PLASMODIUM VIVAX VACCINE COMPOSITIONS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Serial No. 61/614,439, filed on March 22, 2012, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to Plasmodium vivax vaccine compositions.

BACKGROUND OF THE INVENTION

In contrast to reports from the last decade that characterized Plasmodium vivax malaria as a benign infection, P. vivax is now recognized as a cause of severe disease and death [1-3]. Recent data indicate that 130-435 million infections occur annually, and an estimated 2.6 billion people live in areas at risk of P. vivax infection [4]. The relatively low efficacy of the measures currently used to control P. vivax infection demands the development of new strategies to prevent infection [5].

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].

The confirmation that the CS protein can be used as vaccine in humans comes from studies performed with the deadly parasite P. falciparum. 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. The efficacy of the RTS,S ^® formulation was tested in the presence of adjuvant systems (AS), which consist of two different formulations that include monophosphoryl lipid A (MPL, a detoxified form of LPS) and QS21 (saponin purified from Quillaja saponaria) in an oil-in-water emulsion (AS02) or in a liposomal suspension (AS01, [6]).

In Phase II trials performed in naive human volunteers experimentally challenged with P. falciparum sporozoites efficacies ranged from 32% to 47.2%. Immunological studies performed on these vaccinated individuals indicated that protection correlated with the concentration of specific antibodies and the frequency of IFN-γ producing cells as detected by ELISPOT [8]. Preliminary results of an ongoing Phase III trial performed in children in the endemic areas of Africa showed statistically significant efficacy during the period studied retarding the first malaria episode [9]. Parallel studies performed in non-human primates used recombinant adenoviruses in a heterologous prime-boost protocol with the recombinant vaccine RTS,S ^® [10].

As described for the CS proteins of all other species of Plasmodium, 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].

Sequencing of the gene encoding the CS of different strains of P. vivax found the presence of three different alleles at the central repeat domain. These alleles were described in different parts of the world [12-15]. This strain diversity adds to the complexity for the development of a universal vaccine against P. vivax malaria. CS protein designated VK210, or Type 1, and CS protein designated VK247, or Type 2, or Vivax-like are almost identical in their N- and C-terminal domains, but differ in the central repeat region.

A recombinant P. vivax CS protein (PvCS) was expressed and tested as a vaccine with very limited success (Collins et al, 1989, Am. J. Trop. Med. Hyg. 40, 455-64) and was not pursued further. Recently, a Phase I study was carried out with the N-terminal, C-terminal and repeat region of PvCS of the P. vivax Sal 1 parasite and shown to induce some antibody and IFN-γ responses in vaccinated individuals (Herrera et al. 2005, Am. J. Trop. Med. Hyg. 73, 3-9). A recombinant P. vivax vaccine representing PvCS of the two variants VK210 and VK247 has been generated but not yet tried in humans (Bell et al. 2009, Vaccine 27, 1448-53).

SUMMARY OF THE INVENTION

As indicated in the Background section, above, there is a great need in the art to develop new effective strategies for protection against Plasmodium vivax infection. 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.

In one embodiment, the invention provides an isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGDRADGQPAGDRADGQPAGDRADG

QPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADGQPAG

DRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAD

GQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAA

GDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCS

VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 1; PvCs-

VK210).

In a specific embodiment, the protein consists of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGDRA

DGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRADGQP

AGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR

ADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQ

PAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGANAPNEK SVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLET DVCT (SEQ ID NO: 21; His ₆ -PvCS-VK210).

In another embodiment, the invention provides an isolated protein comprising the sequence

PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAA

GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPA

GDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRA

DGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCT (SEQ ID NO: 27; PIC-PvCS-VK210).

In a specific embodiment, the protein consists of the sequence

PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAA

GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPA

GDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRA

DGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCTHHHHHHH (SEQ ID NO: 28; PIC-PvCS-

VK210-His ₆ ).

In another embodiment, the invention provides, an isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGANGAGNQPGANGAGNQPGANGAG

NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPG

ANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA

GNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQP

GANGAGNQPGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCS

VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 2; PvCs-

VK247).

In a specific embodiment, the protein consists of the sequence MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGV F VDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGANG

AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGN

QPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQGQ EGANAPNEK

SVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLET

DVCT (SEQ ID NO: 22; His ₆ -PvCS-VK247).

In another embodiment, the invention provides an isolated protein comprising the sequence

PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANG

AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCT (SEQ ID NO: 31; PIC-PvCS-VK247).

In a specific embodiment, the protein consists of the sequence

PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANG

AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 32; PIC-PvCS-VK247-His ₆ ).

In a further embodiment, the invention provides an isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGAPGANQEGGAAAPGANQEGGAAAP

GANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA

PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ EGGAADRAAGQAAGGNAGGQGQ EGANAPNEKSVKEYLDKVRATVGTE WTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLETDVCT (SEQ ID NO: 3; PvCs -Vivax-like) .

In a specific embodiment, the protein consists of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGAPG

ANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEG

GAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAP

GANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGNAGGQGQNNEGA

NAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLT

LNDLETDVCT (SEQ ID NO: 23; His ₆ -PvCS-Vivax-like).

In another embodiment, the invention provides an isolated protein comprising the sequence

PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA

PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGN

AGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRR

VNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 35; PIC-PvCS-Vivax-like).

In a specific embodiment, the protein consists of the sequence

PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA

PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGN

AGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRR

VNAANKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 36; PlC-PvCS-Vivax-

Iike-His6). In yet another embodiment, the invention provides an isolated protein comprising the sequence

THCGHNVDLSKAI LNGV F VDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGPGDRADGQPAGDRADGQPAGDRAA

GQPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAP

GANQEGGAAAPGANQEGGAAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGDRAAGQAAGGNAGGQGQ EGANAPNEKSVKEYLDKVRATVGTEWTPC

SVTCGVGVRVRRRV AANKKPEDLTLNDLETDVCT (SEQ ID NO: 4; PvCs-

All-CS-Epitopes).

In a specific embodiment, the protein consists of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGPGD

RADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADG

QPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGA

GNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPN

EKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDL

ETDVCT (SEQ ID NO: 24; His ₆ -PvCS-All CS epitopes).

In another embodiment, the invention provides an isolated protein comprising the sequence

PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR

ADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGA

NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAG

GQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVN

AANKKPEDLTLNDLETDVCT (SEQ ID NO: 39; PIC-PvCS-All-CS epitopes).

In a specific embodiment, the protein consists of the sequence PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR ADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQE GGAAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGA 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).

The invention also provides DNA molecules encoding the proteins of the invention.

In one specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 1 (PvCs-VK210), said DNA comprising the sequence

5 '-ACCCACTGCGGCCATAACGTTGACCTGTCTAAAGCGATTAACCTGAACGGTGTTAACT TTAACAAC GTTGATGCCTCCTCCCTGGGTGCCGCGCATGTTGGTCAGAGCGCATCCCGCGGTCGTGGC CTGGGCGAA AACCCGGATGACGAAGAAGGCGACGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCG AAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGCGACCGCGCGGATGGCCAGCCGGCCGGCGACCGT GCGGATGGC CAGCCGGCAGGCGACCGTGCGGATGGTCAGCCGGCCGGTGACCGTGCGGACGGTCAGCCG GCGGGCGAC CGCGCTGCAGGCCAGCCGGCCGGTGACCGCGCCGATGGCCAGCCGGCTGGCGACCGCGCG GATGGTCAG CCGGCGGGTGATCGCGCGGACGGTCAGCCGGCAGGTGATCGCGCAGACGGTCAGCCGGCC GGCGATCGC GCGGCAGGCCAGCCGGCGGGCGACCGTGCGGCCGGTCAGCCGGCGGGTGATCGTGCCGAC GGCCAGCCG GCGGGTGACCGCGCTGCGGGTCAGCCGGCAGGCGACCGCGCGGACGGCCAGCCGGCAGGT GACCGTGCA GCGGGCCAGCCGGCTGGCGACCGTGCCGATGGTCAGCCGGCCGGCGATCGTGCGGCAGGC CAGCCGGCT GGTGACCGTGCCGCGGGTCAGCCGGCAGGTGACCGCGCAGCGGGTCAGGCCGCAGGTGAT CGTGCGGCG GGTCAGGCCGCGGGCGGTAACGCGGGCGGCCAGGGCCAGAACAACGAAGGCGCAAACGCT CCGAACGAA AAATCCGTGAAAGAATACCTGGATAAAGTGCGTGCGACCGTTGGTACCGAATGGACCCCG TGTTCCGTG ACCTGTGGCGTTGGCGTTCGCGTGCGCCGCCGTGTTAACGCAGCGAACAAAAAACCGGAA GATCTGACC CTGAACGATCTGGAAACCGACGTTTGTACCTAA- 3 ' (SEQ ID NO : 5) .

In a further specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 27 (PIC-PvCS-VK210), said DNA comprising the sequence 5' -

CCAAGAGAAAACAAGTTGAAACAGCCAGGAGACAGAGCAGACGGTCAGCCAGCAGGA GACAGAGCAGAT GGACAGCCAGCAGGAGACAGAGCTGATGGTCAACCAGCAGGAGATAGAGCTGACGGTCAG CCTGCTGGA GACAGAGCTGCCGGTCAACCAGCCGGAGATAGAGCAGATGGACAGCCTGCTGGAGATAGA GCCGACGGA CAGCCTGCCGGAGATAGAGCCGATGGTCAACCTGCTGGAGATAGAGCAGACGGTCAACCA GCTGGAGAT AGAGCAGCTGGTCAGCCTGCCGGAGACAGAGCCGCAGGACAGCCAGCCGGAGATAGAGCT GATGGACAA CCAGCGGGAGATCGTGCTGCCGGACAGCCTGCAGGAGATAGAGCCGACGGTCAACCTGCC GGAGATAGA GCTGCCGGACAGCCAGCTGGAGATAGAGCTGATGGTCAACCCGCCGGAGATAGAGCCGCA GGTCAACCA GCCGGAGACAGAGCCGCCGGACAACCAGCCGGAGATCGTGCAGCTGGTCAAGCCGCAGGT GACAGAGCC GCTGGTCAAGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGTGCTAAC GCCCCAAAT GAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGGTACAGAGTGGACC CCTTGTTCC GTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCA GAAGATTTG ACCTTGAACGACTTGGAAACCGATGTCTGCACT-3 ' (SEQ ID NO: 25) .

In another specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 2 (PvCs-VK247), said DNA comprising the sequence 5 ' -ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGTGTGAACTT CAACAAC

GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGCGGTCGT GGCCTGGGTGAA AACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCG AAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGTGCTAACGGTGCAGGTAACCAGCCGGGTGCAAAC GGCGCGGGT AACCAGCCGGGCGCGAACGGCGCTGGCAACCAGCCGGGCGCAAACGGCGCTGGTAACCAG CCGGGCGCC AACGGCGCCGGCAACCAGCCGGGTGCGAACGGCGCCGGTAACCAGCCGGGCGCTAACGGC GCAGGCAAT CAACCGGGCGCGAACGGCGCGGGCAACCAGCCGGGTGCCAACGGCGCAGGCAATCAGCCT GGCGCGAAC GGCGCAGGCAACCAGCCGGGTGCTAACGGCGCAGGAAATCAGCCGGGCGCTAACGGTGCG GGCAATCAA CCGGGTGCCAACGGTGCGGGCAATCAGCCTGGTGCCAACGGCGCAGGAAACCAACCGGGC GCGAACGGT GCGGGTAATCAACCGGGTGCGAATGGCGCTGGCAATCAACCGGGCGCAAATGGCGCGGGA AATCAGCCG GGTGCAAACGGTGCGGGTAATCAGCCCGGTGCCAACGGCGCAGGGAATCAACCTGGCGCT AACGGCGCA GGTAACCAGCCGGGCGGTAACGCGGGTGGCCAGGGCCAGAACAACGAAGGTGCGAACGCA CCGAACGAA AAAAGCGTGAAAGAATACCTGGATAAAGTGCGCGCGACCGTTGGTACCGAATGGACCCCG TGCTCCGTG ACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAACCGGAA GACCTGACC CTGAACGATCTGGAAACCGACGTTTGTACCTAA-3 ' (SEQ ID NO : 6) .

In a further specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 31 (PIC-PvCS-VK247), said DNA comprising the sequence

5' - CCAAGAGAAAACAAGTTGAAACAGCCAGGAGCAAATGGAGCAGGTAACCAGCCAGGAGCA AACGGAGCA GGTAACCAGCCAGGAGCAAACGGTGCTGGAAATCAACCAGGTGCCAACGGTGCAGGAAAT CAGCCTGGA GCTAACGGTGCCGGAAATCAACCAGGTGCAAACGGTGCTGGAAATCAGCCTGGAGCAAAT GGTGCAGGA AATCAACCAGGTGCTAATGGAGCCGGTAACCAGCCTGGAGCCAATGGAGCCGGTAACCAA CCTGGAGCT AATGGAGCTGGTAACCAACCAGGTGCTAATGGTGCTGGTAACCAGCCTGGAGCAAACGGA GCCGGTAAT CAGCCTGGAGCAAACGGTGCCGGTAACCAGCCTGGTGCCAATGGTGCCGGAAATCAGCCT GGTGCTAAT GGAGCCGGAAACCAGCCTGGAGCCAACGGAGCTGGTAACCAGCCTGGTGCAAATGGAGCT GGTAATCAG CCAGGTGCAAATGGAGCCGGAAATCAACCTGGTGCTAATGGAGCAGGTAACCAACCTGGT GCCAATGGA GCTGGAAATCAACCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGAGCAAAC GCTCCAAAT GAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGGTACAGAGTGGACC CCTTGTTCC GTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCA GAAGACCTT ACTTTGAACGACTTGGAAACCGATGTCTGCACT-3 ' (SEQ ID NO: 29) .

