AU709479B2 - Recombinant attentuated canrypox ALVAC virus expression vectors containing heterologous DNA segments encoding malarial gene products - Google Patents

Description

Field of the Invention 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 of these references is found at the end of the specification 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.

Background of the Invention 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 a foreign genetic element in a donor plasmid and homologous 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, 4,772,848 and 4,603,112, and in copending application *ee [n:\libc]01912:MEF -2e 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 15 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; Sambrook et al., 1989).

Second, the isolated plasmid containing the DNA 20 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 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 manufacture of new viral -3genomes 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 sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except 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 -4number 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.

25 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 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 meals and injects the sporozoites into the human blood stream.

Immunity to Plasmodium does develop naturally although repeated infections 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 Plasmodium is also species and stage specific, i.e. one may be immune to P. falciparum but not 15 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 S efforts at developing a malaria vaccine are targeted to three stages in the parasite life cycle the infection i of liver cells by sporozoites, the perpetuation of the 25 blood stage by merozoites, and the transmission to mosquitos by gametes. In most 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 -6et al., 1987), and 105 kD (Banyal and Inselburg, .1985)] and is processed at the time of schizont ruptUreiinto 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 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 of 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). Immunoprecipitation 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 -7et 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 gene corresponding only to FCR3 allele I (Li et al., 1989) whereas the nucleotide sequence of the SERA gene from the FCBR strain is identical to FCR3 allele II (Knapp et al., 1989; Li et al., 1989).

The functional role of SERA during the parasite 15 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 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 -8and 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 strain 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 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 among P. falciparum strains. The partial cDNA 15 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 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).

is expressed during the liver (Renia et al., 1990) and throughout the blood stages of infection (Ardeshir et al., 1987; Bianco et al., 1986), but not by sporozoites (Bianco et al., 1986; Renia et al., 1990).

Experiments with P. falciparum-infected human hepatocyte cultures suggest that Pfhsp70 is expressed on the hepatocyte surface during the liver stage (Renia et al., -9- 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 of the carboxy-terminal 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.

25 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 carboxyterminus, 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 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 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).

AMA1 is a late-stage schizont protein originally isolated from Plasmodium knowlesi infected erythrocytes as a 66 kD protein (PK66). PK66 is processed to 44/42 kD components at the time of merozoite release and these maturation products are associated with 15 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 25 merozoites developing within the segmenting schizont. At schizont rupture, AMAl is processed to a 44/42 kD doublet (Waters et al., 1990). During the invasion of O*o erythrocytes, the 44/42 kD doublet is not carried into the erythrocytes, but remains associated with the invasion interface.

In Plasmodium falciparum, AMA1 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, AMA1 is lost: it cannot be found in the newly infected erythrocyte.

-11- AMA1 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. falciparum protein expressed during the sexual stages of parasite development. This 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 15 midgut and not in the human host (Carter et al., 1988; Kaslow 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 25 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). 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 antibodies in all strains tested, indicating -12that this protein is a good immunogen (Good et als, 1988).

is considered a potential vaccine candidate based on the ability of anti-Pfs25 mAbs to block transmission of the parasite from the vertebrate host to mosquitoes (Kaslow et al., 1989). Immunization of mice with a vaccinia recombinant producing surfaceexpressed 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. falciparum protein expressed by the sporozoite as well as the sexual stages of the 15 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 (Moelans et al., 1991a). The 20 Pfsl6 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 Cterminus.

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 acid substitutions, respectively (Moelans et al., 1991b).

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 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, 1990). Second, sera from adults living in highly endemic regions has been shown to -13recognize the Pfsl6 protein, indicating that it is immunogenic in humans (Moelans et al., 1991a)., Third, polyvalent rabbit sera raised against gametes and gametocytes recognizes Pfsl6 and has high transmission blocking activity. Preliminary studies with two Pfsl6specific mAbs indicate that one of the antibodies has transmission blocking activity (Moelans et al., 1991a).

The P. falciparum circumsporozoite (CS) protein is a 60 kD membrane protein that is uniformly distributed over the sporozoite surface (Nussenzweig et al., 1984).

CS is not expressed at any other stage of the parasite life cycle.

The CS gene consists of an uninterrupted open reading frame of approximately 1200 bp. CS is 15 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 20 can vary among different isolates. Variation in nonrepetitive 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 Plasmodia, 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 antisporozoite immunity. In terms of 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 -14sporozoite-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 immunity. However, 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 15 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 20 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 CD8 CTL epitope on the CS protein (Kumar et al., 1988b), suggesting that such cells may be important for the 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.

Likewise, such malaria recombinant poxvirus is also highly desirable for the production of Plasmodium immunogens in vitro.

Objects of the Invention It is therefore an object of this invention to provide recombinant poxviruses, which viruses express gene products of Plasmodium, and to provide a method of making such recombinant poxviruses.

It is an additional object of this invention to provide for the cloning and expression of Plasmodium coding sequences, particularly SERA, ABRA, Pfhsp70 and AMA1 Plasmodium blood stage antigens as well as Pfs25, Pfsl6 and CS Plasmodium antigens, in a poxvirus vector, particularly vaccinia and an avipox virus such as fowlpox or canarypox virus.

It is another object of this invention 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.

S. 15 These and other objects and advantages of the present invention will become more readily apparent after consideration of the following.

Statement of the 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 20 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 product of the foreign Plasmodium gene. The foreign DNA codes for a SERA, ABRA, AMA1, 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 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 Plasmodium, as well as to methods for inducing such an immunological response in an animal by inoculating the animal with a malaria recombinant poxvirus. The DNA codes for and expresses a SERA, ABRA, AMA1, Pfs25, Pfsl6 or CS Plasmodium gene. A plurality of Plasmodium genes advantageously 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 of canarypox virus.

[N:\LIBFF]00442:MEF 16 According to a first embodiment of the invention, there is provided A recombinant poxvirus containing therein DNA from Plasmodium in a nonessential region of the poxvirus genome, wherein the DNA codes for at least one Plasmodium falciparum antigen selected from the group consisting of SERA, ABRA, Phfsp7, AMA-1, Pfs25, Pfs16, CS and MSA-1 and combinations thereof, and wherein the poxvirus is ALVAC or a poxvirus having the identifying characteristics of ALVAC; the poxvirus being attenuated through serial passage on host cells, purified under agar, and amplified by passage on host cells.

According to a second embodiment of the invention, there is provided A vaccine for inducing an immunological response in a host animal inoculated with said vaccine, said vaccine comprising o1 a carrier and a recombinant poxvirus in accordance with the first embodiment of the invention, wherein said DNA codes for an expresses a Plasmodium falciparum gene selected from the group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof.

According to a third embodiment of the invention, there is provided A method for producing a Plasmodium falciparum immunogen selected from the group consisting of SERA, ABRA, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof, said method comprising infecting a cell in vitro with a recombinant poxvirus in accordance with the first embodiment of the invention.

Brief Description of the Drawings A better understanding of the present invention will be had by referring to the accompanying 20 drawings, in which: FIG. 1 schematically shows the SERA coding sequence; FIG.2 shows the nucleotide (SEQ ID NO: 1) and predicted amino acid (SEQ ID NO: 2) sequence of the SERA cDNA in p 1 26. FIG. 3 shows the nucleotide (SEQ ID NO: 3) and predicted amino acid (SEQ ID NO: 4) 25 sequence of the ABRA cDNA in pABRA-8; FIG. 4 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: 8) sequence of the 3D7 strain AMA1 gene.

