CA2106464A1 - Malaria recombinant poxvirus vaccine - Google Patents

Abstract

What is described is a recombinant poxvirus, such as vaccinia or canarypox virus, containing foreign DNA from Plasmodium. What is also described is a vaccine containing the recombinant poxvirus for inducing an immunological response in a host animal inoculated with the vaccine. Preferred recombinants have attenuated virulence.

Description

S . ~i l MALARIA RECOMBINANT POXVIRUS VACCINE
CRO88-REFERENCE TO RELATED APPLICATIONg This application is a continuation-in-part of -- application Serial No. 07/672,183, filed March 20, 1991, incorporated herein by reference. Reference is also made to application Serial No. 07/847,951, filed March 6, - 1992, entitled "Genetically Engineered Vaccine Strain"
(Attorney Docket No. 454310-2400), also incorporated ' herein by reference.
~ 10 FIELD OF T~E INVENTION ~- -s The present invention relates to a modified -~
; poxvirus and to methods of making and using the same.
, More in particular, the invention relates to recombinant . poxvirus, which virus expresses gene products of a Plasmodium gene, and to vaccines which provide protective immunity against Plasmodium infections.
Several publications are referenced in this application within parentheses. Full citation to these references is found at the end of the specification 20 immediately preceding the claims. These references ~ -relate to the field to which this invention pertains;
~ and, each of these references are hereby incorporated ¦ herein by reference.
BAC~GROUND OF TXE INVEN$ION
~J 25 Vaccinia virus and more recently other ¦ poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking ~ 30 a foreign genetic element in a donor plasmid and -~ hcmologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the ~ ~ vaccinia virus described in U.S. Patent Nos. 4,769,330, r' ~ ~ i 4,772,848 and 4,603,112, and in copending application . ~
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Serial No. 07/537,882, filed June 14, 1990, the disclosures of which are hereby incorporated herein by reference. In this regard reference is also made to copending U.S. application Serial No. 537,890, filed June 14, 1990, also incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA
of the poxvirus has been inserted. Separately, the DNA
gene sequence to be inserted is ligated to a promoter.
The promoter-gene linkage is positioned in the plasmid - construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell and Helinski, 1969; Sa~brook et al., 1989).
Second, the isolated plasmid containing the DNA
gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential ~; 25 region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA
from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the ~
; exogenous DNA is placed. -Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA.
Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufac_ure of new viral ~ ... .
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,~ - . ' W092/16616 ~ PCT/US92/02207 genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sec~ions of DNA in different genomes that are not perfectlv homologous. If one such section is from a first geno~e homologous with a section of another genome excep, for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and - the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA
genetic sequence by the modified infectious virus - requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
The technology of generating vaccinia virus recombinants has recently been extended to other members of the poxvirus family which have a more restricted host range. The avipoxvirus, fowlpox, has been engineered as a recombinant virus expressing the rabies G gene (Taylor et al., 1988a; Taylor et al., 1988b). This recombinant virus is also described in PCT Publication No.
W089/03429. On inoculation of the recombinant into a SUBSTITUTE SHEET

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~l a ~ 4-number of non-avian species an immune response to rabies is elicited which in mice, cats and dogs is protective against a lethal rabies challenge.
Malaria today still remains one of the world's major health problems. It is estimated that 200-300 million malaria cases occur annually while 1-2 million people, mostly children, die of malaria each year.
Malaria in humans is caused by one of four species of the genus Plasmodium - P. falciparum, P. vivax, P. malariae, and P. ovale. Clinically, P. falciparum is the most important human Plasmodium parasite because this species is responsible for most malaria fatalities.
Plasmodium infections begin when sporozoites are injected into the bloodstream by the bite of an - 15 infected female Anopheles mosquito. The liver stage of infection begins when the sporozoites disappear from the - blood stream and invade hepatocytes. Over a 5-7 day period, merozoites develop asexually within the infected - liver cells and are subsequently released into the blood stream where they invade erythrocytes, initiating the blood stage of infection. Parasites in infected '. erythrocytes develop asexually through ring, trophozoite, ~` and schizont stages. The rupture of schizonts releases merozoites which can then infect more red blood cells.
This self-perpetuating cycle of blood stage infection causes the clinical symptoms of malaria.
Some merozoites that infect red blood cells ~ differentiate into male and female gametes. These :~ gametes, which allow sexual reproduction, are subsequently ingested by Anopheles mosquitoes during a blood meal. The female gamete is then fertilized by the male gamete in the mosquito gut and the resultant zygotes :1 invade the gut wall where they undergo asexual division and eventually produce sporozoites which lodge in the mosquito salivary gland. The transmission cycle is completed when the infected mosquito takes other blood Ti T UTE Sn'~EJ

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meals and injects the sporozoites into the human blood stream.
Immunity to Pl asmodium does develop naturally alt~ough repeated lnfections over many years are required. This is probably a result of the antigenic diversity exhibited by some Plasmodium proteins among different parasite isolates. As a consequence, previously infected adults develop very low parasitemias after infection and rarely display clinical symptoms while children under the age of 5 are most susceptible to severe clinical disease. The developed immunity is not long lasting and will decline without reinfection.
Immunity to ~lasmodium is also species and stage specific, i.e. one may be immune to P. falciparum but not P. vivax and to sporozoites but not merozoites.
Malaria control measures have so far relied on drug treatment to control and prevent infections and pesticide use to control mosquito populations. The development of an effective malaria vaccine has become imperative due to the emergence and spread of drug resistant parasites in recent years. Most current efforts at developing a malaria vaccine are targeted to three stages in the parasite life cycle - the infection of liver cells by sporozoites, the perpetuation of the blood stage by merozoites, and the transmission to mosquitos by gametes. In mos~ cases, purified parasite proteins have been utilized as subunit vaccines with variable and generally disappointing results.
SERA, the serine repeat antigen, is a Plasmodium falciparum protein expressed during the blood and liver stages of infection (Szarfman et al., 1988).
In the blood stage, SERA is found in the parasitophorous vacuole and surrounding membranes of trophozoites and schizonts (Chulay et al., 1987; Coppel et al., 1988;
Delplace et al., 1987; Knapp et al., 1989). The SERA
precursor protein has a molecular weight of 126 kD [also described as 140 kD (Perrin et al., 1984), 113 kD (Chulay SlJBSTlTUTE SH~ET

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et al., 1987), and ~05 kD (Banyal and Inselburg, 1985)]
and is processed at the time of schizont rupture into 50, 47, and 18 kD fragments (Delplace et al., 1987; Delplace et al., 1988). The 47 and 18 kD fragments are associated by disulfide bonds to form a 73 kD complex.
Complete SERA genes have been obtained from genomic DNA of the FCR3 and FCBR strains and complete or partial cDNA clones obtained from 5 strains (Bzik et al., 1988; Coppel et al., 1988; Horii et al., 1988; Knapp et al., 1989; Li et al., 1989; Weber et al., 1987). The SERA gene is encoded in four exons separated by three intervening sequences (Knapp et al., 1989; Li et al., 1989). The coding sequence is characterized by two repeat structures; one a series of glycine-rich octamers 15 near the initiation codon and the second a polyserine repeat from which the protein derives its name. The ; predicted amino acid sequence does not contain a hydrophobic transmembrane region. SERA mRNA is 3.6-4.1 Kb long and appears to be quite abundant in late f 20 trophozoites and schizonts (Bzik et al., 1988; Knapp et al., 1989).
Although the data are limited, it appears that SERA is well conserved among strains of P. falciparum.
Comparison or the various genomic and cDNA clones 25 indicates that the majority of the SERA coding sequence ' is invariant in the strains studied. Most nucleotide differences among these strains occur within or around the polyserine repeat and also within the octapeptide repeats (Bzik et al., 1988; Horii et al., 1988; Knapp et al., 1989; Li et al., 1989). The genomic organization of SERA is conserved in 12 strains as studied by Southern analysis (Coppel et al., 1988; Horii et al., 1988; Knapp et al., 1989). Immunoprecipi.ation analysis of ten geographically diverse P. falciparum isolates indicated that the sizes of SERA and its processed fragments are well conserved. Some variation was observed with the 47 kD fragment, which varied in size from 47-50 kD (Bhatia SU13STITUTE SHEE'F

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W092/l66~6 ~ PCT/US92/02207 et al., 1987). This fragment contains the polyserine repeats. Thus, the size variation in the 47 kD fragment is probably due to differences in the polyserine repeats, perhaps different numbers of serine residues.
Interestingly, two SERA alleles have been described in the FCR3 strain - allele I and allele II -whose differences primarily occur within both repeat regions (Li et al., 1989). Southern analysis indicates - that the Honduras I strain contains a SERA qene corresponding only to FCR3 allele I (Li et al., 1989) whereas the nucl~otide sequence of the SERA gene from the FCBR strain is identical to FCR3 allele II (Knapp et al., 1989; Li et 21., 1989).
The functional role of SERA during the parasite life cycle is not known. Recently, homology searches of protein databases have revealed that SERA has significant similarity at and around two active sites found in -~ cysteine proteinases and may therefore be a cysteine proteinase (Higgins et al., 1989). However, it has since been pointed out that although SERA has a cysteine proteinase conformation, it may actually be a serine ~ proteinase due to the presence of a serine at the i putative catalytic site (Eakin et al., 1989; Mottram et al., 1989). Although this has yet to be confirmed experimentally, it may indicate an important role for SERA in the parasite life cycle because it is known that proteases are necessary for the cleavage of some proteins during the blood stage and also that protease inhibitors interrupt the development of the parasite (Debrabant and Delplace, 1989).
ABRA, the acidic basic repeat antigen, is also expressed during both the blood and liver stages of P.
falciparum infection (Szarfman et al., 1988). In infected erythrocytes, ABRA is expressed during the late trophozoite and schizont stages and is found in the parasitophorous vacuole (Chulay et al., 1987; Stahl et al., 1986). ABRA has a molecular weight of 100-102 kD

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and is released from rupturing schizonts (Chulay et al., 1987; Stahl et al., 1986; Weber et al., 1988).
A complete genomic ABRA gene from the CAMP
straln and partial ABRA cDNAs from the FCR3 and FC27 strains have been obtained (Stahl et al., 1986; Weber et al., 1988). The ABRA coding sequence does not contain introns and is characterized by two repeat structures.
The first consists of eight hexapeptide repeats near the center of the coding sequence and the second consists of lo a series of tandem dipeptide and tripeptide repeats, mostly of the amino acid sequences KE and KEE (Stahl et al., 1986; Weber et al., 1988).
Based on limited data, ABRA appears to be well conserved a~ong P. falciparum strains. The partial cDNA
clones from the FCR3 and FC27 strains are almost identical to the CAMP strain genomic ABRA gene. The FCR3 clone differs at four positions and the FC27 clone contains some rearrangements within the carboxy-terminal repeat region as compared to the CAMP ABRA gene (Stahl et al., 1986; Weber et al., 1988). The general genomic organization of ABRA as detected by Southern analysis is conserved in six P. falciparum isolates (Stahl et al., 1986). Additionally, immunoprecipitation analysis 'indicates that the size of ABRA from seven geographically !25 diverse isolates is conserved (Chulay et al., 1987; Stahl et al., 1986).
Pfhsp70 is a Plasmodium falciparum protein that shares significant similarity with members of the mammalian 70 kD heat shock protein family (Ardeshir et al., 1987; Bianco et al., 1986; Newport et al., 1988).
Pfhsp70 is expressed during the liver (Renia et al., 1990) and throughout the blood stages of infection (Ardeshir et al., 1987; Bianco e. al., 1986), but not by sporozoites (Bianco et al., 1986; Renia et al., 1990).
Experiments with P. falciparu~-infected human hepatocyte cultures suggest that Pfhsp70 is expressed on the hepatocyte surface during the liver stage (Renia et al., S~6~TITUTE S~IEET

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_g_ 1990). The localization of Pfhsp70 during the blood ~
stage remains controversial, with exclusively cytoplasmic and merozoite surface locations both reported (Ardeshir et al., 1987; Bianco et al., 1986). Pfhsp70 has a molecular weight of 75 kD (Ardeshir et al., 1987; Bianco et al., 1986; Kumar et al., 1988a), although a molecular weight of 72 kD has also been reported (Dubois et al., 1984; Jendoubi and Pereira da Silva, 1987).
A complete genomic Pfhsp70 gene from the FCR3 strain and partial Pfhsp70 cDNAs from the FC27, Honduras 1, and 7G8 strains have been obtained (Ardeshir et al., 1987; Bianco et al., 1986; Kumar et al., 1988a; Yang et al., 1987). The partial cDNAs encode approximately 40%
of the carboxy-~erminai coding sequence and each initiates at the same nucleotide relative to the complete gene (Ardeshir et al., 1987; Bianco et al., 1986; Kumar et al., 1988a). The carboxy-terminal portion of the coding sequence is characterized by a series of 7-8 tandem repeats, mostly of sequence GGMP (Ardeshir et al., 1987; Bianco et al., 1986; Kumar et al., 1988a; Yang et al., 1987). Pfhsp70 mRNA is 2.8 Kb in size (Kumar et al., 1988a).
Based on limited data, Pfhsp70 appears to be '- well conserved among P. falciparum strains and isolates.
The partial cDNAs from the FC27 and Honduras 1 strains are identical in the coding region and differ from the ;
7G8 partial cDNA at only a few nucleotides. The FCR3 genomic gene is very similar to the cDNAs in its carboxy-terminus, with the only differences being the presence of an additional GGMP repeat and a few nucleotide substitutions. The general genomic organization of the carboxy-terminal region of Pfhsp70 as detected by Southern analysis is conserved in 14 P. falciparum strains (Ardeshir et al., 1987; Kumar et al., 1990).
Also, immunoprecipitation analysis indicates that the size of Pfhsp70 from 20 geographically diverse isolates is conserved (Ardeshir et al., 1987; Jendoubi and Pereira - SH'E~ ~

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WO92t16616 PCT/US92/02207 da Silva, 1987). Some variation of tryptic peptide maps among three strains has been detected, however (Jendoubi and Pereira da Silva, 1987).
The function of Pfhsp70 in the parasite life cycle is not known. However, the induction of Pfhsp70 expression at the two-nuclei stage after sporozoite infection of liver cells has led to the suggestion that this heat shock-like protein may play a role in parasite differentiation (Renia et al., 1990).
AMAl is a late-stage schizont protein originallv isolated from Plasmodium knowlesi infected erythrocytes as a 66 kD protein (PK66). PK66 is processe~ to 44/42 kD components at the time of merozoite release and these maturation products are associated with the merozoite surface. When isolated in native form, PK66 induced inhibitory antibodies and protected rhesus monkeys against a blood-stage challenge (Deans et al., 1988). The Plasmodium falciparum equivalent of PK66 has been isolated by using human antimalarial antibodies (Peterson et al., 1988) or rabbit anti-PK66 polyclonal serum (Thomas et al., 1990), and has also been called PF83.
In Plasmodium knowlesi, AMA1 is synthesized late in schizogony and is distributed at the apex of the merozoites developing within the segmenting schizont. At schizont rupture, AMA1 is processed to a 44/42 kD doublet (Waters et al., 1990). During the invasion of erythrocytes, the 44/42 kD doublet is not carried into the erythrocytes, but remains associated with the invasion interface~
In Plasmodium falciparum, AMAl is located at the apex of the segmented schizont, although a merozoite surface localization cannot be excluded (Peterson et al., 1988). AMA1 is probably first located in the apical complex and then exported to the merozoite surface.
During erythrocyte invasion, AMAl is lost: it cannot be found in the newly infected erythrocyte.

