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
  • A61K2039/525—Virus
  • A61K2039/5256—Virus expressing foreign proteins
• • 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

• the invention relates to Plasmodium vivax vaccine compositions.
• the circumsporozoite (CS) protein is present on the infective stages (sporozoites) of all malaria parasites and is the most abundant protein. It is involved in the initial stages of invasion of the mammalian host's hepatocytes where the parasite continues the life cycle. CS protein is an important target for antibodies, CD4 + and CD8 + T cells that can eliminate the pre-erythrocytic stages of the parasite [6]. These results led to the development of vaccine formulations that elicit high antibody titers against sporozoites and/or increased numbers of CD4 + and CD8 + T cells specific for malaria liver stages [7].
• This vaccine formulation consists of a large C-terminal fragment of the CS protein sequence fused to the Hepatitis B antigen S (conventional hepatitis B vaccine, Engerix B ® ) and expressed as a recombinant protein in Saccharomyces cerevisiae.
• the fusion protein named RTS, when expressed together with antigen S, naturally assembles into viruslike particles called RTS,S.
• AS adjuvant systems
• the primary structure of P. vivax CS protein has three major defined domains ( Figure 1).
• the center of the protein contains a long stretch of amino acid repetitions (central repeat domain). This domain is flanked by highly conserved regions, the N- and C- terminal domains. All three domains can be target of specific antibodies. However, only antibodies directed against the central repeat domain of the molecule have been described as capable of neutralizing the sporozoite infectivity [1 1].
• PvCS P. vivax CS protein
• the present invention addresses these and other needs by providing immunogenic recombinant proteins and recombinant adenoviral vectors which elicit high antibody titers to the circumsporozoite (CS) protein of P. vivax and are useful in vaccines against P. vivax malaria.
• CS circumsporozoite
• the invention provides an isolated protein comprising the sequence
• the protein consists of the sequence
• PAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGANAPNEK SVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLET DVCT SEQ ID NO: 21; His 6 -PvCS-VK210.
• the invention provides an isolated protein comprising the sequence
• NKKPEDLTLNDLETDVCT SEQ ID NO: 27; PIC-PvCS-VK210.
• the protein consists of the sequence
• NKKPEDLTLNDLETDVCTHHHHHHH SEQ ID NO: 28; PIC-PvCS-
• the invention provides, an isolated protein comprising the sequence
• VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT SEQ ID NO: 2; PvCs-
• the protein consists of the sequence MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGV F VDASSLGA
• the invention provides an isolated protein comprising the sequence
• NKKPEDLTLNDLETDVCT SEQ ID NO: 31; PIC-PvCS-VK247).
• the protein consists of the sequence
• NKKPEDLTLNDLETDVCTHHHHHH SEQ ID NO: 32; PIC-PvCS-VK247-His 6 ).
• the invention provides an isolated protein comprising the sequence
• the protein consists of the sequence
• LNDLETDVCT (SEQ ID NO: 23; His 6 -PvCS-Vivax-like).
• the invention provides an isolated protein comprising the sequence
• VNAANKKPEDLTLNDLETDVCT SEQ ID NO: 35; PIC-PvCS-Vivax-like.
• the protein consists of the sequence
• VNAANKKPEDLTLNDLETDVCTHHHHHH SEQ ID NO: 36; PlC-PvCS-Vivax-
• the invention provides an isolated protein comprising the sequence
• the protein consists of the sequence
• ETDVCT SEQ ID NO: 24; His 6 -PvCS-All CS epitopes.
• the invention provides an isolated protein comprising the sequence
• AANKKPEDLTLNDLETDVCT SEQ ID NO: 39; PIC-PvCS-All-CS epitopes.
• the protein consists of the sequence PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR ADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQE GGAAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGA NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAG GQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVN AANKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 40; PIC-PvCS-All-CS epitopes-His6).
• the proteins of the invention can be produced, for example, by heterologous expression in a bacterial host cell (e.g., an E. coli host cell) or in a yeast host cell (e.g., a P. pas tor is host cell).
• a bacterial host cell e.g., an E. coli host cell
• a yeast host cell e.g., a P. pas tor is host cell
• the invention also provides DNA molecules encoding the proteins of the invention.