FIG.6A illustrates the results of restriction digests of plaque derived from the canarypox vaccine pool (CP-1) Kanapox and canarypox virus isolates 1.1.1.1 (ALVAC) 4.1.1.1 and 5.1.1.1 FIG.6B illustrates the results of restriction digests of plaque derived from the fowlpox vaccine pool (FP-1) and fowlpox virus isolates 1.1.1.1 (TROVAC) 2.1.1.1 and 3.1.1.1 [I:\DayLib\LBFF]3 2 I766.docKWW -17-

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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 Plasmodium 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 vitro production of the gene products, for subsequent use of the products as immunogens. The SERA, ABRA, Pfhsp70, and AMA1 P. falciparum blood stage genes were isolated, characterized and inserted into poxvirus, 15 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 methods and materials are employed.

Enzymes, 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-1 Blue and SURE were obtained from Stratagene (La Jolla, CA) and strain NM522 from IBI (New Haven, CT). Plasmid vector pUC19 was obtained from New England Biolabs (Beverly, MA).

Cell Lines and Virus Strains. Vaccinia recombinants containing Plasmodium blood stage genes were generated with the Copenhagen vaccinia strain, or NYVAC

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-18- (vP866) (Tartaglia et al., 1992) vaccinia strain (-having attenuated virulence), or the vP668 vaccinia r 6 combinant as rescuing virus. All vaccinia virus stocks were produced in either Vero (ATCC CCL81) or MRC5 (ATCC CCL71) cells in Eagles MEM 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).

Polymerase Chain Reaction (PCR). The GeneAmp DNA amplification kit (Perkin Elmer Cetus, Norwalk, CT.) was used for PCR (Saiki et al., 1988) according to the manufacturers specifications with custom synthesized oligonucleotides as primers. Reactions were processed in 15 a Thermal Cycler (Perkin Elmer Cetus) with standard conditions (Saiki et al., 1988).

::Construction of P. Falciparum FCR3 Strain Blood Stage cDNA Library. Total RNA from human erythrocytes infected with P. falciparum FCR3 strain was obtained from 20 Dr. P. Delplace (INSERM-U42, 369 rue Jules-Guesde, 59650 Villeneuve-D'Ascq, 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 NaC1, 0.01M 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 (0.01M Tris-Cl, pH 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 RNA was used as a template for the synthesis of first strand cDNA by reverse transcriptase in a reaction primed with oligo(dT) -19- (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 100mM Tris-Cl pH 8.3, 30mM KCl, 6mM MgC1 2 25mM DTT, units RNasin, ImM each dNTP, and 24ug/ml oligo(dT) 12 18 as primer for 2 hours at 42 0 C. 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 20mM Tris-Cl pH 6, MgC12, 10mM (NH 4 2

SO

4 100mM KCl, 500ug/ml BSA, DTT, and 0.1mM each dNTP at 12°C for one hour followed by one hour at room temperature to synthesize second strand 15 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 20 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-1 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 Stage cDNA Clones. The FCR3 strain cDNA library was screened by plaque hybridization with 32 P 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 (Stratagene, La Jolla, CA) in 150mm dishes at a density of 100,000 plaques per dish.

Plaques were transferred to nitrocellulose filters which were then soaked in 1.5M NaCl/0.5M NaOH for 2 minutes, 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 0 C vacuum oven. Filters were prehybridized in 6X SSC, 5X Denhardts, 20mM NaH 2

PO

4 500ug/ml salmon sperm DNA for two hours at 42 0 C. Hybridizations were performed in 0.4% SDS, 6X SSC, 20mM NaH 2

PO

4 500ug/ml salmon sperm DNA for 18 hours at 42 0 C after the addition of 32 P-labelled oligonucleotides. After hybridization, filters were rinsed 3 times with 6X SSC, 0.1% SDS, washed for minutes at room temperature, and washed for 5 minutes at 58°C. Filters were then exposed to X-ray film at -70 0

C.

Plaques hybridizing with oligonucleotide probes were cored from plates and resuspended in SM buffer 15 (100mM NaC1, 8mM MgS0 4 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 32 P-labelled oligonucleotides. Well 20 isolated positive plaques were selected and subjected to two additional rounds of purification as just described.

Isolation of Plasmodium cDNA-containing Plasmids From Positive Phage Clones. Plasmodium cDNAs in the pBluescript plasmid vector were obtained by an in 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 37 0 C. After the addition of 2X YT media NaCI, 1% yeast extract, 1.6% Bacto-tryptone), incubation was continued for 3 hours at 370C followed by minutes at 70 0 C. After centrifugation, filamentous phage particles containing pBluescript phagemid (with cDNA insert) were recovered in the supernatant.

Dilutions of the recovered filamentous phage stock were mixed with XL-1 Blue and plated to obtain colonies -21containing 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 performed 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 and T7 primers or custom synthesized oligodeoxyribonucleotides.

Sequence data were analyzed with the IBI Pustell Sequence Analysis Package, Version 2.02 (International Biotechnologies, New Haven, CT).

Generation of Vaccinia Recombinants Containing 15 P. Falciparum Genes. P. falciparum genes were cloned 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 CO1LW early 20 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 KIL host range gene (Gillard et al., 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 -22- Sall B (Goebel et al., 1990) as template. The left arm was synthesized using synthetic deoxyoligonuc.1.eotides MPSYN267 (SEQ ID NO:9)

GGGCTGAAGCTTGCTGGCCGCTCATTAGACAGCGAATGAGGGAC-

3 I) and MPSYN268 (SEQ ID NO:10) AGATCTCCCGGGCTCGAGTATTAATTATTTTTATTACACCAGAA1AGACGGCTTG AGATC-3') as primers. The right arm was synthesized using synthetic deoxyoligonucleotides MPSYN269 (SEQ ID NO:11)

"TATCCACCGAACATTATTATAAACCTTTG

ATATAC and MPSYN270 (SEQ ID NO:12)

TATCTCGAATTCCCGCGGCTTTATGGACGGACTCTTTTCCCC-

3 I) as primers. The two PCR-derived DNA fragments containing the left and right arms were combined in a further PCR reaction. The resulting product was cut with EcoRI/HindlII and a 0.9kb fragment isolated. The 0.9kb 9*:.fragment was ligated with pUC8 cut with EcoRI/HindlII, resulting in plasmid pSD54l. The polylinker region located at the vaccinia deletion locus was expanded as 20 follows. pSD54l was cut with Bg.lII/XhoI and ligated with annealed complementary synthetic deoxyoligonucleotides MPSYN333 (SEQ ID NO:13) TACCC-3') and MPSYN334 (SEQ ID NO:14)

TCGAGGGTACCGGATCCCCCGGGAATCGATTCGCGATAGCTGATTAGTTTTTGTTAA

CAAAA-3') generating plasmid pSD552. The KlL host range gene was isolated as a 1kb BqlII(partial)/HoaI fragment from plasmid pSD552 (Perkus et al., 1990). pSD552 was cut with BlII/HpaI and ligated with the KIL containing fragment, generating pSD553.

Plasmid pMP13H contains the vaccinia 13L 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 of vaccinia HindIII I, as template and synthetic oligonucleotides MPSYN283 (SEQ ID NO: 15) (5 '-CCCCCCAAGCTTACATCATGCAGTGGTTAAC-3.J) and -23- MPSYN287 (SEQ ID NO:16) (5'-GATTAAACCTAAATAATTGT-3').