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AMAl is highly conserved among different isolates of Plasmodium falciparum: Camp, FCR3, 7G8 Thai TN, FC27 (Thomas et al., 1990). The AMA1 gene is 1866 bp long, no introns have been reported, and it codes for a 622 amino acid protein without repetitive sequences.
This protein has a structure expected for an integral membrane protein: it contains two hydrophobic stretches, one near the N-terminus which may act a signal peptide, and a second located 55 amino acids from the C-terminus.
Pfs25 is a P. falci~arum protein expressed during the sexual stages of parasite development. This 25 kD membrane protein is localized on the surface of zygotes and ookinetes (Vermeulen et al., 1985) and as a consequence is probably only expressed in the mosquito midgut and not in the human host (Carter et al., 1988;
Xaslow et al., 1989).
The Pfs25 gene from the 3D7 clone of P.
: falciparum strain NF54 consists of an uninterrupted open ; reading frame of 654 bp encoding a protein with a predicted molecular weight of 24.1 kD (Kaslow et al., 1988). The predicted amino acid sequence includes a hydrophobic signal peptide at the N-terminus and a short hydrophobic anchor sequence at the C-terminus, consistent with the surface localization of Pfs25. In addition to four potential N-glycosylation sites, the Pfs25 coding sequence contains an organization of predicted cysteine residues that suggests the presence of four tandemly repeated EGF-like domains (Kaslow et al., 1988). Pfs25 is very highly conserved, with only one single-base substitution detected among 8 geographically diverse isolates (Kaslow et al., 1989).
Antibodies to Pfs25 have not been detected in humans from endemic areas, probably because this protein is not expressed in the human host (Carter et al., 1988).
Immunizations of H-2 congenic mouse strains generated anti-Pfs25 antibodies in all strains tested, indicating ., . .~ . . .
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~ J~ x -12-that this protein is a good immunogen (Good et al., 1988).
Pfs25 is considered a potential vaccine candidate based on the ability of anti-Pfs25 mAbs to bloc~ transmission of the parasite from the vertebrate host to mosquitoes (Kaslow et al., 1989). Immunization of mice with a vaccinia recombinant producing surface-expressed Pfs25 also generates transmission blocking antibodies after three inoculations and the generation of such antibodies by vaccinia recombinants is not restricted to particular MHC haplotypes (Kaslow et al., 1991) .
Pfsl6 is a P. falciDarum protein expressed by the sporozoite as well as the sexual stages of the parasite developmental cycle. This 16 kD protein is found on the membrane of intracellular gametocytes and possibly the parasitophorous vacuole membrane, on the outer membrane of extracellular macrogametes, and on the surface of sporozoites (~oelans et al., l99la). The Pfs16 gene is 544 bp in length and the coding sequence is characterized by a putative N-terminal signal sequence, a hydrophobic anchor sequence, and a highly hydrophilic C-terminus.
Pfsl6 is highly conserved among P. falciparum isolates. Of eight strains studied, variation was only found in two isolates which contained two and three amino i acid substitutions, respectively (Moelans et al., l99lb).
Pfsl6 is considered as a vaccine candidate for ' several reasons. First, the expression of Pfsl6 by both sporozoites and sexual stages make this protein attractive for inclusion in a multi-stage vaccine because immunity to it may protect against infection by i sporozoites and transmission by sexual stages. Of note is that in preliminary studies with four Pfsl6-specific mAbs, no in vitro inhibition of sporozoite invasion was detected (Targett, l990). Second, sera from adults living in highly endemic regions has been shown to S~8STI~UTE SHEET

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.L ~ 3 recognize the Pfsl6 protein, indicating that it is immunogenic in humans (Moelans et al., l991a). Third, polyvalent rabbit sera raised against gametes and gametocytes recognizes Pfsl6 and has high transmission bloc~ing activity. Preliminary studies with two ~fsl6-specific mAbs indicate that one of the antibodies has transmission blocking activity (Moelans et al., l991a).
The P. fal ciparum circumsporozoite (CS) protein is a 60 kD me~brane protein that is uniformly distributed over the sporozoite surface (Nussenzweig et al., 1984).
CS is not expressed at anv other stage of the parasite life cycle.
The CS gene consists of an uninterrupted open reading frame of approximately 1200 bp. CS is characterized by a central region consisting of the repeated sequence NANP with a few variant NVDP repeats, flanked by nonrepetitive regions that contain charged residues (Dame et al., 1984). The repetitive NANP
sequences are conserved, although the number of repeats can vary among different isolates. Variation in non-repetitive regions is seen near the amino-terminus due to insertions or deletions, while the carboxy-terminal domain contains only base pair substitutions (Caspers et al., 1989). Of the 412 amino acids of CS, only thirteen positions segregated in three distinct polymorphic regions are known to be variant (Caspers et al., 1989).
Three regions found in the non-repetitive domains are relatively well conserved among species of Pl asmodia, region I in the N-terminal domain and regions II and III
in the C-terminal domain (Lockyer and Holder, 1989).
Both humoral and cell-mediated immune responses to CS appear to play a role in the induction of anti- -sporozoite immunity. In terms o' humoral responses, it has been shown that naturally protected humans contain antibodies to the CS protein and these antibodies increase with age and parallel acquired immunity (Nussenzweig and Nussenzweig, 1989). However, CS and ~ S~

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sporozoite-specific antibody levels in naturally infected adults do not correlate with protection from further infection (Hoffman et al., 1987), suggesting that other factors such as cell mediated immunity may be important in natural i~munity. ~Iowever, several studies have shown that humans can be protected by immunization with irradiated sporozoites (Clyde, 1975; Rieckmann, 1974) and that protection was correlated with antibodies against the CS protein (Nussenzweig et al., 1985). Human vaccine trials with CS-based peptide subunits have demonstrated the ability of such constructs to induce CS-specific antibody responses and to completely protect some vaccinees (Herrington et al., 1987; Ballou et al., 1987).
Cell mediated responses to the CS protein have also been studied. Several T cell epitopes have been identified in the P. falciparum CS protein in man (Good et al., 1987). Interestingly, most human T cell epitopes . occur in polymorphic regions of CS suggesting that parasite mutations and selection have occurred in response to immune pressure from T cells. However, one human T helper epitope, CS.T3, is located in a conserved region of the CS protein and is recognized by human T
cells in association with many different human MHC class II molecules (Sinigagla et al., 1988). Also, sporozoites are able to induce cytotoxic T cells specific for a CD8t ; CTL epitope on the CS protein (Kumar et al., 1988b), suggesting that such cells may be important for the i induction of immunity to P. falciparum.
It can be appreciated that provision of a malaria recombinant poxvirus, and of vaccines which provide protective immunity against Plasmodium infections, or which stimulate an immunological response in a host to Plasmodium immunogens would be a highly desirable advance over the current state of technology.
35 Likewise, such malaria recombinan. poxvirus is also highly desirable for the production of Plasmodium immunogens in vi tro.

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W092/16616 , ~ PCT/US92/0220 OBJECTS OF THE INYENTION
It is therefore an object of this invention to provlde recomblnant poxvlruses, whlch vlruses express gene products of Plasmodium, and to provlde a method of maklng such recomblnant poxvlruses.
It ls an addltional object of thls invention to provlde for the clonlng and expression of Plasmodium coding sequences, particularly SERA, ABRA, Pfhsp70 and AMAl Plasmodium blood stage antigens as well as Pfs25, Pfsl6 and CS Plasmodium antigens, in a poxvirus vector, particularly vaccinia and an avlpox vlrus such as fowlpox or canarypox virus.
It is another object of this inventlon to provide a vaccine which is capable of eliciting malaria antibodies and protective immunity against Plasmodium infection. It is a further object of the invention to provide malaria recombinant poxvirus useful for the production of Plasmodium immunogens, in vivo or in vitro;
and, the recombinant immunogens.
These and other objects and advantages of the present invention will become more readily apparent after consideration of the following.
STATEMENT OF T~E INVENTION
In one aspect, the present invention relates to a recombinant poxvirus containing therein a DNA sequence from Plasmodium in a nonessential region of the poxvirus genome. The poxvirus is advantageously a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus.
According to the present invention, the recombinant poxvirus expresses gene products of the foreign Plasmodium gene. In particular, the foreign DNA
codes for a SERA, ABRA, Pfhsp70, AMAl, Pfs25, Pfsl6 or CS
gene. Advantageously, a plurality of Plasmodium genes are co-expressed in the host by the recombinant poxvirus.
The invention is also directed to the methods of using the malaria recombinant poxvirus for the production of SUBSTITUTE SHEET

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;~ J i~ L -16-Plasmodium gene products, either in vivo or in vitro as well as to the recombinant gene products.
In another aspect, the present invention relates to a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine including a carrier and a recombinant poxvirus containing, in a nonessential region thereof, DNA from Plas,~odiu~, as well as to methods for inducing such an immunological response in an animal by inoculating the animal with a malaria recombinant poxvirus. .hdvant2aeously, the DNA codes for and expresses a SE~, ABRA, Pfhsp70, AMAl, Pfs25, Pfsl6 or CS
?lasmodiu,., ger.e. ~ plurality of Plasmodium genes advanLaseously are co-expressed in the host. The poxvirus used in the vaccine and method according to the present invention is advantageously a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus.
BRIEF DESCRIPTION OF THE DRAWINGS
A better understanding of the present invention will be had by referring to the accompanying drawings, in which:
FIG. l schematically shows the SERA coding sequence;
FIG. 2 shows the nucleotide (SEQ ID NO:l) and predicted amino acld (SEQ ID NO:2) sequence of the SERA
cDNA in pl26.15;
FIG. 3 shows the nucleotide (SEQ ID NO:3) and predicted amino acid (SEQ ID NO:4) sequence of the ABRA
cDNA in pABRA-8;
FIG. ~ shows the nucleotide (SEQ ID NO:5) and predicted amino acid (SEQ ID NO:6) sequence of the Pfhsp70 partial cDNA in pHSP70.2; and FIG. 5 shows the nucleotide (SEQ ID NO:7) and predicted amino acid (SEQ ID NO:~) sequence of the 3D7 strain AMAl gene.