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 1 (PvCs-VK210), said DNA comprising the sequence
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 27 (PIC-PvCS-VK210), said DNA comprising the sequence 5' -
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 2 (PvCs-VK247), said DNA comprising the sequence 5 ' -ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGTGTGAACTTCAACAAC
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 31 (PIC-PvCS-VK247), said DNA comprising the sequence
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 3 (PvCs-Vivax-like), said DNA comprising the sequence
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 35 (PIC-PvCS-Vivax-like), said DNA comprising the sequence
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 4 (PvCs-All-CS-Epitopes), said DNA comprising the sequence
• the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 39 (PIC-PvCS-All-CS epitopes), said
• DNA comprising the sequence 5' -
• the invention also provides vectors (e.g., expression vectors) comprising the
• the invention provides a method for making the recombinant protein immunogens and the protein immunogens produced by said method.
• the protein immunogen is encoded as His 6 tagged protein within Pet28A expression vector and is produced in a E. coli BL21 DE3 host cell and purified by a method comprising the steps of:
• washing the column with Wash buffer 50 mM NaH 2 P0 4 , 300 mM NaCl, 10 % glycerol, pH 6.0
• Elution buffer Wash buffer containing 500 mM imidazole and 2 mM PMSF, pH 6.0
• the protein immunogen is encoded as His 6 tagged protein within pPIC9K P. pastoris expression vector and is produced in a P. pastoris his4 ⁇ host cell and purified by a method comprising the steps of:
• the invention provides an adenoviral vector comprising the sequence
• the invention provides an adenoviral vector encoding a sequence comprising:
• VK210 epitope DRADGQPAG 2 (DRAAGQPAG) 2 DRADGQPAGD (SEQ ID NO: 11),
• VK247 epitope ANGAGNQP G 4 (SEQ ID NO: 12),
• the invention provides an adenoviral vector encoding a protein comprising the sequence
• NDLETDVCT SEQ ID NO: 20; Ad-All-CS-Epitopes.
• adenoviral vectors of the invention can be based, for example, on AdC68 or
• the invention also provides immunogenic and vaccine compositions comprising one or more proteins and/or adenoviral vectors of the invention and a carrier or diluent.
• the immunogenic or vaccine composition comprises (i) a protein comprising SEQ ID NO: 1 (PvCs-VK210) or SEQ ID NO: 27 (PIC-PvCS-VK210), (ii) a protein comprising SEQ ID NO: 2 (PvCs-VK247) or SEQ ID NO: 31 (PIC-PvCS-VK247), (iii) a protein comprising SEQ ID NO: 3 (PvCs- Vivax-like) or SEQ ID NO: 35 (PIC-PvCS-Vivax-like), and (iv) a carrier or diluent.
• the immunogenic or vaccine composition comprises (a) two or more proteins, selected from the group consisting of (i) a protein comprising SEQ ID NO: 1 (PvCs-VK210), (ii) a protein comprising SEQ ID NO: 2 (PvCs-VK247), (iii) a protein comprising SEQ ID NO: 3 (PvCs-Vivax-like), (iv) a protein comprising SEQ ID NO: 4 (PvCs-All-CS-Epitopes), (v) a protein comprising SEQ ID NO: 27 (PIC-PvCS-VK210), (vi) a protein comprising SEQ ID NO: 31 (PIC- PvCS-VK247), (vii) a protein comprising SEQ ID NO: 35 (PIC-PvCS-Vivax-like), and (viii) a protein comprising SEQ ID NO: 39 (PIC-PvCS-All-CS epitopes), and (b) a protein comprising S
• the immunogenic or vaccine composition comprises
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) VK210 epitope (DRADGQPAG) 2 (DRAAGQPAG) 2 DRADGQPAGD (SEQ ID NO: 11), and (iii) P.
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) VK247 epitope (ANGAGNQP G) 4 (SEQ ID NO: 12), and (iii) P. vivax CS protein C-terminal sequence
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) Vivax-like epitope (APGANQEGGAA) 3 (SEQ ID NO: 13), and (iii) P. vivax CS protein C- terminal sequence ANGAGNQPGDRAAGQAAGGNAGGQGQ EGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLETDVCT (SEQ ID NO: 14), and
• the immunogenic or vaccine compositions of the invention can further comprise an adjuvant.