DNA from this reaction was cut with HindIII and RsaI and a 0.1 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 HDaI 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) CCCCCCGAATTCGTCGACGATTGTTCATGATGGCAAGAT-3,) and MPSYN280 (SEQ ID NO:20)

CCCGGGGGATCCCTCGAGGGTACCAAGCTTAATTAATTAAATATTAGTATAAAAAGT

GATTTATTTTT-3') as primers. The right arm was synthesized using MPSYN281 (SEQ ID NO:21)

AAGCTTGGTACCCTCGAGGGATCCCCCGGGTAGCTAGCTAATTTTTCTTTTACGTAT

TATATATGTAATAAACGTTC-3') and MPSYN312 (SEQ ID NO:22) TTTTTTCTGCAGGTAAGTATTTTTAAACTTCTAACACC-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 fragment was gel-purified and ligated into pUC8 cut with EcoRI/HindIII, resulting in plasmid pSD544.

-24- 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., 1990) is replaced by a cloning region consisting of gllII and SmaI sites. To expand the multicloning region, pSD548 was cut with BllII and SmaI and ligated with annealed complementary synthetic oligonucleotides 539A (SEQ ID NO:23)

AGAAAAATCAGTTAGCTAAGATCTCCCGGGCTCGAGGGTACCGGATCCTGATTAGTT

AATTTTTGT-3') and 539B (SEQ ID NO:24)

GATCACAAAAATTAACTAATCAGGATCCGGTACCCTCGAGCCCGGGAGATCTTAGCT

AACTGATTTTTCT-3'). In the resulting plasmid, pSD550, the multicloning region contains BgII, SmaI, XhoI, KDnI and BamHI restriction sites.

15 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 (5'-GGTCGACGGATCCT-3') and MPSYN289 (SEQ ID NO:26) 20 (5'-GATCAGGATCCGTCGACCTGCA-31) resulting in plasmid pSD542.

The pNVQH6C5SPl8 ALVAC C5 insertion vector, which contains 1535 bp upstream of C5, a polylinker containing KEnI, Smal, 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 Clal fragment from pHCOSI containing the C5 region was identified. Sequence analysis of the Clal 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 (SEQ ID NO:27) (5'-ATCATCGAATTCTGAATGTTAAATGTTATACTTG-3') and C5B (SEQ 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 The 404 bp arm was generated by PCR amplification using oligonucleotides C5C (SEQ ID NO:29) (5'-GGGTCTAGAGCGGCCGCTTATAAAGATCTAAAATGCATAATTTC-3') and (SEQ ID (5'-ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3'). This fragment was digested with PstI (within oligo C5DA) and cloned into SmaI/PstI digested pC5LAB generating was digested within the polylinker with Asp718 and NotI, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP26 (SEQ ID NO:31) ATCCCGGGTTTTTATGACTAGTTAATCAC-3') and CP27 (SEQ ID NO:32) CCGGATCCTTTTTATAGCTAATTAGTCAC-3') (containing a disabled Asp718 site, translation stop codons in six reading 20 frames, vaccinia early transcription termination signal (Yuen and Moss, 1987), BamHI, KpnI, 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 (SEQ ID NO:33) (5'-TCGGGATCCGGGTTAATTAATTAGTCATCAGGCAGGGCG-3') and CP31 (SEQ ID NO:34) (5'-TAGCTCGAGGGTACCTACGATACAAACTTAACGGATATCG-3'). The PCR product was digested with BamHI and Xhol (sites created at the 5' and 3' termini by the PCR) and ligated to similarly digested pC5LSP generating pVQH6C5LSP was digested with EcoRI, treated with alkaline phosphatase, ligated to self-annealed oligonucleotide CP29 (SEQ ID NO:35) (5'-AATTGCGGCCGC-3'), digested with -26- NotI and linear purified followed by self-ligation. This procedure introduced a NotI site to generating pNVQH6C5SP18.

The pNC5LSP-5 plasmid, another ALVAC insertion vector, was derived as follows. Plasmid was digested with EcoRI, treated with alkaline phosphatase, ligated to self-annealed oligonucleotide CP29 (SEQ ID NO:35), digested with NotI and linear purified followed by self-ligation. This procedure introduced a NotI site to pC5LSP, generating Insertion plasmid VQCP3L was derived as follows. An 8.5kb canarypox BllII fragment was cloned in the BamHI site of pBS-SK plasmid vector to form Nucleotide sequence analysis revealed a reading frame 15 designated C3. In order to construct a donor plasmid 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 20 (SEQ ID NO:36) (5'-CAGTTGGTACCACTGGTATTTTATTTCAG-31) and RG278 (SEQ ID NO:37)

TATCTGAATTCCTGCAGCCCGGGTTTATAGCTAATTAGTCAAATGTGAGTTAATA

TTAG-3'). Primers for the 3' sequences were RG279 (SEQ ID NO:38) AAGCATACAAGC-3') and RG280 (SEQ ID NO:39) 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 (As718 and EcoRI for left arm and EcoRI and SacI for right arm) which enabled the two arms to ligate into AsE718/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 of plasmid pWW5 with NsiI -27and SspI. A 604 bp fragment of canarypox and DNA was derived by PCR using plasmid pWW5 as template and oligonucleotides CPl6 (SEQ ID NO:40) TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTCTGC-3') and CP17 (SEQ ID NO:41) (5'-TCGCTCGAGTAGGATACCTACCTACTACCTACG-3') The 604 bp fragment was digested with AsR718 and Xho (sites present at the 5' ends of oligonucleotides CP16 and CP17, respectively) and cloned into Asp718-XhoI digested and alkaline phosphatase treated IBI25 (International Biotechnologies, Inc., New Haven, CT) generating plasmid SPC3LA. SPC3LA was digested within IBI25 with EcoRV and within canarypox DNA with NsiI, and ligated to the 908 bp NsiI-SspI fragment generating SPCPLAX which contains 1444 bp of canarypox DNA upstream of the C3 locus. A 2178 bp BqlII-StvI 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 CP19 (SEQ 20 ID NO:42) (5'-TCGCTCGAGCTTTCTTGACAATAACATAG-3') and (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 CPl9 and CP20, respectively) and cloned into SacI-XhoI 25 digested and alkaline phosphatase treated **generating plasmid SPC3RA. To add additional unique sites to the polylinker, pC3I was digested within the polylinker region with EcoRI and Clal, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP12 (SEQ ID NO:44) AATTCCTCGAGGGATCC-3') and CP13 (SEQ ID NO:45) CGGGATCCCTCGAGG-3') (containing an EcoRI sticky end, XhoI site, BamHI site and a sticky end compatible with Clal) generating plasmid SPCP3S. SPCP3S was digested within the canarypox sequences downstream of the C3 locus with Styl and SacI (pBS-SK) and ligated to a 261 bp BqlII-SacI fragment from SPC3RA and the 2178 bp gilII-StvI fragment -28from 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 SPCPLAX 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 NO:46)

CCGGTTAATTAATTAGTTATTAGACAAGGTGAAAACGAAACTATTTGTAGCTTAATT

AATTAGGTCACC-3') and CP24 (SEQ ID NO:47)

CCGGGGTCGACCTAATTAATTAAGCTACAAATAGTTTCGTTTTCACCTTGTCTAATA

ACTAATTAATTAA-3').

The ALVAC C6 insertion vector pC6L contains a 1615 bp SacI/KnI fragment containing the C6 region of ALVAC inserted in the pBS,SK vector (Stratagene, La Jolla, CA). A polylinker region has been introduced 20 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 S(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 Poxvirusexpressed Plasmodium Antigens. 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 antimalaria titers was provided by Dr.