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WO92/16616 ,~ A 1~ PCT/US92/02207 DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to recombinant poxviruses containing therein a DNA sequence from Plasmodium in a nonessential region of the poxvirus genome. The recombinant poxviruses express gene products - of the foreign Plasmodlum gene. For example, P.
falciparum genes were expressed in live recombinant poxviruses. This expression makes these recombinants useful for vaccines, for stimulating an immunological response response to the gene products, or for the in vitrG production of the gene products, e.g., ror subsequent use of the products as immunogens. The S~RA, ABRA, Pfhsp70, and AMA1 P. falciparum blood stage genes were isolated, characterized and inserled into poxvirus, e.g., vaccinia, canarypox, virus recombinants, as well as the Pfs25, Pfsl6, and CS P. falciparum genes. The invention is illustrated by the non-limiting examples : (below), which are not to be considered a limitation of this invention as many apparent variations of which are - 20 possible without departing from the spirit or scope thereof. In the examples herein, the following ~ethods and materials are employed.
Enzvmes. Bacteria and Plasmids. Restriction enzymes and other DNA modifying enzymes were obtained -from Boehringer Mannheim (Indianapolis, IN), New England Biolabs ~Beverly, MA), and BRL Life Technologies Inc.
(Gaithersburg, MD) and used according to manufacturers recommendations, unless otherwise noted. Standard molecular cloning procedures were followed (Sambrook et al., 1989).
; The E. coli strains XL-l Blue and SURE were obtained from Stratagene (La Jolla, CA) and strain ~522 . from IBI (New Haven, CT). Plasmid vector pUC19 was i obtained from New England Biolabs (Beverly, MA).
~ 35 Cell Lines and Virus Strains. Vaccinia ', recombinants containing Plasmodium blood stage genes were i generated with the Copenhagen vaccinia strain, or NYVAC

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W0~2/16616 PCT/US92/02207 i3l~

(vP866) (Tar~aglia et al., 1992) vaccinia strain (having attenuated virulence), or the vP668 vaccinia recombinant as rescuing virus. All vaccinia virus stocks were produced in either Vero (ATCC CCL81) or MRC5 (ATCC CCL71) cells in Eagles ~E.~ medium supplemented with 5-10%
newborn calf serum (Flow Laboratories, McLean, VA).
Canarypox recombinants containing P. falciparum genes were generated with the ALVAC strain (having attenuated virulence) as rescuing virus (Tartaglia et al., 1992).
Polvmerase Chain Reaction (PCR). The GeneAmp DNA ampliric~tion kit (Perkin Elmer Cetus, Norwalk, CT.) was used for ?CR (Saiki et al., 1988) according to the manufacturers specifications with custor, synthesized oligonucleotides as primers. Reactions were processed in a Thermal Cycler (Perkin Elmer Cetus) with standard conditions (Saiki et al., 1988).
Construction of P. Falciparum FCR3 Strain Blood Staae cDNA Li~rarv. Total RNA from human erythrocytes infected with P. falciparum FCR3 strain was obtained from Dr. P. Delplace (INSERM-U42, 369 rue Jules-Guesde, 59650 Villeneuve-D'Asc~, France). Poly-A+ RNA was isolated from this sample by use of oligo(dT) cellulose (Stratagene, La Jolla, CA) as described by Aviv and Leder (Aviv and Leder, 1972) and modified by Kingston (Kingston, 1987). Briefly, total RNA was mixed with oligo(dT) cellulose in Binding buffer (0.5M NaCl, O.OlM
Tris-Cl, pH 7.5) and incubated for 30 minutes at room temperature. Poly-A+ RNA/oligo(dT) cellulose complexes were pelleted by centrifugation and washed 3 times with Binding buffer. Purified poly-A+ RNA was eluted from the oligo(dT) cellulose in Elution buffer (O.OlM Tris-Cl, pH
7.5). A second elution with DEPC-treated dH20 was performed, the eluates were pooled, and the poly-A~ RNA
recovered by ethanol precipitation.
The purified poly-A+ RN.~ was used as a template for the synthesis of first stranc cDNA by reverse transcriptase in a reaction primed with oligo(dT) i T V ~ ~ r~ ' '-'--'~-; ' ., ' - ' ''.', ' ,.. , : -, . . .. ':' . .. .. :... . :: . . ... ,. -:. , ,, : . -: . , : ,: . .. . .

WO 92/16~i16 ~` 1 r ~ ~ r 1 PCI/US92/0220~

(Klickstein and Neve, 1987; Watson and Jackson, 1985).
For this reaction, 12ug poly-A+ RNA was incubated with 105 units AMV reverse transcriptase (Life Sciences) in lOOmM Tris-Cl pH 8.3, 30mM KCl, 6mM MgCl2, 25mM DTT, 80 units RNasin, lmM each dNTP, and 24ug/ml oligo(dT)12-18 as primer for 2 hours at 42C. After organic extractions, double stranded cDNA was obtained by use of DNA
polymerase I and RNase H with first strand cDNA as template (Klickstein and Neve, 1987; Watson and Jackson, 1985). The first strand cDNA was incubated with 25 units DNA polymerase I and 1 unit RNase H in 20m~ Tris-Cl pH 6, 5mM MgCl2, lOmM (NH4)2S04, lOOmM KCl, 500ug/ml BSA, 25~
DTT, and O.lmM each dNTP at 12C for one hour followed by one hour at room temperature to synthesize second strand cDNA. The double stranded cDNA was recovered by organic extractions and ethanol precipitation.
The double-stranded blood stage cDNA was then sequentially treated with T4 DNA polymerase to create blunt ends and EcoRI methylase to protect internal EcoRI
sites. EcoRI linkers were then added followed by digestion with EcoRI and size selection on a 5-25%
sucrose gradient. Fractions containing long cDNAs (1-10 Kb) were pooled and ligated into EcoRI cleaved Lambda ZAPII vector (Stratagene, La Jolla, CA). The resulting phage were packaged and used to infect the XL-l Blue E.
coli strain (Stratagene, La Jolla, CA). The phage were then harvested from these cells and amplified by one additional cycle of infection of XL-1 Blue to produce a high titer FCR3 strain blood stage cDNA library.
Screen of cDNA Library for Plasmodium Blood Staqe cDNA Clones. The FCR3 strain cDNA library was screened by plaque hybridization with 32p end-labelled oligonucleotides derived from published sequences of blood stage genes to detect cDNA. The cDNA library was plaqued on lawns of XL-1 Blue (S.ratagene, La Jolla, CA) in 150mm dishes at a density of 100,000 plaques per dish.
Plaques were transferred to nit-ocellulose filters which - Sl~B~

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~ ~ 3 ~ 20-were then soaked in 1.5M NaCl/0.5M NaOH for 2 minutes, 1.5M NaCl/0.5M Tris-Cl pH 8 for 5 minutes, 0.2M Tris-Cl pH 7.5/2X SSC for one minute, and baked for 2 hours in an 80C vacuum oven. Filters were prehybridized in 6X SSC, 5X Denhardts, 20mM NaH2PO4, 500ug/ml salmon sperm DNA for two hours at 42C. Hybridizations were performed in 0.4%
SDS, 6X SSC, 20mM NaH2PO4, 500ug/ml salmon sperm DNA for 18 hours at 42C after the addition of 32P-labelled oligonucleotides. After hybridi~ation, filters were rinsed 3 ti~es with 6X SSC, 0.1% SDS, washed for 10 minutes at room temperature, and washed 'o- 5 minutes at 58C. Filters were then exposed to X-ray film at -70C.
Plaques hybridlzing with oligonucleotide probes were cored from plates and resuspended in SM buffer (lOOmM NaCl, 8mM MgSO4, 50mM Tris-Cl pH 7.5, 0.01%
gelatin) containing 4% chloroform. Dilutions of such phage stocks were used to infect XL-1 Blue, plaques were transferred to nitrocellulose, and the filters were hybridized with 32P-labelled oligonucleotides. Well 20 isolated positive plaques were selected and subjected to two additional rounds of purification as just described.
Isolation of Plasmodium cDNA-containina Plasmids From Positive Phaae Clones. Plasmodium cDNAs in the pBluescript plasmid vector were obtained by an in r 25 vivo excision protocol developed for use with the lambda ZAPII vector (Stratagene, La Jolla, CA). Briefly, : purified recombinant lambda phage stocks were incubated with XL-1 Blue cells and R408 filamentous helper phage for 15 minutes at 37C. After the addition of 2X YT
30 media (1% NaCl, 1% yeast extract, 1.6% Bacto-tryptone), incubation was continued for 3 hours at 37C followed by 20 minutes at 70C. After centrifugation, filamentous phage particles containing pBluescript phagemid (with cDNA insert) were recovered in the supernatant.
35 Dilutions of the recovered filamentous phage stock were mixed with XL-1 Blue and plated to obtain colonies . :
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WO92/16616 ~-`J~, ; j,1 PCT/US92/02207 containing pBluescript plasmids with Plasmodium cDNA
inserts.
DNA Sequence Analysis of Plasmodium Genes.
Plasmodium genes were obtained in pBluescript or cloned into other plasmid vectors. DNA sequencing was per.ormed with the Sequenase modified T7 polymerase (U.S.
Biochemicals, Cleveland, OH). Sequencing reactions were performed on alkali denatured double stranded plasmid templates (Hattori and Sakaki, 1986) with the T3 anà T7 primers or custom synthesized oligodeoxyribonucleotldes.
Sequence data were analyzed with the IBI Pustell Sequence Analysis Package, Version 2.02 (International Biotechnologies, New Haven, CT).
Generation of Vaccinia Recombinants Containinc P. Falciparum Genes. P. falciparum genes were cloned such that they are placed under the control of poxvirus promoters for expression by vaccinia vectors. The - promoters we have utilized are the vaccinia early/late H6 promotor (Perkus et al., 1989), the Pi or ClOLW early promotor from vaccinia WR (Wachsman et al., 1989), the vaccinia I3L early intermediate promotor (Perkus et al., 1985; Schmitt and Stunnenburg 1988), and the entomopoxvirus 42K early promotor (Gettig et al., unpublished).
P. falciparum genes must then be cloned into vaccinia donor plasmids in preparation for insertion into vaccinia virus. The pCOPCS-5H and pCOPCS-6H donor plasmids have been previously described (Perkus et al., 1991) .
Plasmid pSD553 is a vaccinia deletion/insertion plasmid of the COPAK series. It contains the vaccinia KlL host range gene (Gillard et a_ , 1986) within flanking Copenhagen vaccinia arms, replacing the ATI
region (orfs A25L, A26L; Goebel et al., 1990). pSD553 was constructed as follows. Left and right vaccinia flanking arms were constructed by polymerase chain reaction using pSD414, a pUC8-based clone of vaccinia ~ ,~, . .... . . . . . .

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~ L ~ 22-SalI B tGoebel et al., l990) as template. The left arm was synthesized using synthetic deoxyoligonucleotides MPS~N267 (SEQ ID NO:9) (5'~
GGGCTG~AGCTTGCTGGCCGCTCATTAGACAAGCGAATGAGGGAC-3') and MPSYN268 (SEQ ID NO:10) (5'~
AGATCTCCCGGGCTCGAGTAATTAATTAATTTTTATTACACCAGAAAAGACGGCTTG
AGATC-3') as primers. The right arm was synthesized using synthetic deoxyoligonucleotides MPSYN269 (SEQ ID
NO:ll) (5'-TAATTACTCGAGCCCGGGAGATCTAATTT~ATTTAATTTATATAACTCATTTTTTGAATATAC T-3'~ and MPS''N270 (SEQ ID N0:12) (5'~
TATCTCGAATTCCCGCGGCTTTA.~TGGACGGAACTCTTTTCCCC-3') as primers. The two PCR-derived DNA fragm~n~s containing . the left and right arms were combined in a further PCR
reaction. The resulting product was cut with EcoRI/HindIII and a 0.9kb fragment isolated. The 0.9kb fragment was ligated with pUC8 cut with EcoRI/HindIII, resulting in plasmid pSD541. The polylinker region located at the vaccinia deletion locus was expanded as follows. pSD541 was cut with BqlIItXhoI and ligated with annealed complementary synthetic deoxyoligonucleotides MPSYN333 (SEQ ID NO:13) (5'~
GATCTTTTGTTAACAAAAACTAATCAGCTATCGCGAATCGATTCCCGGGGGATCCGG
TACCC-3') and MPSYN334 (SEQ ID N0:14) (5'~
TCGAGGGTACCGGATCCCCCGGGAATCGATTCGCGATAGCTGATTAGTTTTTGTTAA
CAAAA-3') generating plasmid pSD552. The KlL host range gene was isolated as a lkb aglII(partial)/H~aI fragment , from plasmid pSD552 (Perkus et al., l990). pSD552 was cut with BqlII/HpaI and ligated with the KlL containing fragment, generating pSD553.
Plasmid pMPI3H contains the vaccinia I3L -early/intermediate promoter element (Schmitt and Stunnenberg, 1988) in a pUC8 background. The promoter ` element was synthesized by polymerase chain reaction (PCR) using pMPVCl, a subclone or vaccinia HindIII I, as template and synthetic oligonucleotides MPSYN283 (SEQ ID
N0:15) (5'~CCCCCCAAGCTTACATCr.TGCAGTGGTTAAAC~3') and SUBSTlTUTE SHEET
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DNA from this reaction was cut with HindIII and RsaI and a O.1 kb fragment containing the promoter element was purified. A linker region was assembled by annealing complementary synthetic oligonucleotides MPSYN398 (SEQ ID
NO:17) (5'-ACAATTATTTAGGTTAACTGCA-3') and MPSYN399 (SEQ
ID NO:18) (5'-GTTAACCTAAATAATTGT-3'). The PCR-derived promoter element and the polylinker region were ligated with vector plasmid pUC8 which had been cut with HindIII
and PstI. The resulting plasmid, pMPI3H, contains the I3L
promoter region from positions -100 through -6 relative to the initiation codon, followed by a polylinker region :
containing HpaI, PstI, SalI, BamHI, SmaI and EcoRI sites.
Cleavage with HpaI produces blunt ended DNA linearized at 15 position -6 in the promoter.
The pSD544 insertion vector was derived as follows. pSD456 is a subclone of Copenhagen vaccinia DNA
containing the HA gene (A56R; Goebel et al., 1990) and surrounding regions. pSD456 was used as template in 20 polymerase chain reactions for synthesis of left and right vaccinia arms flanking the A56R ORF. The left arm ,~ was synthesized using synthetic oligodeoxynucleotides MPSYN279 (SEQ ID NO:19) (5'-CCCCCCGAATTCGTCGACGATTGTTCATGATGGCAAGAT-3') and MPSYN280 r 25 (SEQ ID NO:20) (5'-CCCGGGGGATCCCTCGAGGGTACCAAGCTTAATTAATTAAATATTAGTATAAAAAGT
GATTTATTTTT-3') as primers. The right arm was synthesized using MPSYN281 (SEQ ID NO:21) (5'-AAGCTTGGTACCCTCGAGGGATCCCCCGGGTAGCTAGCTAATTTTTCTTTTACGTAT
TATATATGTAATAAACGTTC-3') and MPSYN312 (SEQ ID NO:22) (5'-TTTTTTCTGCAGGTAAGTATTTTTAAAACTTCTAACACC-3') as primers.
Gel-purified PCR fragments for the left and right arms were combined in a further PCR reaction. The resulting product was cut with EcoRI/HindIII. The resulting 0.9 kb 1 35 fragment was gel-purified and ligated into pUC8 cut with EcoRI/HindIII, resulting in plas~id pSD544.

