JPH06505397A - malaria recombinant poxvirus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] References to related applications No. 077, filed March 20, 1991, incorporated herein by reference. This is a continuation application of No. 872.183. In addition, “vaccine strains obtained through genetic engineering” ('Genetically Englneeered Vaccine 5t U.S. patent application filed on March 6, 1992 entitled RA1N”) No. 07/847.951 (Agent Case No. 45310-2400) is also here. Refer to.

field of invention The present invention relates to mutated poxviruses and methods for producing and using the same. Ru. More particularly, the present invention provides expression of gene products of Plasmodium genes. for recombinant poxvirus and Plasmodium (PIasgodfu*) infections. It relates to vaccines that provide protective immunity against.

In this application, certain publications are referenced parenthetically. A complete list of these citations References appear immediately before the claims at the end of the specification. These references are within the field to which the present invention pertains. and each of these citations is hereby incorporated by reference.

Background of the invention Vaccinia virus and more recently other poxviruses have introduced foreign genes. It has been used for input and expression. Infectious pox virus with live foreign genes The basic technique for inserting foreign genetic elements into a donor plasmid is Box DNA sequences located on both sides and present in rescue poxvirus It involves recombination with existing homologous sequences (Piccini et al. ,, 1987).

In particular, recombinant poxviruses are known and US Patent No. 4.7B9.330 No. 4,772.848, No. 4.603.112, and herein incorporated by reference. A pending U.S. patent application filed June 14, 1990 that cites the disclosure for Production of synthetic recombinant vaccinia virus described in Application No. 071537.882 It is manufactured in two steps similar to the manufacturing method. In this regard, I quote here for reference 1 See also co-pending U.S. Patent Application No. 537.890, filed June 14, 990. do.

First, the DNA gene sequence to be inserted into the virus, especially from a non-box source. The oven reading frame is placed in the E.coli plasmid construct and this construct DNA corresponding to the poxvirus DNA section is inserted into the structure. . Apart from this, the DNA sequence to be inserted is ligated to a promoter. promotion Both ends of the protein gene junction are DNAs that flank the box DNA region containing the dispensable locus. It is homologous to the sequence and is flanked by NA. The plasmid construct thus obtained is then amplified by growth in B9 coli bacteria (C1eve11.19 72), Separate (Clewell and Helinskl, 1989; 5aabrook et al., 1989).

The isolated plasmid containing the DNA sequence to be inserted is then added to, for example, a chick embryo line. co-transfected with poxviruses into cell cultures such as fibroblasts. Plasmi By recombination between the homologous box DNA in the virus genome and the virus genome, each poxviruses that have mutated due to the presence of foreign DNA sequences within the dispensable locus. produce. “The term foreign JDNA refers to exogenous DNA, especially from non-box sources. DNA that is a gene that is not normally produced by the genome in which exogenous DNA is placed. It refers to something that encodes a gene.

Genetic recombination generally refers to the exchange of homologous parts between two pieces of DNA. some u RNA can replace DNA in viruses. Homologous parts of nucleic acids are Nucleic acid (DNA or RNA) portions that have the same nucleotide base sequence .

Genetic recombination is the replication or manufacturing of new genomes within infected host cells. can occur naturally. Therefore, there may be two genetic recombinations between the viral genes. host infected with more than one different virus or other genetic construct at the same time. It can occur during the viral replication cycle that occurs in the principal cell. derived from the first genome The second DNA portion has DNA homologous to the DNA of this first virus genome. used interchangeably to form part of the genome of a co-infecting virus.

However, recombination occurs between parts of DNA in different getums that are not completely homologous. But it can happen. One such portion may include e.g. homologous DNA in the first portion. A gene or genetic marker is present that encodes an antigenic determinant to be inserted into the part. recombination even when derived from a first genome that is otherwise homologous to part of another genome. The recombinant product is a gene in the recombinant viral genome. Can be detected by the presence of a label or gene.

Successful expression of the inserted DNA gene sequence by a mutated infectious virus Two conditions are required for this to occur. First, the mutated virus is viable. Insertions must be made within essential regions of the virus so that the virus remains intact. inserted The second condition for expression of the inserted DNA is the appropriate relationship to the inserted DNA. The existence of a promoter with a relationship with This promoter carries the DNA sequence to be expressed. It must be placed upstream.

Techniques for producing recombinant vaccinia viruses have recently become more stringent. It has also been extended to other members of the poxviridae family. AvVoxu The fowlpox virus was used as a recombinant virus expressing the rabies G gene. (Taylor et at,, 19g+1a; Taylor et al , +1988b).

This recombinant virus is also described in PCT Publication No. WO39103429. Ru. By inoculating a large number of non-avian species with the recombinant, it was possible to administer a lethal dose of rabies antigen. The immune response to rabies has been shown to protect mice, cats, and dogs against rabies. It's getting brighter.

Malaria remains one of the world's major health problems today.

There are 200 million to 300 million cases per year, and 1 to 2 million people, mainly children, suffer from malaria each year. It is estimated that he died in A.

Human malaria is caused by four species of the genus Plasmodium, P. falciparum (P., f. alciparaum), P6 Vive 7 Mouse (P, vlvax), P, Ma Lali s- (P, malariae), and P, ovale (P, oval) e) caused by one of the following. Clinically, P. falciparum is the most important. Human Blasmodium is a parasitic fungus. Because this species causes most malaria deaths. This is because it is the cause of

Blasmodium infection is caused by infected female Anopheles 0 sq. It begins when sporozoites are injected into the bloodstream by the bite of a larva. Sporo The hepatic stage of the infection begins when the zoites disappear from the bloodstream and begin to invade liver cells. 5~ Over a period of 7 days, merozoites develop asexually in infected hepatocytes and are then released into the bloodstream. , where it attacks the red blood cells and the blood stage of the infection begins. Parasitism in infected red blood cells The fungus develops asexually through a ring stage, a vegetative stage, and a schizozoite stage. rupture of the meristem merozoites are released, and these can then infect many red blood cells. Ru. This automatic perpetuation cycle of blood stage infection leads to clinical symptoms of malaria. woken up.

Some merozoites that infect red blood cells differentiate into male and female gametes. this Their reproductive bodies are those that enable sexual reproduction, and then during blood feeding Ingested by Ra force. The female gametes are then extracted by the female gametes in the mosquito's digestive tract. After fertilization, the resulting zygote invades the wall of the gastrointestinal tract, where it undergoes asexual division and its As a result, sporozoites are produced, which lodge in the mosquito's salivary glands. Infected mosquitoes Injecting sporozoites into the human bloodstream through blood ingestion completes the transmission cycle do.

Immunity to Blasmodium develops naturally, but after years of repeated infections is necessary. This is likely due to the presence of some plasmodium among different parasitic fungal isolates. This is due to the diversity of antigens exhibited by these proteins. As a result, previously In infected adults, only a very low level of parasitic bacteremia develops after infection, with little clinical evidence. Children under 5 years of age are more likely to develop severe clinical symptoms.

The developed immunity does not last long and declines without reinfection. Also, Immunity to Plasmodium is genus and stage specific, e.g. Luci, even if you are immune to Lum, it is not necessarily immune to P, Viveax, and Sporozo. Immunity to merozoites does not necessarily mean immunity to merozoites.

Until now, malaria control methods have included drug treatment and mosquito recruitment to control and prevent infection. depended on the use of insecticides to control the population. Recent outbreaks of drug-resistant parasitic bacteria and developments have made the development of an effective malaria vaccine an urgent challenge. Ru. The latest attempt to develop a malaria vaccine explores the parasitic life cycle 3 stages: infection of hepatocytes by sporozoites, blood stage by merozoites perpetuation and transmission to mosquitoes by reproductive bodies.

In many cases, purified parasitic white matter was used as a subunit vaccine. However, the results were mixed and many were unsatisfactory.

5ERA, or serine repeat antigen, is a protein expressed during the blood and liver stages of infection. Lasmodia falcivalum protein <Szarfman et al. 198g) o During the blood stage, 5ERA carries trophozoite and mesozoite parasitic bacteria. found in the vacuole and surrounding membrane (Chulay et al., 1987 ; C0ppel et al, 1988; Delplace et a t., 1987; Knapp et al., 1989).

5ERA precursor protein has a molecular weight of 126 kD [:140 kD (Perrin e tal, 1984), 113kD (Chulay et al., In2 ), and 105 kD (Banyal and Iselburg, 1985 ) and is processed into 50.47 and 11D fragments when the meristem ruptures. (Delplace et at, 1987; Delplace e t at, 1988). The 47 and 18 kD fragments are separated by disulfide bonds. are linked together to form a 73 kD complex.

The complete 5ERA gene is derived from the genomic DNA of strains FCl2 and FCBR and from strain 5. obtained from complete or partial cDNA clones derived from (Bzik et al, 1988; Coppel et at, 1988; H oril et al., 1988: Knappet al.

, 1989; Ll et al, 1989; weber et al ,, 1987). The SERA gene is divided into four genes separated by three intervening sequences. (Knapp et al., 1989; Ll etal, 19g9). The coding sequence consists of two repeating structures. characterized. The first is a glycine-rich series of 8 near the start codon. The second one is the polyserine repeat from which the protein gets its name. . The predicted amino acid sequence does not have a hydrophobic transmembrane region, 5 ERA mRNA is 3.8-4. IKb long and polar to late trophozoites and meristems. (Bzik et al., 1988; K Napp et al., 1989).

Although data are limited, 5ERA is fairly constant among P. falciparum strains. It is considered to be something similar. For comparison of various genomic and cDNA clones According to the authors, the 5ERA coding sequence is uniform in the strains studied. shown. Many of the nucleotide differences between these strains are within the polyserine repeats. or occur around and within the octapeptide repeats (Bzik et al. ,, 1988; Horil et at,, 19118; Knapp et al, 19H; Li et at, In2).

The genome organism of 5ERA is uniform in 12 strains according to Southern analysis experiments. (Coppel et al, 19H; Horii et at.

, 1988: Knappet al,, 19g9) 0101fll land Immunoprecipitation analysis of physically distinct P. falciparum revealed that the size and size of 5ERA and its processed fragments were fairly consistent. In the 47kD fragment Some differences were observed in the size of 47-50 kD (Bhatl a et al., 1987). This fragment contains polyserine repeats. did Therefore, the size difference in the 47 kD fragment is probably due to differences within the polyserine repeats, especially the serine repeats. This is thought to be due to the difference in the number of residues.