M. Hommel (Liverpool School of Tropical Medicine) and -29used for expression analysis of recombinants (such as and the AMA-1-containing recombinantsy. 'The ABRA-specific mAb 3D5 and rabbit anti-CS repeat and antirepeatless CS serum were provided by Dr. D. Lanar (WRAIR, Washington, The Pfs25-specific mAb 4B7 was provided by Dr. D. Kaslow (NIAID, NIH). MSA-1-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 cell monolayers were infected with recombinant or parental virus (or mock infected) at an moi of PFU/cell. At one hour post infection, the inoculum was removed and replaced by methionine-free medium 15 supplemented with 35 S-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, 20 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.

Endoqlvcosidase Digestions of Vacciniaexpressed P. Falciparum Antigens. 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.

Example 1 GENERATION OF SERA-CONTAINING VACCINIA VIRUS RECOMBINANT Several lines of evidence suggest the importance of SERA in protective immunity to P.

falciparum. Most importantly, immunization with purified SERA protein partially protects Saimiri monkeys from both 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. falciparum 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 15 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 20 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 FCR3 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 octamer repeat region and serine repeat region The shaded box delineates a KpnI/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 p126.8.

The p126.6 cDNA was isolated from the blood stage cDNA Lambda ZAPII cDNA library by hybridization to a SERA-specific oligonucleotide JAT2 (SEQ ID NO:48) -31- 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) (5'-CACGGATCCATGAAGTCATATATTTCCTT-3') and JAT16 (SEQ ID (5'-GTGAAGCTTAATCCATAATCTTCAATAATT-3') and SERA first strand cDNA template (obtained with oligonucleotide primer JAT17 (SEQ ID NO:51) 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, p126.8, was found by DNA 15 sequence analysis to contain a Tag polymerase error at 9* nucleotide 1357. This error, an A to G substitution, resides within a 315 bp KpnI/NdeI restriction fragment (Figure A second SERA 5' cDNA, p126.9, has no mutations within this KpnI/NdeI fragment. An unmutated 20 5' SERA cDNA was generated by replacing the 315 bp KpnI/NdeI fragment in p126.8 with the analogous fragment from p126.9 to generate p126.14. Full length SERA cDNA uL was generated by ligating the p126.14 5' cDNA as an XmaI/EcoRI fragment into a partial EcoRI/XmaI digested p126.6 vector fragment to generate p126.15 (Figure 1).

The complete nucleotide sequence of the p126.15 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 SERA cDNA from p126.15 as a 3 Kb XmaI/EcoRV -32fragment and ligating the XmaI end into an XmaI/BglII digested pCOPCS-5H vector fragment. DNA polymerase I Klenow fragment was used to fill in the pCOPCS-5H BllII site which was subsequently ligated to the EcoRV end to generate p126.16. In this insertion plasmid, SERA is under the control of the early/late vaccinia H6 promoter (Rosel et al., 1986) and the insertion of this cDNA is directed to the site of a C6L-K1L deletion.

The p126.16 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 15 Vero cells infected at an moi of 10 PFU/cell and pulsed with 35 S-methionine. Immunoprecipitated proteins were S..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 S: 25 domain within the SERA coding sequence (Bzik et al., S, 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 ABRA 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 -33anti-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 isolates.

To this end, cDNA encoding ABRA from the FCR3 P. falciparum strain was isolated and a vaccinia virus 15 recombinant containing the ABRA coding sequence was generated.

Full length ABRA cDNA was generated by PCR with ABRA-specific primers JAT32 (SEQ ID NO:52) CACGGATCCATGATGAACATGAAAATTGTTTTATTC-3') and JAT34 (SEQ 20 ID NO:53) (5'-GTGCTCGAGTTATTTTGATTCTTCAGTTGTCAA-3') and ABRA first strand cDNA template (obtained with primer JAT33 (SEQ ID NO:54) GTGCTCGAGGTTTAATTATTTTGATTCTTCAGTTG-3'). The amplified ABRA cDNA was flanked by BamHI and XhoI restriction sites 25 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 Tag 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 Tag 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 -34position 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 1191, 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-8.

The complete nucleotide sequence of the pABRA-8 composite cDNA, as well as the predicted amino acid 15 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 20 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 ,.25 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 VACCINIA VIRUS RECOMBINANT 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 homologous challenge with blood stage parasites (Dubois et al., 1984). This protection correlates with the development of antibodies against Pfhsp70, as well as a kD parasite protein, in vaccinated monkeys (Dubois et al., 1984; Jendoubi and Pereira da Silva, 1987). Also, expressed on the surface of infected mouse hepatocytes is a target for antibody-dependent cellmediated cytotoxic mechanisms carried out by both spleen cells and nonparenchymal liver cells (Renia et al., 1990). Thus, it can be envisioned that antibodies induced by vaccination could act to limit Plasmodium infection by acting at the liver stage via this mechanism. Additionally, in studies of humans exposed to P. falciparum, both specific antibodies and 15 lymphocyte responsiveness to Pfhsp70 have been detected which indicates that this protein is an immune target i:during natural Plasmodium infections (Kumar et al., 1990). Finally, although the similarity among P.

falciparum and other mammalian heat shock proteins raises 20 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 (Blisnick et al., 1988).

25 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 coding sequence (Ardeshir et al., 1987; Bianco et al., 1986).

One clone, designated pHSP70.2, contains the 3' 966 bp of as compared to the full length Pfhsp70 coding -36sequence (Yang et al., 1987). Other partial cDNAs that were obtained are identical to pHSP70.2.

The nucleotide sequence of the partial cDNA in plasmid pHSP70.2 is shown along with the predicted amino acid sequence in Figure 4. The GGMP 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 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 828 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.

pHSP70.2 was digested with EcoRI, the restriction site filled in with DNA polymerase I Klenow fragment, and further digested with XhoI to liberate the Pfhsp70 cDNA.

This fragment was ligated into plasmid pHES3 which was previously digested with BamHI, treated with Klenow 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 construction introduced four amino acids between the initiator Met and the first amino acid of Pfhsp70 Gly Asp Gln Phe -37- 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 1 Kb 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 Xhol, which contains the remainder of H6. The resulting plasmid, pHSP70.4, contains the full length H6 promoter linked to the Pfhsp70 partial cDNA in the pCOPAK insertion vector.

The pHSP70.4 insertion vector was used as a donor plasmid to insert the partial Pfhsp70 cDNA into 15 vaccinia virus by recombination. A recombinant was isolated, plaque purified, and amplified and the resultant virus designated vP905.

Immunoprecipitation analysis was performed on Vero cells infected at an moi of 10 PFU/cell and pulsed 20 with 35 S-methionine. At 8 hours post infection, cell lysates were harvested and immunoprecipitated with human antimalaria immunoglobulins. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and bands visualized by autoradiography. The antimalaria human immunoglobulins 25 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 GENERATION OF AMAl-CONTAINING VACCINIA VIRUS RECOMBINANT The complete AMA1 gene from the Plasmodium falciparum 3D7 clone was isolated and its nucleotide sequence was determined.

The complete AMA1 gene was generated by PCR with two AMA1 specific oligonucleotides and 3D7 genomic DNA as template. The AMA1 specific sequences of the two oligonucleotides were derived from the PF83 Camp sequence -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 Stop The PCR reaction was processed in a Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) with 40 cycles at 94 0 C for 1 minute, 42 0 C for 1.5 minutes, and 72 0 C for 3 minutes, and a final extension step at 72 0 C for minutes. The PCR product was purified, digested with 15 BamHI and cloned into the HpaI/BamHI plasmid pMPI3H.

The 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 Examle 5 MALARIA RECOMBINANT POXVIRUS VACCINES Recombinant poxviruses containing, in a nonessential region thereof, DNA from Plasmodium provide 25 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 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 of parasite growth and/or -39infectivity 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.