In a further specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 3 (PvCs-Vivax-like), said DNA comprising the sequence

5'-

ACCCACTGCGGTCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGCGTGAAC TTCAACAAC GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGTCGTGGT CTGGGTGAA AACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCG AAAAACCCG CGCGAAAACAAACTGAAACAGCCGGGTGCACCGGGTGCTAACCAGGAAGGTGGCGCTGCA GCACCGGGT GCAAACCAGGAAGGTGGCGCAGCGGCGCCGGGCGCGAACCAGGAAGGTGGCGCGGCAGCT CCGGGTGCC AACCAGGAAGGTGGCGCCGCGGCACCGGGTGCGAATCAAGAAGGTGGCGCGGCGGCGCCG GGCGCAAAT CAAGAAGGTGGCGCAGCAGCGCCGGGCGCAAATCAGGAAGGTGGCGCAGCTGCGCCGGGC GCTAATCAA GAAGGTGGCGCTGCTGCACCGGGTGCGAATCAGGAAGGTGGCGCCGCTGCGCCGGGCGCA AACCAAGAA GGTGGCGCAGCCGCGCCGGGCGCAAACCAGGAAGGTGGCGCAGCCGCACCTGGCGCGAAT CAAGAAGGT GGCGCCGCTGCTCCGGGTGCGAACCAAGAAGGTGGCGCGGCTGCGCCGGGCGCTAACCAA GAAGGTGGC GCTGCGGCGCCGGGCGCTAATCAGGAAGGTGGCGCGGCGGCTCCGGGTGCGAACCAGGAA GGTGGCGCA GCAGATCGTGCAGCTGGTCAGGCAGCGGGTGGCAACGCGGGTGGCCAGGGCCAGAACAAC GAAGGTGCC AACGCGCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGCGCGACCGTTGGT ACCGAATGG ACCCCGTGCTCCGTGACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCAGCT AACAAAAAA CCGGAAGATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA- 3 ' (SEQ ID NO : 7) .

In a further specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 35 (PIC-PvCS-Vivax-like), said DNA comprising the sequence

5' -

CCAAGAGAAAATAAGTTGAAACAGCCAGGTGCCCCAGGAGCAAACCAAGAGGGAGGT GCCGCCGCCCCA GGAGCAAACCAGGAGGGAGGTGCTGCCGCACCAGGAGCCAACCAAGAAGGTGGAGCTGCC GCACCTGGT GCAAATCAGGAGGGTGGAGCTGCCGCACCAGGAGCTAATCAGGAAGGAGGTGCTGCCGCA CCTGGTGCC AACCAAGAGGGAGGTGCTGCCGCACCAGGTGCTAATCAGGAAGGAGGAGCTGCCGCACCA GGTGCCAAC CAAGAGGGTGGTGCTGCCGCACCTGGAGCCAATCAGGAAGGTGGTGCTGCCGCACCAGGC GCAAACCAA GAGGGAGGAGCTGCCGCACCTGGAGCTAATCAGGAAGGCGGTGCTGCCGCACCCGGAGCA AACCAAGAG GGCGGTGCTGCCGCACCTGGCGCTAATCAGGAAGGCGGCGCTGCCGCACCTGGTGCTAAC CAAGAGGGC GGCGCTGCCGCACCAGGTGCAAATCAGGAAGGCGGGGCTGCCGCACCCGGCGCTAACCAA GAGGGTGGA GCTGCCGATAGAGCAGCTGGTCAAGCCGCAGGTGGAAACGCTGGTGGACAGGGTCAGAAC AATGAAGGA GCAAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGAGCTACTGTT GGTACAGAG TGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCT GCCAATAAG AAACCAGAAGACCTTACCTTGAATGACTTGGAGACAGATGTTTGTACC-3' (SEQ ID NO: 33) .

In yet another specific embodiment, the invention provides an isolated DNA encoding the protein comprising SEQ ID NO: 4 (PvCs-All-CS-Epitopes), said DNA comprising the sequence

5'-

ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGTGTGAAC TTCAACAAC GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGTCGTGGT CTGGGTGAA AACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCG AAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGTCCGGGTGATCGTGCAGACGGTCAGCCGGCTGGC GACCGCGCA GACGGTCAGCCGGCAGGTGATCGTGCTGCAGGCCAGCCGGCAGGTGACCGTGCGGCAGGC CAGCCGGCT GGTGATCGCGCCGACGGCCAGCCGGCCGGCGACCGTGCGGACGGTCAGCCGGCTGGTGAT CGTGCAGAC GCACCGGGCGCCAACCAGGAAGGTGGCGCTGCCGCGCCGGGTGCGAACCAGGAAGGTGGC GCAGCTGCA CCGGGTGCAAACCAGGAAGGTGGCGCGGCAGCTGCACCGGGTGCTAACCAGGAAGGTGGC GCCGCAGCT CCGGGTGCCAATCAAGAAGGTGGCGCCGCGGCGCCGGGCGCAAATCAAGAAGGTGGCGCT GCAGCAGCA AACGGTGCAGGTAACCAGCCGGGTGCGAACGGCGCGGGCAACCAGCCGGGTGCTAACGGC GCGGGTAAC CAGCCGGGTGCCAACGGCGCTGGCAACCAGCCGGGCGCGAACGGCGCTGGTAACCAGCCG GGTGATCGT GCTGCGGGTCAGGCAGCAGGTGGCAACGCGGGTGGCCAGGGTCAGAACAACGAAGGTGCT AACGCCCCG AACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGTGCGACCGTTGGTACCGAATGG ACCCCGTGC TCCGTGACCTGTGGCGTTGGTGTGCGCGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAA CCGGAAGAT CTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA- 3 ' (SEQ ID NO : 8) .

In a further specific embodiment, 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' -

CCAAGAGAAAACAAATTGAAGCAACCAGGACCAGGAGATAGAGCAGATGGACAACCA GCAGGAGATAGA GCCGACGGACAGCCAGCAGGAGATAGAGCTGCCGGTCAACCAGCTGGAGACAGAGCAGCT GGTCAGCCT GCTGGAGATAGAGCCGACGGTCAGCCTGCCGGAGATAGAGCTGACGGTCAGCCAGCTGGA GATAGAGCT GACGCCCCTGGTGCTAACCAAGAAGGTGGAGCCGCAGCTCCAGGAGCTAATCAGGAGGGT GGAGCCGCA GCTCCTGGAGCCAATCAGGAAGGAGGTGCCGCAGCTGCCCCAGGTGCTAATCAAGAGGGT GGAGCAGCT GCCCCTGGAGCCAACCAAGAAGGAGGTGCAGCTGCCCCTGGAGCAAACCAGGAAGGTGGA GCAGCTGCC GCAAACGGTGCTGGAAATCAACCTGGTGCCAACGGTGCAGGAAATCAGCCAGGAGCTAAC GGTGCCGGA AATCAACCTGGTGCTAATGGTGCTGGAAATCAGCCAGGAGCAAATGGTGCAGGAAATCAA CCTGGAGAT AGAGCTGCCGGACAGGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGA GCAAACGCT CCTAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGAGCTACTGTTGGTACAGAG TGGACCCCA TGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAG AAACCTGAA GATTTGACTTTGAACGACTTGGAAACCGATGTCTGCACC-3' (SEQ ID NO: 37) .

The invention also provides vectors (e.g., expression vectors) comprising the

DNA molecules of the invention.

In a separate aspect, the invention provides a method for making the recombinant protein immunogens and the protein immunogens produced by said method.

In one embodiment, 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:

(a) culturing E.coli BL21 DE3 host cells comprising the Pet28A expression vector encoding the His ₆ tagged protein at 37°C in LB media containing 30 μg/ml kanamycin; (b) inducing protein expression at OD ₆ oo of 0.6 with 0.1 mM IPTG for 4 hours;

(c) resuspending bacterial pellet in Phosphate buffer (50mM NaH ₂ P04, 300mM NaCl, lmg/ml lysozyme, lmM PMSF, pH 7.0);

(d) lysing bacterial pellet on ice using ultrasonic processor;

(e) centrifuging bacterial lysate at 15000 rpm; (f) taking the supernatant, adding 8M urea and boiling at 100°C for 15 minutes; (g) applying the resulting mixture to a column with Ni ²⁺ -NTA-agarose resin;

(h) washing the column with Wash buffer (50 mM NaH ₂ P0 ₄ , 300 mM NaCl, 10 % glycerol, pH 6.0); (i) eluting bound protein with 30ml of Elution buffer (Wash buffer containing 500 mM imidazole and 2 mM PMSF, pH 6.0);

(j) dialyzing the eluted protein against 20 mM Tris-HCl (pH 8.0) overnight, at 4°C; (k) further purifying protein by ion-exchange chromatography;

(1) incubating fractions containing protein with polymyxin B agarose beads overnight at room temperature, and

(m) dialyzing unbound protein against PBS (pH=7.2) overnight, at 4°C.

In another embodiment, 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:

(a) culturing P. pastoris host cells comprising the pPIC9K expression vector encoding the His ₆ tagged protein overnight at 28-30°C with vigorous shaking (e.g., in a medium comprising 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);

(b) harvesting cells;

(c) resuspending harvested cells in a medium comprising 0.5% v/v methanol (to induce protein expression) and incubating for 72 hours at 28-30°C with vigorous shaking adding methanol to final concentration of 1% v/v every 24 hours;

(d) removing cells by centrifugation at 250 rpm;

(e) concentrating the culture supernatant by ultrafiltration with 30.000 MWCO membrane;

(f) dialyzing the concentrated culture supernatant overnight at 4°C against 20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.2 M NaCl; (g) applying the dialyzed culture supernatant to a column with Ni ²⁺ -NTA-agarose resin previously equilibrated with 20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.5 M NaCl;

(h) eluting bound proteins with a 15 to 400 mM Imidazole gradient in Wash buffer (20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.5 M NaCl, 1 mM PMSF and 10% glycerin);

(i) dialyzing the eluted protein against 20 mM Tris-HCl (pH 8.0) overnight, at 4°C, and

(j) further purifying protein by ion-exchange chromatography.

In a separate embodiment, the invention provides an adenoviral vector comprising the sequence

5'-

TCTAGAGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTC CTGTGGGTGCCC GGGTCCAGAGGAACCCACTGCGGCCACAACGTGGACCTGAGCAAGGCCATCAACCTGAAT GGCGTGAAT TTCAACAATGTGGATGCTAGCTCCCTGGGAGCTGCTCACGTGGGACAGTCTGCTAGCCGC GGAAGGGGA CTGGGAGAGAACCCAGACGATGAGGAGGGCGACGCCAAGAAGAAGAAGGATGGAAAGAAG GCTGAGCCA AAGAACCCCAGGGAGAATAAGCTGAAGCAGCCCGGACCTGGGGACAGAGCCGATGGCCAG CCCGCCGGA GACCGGGCTGATGGACAGCCTGCTGGCGACAGGGCTGCTGGACAGCCAGCTGGGGATAGA GCCGCTGGA CAGCCAGCTGGAGACAGAGCTGATGGACAGCCTGCCGGCGACAGGGCCGATGGACAGCCT GCTGGGGAC AGGGCTGATGCTCCAGGCGCTAACCAGGAGGGAGGAGCTGCTGCCCCTGGAGCCAATCAG GAAGGCGGA GCTGCCGCTCCAGGCGCCAATCAGGAAGGAGGAGCTGCTGCTGCTCCCGGCGCCAATCAG GAGGGCGGA GCCGCTGCCCCTGGCGCCAACCAGGAGGGGGGCGCTGCCGCTCCAGGCGCTAATCAGGAG GGCGGCGCT GCTGCTGCTAACGGAGCTGGAAATCAGCCAGGGGCCAATGGAGCTGGAAATCAGCCTGGC GCTAACGGA GCTGGGAATCAGCCTGGAGCCAACGGCGCCGGCAATCAGCCAGGGGCCAACGGGGCTGGC AATCAGCCC GGCGACAGAGCCGCTGGACAGGCTGCTGGAGGAAACGCTGGAGGACAGGGACAGAACAAT GAGGGGGCC AACGCTCCCAATGAGAAGTCCGTGAAGGAGTACCTGGATAAGGTGAGGGCTACCGTGGGC ACAGAGTGG ACCCCTTGCTCTGTGACATGTGGAGTGGGGGTGAGAGTGAGGAGACGGGTGAACGCCGCT AATAAGAAG CCAGAGGACCTGACCCTGAACGACCTGGAGACAGATGTGTGTACCTGA- 3' (SEQ ID NO : 9) .

In another embodiment, the invention provides an adenoviral vector encoding a sequence comprising:

(i) P. vivax CS protein N-terminal sequence

THCGHNVDLSKAI LNGV F VDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10),

(ii) VK210 epitope (DRADGQPAG) ₂ (DRAAGQPAG) ₂ DRADGQPAGD (SEQ ID NO: 11), (iii) 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 ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14).

In a further embodiment, the invention provides an adenoviral vector encoding a protein comprising the sequence

MGMVQSIQLFLLLLWVPGSRGQPTHCGHNVDLSKAINLNGVNFNNVDASSL

GAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGP

GDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRA

DGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGAN

QEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGAN

APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL

NDLETDVCT (SEQ ID NO: 20; Ad-All-CS-Epitopes).

The adenoviral vectors of the invention can be based, for example, on AdC68 or

AdHu5.

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.

In one specific embodiment, 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. In another specific embodiment, 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 carrier or diluent.

In a further specific embodiment, the immunogenic or vaccine composition comprises

(a) 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. vivax CS protein C-terminal sequence ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14);

(b) 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

ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14);

(c) 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

(d) a carrier or diluent.

The immunogenic or vaccine compositions of the invention can further comprise an adjuvant. Non-limiting examples of 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. In one specific embodiment, the adjuvant is Poly IC.

In conjunction with the disclosed immunogenic compositions, 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. In one embodiment, 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 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 μg/mouse/dose) or a mixture containing His ₆ PvCS-VK210, His ₆ PvCS-VK247 and His ₆ PvCS-Vivax-like protein (30 μg/mouse/dose) or His ₆ PvCS-All-CS-epitopes (30 μg/mouse/dose) in the presence of Poly(I:C) adjuvant (50 μg/mouse/dose).

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 β-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 μg/mouse/dose total) in the presence of Poly(LC) adjuvant (50 μg/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). 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 μg/mouse/dose total) or His ₆ PvCS-All-CS- Epitopes (30 μg/mouse/dose) in the presence of Poly(I:C) adjuvant (50 μg/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).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term "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.

The term "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. It should be noted that to be effective, 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.

The term "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. 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. Preferably, the adjuvant is pharmaceutically acceptable.

Within the meaning of the present invention, 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).

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. The terms "protective 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.

The term "therapeutically effective amount/dose" is used herein interchangeably with the term "immunogenically effective amount/dose" and 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.

The phrase "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. Preferably, as used herein, the term "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.

The term "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. 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.

The term "purified" as used herein 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. For example, a purified recombinant protein is preferably substantially free of host cell or culture components. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, 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.

The term "subject" as used herein 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.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook,

Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold

Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid

Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And

Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I.

Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B.

Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.).

Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad.

Sci. USA 82: 488- 492 (1985), U. S. Patent No. 5,071, 743, Fukuoka et al. , Biochem.

Biophys. Res. Commun. 263 : 357-360 (1999); Kim and Maas, BioTech. 28: 196-198

(2000); Parikh and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff,

BioTech. 13 : 342-346 (1992); Wang et al, BioTech. 19: 556-559 (1995); Wang and

Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999),

U.S. Patents Nos. 5,789, 166 and 5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents Nos. 5,702,931, 5,780,270, and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al, Biotech. 20: 44-46 (1996), Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al, Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al, Meth. Molec. Biol. 67: 209-218.