Interestingly, two 5ERA alleles have been reported in the FCl2 strain.

The differences between these, allele I and allele II, mainly occur within both repeat regions. (Ll et al., 1989). Honduras according to Southern analysis One strain was shown to have the 5ERA gene corresponding only to FCR3 allele I. (Li et al., 1989), whereas 5ERA derived from the FCBR strain The nucleotide sequence of the gene is identical to the FCR3 allele IT (Knapp e t al, , 1989; Ll et at, , 1989).

The function of 5ERA during the life cycle of parasitic fungi is unknown. Recently, egg A homology search of white matter databases revealed that 5ERA is found in cysteine proteinases. have significant similarities in and around the two active sites and therefore It became clear that it could be cysteine proteinase (Hlgglns et al., 1989). However, later on, 5ERA became cysteine. Even if it has a proteinase structure, serine is present at the presumed catalytic point. It has also been pointed out that it may actually be a serine proteinase. (Eakin et al, 1989; Mottra* etal ,, 19g9). Although this has not yet been experimentally confirmed, the life cycle of parasitic fungi This may indicate that 5ERA has an important role in the cycle. Na This is because proteases are needed to split some proteins during the blood phase. Yes, and protease inhibitors are known to interfere with the development of parasitic fungi. (Debrabant and Delplace, 1989).

Also, during both the blood and liver stages of P. falciparum infection, AB RA or acid-base repeat antigen is expressed (Szarfian et al., (1988) o Within infected red blood cells, ABRA is present during the late trophozoite stage and during differentiation. It is expressed during the fission phase and is found in the vacuoles carrying parasitic bacteria (Chulay a t al,, 1987; 5tahl et al,, 198B), A BRA has a molecular weight of 100 to LO2kD and is released from rupturing fission bodies. (Chulay et al, 1987; 5tahl et al, , 1988; Veber et al., 198B).

Complete genome ABRA gene derived from CAMP strain and FCl2 strain and F A partial ABRA cDNA derived from the C27 strain was obtained (Stahl et al. al., 1986; Veberet al., 19H). ABRA Co. The coding sequence does not contain introns and is characterized by two repeats. It will be done.

The first structure consists of eight hexapeptide repeats near the center of the coding sequence; The second structure consists of a series of tandem dipeptide and tripeptide repeats; Much of the structure of 2 consists of the amino acid sequences KE and KEE (Stahl et al. al., 1988: Weber et al., 1988).

Limited data suggest that ABRA is fairly invariant among P. falciparum strains. It is something. Partial cDNA clones derived from Fe12 and FC27 strains were CA The genome of the MP strain is almost identical to the ABRA gene. ABRA gene of CAMP and By comparison, the FCR3 clone differs at four positions and the FC27 clone contains several rearrangements within the carboxy-terminal repeat region (Stahl et al. al,, 1988; Veber et al,, 19f18) o sub-2 The common ABRA organisms detected by analysis are six species of P. falciparum. (Stahl et al., 1988). Also , immunoprecipitation analysis showed that A. The dimensions of the BRA are uniform (Chulay et al., 19117; 5t ahl et at, 198B).

Pfhsp70 has significant similarities to components of the mammalian 70 kD heat shock proteins. It is a Blasmodium falciparum protein that shares the same sex (Ardeshir et al, 1987; Blanco eta;, 1988; ne vport et al., 1988), Pfhsp70 is infected during the liver stage (Renia et al., 1990) and in general during the blood stage (Ar Deshir et al, 1987; Blanco et al, 198B) but not in sporozoites (Bianc o et al, 1988; Ren1a et al, 1990) , Suggestions from experiments with human hepatocyte cultures infected with P. falsivarum According to the authors, Pfhsp70 is expressed on the hepatocyte surface during the liver stage (Ren1a et al., 1990). Regarding the position of Pfhsp70 during the blood stage is controversial, and localization has been reported both in the cytoplasm and on the merozoite surface. (Ardeshlr et al., 1987; Blanco et al., 198B). Pfhsp70 has a molecular weight of 75 kD (Ardeshir et al, L987; Bianco et a l,, 1988; Kumar et al., 1988a), 72kD and It has also been reported that the molecular weight of endoubi and Perelra da 511va, 1987).

Complete genome Pfhsp70 gene derived from FCR3 strain and FC27 strain, Hon. Partial Pfhsp70 cDNAs derived from Jurus 1 and 7G8 strains have been obtained. (Ardeshir et al., 1987; Bianco et al. ,, 1988; Kular et al., 198ga; Yang et al., 1987) cs partial cDNA contains approximately 40% carboxy-terminal code. each encodes an identical sequence for a complete gene. starting from a nucleotide (Ardeshir et al., 19117 : Bianco at al.

, 1986; Kumar et al., 1988a) o coding arrangement. The carboxy-terminal part of the string consists of a series of 7-8 direct sequences, many of which are GGMP sequences. Characterized by sequence repetition (Ardeshlr et al., 1987 ; Bianco et al, 1988: Kusar et al, 1988a; Yang et al., 1987). P　f　h　s　 The size of p70 mRNA is 2.8 Kb (Kumar et al. 1988a).

Limited data suggest that Pfhsp70 is associated with P. falciparum strains and isolates. It is considered to be fairly constant between FC27 strain and Honduras 1 strain origin The original partial Pfhsp70 cDNA is identical in the coding region. , differs by only a few nucleotides from the partial cDNA of 7Gg. . The FCR3 genomic gene is very similar to cDNA at its carboxy terminus. with additional GGMP repeats and several nucleotide substitutions There is only a difference in one point. Pfhsp70 detected by Southern analysis The general genomic organization of the carboxy terminus is found in 14 P, falciparum strains. (Ardeshir et al., 1987; Kumar et at.

, 1990). Immunoprecipitation analysis also shows that geographically diverse The dimensions of Pfhsp70 from 0 isolates are uniform (Ardeshi r et at, 1987; Jendoubi and peretra dasllva, 1987) OL between three types of strains. Some differences were observed in the map of Jendoubi peptides (Jendoubi and Perefra da 5ilva, 1987).

The function of Pfhsp7Q in the parasitic surface life cycle is unknown. deer However, the induction of Pfhsp70 expression during the binucleate stage after sporozoite infection of hepatocytes Studies suggest that this heat-shock-like protein contributes to the differentiation of the parasitic surface. (Renia et al., 1990).

AMAl was originally known as Plassodium know I. As an 88kD protein (PK66) from red blood cells infected with This is an isolated late meristem protein. PK66 is processed during merozoid release. 44/42 kD components, and these ripened products associate with the merozoite surface. I get kicked. PK66, isolated in its native form, induces inhibitory antibodies and induces blood loss in rhesus macaques. Protected against liquid phase counter-doses (Deans et al., 1988). bra The PK66 equivalent in Summodium falciparum is a human antimalarial antibody (Pe Terson et al., 198g) or rabbit anti-PK66 Voliclo PF It is also called 33.

In Blasmodium knowlesi, AMAI is synthesized at the late stage of schizogony and separated. It is distributed at the apex of the merozoite that develops within the meristem during the fissure. due to rupture of the mesotide AMA 1 is processed and becomes a 44/42 kD duplex (Waters et al. al., 1990). During the invasion of red blood cells, the 44/42 kD duplex is present in red blood cells. It is not translocated to the surface and remains attached to the entry interface.

In Blasmodium falciparum, AMAI is located at the apex of the divided meristem. but may also be located on the merozoite surface (Peterson et al. al,, 198g). AMA 1 is probably first located in the vertex complex and then transported to the merozoite surface. AMAI becomes invisible during red blood cell invasion and cannot be found in newly infected red blood cells.

AMAI has different bras such as CampSFCR3, 7G8 Thailand TNSFC27. It is extremely consistent among Summodium falsivarum isolates (Thomas falciparum et al., 1990).

The AMA1 gene has a length of 1888 bp, and no introns have been reported. It encodes a protein consisting of 822 amino acids with no repetitive sequences. child The protein has a structure expected for an essential membrane protein. That is, two hydrophobic The first part is located near the N-terminus and acts as a signal peptide. The second portion is located 55 amino acids from the C-terminus.

Pfs25 is a P falciparum protein expressed during the sexual phase of parasitic surface development. Ru. This 25kD membrane protein is located on the surface of the conjugate auxinate (V ermeulen et al., 1985), and as a result, probably and not in the human host (Carter et al. Kaslov et al., 1989).

The Pfs25 gene derived from the 3D7 clone of P. falciparum NFS4 strain is An 854 bp non-interrupted sequence encodes a protein with a predicted molecular weight of 24.1 kD. Consists of an open reading frame (Kaslow et at, 198 g) o The predicted amino acid sequence has a hydrophobic signal peptide at the N-terminus and a C-terminal It has a short hydrophobic anchor sequence on the surface, consistent with the surface arrangement of Pfs25. . In addition to four potential N-glycosylation sites, the Pfs25 coding sequence contains Contains the predicted cysteine residue organism and contains four tandemly repeated EG The existence of an F-like region is suggested (Kaslow et al., 1988). P fs25 is highly invariant and has been shown to be highly invariant in eight geographically diverse isolates. Only one single base substitution was detected (Kaslov et al. , 1989).

Antibodies against Pfs25 have not been detected in humans in endemic areas. This is o This is probably because this protein is not expressed in the human host (Carter et al., 1988), by immunization of the H-2 congenic mouse strain. Anti-Pfs25 antibodies were produced in all systems tested, indicating that this protein is a good immunogen. It has been suggested that there is (Good et al., 198Jl).

Anti-Pfs25 mAbs demonstrate the ability to block parasitic transmission from vertebrate hosts to mosquitoes. Therefore, Pfs25 is considered a potential vaccine candidate (Kas low et at, 1989). Additionally, the surface produced by the vaccinia recombinant Mice immunized with expressed Pfs25 also produced infection-blocking antibodies after three vaccinations; The production of antibodies by such vaccinia recombinants is limited to specific MHC haplotypes. (Kaslow et al., 1991).

Pfs16 is expressed by sporozoites and the sexual phase of the parasitic fungus developmental cycle P, falciparum protein.