• adjuvants useful in the immunogenic and vaccine composition of the invention include Poly IC, Poly ICLC, Poly IC 12 U, alum, aluminum salts, MF59, QS-21, monophosphoryl lipid A (MPL), AS01 (e.g., AS01A, AS01B, AS01C, AS01D, AS01E), AS02 (e.g., AS02A, AS02B, AS02C, AS02D), AS03, AS04, AS 15, a-tocopherol, flagellin, flagellin-Ag fusion proteins, imiquimods, CpG oligodeoxynuceotides, IC31, QB10, CAF01, ISCOMS, ISCOMATRIX, and any combination thereof.
• the adjuvant is Poly IC.
• the invention provides a method for inducing an immune response (e.g., a protective immune response) to P. vivax circumsporozoite (CS) protein in a subject (e.g., human) comprising administering to the subject an effective amount of an immunogenic composition of the invention.
• the method for inducing an immune response to P. vivax CS protein in a subject comprises (i) administering to the subject an effective amount of the immunogenic composition comprising an adenoviral vector (as a prime) followed by (ii) one or more boosts comprising administering to the subject an effective amount of the immunogenic composition comprising one or more protein immunogens.
• the invention also provides methods for protecting a subject from P. vivax malaria, which methods comprise administering to the subject an effective amount of vaccine compositions disclosed herein.
• FIG 1 is a schematic representation of the CS protein of P. vivax (PvCS).
• Figure 2 is SDS-PAGE analysis of purified recombinant proteins Hise-PvCS- VK210 (lane A), His 6 -PvCS-VK247 (lane B), His 6 -PvCS-Vivax-like (lane C), and His6-PvCS-All-CS epitopes (lane D).
• Figure 3A shows the timeline of mouse immunizations with recombinant proteins and serum antibody titers determination.
• Mice were immunized subcutaneously with each individual protein (10 ⁇ g/mouse/dose) or a mixture containing His 6 PvCS-VK210, His 6 PvCS-VK247 and His 6 PvCS-Vivax-like protein (30 ⁇ g/mouse/dose) or His 6 PvCS-All-CS-epitopes (30 ⁇ g/mouse/dose) in the presence of Poly(I:C) adjuvant (50 ⁇ g/mouse/dose).
• each individual protein (10 ⁇ g/mouse/dose) or a mixture containing His 6 PvCS-VK210, His 6 PvCS-VK247 and His 6 PvCS-Vivax-like protein (30 ⁇ g/mouse/dose) or His 6 PvCS-All-CS-epitopes (30 ⁇ g/mouse/dose) in the presence
• Figures 3B-D show the antibody titers of immunized and control mice to the recombinant proteins His 6 -PvCS-VK210 (B), His 6 -PvCS-VK247 (C), His 6 -PvCS- Vivax-like (D).
• Figures 4A-B show IgG antibody titers to the recombinant proteins Hisg- PvCS-VK210, His 6 -PvCS-VK247, and Hisg-PvCS-Vivax-like in mice five weeks after immunization sub-cutaneously with a single dose of PvCS-AdC68 (groups 2, 4 and 5) or a control adenovirus expressing ⁇ -galactosidase (groups 1 and 3).
• mice from groups 3 to 5 were subsequently boosted subcutaneously with a mixture containing His 6 PvCS-VK210, His 6 PvCS-VK247 and His 6 PvCS-Vivax-like proteins (30 ⁇ g/mouse/dose total) in the presence of Poly(LC) adjuvant (50 ⁇ g/mouse/dose).
• Asterisks denote that mice injected with 2xl0 10 or 10 9 viral particles of PvCS-AdC68 (groups 2, 4 and 5) presented serum antibody titers significantly higher than control mice injected with 2x10 viral particles of control adenovirus (groups 1 and 3, P ⁇ 0.01).
• mice from groups 4 and 5 After boosting the antibody titers of mice from groups 4 and 5 (primed with 2xl0 10 or 10 9 viral particles of PvCS-AdC68, respectively) presented antibody titers to all P. vivax recombinant CS proteins significantly higher than mice from group 3 (P ⁇ 0.01 indicated by crosses).
• Figure 5A shows the timeline of mouse immunizations with recombinant proteins and serum antibody titers determination.
• Mice were immunized subcutaneous ly with a mixture containing His 6 PvCS-VK210, His 6 PvCS-VK247 and His 6 PvCS-Vivax-like proteins (30 ⁇ g/mouse/dose total) or His 6 PvCS-All-CS- Epitopes (30 ⁇ g/mouse/dose) in the presence of Poly(I:C) adjuvant (50 ⁇ g/mouse/dose) or Complete Freuds Adjuvant-CFA (1 : 1 emulsions).