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Plasmid pSD550 was derived as follows. Plasmid pSD548 (Tartaglia et al., 1992) is a vaccinia vector plasmid in which the I4L ORF (Goebel et al., l99O) is replaced by a cloning region consisting of BglII and SmaI
sites. To expand the multicloning region, pSD54~ was cut with BqlII and SmaI and ligated with annealed complementary synthetic oligonucleotides 539A (SEQ ID
NO:23) (5'-AGAAAAATCAGTTAGCTAAGATCTCCCGGGCTCGAGGGTACCGGATCCTGATTAGTT
AATTTTTGT-3') and 539B (SEQ ID NO:24) (5'-GATCACAAAAATTAACTAATCAGGATCCGC-TACCCTCGAGCCCGGGAGATCTTAGCT
AACTGATTTTTCT-3'). In the resulting plasmid, pSD5~0, tne multicloning region contains BqlII, Smal, X:noI, K~nI and BamHI restriction sites.
Plasmid pSD542 was derived as follows. To modify the polylinker region, plasmid pSD513 (Tartaglia et al., 1992) was cut with PstI/BamHI and ligated with annealed synthetic oligonucleotides MPSYN288 (SEQ ID
NO:25) (5'-GGTCGACGGATCCT-3') and MPSYN289 (SEQ ID NO:26) (5'-GATCAGGATCCGTCGACCTGCA-3') resulting in plasmid pSD542.
The pNVQH6C5SP18 ALVAC C5 insertion vector, which contains 1535 bp upstream of C5, a polylinker containing KpnI, SmaI, XbaI, and NotI sites, and 404 bp of canarypox DNA (31 bp of C5 coding sequence and 373 bp of downstream sequence) was derived in the following manner. A genomic library of canarypox DNA was constructed in the cosmid vector puK102, probed with pRW764.5 and a clone containing a 29 kb insert identified (pHCOS1). A 3.3 kb ClaI fragment from pHCOS1 containing the C5 region was identified. Sequence analysis of the ClaI fragment was used to extend the sequence in from nucleotides 1-1372. The C5 insertion vector was constructed as follows. The 1535 bp upstream sequence was generated by PCR amplification using oligonucleotides C5A (SEQ ID NO:27) (5'-ATCATCGAATTCTGAATGTTAAATGTTATACTTG-3') and C5B (SEQ

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WO92/~6616 L,, ~ P~/US92/02207 3~

ID NO:28) (5'-GGGGGTACCTTTGAGAGTACCACTTCAG-3') and purified genomic canarypox DNA as template. This fragment was digested with EcoRI (within oligo C5A) and cloned into EcoRI/SmaI digested pUC8 generating pC5LAB.
The 404 bp arm was generated by PCR amplification using oligonucleotides C5C (SEQ ID NO:29) (5'-GGGTCTAGAGCGGCCGCTTATAAAGATCTAAAATGCATAATTTC-3') and C5DA (SEQ ID NO:30) (5'-ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3'). This fragment was digested with PstI (within oligo C5DA) and cloned into SmaI/PstI digested pC5LAB generating pC5L.
pC5L was digested within the polylinker with As~718 and NotI, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP26 (SEQ ID NO:31) (5'-GTACGTGACTAATTAGCTATAAAAAGGATCCGGTACCCTCGAGTCTAGAATCG
ATCCCGGGTTTTTATGACTAGTTAATCAC-3') and CP27 (SEQ ID NO:32) (5'-GGCCGTGATTAACTAGTCATAAAAACCCGGGATCGATTCTAGACTCGAGGGTA
CCGGATCCTTTTTATAGCTAATTAGTCAC-3') (containing a disabled As~718 site, translation stop codons in six reading frames, vaccinia early transcription termination signal -~; (Yuen and Moss, 1987), BamHI, K~nI, XhoI, XbaI, ClaI, and SmaI restriction sites, vaccinia early transcription termination signal, translation stop codons in six reading frames, and a disabled NotI site, generating plasmid pC5LSP. The early/late H6 vaccinia virus promoter (Perkus et al., 1989) was derived by PCR from a plasmid containing the promoter using oligonucleotides CP30 (SEQ ID NO:33) (5'-TCGGGATCCGGGTTAATTAATTAGTCATCAGGCAGGGCG-3') and CP31 (SEQ ID NO:34) (5'-TAGCTCGAGGGTACCTACGATACAAACTTAACGGATATCG-3'). The PCR product was digested with BamHI and XhoI (sites created at the 5' and 3' termini by the PCR) and ligated to similarly digested pC5LSP generating pVQH6C5LSP.
pVQH6C5LSP was digested with EcoRI, treated with alkaline phosphatase, ligated to self-annealed oligonucleotide CP29 (SEQ ID NO:35) (5'-AATTGCGGCCGC-3'), digested with `SUB-~iTI ~ ~,T-~

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WO92/16616 ~ 26- PCT/US92/02207 NotI and linear purified followed by self-ligation. This procedure introduced a NotI site to pVQH6C5LSP, generating pNVQH6C5SP18.
The pNC5LSP-5 plasmid, another ALVAC C5 insertion vector, was derived as follows. Plas~id pC5LSP
was digested with EcoRI, treated with alkaline phosphatase, ligated to self-annealed oliaonucleotide CP29 (SEQ ID NO:35), digested with NotI and linear purified followed by self-ligation. This proczdure introduced a NotI site to pC5LSP, generating pNC5LSP-5.
Insertion plasmid VQCP3L was der ved as follows. An 8.5kb canarypox BalII fragment was cloned in the BamHI site of pBS-SK plasmid vector to form p~
Nucleotide sequence analysis revealed a reading frame designated C3. In order to construct a donor plas~id for insertion of foreign genes into the C3 locus with the complete excision of the C3 open reading frame, PCR
primers were used to amplify the 5' and 3' sequences relative to C3. Primers for the 5' sequence were RG277 (SEQ ID NO:36) (5'-CAGTTGGTACCACTGGTATTTTATTTCAG-3') and RG278 (SEQ ID NO:37) (5'-TATCTGAATTCCTGCAGCCCGGGTTTTTATAGCTAATTACTCAAATGTGAGTTAATA
TTAG-3'). Primers for the 3' sequences were RG279 (SEQ
ID NO:38) (5'TCGCTGAATTCGATATCAAGCTTATCGATTTTTATGACTAGTTAATCAAATAAA
AAGCATACAAGC-3') and RG280 (SEQ ID NO:39) (5'-TTATCGAGCTCTGTAACATCAGTATCTAAC-3'). The primers were designed to include a multiple cloning site flanked by vaccinia transcriptional and translational termination signals. Also included at the 5'-end and 3'-end of the left arm and right arm were appropriate restriction sites (As~718 and EcoRI for left arm and EcoRI and SacI for right arm) which enabled the two arms to ligate into ~p718/SacI digested pBS-SK plasmid vector. The resultant plasmid was designated as pC3I. A 908 bp fragment of canarypox DNA, immediately upstream of the C3 locus was obtained by digestion c' plasmid pWW5 with NsiI
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W0 92/16616 , ~ PCT/US92/02207 and Ss~I. A 604 bp fragment of canarypox and DNA was derived by PCR using plasmid pWW5 as template and oligonucleotides CPl6 (SEQ ID NO:40) (5'-TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTCTGC-3') and CP17 (SEQ
ID N0:41) (5'-TCGCTCGAGTAGGATACCTACCTACTACCTACG-3'). The 604 bp frag~ent was digested with Asp718 and XhoI (sites present at the 5' ends of oligonucleotides CP16 and CP17, respectively) and cloned into As~718-XhoI digested and alkaline phosphatase treated IBI25 (International - 10 Biotechnologies, Inc., New Haven, CT) generating plasmid SPC3LA. SPC3LA was digested within IBI2S with EcoRV and within canarypox DNA with NsiI, and ligated to the 908 bp NsiI-Ss~I fragment generating SPCPLAX which contains 1444 bp of canarypox DNA upstream of the C3 locus. A 2178 bp BqlII-~yI fragment of canarypox DNA was isolated from plasmids pXX4 (which contains a 6.5 kb NsiI fragment of canarypox DNA cloned into the PstI site of pBS-SK. A 279 bp fragment of canarypox DNA was isolated by PCR using plasmid pXX4 as template and oligonucleotides CPl9 (SEQ
ID NO:42) (5'-TCGCTCGAGCTTTCTTGACAATAACATAG-3') and CP20 (SEQ ID NO:43) (5'-TAGGAGCTCTTTATACTACTGGGTTACAAC-3').
The 279 bp fragment was digested with XhoI and SacI
^ (sites present at the 5' ends of oligonucleotides CP19 and CP20, respectively) and cloned into SacI-XhoI
digested and alkaline phosphatase treated IBI25 generating plasmid SPC3RA. To add additional unique sites to the polylinker, pC3I was digested within the polylinker region with Eco~I and ClaI, treated with ~ alkaline phosphatase and ligated to kinased and annealed r 30 oligonucleotides CP12 (SEQ ID N0:44) (5'-AATTCCTCGAGGGATCC-3') and CP13 (SEQ ID N0:45) (5'-CGGGATCCCTCGAGG-3') (containing an EcoRI sticky end, XhoI
site, BamHI site and a sticky end compatible with ClaI) generating plasmid SPCP3S. SPCP3S was digested within the canarypox sequences downstream of the C3 locus with StyI and SacI (pBS-SK) and ligated to a 261 bp BalII-SacI
fragment from SPC3RA and the 217~ bp BqlII-StvI fragment .. SUE3'~ T

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WO92/16616 rCTtUS92/02207 ~ 28-from pXX4 generating plasmid CPRAL containing 2572 bp of canarypox DNA downstream of the C3 locus. SPCP3S was digested within the canarypox sequences upstream of the C3 locus with Asp718 (in pBS-SK) and AccI and ligated to a 1436 bp Asp718-AccI fragment from SPCPLA~ generating plasmid CPLAL containing 1457 bp of canarypox DNA
upstream of the C3 locus. The derived plasmid was designated as SPCP3L. VQCPCP3L was derived from pSPCP3L
by digestion with XmaI, phosphatase treating the linearized plasmid, and ligation to annealed, kinased oligonucleotides CP23 (SEQ ID N0:46) (5'-CCGGTTAATTAATTAGTTATTAGACAAGGTGAAAACGAAACTATTTGTAGCTT.~TT
- AATTAGGTCACC-3') and CP24 (SEQ ID N0:47) (~'-CCGGGGTCGACCTAATTAATTAAGCTACAAATAGTTTCGTTTTCACCTTGTCTAATA
ACTAATTAATTAA-3').
The ALVAC C6 insertion vector pC6L contains a 1615 bp SacI/K~nI fragment containing the C6 region of ALVAC inserted in the pBS,SK vector (Stratagene, La ~olla, CA). A polylinker region has been introduced approximately at position 400 of the C6 sequence which contains translational stops in six reading frames, early transcriptional termination signals in both directions, and a series of restriction enzyme sites for cloning (SmaI, PstI, XhoI, and EcoRI).
Transfection of insertion vectors into tissue ' culture cells infected with rescuing pox virus (Copenhagen vaccinia virus, NYVAC, ALVAC) and the identification of recombinants by in situ hybridization was as previously described (Piccini et al., 1987).
Immunoprecipitation Analysis of ~oxvirus-;~ expressed Plasmodium Antiaens. Rabbit anti-SERA serum for expression analysis of the SERA-containing recombinants was provided by Dr. P. Delplace (INSERM-U42, 369 rue Jules-Guesde, 59650 Villeneuve-D'Ascq, France).
A pool of antimalaria human immunoglobulins from African donors with high antimala~ia titers was provided by Dr.
M. Hommel (Liverpool School of Tropical Medicine) and .

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.. . ,. . . . : . : , . . ~ . - . : .

WO92/16616 ~ ~,Jj1,,3,~ PCT/US92/02207 used for expression analysis of recombinants (such as Pfhsp70- and the AMA-1-containing recombinants). The ABRA-specific mAb 3D5 and rabbit anti-CS repeat and anti-repeatless CS serum were provided by Dr. D. Lanar (WRAIR, Washington, D.C.). The Pfs25-specific mAb 4B7 was provided by Dr. D. Kaslow (NIAID, NIH). MSA-l-specific rabbit serum was provided by Dr. S. Chang (University of Hawaii).
Immunoprecipitations were performed essentially as described previously (Taylor et al., 1990). Briefly, Vero ceil monolayers were infected with recombinant or parental virus (or mock infected) at an moi of 10 PFU/cell. At one hour post infection, the inoculum was removed and replaced by methionine-free medium supplemented with 35S-methionine. At 8 hours post infection, cells were lysed under non-denaturing conditions by the addition of buffer A (Stephenson and ter Meulen, 1979) and immunoprecipitation performed using anti-serum and protein A-Sepharose CL-4B (Pharmacia, Piscataway, NJ) as described (Taylor et al., 1990).
Immunoprecipitates were solubilized in Laemmli disrupting solution (Laemmli, 1970) prior to analysis by denaturing polyacrylamide gel electrophoresis and autoradiography.
Endoalycosidase Diaestions of Vaccinia-ex~ressed P. Falciparum Antiaens. After immunoprecipitation, peptides from recombinant-infected Vero cells and culture supernatants were digested with endoglycosidase H (endo H) and glycopeptidase F (PNGase F) as described (Mason, 1989). The digested glycoproteins were subsequently analyzed by denaturing polyacrylamide gel electrophoresis.
ExamDle 1 - GENERATION OF SERA-CONTAINING
VACCINIA VIRUS RECONBINANT
Several lines of evidence suggest the importance of SERA in protective immunity to P.
falciparum. Most importantly, i~munization with purified SERA protein partially protects Saimiri monkeys from both 3~.~,-'.T~ T-S.~.``- , _, , . . , ~ .. . . . . . . .
~ . , . . . . , -,: ., ., , .. . , - ..
. . .. - . . , '.- ''~ ,: . ,. . ' :

'.. :: , , ' ' : . .: . ' : . : ' : . -: - : - . - :. - .- :;,, , : . .. .
.. : : .:, . ., - ~ .. , . , : . . .
. . . ,. : . ;: . . : ., : : -. ' ' ~ :: - . , ' . : . . : , : '- . : :
- . . . . : . .. ..