This IBkD protein is found on the membrane of intracellular gametocytes and is probably It is also found on the outer macrogametes and on the sporozoite surface (Moelans et al. al,, 1991a) o P f s 16 genes have a length of 544 bp The coding sequence includes a predicted N-terminal signal sequence, a hydrophobic anchor sequence, and a and a highly hydrophilic C-terminus.

Special table 16-505397 (7) Pfs16 is highly invariant among P. falciparum isolates. researched 8 Among the different strains, differences were observed in only two isolates; and only contained three acid substitutions (Moelans et al. 1991b).

Pfs16 is considered a vaccine candidate for several reasons. First, Sporo Because Pfs16 is expressed by both zoites and sexual stages, this protein is a multistep protein. This makes them attractive for inclusion in vaccines. Because for this Immunity may protect against sporozoite infection and sexual transmission It is from. Of note, using four types of Pfs16-specific mAbs, In preliminary experiments, no inhibition of sporozoite invasion in vitro was detected. (Targett, 1990). Second, areas with extremely high endemic diseases Serum derived from adults living in the United States has been shown to recognize Pfs16; is suggested to be an immunogen in humans (Moelans at al. , 1991a). Third, stimulated polyvalent tumors against gametes and gametocytes Heron serum recognizes Pfs16 and has a high ability to inhibit infection. 2N class P Preliminary experiments using mAbs specific for fs16 indicate that the antibody 1 have the ability to inhibit transmission (Moelans et al., 1991a ).

The ring sporozoite protein (CS) of P. falciparum is an 80 kD membrane protein. and are uniformly distributed on the sporozoite surface (Nussenzweig et al. al,, 19g4). CS is not expressed at other stages of the parasitic fungus life cycle. do not have.

The C8 gene consists of an uninterrupted open reading frame of approximately 1200 bp. . C8 consists of a central region consisting of the repeat sequence NANP and some heterologous NVDP repeats; characterized by non-repetitive regions containing charged residues flanked by (D ame et al, 19g4). Repetitive NANP sequences are invariant However, the number of repeats can vary between different isolates. Differences in non-repetitive areas Differences are observed near the amino terminus due to insertions or deletions, whereas differences in the carboxyl The terminal region contains only base pair substitutions (Caspers et al., 1989). Of the 412 amino acids of C8, they are separated by three distinct polymorphic regions. It is known that only 13 positions differ (Caspers et al. al., 1989). In the non-repetitive region among Plasmodium spp. Three areas were found to be fairly constant. These are in the N-terminal region Area I in the C-terminal region and Areas 11 and III in the C-terminal region (Lock er and Holder, 1989).

Both humoral and cellular immune responses to C8 induce anti-sporozoite immunity. It is thought that this contributes to In humoral reactions, naturally protected humans have C It has antibodies against 8 proteins, and these antibodies increase with age. Parallel to target immunity (Nussenzvelg and Nussenzwelg , 1989). However, C8 levels and sporozoites in naturally infected adults Hoff-specific antibody levels do not correlate with protection against further infection (Hoff Man et al., 1987), other factors such as cell-mediated immunity contribute to innate immunity. It is suggested that this may be important. However, some studies have revealed They found that humans were protected by immunization with irradiated sporozoites. (Clyde, 1975; Rleclvann, 1974 ), this protection was correlated with antibodies against the C8 protein (Nussenzvei g et al., 19115). Using CS-based peptide subunits Human vaccine trials have shown that such structures induces a specific antibody response and provides complete protection for some vaccines. (Herrington et at, 1987; Ba1lou et al., 1987).

Cellular responses to C8 protein were also studied. Human P, falciparum Several T cell epitopes were identified in the C8 protein (Good et al., 1987).

Interestingly, many human T cell epitopes occur in the C8 polymorphic region. , mutation and selection of parasitic bacteria occur in response to T cell-derived immune pressure. It is suggested.

However, one of the human T helper epidomes, CS, T3, is a C8 protein. It is located in the constant region of the human body and is associated with many different human MHC class 11 molecules. recognized by human T cells (Sinigagla et al., 1999). 8g). In addition, sporozoites specifically target the CD8+ CTL epitope on the C8 protein. can induce heterogeneous cytotoxic T cells (Kumar et al., 1999). 88b), such cells are effective in inducing immunity against P. falciparum. It is suggested that it may be important.

Provision of malaria recombinant poxvirus and protection against plasmodium infection provide protective immunity or an immune response to the Blasmodium immunogen in the host. Providing a vaccine that stimulates the It is thought to bring about sex. Similarly, such malaria recombinant pox viruses are also highly desirable for in vitro production of plasmodium immunogens. Therefore, it is an object of the present invention to develop recombinant organisms expressing the gene products of Plasmodium to provide poxviruses, and to provide such recombinant poxviruses. The purpose is to provide a manufacturing method.

An additional object of the present invention is that poxvirus vectors, in particular vaccinia and fowlpox, can also be or within avivox viruses such as canarypox virus, especially 5 ERA, ABRASPfhsp70 and AMAI plasmodium blood stage antigen. and plasmodium antigens such as Pfs25, Pfs16 and CS plasmodium antigens. The aim is to clone and express the Zium coding sequences.

Another object of the invention is to provide malaria antibodies and protection against Plasmodium infection. The objective is to provide a vaccine that can induce immunity. Further aspects of the invention The purpose is to be useful for the production of plasmodium immunogens in vivo or in vitro. Our objective is to provide a malaria recombinant poxvirus and recombinant immunogen. .

These and other objects and advantages of the present invention will be apparent from consideration of the following: It will become clear easily.

Disclosure of invention In one embodiment, the present invention provides plasmonoma in essential regions of the poxvirus genome. The present invention relates to a recombinant poxvirus containing a DNA sequence derived from Z. pox The virus is preferably vaccinia virus or fowlpox virus or canary virus. It is an avivox virus, similar to the pox virus.

According to the present invention, the recombinant poxvirus is derived from a foreign Plasmodium gene. Express the product. In particular, foreign DNAs include 5ERA, ABRASPfhsp70, Encodes AMAI, Pfs25, Pfs16 or CS gene.

Preferably, more than one Plasmodium gene is harbored by the recombinant poxvirus. Co-expressed within the main. Furthermore, the present invention uses a malaria recombinant poxvirus. Plasmodium gene products either in vivo or in vitro and recombinant gene products.

According to another aspect, the present invention provides a method for inducing an immune response in an inoculated host animal. Regarding vaccines for children. This vaccine contains a carrier and a recombinant poxvirus. The recombinant poxvirus contains Plasmodium-derived DNA in its essential region. Contains A. The present invention also provides a method for inoculating animals with a malaria recombinant virus. and a method for inducing such an immune response in an animal. preferably DNA is 5ERA, ABRASPfhsp70, AMAI, Pfs25, Pf Encodes or expresses the s16 or CS plasmodium gene. preferably allows multiple Blasmodium genes to be expressed simultaneously in the host. Vaccine of the invention and the poxvirus used in the method is preferably vaccinia virus or is an avivox virus such as fowl pox virus or canary pox virus. be.

Brief description of the drawing The present invention will be better understood by referring to the following accompanying drawings. .

Figure 1 is a schematic diagram showing the 5ERA coding sequence, Figure 2 is on p126.15. Nucleotides of 5ERA cDNA (SEQ ID NO: 1) and predictions Figure 3 shows the amino acid (SEQ ID NO: 2) sequence of pABRA. -8 ABRA cDNA nucleotide (SEQ ID NO: 3) and Figure 4 shows the predicted amino acid (SEQ ID NO: 4) sequence. i (nucleotides of partial cDNA of Pfhsp70 in SP70.2 ( SEQ ID NO: 5) and predicted amino acids (SEQ ID NO: 6) ) Diagram showing the array, Figure 5 shows the nucleotides (SEQ ID NO near) of the 3D7 strain AMA1 gene. and the predicted amino acid (SEQ ID NO: 8) sequence.

Detailed description of the invention The present invention provides DNA sequences derived from Plasmodium in essential regions of the poxvirus genome. Recombinant poxvirus containing the sequence. Recombinant poxviruses are foreign Expressing the gene product of a Blasmodium gene. For example, P. falciparum The gene was expressed in a living recombinant poxvirus. This expression causes this These recombinants can be used as vaccines to stimulate an immune response against the gene product. or in vitro, for example for subsequent use of the product as an immunogen. Useful for the production of gene products.

5ERASABRA, Pfhsp70, and AMAI P, falciparum blood Liquid phase genes have been isolated and classified, e.g. vaccinia, canarypox and virus. Poxviruses such as Pfs25, Pfs16, and C P of 8 was inserted into the falciparum gene.

The invention is illustrated by non-limiting examples (below), which do not limit the invention. This invention should not be construed as limiting, and should not be construed as limiting the spirit or scope of the invention. However, many changes are possible. In the embodiments of the present invention, the following methods and and raw materials were used.

Enzymes, bacteria, and plasmids. Restriction enzymes and other DNA modifying enzymes are Ger Mannheim [Boehringer Hanheii (Indlanap olis, IN)], Ninny England Biolabs [New Engl. and Blolabs (Beverly, MA)], and BRL Li Futechnology Inc. [BRL Life Technology 1nc, (Gaithersburg, MD)] and manufactured unless otherwise noted. It was used according to the recommendations of the author. Standard molecular cloning methods were then performed ( Sambrook et at, 1989).

E. coli XL-1 blue and 5URE strains were treated with stratazine [Stratage (La Jolla, CA)], and the NM522 strain was obtained from IB I (New Heaven, CT). Plasmid vector pUc 19 is two-year-old England Biolabs [New England Blol] ABS (Beverly, MA)].

Cell lines and virus strains. Vaccinia group containing the plasmodium blood stage gene The replacement is Copenhagen vaccinia strain or NYV (with diminished national power) AC (vP866) (Tartaglia et al, 1992) Rescue breeding using the senior strain or the vP668 vaccinia recombinant. Generated as a rule. All vaccinia virus shocks are 5-10% de novo. Add live serum (Plow Laboratories, McLean, VA). Vero cells (ATCCCCCL81) in Eagles MEM medium supplemented with were generated in either MRC5 (ATCC CLL71) cells. P, fal'Cie The canarypox recombinant containing palmus (with diminished strength) ALVAC strain was used to generate a rescue virus (Tartaglla et al. al., 1992).