• Figures 5B-D show the antibody titers of immunized and control mice to the recombinant proteins His 6 -PvCS-VK210 (B), His 6 -PvCS-VK247 (C), His 6 -PvCS- Vivax-like (D).
• Figure 6 is SDS-PAGE analysis of purified recombinant proteins PIC-PvCS- VK210-His 6 (lane A), PIC-PvCS-VK247-His 6 (lane C), PIC-PvCS-Vivax-like-His 6 (lane D), and PIC-PvCS-All-CS epitopes-His 6 (lane E).
• immunogenic means that an agent is capable of eliciting a humoral or cellular immune response, and preferably both.
• An immunogenic entity is also antigenic.
• An immunogenic composition is a composition that elicits a humoral or cellular immune response, or both, when administered to an animal having an immune system.
• vaccine refers to a composition (e.g., a recombinant protein or a recombinant adenoviral vector with or without an adjuvant) that can be used to elicit protective immunity in a recipient.
• a vaccine of the invention can elicit immunity in a portion of the immunized population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response. This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., due to treatment with chemotherapy or use of immunosuppressive drugs).
• Vaccine efficacy can be established in animal models.
• adjuvant refers to a compound or composition that augments the host's immune response to another antigen (e.g., a recombinant protein or a recombinant adenoviral vector) when administered conjointly with that antigen.
• another antigen e.g., a recombinant protein or a recombinant adenoviral vector
• Adjuvants useful in the vaccine compositions of the present invention include, but are not limited to, Poly IC, Poly ICLC, and Poly IC12U, alum, aluminum salts, MF59, QS-21, monophosphoryl lipid A (MPL), AS01 (e.g., AS01A, AS01B, AS01C, AS01D, AS01E), AS02 (e.g., AS02A, AS02B, AS02C, AS02D, AS02D), AS03, AS04, AS15, a-tocopherol, flagellin, flagellin-Ag fusion proteins, imiquimods, CpG oligodeoxynuceotides, IC31, QB 10, CAF01, ISCOMS, ISCOMATRIX, and any combination thereof.
• the adjuvant is pharmaceutically acceptable.
• the term “conjoint administration” is used to refer to administration of components (e.g., an immune adjuvant and an antigen or two or more different antigens) simultaneously in one composition, or simultaneously in different compositions, or sequentially within a specified time period (e.g., 24 hours).
• components e.g., an immune adjuvant and an antigen or two or more different antigens
• the term "protect” is used herein to mean prevent or treat, or both, as appropriate, development or continuance of a disease in a subject. This term encompasses partial, enhanced, or full protection of subjects who have not previously been exposed to P. vivax, or have been exposed, but are not fully protected. The term encompasses reducing the chance of developing a P. vivax infection, reducing the chance of becoming ill when one is infected, reducing the severity of the illness (e.g., fever), when one becomes infected, reducing the concentration of parasites in the infected person, or reducing mortality or morbidity from malaria when one is exposed to malaria parasites. In many cases even partial protection is beneficial.
• This term encompasses partial, enhanced, or full protection of subjects who have not previously been exposed to P. vivax, or have been exposed, but are not fully protected. The term encompasses reducing the chance of developing a P. vivax infection, reducing the chance of becoming ill when one is infected, reducing
• protection immune response or “protective immunity” comprise a humoral (antibody) immunity or cellular immunity, or both, effective to, e.g., eliminate or reduce the load of a pathogen (e.g., P. vivax) or produce any other measurable alleviation of the infection in an immunized (vaccinated) subject.
• a pathogen e.g., P. vivax
• terapéuticaally effective amount/dose refers to that quantity of an immunogenic or vaccine composition comprising an antigen (e.g., a recombinant protein or a recombinant adenoviral vector) that is sufficient to produce a protective immune response upon administration to a mammal.
• an antigen e.g., a recombinant protein or a recombinant adenoviral vector
• pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier applied to immunogenic or vaccine compositions of the invention refers to a diluent, excipient, or vehicle with which an antigen (e.g., a recombinant protein or a recombinant adenoviral vector) is administered.
• an antigen e.g., a recombinant protein or a recombinant adenoviral vector
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution, saline solutions, and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin, 18th Edition.
• purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained.
• a purified recombinant protein is preferably substantially free of host cell or culture components.