Recombinant Protein Immunogens of the Invention

The invention provides the following isolated recombinant protein immunogens.

An isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGDRADGQPAGDRADGQPAGDRADG

QPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADGQPAG

DRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAD

GQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAA

GDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCS

VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 1; PvCs-

VK210).

An isolated protein consisting of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGDRA

DGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRADGQP

AGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR

ADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQ

PAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGANAPNEK

SVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLET

DVCT (SEQ ID NO: 21; His ₆ -PvCS-VK210).

An isolated protein comprising the sequence PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAA

GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPA

GDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRA

DGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQ

GQ EGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AA

NKKPEDLTLNDLETDVCT (SEQ ID NO: 27; PIC-PvCS-VK210).

An isolated protein consisting of the sequence

PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAA

GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPA

GDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRA

DGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCTHHHHHHH (SEQ ID NO: 28; PIC-PvCS-

VK210-His ₆ ).

An isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGANGAGNQPGANGAGNQPGANGAG

NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPG

ANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA

GNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQP

GANGAGNQPGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCS

VTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 2; PvCs-

VK247).

An isolated protein consisting of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGANG AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGN QPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQGQNNEGANAPNEK SVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AANKKPEDLTLNDLET DVCT (SEQ ID NO: 22; His ₆ -PvCS-VK247).

An isolated protein comprising the sequence

PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANG

AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCT (SEQ ID NO: 31; PIC-PvCS-VK247).

An isolated protein consisting of the sequence

PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANG

AGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQ

GQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAA

NKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 32; PIC-PvCS-VK247-His ₆ ).

An isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGAPGANQEGGAAAPGANQEGGAAAP GANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ EGGAADRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTE WTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 3; PvCs -Vivax-like) .

An isolated protein consisting of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGAPG

ANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEG GAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAP GANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE GGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGNAGGQGQ EGA NAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRV AANKKPEDLT LNDLETDVCT (SEQ ID NO: 23; His ₆ -PvCS-Vivax-like).

An isolated protein comprising the sequence

PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA

PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGN

AGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRR

VNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 35; PIC-PvCS-Vivax-like).

An isolated protein consisting of the sequence

PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAA

PGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGN

AGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRR

VNAANKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 36; PlC-PvCS-Vivax-

An isolated protein comprising the sequence

THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGPGDRADGQPAGDRADGQPAGDRAA

GQPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAP

GANQEGGAAAPGANQEGGAAAAPGANQEGGAAAPGANQEGGAAAPGANQ

EGGAAAANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ

PGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPC SVTCGVGVRVRRRV AANKKPEDLTLNDLETDVCT (SEQ ID NO: 4; PvCs- All-CS-Epitopes).

An isolated protein consisting of the sequence

MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA

AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGPGD

RADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADG

QPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQE

GGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGA

GNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPN

EKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDL

ETDVCT (SEQ ID NO: 24; His ₆ -PvCS-All CS epitopes).

An isolated protein comprising the sequence

PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR

ADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGA

NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAG

GQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVN

AANKKPEDLTLNDLETDVCT (SEQ ID NO: 39; PIC-PvCS-All-CS epitopes).

An isolated protein consisting of the sequence

PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDR

ADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQE

GGAAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGA

NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAG

GQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVN

AANKKPEDLTLNDLETDVCTHHHHHH (SEQ ID NO: 40; PIC-PvCS-All-CS epitopes-His6).

In conjunction with the protein immunogens, the invention provides DNA molecules encoding such protein immunogens. Specific non-limiting examples of such DNA molecules are as follows. An isolated DNA comprising the sequence

5 ' - ACCCACTGCGGCCATAACGTTGACCTGTCTAAAGCGATTAACCTGAACGGTGTTAACTTT AACAAC GTTGATGCCTCCTCCCTGGGTGCCGCGCATGTTGGTCAGAGCGCATCCCGCGGTCGTGGC CTGGGCGAAAACCCG GATGACGAAGAAGGCGACGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCGAAAAAC CCGCGTGAAAACAAA CTGAAACAGCCGGGCGACCGCGCGGATGGCCAGCCGGCCGGCGACCGTGCGGATGGCCAG CCGGCAGGCGACCGT GCGGATGGTCAGCCGGCCGGTGACCGTGCGGACGGTCAGCCGGCGGGCGACCGCGCTGCA GGCCAGCCGGCCGGT GACCGCGCCGATGGCCAGCCGGCTGGCGACCGCGCGGATGGTCAGCCGGCGGGTGATCGC GCGGACGGTCAGCCG GCAGGTGATCGCGCAGACGGTCAGCCGGCCGGCGATCGCGCGGCAGGCCAGCCGGCGGGC GACCGTGCGGCCGGT CAGCCGGCGGGTGATCGTGCCGACGGCCAGCCGGCGGGTGACCGCGCTGCGGGTCAGCCG GCAGGCGACCGCGCG GACGGCCAGCCGGCAGGTGACCGTGCAGCGGGCCAGCCGGCTGGCGACCGTGCCGATGGT CAGCCGGCCGGCGAT CGTGCGGCAGGCCAGCCGGCTGGTGACCGTGCCGCGGGTCAGCCGGCAGGTGACCGCGCA GCGGGTCAGGCCGCA GGTGATCGTGCGGCGGGTCAGGCCGCGGGCGGTAACGCGGGCGGCCAGGGCCAGAACAAC GAAGGCGCAAACGCT CCGAACGAAAAATCCGTGAAAGAATACCTGGATAAAGTGCGTGCGACCGTTGGTACCGAA TGGACCCCGTGTTCC GTGACCTGTGGCGTTGGCGTTCGCGTGCGCCGCCGTGTTAACGCAGCGAACAAAAAACCG GAAGATCTGACCCTG AACGATCTGGAAACCGACGTTTGTACCTAA- 3 ' (SEQ ID NO: 5) .

An isolated DNA comprising the sequence

5' -

CCAAGAGAAAACAAGTTGAAACAGCCAGGAGACAGAGCAGACGGTCAGCCAGCAGGA GACAGAGCAGATGGACAGC CAGCAGGAGACAGAGCTGATGGTCAACCAGCAGGAGATAGAGCTGACGGTCAGCCTGCTG GAGACAGAGCTGCCGG TCAACCAGCCGGAGATAGAGCAGATGGACAGCCTGCTGGAGATAGAGCCGACGGACAGCC TGCCGGAGATAGAGCC GATGGTCAACCTGCTGGAGATAGAGCAGACGGTCAACCAGCTGGAGATAGAGCAGCTGGT CAGCCTGCCGGAGACA GAGCCGCAGGACAGCCAGCCGGAGATAGAGCTGATGGACAACCAGCGGGAGATCGTGCTG CCGGACAGCCTGCAGG AGATAGAGCCGACGGTCAACCTGCCGGAGATAGAGCTGCCGGACAGCCAGCTGGAGATAG AGCTGATGGTCAACCC GCCGGAGATAGAGCCGCAGGTCAACCAGCCGGAGACAGAGCCGCCGGACAACCAGCCGGA GATCGTGCAGCTGGTC AAGCCGCAGGTGACAGAGCCGCTGGTCAAGCAGCTGGTGGAAACGCTGGTGGACAGGGTC AGAACAATGAAGGTGC TAACGCCCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGG TACAGAGTGGACCCCT TGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAG AAACCAGAAGATTTGA CCTTGAACGACTTGGAAACCGATGTCTGCACT-3 ' (SEQ ID NO: 25) .

An isolated DNA comprising the sequence 5 ' - ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGTGTGAACTTC AACAAC

GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGCGGTCGT GGCCTGGGTGAAAACCCG GATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCGAAAAAC CCGCGTGAAAACAAA CTGAAACAGCCGGGTGCTAACGGTGCAGGTAACCAGCCGGGTGCAAACGGCGCGGGTAAC CAGCCGGGCGCGAAC GGCGCTGGCAACCAGCCGGGCGCAAACGGCGCTGGTAACCAGCCGGGCGCCAACGGCGCC GGCAACCAGCCGGGT GCGAACGGCGCCGGTAACCAGCCGGGCGCTAACGGCGCAGGCAATCAACCGGGCGCGAAC GGCGCGGGCAACCAG CCGGGTGCCAACGGCGCAGGCAATCAGCCTGGCGCGAACGGCGCAGGCAACCAGCCGGGT GCTAACGGCGCAGGA AATCAGCCGGGCGCTAACGGTGCGGGCAATCAACCGGGTGCCAACGGTGCGGGCAATCAG CCTGGTGCCAACGGC GCAGGAAACCAACCGGGCGCGAACGGTGCGGGTAATCAACCGGGTGCGAATGGCGCTGGC AATCAACCGGGCGCA AATGGCGCGGGAAATCAGCCGGGTGCAAACGGTGCGGGTAATCAGCCCGGTGCCAACGGC GCAGGGAATCAACCT GGCGCTAACGGCGCAGGTAACCAGCCGGGCGGTAACGCGGGTGGCCAGGGCCAGAACAAC GAAGGTGCGAACGCA CCGAACGAAAAAAGCGTGAAAGAATACCTGGATAAAGTGCGCGCGACCGTTGGTACCGAA TGGACCCCGTGCTCC GTGACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAACCG GAAGACCTGACCCTG AACGATCTGGAAACCGACGTTTGTACCTAA- 3' (SEQ ID NO : 6) .

An isolated DNA comprising the sequence 5' -

CCAAGAGAAAACAAGTTGAAACAGCCAGGAGCAAATGGAGCAGGTAACCAGCCAGGA GCAAACGGAGCAGGTAACC AGCCAGGAGCAAACGGTGCTGGAAATCAACCAGGTGCCAACGGTGCAGGAAATCAGCCTG GAGCTAACGGTGCCGG AAATCAACCAGGTGCAAACGGTGCTGGAAATCAGCCTGGAGCAAATGGTGCAGGAAATCA ACCAGGTGCTAATGGA GCCGGTAACCAGCCTGGAGCCAATGGAGCCGGTAACCAACCTGGAGCTAATGGAGCTGGT AACCAACCAGGTGCTA ATGGTGCTGGTAACCAGCCTGGAGCAAACGGAGCCGGTAATCAGCCTGGAGCAAACGGTG CCGGTAACCAGCCTGG TGCCAATGGTGCCGGAAATCAGCCTGGTGCTAATGGAGCCGGAAACCAGCCTGGAGCCAA CGGAGCTGGTAACCAG CCTGGTGCAAATGGAGCTGGTAATCAGCCAGGTGCAAATGGAGCCGGAAATCAACCTGGT GCTAATGGAGCAGGTA ACCAACCTGGTGCCAATGGAGCTGGAAATCAACCTGGTGGAAACGCTGGTGGACAGGGTC AGAACAATGAAGGAGC AAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGG TACAGAGTGGACCCCT TGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCCAATAAG AAACCAGAAGACCTTA CTTTGAACGACTTGGAAACCGATGTCTGCACT-3 ' (SEQ ID NO: 29) . An isolated DNA comprising the sequence

5 ' - ACCCACTGCGGTCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGCGTGAACTTC AACAAC GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGTCGTGGT CTGGGTGAAAACCCG GATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCGAAAAAC CCGCGCGAAAACAAA CTGAAACAGCCGGGTGCACCGGGTGCTAACCAGGAAGGTGGCGCTGCAGCACCGGGTGCA AACCAGGAAGGTGGC GCAGCGGCGCCGGGCGCGAACCAGGAAGGTGGCGCGGCAGCTCCGGGTGCCAACCAGGAA GGTGGCGCCGCGGCA CCGGGTGCGAATCAAGAAGGTGGCGCGGCGGCGCCGGGCGCAAATCAAGAAGGTGGCGCA GCAGCGCCGGGCGCA AATCAGGAAGGTGGCGCAGCTGCGCCGGGCGCTAATCAAGAAGGTGGCGCTGCTGCACCG GGTGCGAATCAGGAA GGTGGCGCCGCTGCGCCGGGCGCAAACCAAGAAGGTGGCGCAGCCGCGCCGGGCGCAAAC CAGGAAGGTGGCGCA GCCGCACCTGGCGCGAATCAAGAAGGTGGCGCCGCTGCTCCGGGTGCGAACCAAGAAGGT GGCGCGGCTGCGCCG GGCGCTAACCAAGAAGGTGGCGCTGCGGCGCCGGGCGCTAATCAGGAAGGTGGCGCGGCG GCTCCGGGTGCGAAC CAGGAAGGTGGCGCAGCAGATCGTGCAGCTGGTCAGGCAGCGGGTGGCAACGCGGGTGGC CAGGGCCAGAACAAC GAAGGTGCCAACGCGCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGCGCG ACCGTTGGTACCGAA TGGACCCCGTGCTCCGTGACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCA GCTAACAAAAAACCG GAAGATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA- 3' (SEQ ID NO: 7) .

An isolated DNA comprising the sequence

5' -

CCAAGAGAAAATAAGTTGAAACAGCCAGGTGCCCCAGGAGCAAACCAAGAGGGAGGT GCCGCCGCCCCAGGAGCAA ACCAGGAGGGAGGTGCTGCCGCACCAGGAGCCAACCAAGAAGGTGGAGCTGCCGCACCTG GTGCAAATCAGGAGGG TGGAGCTGCCGCACCAGGAGCTAATCAGGAAGGAGGTGCTGCCGCACCTGGTGCCAACCA AGAGGGAGGTGCTGCC GCACCAGGTGCTAATCAGGAAGGAGGAGCTGCCGCACCAGGTGCCAACCAAGAGGGTGGT GCTGCCGCACCTGGAG CCAATCAGGAAGGTGGTGCTGCCGCACCAGGCGCAAACCAAGAGGGAGGAGCTGCCGCAC CTGGAGCTAATCAGGA AGGCGGTGCTGCCGCACCCGGAGCAAACCAAGAGGGCGGTGCTGCCGCACCTGGCGCTAA TCAGGAAGGCGGCGCT GCCGCACCTGGTGCTAACCAAGAGGGCGGCGCTGCCGCACCAGGTGCAAATCAGGAAGGC GGGGCTGCCGCACCCG GCGCTAACCAAGAGGGTGGAGCTGCCGATAGAGCAGCTGGTCAAGCCGCAGGTGGAAACG CTGGTGGACAGGGTCA GAACAATGAAGGAGCAAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGT CAGAGCTACTGTTGGT ACAGAGTGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTT AACGCTGCCAATAAGA AACCAGAAGACCTTACCTTGAATGACTTGGAGACAGATGTTTGTACC-3 ' (SEQ ID NO: 33) .