Polymerase chain reaction (PCR) o GeneAmp-DNA amplification kit (P erkln Elser Cetus, Norvalk, CT) from the manufacturer. PC using custom synthesized oligonucleotides as primers according to specifications. (Saiklst al., 198B), the reaction was thermal φCycler - [ThersalCycler　(Perkln　Elser　C etus) under standard conditions (Saiki et at, 19 88).

Formation of P. falciparum FCRB strain blood stage library.

P, total RNA derived from human erythrocytes infected with P. falciparum FCR3 strain. Plus Exp ± [Dr, P, Delplace (INSERM-U42.36 9 rue Jules-Guesde, 59650 Villeneuve -D'Ascq, France). Avlv and Leder (Avlv and Leder, 1972) and Kixton (Klng st.

Oligo(dT) cellulose (Str atagene, La Jolla, CA) from this sample. -A” RNA was isolated. Briefly, total RNA was separated into binding buffer y (0 The oligo( dT) and incubated with cellulose for 30 minutes at room temperature. Poly-A”R The NA/oligo(dT) cellulose complex was pelleted by centrifugation and bound to the binding buffer. Washed three times with buffer. Elution buffer y (0.01M Tr i s-CI, p Elute purified poly-A” RNA from oligo(dT) cellulose in H-7,5) did. Perform a second elution with DEPC-treated dH,O and pool the eluents. , poly-A'' RNA was recovered by ethanol precipitation.

First strand cDNA by reverse transcriptase in an oligo(dT) primed reaction Purified poly-A+RNA was used as a template for synthesis (K11ckste1n an d Neve, 19117; Vatson and Jackson, 1 985). For this reaction, 12 ug of poly-A'' RNA was added at pH-8,3 and 105 units of AMV reverse transcriptase (Llfe) in 100 sH Tris-CI. 5 sciences) 30mM KCI.

6sM MgCl, 25mM DTT, 80 units of RNazine, 1 piece each. of dNTPs, and 24ug/ml of oligo(dT) used as primer. Incubated with 1z-+s for 2 hours at 42°C. After organic extraction, the first Using DNA polymerase I and RNase H using single-stranded cDNA as a template Double-stranded cDNA was obtained by (Klickstein and Neve, 1987; Watson and Jackson, 1985). Sunawa First, the first strand cDNA was dissolved in 25 units of Tris-CI at pH-6 and 20 sH. of DNA polymerase I and 1 unit of RNase H15wM MgC12,1 0mM CNHa’) 2SOs, 1001% KCl, 500 With ug/sl BSA, 255M DTT, and 0°isM each dNTP. Synthesize the second strand cDNA by incubating for 1 hour at 12°C and then 1 hour at room temperature. accomplished. Double-stranded cDNA was recovered by solvent extraction and ethanol precipitation.

This double-stranded blood stage cDNA was then treated sequentially with T4 DNA polymerase to create two Full chain ends were formed and the internal EcoR1 site was protected with EcoRI methylase. So After adding EcoRI linker, digest with EcoRI and reduce to 5-25% Dimension selection was performed on the loin slope. Fragments containing long cDNAs (1-10 Kb) Lambda ZAP II vector ( Stratagene, LaJolla, CA). obtained The phages were collected and E. coli XL-1 blue strain (Stratagene, L a Jolla, CA). Then from these cells Collect the phage and amplify it by infecting it again with XL-1 Blue. , generated a high titer FCRB strain blood stage C DNA library.

cDNA Library Sfree for Plasmodium Blood Stage cDNA Clones ring. FCRB strain cDNA library derived from published blood stage gene sequences Bractu1 hybridization with oligonucleotides labeled at the 32p end to -CDNA was screened to detect cDNA. cDNA library XL-1 Blue (Stratagene, La Jolla, CA) A density of 100,000 plaques/dates on the bacterial flora. It was distributed as follows. The braque was transferred to a nitrocellulose filter and then 1. 5M NaCl 10, 5M NaOH for 2 min, 1.5M NaCl/pH-8 Katsu 0.5M Tris-CI for 5 minutes, pH-7.5-) 0.2M T Immerse in r i s-CI/2X SCC for 1 minute, then soak in vacuum ozone at 80℃ for 2 hours. It was heated for a while. The filters are 6X SSC, 5X Denhard. ts), 20mM NaH4F 04% 500ug/II salmon sperm DN Prehybridization was carried out in A for 2 hours at 42°C. Hybridization After addition of S-labeled oligonucleotide at 32p, 0.4% SDS, 6X of S CC, 205 M NaH2PO, and 500 ug/ml salmon sperm D The test was carried out in NA at 42°C for 18 hours. After hybridization, filter Rinse 3 times with 6X SSc, o, i% SDS and wash for 10 min at room temperature, 58 Washed at ℃ for 5 minutes. The filter was then exposed to X-ray film at -70”C. I did it.

Remove plaques that hybridize with the oligonucleotide probe from the dish. Take a sample and add it to SM buffer containing 4% chloroform (100mM Na). C1s 8mM MgSO4, pH=7.5 and 50sH Tris-CI, 0.01% gelatin). Dilution of such phage stocks Plaques were transferred to nitrocellulose and plated with The filter was hybridized with a 32p-labeled oligonucleotide. good Positive plaques that were well isolated were selected and the above purification process was repeated two more times. .

Isolation of Plasmodium cDNA-containing plasmids from positive phage clones. La Muda ZAP II vector (Stragene.

In Vivo Ablation Protocol Developed for Use in La Jolla, CA) pBluescript (pBluescrfpt> in plasmid vector) Plasmodium cDNA was obtained. Briefly, purified recombinant lambda image stock with XL-1 blue cells and R408 filamentous helvaphage. Incubated at 87°C for 15 minutes. 2X YT medium (1% NaCl, 1% yeast extract, 1.6% bactotributone), then incubation. The reaction was continued for 3 hours at 37°C and then for 20 minutes at 70°C. centrifugation After that, pBluescript phagemid (with cDNA insert) Filamentous phage particles containing sld) were collected from the supernatant. Recovered linear fiber dilutions of the plasmid stock were mixed with XL-1 Blue and plated. Colonies containing the pBluescript plasmid with a mu cDNA insert were Obtained.

DNA sequence analysis of Blasmodium genes. The plasmodium gene is p bluesc. or cloned into another plasmid vector. Ta. DNA sequencing was performed using sequencease modified T7 polymerase (U, S, Bloeheaicals, C1eveland, OR) It was. Sequencing reactions were performed using T3 and T7 primers or custom synthesized Alkaline-denatured double-stranded plus oligodeoxyribonucleotides It was performed on a mid-mold (Hattori and 5akak1.lH&). Distribution Column data was collected using IBI Pastel Sequence Analysis Package (IBI Pu5t). ell 5equence Analysis Package)Version 2.02 (International Blotechnologies, New Haven, CT).

P, Formation of vaccinia recombinants containing the falciparum gene. P. Falcipa The rum gene is a poxvirus promoter for expression by vaccinia vectors. cloned to be under the control of. The promoter used by the inventor was Kushina early/late H16 Bromota (Perkuset at, 19g9 ), Vaccinia 13L first Pi or Cl0LW initial promoter (Wach Sman et al., 1989), vaccinia 13L early intervening promoter Perkus et at, 1985; Schmitt and S 198g) and entomopoxvirus 42. promoter (Gettig et al., unpublished). Ru.

The P. falciparum gene was then prepared for insertion into vaccinia virus. Therefore, it must be cloned into a vaccinia donor plasmid. pcOP cs-5E and pcOPcs-6H donor plasmids have been previously reported. (Perkus et al., 1991).

Plasmid pSD553 is a C0PAK-based vaccinia deletion/insertion plasmid. Ru. This plasmid was inserted into vaccinia K flanked by Copenhagen vaccinia arms. IL host range gene (Gllard et al., 198B) was inserted into the ATI region. area (orfs A25L, A28L; Goebel at al, 19 90).

pSD553 was formed as follows. The left and right vaccinia lateral arms are Senior 5 a I I B (Goebel et al.

, 1990) (7) p UC8 h-7, (Dya-n t'J le p S D4 140 as a template by polymerase chain reaction. The left arm Synthetic deoxyoligonucleotide MPSYN267 (SEQ ID NO: 9) (5'-GGGCTGAAGCTTGCTGGCCGCTCATTAGACAA GCGAATGAGGAC-3') and MP S YN268 (SEQ ID No.: 10) (5’-AGATCTCCCGGGCTCGAGTAA TTAATTAATTTTTTATTACACCAGAAAAGACGGCTTG AGATC-3') was used as a primer. The right arm is synthetic deoki Thioligonucleotide MPSYN269 (SEQ ID NO: 11) (5'- TAATTACTCGAGCCCGGGAGATCTAAATTTAATTTAA TTTATATAACTCATTTTTTTGAATATACT-3') and and MPSYN270 (SEQ ID NO: 12) (5’-TATCTCGA ATTCCCGCGGCTTTAATGGACGGAACTCTTTTCCC -3') was synthesized using as a primer. Two PCs containing left and right arms The R-derived DNA fragments were combined in a new PCR reaction. The resulting product was labeled with EcoRI The /H piece was ligated with pUC8 cut with EcoRI/HindII1. , plasmid psD541 was obtained. Polyrin located at the vaccinia deletion locus The power area has been expanded as follows. Turn off psD541 with Bg 111/Xho I Cleaved and annealed complementary synthetic deoxyoligonucleotide MPSYN33B ( SEQ ID NO: 13) (5’-GATCTTTTGTTAACAAAA ACTAATCAGCTATCGCGAATCGATTCCCGGGGGGATC CGGTACCC-3') and MPSYN334 (SEQID NO: 1 4) (5’-TCGAGGGTACCGGATCCCCCGGGAATCGA TTCGCGATAGCTGATTAGTTTTTTGTTAACAAAA-3' ) to form plasmid pSD552. Plasmid pSD55 2 to the KIL host range gene as a lkb Bglll (partial)/HpaI fragment. (Perkuset at, 1990), pSD552 was isolated from B g±II/Hpal and ligated with the KIL-containing fragment to form pSD553. accomplished.