Example 6 MODIFICATIONS OF SERA GENE SERA. We have previously derived a SERAcontaining vaccinia recombinant designated vP870 (Example This recombinant contains full length SERA cDNA from the FCR3 isolate regulated by the vaccinia H6 promotor and inserted at the site of a C6L-K1L deletion.

Immunoprecipitation studies have demonstrated that a SERA peptide of 136 kD is secreted from vP870-infected Vero cells. A series of intracellular SERA peptides of 135, 15 122, 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 20 by the entomopox 42K promotor, which are described here.

Linkage 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 (TsNT) and a series of restriction 25 sites (XhoI, SmaI, SacI) immediately after the TAA termination codon. This was accomplished by PCR with oligonucleotides JAT51 (SEQ ID NO:58) TAGAATCTGCAGGAACTTCAA-3'), JAT52 (SEQ ID NO:59)

CTACACGAGCTCCCGGGCTCGAGATAAAAATTATACATAACAGAAATAACATTC-

and plasmid p126.16 (Example 1) as template. The resulting -300 bp amplified fragment was cloned as a PstI/SacI fragment into p126.16 digested with PstI and SacI to generate p126.17.

The 5' end of the SERA cDNA in p126.17 was modified to place several restriction sites (HindIII, SmaI, BamHI) and the 42K entomopox promotor before the ATG initiation codon. This was accomplished by PCR with oligonucleotides JAT53 (SEQ ID NO:60)

CTAGAGAAGCTTCCCGGGATCCTCAAAATTGAAAATATATAATTACAATATAAAATG

AAGTCATATATTTCCTTGT-3'), JAT54 (SEQ ID NO:61) ACTTCCGGGTTGACTTGCT-3'), and plasmid p126.16 as template.

The resulting -250 bp amplified fragment was cloned as a HindIII/HindII fragment into p126.17 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, SmaI, SacI) ends.

Generation of a donor plasmid for insertion of SERA at the ATI site. The 42K promotor/SERA cassette was 15 isolated from p126.18 as a BamHI/XhoI fragment and cloned into a BamHI/XhoI digested pSD553 vector fragment. The resulting plasmid, designated p126.ATI, targets the insertion of 42K/SERA into the ATI site.

Construction of an ATI donor plasmid containing 20 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 25 fragment was derived by PCR with primers JPW14126 (SEQ ID SNO:62)

GGCTATCCATCAAATGGTACAACTGGTGAACAAGAAAGTCTTCCTGCTAATGGACCT

GATTCCCC-3'), JPW15126 (SEQ ID NO:63) TAGTATACTAGTAAATGGGGT-3'), and plasmid p126.ATI as template. The resulting fragment was digested with SpeI/PflMI and cloned into an SpeI/PflMI digested p126.ATI vector fragment to generate p126.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 -41transmembrane 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/BllII EBV gp340 transmembrane anchor fragment, and a SmaI/BamHI digested vector 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 was amplified with primers Pstl26 (SEQ ID NO:64) (5'-GCATTAGAATCTGCAGGAAC-3'), Sacl26 (SEQ ID N0:65) and plasmid p126.18 as template. This primer pair 15 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-l. 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.

25 Generation of SERA-containing vaccinia recombinants. The SERA-containing donor plasmids described above were used to insert the various forms of SERA into the ATI site of NYVAC K1L) by recombination.

The p126.ATI donor plasmid was used to generate vP1039- (42K/SERA), p126.RPLS to generate vP1040 (42K/SERA, serine-repeatless), and pl26/anchor-1 to generate vP1023 (42K/SERA EBV gp340 anchor).

Example 7 EXPRESSION OF SERA BY VACCINIA

RECOMBINANTS

Glycosylation and biosynthesis of vP870expressed SERA. The expression of intracellular

SERA

peptides of 135, 122, and 110 kD and a 136 kD secreted -42- SERA peptide by vP870 (H6/SERA) has been described previously. We have performed additional studies to further characterize SERA expression by vP870. Pulsechase 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 examined. Both secreted and intracellular vP870expressed SERA peptides are glycosylated, as determined by endoglycosidase digestion. However, the nature of Nlinked sugars differs in that intracellular SERA contains only simple N-linked oligosaccharides whereas the Nlinked carbohydrates on secreted SERA have been converted to complex form.

SERA expression by vP1039, vPl040, and vP1023.

The expression of SERA by vP1039 (42K/SERA) is equivalent to that of vP870 (H6/SERA) as detected by immunoprecipitation with SERA-specific rabbit antiserum.

vPl040 (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 25 by vP870 but no secreted SERA is produced, consistent with the inclusion of the gp340 transmembrane domain in this construct.

Example a IMMUNOGENICITY OF VACCINIA-EXPRESSED

SERA

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 flea -43expressed 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.

~mpi EEAINO OO LBI O Example 9 GENERATION OF A DONOR PLASMID FOR INSERTION OF AMA-1 AT THE HA SITE The complete AMA-1 gene from the NF54/3D7 clo 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 4).

ne

CCC...

C

*c 9* 15 The I3L/AMA-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 resulting plasmid, designated p544AMA-1, targets the insertion of I3L/AMA-1 into the HA site.

Example 1 GENERATION OF AN AMA-I-CONTAINING

VACCINIA

RECOMBINANT

The p544AMA-1 donor plasmid was used to insert I3L/AMA-1 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 vP1018-infected cells was demonstrated by immunofluorescence analysis with a pool of human antimalarial 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.

Example 12 GENERATION OF AN ABRA-CONTAINING

VACCINIA

RECOMBINANT

An ABRA-containing vaccinia recombinant designated vP947 (see Example 2) contains vaccinia H6- -44promoted ABRA cDNA from the FCR3 isolate inserted at the ATI site of NYVAC

KIL).

The pABRA-8 donor plasmid (see Example 2) was used to insert H6/ABRA into the ATI site of NYVAC

KIL)

by recombination. The resulting vaccinia recombinant was designated vP1052.

Example 13 EXPRESSION OF ABRA BY vP947 AND VP1052 The expression of ABRA in vP947 and VP1052infected cells was demonstrated by immunofluorescence with the ABRA-specific mAb 3D5 (provided by WRAIR).

However, no product was detected by immunoprecipitation with this antibody. Analysis of transient expression from the pABRA-8 donor plasmid in NYVAC-infected cells suggests that ABRA is being expressed by the donor 15 plasmid as detected by immunofluorescence analysis and immunoprecipitation with mAb Example 14 AMPLIFICATION AND CLONING OF The Pfs25 gene from NF54/3D7 in plasmid pNF4.13 (Kaslow et al., 1988) was amplified by PCR with the 20 Pfs25-specific primers JAT61 (SEQ ID NO:66) TAATCATGAATAAACTTTACAGTTTG-3'), JAT62 (SEQ ID NO:67) 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 HpaIa/PstI-digested pMPI3H vector fragment.

The resulting plasmid, pPfs25.1, contains the 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 GENERATION OF A DONOR PLASMID

FOR

INSERTION OF Pfs25 AT THE I4L SITE The I3L/Pfs25 cassette was isolated from pPfs25.1 as a 750 bp blunt/BqlII fragment and cloned into a SmaI/BalII-digested pSD550 vector fragment. The resulting donor plasmid, pPfs25.2, targets insertion of I3L/Pfs25 into the I4L site.

Example 16 GENERATION OF A Pfs25-CONTAINING

VACCINIA

RECOMBINANT

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 mAb 4B7. This surface expression is consistent with the presence of a hydrophobic transmembrane domain in Two Pfs25 peptides of 25 and 28 kD were expressed in vP1085-infected cells as detected by immunoprecipitation with 4B7.