An isolated DNA comprising the sequence 5 ' - ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGTGTGAACTTC AACAAC

GTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGTCGT GGTCTGGGTGAAAACCCG GATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCGAAAAAC CCGCGTGAAAACAAA CTGAAACAGCCGGGTCCGGGTGATCGTGCAGACGGTCAGCCGGCTGGCGACCGCGCAGAC GGTCAGCCGGCAGGT GATCGTGCTGCAGGCCAGCCGGCAGGTGACCGTGCGGCAGGCCAGCCGGCTGGTGATCGC GCCGACGGCCAGCCG GCCGGCGACCGTGCGGACGGTCAGCCGGCTGGTGATCGTGCAGACGCACCGGGCGCCAAC CAGGAAGGTGGCGCT GCCGCGCCGGGTGCGAACCAGGAAGGTGGCGCAGCTGCACCGGGTGCAAACCAGGAAGGT GGCGCGGCAGCTGCA CCGGGTGCTAACCAGGAAGGTGGCGCCGCAGCTCCGGGTGCCAATCAAGAAGGTGGCGCC GCGGCGCCGGGCGCA AATCAAGAAGGTGGCGCTGCAGCAGCAAACGGTGCAGGTAACCAGCCGGGTGCGAACGGC GCGGGCAACCAGCCG GGTGCTAACGGCGCGGGTAACCAGCCGGGTGCCAACGGCGCTGGCAACCAGCCGGGCGCG AACGGCGCTGGTAAC CAGCCGGGTGATCGTGCTGCGGGTCAGGCAGCAGGTGGCAACGCGGGTGGCCAGGGTCAG AACAACGAAGGTGCT AACGCCCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGTGCGACCGTTGGT ACCGAATGGACCCCG TGCTCCGTGACCTGTGGCGTTGGTGTGCGCGTTCGTCGCCGTGTTAACGCTGCCAACAAA AAACCGGAAGATCTG ACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA- 3' (SEQ ID NO : 8) .

An isolated DNA comprising the sequence 5' -

CCAAGAGAAAACAAATTGAAGCAACCAGGACCAGGAGATAGAGCAGATGGACAACCA GCAGGAGATAGAGCCGACG GACAGCCAGCAGGAGATAGAGCTGCCGGTCAACCAGCTGGAGACAGAGCAGCTGGTCAGC CTGCTGGAGATAGAGC CGACGGTCAGCCTGCCGGAGATAGAGCTGACGGTCAGCCAGCTGGAGATAGAGCTGACGC CCCTGGTGCTAACCAA GAAGGTGGAGCCGCAGCTCCAGGAGCTAATCAGGAGGGTGGAGCCGCAGCTCCTGGAGCC AATCAGGAAGGAGGTG CCGCAGCTGCCCCAGGTGCTAATCAAGAGGGTGGAGCAGCTGCCCCTGGAGCCAACCAAG AAGGAGGTGCAGCTGC CCCTGGAGCAAACCAGGAAGGTGGAGCAGCTGCCGCAAACGGTGCTGGAAATCAACCTGG TGCCAACGGTGCAGGA AATCAGCCAGGAGCTAACGGTGCCGGAAATCAACCTGGTGCTAATGGTGCTGGAAATCAG CCAGGAGCAAATGGTG CAGGAAATCAACCTGGAGATAGAGCTGCCGGACAGGCAGCTGGTGGAAACGCTGGTGGAC AGGGTCAGAACAATGA AGGAGCAAACGCTCCTAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGAGCTAC TGTTGGTACAGAGTGG ACCCCATGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCA AATAAGAAACCTGAAG ATTTGACTTTGAACGACTTGGAAACCGATGTCTGCACC-3 ' (SEQ ID NO: 37) . The invention also provides methods for preparing the above protein immunogens and immunogens produced by such methods.

In one specific embodiment, 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:

(a) culture E.coli BL21 DE3 host cells comprising the pET28a expression vector encoding the His ₆ tagged protein at 37°C in LB media containing 30 μg/ml kanamycin;

(b) induce protein expression at OD ₆ oo of 0.6 with 0.1 mM IPTG for 4 hours;

(c) resuspend bacterial pellet in Phosphate buffer (50mM NaH ₂ P0 ₄ , 300mM NaCl, lmg/ml lysozyme, lmM PMSF, pH 7.0);

(d) lyse bacterial pellet on ice using ultrasonic processor;

(e) centrifuge bacterial lysate at 15000 rpm;

(f) take the supernatant, add 8M urea and boil at 100°C for 15 minutes;

(g) apply the resulting mixture to a column with Ni ²⁺ -NTA-agarose resin;

(h) wash the column with Wash buffer (50 mM NaH ₂ P0 ₄ , 300 mM NaCl, 10 % glycerol, pH 6.0);

(i) elute bound protein with 30ml of Elution buffer (Wash buffer containing 500 mM imidazole and 2 mM PMSF, pH 6.0);

(j) dialyze the eluted protein against 20 mM Tris-HCl (pH 8.0) overnight, at 4°C; (k) further purify protein by ion-exchange chromatography;

(1) incubate fractions containing protein with polymyxin B agarose beads overnight at room temperature, and

(m) dialyze unbound protein against PBS (pH=7.2) overnight, at 4°C. In another embodiment, 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:

(a) culture P. pastoris host cells comprising the pPIC9K expression vector encoding the His ₆ tagged protein overnight at 28-30°C with vigorous shaking in a medium comprising 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;

(b) harvest cells;

(c) to induce protein expression, resuspend harvested cells in a medium comprising 0.5% v/v methanol and incubate for 72 hours at 28-30°C with vigorous shaking adding methanol to final concentration of 1% v/v every 24 hours;

(d) remove cells by centrifugation at 250 rpm;

(e) concentrate the culture supernatant by ultrafiltration with 30.000 MWCO membrane;

(f) dialyze the concentrated culture supernatant overnight at 4°C against 20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.2 M NaCl;

(g) apply the dialyzed culture supernatant to a column with Ni ²⁺ -NTA-agarose resin previously equilibrated with 20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.5 M NaCl;

(h) elute bound proteins with a 15 to 400 mM Imidazole gradient in Wash buffer (20 mM NaH ₂ P0 ₄ buffer, pH 8.0, 0.5 M NaCl, 1 mM PMSF and 10% glycerin);

(i) dialyze the eluted protein against 20 mM Tris-HCl (pH 8.0) overnight, at 4°C, and

(j) further purify protein by ion-exchange chromatography. Recombinant Adenoviral Vectors of the Invention

The invention also provides various immunogenic adenoviral vectors useful in immunogenic and vaccine compositions of the invention.

In one embodiment, the invention provides an adenoviral vector comprising the sequence

5 ^' -

TCTAGAGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTC CTGTGGGTGCCCGG GTCC

AGAGGAACCCACTGCGGCCACAACGTGGACCTGAGCAAGGCCATCAACCTGAATGGC GTGAATTTCAACAA TGTG

GATGCTAGCTCCCTGGGAGCTGCTCACGTGGGACAGTCTGCTAGCCGCGGAAGGGGA CTGGGAGAGAACCC AGAC

GATGAGGAGGGCGACGCCAAGAAGAAGAAGGATGGAAAGAAGGCTGAGCCAAAGAAC CCCAGGGAGAATAA GCTG AAGCAGCCCGGACCTGGGGACAGAGCCGATGGCCAGCCCGCCGGAGACCGGGCTGATGGA CAGCCTGCTGG CGAC

AGGGCTGCTGGACAGCCAGCTGGGGATAGAGCCGCTGGACAGCCAGCTGGAGACAGA GCTGATGGACAGCC TGCC

GGCGACAGGGCCGATGGACAGCCTGCTGGGGACAGGGCTGATGCTCCAGGCGCTAAC CAGGAGGGAGGAGC TGCT

GCCCCTGGAGCCAATCAGGAAGGCGGAGCTGCCGCTCCAGGCGCCAATCAGGAAGGA GGAGCTGCTGCTGC TCCC

GGCGCCAATCAGGAGGGCGGAGCCGCTGCCCCTGGCGCCAACCAGGAGGGGGGCGCT GCCGCTCCAGGCGC TAAT CAGGAGGGCGGCGCTGCTGCTGCTAACGGAGCTGGAAATCAGCCAGGGGCCAATGGAGCT GGAAATCAGCC TGGC

GCTAACGGAGCTGGGAATCAGCCTGGAGCCAACGGCGCCGGCAATCAGCCAGGGGCC AACGGGGCTGGCAA TCAG

CCCGGCGACAGAGCCGCTGGACAGGCTGCTGGAGGAAACGCTGGAGGACAGGGACAG AACAATGAGGGGGC CAAC

GCTCCCAATGAGAAGTCCGTGAAGGAGTACCTGGATAAGGTGAGGGCTACCGTGGGC ACAGAGTGGACCCC TTGC

TCTGTGACATGTGGAGTGGGGGTGAGAGTGAGGAGACGGGTGAACGCCGCTAATAAG AAGCCAGAGGACCT GACC CTGAACGACCTGGAGACAGATGTGTGTACCTGA- 3 ' ( SEQ ID NO : 9 ) .

In another embodiment, the invention provides an adenoviral vector encoding a sequence comprising:

(i) P. vivax CS protein N-terminal sequence THCGHNVDLSKAI LNGV F VDASSLGAAHVGQSASRGRGLGENPDDEE GDAKKKKDGKKAEPKNPRENKLKQPGP (SEQ ID NO: 10),

(ii) VK210 epitope (DRADGQPAG) ₂ (DRAAGQPAG) ₂ DRADGQPAGD (SEQ ID NO: 11),

(iii) 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

ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14).

In yet another embodiment, the invention provides an adenoviral vector encoding a protein comprising the sequence

MGMVQSIQLFLLLLWVPGSRGQPTHCGHNVDLSKAINLNGVNFNNVDASSL

GAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGP

GDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRA

DGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGAN

QEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGAN

APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL

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.

Immunogenic and Vaccine Compositions of the Invention

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.

In one specific embodiment, 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.

In another specific embodiment, 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 carrier or diluent. In a further specific embodiment, the immunogenic or vaccine composition comprises

(a) 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. vivax CS protein C-terminal sequence ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14);

(b) 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

ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRAT VGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT (SEQ ID NO: 14);

(c) 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

(d) a carrier or diluent. Methods of formulating immunogenic and vaccine compositions are well- known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Company: 1990)). 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. Such 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. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference.

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. In general, 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. Examples of 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).

For better storage, the immunogenic and vaccine compositions may be preserved, cryopreserved, lyophilized, refrigerated, or the like. A kit 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.

As a vaccine, 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.

Methods, Doses and Administration

In conjunction with immunogenic and vaccine compositions, 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.

Also disclosed are methods for protecting a subject from P. vivax malaria, which methods comprise administering to the subject an effective amount of vaccine compositions disclosed herein.

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. For such methods, 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. Moreover, 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.

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. 34: 241-251); interferon gamma ELISPOT assays, and skin tests for cell mediated immunity (Chang et al. (1993) Cancer Res. 53: 1043-1050). See also methods provided in Coligan et al. (Ed.) (2000) Current Protocols in Immunology, Vol. 1, Wiley & Sons.

Methods for determining therapeutically/immunogenically effective amount/dose of the immunogenic and vaccine compositions are known in the art and involve initial testing in animal models of P. vivax infection (e.g., Aotus monkeys, see Jones et al. (2000) Am. J. Trop. Med. Hyg., 62: 675-680). 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. vivax infection, and other relevant factors. It is expected that 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 μg/dose, more particularly from about 1.0 to 100 μg/dose, most preferably from about 10 to 50 μg/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.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

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:

PvCS-VK210 5 ' -

AGCCATATGACCCACTGCGGCCATAACGTTGACCTGTCTAAAGCGATTAACCTGAAC GGTGTTAACTTT AACAACGTTGATGCCTCCTCCCTGGGTGCCGCGCATGTTGGTCAGAGCGCATCCCGCGGT CGTGGCCTG GGCGAAAACCCGGATGACGAAGAAGGCGACGCGAAAAAGAAAAAAGACGGTAAAAAAGCG GAACCGAAA AACCCGCGTGAAAACAAACTGAAACAGCCGGGCGACCGCGCGGATGGCCAGCCGGCCGGC GACCGTGCG GATGGCCAGCCGGCAGGCGACCGTGCGGATGGTCAGCCGGCCGGTGACCGTGCGGACGGT CAGCCGGCG GGCGACCGCGCTGCAGGCCAGCCGGCCGGTGACCGCGCCGATGGCCAGCCGGCTGGCGAC CGCGCGGAT GGTCAGCCGGCGGGTGATCGCGCGGACGGTCAGCCGGCAGGTGATCGCGCAGACGGTCAG CCGGCCGGC GATCGCGCGGCAGGCCAGCCGGCGGGCGACCGTGCGGCCGGTCAGCCGGCGGGTGATCGT GCCGACGGC CAGCCGGCGGGTGACCGCGCTGCGGGTCAGCCGGCAGGCGACCGCGCGGACGGCCAGCCG GCAGGTGAC CGTGCAGCGGGCCAGCCGGCTGGCGACCGTGCCGATGGTCAGCCGGCCGGCGATCGTGCG GCAGGCCAG CCGGCTGGTGACCGTGCCGCGGGTCAGCCGGCAGGTGACCGCGCAGCGGGTCAGGCCGCA GGTGATCGT GCGGCGGGTCAGGCCGCGGGCGGTAACGCGGGCGGCCAGGGCCAGAACAACGAAGGCGCA AACGCTCCG AACGAAAAATCCGTGAAAGAATACCTGGATAAAGTGCGTGCGACCGTTGGTACCGAATGG ACCCCGTGT TCCGTGACCTGTGGCGTTGGCGTTCGCGTGCGCCGCCGTGTTAACGCAGCGAACAAAAAA CCGGAAGAT CTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC - 3 ' (SEQ ID NO: 15)