Plasmid pMP 13H is the first vaccinia 13L in pUC8 background. Contains phase/late promoter elements (Schmitt and Stunn enberg, 198g) Synthesis using promoter element pMPVc1 as a template Oligonucleotide MPSYN283 (SEQ ID NO: 15) (5'-C CCCCCAAGCTTACATCATGCAGTGGTTAAAC-3') and MPSYN287 (SEQ ID No: 16) (5’-GATTA Polymerase chain reaction (P CR). The DNA obtained in this reaction was prepared using Hindl II and Rsal and the 0, lkb fragment containing the promoter element was purified. Supplementary synthesis ori Gonucleotide MPSYN398 (SEQ ID NO: 17) (5'-ACA ATTATTTAGGTTAACTGCA-3') and MPSYN399 (SEQ ID NO: 18) (5’-GTTAACCTAAATAATTG The linker region was assembled by annealing T-3'). P The CR-derived promoter element and polylinker region were combined with Hindl II and Pst It was ligated with vector plasmid pUC8 cut with I. Plasmi obtained in this way pMP I 3H has a 23L protein located at -100 to -6 relative to the start codon. It has a motor region, followed by HpaI and PstISSal promoters. A double-stranded terminal DNA linearized at the 16th position is generated.

The pSD544 insertion vector was obtained as follows. pSD456 contains the HA gene ( ＾56R; Goebel et at, 1990) and the surrounding area. This is a subclone of Copenhagen vaccinia DNA. A36RORF p as a polymerase chain reaction template for the synthesis of the sandwiching left and right vaccinia arms. SD456 was used. The left arm is a synthetic oligodeoxynucleotide MPSYN2 79 (SEQ ID No: 19) (5’-CCCCCCGAATTCGT CGACGATTGTTCATGATGGCAAGAT-3') and MPS YN280 (SEQ ID NO: 20) (5'-CCCGGGGGATCC TCGAGGGTACCAAAGCTTAATTAATTAAATATTAGTA TAAAAAAGTGATTTATTTTTT-3') was used as a primer. accomplished. The right arm is a synthetic oligodeoxynucleotide MPSYN281 (SEQ ID NO:21>(5’-AAGCTTGGTACCCTCGAGGGA TCCCCCGGGTAGCTAGCTAATTTTTCTTTTACGTAT TATATATGTAATAAAACGTTC-3') and MPSYN312 (SEQ IDN0: 22) (5’-TTTTTTCTGCAC; GT AAGTATTTTTTAAAACTTCTAACACC-3') as a primer. It was synthesized using Gel-purified left and right arm PCR fragments can be used in a new PCR reaction. Combined by The obtained product was cleaved with EcoRI/HindII did. The obtained 0.9 kb fragment was gel purified and preliminarily purified with EcoRI/HindlII. The plasmid pSD544 was obtained by ligation into pUCg which had been cut with .

Plasmid psD550 was obtained as follows. Plasmid p SD 54 8 (Tartaglia et at, 1992) is BglII and Σ I4L 0RF (Goebel et at., 1990) is replaced with a vaccinia vector plasmid.

To expand the multiple cloning region, pSD548 was cloned into Bgll and Sm Complementary synthetic oligonucleotide cut with aI and annealed)’MPSYN539 A (SEQ ID NO: 23) (5’-AGAAAAAATCAGTTAG CTAAGATCTCCCGGGCTCGAGGGTACCGGATCCTGA TTAGTTAATTTTGT-3') and 539B (SEQ ID N O:24>(5'-GATCACAAAAATTAACTAATCAGGATC CGGTACCCTCGAGCCCGGGAGATCTTAGCTAACTGA TTTTTTCT-3'). The thus obtained plasmid psD The multiple cloning regions in 550 are BglII, Gyoukoal, ” and a BamHI restriction site.

Plasmid pSD542 was obtained as follows. To change the polylinker area Plasmid pSD513 (TartaglIa et al., 1992) was cut with PstI/BamHI and annealed. Otido MPSYN288 (SEQ ID NO: 25) (5'-GGTCGA CGGATCCT-3') and MPSYN289 (SEQ ID NO: 26) (5'-GATCAGGATCCGTCGACCTGCA-3') plasmid pSD542 was obtained.

Kpnl, Sma1, 1535 bp upstream of C5. %Xba　I, and Not A polylinker containing 1 part of the polylinker and 404 bp of canarypox DNA (31 bp of pNVQH6C containing the 05 coding sequence and 373 bp downstream sequence) 5SP18 ALVACC5 insertion vector was obtained as follows. pRW746 ．． 5 and the identified clone containing the 29 kb insert (pHCO8I). Genomic library of canarypox DNA in cosmid vector pUK102 as a library. formed a li.

A 3.3 kb C1ar fragment from pHcOs1 containing the C5 region was identified. . The sequence was expanded from nucleotides 1-1372 using C1al fragment analysis.

The C5 insertion vector was formed as follows. The 1535 bp upstream sequence is an oligo Nucleotide C5A (SEQ ID NO: 27) (5'-ATCATCGA ATTCTGAATGTTAAATGTTATACTTG-3') and C 5B (SEQ IDN0:28) (5’-GGGGGTACCTTTGAG AGTACCACTTCAG-3') and purified genomic canary acid DNA were used as templates. Formed by PCR amplification and digested with EcoRI/Σmal to be used as template. It was cloned into pUC8 to obtain pC5LAB. The 404bp arm is I)’: f5Z’ZLiyFtide C5C (SEQ ID NO: 29) (5’ -GGGTCTAGAGCGGCCGCTTAAAAGATCTAAAATG CATAATTTC-3') Oyobi C3DA (SEQ ID NO: 30) ( 5'-ATCATCCTGCAGGTATTCTAAAACTAGGAATAGA PCR amplification using TG-3') was performed and cloned into a trowel shape to obtain pC5L. Ta. pC5L is digested with Te Asp 718o and No. treated with alkaline phosphatase, kinased and annealed f::4- 1) Gonuk I, #chiFCP26C8EQ ID NO: 31) (5’-GTA CGTGACTAATTAGCTATAAAAAGGGATCCGGTACCCT CGAGTCTAGAATCGATCCCGGGTTTTTATGACTAGT TAATCAC-3') and (disabled Asp718 site, 6 leads Translation stop codon consisting of a n and Mo5s.

Positive signal, translation stop core (SEQ ID N) consisting of six reading frames O:32) (5'-GGCCGTGATTAACTAGTCATAAAAAC CCGGGATCGATTCTAGACTCGAGGGGTACCGGATCCT TTTTATAGCTAATTAGTCAC-3') and plasmid. pC5LSP was formed. Early/late H6 vaccinia virus promoter (P Erkus et al., 1989) used the oligonucleotide CP30 (S EQ IDN0:33) (5’-TCGGGATCCGGGTTAATTAA TTAGTCATCAGGCAGGGCG-3') and CF3I (SEQ ID NO: 34) (5'-TAGCTCGAGGGTACCTACGATAC Promoted by PCR using AAAACTTAACGGATATCG-3') obtained from a plasmid containing the data. PCR products are BamHI and Xhol (sites formed at the 5' and 3' ends by PCH), and similarly digested pC5LSP was ligated to form pVQH6C5LSp. pVQ H6C5LSP was digested with EcoRI, treated with alkaline phosphatase, and Self-annealed oligonucleotide CP29 (SEQ ID NO: 35 ) (5’-AATTGCGGCCGC-3’) and erased with NotI. and linear purification followed by autoligation. By this method pV Not1 site was introduced into QH6C5LSP, forming pNVQH6c5sP18 It was done.

1-) 17) The pNC5LSP-5 plasmid, which is the ALVACC5 vector, is Obtained as follows. Plasmid pC5LSP was digested with EcoRI and Oligonucleotide CP29 treated with phosphatase and self-annealed (SEQ ID NO: 35) (5'-AATTGCGGCCGC-3') ligated, digested with NotI, linearly purified, and then subjected to autoligesidin. Ta. By this method, the No.±.1 site was introduced into pC5LSP, and pNC5LSP -5 was formed.

Insertion plasmid VQCP3L was obtained as follows.

The 8.5 kb Canarypox l±II fragment was inserted into the Ba of the pBS-5K plasmid vector. Cloned into the mH1 site to form pWW5. By nucleotide sequence analysis A reading frame called C3 was revealed. 03 O in 03 seat To insert a foreign gene that completely excises the leading frame PCR primers were used to generate a donor plasmid for C3. ' and 3' sequences were amplified. The primer for the 5' sequence was RG277 (SE Q ID NO: 36) (5'-CAFTTGGTACCACTGGTATTT TATTTCAG-3') and RG278 (SEQ ID NO: 37) (5 ’　-TATCTGAATTCCTGCAGCCCGGGTTTTTATAGC TAATTAGTCAAATGTGAGTTAATTAG-3') Ta. The primer for the 3' sequence was RG279 (SEQ ID NO: 3g) (5 ’　-TCGCTGAATTCGATATCAAGCTTATCGATTTTTT ATGACTAGTTAATCCAAATAAAAAGCATACAAGC-3' ) and RG280 (SEQ ID NO: 39) (5'-TTATCGA GCTCTGTAACATCAGTATCTAAC-3'). Primer contains multiple cloning sites flanked by vaccinia transcription and translation signals. was configured. In addition, appropriate restriction sites are placed at the 5' and 3' ends of the left and right arms. (Asp718 and Ec on the left arm.