• substantially free is used operationally, in the context of analytical testing of the material.
• purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and still more preferably at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
• subject refers to an animal susceptible to P. vivax infection, preferably a mammal (e.g., rodent such as mouse). In particular, the term refers to humans.
• the term "about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
• the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
• the invention provides the following isolated recombinant protein immunogens.
• An isolated protein comprising the sequence PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAA
• NKKPEDLTLNDLETDVCT SEQ ID NO: 27; PIC-PvCS-VK210.
• NKKPEDLTLNDLETDVCTHHHHHHH SEQ ID NO: 28; PIC-PvCS-
• VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT SEQ ID NO: 2; PvCs-
• NKKPEDLTLNDLETDVCT SEQ ID NO: 31; PIC-PvCS-VK247).
• NKKPEDLTLNDLETDVCTHHHHHH SEQ ID NO: 32; PIC-PvCS-VK247-His 6 ).
• VNAANKKPEDLTLNDLETDVCT SEQ ID NO: 35; PIC-PvCS-Vivax-like.
• VNAANKKPEDLTLNDLETDVCTHHHHHH SEQ ID NO: 36; PlC-PvCS-Vivax-
• ETDVCT SEQ ID NO: 24; His 6 -PvCS-All CS epitopes.
• AANKKPEDLTLNDLETDVCT SEQ ID NO: 39; PIC-PvCS-All-CS epitopes.
• AANKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 40; PIC-PvCS-All-CS epitopes-His6).
• the invention provides DNA molecules encoding such protein immunogens.
• DNA molecules encoding such protein immunogens. Specific non-limiting examples of such DNA molecules are as follows. An isolated DNA comprising the sequence
• the protein immunogen is encoded as His 6 tagged protein within pET28a expression vector and is produced in a E. coli BL21 DE3 host cell and purified by a method comprising the steps of:
• wash the column with Wash buffer 50 mM NaH 2 P0 4 , 300 mM NaCl, 10 % glycerol, pH 6.0;
• Elution buffer Wash buffer containing 500 mM imidazole and 2 mM PMSF, pH 6.0
• the protein immunogen is encoded as His 6 tagged protein within pPIC9K P. pastoris expression vector and is produced in a P. pastoris his4 ⁇ host cell and purified by a method comprising the steps of:
• the invention also provides various immunogenic adenoviral vectors useful in immunogenic and vaccine compositions of the invention.
• the invention provides an adenoviral vector comprising the sequence
• the invention provides an adenoviral vector encoding a sequence comprising:
• VK210 epitope DRADGQPAG 2 (DRAAGQPAG) 2 DRADGQPAGD (SEQ ID NO: 11),
• VK247 epitope (ANGAGNQP G) 4 (SEQ ID NO: 12), (iii) Vivax-like epitope (APGANQEGGAA) 3 (SEQ ID NO: 13), and (iv) P. vivax CS protein C-terminal sequence
• the invention provides an adenoviral vector encoding a protein comprising the sequence
• NDLETDVCT SEQ ID NO: 20; Ad-All-CS-Epitopes.
• the adenoviral vectors of the invention can be based on various adenoviruses.
• Non-limiting examples of useful adenoviruses include AdC68 and AdHu5.
• the invention provides various immunogenic and vaccine compositions comprising one or more of the above-described protein immunogens and/or adenoviral vectors of the invention and a carrier or diluent.
• the immunogenic or vaccine composition comprises (i) a protein comprising SEQ ID NO: 1 (PvCs-VK210) or SEQ ID NO: 27 (PIC-PvCS-VK210), (ii) a protein comprising SEQ ID NO: 2 (PvCs-VK247) or SEQ ID NO: 31 (PIC-PvCS-VK247), (iii) a protein comprising SEQ ID NO: 3 (PvCs- Vivax-like) or SEQ ID NO: 35 (PIC-PvCS-Vivax-like), and (iv) a carrier or diluent.
