Classifications

• • 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
• • 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
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
• • 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
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
• • 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
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
• • 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

• This invention provides a method for inoculating a vertebrate host against malaria by administering to the host a live rodent Plasmodium organism and exploiting its cross-species protection potential.
• the invention further provides a vaccine composition comprising a rodent Plasmodium organism that is genetically engineered to express immunospecific single (liver, blood or gametocyte) or multi (liver and blood, or liver and gametocyte, or blood and gametocyte, or liver and blood and gametocyte) stage immuneeffectors from one or several human Plasmodium parasites.
• the invention also provides for production of a vaccine composition, by suspending wild-type or genetically modified rodent Plasmodium organisms in a suitable pharmaceutically acceptable carrier solution.
• An alternative to subunit vaccine candidates is the use of a whole-organism approach.
• Such a strategy is based on the generation of immunity by attenuated sporozoites, the Plasmodium form that is injected by an infected mosquito into its vertebrate host.
• EEFs Plasmodium exoerythrocytic forms
• a few decades ago it was shown that sterile protection of humans could be achieved through the injection P. falciparum radiation- attenuated sporozoites (RAS) [4].
• Plasmodium CS is the immunodominant protective antigen in both RAS and GAS [7] and previous studies have shown that protection could be achieved by immunization with CS alone. However, it is also clear that CS is not the sole immunogen at play in the immunity triggered by a whole-organism approach [8, 9] .
• the present invention proposes an alternative strategy for the development of a pre-erythrocytic, whole organism vaccine against malaria, based on the use of rodent parasites as primary immunizing vectors.
• Rodent Plasmodium organisms can be used to induce an immune response in human hosts capable of protecting against infection with human- infective Plasmodium spp . , by exploiting the cross-species protection potential created by homologous molecules of the rodent parasite.
• the immunizing potential of these rodent Plasmodium organisms can be enhanced by introducing selected immunogenic antigens through genetic engineering.
• rodent Plasmodium organisms can carry antigens of human-infective Plasmodium spp. to elicit a specific protective immune response against human Plasmodium parasites, in addition to their natural cross- species protective capability, provided by evolutionarily conserved molecules.
• the present invention describes a live rodent Plasmodium organism for use against human malaria.
• a preferred embodiment of the present invention provides the live rodent Plasmodium organism genetically engineered to express genes or gene sections of one or more species of human Plasmodium parasites, for use as immunospecific single- or multi-stage immunoeffectors against human malaria .
• the previous live rodent Plasmodium organism is any member of the protozoan genus Plasmodium whose natural host is a rodent, including P. berghei , P. yoeli, P. vinckei and P. chabaudi .
• a preferred embodiment of the present invention provides the use of the live rodent Plasmodium organism, wherein the single or multistage immunoeffectors are liver-stage antigens such as circumsporozoite protein (CS) , liver stage antigen 1 (LSA-1), thrombospondin-related adhesion protein (TRAP) , and liver stage antigen 3 (LSA-3) or blood-stage antigens such as erythrocyte-binding antigen-175 (EBA-175) , apical membrane antigen-1 (AMA-I), merozoite surface protein 1 (MSP-1), Duffy-binding protein (DBP) , and reticulocyte-binding proteins (RBPs) , or gametocyte- specific antigens such as p48/45, or other immunogenic antigens .
• liver-stage antigens such as circumsporozoite protein (CS) , liver stage antigen 1 (LSA-1), thrombospondin-related adhesion protein (TRAP) , and liver stage antigen 3
• the human malaria is caused by any of the human Plasmodium parasite from the protozoan genus Plasmodium including P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi .
• a preferred embodiment of the present invention provides the use of the live rodent Plasmodium organism, for the manufacture of an admixture with a pharmaceutically acceptable carrier.
• the live rodent Plasmodium organism is used for the manufacture of a vaccine against malaria.
• Figure 1 Rodent Plasmodium parasites infection and development within human hepatic cells .