RI, the right arm contains EcoRI and 5acl), and these The two arms are pBS-5K plus digested with Δ5p718/SacI. It could now be ligated into midvector. The plasmid thus obtained The code was named pC31. 908 bp fragment of canarypox DNA immediately upstream of locus 03 The fragment was obtained by digesting plasmid pWW5 with approximately 1±l and 5 spl. . Canarypox and ao4bp fragments of DNA were produced using plasmid pWW5 as a template. , oligonucleotide CP16 (SEQ ID NO: 40) (5'-TC CGGTACCGCGGCCGCAGATATTGTTAGCTTCTGC- 3') and CP17 (SEQ ID NO: 41) (5'-TCGCTCG P using AGTAGGATACCTACCTACTACCTACG-3') Obtained by CH. The 604 bp fragment contains mu1λ718 and Xhol (oligonuclease). Digested with the sites present at the 5' ends of leotide CP16 and CP17, respectively). , Δ5p718-digested with XhoI and treated with alkaline phosphatase. I B I 25 (International Biotechnolog ies, Inc., New Haven, CT) and Sumid 5PC3LA was formed. 5PC3LAHa I B I E in 25 C0Rv, digested with N1±l in canarypox DNA, resulting in a 908 bp Ns± 1444 bp of canarypox DNA upstream of the 03 locus. 5PCPLAX containing the following was formed. 2178 bp canarypox DVA<7) The BglI l-5tyl fragment (isolated from pBS-8K. Using plasmid pXX4 as a template, the Leapox DNA fragment was added to the oligonucleotide CP. 19 (SEQ ID NO: 42) (5’-TCGCTCGAGCTTTTCT TGACAATAACATAG-3') and CP2O (SEQ ID NO. :43) (5'-TAGGAGCTCTTTACTACTGGGTTAC Separated by PCH using AAC-3'). The 279bp fragment was and 5acl (at the 5' end of oligonucleotides CP19 and CP2O) 5acl-Xhol and is digested with alkaline phosphate. Cloned into sphatase-treated IBI25 and plasmid 5PC3RA was formed. To add additional unique sites to the polylinker, pC31 was Digested with EcoRI and 1al in the lysine power region and alkaline phosphatide. atase-treated, kinased and annealed oligonucleotide C P12 (SEQ ID No: 44) (5’-AATTCCTCGAGGG ATCC-3') and (EcoRI adjacent end, XhoI site, BamH1 site , and a cohesive end compatible with C1al) CP13 (SEQ I D NO: 45) (5'-CGGGATCCCTCGAGG-3') to form plasmid 5PCPBS. 5PCP3S is a canary downstream of the 03 locus Σty in the smallpox array! and 5ac1 (pBS-SK), 5P The 261 bp I±ll-1 Earthwork I fragment from C3RA and the 2 from pXX4 Ligated to the 178bp Bg±ll-8tyI fragment, 2572b downstream of the 03 locus. A plasmid CPRAL containing canarypox DNA of p. 5PCP3 S is Δ5p718 (in pBS-SK) within the canarypox sequence upstream of the 03 locus. and Δcanarypox DNA-containing plasmid CPLAL was formed. instructor The resulting plasmid was named 5PCP3L. pSPCP3L in Xmal The digested, linearized plasmid is treated with phosphatase, annealed and Kinase-treated oligonucleotide CP23 (SEQID NO: 46) (5’-CCGGTTAATTAATTAGTTATTAGACAAGGTG AAAACGAAAACTATTTGTAGCTTAATTAATTAGGTCA CC-3') and CP24 (SEQ ID No: 47) (5'-CC GGGGTCGACCTAATTAATTAAGCTACAAAATAGTTTC GTTTTCACCTTGTCTAATAACTAATTAATTAA-3') VQCPCP3L was obtained by ligacydin with.

The ALVACC6 insertion vector is a 1815 bp S a c I / K pnl fragment. This fragment contains pBS, SK vector (Stragene, La Jol! a, Contains the C6 region of ALVAC inserted into CA).

A polylyline force region was introduced around position 400 of the C6 sequence. This position has six links. a translation stop region consisting of a coding frame, a bidirectional early transcription termination signal, and a sequence of Restriction enzyme site for cloning (SmaI, PstIsXhoI s and EcoRI).

Rescue pox virus (Copen/Han-gen vaccinia virus, N Insertion of the inserted vector into tissue culture cells infected with YVAC, ALVAC) Identification of recombinants by fection and in situ hybridization has already been done. (Plcclnl et al., 1987).

Immunoprecipitation analysis of plasmodium antigens expressed by poxviruses. 5ER Rabbit anti-5ERA sera for expression analysis of A-containing recombinants were P, Del Plus Bo ± [ Dr, P, Delplace (INSERM-U42.389 rue Jutes-Guesde, 59850 Villaneuve-D'asc q, France)]. Aphids with high antimalarial potency Antimalarial human immunoglobulin derived from a human donor was produced by M. Hommel, Hiroshi [Ko, M, Hou+el (Llverpool 5chool or Tropl Cal Medicine)], (Pfhsp70 and AMA-1 was used for expression analysis of recombinants (such as those containing ABRA specific mAb3D5 and anti-C8 repeat and anti-repeat C8 sera.

Dr. D. Lanar (iAIR, Washington) , D, C,)]. Pfs25-specific mAb 4B7 beam , to Hiroshi Kaslow [Dr, D, Kaslow (NIAID, NIH)] Provided by. Rabbit serum specific for MSA-1 was obtained from S. Chang Bo± [Dr. S. Chang (University of Hawaii)] provided.

Immunoprecipitation was performed using a method previously reported (Taylor et al., 1990). ). Briefly, Vero cell monolayers are injected with recombinants or mother cells. Infection (or mock infection) was carried out at an moi of 0 PFU/cell. 1 hour after infection The inoculum was removed and replaced with methionine-free medium supplemented with 353-methionine. . Eight hours after infection, buff y A (Stephenson and ter Cells were lysed under nondenaturing conditions by the addition of Antisera and Pharose CL-4B (Pharsacia, Plscatavay, Immunoprecipitation was performed using NJ). Immunoprecipitates were prepared by Laemmli (Laemm II) After dissolving in cleavage solution (Laessli, 1970), modified polyacrylic Analyzed by midgel electrophoresis and autoradiography.

Peptides derived from ero cells and culture supernatants were prepared using a reported method (Maso Endoglycosidase H (Endo H) and glycopene Digested with putidase F (PNGase F). The digested glycoproteins are then Analyzed by denaturing polyacrylamide gel electrophoresis.

Several lines of evidence suggest that 5ERA is effective against P. falciparum. important for protective immunity.

Most importantly, immunization with purified 5ERA protein ) to partially protect monkeys from countervailing doses of both heterologous and homologous blood-stage parasites. (Delplace et at,.

1988; Perrin et al., 19g4). Also, 5ER Antisera and mAbs specific for A inhibit bacterial invasion and growth in vitro. (Banyal and Inselburg, 1985; De lplace et at, 1988: Perrin et at,, 19g4) has also been revealed. 5ERA and anti-5ERA antibodies also found in immune complexes formed in vitro upon rupture in the presence of epizootic serum. (Chulay et al., 1987; Ly.

n et al, 1989), S E RA is P, falciparum liver. Expressed both during the visceral and blood stages (Szarf'anet a T., 1988), vaccine-induced anti-5ERA immunity is produced in infected hepatocytes. It can be considered that the spread of blood-stage infections can be limited by the use of blood-stage infections.

These results have generated interest in 5ERA as a potential vaccine candidate. Ru.

For this purpose, the cDNA encoding 5ER from P. falciparum FCRB strain A and vaccinia virus recombinants containing the 5ERA coding sequence were isolated. Formed.

The 5ERA full-length precursor is expressed in cells infected with this recombinant and released into the culture medium. I set it.

P. falciparum strain FCRB, duplication of the 5ERA coding sequence c. DNA clones were isolated.

FIG. 1 shows a schematic representation of the 5ERA coding sequence below the scale. point The box-shaped part contains the leading peptide (L), the octameric repeat region (8-R), and the This is the repeated region (S-R). The solid box-shaped part is peλn I / N d e I restriction fragment is shown. The location of the 5ERA cDNA clone is relative to the coding sequence. It is shown as The asterisk (ne) indicates the position of the point mutation in clone p126.8. show.

The cDNA of p126.6 is included in the blood stage cDNA lambda ZAPII cDNA library. 5ERA-specific oligonucleotide JAT2 (SEQ ID NO: 48 ) (5'-GTCTCAGAACGTGTTCATGT-3') and hybrida It was separated by lysis. This oligonucleotide is a 5ERA copolymer. It was obtained from the 3' end of the coding sequence (Bzlk et al., 1 988; Knapp et al.

1989). Clones derived from the 5' end of the 5ERA coding sequence were primed with primer J. AT15 (SEQ ID NO: 49) (5’-CACGGATCCATG AAGTCATATATTCCTT-3') and JAT16 (SEQ ID No: 50) (5’-GTGAAGCTTAATCCATAATCTT CAATAATT-3') and 5ERA first strand template [oligonucleotide template] Lima JAT17 (SEQ ID NO: 51) (5'-GTGAAGCTT obtained using TTATACATAACAGAAATAACA-3') was obtained using PCR and cloned into pUc19. These 1923 bp c The DNA is 31 bp 3' from the start codon to the internal EcoR1 site (position 1892) expanded to. DNA sequence analysis revealed that one such cDNA, p12 6.8 may contain a Taq polymerase error at nucleotide 1357. Admitted. This error is a substitution of A to G, and the 315bp KpnI/N located within the del restriction fragment (Figure 1). The second 5ERA5' cDNA p126.9 is this 5ldanl/ordinary! ■No mutations within the fragment. Unhittable Natural mutation 5' S E RA cDNA Alp126.8 middle 315 bp (7) K Replace the p　n　　/Nd　e　I fragment with the homologous fragment from p126.9, p It was obtained by forming 126°14. The full-length 5ERA cDNA is p126. The 5' cDNA of 14 was converted into a Kasa Futami I/Tansho oRI fragment, and partially XmaI/ ligated into the EcoRI-digested p126.6 vector fragment. 15 (FIG. 1).

Complete nucleotide sequence and predicted p126.15SERA cDNA insert The amino acid sequence obtained is shown in FIG.

This cDNA is a 2955 bp open sequence encoding 984 amino acids. These amino acids contain the 5ER coding frame in the FCRB strain. Identical to the A allele 11 gene and the 5ERA gene of FCBR (Knapp et al.

1989; Li et al., 1989), in Figure 2, the leading page Peptides are underlined and octapeptide repeat regions are underlined and placed in parentheses. The serine repeat region is clearly marked in bold.

Vaccinia donor plasmid contains 3K 5ERA cDNA from p126.15. The XmaI/EcoRV fragment of ligated into the pcOPcs-5H vector fragment that had been digested with It was formed by pc using DNA polymerase I Klenow fragment Fill in the Bg11I site of OPcs-5H and then insert this into the EcoRV end. Ligated to form p126.16. In this insertion plasmid, 5E RA is under the control of the early/late vaccinia H6 promoter (Rosel et al. al,, 198B>, this cDNA insertion is directed towards the C6L-KIL deletion site. Can be attached.