• the immunogenic or vaccine composition comprises (a) two or more proteins, selected from the group consisting of (i) a protein comprising SEQ ID NO: 1 (PvCs-VK210), (ii) a protein comprising SEQ ID NO: 2 (PvCs-VK247), (iii) a protein comprising SEQ ID NO: 3 (PvCs-Vivax-like), (iv) a protein comprising SEQ ID NO: 4 (PvCs-All-CS-Epitopes), (v) a protein comprising SEQ ID NO: 27 (PIC-PvCS-VK210), (vi) a protein comprising SEQ ID NO: 31 (PIC- PvCS-VK247), (vii) a protein comprising SEQ ID NO: 35 (PIC-PvCS-Vivax-like), and (viii) a protein comprising SEQ ID NO: 39 (PIC-PvCS-All-CS epitopes), and (b) a protein comprising S
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) VK210 epitope (DRADGQPAG) 2 (DRAAGQPAG) 2 DRADGQPAGD (SEQ ID NO: 11), and (iii) P.
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) VK247 epitope (ANGAGNQP G) 4 (SEQ ID NO: 12), and (iii) P. vivax CS protein C-terminal sequence
• a protein comprising (i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10), (ii) Vivax-like epitope (APGANQEGGAA) 3 (SEQ ID NO: 13), and (iii) P. vivax CS protein C- terminal sequence ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14), and
• Such vaccines may be for administration by oral, parenteral (intramuscular, intraperitoneal, or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
• compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into Immune Stimulating Complexes together with saponins, for example QuilA (ISCOMS), or into liposomes.
• buffer content e.g., Tris-HCl, acetate, phosphate
• anti-oxidants e.g., ascorbic acid, sodium metabisulfite
• preservatives e.g., Thimersol, benzyl alcohol
• Useful solid oral dosage forms can include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules.
• a description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by reference.
• the formulation will include the therapeutic agent and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
• Immunogenic and vaccine compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants, preserving, wetting, emulsifying, and dispersing agents. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
• Immunogenic and vaccines compositions of the invention may be administered in conjunction with one or more additional immunogenic or vaccine compositions described herein and/or one or more additional active ingredients, pharmaceutical compositions, or vaccines (e.g., compositions comprising other malaria antigens, such as, e.g., DBP, PvTRAP, PvMSPl, PvMSP2, PvMSP3, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PvAMAl, RBP, and any combinations thereof).
• additional immunogenic or vaccine compositions described herein e.g., compositions comprising other malaria antigens, such as, e.g., DBP, PvTRAP, PvMSPl, PvMSP2, PvMSP3, PvMSP4, PvMSP5, PvMSP6, PvMSP7, PvMSP8, PvMSP9, PvAMAl, RBP, and any combinations thereof).
• kits may additionally comprise carrier, either in combination with or separate from the pharmaceutical composition.
• a kit may additionally comprise means for delivery of the pharmaceutical composition, such as syringe and needle or microneedle.
• the PvCS recombinant proteins and recombinant adenoviruses are preferably delivered with an adjuvant.
• Adjuvants useful in the vaccine compositions of the present invention include, but are not limited to, Poly IC, Poly ICLC, and Poly IC12U, alum, aluminum salts, MF59, QS-21, monophosphoryl lipid A (MPL), AS01 (e.g., AS01A, AS01B, AS01C, AS01D, AS01E), AS02 (e.g., AS02A, AS02B, AS02C, AS02D, AS02D), AS03, AS04, AS 15, a-tocopherol, flagellin, flagellin-Ag fusion proteins, imiquimods, CpG oligodeoxynuceotides, IC31, QB10, CAF01, ISCOMS, ISCOMATRIX, and any combination thereof.
• MPL monophosphoryl lipid A
• AS01 e.g., AS01
• the invention provides methods for inducing an immune response (e.g., a protective immune response) to P. vivax CS protein (PvCS) in a subject (e.g., human) comprising administering to the subject an effective amount of any of the above-described immunogenic or vaccine compositions.
• an immune response e.g., a protective immune response
• PvCS P. vivax CS protein
• the subject to which the vaccine is administered in accordance with these methods may be any human or non-human animal susceptible to infection with the malaria parasite.
• administration can be oral, parenteral (e.g., parenteral injection such as intraperitoneal, subcutaneous, or intramuscular injection), intranasal, intramuscular, or any one or more of a variety of well-known administration routes.
• parenteral e.g., parenteral injection such as intraperitoneal, subcutaneous, or intramuscular injection
• intranasal e.g., intranasal, intramuscular, or any one or more of a variety of well-known administration routes.
• the administration may be by continuous infusion or by single or multiple boluses.
• the effectiveness of protection from P. vivax malaria may be readily ascertained by the skilled practitioner by evaluation of infection in red blood cells (erythrocytes) or clinical manifestations associated with malarial infection, for example fatigue, headache, elevated temperature, and coma.