WO 92/16616 PCr/US92/02207 n~
h i V ~ 3 ~

heterologous and homologous challenge with blood stage parasites (Delplace et al., 1988; Perrin et al., 1984).
Additionally, SERA-specific antisera and mAbs have been shown to inhibit parasite invasion and growth in vitro (Banyal and Inselburg, 1985; Delplace et al., 1985;
Delplace et al., 1987; Perrin et al., 1984). SERA, and anti-SERA antibodies, are also found in immune complexes that form in vitro when schizonts rupture in the presence of immune serum (Chulay et al., 1987; Lyon et al., 1989).
Because SERA is expressed during both the liver and blood stages of P. falcipar~m infection (Szarfman et al., 1988), it can be envisioned that vaccine-induced anti-SERA immunity may limit the spread of blood stage infection by acting on infected liver cells. These results have generated an interest in SERA as a potential vaccine candidate.
To this end, cDNA encoding SERA from the FCR3 P. falciparum . strain was isolated and a vaccinia virus recombinant containing the SERA coding sequence was generated. The full length SERA precursor protein was expressed in cells infected with this recombinant and released into the culture medium.
Overlapping cDNA clones spanning the SERA
coding sequence were isolated from the PCR3 strain of Plasmodium falciparum.
Referring now to Figure 1, a schematic ; representation of the SERA coding sequence is shown below the scale. Dotted boxes represent the leader peptide (L), octamer repeat region (8-R), and serine repeat region (S-R). The shaded box delineates a ~EaI/NdeI
restriction fragment. The location of SERA cDNA clones is shown in relation to the coding sequence. The star -(*) indicates the position of a point mutation in clone pl26.8.
The pl26.6 cDNA was isolated from the blood stage cDNA Lambda ZAPII cDNA library by hybridization to a SERA-specific oligonucleotide JAT2 (SEQ ID NO:48) (5'-SIJB~;T3~ 1 ~ S; ~EE~ -- - . ~ . - ~ . , . ....................... . -, . :

-, .~ , . : .

WO92/16616 ~ /' PCT/US92/02207 GTCTCAGAACGTGTTCATGT-3'), which was derived from the 3' end of the SERA coding sequence (Bzik et al., 1988; Knapp et al., 1989). Clones derived from the 5' end of the SERA coding sequence were obtained by PCR with primers JAT15 (SEQ ID NO:49) (s~-CACGGATcCATGAAGTCATATATTTCCTT-3') and JAT16 (SEQ ID
NO:50) (5~-GTGAAGCTTAATCCATAATCTTCAATAATT-3') and SERA first strand cDNA template (obtained with oligonucleotide primer JAT17 (SEQ ID NO:51) (5'-GTGAAGCTTTTATACATAACAGAAATAACA-3') and were cloned into pUC19. These 1923 bp cDNAs extend from the initiation codon to a point 31 bp 3' of the internal EcoRI site (position 1892). One such cDNA, pl26.8, was found by DNA
sequence analysis to contain a Taq polymerase error at nucleotide 1357. This error, an A to G substitution, - resides within a 315 bp K~nI/NdeI restriction fragment (Figure 1). A second SERA 5' cDNA, pl26.9, has no mutations within this K~nI/NdeI fragment. An unmutated 20 5' SERA cDNA was generated by replacing the 315 bp - KpnI/NdeI fragment in pl26 .8 with the analogous fragment from pl26 . 9 to generate pl26. 14 . Full length SERA cDNA
was generated by ligating the pl26.14 5' cDNA as an XmaI/EcoRI fragment into a partial EcoRI/XmaI digested 25 pl26 . 6 vector fragment to generate pl26. 15 (Figure 1).
The complete nucleotide sequence of the pl26. 15 SERA cDNA insert, as well as the predicted amino acid sequence, is shown in Figure 2. This cDNA contains a 2955 bp open reading frame encoding 984 amino acids that is identical to the SERA allele II gene in the FCR3 strain and the FCBR SERA gene (Knapp et al., 1989; Li et al., 1989). The leader peptide is underlined, the octapeptide repeat region is underlined in bold and enclosed in brackets and the serine repeat region is highlighted in bold in Figure 2.
A vaccinia donor plasmid was constructed by isolating SER~ cDNA from pl26.15 as a 3 Kb XmaI/EcoRV

,..... , . . , . . . . ~ . . . :

~ . . , , ~ , . . . . .

, . . -:
.. . . .

~ 1 3 ~ 32-fragment and ligating the XmaI end into an XmaI/BalII
digested pCOPCS-5H vector fragment. DNA polymerase I
Klenow fragment was used to fill in the pCOPCS-5H BalII
site which was subsequently ligated to the EcoRV end to generate pl26.16. In this insertion plasmid, SERA is under the control of the early/late vaccinia ~6 promoter (Rosel et al., 1986) and the insertion of this cDNA is directed to the site of a C6L-KlL deletion.
The pl26.16 insertion vector was used as a donor plasmid to insert SERA into vaccinia virus by recombination. A SERA-containing recombinant was isolated, plaque purified, and amplified and the resultant virus designated vP870.
Immunoprecipitation analysis was performed on Vero cells infected at an moi of 10 PFU/cell and pulsed with 35S-methionine. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and bands visualized by autoradiography. Expression of SERA by vP870 in Vero cells can be detected by immunoprecipitation with SERA-`- 20 specific rabbit antiserum. At 8 hours post-infection, the anti-SERA reagent detects a high molecular weight ~ SERA protein in the culture medium indicating that it is - released from infected cells. This result is consistent with the absence of a putative hydrophobic transmembrane domain within the SERA coding sequence (Bzik et al., 1988; Knapp et al., 1989; Li et al., 1989). Smaller SERA-specific peptides remain cell-associated at this timepoint.
Example 2 - GENERATION OF ABRA-CONTAINING
VACCINIA VIRUS RECOMBINANT
The functional role of ABRA in the parasite life cycle is unknown. Despite this, several studies suggest the importance of A8RA as a vaccine candidate. -First, an ABRA-specific mAb inhibits the release of merozoites from rupturing schizonts and results in immune ¦ complex formation, thus preventing the spread of infection in vitro (Chulay et al., 1987). ABRA, and "
SU3~ T

,';' ' ~ ., . '. ' :, . ' '' , . , . '~: ' ' - . ... ' . . ,: . ., ' .

~ r ~,~
~ .i ~, i .. ~J ' anti-ABRA antibodies, are also found in such immune complexes formed in vitro when schizonts rupture in the presence of immune serum (Chulay et al., 1987). Because ABRA is expressed during both the liver and blood stages of P. falciparum infection (Szarfman et al., 1988), it can be envisioned that vaccine-induced anti-ABRA immunity may limit the spread of blood stage infection by acting on infected liver cells. Finally, the apparent conservation of ABRA (Chulay et al., 1987; Stahl et al., 1986; Weber et al., 1988) suggests that immunity to this protein might confer protection against numerous isola.es.
To this end, cDNA encoding ABRA from the FCR3 P. falciparum strain was isolated and a vaccinia virus recombinant containing the ABRA coding sequence was generated.
Full length ABRA cDNA was generated by PCR with ABRA-specifio primers JAT32 (SEQ ID NO:52) (5'-CACGGATCCATGATGAACATGAAAATTGTTTTATTC-3') and JAT34 (SEQ
ID NO:53) (5'-GTGCTCGAGTTATTTTGATTCTTCAGTTGTCAA-3') and ABRA first strand cDNA template (obtained with primer JAT33 (SEQ ID NO:54) (5'-GTGCTCGAGGTTTAATTATTTTGATTCTTCAGTTG-3'). The amplified ABRA cDNA was flanked by BamHI and XhoI restriction sites due to their inclusion in primers JAT32 and JAT34, respectively. This allowed the cloning of ABRA as a BamHI/XhoI fragment into the vaccinia donor plasmid - pCOPAK-H6. Clones containing ABRA cDNA derived from two independent PCRs were obtained to control for Taa polymerase errors. Two such clones are pABRA-2 and pABRA-4.
Complete nucleotide sequence analysis of pABRA-

2 and pABRA-4 revealed one amino acid-altering Taq polymerase error each in pABRA-2 (at position 1580, an A
insertion) and pABRA-4 (at position 140, a C for T
substitution). Additionally, pABRA-4 contains a deletion of an A residue within a poly-A stretch beginning at ~ . .
.
: .: , '. . '. ' :- . . - :- : , : .
;: . - . :. .. .. :: . : -, : ~; : .
:. .

. . . . . . ..
:-: - - . -. . :
~. :. .:

WO92/16616 PCT/USg2/02207 ~ 0~ 34-position 583 of the insert. This deletion has been previously reported for a partial FCR3 strain ABRA cDNA
clone (Weber et al., 1988).
A composite ABRA cDNA consisting of segments from the pABRA-2 and pABRA-4 inserts was derived in order to correct the deletion and polymerase errors. A 2460 bp NdeI fragment was isolated from pABRA-2 which extended from the internal ABRA NdeI site at position ll9l, through the 5' end of the ABRA insert, to an NdeI site in the right flanking arm of pCOPAK H6. This fragment was inserted into pABRA-4, from which the 2460 bp NdeI
fragment had been removed, to generate pABRA-~.
The complete nucleotide sequence of the pABRA-8 composite cDNA, as well as the predicted amino acid sequence, is shown in Figure 3. This cDNA contains a 2223 bp open reading frame encoding 740 amino acids. The nucleotide and predicted amino acid sequences of the ABRA
cDNA in pABRA-8 are shown. The leader peptide is underlined, the hexapeptide repeat region is underlined in bold and enclosed in brackets and the dipeptide/tripeptide repeat region is highlighted in bold and enclosed in brackets in Figure 3.
The ABRA cDNA in the pABRA-8 insertion vector, which contains pCOPAK vector sequences, is under the control of the vaccinia H6 promoter (Rosel et al., 1986) and its insertion is directed to the ATI site.
The pABRA-8 insertion vector was used as a donor plasmid to insert ABRA into vaccinia virus by recombination. An ABRA-containing recombinant was isolated, plaque purified, and amplified and the resultant virus designated vP947.
Example 3 - GENERATION OF Pfhsp70-CONTAINING
VACCINIA VIRUS RECO~BINANT
Several studies suggest the importance of ;
Pfhsp70 as a potential vaccine candidate. First, immunization of Saimiri monkeys with a protein fraction containing Pfhsp70 results in partial protection from . . . . . - - ~ . .. ,, . , . . : . - - :, .

WO92/16616 ~ 1 V 0'i~t PCT/US92/0220 homologous challenge with blood stage parasites (Dubois et al., 1984). This protection correlates with the development of antibodies against Pfhsp70, as well as a 90 ~D parasite protein, in vaccinated monkeys (Dubois et al., 1984; Jendoubi and Pereira da Silva, 1987). Also, Pfhsp70 expressed on the surface of infected mouse hepatocytes is a target for antibody-dependent cell-mediated cytotoxic mechanisms carried out by both spleen cells and nonparenchymal liver cells (Renia et al., 1990). Thus, it can be envisioned that anti-Pfhsp70 antibodies induced by vaccination could act to limit Plasmodlum infection by acting at the liver stage via this mechanism. Additionally, in studies of humans exposed to ~. falciparum, both specific antibodies and lymphocyte responsiveness to Pfhsp70 have been detected which indicates that this protein is an immune target during natural Pl~smodium infections (Kumar et al., 1990). Finally, although the similarity among P.
falciparum and other mammalian heat shock proteins raises the possibility of autoimmune complications (Mattei et al., 1989), results with vaccinated monkeys indicate that their humoral immune responses are preferentially directed against non-conserved regions of Pfhsp70 (Blisnick et al., 1988).
A partial cDNA encoding the carboxy terminus of Pfhsp70 from the FCR3 P. falciparum strain was isolated and a vaccinia recombinant that expresses this cDNA was generated.
Partial Pfhsp70 cDNA clones were isolated from the lambda ZAPII blood stage cDNA library by hybridization to the Pfhsp70-specific oligonucleotide HSP3 (SEQ ID NO:55) (5'-CCAGGAGGTATGCCCGGAGCAGG-3'), which is derived from the 3' end of the Pfhsp70 c~ding j sequence (Ardeshir et al., 1987; Bianco et al., 1986).
One clone, designated pHSP70.2, contains the 3' 966 bp of Pfhsp70 as compared to the fu'l length Pfhsp70 coding :~UP~

" . . .: . . . . . .
,,, . :- . . . . : .
,~ . ~ . - . ... .. .

