PfCS were verified by denaturing gel electrophoresis (Supplemental Figure 1-2B) and then transfected into mammalian cells for validation.
  • the repRNA-CS vaccines were formulated with a LION oil-in-water emulsion ( Figure IB) 4 .
  • the LION/repRNA vaccine platform utilizes an ad-mixture formulation of LION, a highly stable cationic (DOTAP) squalene emulsion embedded in a hydrophobic oil phase (as in Figure 1-2B) that can be manufactured independently of the RNA component and combined by a simple mixing step, e.g., at 1 : 1 (v/v), with the repRNA-construct for example 30 minutes prior to immunization.
  • DOTAP highly stable cationic
  • mice were immunized with an IM prime of 5 pg of LION/repRNA followed by a homologous boost 14 days later (Supplemental Figure 2-2A).
  • the mice either received LION/repRNA vaccines for each antigen (PyCS or PfCS; 5 mg per antigen) or the combination of two antigens (PyCS and PfCS, or PfCS and repRNA-GFP (green-fluorescent protein) control; 2.5 mg per antigen).
  • a repRNA- GFP construct was used here as a non-malaria coding antigen control.
  • Antibody responses were evaluated by ELTSA following the prime (day 13) and boost (day 29) against peptides containing their corresponding CS tandem repeat sequence ( Figure 2- 2B).
  • mice from the LION/repRNA-PyCS cohort were then challenged 3 weeks post-boost with an intravenous injection of 1000 wild-type (WT) Py 17XNL spz freshly dissected from mosquito salivary glands.
  • WT wild-type
  • mice and the mice primed with the control LION/repRNA- PfCS did not recognize the PyCS epitope by ELISPOT.
  • mice were primed IM with repRNA-PyCS (1 mg or 5 mg) 14 or 5 days prior to a PyRAS trapping dose (Figure 3-2A). All mice immunized with this heterologous vaccination as shown by the 14-day vaccine regimen using 5mg of repRNA-PyCS as prime dose and 25,000 RAS trapping dose remained healthy throughout the vaccination study (Supplemental Figure 3-2B).
  • mice immunized with both 14-day regimens have higher antibody titers than mice immunized with a 5 -day regimen of repRNA-PyCS and the irrelevant repRNA-PfCS regimen with RAS cohort (Trap alone, Figure 3-2B).
  • mice were immunized with the different immunization schedules (5mg vs Img as priming dose in a prime-and-trap 14-day vs 5-day regimen; 5mg prime-boost or 25,000 spz trap alone regimen) as described above ( Figure 4-2 A). Livers were collected 28 days after the last immunization from two independent experiments, and lymphocytes were isolated, purified, and stained for flow cytometry as described previously 1 .
  • mice with breakthrough parasitemia in those immunized cohorts saw a consistent 2 to 3-day delay in the onset of blood-stage with a reduced peak of the parasitemia (Figure 5-2C).
  • Two weeks post-challenge sera was collected, and total IgG levels were quantified by ELISA.
  • the total IgG titer post-challenge was similar in all immunized cohorts (prime-and-trap vs trap alone) indicating a recall of the CS-specific humoral immune response following sporozoite challenge ( Figure 5 -2D).
  • mice were immunized 5 days apart with 2 injections of 5mg rep-RNA-PyCS as the homologous prime-boost cohort. Even though this experiment was not replicated independently, Applicants observed similar results than previously reported, i.e., the homologous prime-boost repRNA-PyCS cohort showed no protection following challenge ( Figures 6-2B, 6-2C, orange data), and was indistinguishable from Applicant’s control infectivity cohort ( Figures 6-2B, 6-2C, black data).
  • the prime-and-trap cohort (repRNA-PyCS+RAS) were compared to a prime-boost repRNA-PyCS cohort, a trap cohort (repRNA-PvCS +RAS), a double trap (RAS+RAS) cohort and infectivity control cohort.
  • a prime-boost repRNA-PyCS cohort a trap cohort (repRNA-PvCS +RAS), a double trap (RAS+RAS) cohort and infectivity control cohort.
  • Both trap and prime-boost cohorts had significantly lower IgG titers compared to the double RAS or the prime-and- trap cohorts, which both have equivalent and strong anti-CS antibody titers ( Figure 7- 2B).