PvCS-VK247

5 ' -

AGCCATATGACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAAC GGTGTGAACTTC AACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGCGGT CGTGGCCTG GGTGAAAACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCC GAACCGAAA AACCCGCGTGAAAACAAACTGAAACAGCCGGGTGCTAACGGTGCAGGTAACCAGCCGGGT GCAAACGGC GCGGGTAACCAGCCGGGCGCGAACGGCGCTGGCAACCAGCCGGGCGCAAACGGCGCTGGT AACCAGCCG GGCGCCAACGGCGCCGGCAACCAGCCGGGTGCGAACGGCGCCGGTAACCAGCCGGGCGCT AACGGCGCA GGCAATCAACCGGGCGCGAACGGCGCGGGCAACCAGCCGGGTGCCAACGGCGCAGGCAAT CAGCCTGGC GCGAACGGCGCAGGCAACCAGCCGGGTGCTAACGGCGCAGGAAATCAGCCGGGCGCTAAC GGTGCGGGC AATCAACCGGGTGCCAACGGTGCGGGCAATCAGCCTGGTGCCAACGGCGCAGGAAACCAA CCGGGCGCG AACGGTGCGGGTAATCAACCGGGTGCGAATGGCGCTGGCAATCAACCGGGCGCAAATGGC GCGGGAAAT CAGCCGGGTGCAAACGGTGCGGGTAATCAGCCCGGTGCCAACGGCGCAGGGAATCAACCT GGCGCTAAC GGCGCAGGTAACCAGCCGGGCGGTAACGCGGGTGGCCAGGGCCAGAACAACGAAGGTGCG AACGCACCG AACGAAAAAAGCGTGAAAGAATACCTGGATAAAGTGCGCGCGACCGTTGGTACCGAATGG ACCCCGTGC TCCGTGACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAA CCGGAAGAC CTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC - 3 ' (SEQ ID NO: 16)

PvCS- Vivax-like

5 ' - AGCCATATGACCCACTGCGGTCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGC GTGAACTTC AACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGT CGTGGTCTG GGTGAAAACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCG GAACCGAAA AACCCGCGCGAAAACAAACTGAAACAGCCGGGTGCACCGGGTGCTAACCAGGAAGGTGGC GCTGCAGCA CCGGGTGCAAACCAGGAAGGTGGCGCAGCGGCGCCGGGCGCGAACCAGGAAGGTGGCGCG GCAGCTCCG GGTGCCAACCAGGAAGGTGGCGCCGCGGCACCGGGTGCGAATCAAGAAGGTGGCGCGGCG GCGCCGGGC GCAAATCAAGAAGGTGGCGCAGCAGCGCCGGGCGCAAATCAGGAAGGTGGCGCAGCTGCG CCGGGCGCT AATCAAGAAGGTGGCGCTGCTGCACCGGGTGCGAATCAGGAAGGTGGCGCCGCTGCGCCG GGCGCAAAC CAAGAAGGTGGCGCAGCCGCGCCGGGCGCAAACCAGGAAGGTGGCGCAGCCGCACCTGGC GCGAATCAA GAAGGTGGCGCCGCTGCTCCGGGTGCGAACCAAGAAGGTGGCGCGGCTGCGCCGGGCGCT AACCAAGAA GGTGGCGCTGCGGCGCCGGGCGCTAATCAGGAAGGTGGCGCGGCGGCTCCGGGTGCGAAC CAGGAAGGT GGCGCAGCAGATCGTGCAGCTGGTCAGGCAGCGGGTGGCAACGCGGGTGGCCAGGGCCAG AACAACGAA GGTGCCAACGCGCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGCGCGACC GTTGGTACC GAATGGACCCCGTGCTCCGTGACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAAC GCAGCTAAC AAAAAACCGGAAGATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGA TCC - 3 ' (SEQ ID NO: 17)

PvCS-All CS epitopes 5 ' -

AGCCATATACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACG GTGTGAACTTCA ACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGCCAGTCCGCATCTCGTGGTC GTGGTCTGG GTGAAAACCCGGATGACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCG AACCGAAAA ACCCGCGTGAAAACAAACTGAAACAGCCGGGTCCGGGTGATCGTGCAGACGGTCAGCCGG CTGGCGACC GCGCAGACGGTCAGCCGGCAGGTGATCGTGCTGCAGGCCAGCCGGCAGGTGACCGTGCGG CAGGCCAGC CGGCTGGTGATCGCGCCGACGGCCAGCCGGCCGGCGACCGTGCGGACGGTCAGCCGGCTG GTGATCGTG CAGACGCACCGGGCGCCAACCAGGAAGGTGGCGCTGCCGCGCCGGGTGCGAACCAGGAAG GTGGCGCAG CTGCACCGGGTGCAAACCAGGAAGGTGGCGCGGCAGCTGCACCGGGTGCTAACCAGGAAG GTGGCGCCG CAGCTCCGGGTGCCAATCAAGAAGGTGGCGCCGCGGCGCCGGGCGCAAATCAAGAAGGTG GCGCTGCAG CAGCAAACGGTGCAGGTAACCAGCCGGGTGCGAACGGCGCGGGCAACCAGCCGGGTGCTA ACGGCGCGG GTAACCAGCCGGGTGCCAACGGCGCTGGCAACCAGCCGGGCGCGAACGGCGCTGGTAACC AGCCGGGTG ATCGTGCTGCGGGTCAGGCAGCAGGTGGCAACGCGGGTGGCCAGGGTCAGAACAACGAAG GTGCTAACG CCCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGTGCGACCGTTGGTACCG AATGGACCC CGTGCTCCGTGACCTGTGGCGTTGGTGTGCGCGTTCGTCGCCGTGTTAACGCTGCCAACA AAAAACCGG AAGATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC - 3 ' (SEQ ID NO : 18)

Bold and underlined regions represent the sites for Ndel (CATATG) and

BamHl (GGATCC). These genes were subcloned into pET28a expression vector (Novagen). The recombinant proteins were expressed and purified as described below. Recombinant E. coli BL21 DE3 (Novagen) transformed with each recombinant plasmid was cultivated at 37°C in flasks containing Luria broth (LB) and kanamycin (30 μg/ml). Protein expression was induced at an OD ₆ oo of 0.6 with 0.1 mM IPTG (Invitrogen) for 4 hours. After centrifugation, bacteria were lysed on ice with the aid of an ultrasonic processor (Sonics and Materials TNC Vibra Cell VCX 750; 2 cycles of 5 min) in a phosphate buffer (50mM NaH ₂ P0 ₄ , 300mM NaCl, pH 7.0) with 1.0 mg/ml lysozyme (Sigma) and ImM PMSF (Sigma). Bacterial lysate was centrifuged, and the supernatant was resuspended in 8M Urea (Invitrogen). After boiling at 100°C for 15 min, the proteins were applied to a column with Ni ²⁺ -NTA- agarose resin (Quiagen). After several washes with Wash buffer (50 mM NaH ₂ P0 ₄ , 300 mM NaCl, 10 % glycerol, pH 6.0), bound proteins were eluted with with 30ml of Elution buffer (Wash buffer containing 500 mM imidazole (Sigma) and 2 mM PMSF, pH 6.0). The eluted protein was dialysed against 20 mM Tris-HCl (pH 8.0) overnight, at 4°C, and the recombinant proteins were purified by ion-exchange chromatography using a Resource Q column (GE Healthcare) coupled to an FPLC system (GE Healthcare). Proteins were eluted using a NaCl gradient 0-lM. Fractions containing the recombinant proteins with a high degree of purity were pooled and incubated with polymyxin B agarose beads (Sigma) overnight at room temperature. Unbound material was dialysed against phosphate-buffered saline (PBS/pH=7.2) overnight, at 4°C. Protein concentration was determined with the Bradford assay and by SDS-PAGE analyses (see Figure 2).

For the characterization of the immunogenic properties of these recombinant proteins, six- to eight-week-old female C57BL/6 (H-2 ^b ) mice purchased from the Federal University of Sao Paulo, Brazil, were used. Mice were immunised three times, three weeks apart, subcutaneously in the two hind footpads, using a final volume of 50 μΐ in each footpad (first dose) and a final volume of 100 μΐ at the base of the tail (second and third dose). For each dose, 10 μg of the recombinant proteins His ₆ PvCS-VK210 (SEQ ID NO: 21) or His ₆ PvCS-VK247 (SEQ ID NO: 22) or His ₆ PvCS-Vivax-like (SEQ ID NO: 23) were used. Mice immunized with the recombinant protein His ₆ PvCS-All-CS epitopes (SEQ ID NO: 24) received doses of 30 μg each. Proteins were administered in the presence of 50 μg of Poly(I:C)-LMW (Invivogene).

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, either individually or admixed, 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:

5 ' - TCTAGAGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTGCTCCTCCTG TGGGTGCCC GGGTCCAGAGGAACCCACTGCGGCCACAACGTGGACCTGAGCAAGGCCATCAACCTGAAT GGCGTGAAT TTCAACAATGTGGATGCTAGCTCCCTGGGAGCTGCTCACGTGGGACAGTCTGCTAGCCGC GGAAGGGGA CTGGGAGAGAACCCAGACGATGAGGAGGGCGACGCCAAGAAGAAGAAGGATGGAAAGAAG GCTGAGCCA AAGAACCCCAGGGAGAATAAGCTGAAGCAGCCCGGACCTGGGGACAGAGCCGATGGCCAG CCCGCCGGA GACCGGGCTGATGGACAGCCTGCTGGCGACAGGGCTGCTGGACAGCCAGCTGGGGATAGA GCCGCTGGA CAGCCAGCTGGAGACAGAGCTGATGGACAGCCTGCCGGCGACAGGGCCGATGGACAGCCT GCTGGGGAC AGGGCTGATGCTCCAGGCGCTAACCAGGAGGGAGGAGCTGCTGCCCCTGGAGCCAATCAG GAAGGCGGA GCTGCCGCTCCAGGCGCCAATCAGGAAGGAGGAGCTGCTGCTGCTCCCGGCGCCAATCAG GAGGGCGGA GCCGCTGCCCCTGGCGCCAACCAGGAGGGGGGCGCTGCCGCTCCAGGCGCTAATCAGGAG GGCGGCGCT GCTGCTGCTAACGGAGCTGGAAATCAGCCAGGGGCCAATGGAGCTGGAAATCAGCCTGGC GCTAACGGA GCTGGGAATCAGCCTGGAGCCAACGGCGCCGGCAATCAGCCAGGGGCCAACGGGGCTGGC AATCAGCCC GGCGACAGAGCCGCTGGACAGGCTGCTGGAGGAAACGCTGGAGGACAGGGACAGAACAAT GAGGGGGCC AACGCTCCCAATGAGAAGTCCGTGAAGGAGTACCTGGATAAGGTGAGGGCTACCGTGGGC ACAGAGTGG ACCCCTTGCTCTGTGACATGTGGAGTGGGGGTGAGAGTGAGGAGACGGGTGAACGCCGCT AATAAGAAG CCAGAGGACCTGACCCTGAACGACCTGGAGACAGATGTGTGTACCTGAGGTACC - 3 ' (SEQ ID NO : 19)

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.

The immunogenicity of the recombinant adenovirus PvCS-AdC68 was tested by the intra-muscular immunization of groups of C57BL/6 mice (n=6) with 2xl0 ¹⁰ or 10 ⁹ viral particles as described in the reference 18. Five weeks after a single dose, 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 (groups 2, 4 and 5) presented serum antibody titers significantly higher than control mice injected with 2xl0 ¹⁰ viral particles of control adenovirus expressing the unrelated protein β-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 μg of each protein) in the presence of 50 μg of Poly(LC). After boosting, 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). These results indicate that injection of 2xl0 ¹⁰ or 10 ⁹ viral particles of PvCS-AdC68 successfully primed these mice for a subsequent immunization with recombinant proteins administered in Poly(I:C).

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:

PIC-PvCS- VK210-His ₆ Coding sequence

CCAAGAGAAAACAAGTTGAAACAGCCAGGAGACAGAGCAGACGGTCAGCCAGCAGGA GACAGAGCAGAT GGACAGCCAGCAGGAGACAGAGCTGATGGTCAACCAGCAGGAGATAGAGCTGACGGTCAG CCTGCTGGA GACAGAGCTGCCGGTCAACCAGCCGGAGATAGAGCAGATGGACAGCCTGCTGGAGATAGA GCCGACGGA CAGCCTGCCGGAGATAGAGCCGATGGTCAACCTGCTGGAGATAGAGCAGACGGTCAACCA GCTGGAGAT AGAGCAGCTGGTCAGCCTGCCGGAGACAGAGCCGCAGGACAGCCAGCCGGAGATAGAGCT GATGGACAA CCAGCGGGAGATCGTGCTGCCGGACAGCCTGCAGGAGATAGAGCCGACGGTCAACCTGCC GGAGATAGA GCTGCCGGACAGCCAGCTGGAGATAGAGCTGATGGTCAACCCGCCGGAGATAGAGCCGCA GGTCAACCA GCCGGAGACAGAGCCGCCGGACAACCAGCCGGAGATCGTGCAGCTGGTCAAGCCGCAGGT GACAGAGCC GCTGGTCAAGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGTGCTAAC GCCCCAAAT GAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGGTACAGAGTGGACC CCTTGTTCC GTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCA GAAGATTTG ACCTTGAACGACTTGGAAACCGATGTCTGCACTCACCACCACCACCACCACCATTAG (SEQ ID NO: 26)

Protein PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRADG QPAGDRADG QPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDR ADGQPAGDR AAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQ NNEGANAPN EKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHH HHHH ( SEQ I D NO : 28)

PIC-PvCS- VK247 -His ₆

Coding sequence

CCAAGAGAAAACAAGTTGAAACAGCCAGGAGCAAATGGAGCAGGTAACCAGCCAGGA GCAAACGGAGCA GGTAACCAGCCAGGAGCAAACGGTGCTGGAAATCAACCAGGTGCCAACGGTGCAGGAAAT CAGCCTGGA GCTAACGGTGCCGGAAATCAACCAGGTGCAAACGGTGCTGGAAATCAGCCTGGAGCAAAT GGTGCAGGA AATCAACCAGGTGCTAATGGAGCCGGTAACCAGCCTGGAGCCAATGGAGCCGGTAACCAA CCTGGAGCT AATGGAGCTGGTAACCAACCAGGTGCTAATGGTGCTGGTAACCAGCCTGGAGCAAACGGA GCCGGTAAT CAGCCTGGAGCAAACGGTGCCGGTAACCAGCCTGGTGCCAATGGTGCCGGAAATCAGCCT GGTGCTAAT GGAGCCGGAAACCAGCCTGGAGCCAACGGAGCTGGTAACCAGCCTGGTGCAAATGGAGCT GGTAATCAG CCAGGTGCAAATGGAGCCGGAAATCAACCTGGTGCTAATGGAGCAGGTAACCAACCTGGT GCCAATGGA GCTGGAAATCAACCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGAGCAAAC GCTCCAAAT GAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACTGTTGGTACAGAGTGGACC CCTTGTTCC GTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCA GAAGACCTT ACTTTGAACGACTTGGAAACCGATGTCTGCACTCACCACCACCACCACCACTAA ( SEQ I D NO : 30)

Prote in

PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN GAGNQPGANGAG NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN GAGNQPGAN GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQGQ NNEGANAPN EKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHH HHH ( SEQ I D NO : 32)