Using the p126.16 insertion vector as a donor plasmid, the 5E RA was inserted into vaccinia virus. Isolate the 5ERA-containing recombinant and The virus was purified and propagated, and the resulting virus was named vP870.

35S-methyl on Vero cells infected with an moi of 10 PFU/cell. Immunoprecipitation analysis was performed by pulsing with onin.

Immunoprecipitated proteins were resolved by 10% 5DS-PAGE and autoradiographed. The band was visualized using Expression of 5ERA by vP870 in Vero cells It can be detected by rabbit antiserum specific for 5ERA. 8 hours of infection Later, high molecular weight 5ERA protein was detected in the medium using an anti-5ERA reagent, and this is shown to be released from infected cells. This result shows that the 5ERA chordin consistent with the absence of a predicted hydrophobic transmembrane region in the sequence. (Bzik et al, 1988; xnapp et al, 1 989; Ll et al., 1989), at this point the smaller 5 The ERA-specific peptide remains associated with the cell.

The functional role of ABRA in the life cycle of parasitic fungi is unknown. Also Nevertheless, some studies suggest that ABRA is a potential vaccine candidate. It is important as a supplement. First, ABRA-specific mAbs are isolated from rupturing meristems. merozoite release, resulting in the formation of immune complexes and, therefore, Preventing the spread of infection in vitro (Chulay et al., 1987 ), ABRA and anti-ABRA antibodies also rupture in the presence of immune serum. are often found in such immune complexes formed in vitro (Chula y et al, 1987), A B RA is P, falciparum liver Expressed both during the visceral and blood stages (Szarf'wan et al. al, , 118g), vaccine-induced anti-ABRA immunity affects infected hepatocytes. It is thought that the spread of blood-stage infections can be limited by the action of . Also, the apparent invariance of ABRA (Chulay et al., 1987; 5tahl et al, 1986;'1ieber et al, (1988) suggested that immunity to this protein is due to a number of factors. It can provide protection against detachment.

For this purpose, the cDNA encoding ABR from P. falciparum FCRB strain A vaccinia virus recombinant containing the ABRA coding sequence was isolated. Formed.

The full-length ABRA cDNA was synthesized with the ABRA-specific primer JAT32 (SEQ ID NO: 52) (5’-CACGGATCCATGATGAAACATGAAA ATTGTTTATTC-3') and JAT34 (SEQ IDN0: 53) (5’-GTGCTCGAGTTATTTTGATTCTTCAGT TGTCAA-3”) and ABRA first strand cDNA template [primer JAT3B (SEQID NO:54) (5’-GTGCTCGAGGTTTAAT By PCR using TATTTTGATTCTTCAGTTG-3') Formed. The amplified ABRA cDNA was digested with BamHI and XhoI restriction sites. I was caught.

This is because these restriction sites are included in JAT32 and JAT34, respectively. Because there is. This allows ABRA to be made into a vaccine as a BamHI/Xhol fragment. Allows cloning into the senior donor plasmid pcOPAK-H6 became. Clones containing ABRA cDNA were obtained from two separate PCRs and T Served as a control for aq polymerase errors. Two of these loans are pABR A-2 and pABRA-4.

Complete nucleotide sequence analysis of pABRA-2 and pABRA-4 revealed that pA BRA-2 (A insertion at position 1580) and pABRA-4 (A insertion at position 140) (replacement of T by C) in each case. - was discovered. In addition, pABRA-4 has a polyA stretch starting from position 583 of the insertion site. Contains an A residue deletion within the long region. This deletion is a partial FCR3 strain ABRAc DNA clones have already been reported (Weber et al., 1 9H).

pABRA-2 and pABR to collect deletions and polymerase errors. A complex ABRA cDNA consisting of a segment of the insert of A-4 was obtained. pABR 5 of the ABRA insert from the internal ABRA Nde1 site at position 1191. ’ end to the ΣdeI site in the right arm of pcOPAK H6, From this, a 2460 bp ΣdeI fragment was isolated. This fragment was pre-installed at 2480b The Ndel fragment of p was inserted into pABRA-4 from which it had been removed, and pABRA-8 was formed.

Complete nucleotide sequence and predicted nucleotide sequence of pABRA-8 complex cDNA The amino acid sequence is shown in FIG. This CDNA encodes 740 amino acids It contains an open reading frame of 2228 bp. pABRA-8 Nucleotide and predicted amino acid sequence of A B RA c D N A in Indicates a column. In Figure 3, the lead peptide is underlined and the hexapeptide reaction Repeat regions are underlined and in parentheses, and dipeptide/tripeptide repeat regions are marked with bold underlines and parentheses. They are clearly marked in bold and in parentheses.

In the pABRA-8 insert vector with the pcOPAK vector sequence, the ABR AcDNA is under the control of the vaccinia H6 promoter (Rosel et al. L., 1988), whose insertion is directed to the AT1 site.

Using the pABRA-8 insertion vector as a donor plasmid, AB RA was inserted into vaccinia virus. Isolate ABRA-containing recombinants and The virus was purified and amplified, and the resulting virus was named vP947.

Some studies suggest that Pfhsp70 is a potential vaccine. Important as a candidate. First, immunotherapy by protein fragments containing Pfhsp70 The epidemic is caused by the Saimiri monkeys being partially affected by homologous counter-doses of blood-stage parasitic bacteria. protect (Dubols et al., 1984) o This protection is provided by vaccines. Anti-Pfhsp70 antibody and 90 kD parasite burden in inoculated monkeys It is correlated with development (Dubols et al., 1984; Jendou bi and Perelra da 5ilva, 1987) o Also, infection Pfhsp70 expressed on hepatocytes of spleen cells and non-parenchymal liver cells is a target of antibody-dependent cell-mediated cytotoxicity mechanisms carried out by both Ren and Ren. Ia et al., 1990).

Therefore, anti-Pfhsp70 antibodies induced by vaccination may be responsible for this mechanism. A substance that could limit Plasmodium infection by acting on the liver stage through the mechanism it is conceivable that.

Additionally, studies of humans exposed to P. falciparum have shown that Pfhsp70 However, both specific antibodies and lymphocyte reactivity were detected, indicating that this protein is naturally shown to be an immune target during Plasmodium infection (Ku*ar e tal, 1990). In addition, P. falciparum and other mammalian heat Similarity of Yottsuku proteins 4 brings about the possibility of autoimmune complications (Ma ttei et al., 1989), experimental results on vaccinated monkeys. have shown that the humoral immune response is preferentially dependent on Pfhsp70. (B11nsnick et al., 198 g).

P, the part encoding the carboxy terminus derived from M. falciparum FCRB strain. A cotton cDNA was isolated and a recombinant cotton cDNA expressing this cDNA was formed.

Partial Pfhsp70 cDNA clone is a lambda ZAPII blood stage cDNA clone. Pfhsp70-specific oligonucleotide HSP3 (SEQ ID No: 55) (5’-CCAGGAGGTATGCCCGGAGCA It was separated by hybridization to GG-3'). This oligonuc Reotide is derived from the 3' end of the Pfhsp70 coding sequence (Ardes hir et al, 1987; Bianco et al, 198 B) One clone, named OpHS P2O,2, was a full-length Pfhsp7 0 coding sequence, it contains 988 bp of Pfhsp70 in the 3' (Yang et al., 1987) o Other partial Pf obtained hsp70 was identical to pH5P70.2.

Partial plasmid Pfhsp70 cDNA in plasmid pH5P70.2 The nucleotide sequence is shown in Figure 4 along with the predicted amino acid sequence. In Figure 4 , GGMP repeats are bolded, underlined, and in parentheses.

This cDNA is a 948 bp open-leaved cDNA encoding 315 amino acids. contains the already published complete FCR3 strain Pf Almost identical to the homologous region of the hsp70 gene (Yang et al., 1987) It is one. The partial loan contains two single nucleotide substitutions (nucleotide 82 Substitution of C by G at position 8, substitution of A by G at position 844) was observed. This allows for amino acid substitutions (Ile by Met, Gly by Set substitution) occurs. In addition, partial cDNAs, with two exceptions, are Fe 12 and two previously reported P f h s p derived from Honduras 1 strain. 70 cDNA (Ardeshir et al.

1987; Bianco et al., 198B). p The H5P70.2 insert consists of four amino acids at the 3' end of the coding sequence. an extra copy of the repeat unit and an insert starting at nucleotide 712. Contains an ATT to GAA substitution.

To form a Pfhsp70-containing vaccinia insertion vector, the Pfhsp70 portion The cDNA was first placed under the control of the vaccinia H6 promoter. pH5P70.2 was digested with EcoRI, the restriction positions were filled in with DNA polymerase I Klenow fragment, Pfhsp70 cDNA was released by rough digestion with XhoI. This fragment is had been digested with BamHI, treated with Klenow fragment, and digested with Xhol. Ligate into plasmid pHESB. The plasmid thus obtained, pH5 P70.3 contains the Pfhsp70 partial cDNA, which contains the H6 bromo The frame is attached to the ATG start codon supplied by the pklsE3 vector. It was inserted in the system. This structure allows initiators Met and Pfhs Gly (G), Asp (D), Gin ( Four amino acids were introduced: Q), Phe (F).

The pcOPAK plasmid was then used to generate the H6-promoted partial Pf hsp70 cDNA can be inserted into vaccinia at the AT1 site. (to replace oven reading frames A25L and A26L) (see Goebel et al., 1990), the vaccinia insertion vector Formed. First, an approximately 1 kb NruI/Xho1 fragment was extracted from pH3P70.3. separated. This fragment contains the 24 bp H6 bromotor located 3' and the Pfh containing sp70 and remaining H6 digested with Nrul and XhoI. It was ligated to pcOPAK-H6-0 with the same content. The plasmid thus obtained The code pH8P70.4 is linked to Pfhsp70 in the pcOPAK insertion vector. Contains a full-length H6 bromotor.

Using the pH5P70.4 insertion vector as a donor plasmid, the partial The partial Pfhsp70 cDNA was inserted into vaccinia virus. Pfhsp 70-containing recombinants were isolated, plaque purified, amplified, and the resulting virus was transformed into vP9. It was named 05.