S* 15 Example 18 AMPLIFICATION AND CLONING Of Pfsl6 The complete Pfsl6 gene was generated by PCR using P. falciparum NF54 clone 3D7 genomic DNA as template and the Pfsl6 specific oligonucleotides C040 (SEQ ID NO:68) (5'-TAATCATGAATATTCGAAAGTTC-31) and C041 20 (SEQ ID NO:69) (5'-GCGAATTCATAAAAATTAAGAATCATCTCCTTC-3 which were derived from the NF54 sequence (Moelans et al., 1991a), 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, pPfsl6.1, 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., 1991a).

Example 19 GENERATION OF A DONOR PLASMID FOR INSERTION OF Pfsl6 AT THE TK SITE The I3L/Pfsl6 cassette was isolated from pPfsl6.1 as a 600 bp blunt-ended fragment (HindIII/EcoRI 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.

-46- Example 20 GENERATION OF A PfSl6-CONTAINING

VACCINIA

RECOMBINANT

The pPfsl6.2 donor plasmid was used to insert I3L/Pfsl6 into the TK site of NYVAC by recombination.

Purified recombinants were isolated and designated H3xxl, H3xx2, H3xx3, and H3xx4.

Example 21 EXPRESSION ANALYSIS OF Pfsl6-CONTAINING

RECOMBINANTS

The pool of human anti-malarial Igs did not detect Pfsl6 expression in H3xx4-infected cells by immunofluorescence analysis. Pfsl6 expression was also not detected with this serum by immunoprecipitation analysis of cells infected with H3xxl, H3xx2, H3xx3, and H3xx4. Although this human serum contains antibodies 15 reactive with vaccinia-expressed MSA-1, SERA, and AMA-1, 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, 20 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 1091 results in an amino acid change from er 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 (TNT) 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 (SEQ ID NO:70) (5'-GAAAGCTTCTTTATTCTATAC-3') and (SEQ ID NO:71) (5'-CCTCAACAAATAGGAAGGAAG-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' of the transcriptional termination signal and -47has an altered nucleotide sequence which eliminates that signal without changing the amino acid sequence. After digestion with HindIII, this HindIII/HaeIII fragment was ligated with a 1,058 bp HaeIII/KEnI fragment containing the remainder of the CS coding sequence and a HindIII/KPnI-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 plasmid for insertion of CS into vaccinia. A 1,100 bp NruI/KDnI 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/KpnI-digested 15 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-K1L deletion.

Example 23 MODIFICATION OF THE CS CODING SEQUENCE Derivation of a leader-minus CS construct. A 20 CS construct lacking the N-terminal leader sequence was derived to determine if the expected alteration of intracellular transport would affect the induction of S* immunological responses to CS. Prior to removal of the leader sequence, the H6/CS cassette was subcloned from 25 p542MLF-CS (H6/CS cassette cloned as a BamHI/BgllII 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 -110 bp NruI/BstXI fragment from pMLF-CS.24, within which the leader sequence is located, and replacing this fragment with an analogous NruI/BstXI fragment that contains a precise deletion of the leader sequence. This "deleted" fragment was derived by annealing oligonucleotides NruMLFCS (SEQ ID NO:72)

GATTATCGCGATATCCGTTAAGTTTGTATCGTAATGCAGGAATACCAGTGCTATGGA

AGTTCGTCAAAC-3') and NruMFCSR (SEQ ID NO:73) -48- GTTTGACGA ACTTCCATAG CA T G A T C G 'C T A G T c A C T A G A

ATCGCGATATC-

3 1) followed by digestion with Nrul and BstX1. The resulting plasmid, PMLFCS.2.

24 contains a leader-minus Cs gene linked to H6.

Generation of a don or_, 1iasmidf orinsert i on o~f leader-minus CS ,nto vaccinI. A 1,040 bp NruI/X~nj fragmnent was isolated from pMLFCS.2.

2 4 which contained the 3' end of the H6 promotor linked to the leader-minus CS coding sequence. This fragment was cloned into an NrIKn-dgse PCOPCS... vector fragment. The resulting donor plasmid, PMLF-CS.3, contains the regenerated H6 promotor linked to leader-minus CS. and targets insertion to the site of a C6L-KIL deletion.

ExamrLe 24 GENERA~TION or CS-CONTAINING

VACCINIA

RECOMB

INANTS

TeCS-containing donor plasmidsdecid :::*above were used to insert cs at the site of a C6L-KL deletion in VP668 by recombination. The pCOPCS-CS donor plasmid was used to generate VP868 (H6/CS) and P?4LF-CS.3 to generate vP1os 6 (H/ede-iu

CS).

EXPRESSION OF CS BY N P0 6 The expression of CS by VP8.68 was demonstrated by both iflllunofluorescenc and immunoprecipitatio.C was expressed on the surface of vP868-infected Vero cells as determined by ilfllunofluorescence analysis with rabbit .:anti-CS repeat and at-eals CS serum. The anti- repeatless sera detects two CS proteins of 60 and 56 kD by inununoprecipitatio of vP8 6 8-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 inuunoprecipitatio 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 19 amino acids is predicted to have a molecular weight of -49- 2.1 kD) may not be cleaved efficiently in vacciniainfected 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 VACCINIA-EXPRESSED CS To study the immunogenicity of vacciniaexpressed CS, two rabbits were immunized intradermally with 10 8 PFU of vP868 and boosted with the same dose at 3, 6, and 9 weeks post-inoculation. ELISA titers to CS peptides derived from NF54/3D7 that correspond to the repeat 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 15 as strong to the flanking regions as to the repeats.

Primary T cell responses were studied by injecting vP868 into mice and analyzing in 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 25 with vP868 can stimulate CS-specific cytotoxic T-cells in vitro and also can serve as targets for such CTLs.

Example 27 VACCINIA RECOMBINANTS CONTAINING MULTIPLE P. FALCIPARUM GENES CS AND SERA Generation of CS/SERA-containing TK donor plasmid. To generate a donor plasmid containing both CS and SERA, the 42K/SERA cassette was isolated from p126.18 (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 "headto-head," with opposite transcriptional orientations) and directs insertion to the vaccinia TK site.

Generation of CS SERA double recombinant. The pl 2 6/CS-TK2 donor plasmid was used to insert 42 K/SERA and H6/CS at the TK site of NYVAC by recombination. The resulting vaccinia recombinant was designated vPh007.

Exression of C and SERA b VP100 7 The expression of both SERA and CS in VPl007-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.

Immunization of rabbits with VPl00 7 Two rabbits were immunized Subcutaneously with 108 PFU of Pl00 7 and boosted With the same dose at 3, 6, and 9 weeks Post-inoculation. Serum was collected prior to immunization and every week thereafter beginning at week through week 12. The preinoculation serum does not contain antibodies pecie s e r u m d o e s n o t contain antibodies specific to SERA or CS whereas the Post inoculation serum does contain the antibodies.

Accordingly, VP100 7 is useful to stimulate an immune response, e.g.as a vaccine, or for in vitro expression (production) of SERA and CS. expr Exa.e28 VACCINIA RECOMBINANTS CONTAINING

MULTIPLE

P. ALCPARUM GENES CSERAND M Generation ofAND

MSA-

2 5 er on ERA MSA- tri le recombinant.

The pn 26 /CS-TK2 donor plasmid was used to insert 42

K/SERA

*and H6/CS at the TK site of vP924 by recombination.

vP924 contains the P. falciparum MSA-1 gene promoted by vaccinia H6 and inserted at the ATI ite f NYVAC. The resulting CS/SERA/MSA-1-containing The as designated vP967, cotaining vaccinia recombinant was designated VP967.