PIC-PvCS-Vivax-like-His ₆

Coding sequence

CCAAGAGAAAATAAGTTGAAACAGCCAGGTGCCCCAGGAGCAAACCAAGAGGGAGGT GCCGCCGCCCCA GGAGCAAACCAGGAGGGAGGTGCTGCCGCACCAGGAGCCAACCAAGAAGGTGGAGCTGCC GCACCTGGT GCAAATCAGGAGGGTGGAGCTGCCGCACCAGGAGCTAATCAGGAAGGAGGTGCTGCCGCA CCTGGTGCC AACCAAGAGGGAGGTGCTGCCGCACCAGGTGCTAATCAGGAAGGAGGAGCTGCCGCACCA GGTGCCAAC CAAGAGGGTGGTGCTGCCGCACCTGGAGCCAATCAGGAAGGTGGTGCTGCCGCACCAGGC GCAAACCAA GAGGGAGGAGCTGCCGCACCTGGAGCTAATCAGGAAGGCGGTGCTGCCGCACCCGGAGCA AACCAAGAG GGCGGTGCTGCCGCACCTGGCGCTAATCAGGAAGGCGGCGCTGCCGCACCTGGTGCTAAC CAAGAGGGC GGCGCTGCCGCACCAGGTGCAAATCAGGAAGGCGGGGCTGCCGCACCCGGCGCTAACCAA GAGGGTGGA GCTGCCGATAGAGCAGCTGGTCAAGCCGCAGGTGGAAACGCTGGTGGACAGGGTCAGAAC AATGAAGGA GCAAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGAGCTACTGTT GGTACAGAG TGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCT GCCAATAAG AAACCAGAAGACCTTACCTTGAATGACTTGGAGACAGATGTTTGTACCCACCACCACCAC CACCACTAA

( SEQ I D NO : 34)

Prote in

PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPG ANQEGGAAAPGA NQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGG AAAPGANQE GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGNA GGQGQNNEG ANAPNEKSVKEYLDKVRATVGTEWT PCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTHHHHHH

( SEQ I D NO : 36) PIC-PvCS-All-CS epitopes-His ₆

Coding sequence

CCAAGAGAAAACAAATTGAAGCAACCAGGACCAGGAGATAGAGCAGATGGACAACCA GCAGGAGATAGA GCCGACGGACAGCCAGCAGGAGATAGAGCTGCCGGTCAACCAGCTGGAGACAGAGCAGCT GGTCAGCCT GCTGGAGATAGAGCCGACGGTCAGCCTGCCGGAGATAGAGCTGACGGTCAGCCAGCTGGA GATAGAGCT GACGCCCCTGGTGCTAACCAAGAAGGTGGAGCCGCAGCTCCAGGAGCTAATCAGGAGGGT GGAGCCGCA GCTCCTGGAGCCAATCAGGAAGGAGGTGCCGCAGCTGCCCCAGGTGCTAATCAAGAGGGT GGAGCAGCT GCCCCTGGAGCCAACCAAGAAGGAGGTGCAGCTGCCCCTGGAGCAAACCAGGAAGGTGGA GCAGCTGCC GCAAACGGTGCTGGAAATCAACCTGGTGCCAACGGTGCAGGAAATCAGCCAGGAGCTAAC GGTGCCGGA AATCAACCTGGTGCTAATGGTGCTGGAAATCAGCCAGGAGCAAATGGTGCAGGAAATCAA CCTGGAGAT AGAGCTGCCGGACAGGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGA GCAAACGCT CCTAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGAGCTACTGTTGGTACAGAG TGGACCCCA TGTTCCGTCACTTGCGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAG AAACCTGAA GATTTGACTTTGAACGACTTGGAAACCGATGTCTGCACCCACCACCACCACCACCATTAG (SEQ ID NO: 38)

Protein

PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAG DRADGQPAGDRA DAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQEGGAAAPGANQEGGAAAPG ANQEGGAAA ANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQ GQNNEGANA PNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTH HHHHH

(SEQ ID NO: 40)

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. In order to transform P. pastoris GS1 15 strain (his4 ^~ ) by electroporation, 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.

The expression and purification of the recombinant proteins was performed as described below. 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. 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 h. After induction for 72 h, cells were removed by centrifugation, the culture supernatant was concentrated by ultrafiltration with an Amicon Ultracel with 30.000 MWCO membrane (Millipore) and extensively dialyzed at 4°C against sodium phosphate buffer 20 mM, pH 8.0, 0.2 M NaCl. The supernatant was applied to a column with Ni ²⁺ -NTA-Agarose resin (Quiagen) previously equilibrated (sodium phosphate buffer 20 mM, pH 8.0, 0.5 M NaCl). 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.

REFERENCES

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[10] Rodriguez A, Mintardjo R, Tax D, Gillissen G, Custers J, Pau MG, Klap J, Santra S, Balachandran H, Letvin NL, Goudsmit J, Radosevic K.Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys. Vaccine. 2009 Oct 19; 27(44):6226-33.

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LIST OF SEQUENCES:

SEQ NAME SEQUENCE ID

1 PvCs-VK210 THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGDRADGQPAGDRADGQPAGDRADGQP AGDRADGQPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAD GQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGD RAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQA AGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRV RRRVNAANKKPEDLTLNDLETDVCT

2 PvCs-VK247 THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE

GDAKKKKDGKKAEPKNPRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQ PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA GNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGA NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ PGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRV RRRVNAANKKPEDLTLNDLETDVCT

3 PvCs-Vivax- THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE like GDAKKKKDGKKAEPKNPRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPG

ANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGA AAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQ EGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAADR AAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCG VGVRVRRRVNAANKKPEDLTLNDLETDVCT

4 PvCs-All-CS- THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE Epitopes GDAKKKKDGKKAEPKNPRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAG

QPAGDRAAGQPAGDRADGQPAGDRADGQPAGDRADAPGANQEGGAAAPGAN QEGGAAAPGANQEGGAAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAA AANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAG QAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGV RVRRRVNAANKKPEDLTLNDLETDVCT

5 PvCs-VK210 ACCCACTGCGGCCATAACGTTGACCTGTCTAAAGCGATTAACCTGAACGGT

GTTAACTTTAACAACGTTGATGCCTCCTCCCTGGGTGCCGCGCATGTTGGT CAGAGCGCATCCCGCGGTCGTGGCCTGGGCGAAAACCCGGATGACGAAGAA GGCGACGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCGAAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGCGACCGCGCGGATGGCCAGCCGGCC GGCGACCGTGCGGATGGCCAGCCGGCAGGCGACCGTGCGGATGGTCAGCCG GCCGGTGACCGTGCGGACGGTCAGCCGGCGGGCGACCGCGCTGCAGGCCAG CCGGCCGGTGACCGCGCCGATGGCCAGCCGGCTGGCGACCGCGCGGATGGT CAGCCGGCGGGTGATCGCGCGGACGGTCAGCCGGCAGGTGATCGCGCAGAC GGTCAGCCGGCCGGCGATCGCGCGGCAGGCCAGCCGGCGGGCGACCGTGCG GCCGGTCAGCCGGCGGGTGATCGTGCCGACGGCCAGCCGGCGGGTGACCGC GCTGCGGGTCAGCCGGCAGGCGACCGCGCGGACGGCCAGCCGGCAGGTGAC CGTGCAGCGGGCCAGCCGGCTGGCGACCGTGCCGATGGTCAGCCGGCCGGC GATCGTGCGGCAGGCCAGCCGGCTGGTGACCGTGCCGCGGGTCAGCCGGCA GGTGACCGCGCAGCGGGTCAGGCCGCAGGTGATCGTGCGGCGGGTCAGGCC GCGGGCGGTAACGCGGGCGGCCAGGGCCAGAACAACGAAGGCGCAAACGCT CCGAACGAAAAATCCGTGAAAGAATACCTGGATAAAGTGCGTGCGACCGTT GGTACCGAATGGACCCCGTGTTCCGTGACCTGTGGCGTTGGCGTTCGCGTG CGCCGCCGTGTTAACGCAGCGAACAAAAAACCGGAAGATCTGACCCTGAAC GATCTGGAAACCGACGTTTGTACCTAA

6 PvCs-VK247 ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGT

GTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGC CAGTCCGCATCTCGCGGTCGTGGCCTGGGTGAAAACCCGGATGACGAAGAA GGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCGAAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGTGCTAACGGTGCAGGTAACCAGCCG GGTGCAAACGGCGCGGGTAACCAGCCGGGCGCGAACGGCGCTGGCAACCAG CCGGGCGCAAACGGCGCTGGTAACCAGCCGGGCGCCAACGGCGCCGGCAAC CAGCCGGGTGCGAACGGCGCCGGTAACCAGCCGGGCGCTAACGGCGCAGGC SEQ NAME SEQUENCE

ID

AATCAACCGGGCGCGAACGGCGCGGGCAACCAGCCGGGTGCCAACGGCGCA GGCAATCAGCCTGGCGCGAACGGCGCAGGCAACCAGCCGGGTGCTAACGGC GCAGGAAATCAGCCGGGCGCTAACGGTGCGGGCAATCAACCGGGTGCCAAC GGTGCGGGCAATCAGCCTGGTGCCAACGGCGCAGGAAACCAACCGGGCGCG AACGGTGCGGGTAATCAACCGGGTGCGAATGGCGCTGGCAATCAACCGGGC GCAAATGGCGCGGGAAATCAGCCGGGTGCAAACGGTGCGGGTAATCAGCCC GGTGCCAACGGCGCAGGGAATCAACCTGGCGCTAACGGCGCAGGTAACCAG CCGGGCGGTAACGCGGGTGGCCAGGGCCAGAACAACGAAGGTGCGAACGCA CCGAACGAAAAAAGCGTGAAAGAATACCTGGATAAAGTGCGCGCGACCGTT GGTACCGAATGGACCCCGTGCTCCGTGACCTGTGGCGTTGGTGTGCGTGTT CGTCGCCGTGTTAACGCTGCCAACAAAAAACCGGAAGACCTGACCCTGAAC GATCTGGAAACCGACGTTTGTACCTAA

7 PvCs-Vivax- ACCCACTGCGGTCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGC like GTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGC

CAGTCCGCATCTCGTGGTCGTGGTCTGGGTGAAAACCCGGATGACGAAGAA GGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCGAAAAACCCG CGCGAAAACAAACTGAAACAGCCGGGTGCACCGGGTGCTAACCAGGAAGGT GGCGCTGCAGCACCGGGTGCAAACCAGGAAGGTGGCGCAGCGGCGCCGGGC GCGAACCAGGAAGGTGGCGCGGCAGCTCCGGGTGCCAACCAGGAAGGTGGC GCCGCGGCACCGGGTGCGAATCAAGAAGGTGGCGCGGCGGCGCCGGGCGCA AATCAAGAAGGTGGCGCAGCAGCGCCGGGCGCAAATCAGGAAGGTGGCGCA GCTGCGCCGGGCGCTAATCAAGAAGGTGGCGCTGCTGCACCGGGTGCGAAT CAGGAAGGTGGCGCCGCTGCGCCGGGCGCAAACCAAGAAGGTGGCGCAGCC GCGCCGGGCGCAAACCAGGAAGGTGGCGCAGCCGCACCTGGCGCGAATCAA GAAGGTGGCGCCGCTGCTCCGGGTGCGAACCAAGAAGGTGGCGCGGCTGCG CCGGGCGCTAACCAAGAAGGTGGCGCTGCGGCGCCGGGCGCTAATCAGGAA GGTGGCGCGGCGGCTCCGGGTGCGAACCAGGAAGGTGGCGCAGCAGATCGT GCAGCTGGTCAGGCAGCGGGTGGCAACGCGGGTGGCCAGGGCCAGAACAAC GAAGGTGCCAACGCGCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAA GTGCGCGCGACCGTTGGTACCGAATGGACCCCGTGCTCCGTGACCTGTGGC GTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCAGCTAACAAAAAACCGGAA GATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA

8 PvCs-All-CS- ACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACCTGAACGGT Epitopes GTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCACACGTTGGC

CAGTCCGCATCTCGTGGTCGTGGTCTGGGTGAAAACCCGGATGACGAAGAA GGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCGAAAAACCCG CGTGAAAACAAACTGAAACAGCCGGGTCCGGGTGATCGTGCAGACGGTCAG CCGGCTGGCGACCGCGCAGACGGTCAGCCGGCAGGTGATCGTGCTGCAGGC CAGCCGGCAGGTGACCGTGCGGCAGGCCAGCCGGCTGGTGATCGCGCCGAC GGCCAGCCGGCCGGCGACCGTGCGGACGGTCAGCCGGCTGGTGATCGTGCA GACGCACCGGGCGCCAACCAGGAAGGTGGCGCTGCCGCGCCGGGTGCGAAC CAGGAAGGTGGCGCAGCTGCACCGGGTGCAAACCAGGAAGGTGGCGCGGCA GCTGCACCGGGTGCTAACCAGGAAGGTGGCGCCGCAGCTCCGGGTGCCAAT CAAGAAGGTGGCGCCGCGGCGCCGGGCGCAAATCAAGAAGGTGGCGCTGCA GCAGCAAACGGTGCAGGTAACCAGCCGGGTGCGAACGGCGCGGGCAACCAG CCGGGTGCTAACGGCGCGGGTAACCAGCCGGGTGCCAACGGCGCTGGCAAC CAGCCGGGCGCGAACGGCGCTGGTAACCAGCCGGGTGATCGTGCTGCGGGT CAGGCAGCAGGTGGCAACGCGGGTGGCCAGGGTCAGAACAACGAAGGTGCT AACGCCCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAGTGCGTGCG ACCGTTGGTACCGAATGGACCCCGTGCTCCGTGACCTGTGGCGTTGGTGTG CGCGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAACCGGAAGATCTGACC CTGAACGATCTGGAAACCGACGTTTGTACCTAA

9 Ad TCTAGAGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTG

CTCCTCCTGTGGGTGCCCGGGTCCAGAGGAACCCACTGCGGCCACAACGTG GACCTGAGCAAGGCCATCAACCTGAATGGCGTGAATTTCAACAATGTGGAT GCTAGCTCCCTGGGAGCTGCTCACGTGGGACAGTCTGCTAGCCGCGGAAGG GGACTGGGAGAGAACCCAGACGATGAGGAGGGCGACGCCAAGAAGAAGAAG GATGGAAAGAAGGCTGAGCCAAAGAACCCCAGGGAGAATAAGCTGAAGCAG CCCGGACCTGGGGACAGAGCCGATGGCCAGCCCGCCGGAGACCGGGCTGAT SEQ NAME SEQUENCE