  • replicon RNA replicon RNA
  • Applicants induced a strong humoral response in Balb/cJ and C57B1/6 mice when given 2 weeks apart or on the same-day compared to the trapping dose alone, and observed a strain-specific CD8 T cell response in Balb/cJ mice. Additionally, Applicants found a 2-3 -day delay in the blood-stage parasitemia compared to the control groups, indicating that some level of protection was conferred.
  • Applicant’s prime-and-trap vaccine approach has the advantage that immunized patients developed humoral and cellular immunities without waning down the CD8 + T cells achieved by the injection of the RAS (or otherwise attenuated WO) trapping dose, and inducing the ability to shift the antibody profile toward a balanced Thl/Th2 humoral immune response.
  • RAS or otherwise attenuated WO
  • the Examples herein demonstrate a multi-component vaccination approach for the concurrent induction of humoral and T cell immunities using a repRNA-CS formulated with LION nanoparticle and PyRAS targeting the liver.
  • the chosen antigen for Applicant’s vaccine design is the full length circumsporozoite (CS) protein from Plasmodium, as described in detail in Supplemental Figure 1-2.
  • This antigen contains an immunodominant and protective CD8 + T cell epitope specific to the H-2K d (Balb/cJ)-restricted genetic background, and two distinct dominants for CD8 + epitopes in the H2K b /D b (C57BL/6)-restricted genetic background.
  • VEE Venezuelan equine encephalitis
  • RNA was purified via lithium chloride precipitation, followed by capping with a capping kit (New England Biolabs) as described 4 .
  • RNA was further purified and stored in -80° C until use. Denatured repRNA were verified by electrophoresis in a 1% agarose gel.
  • the LION formulation has inorganic SPIO nanoparticles within a hydrophobic squalene core to enhance formulation stability.
  • LION particles were manufactured by combining the iron oxide nanoparticles with the oil phase (squalene, Span 60, and DOTAP) while the aqueous phase, containing Tween 80 and sodium citrate dihydrate solution in water, was prepared separately.
  • the oil and aqueous phases were then mixed and emulsified then processed by passaging through a microfluidizer to reach 50 ⁇ 5 nm with a 0.2 polydispersity index.
  • the microfluidized LION was terminally filtered with a 200-nm pore-size polyethersulfone filter and stored at 2° to 8°C.
  • BHK cells American Type Culture Collection (ATCC) were transfected with repRNA or mock transfected using a OptiMEM (Gibco) and Expifectamine transfection kit (ThermoFisher). Cells were scrapped off and lysed with RIPA buffer 24-48 hours later, and lysates were analyzed by SDS-polyacrylamide gel electrophoresis and by Western blot.
  • ATCC American Type Culture Collection
  • OptiMEM Gibco
  • Expifectamine transfection kit ThermoFisher
  • mice and C57B1/6 mice Female Balb/cJ mice and C57B1/6 (B6) mice, six to eight weeks old, were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained under pathogen-free conditions in animal facilities and were fed with autoclaved food ad libitum. Mice were housed and cared for in standard IACUC- approved animal facilities from Bloodworks Northwest and used in compliance with lACUC-approved protocols.
  • RNA was mixed with LION and injected into the mice intramuscularly (IM) using a total of 50pl (25 pl in each leg).
  • IM mice intramuscularly
  • a two-vial formulation method was performed as described 4 and Applicant’s immunization protocol and timeline are described in each respective figure.
  • Wild-type Py (17XNL strain) sporozoites were prepared by cyclical transmission in Balb/cJ mice and Anopheles stephensi mosquitoes at the Seattle Children’s Center for Global Infectious Disease Research Insectary (Seattle, WA, USA).
  • Female 6- to 8-week-old SW mice were injected with blood-stage Py 17XNL WT parasites to begin the growth cycle and used to feed female Anopheles stephensi mosquitoes.
  • salivary gland sporozoites were isolated and harvested as previously described 9 .
  • Infectious sporozoite for challenge were prepared in an equivalent manner but without irradiation. All experimental and control mice were challenged with live Py 17XNL sporozoites.