Expression e o f CR SERAR and MSA-1 96.

Expression of SERA, and SA- was detected in vP967infected cells by imunoprecipitation with the appropriate serological reagents and was equivalent to that observed with theappropriate single recombinants.

Immunization o f rabbits with P96 7 Two rabbits were immunized subcutaneously with 108 PFU of -51vP967 and are boosted with the same dose at 3, 6, and 9 weeks post-inoculation. Serum was collected prior to immunization andare collected every week thereafter beginning at week 2 through week 12. The preinoculation serum does not contain antibodies specific to CS, SERA or MSA-1 whereas the post inoculation serum does contain the antibodies. Accordingly, vP967 is useful to stimulate an immune response, as a vaccine, or for in vitro expression (production) of CS, SERA and MSA-1.

Example 29 VACCINIA RECOMBINANTS CONTAINING MULTIPLE P. FALCIPARUM GENES CS, SERA, MSA-1 AND AMA-1 Generation of CS/SERA/MSA-1/AMA-1 quadruple recombinant. The p544AMA-1 donor plasmid was used to 15 insert I3L/AMA-1 at the HA site of vP967 (CS/SERA/MSA-1 in NYVAC) by recombination. From the isolation and purification of a recombinant, the resulting NYVAC recombinant vP1108 contains the CS, SERA, MSA-1 and AMA-1 genes; and immunoprecipitation studies show expression 20 thereof.

Example 30 ALVAC RECOMBINANTS CONTAINING P.

FALCIPARUM GENES CS Pfs25, SERA, pfsl6, and AMA-1 Construction of a donor plasmid for insertion of CS at the C5 site. A 1,100 bp NruI/KDnI 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/KDnI-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-containing 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.

-52- Construction of a donor plasmid for insertion of Pfs25 at the C5 site. An I3L/Pfs25 cassette was isolated from pPfs25.1 as a 750 bp BamHI/B.lII 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 a Pfs25-containing

ALVAC

recombinant. The pPfs25.3 donor plasmid was used to insert I3L/Pfs25 into the C5 site of ALVAC by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-Pfs25) shows that it contains the gene.

o*o Construction of a donor plasmid for insertion of SERA at the C3 site. A 42K/SERA cassette was isolated 15 from p126.ATI as a BamHI/XhoI fragment and cloned into a BamHI/XhoI-digested pVQCP3L vector fragment. The resulting donor plasmid, p126.C3, targets insertion of 42K/SERA into'the C3 site.

Generation of a SERA-containing

ALVAC

20 recombinant. The p126.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.

25 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-containinq

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.

Construction of a donor plasmid for insertion of AMA-1 at the C6 site An I3L/AMA-1 cassette was isolated from p731AMA-1 as a 2,000 bp blunt-ended fragment (HindIII digestion followed by Klenow fill-in and SmaI digestion) and cloned into a Smal-digested pC6L vector fragment. The resulting plasmid, designated pC6AMA- 1, targets the insertion of I3L/AMA-1 at the C6 site.

Generation of AMA-1-containing ALVAC recombinant The pC6AMA-1 donor plasmid is used to insert I3L/AMA-1 into the C6 site of ALVAC by recombination. The isolation and purification of the ALVAC recombinant (ALVAC-AMA1) 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 Spossible without departing from the spirit or scope of the present invention.

Example 31 15 A study was undertaken to additionally demonstrate that ALVAC is different from, has unique properties over, and is superior to Kanapox.

Stocks of each of Kanapox, ALVAC and the two sister plaque isolates were sonicated and diluted to 3.5 X 106 pfu per ml. Eight replicate dishes each containing a monolayer of 7 X 106 CEF or QT35 cells (Primary chick embryo fibroblast cells derived from 10-11 day old embryos of SPF origin; a continuous cell line derived from chemically induced tumors of Japanese quail, Moscovici et al., 1977, "Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail," Cell 11:95- 103) were inoculated with 200 ul aliquots giving an imput multiplicity of 0.1 pfu per cell.

After incubation for 1 hour at 37 C, the inoculum was removed and the monolayer washed twice to remove unadsorbed virus. At this time the medium was replaced and four of the dishes B, C, and D) frozen to represent time zero samples. The remaining four samples in each set were incubated for a further 72 hours at which time they were also frozen. The inocula were titrated on the day of the assay to confirm the correct input multiplicity. Actual input multiplicities fell within the range of 0.06 to 0.3 pfu per cell.

On the day of titration, the samples were thawed, the monolayer scraped into the supernatant and collected, and two of the replicates from each set were pooled (A with B, C with All samples were then sonicated to disrupt viral aggregates and titered on CEF cells.

The results of growth on CEF and QT35 cells are shown in Tables 1 and 2, respectively. ALVAC displayed a mean lower yield on CEF cells. ALVAC also displayed a lower yield on QT35 cells; and, this lower yield was apparent in both replicates and was confirmed on repeat titration.

[N:\LEBFF]00489:ANB 54 TABLE 1.

Viral yield canarypox vaccine stock and plaque isolates from primary CEF cells.

O hours 72 hours Replicate 1 Replicate 2 Mean Replicate 1 Replicate 2 Mean Kanapox 4.5 4.1 4.3 7.8 7.6 7.7 ALVAC 4.1 4.1 4.1 7.4 7.3 7.4 "sister" 4.0 4.3 4.2 7.5 7.5 (plaque isolate 4) "sister" 4.4 4.1 4.3 7.5 7.6 (plaque isolate TABLE 2.

Viral yield canarypox vaccine stock and plaque isolates from QT35 cells.

0 hours 72 hours Replicate 1 Replicate 2 Mean Replicate 1 Replicate 2 Mean Kanapox 4.2 4.3 4.3 6.1 6.0 6.1 ALVAC 4.0 4.1 4.1 5.

5 b 5 .6b 5.6 "sister" 4.4 4.3 4.4 6.1 6.2 6.2 (plaque isolate 4) "sister" 4.2 4.6 4.4 6.3 6.6 (plaque isolate 0U a: Titers expressed as log10 pfu ml of culture b: As this titer was lower than other samples, numbers shown are the mean of two titrations.

both replicates were re-titered. The [N:\LIBFF]00489:ANB In view of these additional results, ALVAC is different from, has unique properties over, and is superior to Kanapox. It is not unprecedented for a vaccine preparation, such as Kanapox, to contain multiple variants (see Hahn et al., PNAS USA 84:2019-2023 (1987)). For instance, heterogeneity in a poxvirus vaccine pool was recently documented by Boursnell et al., Vaccine 14:1485-94, 1996 which shows that the art is only now recognizing that vaccine strain poxviruses can contain variants with different virulence properties. ALVAC achieves biological consistency and consistently effective recombinant products. When Kanapox is used in deriving recombinants, there is no control over the virus background into which the foreign gene is inserted; and therefore, the properties of the resultant recombinant remain in question (cf. Lee et al., J. Virol.

66(5):2617-30 (1992) (studies with vaccinia virus which illustrate that not all vaccinia genetic backgrounds are equivalent as immunization vehicles)). ALVAC provides certainty with respect to its virus background, its properties related to virulence, and its functioning as an immunization vehicle.

15 Example 32 ALVAC is an attenuated canarypox virus wherein the canarypox virus is attenuated through more than 200 serial passages on chick embryo fibroblasts, a master seed therefrom was subjected to four successive plaque purifications under agar, from which a plaque clone was amplified through five additional passages. ALVAC has been accorded 20 a reduced level of biological containment by the National Institutes of Health Recombinant Advisory Committee (BS1 from BS2). Indeed, ALVAC is patentably distinct from prior canarypox viruses, because it has been shown that it, like TROVAC and NYVAC, has the lowest containment level of any poxvirus.