ID

GGACAGCCTGCTGGCGACAGGGCTGCTGGACAGCCAGCTGGGGATAGAGCC GCTGGACAGCCAGCTGGAGACAGAGCTGATGGACAGCCTGCCGGCGACAGG GCCGATGGACAGCCTGCTGGGGACAGGGCTGATGCTCCAGGCGCTAACCAG GAGGGAGGAGCTGCTGCCCCTGGAGCCAATCAGGAAGGCGGAGCTGCCGCT CCAGGCGCCAATCAGGAAGGAGGAGCTGCTGCTGCTCCCGGCGCCAATCAG GAGGGCGGAGCCGCTGCCCCTGGCGCCAACCAGGAGGGGGGCGCTGCCGCT CCAGGCGCTAATCAGGAGGGCGGCGCTGCTGCTGCTAACGGAGCTGGAAAT CAGCCAGGGGCCAATGGAGCTGGAAATCAGCCTGGCGCTAACGGAGCTGGG AATCAGCCTGGAGCCAACGGCGCCGGCAATCAGCCAGGGGCCAACGGGGCT GGCAATCAGCCCGGCGACAGAGCCGCTGGACAGGCTGCTGGAGGAAACGCT GGAGGACAGGGACAGAACAATGAGGGGGCCAACGCTCCCAATGAGAAGTCC GTGAAGGAGTACCTGGATAAGGTGAGGGCTACCGTGGGCACAGAGTGGACC CCTTGCTCTGTGACATGTGGAGTGGGGGTGAGAGTGAGGAGACGGGTGAAC GCCGCTAATAAGAAGCCAGAGGACCTGACCCTGAACGACCTGGAGACAGAT GTGTGTACCTGA

10 P. vivax CS THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEE protein N- GDAKKKKDGKKAEPKNPRENKLKQPGP

terminal

sequence

11 VK210 DRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGD epitope

12 VK247 ANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPG

epitope

13 Vivax-like APGANQEGGAAAPGANQEGGAAAPGANQEGGAA

epitope

14 P. vivax CS ANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKVRATVG protein C- TEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT terminal

sequence

15 PvCS-VK210 AGCCATATGACCCACTGCGGCCATAACGTTGACCTGTCTAAAGCGATTAAC

CTGAACGGTGTTAACTTTAACAACGTTGATGCCTCCTCCCTGGGTGCCGCG CATGTTGGTCAGAGCGCATCCCGCGGTCGTGGCCTGGGCGAAAACCCGGAT GACGAAGAAGGCGACGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCG AAAAACCCGCGTGAAAACAAACTGAAACAGCCGGGCGACCGCGCGGATGGC CAGCCGGCCGGCGACCGTGCGGATGGCCAGCCGGCAGGCGACCGTGCGGAT GGTCAGCCGGCCGGTGACCGTGCGGACGGTCAGCCGGCGGGCGACCGCGCT GCAGGCCAGCCGGCCGGTGACCGCGCCGATGGCCAGCCGGCTGGCGACCGC GCGGATGGTCAGCCGGCGGGTGATCGCGCGGACGGTCAGCCGGCAGGTGAT CGCGCAGACGGTCAGCCGGCCGGCGATCGCGCGGCAGGCCAGCCGGCGGGC GACCGTGCGGCCGGTCAGCCGGCGGGTGATCGTGCCGACGGCCAGCCGGCG GGTGACCGCGCTGCGGGTCAGCCGGCAGGCGACCGCGCGGACGGCCAGCCG GCAGGTGACCGTGCAGCGGGCCAGCCGGCTGGCGACCGTGCCGATGGTCAG CCGGCCGGCGATCGTGCGGCAGGCCAGCCGGCTGGTGACCGTGCCGCGGGT CAGCCGGCAGGTGACCGCGCAGCGGGTCAGGCCGCAGGTGATCGTGCGGCG GGTCAGGCCGCGGGCGGTAACGCGGGCGGCCAGGGCCAGAACAACGAAGGC GCAAACGCTCCGAACGAAAAATCCGTGAAAGAATACCTGGATAAAGTGCGT GCGACCGTTGGTACCGAATGGACCCCGTGTTCCGTGACCTGTGGCGTTGGC GTTCGCGTGCGCCGCCGTGTTAACGCAGCGAACAAAAAACCGGAAGATCTG ACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC

16 PvCS-VK247 AGCCATATGACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAAC

CTGAACGGTGTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCA CACGTTGGCCAGTCCGCATCTCGCGGTCGTGGCCTGGGTGAAAACCCGGAT GACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCG AAAAACCCGCGTGAAAACAAACTGAAACAGCCGGGTGCTAACGGTGCAGGT AACCAGCCGGGTGCAAACGGCGCGGGTAACCAGCCGGGCGCGAACGGCGCT GGCAACCAGCCGGGCGCAAACGGCGCTGGTAACCAGCCGGGCGCCAACGGC GCCGGCAACCAGCCGGGTGCGAACGGCGCCGGTAACCAGCCGGGCGCTAAC GGCGCAGGCAATCAACCGGGCGCGAACGGCGCGGGCAACCAGCCGGGTGCC SEQ NAME SEQUENCE

ID

AACGGCGCAGGCAATCAGCCTGGCGCGAACGGCGCAGGCAACCAGCCGGGT GCTAACGGCGCAGGAAATCAGCCGGGCGCTAACGGTGCGGGCAATCAACCG GGTGCCAACGGTGCGGGCAATCAGCCTGGTGCCAACGGCGCAGGAAACCAA CCGGGCGCGAACGGTGCGGGTAATCAACCGGGTGCGAATGGCGCTGGCAAT CAACCGGGCGCAAATGGCGCGGGAAATCAGCCGGGTGCAAACGGTGCGGGT AATCAGCCCGGTGCCAACGGCGCAGGGAATCAACCTGGCGCTAACGGCGCA GGTAACCAGCCGGGCGGTAACGCGGGTGGCCAGGGCCAGAACAACGAAGGT GCGAACGCACCGAACGAAAAAAGCGTGAAAGAATACCTGGATAAAGTGCGC GCGACCGTTGGTACCGAATGGACCCCGTGCTCCGTGACCTGTGGCGTTGGT GTGCGTGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAACCGGAAGACCTG ACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC

17 PvCS-Vivax- AGCCATATGACCCACTGCGGTCATAACGTGGATCTGTCTAAAGCGATCAAC like CTGAACGGCGTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCA

CACGTTGGCCAGTCCGCATCTCGTGGTCGTGGTCTGGGTGAAAACCCGGAT GACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCGGAACCG AAAAACCCGCGCGAAAACAAACTGAAACAGCCGGGTGCACCGGGTGCTAAC CAGGAAGGTGGCGCTGCAGCACCGGGTGCAAACCAGGAAGGTGGCGCAGCG GCGCCGGGCGCGAACCAGGAAGGTGGCGCGGCAGCTCCGGGTGCCAACCAG GAAGGTGGCGCCGCGGCACCGGGTGCGAATCAAGAAGGTGGCGCGGCGGCG CCGGGCGCAAATCAAGAAGGTGGCGCAGCAGCGCCGGGCGCAAATCAGGAA GGTGGCGCAGCTGCGCCGGGCGCTAATCAAGAAGGTGGCGCTGCTGCACCG GGTGCGAATCAGGAAGGTGGCGCCGCTGCGCCGGGCGCAAACCAAGAAGGT GGCGCAGCCGCGCCGGGCGCAAACCAGGAAGGTGGCGCAGCCGCACCTGGC GCGAATCAAGAAGGTGGCGCCGCTGCTCCGGGTGCGAACCAAGAAGGTGGC GCGGCTGCGCCGGGCGCTAACCAAGAAGGTGGCGCTGCGGCGCCGGGCGCT AATCAGGAAGGTGGCGCGGCGGCTCCGGGTGCGAACCAGGAAGGTGGCGCA GCAGATCGTGCAGCTGGTCAGGCAGCGGGTGGCAACGCGGGTGGCCAGGGC CAGAACAACGAAGGTGCCAACGCGCCGAACGAAAAAAGCGTGAAAGAATAC CTGGACAAAGTGCGCGCGACCGTTGGTACCGAATGGACCCCGTGCTCCGTG ACCTGTGGCGTTGGTGTGCGTGTTCGTCGCCGTGTTAACGCAGCTAACAAA AAACCGGAAGATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAA CGCGGATCC

18 PvCS-All CS AGCCATATACCCACTGCGGCCATAACGTGGATCTGTCTAAAGCGATCAACC epitopes TGAACGGTGTGAACTTCAACAACGTTGACGCATCTAGCCTGGGTGCAGCAC

ACGTTGGCCAGTCCGCATCTCGTGGTCGTGGTCTGGGTGAAAACCCGGATG ACGAAGAAGGCGATGCGAAAAAGAAAAAAGACGGTAAAAAAGCCGAACCGA AAAACCCGCGTGAAAACAAACTGAAACAGCCGGGTCCGGGTGATCGTGCAG ACGGTCAGCCGGCTGGCGACCGCGCAGACGGTCAGCCGGCAGGTGATCGTG CTGCAGGCCAGCCGGCAGGTGACCGTGCGGCAGGCCAGCCGGCTGGTGATC GCGCCGACGGCCAGCCGGCCGGCGACCGTGCGGACGGTCAGCCGGCTGGTG ATCGTGCAGACGCACCGGGCGCCAACCAGGAAGGTGGCGCTGCCGCGCCGG GTGCGAACCAGGAAGGTGGCGCAGCTGCACCGGGTGCAAACCAGGAAGGTG GCGCGGCAGCTGCACCGGGTGCTAACCAGGAAGGTGGCGCCGCAGCTCCGG GTGCCAATCAAGAAGGTGGCGCCGCGGCGCCGGGCGCAAATCAAGAAGGTG GCGCTGCAGCAGCAAACGGTGCAGGTAACCAGCCGGGTGCGAACGGCGCGG GCAACCAGCCGGGTGCTAACGGCGCGGGTAACCAGCCGGGTGCCAACGGCG CTGGCAACCAGCCGGGCGCGAACGGCGCTGGTAACCAGCCGGGTGATCGTG CTGCGGGTCAGGCAGCAGGTGGCAACGCGGGTGGCCAGGGTCAGAACAACG AAGGTGCTAACGCCCCGAACGAAAAAAGCGTGAAAGAATACCTGGACAAAG TGCGTGCGACCGTTGGTACCGAATGGACCCCGTGCTCCGTGACCTGTGGCG TTGGTGTGCGCGTTCGTCGCCGTGTTAACGCTGCCAACAAAAAACCGGAAG ATCTGACCCTGAACGATCTGGAAACCGACGTTTGTACCTAACGCGGATCC

19 Ad TCTAGAGCCGCCACCATGGGAATGCAGGTGCAGATCCAGAGCCTGTTTCTG

CTCCTCCTGTGGGTGCCCGGGTCCAGAGGAACCCACTGCGGCCACAACGTG GACCTGAGCAAGGCCATCAACCTGAATGGCGTGAATTTCAACAATGTGGAT GCTAGCTCCCTGGGAGCTGCTCACGTGGGACAGTCTGCTAGCCGCGGAAGG GGACTGGGAGAGAACCCAGACGATGAGGAGGGCGACGCCAAGAAGAAGAAG GATGGAAAGAAGGCTGAGCCAAAGAACCCCAGGGAGAATAAGCTGAAGCAG CCCGGACCTGGGGACAGAGCCGATGGCCAGCCCGCCGGAGACCGGGCTGAT SEQ NAME SEQUENCE

ID

GGACAGCCTGCTGGCGACAGGGCTGCTGGACAGCCAGCTGGGGATAGAGCC GCTGGACAGCCAGCTGGAGACAGAGCTGATGGACAGCCTGCCGGCGACAGG GCCGATGGACAGCCTGCTGGGGACAGGGCTGATGCTCCAGGCGCTAACCAG GAGGGAGGAGCTGCTGCCCCTGGAGCCAATCAGGAAGGCGGAGCTGCCGCT CCAGGCGCCAATCAGGAAGGAGGAGCTGCTGCTGCTCCCGGCGCCAATCAG GAGGGCGGAGCCGCTGCCCCTGGCGCCAACCAGGAGGGGGGCGCTGCCGCT CCAGGCGCTAATCAGGAGGGCGGCGCTGCTGCTGCTAACGGAGCTGGAAAT CAGCCAGGGGCCAATGGAGCTGGAAATCAGCCTGGCGCTAACGGAGCTGGG AATCAGCCTGGAGCCAACGGCGCCGGCAATCAGCCAGGGGCCAACGGGGCT GGCAATCAGCCCGGCGACAGAGCCGCTGGACAGGCTGCTGGAGGAAACGCT GGAGGACAGGGACAGAACAATGAGGGGGCCAACGCTCCCAATGAGAAGTCC GTGAAGGAGTACCTGGATAAGGTGAGGGCTACCGTGGGCACAGAGTGGACC CCTTGCTCTGTGACATGTGGAGTGGGGGTGAGAGTGAGGAGACGGGTGAAC GCCGCTAATAAGAAGCCAGAGGACCTGACCCTGAACGACCTGGAGACAGAT GTGTGTACCTGAGGTACC

20 Ad-All-CS- MGMVQSIQLFLLLLWVPGSRGQPTHCGHNVDLSKAINLNGVNFNNVDASSL Epitopes GAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGPG

DRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQ PAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQEGGA AAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGAGNQPG ANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEY LDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

21 His ₆ -PvCS- MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA VK210 AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGDRAD

GQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGD RADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQP AGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAA GQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLDKV RATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

22 His ₆ -PvCS- MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA VK247 AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGANGA

GNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGA NGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQ PGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGA GNQPGANGAGNQPGANGAGNQPGGNAGGQGQNNEGANAPNEKSVKEYLDKV RATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

23 His ₆ -PvCS- MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA Vivax-like AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGAPGA

NQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAA APGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQE GGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPG ANQEGGAAAPGANQEGGAADRAAGQAAGGNAGGQGQNNEGANAPNEKSVKE YLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

24 His ₆ -PvCS- MGSSHHHHHHSSGLVPRGSHMTHCGHNVDLSKAINLNGVNFNNVDASSLGA All CS AHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGPGDR epitopes ADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQPA

GDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAAPGANQEGGAAA PGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGNQPGANGAGNQPGAN GAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEGANAPNEKSVKEYLD KVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCT

25 PIC-PvCS- CCAAGAGAAAACAAGTTGAAACAGCCAGGAGACAGAGCAGACGGTCAGCCA VK210 GCAGGAGACAGAGCAGATGGACAGCCAGCAGGAGACAGAGCTGATGGTCAA