~ 36-sequence (Yang et al., 1987). Other partial Pfhsp70 cDNAs that were obtained are identical to p~SP70.2.
The nucleotide sequence of the partial Pfhsp70 cDNA in plasmid pHSP70.2 is shown along with the 5 predicted amino acid sequence in Figure 4. The GG~P
repeats are underlined in bold and enclosed in brackets in Figure 4.
This cDNA contains a 948 bp open reading frame encoding 315 amino acids that is almost identical to the 10 analogous region of the complete FCR3 strain Pfhsp70 gene published previously (Yang et al., 1987). Two single nucleotide substitutions are found in the partial clone (nucleotide position 82$ - G for C, position 844 - G for A) that result in amino acid substitutions (Met for Ile 15 and Gly for Ser, respectively). The partial cDNA is also almost identical to two published partial Pfhsp70 cDNAs from the FC27 and Honduras 1 strains (Ardeshir et al., 1987; Bianco et al., 1986) with two exceptions. The pHSP70.2 insert contains an extra copy of a four amino -. 20 acid repeat unit at the 3' end of the coding sequence and .' an ATT to GAA substitution starting at nucleotide 712 of the insert.
To generate a Pfhsp70-containing vaccinia insertion vector, the Pfhsp70 partial cDNA was first 25 placed under the control of the vaccinia H6 promoter.
j pHSP70.2 was digested with EcoRI, the restriction site 'l filled in with DNA polymerase I Klenow fragment, and further digested with XhoI to liberate the Pfhsp70 cDNA.
t This fragment was ligated into plasmid pHES3 which was 30 previously digested with BamHI, treated with Xlenow fragment, and digested with XhoI. The resulting plasmid, ~ pHSP70.3, contained the Pfhsp70 partial cDNA coupled to ; the H6 promoter and inserted in frame to an ATG
initiation codon provided by the pHES3 vector. This 35 construction introduced four amino acids between the initiator Met and the first amino acid of Pfhsp70 - Gly (G), Asp (D), Gln (Q), Phe (F).

SUBSTITI,7 T E S~EET . -i ~

.. ~, ~ .. . .. .. . .

` . ... - - ~ . . . , - . . . . . . . - . , . .. . . . . . . .. ;~ . . . ...... .
.. .... . .. ,. . .. - . ... . . . . . .... . . .

WO92/16616 ~ PCT/US92/02207 h ,' j ' 1~

A vaccinia insertion vector was next constructed with the pCOPAK plasmid such that the H6 promoted partial Pfhsp70 cDNA could be inserted into vaccinia at the ATI site (replacing open reading frames A25L and A26L, see reference Goebel et al., 1990).
First, an approximately l Xb NruI/XhoI fragment was isolated from pHSP70.3. This fragment, which contains the 3' 24 bp of the H6 promoter and the Pfhsp70 cDNA, was ligated to pCOPAK-H6-0 digested with NruI and XhoI, which contains the remainder of H6. The resulting plasmid, pHSP70.~, contains the full length ~6 promoter linked to the Pfhsp70 partial cDNA in the pCOPAX insertion vector.
The pHSP70.~ insertion vector was used as a --donor plasmid to insert the partial Pfhsp70 cDNA into vaccinia virus by recombination. A Pfhsp70-containing ~ recombinant was isolated, plaque purified, and amplified v and the resultant virus designated vP905.
Im~unoprecipitation analysis was performed on Vero cells infected at an moi of l0 PFU/cell and pulsed with 35S-methionine. At 8 hours post infection, cell lysates were harvested and immunoprecipitated with human antimalaria immunoglobulins. Immunoprecipitated proteins were resolved by l0~ SDS-PAGE and bands visualized by ' autoradiography. The antimalaria human immunoglobulins specifically immunoprecipitate a peptide of approximately 32 kD from lysates of vP905-infected Vero cells. The size of this peptide is consistent with the size of the partial Pfhsp70 cDNA contained in vP905.
Example 4 - GENERA~ION OF AMAl-CONTAINING
VACCINIA VIRUS RECOMBINANT
The complete AMAl gene from the Plasmodlum falciparum 3D7 clone was isolated and its nucleotide -sequence was determined.
The complete AMAl gene was generated by PCR
with two AMAl specific oligonucleotides and 3D7 genomic DNA as template. The AMAl specific sequences of the two oligonucleotides were derived from the PF83 Camp sequence ~ ~ T ~ 3~

~ !~ U~ 38-(Thomas et al., 1990). The exact composition of the two oligonucleotides was as follows:
C014 (SEQ ID NO:56): TAATCATGAGAAAATTATACTGCG
(SEQ ID NO:74): M R K L C V
C015 (SEQ ID NO:57):
TGAGGATCCATAAAAATTAATAGTATGGTTTTTCCATC
BamHI 8top The PCR reaction was processed in a Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) with 40 cycles at 94C for 1 minute, 42C for 1.5 minutes, and 72C for

3 minutes, and a final extension step at 72C for 5 minutes. The PCR product was purified, digested with BamHI and cloned into the HPaI/BamHI plasmid pMPI3H.
ThP complete nucleotide sequence was determined using customized oligonucleotides. Two independent clones were sequenced and when differences were found a third clone was sequenced. The complete nucleotide and corresponding amino acid sequences are presented Figure 5.
ExamDle 5 - MALARIA RECOMBINANT POXVIRU8 VACCINE8 Recombinant poxviruses containing, in a nonessential region thereof, DNA from Plasmodium provide advantages as vaccines for inducing an immunological response in a host animal. One can readily appreciate that a variety of foreign genes from Plasmodium can be utilized in the recombinant poxvirus vectors. Moreover, one can readily appreciate that the recombinant poxviruses can contain DNA coding for and expressing two or more Plasmodium genes. Furthermore, one can readily i appreciate that additional poxviruses beyond those cited in this application, for example avipox and canarypox viruses, can be utilized as malaria recombinant poxvirus vaccine vectors.
Recombinant vaccines coding for and expressing Plasmodium antigens having demonstrated protection in primate model systems, expression during blood and liver stages, in vitro neutralization o parasite growth and/or SUB~T~' ~ r c~

WO92/16616 ~ ;J,' PCT/US92/02207 infectivity by specific serological reagents would be advantageous candidates for inducing an immunological response in a host animal. Conservation of amino acid sequences of the antigens of interest among isolates and strains may also be advantageously taken into account~
ExamDle 6 - MODIFIC~TlONg OF 8ERA GENE
SER~. We have previously derived a SERA-containing vaccinia recombinant designated vP870 (Example 1). This recombinant contains full length SERA cDNA from the FCR3 isolate regulated by the vaccinia H6 promotor and inserted 2t the site of a C6L-KlL deletion.
Immunoprecipitation studies have demonstrated that a SERA
peptide of ;35 kD is secreted from vP870-infected Vero cells. A serles of intracellular SERA peptides of 135, 122, and 110 kD are also expressed in such cells. We have also further characterized the expression of SERA by vP870 (see Examples 7 and 8, below).
In addition to expressing SERA promoted by H6, we have also generated modified SERA constructs promoted by the entomopox 42K promotor, which are described here.
. Linkaae with 42K entomopoX promotor and modification of 3' end. The 3' end of the SERA cDNA was , modified to place a vaccinia early transcription - termination signal (T5NT) and a series of restriction sites (XhoI, SmaI, SacI) immediately after the TAA
termination codon. This was accomplished by PCR with oligonucleotides JAT51 (SEQ ID NO:58) (5'-j TAGAATCTGCAGGAACTTCAA-3'), JAT52 (SEQ ID NO:59) (5'-CTACACGAGCTCCCGGGCTCGAGATAAAAATTATACATAACAGAAATAACATTC-3'), and plasmid pl26.16 (Example 1) as template. The resulting -300 bp amplified fragment was cloned as a PstI/SacI fragment into pl26.16 digested with PstI and SacI to generate pl26.17.
The 5' end of the SERA cDNA in pl26.17 was modified to place several restriction sites (HindIII, SmaI, BamHI) and the 42K ento~opox promotor before the ATG initiation codon. This was accomplished by PCR with .. , . ' ' . . ~ , . . ' . '. '. ' :: . , ., . ' . ,. , , , :, ! .

W092/l66l6 PCT/US92/02207 2 i U ~ 40-oligonucleotides JAT53 (SEQ ID NO:60) (5'-CTAGAGAAGCTTCCCGGGATCCTCAAAATTGAAAATATATAATTACAATATAAAATG
AAGTCATATATTTCCTTGT-3'), JAT54 (SEQ ID NO:61) (5'-ACTTCCGGGTTGACTTGCT-3'), and plasmid pl26.16 as template.
The resulting -250 bp amplified fragment was cloned as a HindIII/HindII fragment into pl26.17 digested with HindIII and HindII to generate pl26.18. This plasmid contains a cassette consisting of the SERA cDNA
controlled by the 42K entomopox promotor, with a vaccinia early transcription termination signal, and flanked by restriction sites at the 5' (HindIII, SmaI, BamHI) and 3' - (XhoI, S~aI, SacI) ends.
Generation of a donor plasmid for insertion of SERA at the ATI site. The 42K promotor/SERA cassette was isolated from pl26.1~3 as a BamHI/XhoI fragment and cloned into a BamHI/XhoI digested pSD553 vector fragment. The resulting plasmid, designated pl26.ATI, targets the insertion of 42K/SERA into the ATI site.
Construction of an ATI donor plasmid containing serine-repeatless SERA cDNA. A SERA cDNA lacking the ~`~ serine repeat region was derived by replacing a 354 bp SpeI/PflMI fragment of SERA, which contains the repeats, with an analogous PCR generated fragment from which the serine repeats have been precisely deleted. This deleted fragment was derived by PCR with primers JPW14126 (SEQ ID
NO:62) (5'-GGCTATCCATCAAATGGTACAACTGGTGAACAAGAAAGTCTTCCTGCTAATGGACCT
GATTCCCC-3'), JPW15126 (SEQ ID NO:63) (5'-TAGTATACTAGTAAATGGGGT-3'), and plasmid pl26.ATI as template. The resulting fragment was digested with SeI/PflMI and cloned into an S~eI/PflMI digested pl26.ATI vector fragment to generate pl26.RPLS. This donor plasmid directs the insertion of the 42K/SERA
serine-repeatless cassette at the ATI site.
Construction of a SERA cDNA containing a transmembrane anchor. A hybrid SERA gene was generated which contains the SERA coding sequence linked to the ~ . ,. . ;., ` . , -, - . : :
- . ' - , :: ' ' .- :
.. .. . . . .

WO92/16616 ~ PCT/US92/02207 transmembrane anchor sequence of Epstein-Barr virus gp340. A 2780 bp SmaI/PstI 42K/truncated SERA fragment (lacking the 3' 279 bp of the coding sequence), a 130 bp PstI/BqlII EBV gp340 transmembrane anchor fragment, and a SmaI/BamHI digested vec~or fragment were ligated to generate pINT126/anchor. This plasmid contains the gp340 transmembrane domain linked to the truncated SERA
sequence. The full length SERA coding sequence was then regenerated by inserting a PCR-generated 3' SERA fragment between the truncated SERA sequence and the gp340 anchor.
The 3' fragment -~as amplified with primers Pstl26 (SEQ ID
NO:64) (5'-G~ATTAGAATCTGCAGGAAC-3'), Sacl26 (SEQ ID
NO:65~ (5'-TTGTCAGTACTGCA~GAGCTCTACATAACAGAAATAACATTCG-3/), and plasmia pl26.18 as template. Inis primer pair replaces the TAA termination codon with SacI and PstI
sites, which add the amino acids Glu and Leu between the end of the SERA coding sequence and the gp340 transmembrane~domain. The amplified fragment was then digested with PstI and cloned into PstI-digested 20 pINT126/anchor to generate pl26/anchor-1. This plasmid contains, under the control of the entomopox 42K
promotor, the full length SERA coding sequence linked to the EBV gp340 transmembrane domain and targets insertion to the ATI site.
Generation of SERA-containinq vaccinia recombinants. The SERA-containing donor plasmids described above were used to insert the various forms of SERA into the ATI site of NYVAC (+ ~lL) by recombination.
The pl26.ATI donor plasmid was used to generate vP1039 30 (42K/SERA), pl26.RPLS to generate vPl040 (42K/SERA, serine-repeatless), and pl26/anchor-l to generate vPl023 (42K/SERA + EBV gp340 anchor).
Example 7 - EXPRESSION OF SERA BY VACCINIA
RECOMBINANTS
Glvcosvlation and biosynthesis of vP870-expressed SERA. The expression of intracellular SERA
peptides of 135, 122, and 110 kD and a 136 kD secreted . - : . . - : , . .
-. .
- " : ' :. : ' ,- .-". . : ,- , .. : ., - , , . . . . , . :
: . ,- .. . , , . : , - . . - : . . : . .: - ~ -- - ": ' , ' ' , ~ ,: , ' .