• Figure 2a and 2b Rodent Plasmodium parasites infection of human erythrocytes in vivo.
• Bl Average percentage of infected erythrocytes from either human or murine origin, in chimeric and non-chimeric mice infected with Pb(WT) or P. falciparum, across time; Bl) Average percentage of infected human erythrocytes (full line) as well as signal background obtained on the expected region for infected mouse erythrocytes (small dotted line) in chimeric mice engraphed with human erythrocytes infected with P. falciparum.
• B2 Average percentage of infected mouse erythrocytes (long dotted line) as well as signal background obtained on the expected region for infected human erythrocytes (small dotted line) in non-chimeric mice infected with P. berghei ; B3) Average percentage of infected human erythrocytes (full line) as well as average percentage of infected mouse erythrocytes (long dotted line) in chimeric mice engraphed with human erythrocytes infected with P. berghei; CI and C2) Erythrocyte populations obtained after magnetic separation of imE and ihE from chimeric mice engraphed with human erythrocytes infected with P. berghei.
• Figure 4 Genetically modified rodent Plasmodium parasites expressing human Plasmodium parasite antigens.
• mice infected with sporozoites from the different parasite lines were analyzed by ELISA to assay IgG and IgM responses induced against the circumsporozoite protein (CS) in mice challenged with either mock infected salivary-gland injected mice (SG) or mice infected with wild-type P. berghei (WT) or the genetically modified PbCS P f. (PfCS) ; Titers are expressed in arbitrary units of fluorescence (AU) as the highest dilution of sera tested that gave a positive staining; A) IgG and IgM responses induced against P. falciparum-CS ; B) IgG and IgM responses induced against P.
• CS circumsporozoite protein
• berghei-CS C) ELISPOT analysis of CS-specific T-cell responses in mice challenged with either mock infected salivary-gland injected mice (SG) or mice infected with wild-type P. berghei (WT) or the genetically modified PbCS P f. (PfCS); the number of IFN- ⁇ secreting cells in 10 A 6 splenocytes is presented; D) Binding affinity of immune sera from mice challenged with either mock infected salivary-gland injected mice (SG) or mice infected with wild-type P. berghei (WT) or the genetically modified PbCS Pf . (PfCS) to wild-type P.
• berghei sporozoites or genetically modified PbCSp f or P. falciparum sporozoites
• E Gliding motility inhibition assays of P. falciparum sporozoites in the presence of sera from mice challenged with either mock infected salivary-gland injected mice (SG) or mice infected with wild-type P. berghei (WT) or the genetically modified PbCSp f . (PfCS);
• F Hepatic infectivity inhibition assays of P. falciparum in the presence of sera from mice challenged with either mock infected salivary-gland injected mice (SG) or mice infected with wild-type P. berghei (WT) or the genetically modified PbCS Pf (PfCS) .
• the present invention relates to an alternative approach to malaria vaccination that combines unprecedented versatility with high efficacy, whilst ensuring complete safety.
• rodent malaria sporozoites spz
• rodent malaria parasites are an extremely safe antigen delivery platform because they are completely unable to complete their life cycle inside human erythrocytes, which renders then unable to cause a disease- triggering human blood-stage infection
• rodent malaria parasites can induce a high level of cross-species protection against human Plasmodium species because they possess conserved molecules that can be recognized by the human immune system
• Genetically engineered rodent Plasmodium parasites are highly immunogenic, being able to trigger specific immune responses against an engineered human Plasmodium antigen capable of recognizing human Plasmodium parasites and inhibiting human Pla
• rodent Plasmodium organism or rodent Plasmodium parasite any member of the protozoan genus Plasmodium whose natural host is a rodent, including the four known species, P. berghei , P. yoelii, P. vinckei and P. chabaudi .
• human Plasmodium organism or human Plasmodium parasite is meant any member of the protozoan genus Plasmodium known to cause human malaria, including P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi .
• a rodent Plasmodium organism-based malaria vaccine In order to be safe, a rodent Plasmodium organism-based malaria vaccine must ensure that the immunizing parasite is unable to cause disease in humans. This requires that rodent Plasmodium merozoites are unable to effectively invade and multiply inside human red blood cells (RBCs.
• RBCs red blood cells
• This system can be coupled with the use of the nuclear SYTO-16 and the mouse-specific TER- 119 dyes to distinguish infected from non-infected cells and human from rodent erythrocytes, respectively, and thereby monitor infection of either type of cell by flow cytometry (Fig. 2A1, 2A2, 2A3) .
• mice Five days after the initiation of chloroquine treatment, the mice were sacrificed and immune serum was obtained from collected blood. Pre-immune serum from uninfected mice and serum from mice immunized with wild-type P. berghei were obtained and used as controls in these experiments. Antibodies against P. falciparum CS in the serum were quantified by ELISA (Fig. 5A, 5B) . Our results show that mice immunized with the PbCS Pf parasite produced significant amounts of this antibody, showing that immunization of rodents with PbCS Pf elicited the generation of antibodies directed against P. falciparum CS, which are known to mediate protection against the human-infective parasite [ 13 ] .
• CelTOS for cell-traversal protein for ookinetes and sporozoites, which has been recently identified as a target antigen for a pre- erythrocytic vaccine, based on its ability to induce protective immunity through humoral and cellular immune responses.
• CelTOS is highly conserved among the Plasmodium species and immunization with pre-erythrocytic antigen CelTOS from P. falciparum has been shown to elicit cross- species protection against a heterologous challenge with P. berghei , suggesting that the reverse effect might also take place .
• the immunogenicity of rodent Plasmodium parasites can be enhanced by genetic engineering, effectively turning them into platforms for the delivery of immunogenic antigens of human-infective Plasmodium species, capable of eliciting highly efficient specific immune responses.
• more than one antigen already known or hitherto unknown, can be introduced, for either single or multiple stages of the Plasmodium life cycle, and for a single or multiple species of human Plasmodium organisms, because such redundancy may ensure an additional degree of protection against infection of parasitemia.
• PbCSPf itself has the drawback that it produces relatively low numbers of salivary gland sporozoites and lower hepatic infectivity than wild-type P. berghei. This is most likely due to the absence of the endogenous CS, coupled with an inappropriate conformation of the P. falciparum protein in this parasite.
• several alternatives may be envisaged in order to improve the infection yields of PbCSPf, such as the heterologous expression of P. falciparum CS in a neutral locus, under the control of the endogenous CS promoter, in addition rather than as a replacement of the endogenous protein.
• rodent parasites as "piggy-backs" for human malaria genes can be extended to include antigens other than CS .
• antigens other than CS include antigens other than CS .
• TRIP thrombospondin-related adhesion protein
• LSA-1 liver stage antigen 1
• sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation.
• Or strategy also allows for the inclusion of blood-stage antigens.
• Apical membrane antigen I (AMA-I)
• EBA-175) erythrocyte-binding antigen-175
• MSP-1 merozoite surface protein 1
• AMA-I AMA-I
• EBA-175 erythrocyte-binding antigen-175
• MSP-1 merozoite surface protein 1
• this approach can be used to introduce a transmission blocking component in the pseudo-attenuated vaccine, by engineering gametocyte-specific genes, such as p48/45, into the rodent Plasmodium platform.
• P. vivax antigens such as CS, orthologs of the P. falciparum genes listed above, Duffy-binding protein (DBP) , or reticulocyte-binding proteins (RBPs) , in the immunogenic platform.
• DBP Duffy-binding protein
• RBPs reticulocyte-binding proteins
• Prudencio M Rodriguez A and Mota MM. The silent path to thousands of merozoites: the Plasmodium liver stage. Nat Rev Microbiol 2006; 4:849-56.

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