CCAGCAGGAGATAGAGCTGACGGTCAGCCTGCTGGAGACAGAGCTGCCGGT CAACCAGCCGGAGATAGAGCAGATGGACAGCCTGCTGGAGATAGAGCCGAC GGACAGCCTGCCGGAGATAGAGCCGATGGTCAACCTGCTGGAGATAGAGCA GACGGTCAACCAGCTGGAGATAGAGCAGCTGGTCAGCCTGCCGGAGACAGA GCCGCAGGACAGCCAGCCGGAGATAGAGCTGATGGACAACCAGCGGGAGAT CGTGCTGCCGGACAGCCTGCAGGAGATAGAGCCGACGGTCAACCTGCCGGA SEQ NAME SEQUENCE

ID

GATAGAGCTGCCGGACAGCCAGCTGGAGATAGAGCTGATGGTCAACCCGCC GGAGATAGAGCCGCAGGTCAACCAGCCGGAGACAGAGCCGCCGGACAACCA GCCGGAGATCGTGCAGCTGGTCAAGCCGCAGGTGACAGAGCCGCTGGTCAA GCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGTGCTAAC GCCCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACT GTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGA GTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCAGAAGATTTGACCTTG AACGACTTGGAAACCGATGTCTGCACT

26 PIC-PvCS- CCAAGAGAAAACAAGTTGAAACAGCCAGGAGACAGAGCAGACGGTCAGCCA VK210-His ₆ GCAGGAGACAGAGCAGATGGACAGCCAGCAGGAGACAGAGCTGATGGTCAA

CCAGCAGGAGATAGAGCTGACGGTCAGCCTGCTGGAGACAGAGCTGCCGGT CAACCAGCCGGAGATAGAGCAGATGGACAGCCTGCTGGAGATAGAGCCGAC GGACAGCCTGCCGGAGATAGAGCCGATGGTCAACCTGCTGGAGATAGAGCA GACGGTCAACCAGCTGGAGATAGAGCAGCTGGTCAGCCTGCCGGAGACAGA GCCGCAGGACAGCCAGCCGGAGATAGAGCTGATGGACAACCAGCGGGAGAT CGTGCTGCCGGACAGCCTGCAGGAGATAGAGCCGACGGTCAACCTGCCGGA GATAGAGCTGCCGGACAGCCAGCTGGAGATAGAGCTGATGGTCAACCCGCC GGAGATAGAGCCGCAGGTCAACCAGCCGGAGACAGAGCCGCCGGACAACCA GCCGGAGATCGTGCAGCTGGTCAAGCCGCAGGTGACAGAGCCGCTGGTCAA GCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGTGCTAAC GCCCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACT GTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGA GTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCAGAAGATTTGACCTTG AACGACTTGGAAACCGATGTCTGCACTCACCACCACCACCACCACCATTAG

27 PIC-PvCS- PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAG VK210 QPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDR

AAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPA GDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGAN APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL NDLETDVCT

28 PIC-PvCS- PRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAG VK210-His ₆ QPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDR

AAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPA GDRAAGQPAGDRAAGQPAGDRAAGQAAGDRAAGQAAGGNAGGQGQNNEGAN APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL NDLETDVCTHHHHHHH

29 PIC-PvCS- CCAAGAGAAAACAAGTTGAAACAGCCAGGAGCAAATGGAGCAGGTAACCAG VK247 CCAGGAGCAAACGGAGCAGGTAACCAGCCAGGAGCAAACGGTGCTGGAAAT

CAACCAGGTGCCAACGGTGCAGGAAATCAGCCTGGAGCTAACGGTGCCGGA AATCAACCAGGTGCAAACGGTGCTGGAAATCAGCCTGGAGCAAATGGTGCA GGAAATCAACCAGGTGCTAATGGAGCCGGTAACCAGCCTGGAGCCAATGGA GCCGGTAACCAACCTGGAGCTAATGGAGCTGGTAACCAACCAGGTGCTAAT GGTGCTGGTAACCAGCCTGGAGCAAACGGAGCCGGTAATCAGCCTGGAGCA AACGGTGCCGGTAACCAGCCTGGTGCCAATGGTGCCGGAAATCAGCCTGGT GCTAATGGAGCCGGAAACCAGCCTGGAGCCAACGGAGCTGGTAACCAGCCT GGTGCAAATGGAGCTGGTAATCAGCCAGGTGCAAATGGAGCCGGAAATCAA CCTGGTGCTAATGGAGCAGGTAACCAACCTGGTGCCAATGGAGCTGGAAAT CAACCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGAGCAAAC GCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACT GTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGA GTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCAGAAGACCTTACTTTG AACGACTTGGAAACCGATGTCTGCACT

30 PIC-PvCS- CCAAGAGAAAACAAGTTGAAACAGCCAGGAGCAAATGGAGCAGGTAACCAG VK247-His ₆ CCAGGAGCAAACGGAGCAGGTAACCAGCCAGGAGCAAACGGTGCTGGAAAT

CAACCAGGTGCCAACGGTGCAGGAAATCAGCCTGGAGCTAACGGTGCCGGA AATCAACCAGGTGCAAACGGTGCTGGAAATCAGCCTGGAGCAAATGGTGCA GGAAATCAACCAGGTGCTAATGGAGCCGGTAACCAGCCTGGAGCCAATGGA GCCGGTAACCAACCTGGAGCTAATGGAGCTGGTAACCAACCAGGTGCTAAT GGTGCTGGTAACCAGCCTGGAGCAAACGGAGCCGGTAATCAGCCTGGAGCA SEQ NAME SEQUENCE

ID

AACGGTGCCGGTAACCAGCCTGGTGCCAATGGTGCCGGAAATCAGCCTGGT GCTAATGGAGCCGGAAACCAGCCTGGAGCCAACGGAGCTGGTAACCAGCCT GGTGCAAATGGAGCTGGTAATCAGCCAGGTGCAAATGGAGCCGGAAATCAA CCTGGTGCTAATGGAGCAGGTAACCAACCTGGTGCCAATGGAGCTGGAAAT CAACCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGAGCAAAC GCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGATAAGGTCAGAGCTACT GTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGCGGTGTTGGTGTTAGA GTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCAGAAGACCTTACTTTG AACGACTTGGAAACCGATGTCTGCACTCACCACCACCACCACCACTAA

31 PIC-PvCS- PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAG VK247 NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQP GANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQGQNNEGAN APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL NDLETDVCT

32 PIC-PvCS- PRENKLKQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAG VK247-His ₆ NQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGAN

GAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQP GANGAGNQPGANGAGNQPGANGAGNQPGANGAGNQPGGNAGGQGQNNEGAN APNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTL NDLETDVCTHHHHHH

33 PIC-PvCS- CCAAGAGAAAATAAGTTGAAACAGCCAGGTGCCCCAGGAGCAAACCAAGAG Vivax-like GGAGGTGCCGCCGCCCCAGGAGCAAACCAGGAGGGAGGTGCTGCCGCACCA

GGAGCCAACCAAGAAGGTGGAGCTGCCGCACCTGGTGCAAATCAGGAGGGT GGAGCTGCCGCACCAGGAGCTAATCAGGAAGGAGGTGCTGCCGCACCTGGT GCCAACCAAGAGGGAGGTGCTGCCGCACCAGGTGCTAATCAGGAAGGAGGA GCTGCCGCACCAGGTGCCAACCAAGAGGGTGGTGCTGCCGCACCTGGAGCC AATCAGGAAGGTGGTGCTGCCGCACCAGGCGCAAACCAAGAGGGAGGAGCT GCCGCACCTGGAGCTAATCAGGAAGGCGGTGCTGCCGCACCCGGAGCAAAC CAAGAGGGCGGTGCTGCCGCACCTGGCGCTAATCAGGAAGGCGGCGCTGCC GCACCTGGTGCTAACCAAGAGGGCGGCGCTGCCGCACCAGGTGCAAATCAG GAAGGCGGGGCTGCCGCACCCGGCGCTAACCAAGAGGGTGGAGCTGCCGAT AGAGCAGCTGGTCAAGCCGCAGGTGGAAACGCTGGTGGACAGGGTCAGAAC AATGAAGGAGCAAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGAC AAGGTCAGAGCTACTGTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGC GGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCA GAAGACCTTACCTTGAATGACTTGGAGACAGATGTTTGTACC

34 PIC-PvCS- CCAAGAGAAAATAAGTTGAAACAGCCAGGTGCCCCAGGAGCAAACCAAGAG

Vivax-like- GGAGGTGCCGCCGCCCCAGGAGCAAACCAGGAGGGAGGTGCTGCCGCACCA

His ₆ GGAGCCAACCAAGAAGGTGGAGCTGCCGCACCTGGTGCAAATCAGGAGGGT

GGAGCTGCCGCACCAGGAGCTAATCAGGAAGGAGGTGCTGCCGCACCTGGT GCCAACCAAGAGGGAGGTGCTGCCGCACCAGGTGCTAATCAGGAAGGAGGA GCTGCCGCACCAGGTGCCAACCAAGAGGGTGGTGCTGCCGCACCTGGAGCC AATCAGGAAGGTGGTGCTGCCGCACCAGGCGCAAACCAAGAGGGAGGAGCT GCCGCACCTGGAGCTAATCAGGAAGGCGGTGCTGCCGCACCCGGAGCAAAC CAAGAGGGCGGTGCTGCCGCACCTGGCGCTAATCAGGAAGGCGGCGCTGCC GCACCTGGTGCTAACCAAGAGGGCGGCGCTGCCGCACCAGGTGCAAATCAG GAAGGCGGGGCTGCCGCACCCGGCGCTAACCAAGAGGGTGGAGCTGCCGAT AGAGCAGCTGGTCAAGCCGCAGGTGGAAACGCTGGTGGACAGGGTCAGAAC AATGAAGGAGCAAACGCTCCAAATGAAAAGTCTGTTAAAGAGTACTTGGAC AAGGTCAGAGCTACTGTTGGTACAGAGTGGACCCCTTGTTCCGTCACTTGC GGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCCAATAAGAAACCA GAAGACCTTACCTTGAATGACTTGGAGACAGATGTTTGTACCCACCACCAC CACCACCACTAA

35 PIC-PvCS- PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEG Vivax-like GAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGA

NQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAA APGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGNAGGQGQN NEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKP SEQ NAME SEQUENCE

ID

EDLTLNDLETDVCT

36 PIC-PvCS- PRENKLKQPGAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEG

Vivax-like- GAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGA

His ₆ NQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAA

APGANQEGGAAAPGANQEGGAAAPGANQEGGAADRAAGQAAGGNAGGQGQN NEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKP EDLTLNDLETDVCTHHHHHH

37 PIC-PvCS- CCAAGAGAAAACAAATTGAAGCAACCAGGACCAGGAGATAGAGCAGATGGA

All-CS CAACCAGCAGGAGATAGAGCCGACGGACAGCCAGCAGGAGATAGAGCTGCC epitopes GGTCAACCAGCTGGAGACAGAGCAGCTGGTCAGCCTGCTGGAGATAGAGCC

GACGGTCAGCCTGCCGGAGATAGAGCTGACGGTCAGCCAGCTGGAGATAGA GCTGACGCCCCTGGTGCTAACCAAGAAGGTGGAGCCGCAGCTCCAGGAGCT AATCAGGAGGGTGGAGCCGCAGCTCCTGGAGCCAATCAGGAAGGAGGTGCC GCAGCTGCCCCAGGTGCTAATCAAGAGGGTGGAGCAGCTGCCCCTGGAGCC AACCAAGAAGGAGGTGCAGCTGCCCCTGGAGCAAACCAGGAAGGTGGAGCA GCTGCCGCAAACGGTGCTGGAAATCAACCTGGTGCCAACGGTGCAGGAAAT CAGCCAGGAGCTAACGGTGCCGGAAATCAACCTGGTGCTAATGGTGCTGGA AATCAGCCAGGAGCAAATGGTGCAGGAAATCAACCTGGAGATAGAGCTGCC GGACAGGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGA GCAAACGCTCCTAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGA GCTACTGTTGGTACAGAGTGGACCCCATGTTCCGTCACTTGCGGTGTTGGT GTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCTGAAGATTTG ACTTTGAACGACTTGGAAACCGATGTCTGCACC

38 PIC-PvCS- CCAAGAGAAAACAAATTGAAGCAACCAGGACCAGGAGATAGAGCAGATGGA All-CS CAACCAGCAGGAGATAGAGCCGACGGACAGCCAGCAGGAGATAGAGCTGCC epitopes- GGTCAACCAGCTGGAGACAGAGCAGCTGGTCAGCCTGCTGGAGATAGAGCC His ₆ GACGGTCAGCCTGCCGGAGATAGAGCTGACGGTCAGCCAGCTGGAGATAGA

GCTGACGCCCCTGGTGCTAACCAAGAAGGTGGAGCCGCAGCTCCAGGAGCT AATCAGGAGGGTGGAGCCGCAGCTCCTGGAGCCAATCAGGAAGGAGGTGCC GCAGCTGCCCCAGGTGCTAATCAAGAGGGTGGAGCAGCTGCCCCTGGAGCC AACCAAGAAGGAGGTGCAGCTGCCCCTGGAGCAAACCAGGAAGGTGGAGCA GCTGCCGCAAACGGTGCTGGAAATCAACCTGGTGCCAACGGTGCAGGAAAT CAGCCAGGAGCTAACGGTGCCGGAAATCAACCTGGTGCTAATGGTGCTGGA AATCAGCCAGGAGCAAATGGTGCAGGAAATCAACCTGGAGATAGAGCTGCC GGACAGGCAGCTGGTGGAAACGCTGGTGGACAGGGTCAGAACAATGAAGGA GCAAACGCTCCTAATGAAAAGTCTGTTAAAGAGTACTTGGACAAGGTCAGA GCTACTGTTGGTACAGAGTGGACCCCATGTTCCGTCACTTGCGGTGTTGGT GTTAGAGTTAGAAGAAGAGTTAACGCCGCAAATAAGAAACCTGAAGATTTG ACTTTGAACGACTTGGAAACCGATGTCTGCACCCACCACCACCACCACCAT TAG

39 PIC-PvCS- PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRA

All-CS DGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGA epitopes AAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGN

QPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEG ANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDL TLNDLETDVCT

40 PIC-PvCS- PRENKLKQPGPGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRA All-CS DGQPAGDRADGQPAGDRADAPGANQEGGAAAPGANQEGGAAAPGANQEGGA epitopes- AAAPGANQEGGAAAPGANQEGGAAAPGANQEGGAAAANGAGNQPGANGAGN His ₆ QPGANGAGNQPGANGAGNQPGANGAGNQPGDRAAGQAAGGNAGGQGQNNEG

ANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDL TLNDLETDVCTHHHHHH * * *

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.