O g2/16616 PCI/US92/02207 n, r~ I~ 7 i U U '~

SERA peptide by vP870 (H6/SERA) has been described previously. We have performed additional studies to further characterize SERA expression by vP870. Pulse-chase studies suggest that the smaller MW intracellular polypeptides are biosynthetic intermediates of SERA
because the size of these smaller peptides increases during chase, eventually resulting in secretion. It has been implied that SERA expressed by parasites is not glycosylated, although this has not been rigorously 10 examined. Both secreted and intracellular vP870- -expressed SERA peptides are glycosylated, as determined by endoglycosidase digestion. However, the nature of N-linked sugars dif.ers in that intracellular SERA contains only simple N-linked oligosaccharides whereas tAe N-linked carbohydrates on secreted SERA have been converted to complex form.
SERA ex~ression bv vP1039, vP1040, and vP1023.
The expression of SERA by vP1039 (42K/SERA) is equivalent to tha~ of vP870 (H6/SERA) as detected by immunoprecipitation with SERA-specific rabbit antiserum, vP1040 (42K/SERA, serine-repeatless) expresses secreted and intracellular SERA peptides of 126 and 124 kD, respectively, vP1023 (42K/SERA + anchor) expresses intracellular SERA peptides equivalent to those expressed by vP870 but no secreted SERA is produced, consistent with the inclusion of the gp340 transmembrane domain in this construct.
Exam~le 8 - IMMUNOGENICITY OF VACCINIA-EXPRES8ED 8ERA
We have immunized rabbits with vP870 (H6/SERA) , and their sera has been analyzed by D. Camus (Lille, France). Rabbit anti-vP870 sera reacts with parasitized erythrocytes by immunofluorescence analysis in a manner that is indistinguishable from anti-SERA reagents. The rabbit sera also immunoprecipitates authentic 126 kD SERA
precursor and reacts with the authentic SERA precursor and processed SERA fragments of 73 kD and 50 kD by Western analysis. These studies indicate that when ' ITlJT~ Sl tEET

WO92/16616 ,,~ PCT/US92/02207 expressed by vaccinia virus, SERA can stimulate humoral immunity in rabbits that is reactive with SERA derived from blood stage parasites and further that the glycosylation of SERA does not impair the immune response to this protein.
Example 9 - GENERATION OF A DONOR PLASNID FOR
INSERTION OF ~YA-1 AT TXE ~A 8IT~
The complete AMA-l gene from the NF54/3D7 clone was isolated by PCR. The amplified PCR fragment was cloned into vector pMPI3H, which placed AMA-1 under the control of the vaccinia I3L promotor, to generate p731AMA-1. The complete AMA-1 nucleotide sequence was determined, and has been presented previously (see Example ~).
The I3L/~A-1 cassette was isolated from p731AMA-1 as a 2,000 bp HindIII/BamHI fragment and cloned into a HindIII/BamHI-digested pSD544 vector fragment.
The resul~ing plasmid, designated p544AMA-l, targets the insertion of I3L/AMA-l into the HA site.
Example 10 - GENERATION OF AN AMA-l-CONTAINING VACCINIA
RECOMBINANT
The p544AMA-1 donor plasmid was used to insert I3L/AMA-l into the HA site of NYVAC by recombination.
The resulting vaccinia recombinant was designated vP1018.
Example 11 - EXPRESSION OF AMA-1 BY vP1018 The expression of AMA-1 on the surface of vPl018-infected cells was demonstrated by immunofluorescence analysis with a pool of human anti-malarial Igs. This reagent also immunoprecipitated a cell associated protein of approximately 83 kD from vP1018-infected MRC-5 cells. Interestingly, an AMA-1 peptide of ~90 kD was released from infected cells.
ExamDle 12 - GENERATION OF AN ABRA-CONTAINING VACCINIA
RECOMBINANT
An ABRA-containing vaccinia recombinant designated vP947 (see Example 2) contains vaccinia H6-SUBSTITUTE SHEET

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-. - .. - ~ - ` . .. . ..

".. ,.,. , . ,. . , . . .. , .-. ~ .

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. . , - . ~

~ f ~
~. J ~ .t -44-promoted ABRA cDNA from the FCR3 isolate inserted at the ATI site of NYVAC (+ KlL).
The pABRA-8 donor plasmid (see Example 2) was used to insert H6/ABRA into the ATI site of NYVAC ~+ KlL) by reco~bination. The resulting vaccinia recombinant was designated vP1052.
ExamDle 13 - E~PRESSION OF ABRA BY vPs47 AND vP1052 The expression of ABRA in vP947 and vP1052-infected cells was demonstrated by immunofluorescence with the ABRA-specific mAb 3D5 (provided by ~RAIR).
However, no prGduct was detected by immunoprecipitation with this antibody. Analysis of transient expression f`rom the pAB~a-8 donor plasmid in NYVAC-infected cells suggests that ABR~. is being expressed by the donor plasmid as detected by immunofluorescence analysis and immunoprecipitation with mAb 3D5.
ExamDle 14 - AMPLIFICATION AND CLONING OF Pfs25 The Pfs25 gene from NF54/3D7 in plasmid pNF4.13 (Kaslow et al., 1988) was amplified by PCR with the Pfs25-specific primers JAT61 (SEQ ID NO:66) (5'-TAATCATGAATAAACTTTACAGTTTG-3'), JAT62 (SEQ ID NO:67) (5'-' GGATCCTCGAGCTGCAGATCTATAAAAATTACATTATAAAAAAGCATAC-3'), and plasmid pNF4.13 as template. The ~650 bp amplified , fragment, with a 5' blunt end, was digested with PstI and , 25 cloned into a HpaI/PstI-digested pMPI3H vector f,ragment. ' The resulting plasmid, pPfs25.1, contains the Pfs25 coding sequence linked to the vaccinia I3L promotor.
- Sequence analysis was performed to ensure that no Taq polymerase errors were introduced during amplification.
Example 15 - GENERA~ION OF A DONOR PLASMID FOR
INSERTION OF Pfs25 AT THE I4L 81TE
The I3L/Pfs25 cassette was isolated from pPfs25.1 as a 750 bp blunt/BqlII fragment and cloned into a SmaI/BqlII-digested pSD550 vector fragment. The resulting donor plasmid, pPfs25.2, targets insertion of I3L/Pfs25 into the I4L site.
. .

BSTITuTE SHEET

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,' :~ ' ' . ' ' ., . : ~. . ' ' ' , .
, ' ' . . , ' . '. ' . ,' :

`' . ' . : ' '; ' . .'' " ' ' ':' ' .' " ; ~ : , ~ ~ r. ~
~ .1 'J ~, .. .
-~5-Example 16 - GENE~ATION OF ~ Pfs25-CONTAINING V~CCINIA
RECONBIN~NT
The pPfs25.2 donor plasmid was used to insert I3L/Pfs25 into the I4L site of NYVAC by recombination.
The resulting vaccinia recombinant was designated vP1085.
Example 17 - EXPRESSION OF Pfs25 BY vP1085 The expression of Pfs25 on the surface of vP1085-infected cells was demonstrated by immunofluorescence analysis with the Pfs25-specific ~b 4B7. This surface expression is consistent with the presence of a hydrophobic transmembrane domain in P.s25.
Two Pfs25 peptides of 25 and 28 kD were expressed in vPl085-infected cells as detected by immunoprecipitation with 4B7.
ExamDle 18 - AMPLI~IC~TION ~ND C~O~ING Of ~fs~6 The complete Pfsl6 gene was generated by PCR
using P. falciparum NF54 clone 3D7 genomic DNA as template and the Pfs16 specific oligonucleotides C040 (SEQ ID NO:68) (5'-TAATCATGAATATTCGAAAGTTC-3') and C041 (SEQ ID NO:69) (5'-GCGAATTCATAAAAATTAAGAATCATCTCCTTC-3'), which were derived from the NF54 sequence (Moelans et al., l991a), as primers. The -500 bp amplified fragment, with a 5' blunt end, was digested with EcoRI and cloned into a HpaI/EcoRI-digested pMPI3H vector fragment. The resulting plasmid, pPfs16.1, contains the Pfsl6 coding sequence linked to the vaccinia I3L promotor. The amplified NF54/3D7 Pfsl6 sequence is identical to the published NF54 sequence (Moelans et al., l991a).
Example 19 - GENERATION OF A DONOR PL~SMID FOR
INSERTION OF Pfs16 AT THE TR 8ITE
The I3L/Pfsl6 cassette was isolated from pPfsl6.1 as a 600 bp blunt-ended fragment (HindIIIjEcoRI
digestion followed by Klenow fill-in) and cloned into a HincII-digested pSD542 vector fragment. The resulting donor plasmid, pPfsl6.2, targets insertion of I3L/Pfsl6 into the TK site.

. . . - : - . : .: :: : :. : :,; .
~ . -: - . . :: . : .

Example 20 - GENERATION OF ~ Pfs16-CONTAINING VACCINIA
RECOMBINANT
The pPfsl6.2 donor plasmid was used to insert I3L/Pfsl6 into the TK site of NYVAC by recombination.
Purified reco~binants were isolated and designated H3xxl, H3xx2, H3x~3, and H3xx4.
Example 2~ - 2~P~3~SION ~NALYSIS OE Pfsl6-CONTAINING
RECOM~3INA~TS
The pool of human anti-malarial Igs did not detect Pfsl6 exp~ession in H3xx4-infected cells by immuno~'us~oscen_o analysis. Pfsl6 expression was also not detec~ed -with ~his serum by immunoprecipitation analysis of cells infected with H3xxl, H3xx2, H3xx3, and H3xx~. Alcnougn ~nis human serum contains antibodies l~ reactive with vaccinia-expressed MSA-l, SERA, and AMA-l, -it may not contain antibodies to Pfsl6.
Example 22 - CLONING OF THE CS GENE
A CS construct derived from the 3D7 clone of '-the NF54 P. falciparum isolate (provided by Dr. D. Lanar, WRAIR) differs from the published CS sequence of NF54 (Caspers et al., 1989) in that nine repeat units have been deleted (repeats #20-28) and a base change from C to T at position l09l results in an amino acid change from Ser to Phe. In the plasmid containing this construct, pCOPCS-6H-CS, CS is linked to the vaccinia H6 promotor.
Modification of a vaccinia early transcription termination signal. This CS sequence contained a vaccinia early transcription termination signal (T5NT) located near the 5' end of the coding sequence. PCR was used to modify this termination signal without altering the amino acid sequence. A fragment of -160 bp was amplified with pCOPCS-6H-CS as template and primers H6.5 (SEQ ID NO:70) (5'-GAAAGCTTCTTTATTCTATAC-3') and CS.5 (SEQ ID NO:7l) (5'-CCTCAACAAATAGG.iAGGAAG-3'). This fragment extends from the 5' end of the H6 promotor (and introduces a HindIII site for cloning) to a HaeIII site located 3~ Oc the transcripticna' termination signal and SUBSTIT~E S~FET

,. . . . -. . . ... - ". , . . . . . . . . .. . . ... ... . . . .

.
., . . "

r ~
WO92/16616 ~ . PCT/US92/02207 has an altered nucleotide sequence which eliminates that signal without changing the amino acid sequence. After digestion with HindIII, this HindIII/~aeIII fragment was ligated with a l,058 bp HaeIII/KpnI fragment containing the remainder of the CS coding sequence and a HindIII/~E_I-digested pIBI25 (International Biotechnologies, Inc., New Haven, CT) vector fragment.
The resulting plasmid, designated pIBI25-CS, contains the full length CS gene linked to the H6 promotor.
Generation of a donor ~lasmid for insertion of CS into vaccinia. A l,l00 bp NruI/K~nI fragment was isolated from pIBI25-CS which contained the 3' end of the H6 promotor linked to the CS coding sequence. This fragment was cloned into an NruI/~E~I-digested pCOPCS-5H
vector fragment. The resulting donor plasmid, pCOPCS-CS, contains the regenerated H6 promotor linked to CS and targets insertion to the site of a C6L-KlL deletion.
~xample 23 - MODIFICATION OF THE C8 CODING ~EOUENC~
- Derivation of a leader-minus CS construct. A
CS construct lacking the N-terminal leader sequence was derived to determine if the expected alteration of intracellular transport would a~fect the induction of immunological responses to CS. Prior to removal of the leader sequence, the H6/CS cassette was subcloned from p542MLF-CS (H6/CS cassette cloned as a BamHI/BalII
fragment in the BamHI site of pSD542) as a PstI/SalI
fragment into pIBI24 (International Biotechnologies, Inc., New Haven, CT) to generate pMLF-CS.24. The leader sequence was then deleted by removing an -ll0 bp NruI/BstXI fragment from pMLF-CS.24, within which the leader sequence is located, and replacing this fragment with an analogous NruI/BstXI .ragment that contains a precise deletion of the leader sequence. This "deleted"
fragment was derived by annealing oligonucleotides NruMLFCS (SEQ ID NO:72) (5'-GATTATCGCGATATCCGTTAAGTTTGTATCGTAATGCAGGAATACCAGTGCTATGGA
AGTTCGTCAAAC-3') and NruMFCSR (S~Q ID NO:73) (5'-:
SuBsTlTuTE SHEET

. . . . ... - ~ . : ~ . . ... .... ....... -. .. .. - . . .

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- ,.,.~ .. ,. .. - . .. . . . . . . ..

- . . .. . . .

r~
.t J ,~r ~ 4 8 ~

GTTTGACGAACT~CCATAGCACTGGTATTCCTGCATTACGATACAAACTTAACGGAT
ATCGCGATAATC-3') followed by digestion with NruI and BstXI. The resulting plasmid, pMLFCS.2.24, contains a leader-minus CS gene linked to H6.
Generation of a donor plasmid for insert on of leader-minus CS into vaccinia. A l,040 bp NruI/KpnI
fragment was isolated from pMLFCS.2.24 which contained - the 3' end of the H6 promotor linked to the leader-minus CS coding sequence. This fragment was cloned into an NruI/KpnI-digested pCOPCS-5H vector fragment. The resulting donor plasmid, p-~LF-CS.3, contains the regenerated H6 promotor linked to leader-minus CS and targets insertion 'o the site o. a C6L-XlL deletion.
Example 24 - G~A~ION O~ CS-CONTAI~I~G VACCINIA
- 15 ~ECOMBINA~TS
The CS-containing donor plasmids described above were used to insert CS at the site of a C6L-KlL
deletion in vP668 by recombination. The pCOPCS-CS donor plasmid was used to generate vP868 (H6/CS) and pMLF-CS.3 ) 20 to generate vPl056 (H6/leader-minus CS).
Ex~mple 25 - EXPRES8ION OF CS 8Y ~P868 AND VPl056 The expression of CS by vP868 was demonstrated by both immunofluorescence and immunoprecipitation. CS
~ was expressed on the surface of vP868-infected Vero cells ; 25 as determined by immunofluorescence analysis with rabbit anti-CS repeat and anti-repeatless CS serum. The anti-repeatless sera detects two CS proteins of 60 and 56 kD
by immunoprecipitation of vP868-infected Vero cell lysates. The expression of a doublet is consistent with the results of others who have expressed CS from vaccinia (Cheng et al., 1986). A doublet was also detected by immunoprecipitation of vPl056-infected cell lysates.
However, the molecular weights of these peptides are slightly smaller than those expressed by vP868 (58.5 and 54.5 kD versus 60 and 56 kD, respectively). This result suggests that the leader sequence of CS (which at l9 -amino acids is predicted to have 2 molecular weight of 3~T, . ,. ' `_ . . ,........... . . ~ ' . ' - ~ .::
-. . ' : : . ' : - -- .
. ~ . - . :

,..... '; '~ .. ~ ,' . . .. .