To further illustrate this we refer to Figures 6A and 6B, wherein genomic DNA was 25 isolated from plaque isolates: Fowlpox vaccine strain FP-1 (V) Plaque isolates FP 1.1.1.1 (TROVAC) FP 2.1.1.1 FP 3.1.1.1 Canarypox vaccine strain CP-1 (Kanapox) Plaque isolates CP 1.1.1.1 (ALVAC) CP 4.1.1.1 CP 5.1.1.

DNA from the plaques 1.1.1.1, 4.1.1.1, 5.1.1.1. and from the vaccine pool CP-1 were digested with HindIII, BamHI and EcoRI and electrophoresed on a 0.8% agarose gel for 24 hours. The results are shown in Figure 6A. Heterogeneity in the fragmentation profiles both within the plaque isolates and as compared to the vaccine pool was apparent in the HindIII and EcoRI restriction digests. Submolar bands evident in the [N:\LmFF]0489:ANB digestion of the genomic DNA derived from the vaccine pool appeared as molar bands in some of the plaque isolates, indicating that the vaccine pool was a mixed population.

Similarly, DNA from the plaques 1.1.1.1, 2.1.1.1, 3.1.1.1. and from the vaccine pool FP-1 were digested with HindIII, BamHIl, PstI and XhoI and electrophoresed on a 0.8% agarose gel for 24 hours. Heterogeneity both within the plaque isolates and as compared to the vaccine pool was apparent following the BamHI digestion and electrophoresis for 24 hours (Figure 6B). No differences were apparent following digestion with the other enzymes.

The result indicates that both the original fowlpox and canarypox (Kanapox) vaccine pools used to derive the ALVAC and TROVAC isolates were genotypically mixed populations based on restriction enzyme analysis. Therefore, from restriction analyses, the Rentschler strain canarypox virus (Kanapox), represents a mixture of viral variants; that is, Kanapox, from which ALVAC was derived, was a mixed population. It is not unprecedented for a vaccine preparation, such as Kanapox, to contain multiple variants (see Hahn et ALVAC is not a mixed population. As such, ALVAC has several unique properties which are not shared by Kanapox, for instance: ALVAC has a uniform genetic background. This property provides ALVAC with consistency; a unique feature of being useful for preparing vector-based vaccines.

Consistency is critical for quality control and regulatory considerations. This property of 20 consistency of ALVAC provides ALVAC with the ability to pass quality control and regulatory considerations, be useful in the development of vector-based vaccines with predicted genetic properties.

Biological consistency is controlled using ALVAC to derive recombinants. Kanapox does not provide biological consistency. Indeed, Kanapox cannot consistently provide an effective recombinant product. Biological consistency and a consistently effective recombinant product are critical; for instance, for a consistent biological profile with respect to virulence, with regard to virus/host interactions, and ultimately for use as an immunization vehicle. ALVAC achieves biological consistency and consistently effective recombinant products. When Kanapox is used in deriving recombinants, there is no control over the virus background into which the foreign gene is inserted; and therefore, the properties of the resultant recombinant remain in question (cf. Lee et al. (studies with vaccinia virus which illustrate that not all vaccinia genetic backgrounds are equivalent as immunization vehicles)). ALVAC provides certainty with respect to its virus background, its properties related to virulence, and its functioning as an immunization vehicle.

Given the teachings of the starting materials, the repeated serial passage and the plaque purification procedures, ALVAC and TROVAC and their "sister" clones will always be obtained when the procedures disclosed herein are followed. The estimate of probability of success is 100%, since the starting materials are commercially available, and the passage and purification procedures are inherently repeatable.

[N:\LIBFF]00489:ANB 57 When ALVAC and its sisters, or TROVAC and its sisters have been obtained, one need only then select the virus with least replication or highest attenuation. Given that ALVAC and TROVAC each have only two sisters (such that if the choice was one of pure statistics, without further screening the chance of selecting ALVAC or TROVAC is one in three); and, given the teachings in the specification of attenuated virulence (such that one would naturally screen, from the specification's teachings, for the most attenuated isolate).

[N:\LBFF]00489:ANB

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64 The claims defining the invention are as follows: 1. A recombinant poxvirus containing therein DNA from Plasmodium in a nonessential region of the poxvirus genome, wherein the DNA codes for at least one Plasmodium falciparum antigen selected from the group consisting of SERA, ABRA, Phfsp70, AMA-1, Pfs25, Pfs16, CS and MSA-1 and combinations thereof, and wherein the poxvirus is ALVAC or a poxvirus having the identifying characteristics of ALVAC; the poxvirus being attenuated through serial passage on host cells, purified under agar, and amplified by passage on host cells.

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

1o 3. The recombinant poxvirus of claim 2 wherein said coding sequence is a Plasmodium falciparum coding sequence selected from the group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs16, CS and MSA 1 and combinations thereof.

4. The recombinant poxvirus of any one of claims 1 to 3 wherein the poxvirus is ALVAC canarypox virus.

Is 5. The recombinant poxvirus of any one of claims 1 to 4 which is ALVAC-CS, ALVAC- ALVAC-SERA, ALVAC-Pfs16 or ALVAC-AMA1.

6. A vaccine for inducing an immunological response in a host animal inoculated with said vaccine, said vaccine comprising a carrier and a recombinant poxvirus of any one of claims 1 to 5, wherein said DNA codes for and expresses a Plasmodium falciparum gene selected from the 20 group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof.

7. The vaccine of claim 6 wherein the poxvirus is ALVAC canarypox virus.

8. The vaccine of claim 6 wherein the poxvirus is ALVAC-CS, ALVAC- Pfs25, ALVAC- SERA, ALVAC- Pfsl6 or ALVAC-AMA 1.

25 9. A method for producing a Plasmodium falciparum immunogen selected from the group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof, said method comprising infecting a cell in vitro with a recombinant poxvirus of any one of I claims 1 to o 10. The method of claim 9 wherein the poxvirus is ALVAC-CS, ALVAC- Pfs25, ALVAC- SERA, ALVAC- Pfsl6 or ALVAC-AMA 1.

11. A Plasmodium falciparum immunogen produced in accordance with the method of claim 9 or 12. A recombinant poxvirus containing therein DNA from Plasmodium in a nonessential region of the poxvirus genome, the poxvirus being attenuated through serial passage on host cells, [I\DayLib\LIBFF]321766.doc.KWW

Claims (3)

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 as defined in claim 12.

14. A method for producing a Plasmodium falciparum immunogen selected from the group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof, said method comprising infecting a cell in vitro with a recombinant poxvirus as defined in claim 12 or a vaccine as defined in any one of claims 6, 7, 8 or 13. A Plasmodium falciparum immunogen produced in accordance with the method of 1i claim 14.

16. A vaccine for inducing an immunological response in a host animal inoculated with said vaccine, said vaccine comprising a carrier and a recombinant poxvirus of claim 12, wherein said DNA codes for and expresses a Plasmodium falciparum gene selected from the group consisting of SERA, ABRA, Pfhsp70, AMA 1, Pfs25, Pfs16, CS and MSA 1 and combinations thereof. .I •Dated 6 July, 1999 Virogenetics Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON eq SCOW l:\UDayLib\LIBI']321766 doc KWW M ~bsract ~~4fv 4~ ZI~ 4 1 CA-' 1(5do -C 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 contaiming the recombinant poxvirus for inducing an immunological response in a host animal inoculated with the vaccine. Preferred recombinants have attenuated virulence. see., [N:\LIBFFJO488:MCN