• red blood cells erythrocytes
• clinical manifestations associated with malarial infection for example fatigue, headache, elevated temperature, and coma.
• Generation of an immune response in a subject can be measured by standard tests of humoral and cellular immunity including, but not limited to, the following: direct measurement of peripheral blood lymphocytes by means known to the art; natural killer cell cytotoxicity assays (Provinciali et al (1992) J. Immunol. Meth. 155: 19-24), cell proliferation assays (Vollenweider et al. (1992) J. Immunol. Meth. 149: 133-135), immunoassays of immune cells and subsets (Loeffler et al. (1992) Cytom. 13: 169-174; Rivoltini et al. (1992) Can. Immunol. Immunother.
• the therapeutically/immunogenically effective amount/dose of the immunogenic and vaccine compositions of the invention varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of P.
• the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 ⁇ g/dose, more particularly from about 1.0 to 100 ⁇ g/dose, most preferably from about 10 to 50 ⁇ g/dose.
• the dosing schedule may vary, depending on the circulation half-life and the formulation used.
• Useful immunogenic/vaccination regimens of the invention may include prime/boost, preferably combining an adenoviral vector as a prime and one or more protein immunogens as one or several boosts.
• Example 1 Purification to homogeneity and immunogenicity of four bacterial recombinant proteins based on the sequence of P. vivax CS proteins. Recombinant bacterial proteins representing the different allelic forms of the
• P. vivax were expressed and purified as described below. These recombinant proteins are denominated His 6 -PvCS-VK210, His 6 -PvCS-VK247, His 6 --PvCS-Vivax-like and His 6 -PvCS-All-CS epitopes.
• Codon optimized synthetic genes were purchased from Genescript Inc. containing the following sequences of nucleotides:
• mice purchased from the Federal University of Sao Paulo, Brazil.
• Mice were immunised three times, three weeks apart, subcutaneously in the two hind footpads, using a final volume of 50 ⁇ in each footpad (first dose) and a final volume of 100 ⁇ at the base of the tail (second and third dose).
• Serum anti-PvCS antibodies were detected by ELISA essentially as described previously [16].
• the recombinant proteins employed as the solid phase bound antigen were His 6 PvCS-VK210, His 6 PvCS-VK247 or His 6 PvCS-Vivax-like.
• a peroxidase conjugated goat anti-mouse IgG (Sigma) was applied at a final dilution of 1 : 1,000, while the mice sera were tested at serial dilutions starting from 1 :200.
• Specific anti- PvCS titers were determined as the highest dilution yielding an OD4 9 2 higher than 0.1. The results were presented as mean ⁇ SD. Statistical analyses were performed using one-way ANOVA followed by Tukey HSD test.
• mice immunized with the recombinant proteins developed significant specific IgG titers. Maximal IgG antibody titers were achieved after the third immunising dose. These titers were as high as the titers obtained after immunization with Complete Freund's Adjuvant (Sigma) and were maintained until 183 days after the first immunizing dose (see Figures 3 and 5). These antibodies were also capable of recognizing fixed sporozoites of P. vivax by immunofluorescence assay.
• Example 2 Generation of replication-deficient recombinant Human Adenovirus Type 5 and Chimpanzee Adenovirus Type 68 expressing the P. vivax CS protein.
• Codon optimized synthetic gene was purchased from Genscript Inc. containing the following sequence of nucleotides:
• Bold and underlined regions represent the sites for Xbal (TCTAGA) and Kpnl (GGTACC).
• the gene was subcloned into the plasmid pShuttle (Clontech).
• the expression cassette was released by digestion with I-Ceul and PI-SceI, and then cloned into the Ad molecular clone AdC68 or AdHu5 that had been digested with the same enzymes.
• Recombinant Ad vectors were generated by rescue in packaging cells such as HEK 293 cells. Details on the generation of the recombinant adenoviruses, growth and titration can be found in reference 17. The presence of the recombinant P.
• vivax protein expressed by the recombinant adenovirus were confirmed by immunoblot analyses using monoclonal antibodies to the PvCS-VK210 or His 6 PvCS-VK247.
• the expression of the recombinant CS protein was also confirmed by the ability of the recombinant adenoviruses to elicit specific antibodies (see results below).