WO 92/16616 rcrtus92to2207 f~ i ù ~ `i ~;.1 2.1 kD) may not be cleaved efficiently in vaccinia-infected cells. This difference is deemed minor and does not alter the usefulness of the product expressed or the recombinant from which it is expressed.
Example 26 - IMMUNOGENICITY OF VACCI~IA-E æ RESS~D CS
To study the immunogenicity of vaccinia-expressed CS, two rabbits were immunized intradermally with lo8 PFU of vP868 and boosted with the same dose at 3, 6, and 9 weeks post-inoculation. ELISA titers to CS
peDtides derived from NF54/3D7 that correspond to the reLeat region and unique sequences in the flanking nonrepetitive regions were determined. Immunization of rabbits with vP868 induces antibodies to both the repeats and the flanking regions, although the response was not as strong to the flanking regions as to the repeats.
Primary T cell responses were studied by injecting vP868 into mice and analyzing ln vitro - -proliferation with a peptide corresponding to amino acids 368-390 of CS. A significant T cell proliferative response was detected with spleen cells harvested 7 days after inoculation.
Studies performed with T-cells from humans immunized with irradiated sporozoites and protected from sporozoite challenge demonstrated that cells infected with vP868 can stimulate CS-specific cytotoxic T-cells in vitro and also can serve as targets for such CTLs.
ExamDle 27 - VACCINIA RECOMBINANTS CONTAINING MULTIPLE
P. FALCIPARUM GENES CS AND SERA
Generation of CS/SERA-containina TK donor ~lasmid. To generate a donor plasmid containing both CS
and SERA, the 42K/SERA cassette was isolated from pl26.18 (see above) as a 3,000 bp BamHI/XhoI fragment and cloned into a BamHI/XhoI-digested p542MLF-CS (see above) vector fragment. The resulting donor plasmid, pl26/CS-TK2, contains 42K/SERA and H6/CS (promoters positioned "head-to-head," with opposite transcriptional orientations~ and directs insertion to the vaccinia TK site.

~;U STi~3 ,t ~

- ., .. ~ .,. . - . - .. - -.: , - . , . - . . . . ..
: . . - . .- : . , - -- , . ::. - , , .. ~ ~ - . - : -.
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. : : ~ . , - .-, - . :
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WO92/166l6 ~ l'CT/US92/02207 J l ~ /3 Generation of CS/SERA double recombinant. The pl26/CS-TK2 donor plasmid was used to insert 42K/SERA and H6/CS at the TK site of NYVAC by recombination. The resulting vaccinia recombinant was designated vP1007.
Expression of CS and SERA by vP1007. The expression of both SERA and CS in vP1007-infected cells was demonstrated by immunoprecipitation with SERA-specific rabbit serum and anti-repeatless CS serum, respectively, and was equivalent to that observed with the appropriate single recombinants.
Imm~nizarion of rakbits w~'h vP1007. Twc rabbits were immunized subcu,aneously with lo8 PFU or vP1007 and boosted with the same dose at , 6, and 9 weeks post-inoculation. Serum was collected prior to immunization and every wee~ thereaf~er beginning at week 2 through week 12. The preinoculation serum does not contain antibodies specific to SERA or CS whereas the post inoculation serum does contain the antibodies.
Accordingly, vP1007 is useful to stimulate an immune response, e.g., as a vaccine, or for in vitro expression (production) of SERA and CS.
Example 28 - VACCINIA RECONBINANT8 CONTAINING NULTIPLE
P. FALCIPARUM GENES CS. SERA AND MSA-1 Generation of CStSERA/~S.~-1 tri~le recombinant.
The pl26/CS-TK2 donor plasmid was used to insert 42K/SERA
and H6/CS at the TK site of vP924 by recombination.
vP924 contains the D. falclpa-um MSA-1 gene promoted by -vaccinia H6 and inserted at the ATI site of NYVAC. The resulting CS/SERA/MSA-l-containing vaccinia recombinant was designated vP967.
ExDression of CS. SERA. and MSA-1 by vP967.
Expression of CS, SERA, and MSA-1 was detected in vP967-infected cells by immunoprecipitation with the appropriate serological reagents and was equivalent to !35 that observed with the appropriate single recombinants.
Immunization of rabbits with vP967. Two rabbits were immunized subcutaneously with 108 PFU of ~ ;~ ''' I -'' ~ -- ~ ` S ~ , L

.: ' ' ; . . I ' ' . - . .
; ~ . ' . ' ~ . ' . .:

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. ' . ' . ' ': ' `
"~ : ' . '' ' : . ~ `
' : . ' ,. . ' , WO92/16616 ~ PCT/US92/02207 vP967 and are boosted with the same dose at 3, 6, and 9 weeks post-inoculation. Serum was collected prior to immunization and are collected every week thereafter beginning at week 2 through week 12. The preinoculation serum does not contain antibodies specific to CS, SERA or MSA-l whereas the post inoculation serum does contain the antibodies. Accordingly, vP967 is useful to stimulate an immune response, e.g., as a vaccine, or for in vitro expression (production) of CS, SERA and MSA-l.
Example 29 - VACCINIA RECONBINANT8 CONTAINING
MULTIPLE P. FALCIPARUM GENES CS, SERA. MSA-l AND ~MA-l Generation of CS/SERA/MSA-l/AMA-l quadru~le recombinant. The p544AMA-l donor plasmid was used to insert I3L/AMA-l at the HA site of vP967 (CS/SERA/MSA-l in NYVAC) by recombination. From the isolation and purification of a recombinant, the resulting NYVAC
recombinant vPl108 contains the CS, SERA, MSA-l and AMA-l genes; and immunoprecipitation studies show expression thereof.
Exam~le 30 - ALVAC RECOM~INANT8 CONTAINING P.
FALCIPAR~M GEN~B CS Pfs25, SERA, ~f~16. ~nd AMA-l - -Construction of a donor plasmid for insertion of CS at the C5 site. A l,lO0 bp NruI/KpnI fragment was isolated from pCOPCS-CS which contained the 3' end of the H6 promotor linked to the CS coding sequence. This fragment was cloned into an NruI/KpnI-digested pNVQH6C5SP-18 vector fragment. The resulting donor plasmid, pMLF-CS.4, contains the regenerated H6 promotor linked to CS and targets insertion to the C5 site. ~ -Generation of a CS-containina ALVAC
recombinant. The pMLF-CS.4 donor plasmid was used to insert H6/CS into the C5 site of ALVAC (canarypox CPpp having attenuated virulence) by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-CS) shows that it contains the gene.

SUBSTITUTE S~EET

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; .. . ., .,. ., .. ........ .. ..... ,... . . ..... ~ '.. .. . .. ~ . .... - .

h ~ 52--Construction of a donor ~lasmid for insertion of Pfs25 at the C5 site. An I3L/Pfs25 cassette was isolated from pPfs25.l as a 750 bp BamHI/BalII fragment and cloned into a BamHI-digested pNC5LSP-5 vector fragment. The resulting donor plasmid, pPfs25.3, targets insertion of I3L/Pfs25 into the C5 site.
Generation of 2 Pfs25-containina ALVAC
recombinant. The pPfs25.3 donor plasmid was used to insert I3LtPfs25 into the C5 site of ALV.~C by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-~fs25) shows tha' it contains the gene.
Construction of a donor plasmi_ for insert~or of SERA at the C3 site. A 42K/SERA cassette was isolated from pl26.ATI as a BamHI/XhoI fragment and cloned into a BamHI/XhoI-digested pVQCP3L vector fragment. The resulting donor plasmid, pl26.C3, targets insertion of 42K/SERA into the C3 site.
Generation of a SERA-containinq ALVAC
recombinant. The pl26.C3 donor plasmid was used to insert 42K/SERA into the C3 site of ALVAC by recombination. The isolation and purification of the - ALVAC recombinant (ALVAC-SERA) shows that it contains the gene.
Construction of a donor plasmid for insertion of Pfsl6 at the C3 site. An I3L/Pfsl6 cassette was isolated from pPfsl6.2 as a XhoI/BamHI fragment and cloned into a XhoI/BamHI-digested pVQCP3L vector fragment. The resulting donor plasmid, pPfsl6.C3, targets insertion of I3L/Pfsl6 at the C3 site.
Generation of Pfsl6-containina ALVAC
recombinant. The pPfsl6.C3 donor plasmid is used to insert I3L/Pfsl6 into the C3 site of ALVAC by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-Pfsl6) shows that it contains the gene.

, . ,. - . : . ... -. :
~ . . . . ,: , . . . ~ :
- : .: ................ :' . : , .
' .' . : : :' ' : '. ' , ': ' , ' . , ,, ~

WO92/16616 ~ PCT/US92/02207 Construction of a donor Plasmid for insertion of AMA-l at the C6 site. An I3L/AMA-l cassette was isolated from p731AMA-l as a 2,000 bp blunt-ended fragment (HindIII digestion followed by Klenow fill-in and SmaI digestion) and cloned into a SmaI-digested pC6L
vector fragment. The resulting plasmid, designated pC6AMA-l, targets the insertion of I3L/AMA-l at the C6 site.
Generation of AMA-l-containina ALVAC
recombinant. The pC6AMA-l donor plasmid is used to insert I3L/AMA-l into the C6 site of ALVAC by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-AMAl) shows that it contains the gene.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set ; forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

SUBSTITUTE S~tEET :~

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, W092/16616 PCT/US92/02tO7 ~ ~ ~S4~
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' ..... . , ,. .. . .. . . . .. ~. .. . .. . . . . . .

j":., .. .- .. - . : : .. . - ~ .: ~
... . .

.. . : . .
, ~ ., . ;

~ ., ~ . , , ., . : .

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.~ -, ... .
.. .. .
~, , , ~ - `', '' , ' , ' ~ , . . , : ' .
, ' `: ~ . ' . . " ' - " . .- ' . .: ' ' ' .

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Claims (26)

-60_ WHAT IS CLAIMED IS:

1. A recombinant poxvirus containing therein DNA from Plasmodium in a nonessential region of the poxvirus genome.

2. A recombinant poxvirus as in claim 1 wherein said DNA codes for a Plasmodium falciparum Plasmodium gene.

3. A recombinant poxvirus as in claim 2 wherein said Plasmodium falciparum gene is selected from the group consisting of SERA, ABRA, Pfhsp70, AMA1, Pfs25, Pfsl6, CS and MSA1 and combinations thereof.

4. A recombinant poxvirus as in claim 1 wherein said DNA is expressed in a host by the production of a Plasmodium coding sequence.

5. A recombinant poxvirus as in claim 4 wherein said coding sequence is a Plasmodium falciparum coding sequence selected from the group consisting of SERA, ABRA, Pfhsp70, AMAl, Pfs25, Pfsl6, CS and MSAl and combinations thereof.

6. A recombinant poxvirus as in claim 1 wherein the poxvirus is a vaccinia virus.

7. A recombinant poxvirus as in claim 6 wherein the vaccinia virus has attenuated virulence.

8. A recombinant poxvirus as in claim 7 wherein the vaccinia virus is a NYVAC vaccinia virus.

9. A recombinant poxvirus as in claim 1 wherein the poxvirus is a canarypox virus.

10. A recombinant poxvirus as in claim 9 wherein the canarypox virus has attenuated virulence.

11. A recombinant poxvirus as in claim 10 wherein the canarypox virus is an ALVAC canarypox virus.

12. A recombinant poxvirus as claimed in claim 1 which is .

13. A vaccine for inducing an immunological response in a host animal inoculated with said vaccine, said vaccine comprising a carrier and a recombinant poxvirus containing, in a nonessential region thereof, DNA from Plasmodium.

14. A vaccine as in claim 13 wherein said DNA
codes for and expresses a Plasmodium falciparum Plasmodium gene.

15. A vaccine as in claim 14 wherein said Plasmodium falciparum gene is selected from the group consisting of SERA, ABRA, Pfhsp70, AMA1, Pfs25, Pfs16, CS
and MSA1 and combinations thereof.

16. A vaccine as in claim 13 wherein the poxvirus is a vaccinia virus.

17. A vaccine as in 16 wherein the vaccinia virus has attenuated virulence.

18. A vaccine as in claim 17 wherein the vaccinia virus is a NYVAC vaccinia virus.

19. A vaccine as in claim 13 wherein the poxvirus is a canarypox virus.

20. A vaccine as in claim 19 wherein the canarypox virus has attenuated virulence.

21. A vaccine as in claim 20 wherein the canarypox virus is an ALVAC canarypox virus.

22. A vaccine as claimed in claim 13 wherein the poxvirus is or .

23. A method for producing a Plasmodium falciparum immunogen selected from the group consisting of SERA, ABRA, Pfhsp70, AMA1, Pfs25, Pfsl6, CS and MSA1 and combinations thereof, said method comprising infecting a cell in vitro with a recombinant poxvirus containing, in a nonessential region thereof, DNA which codes for and expresses the immunogen.

_62-

24. The method of claim 23 wherein the poxvirus is a vaccinia virus.

25. The method of claim 23 wherein the poxvirus is a canarypox virus.

26. The method of claim 23 wherein the poxvirus is .