  • Livers were perfused with 10 ml PBS with 2 mM EDTA by injection into the portal vein, with outlet drainage via the inferior vena cava. Gall bladder was removed, and livers were placed in 5 ml RPMI 1640 supplemented with glutamine with 5% FBS on ice to ensure cell survival. Livers were mashed through a 200-mm mesh filter (pluriSelect, San Diego, CA) with the back of a 3-ml syringe plunger. The mesh filter and plunger were washed with FBS -/glutamine supplemented RPMI 1640.
  • Cell suspension was spun at 80 3 g for 1 min at 4°C without braking; supernatants were collected and transferred to a clean 50-ml conical tube where they were spun at 500 3 g for 8 min at 4°C.
  • the cell pellet was resuspended in 10 ml room temperature 35% Percoll (GE Healthcare Life Sciences) in HBSS (Life Technologies) supplemented with 100 U heparin and spun at room temperature at 900 3 g for 25 min with no brake.
  • Spleens were harvested and splenocytes separated from Balb/cJ mice at 28 days post immunization. A total of lxl0E5 splenocytes were combined with SYVPSAEQI peptide (1 mg/ml final) (Genemed Synthesis) for murine IFNy ELISPOT (eBioscience), cultured for 18 h at 37 °C and developed following manufacturer guidelines. The percentage of antigen-specific T cells was calculated based on the spot-forming units counted in each well divided by the total number of splenocytes applied to each well.
  • Breakthrough to blood stage patency was assessed by Giemsa-stained thin blood smear starting at day 4 after challenge and ending at day 21, at which time a negative smear was attributed to complete protection.
  • Liver burden was detected by qRT-PCR from harvested liver mice 44hr post-challenge 1,1() .
  • Mice immunized with P. falciparum- or P. vivax-repRNA-CS and challenged with live P. yoelii spz were used as controls.
  • Sterile protection was defined as being blood smear negative, and the Kaplan-Meier curves illustrate the time to 1% parasitemia during days 4-21 after challenge with 17XNL strain P.yoelii live spz.
  • Mouse GAPDH was amplified with cDNA using gap dh-fwd: (CCTCAACTACATGG TTTACAT) and gap dh-rev: (GCTCCTGGAAGATGGTGATG) primers. All qRT-PCR amplification cycles was performed at 95 °C for 30s for DNA denaturation and at 60°C for 4min for primer annealing and extension.

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Abstract

L'invention concerne des systèmes, des compositions, des dispositifs, des procédés, etc. qui fournissent des réponses immunologiques anti-paludisme améliorées comprenant la production, la fourniture et l'administration de vaccins comprenant des molécules d'ARN spécifiques telles qu'un ARN réplicon auto-répliquant (ARNrép) codant pour des protéines de Plasmodium telles que la protéine P.yoelii (Py) CS (CSP), y compris, dans certains modes de réalisation, des protéines sensiblement cibles codant pour des antigènes cibles, par exemple un CSP entier ou sensiblement entier dans l'ARNrép. Les intervalles de première immunisation et piégeage (prime-and-trap) pour l'administration du vaccin peuvent comprendre l'administration d'une dose unique d'un composant nanoporteur à émulsion huile dans l'eau non encapsulant l'ARNrép (par exemple LIONTM) suivie de l'administration de 3 ou 2 doses seulement, voire d'une seule dose, du composant WO (par exemple RAS ou WO génétiquement atténué) à 0 jour (le même jour) ou 1, 2, 3, 4, 5, 10, 14, 15 jours ou 28 jours plus tard.
EP23935577.9A 2023-04-22 2023-04-24 Compositions, dispositifs, systèmes et procédés se rapportant à la vaccination et à la protection stérile contre le paludisme Pending EP4511058A1 (fr)

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US20030104003A1 (en) * 2001-05-25 2003-06-05 James Anthony A. Novel surface protein of the malaria parasite plasmodium falciparum
SI2759306T1 (sl) * 2008-12-09 2016-05-31 Coley Pharmaceutical Group, Inc. Imunostimulacijski oligonukleotidi
WO2019140136A1 (fr) * 2018-01-10 2019-07-18 University Of Washington Méthodes et schémas posologiques de vaccination antipaludique
BR112021009422A2 (pt) * 2018-12-21 2021-10-26 Curevac Ag Rna para vacinas contra malária
CN114901360A (zh) * 2019-12-20 2022-08-12 库瑞瓦格股份公司 用于递送核酸的新型脂质纳米颗粒

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