• Example 3 Vaccination with replication-deficient recombinant adenoviruses expressing the P. vivax CS protein can prime for subsequent boost with the 3 recombinant CS proteins of P. vivax Poly(IC) adjuvant.
• mice immunized with PvCS-AdC68 presented specific IgG antibodies to the recombinant proteins (see Figures 4A and 4B).
• mice injected with 2xl0 10 or 10 9 viral particles of PvCS-AdC68 presented serum antibody titers significantly higher than control mice injected with 2xl0 10 viral particles of control adenovirus expressing the unrelated protein ⁇ -galactosidase (groups 1 and 3, P ⁇ 0.01).
• mice from groups 3 to 5 were subsequently boosted subcutaneously with a mixture containing His 6 PvCS-VK210, His 6 PvCS-VK247 and His 6 PvCS-Vivax-like (10 ⁇ g of each protein) in the presence of 50 ⁇ g of Poly(LC).
• mice from groups 4 and 5 (primed with 2xl0 10 or 10 9 viral particles of PvCS-AdC68, respectively) presented antibody titers to all P. vivax recombinant CS proteins significantly higher than mice from group 3 (P ⁇ 0.01 indicated by crosses).
• Example 4 Expression of four recombinant proteins based on the sequence of P. vivax CS proteins in Pichia pastoris.
• Recombinant proteins representing the different allelic forms of the P. vivax were expressed in Pichia pastoris and purified as described below. These recombinant proteins and their coding sequences are as follows:
• the synthetic genes encoding the above proteins were synthetized by GenScript USA Inc. (Piscataway, NJ) with codon-optimization to improve the expression in Pichia pastoris.
• the constructs were designed with appropriate restriction sites and a carboxyl-terminal His 6 tag to enable purification.
• the synthetic gene delivered cloned in pUC57 vector was obtained by digestion with Notl enzyme mix (New England Biolabs) and subcloned into the Notl site of the P. pastoris expression vector pPIC9K (Invitrogen).
• This expression vector contains the nucleotide sequence encoding the a-factor signal peptide of Saccharomyces cerevisiae for protein secretion, the AOX1 promoter for control of gene expression, and the HIS4 gene for selection of the recombinant yeast clones.
• a clone was selected containing the PvCS in the correct orientation.
• the plasmids were linearized with Sail. High copy number transformants of P. pastoris were isolated by screening for high G418 (Sigma) resistance. Mut + clones were selected and used for further expression and purification studies.
• a Mut + transformant was initially grown overnight in 200 ml of BMGY medium (1% w/v yeast extract, 2% w/v peptone, 1.34% w/v yeast nitrogen base without amino acids, 4 x 10 ⁇ 5 % w/v biotin, 1% w/v glycerol, 0.1 M potassium phosphate, pH 6.0) at 28-30°C with vigorous shaking.
• BMGY medium 1% w/v yeast extract, 2% w/v peptone, 1.34% w/v yeast nitrogen base without amino acids, 4 x 10 ⁇ 5 % w/v biotin, 1% w/v glycerol, 0.1 M potassium phosphate, pH 6.0
• Cells were harvested, resuspended in 2 L of BMMY (BMGY with glycerol replaced by 0.5% v/v methanol) and incubated again for 72 h.
• Methanol was added at a final concentration of 1% v/v every 24
• Bound proteins were eluted with a 15 to 400 mM Imidazole (Sigma) gradient in wash buffer (sodium phosphate buffer 20 mM, pH 8.0, 0.5 M NaCl, 1 mM PMSF and 10%> glycerin). Fractions were analyzed by SDS-PAGE and stained with Coomassie blue. Fractions containing the recombinant proteins with a high degree of purity (see Figure 6) were pooled and dialyzed against PBS. The protein concentration was determined by the Bradford method (BioRad) using bovine serum albumin (BSA, Sigma) as the standard.
• BSA bovine serum albumin
• Kester KE Cummings JF, Ofori-Anyinam O, Ockenhouse CF, Krzych U, Moris P, Schwenk R, Nielsen RA, Debebe Z, Pinelis E, Juompan L, Williams J, Dowler M, Stewart VA, Wirtz RA, Dubois MC, Lievens M, Cohen J, Ballou WR, Heppner DG Jr; RTS,S Vaccine Evaluation Group. Randomized, double-blind, phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associates of protection. J Infect Dis. 2009 Aug l ; 200(3):337-46.

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