3 on Vero cells infected with an moi of 10 PFU/cell! S-Methi Immunoprecipitation analysis was performed by pulsing with onin.

Eight hours after infection, cell lysates were collected and immunoprecipitated with human antimalarial immunoglobulin. I went down. Immunoprecipitated proteins were lysed by 10% 5DS-PAGE and autotransferred. The band was visualized using radiography. Human antimalarial immunoglobulin is specific An approximately 32 kD peptide from vP905-infected Vero cell lysates was immunoprecipitated with let The dimensions of this peptide represent the partial Pfhsp70 contained in vP905. corresponds to the dimensions of

Isolation of the complete AMAI gene from the Blasmodium falciparum 3D7 clone The nucleotide sequence was determined.

The complete AMAI gene was constructed using two AMAI-specific oligonucleotides, It was formed by PCR using 3D7 genomic DNA as a template. two oligonucleotides The AMAI-specific sequence of Otide is the PF83Camp sequence (Thomas et al. al., 1990). The exact composition of the two oligonucleotides is as follows: It is as below.

The PCR reaction was carried out in a thermal cooler [Therg+al Cooler (Perkl (Elmer Cetus, Norvalk, CT)] at 94°C. 40 cycles of 1 minute at 42°C, 1.5 minutes at 72°C, and 3 minutes at 72°C. cells and a final extension step of 5 minutes at 72°C.

The complete nucleotide sequence was determined using customized oligonucleotides. . Sequence two separate clones and use a third clone if differences are observed. Arranged. The complete nucleotide and corresponding amino acid sequences are shown in Figure 5. A recombinant poxvirus containing DNA derived from Blasmodium is a host animal. It offers advantages as a vaccine for inducing an immune response in the body. easy to understand As can be seen, various foreign genes derived from Plasmodium have been implicated in recombinant poxviruses. Can be used in vectors. Also, for ease of understanding, recombinant poxvirus The plasma contains DNA encoding and expressing two or more Blasmodium genes. may have. In addition, for ease of understanding, additional , e.g. poxviruses such as Avivox virus and Canarypox virus. It can be used as a recombinant poxvirus vaccine vector for Smalaria.

Demonstrated protection in primate model systems, expression during blood and liver stages, and Neutralization of parasitic growth in vitro and/or infectivity by specific serum reagents A recombinant vaccine that encodes and expresses a plasmodium antigen that It would be an advantageous candidate for inducing an immune response in the body. The target antigen is Having an amino acid sequence that is invariant between isolates and strains is also considered an advantage. be done.

A near recombinant was already obtained (Example 1). This recombinant has the vaccinia H6 promoter from an FCR3 isolate controlled by and inserted into the C6L-KIL deletion site. Contains the full-length 5ERA cDNA. Immunoprecipitation experiments showed that , a 136-kD 5ERA peptide was isolated from vP870-infected Vero cells. . Also, 135.122. This series of intracellular 5ERA peptides of ILOkD expressed in such cells. The present inventor further demonstrated that the expression of 5ERA by vP870 (see Examples 7 and 8 below).

In addition to expressing 5ERA promoted by H6, the inventors here Changes Promoted by the Promoter to Entomo Box 42 Explained in 5 ERA constructs were also formed.

Terminal changes. The 3' end of the 5ERA cDNA was changed and a word was inserted immediately after the TAA stop codon. Kushina early termination signals (T, NT) and a series of restriction sites (mutations of, 1 to 1 Once acl) was placed. This is oligonucleotide JAT51 (SEQID NO: 5g) (5'-TAGAATCTGCAGGAACTTCAA-3' ), JAT52 (SEQ IDN0:59) (5'-CTACACGAG CTCCCGGGGCTCGAGATAAAAATTATACATAACAGAA ATAACATTC-3'), and plasmid p126.16 as template. Obtained by PCH using (Example 1). The obtained approximately 300 bp amplified fragment was p1 that had been digested with L1±1 and ΣacI as a PstI/5acI fragment 26.16 to form p126.17.

The 5' end of the 5ERA cDNA in p126.17 was changed to replace the ATG start codon. Some restriction sites before (and Saturday dllll, Sma Is BamHI) and Entomopox virus was placed on 42. This is the oligonucleotide JAT5 3 (SEQ ID NO: 60) (5'-CTAGAGAAGCTTCCCGG GATCCTCAAAATTGAAAATATATAATAATTACAATATAA AATGAAGTCATATATTTCCTGT-3'), JAT54 (SEQ ID NO: 61) (5’-ACTTCCGGGTTGACTTG CT-3'), and PCH using plasmid p126.16 as template Obtained by. The obtained approximately 250 bp amplified fragment was transformed into HindllI/HindI p126.I fragment, which had been digested with Hindll and Hindll. p126.18 was cloned into p126.17. This plasmid is Contains a cassette consisting of 5ERA cDNA controlled by the Tomobox promoter. This is accompanied by a vaccinia early transcription termination signal, and the 5′ end (HindI II, Smal, BamBI) and 3' end (Xho I, Sma I, 5a cI) is flanked by restriction sites.

Formation of a donor plasmid for insertion of 5ERA into the AT1 site. Promo to 42 Data/5ERA cassette as BamH1/XhoI fragment from p126.18. In the pSD553 vector fragment that had been isolated and digested with Dando II/XI and oI. was cloned into. The resulting plasmid was named p126, ATI and AT This directs the insertion of 42/5ERA into one site.

Formation of an ATI donor plasmid containing a 5ERA cDNA without serine repeats.

The 354 bp 5pel/Pf1MI fragment of 5ERA with serine repeats was replacement with a homologous PCR-generated fragment in which the repeats have been precisely deleted. 5ERA cDNA without the serine repeat region was obtained. This deleted fragment Imma JPW14126 (SEQ ID NO: 62) (5’-GGCATC CATCAAATGGTACAACTGGTGAACAAGAAAGTCTTC CTGCTAATGGACCTGATTCCCC-3'), JPW15126 ( SEQ ID NO: 63) (5’-TAGTATACTAGTAAATG GGGT-3'), and plasmid P126. as a template. AT left p 126. p126.cloned into the ATI vector fragment. RPLS was formed.

This donor plasmid contains an ATI 42/5ERA serine repeat cassette insertion. Orient to the part.

5 ERA coding sequences linked to 340 transmembrane anchor sequences A hybrid 5ERA gene containing the 5ERA gene was formed. 2780bp SmaI /Pstl　42/truncated 5ERA fragment (3'E279bp (no coding sequence), 130bp Pstl/BgllIEBV gp340) transmembrane anchor fragment and digested with Smal/BamHI The resulting vector sequence was ligated to form pINT/anchor. this plus Mid is a gp340 transmembrane linked to a truncated 5ERA sequence. Contains run regions. Then, the truncated 5ERA sequence and gp34 By inserting a PCR-derived 3'5 ERA fragment between the 0 anchor and the full-length The 5ERA coding sequence was reconstructed. The 3' fragment is ply vPst126 ( SEQ ID NO: 64) (5’-GCATTAGAATCTGCAGGA AC-3'), 5ac126 (SEQ ID NO: 65) (5'-T TGTCAGTACTGCAGGAGCTCTACATAACAGAAAATAA CATTCG-3') and using plasmid p126.18 as a template. and amplified it. This primer pair connects the TAA stop codon to 5acl and PstI. position, thereby aligning the end of the 5ERA coding sequence with gp340) Amino acids Glu and Leu are added between the smembrane region. amplified The fragment was then digested with PstI and the PstI predigested plNT126 /anchor, forming p126/anchor1. This plasmid EBV gp 340) runs on Entomobox 42 under the control of the promoter. Contains the full-length 5ERA coding sequence linked to the smenplan region, Direct the insertion to the AT1 site.

Formation of 5ERA-containing vaccinia recombinants. The 5ERA-containing donor plasmid described above By using and recombining various shapes of 5ERA, NYVAC (+KIL) AT It was inserted into the I site. p126. vP1039 (42 / 5ERA), p126. Vp1040 using RPLS (42 to 5ERA , serine no repeat), vP1023 (42/5 using P126/anchor 1) ERA and EBV gp340 anchors) were formed respectively.

Example 7 - Expression of 5ERA by vaccinia recombinant Glycosylation and biosynthesis of 5ERA. By vP870 (H6/5ERA) 135.122, and the intracellular 5ERA peptide of 110 kD and 186 kD Expression of the secreted 5ERA peptide of D was previously reported.

The present inventor conducted additional studies to analyze the expression of 5ERA by vP870. did. Pulse-chase experiments suggest that lower molecular weight poly The peptide is a biosynthetic intermediate for the 5ERA polypeptide. Because these molecules The size of the smaller polypeptide increases during the chase and eventually leads to secretion. This is because Expressed by parasitic fungi, although not rigorously tested 5ERA is believed to be non-glycosylated. endoglycosidase Secretory and intracellular expression expressed by vP870 as measured by digestion. The 5ERA peptide is glycosylated. However, the nature of N-linked sugars Whereas intracellular 5ERA only has a single N-linked oligosaccharide, secreted 5ERA The N-linked carbohydrates on ERA differ in that they are converted to a complex form. There is.

Expression of 5ERA by vP1039, vP1040, vP1023. to 5ERA vP, as detected by immunoprecipitation with specific rabbit antiserum. Expression of 5ERA by 1039 (42/5ERA) is similar to vP870 (H6/5E RA). VP1040 (42 to 5 ERA, no serine repeats) are a 128 kD secreted 5ERA peptide and a 124 kD intracellular peptide. Express.

vP102B (42/5ERA+anchor) is expressed by vP870 expresses an intracellular 5ERA peptide equivalent to that of the Consistent with this construct encompassing the gp34cl lance membrane region, There is.

The present inventor immunized rabbits with vP870 (H6/5ERA) and , Kami, [D, C11u3 (Llle, France)] It was done. Immunofluorescence analysis showed that rabbit anti-vP870 serum It reacts with blood cells indistinguishably from anti-5ERA reagents. Also, Westa rabbit serum immunoprecipitated the authentic 128 kD 5ERA precursor. authentic 5ERA precursor and processed 73kD and 50kD 5ERA React with fragments. These experiments indicate that 5ERA is When expressed by a virus, it can stimulate humoral immunity in rabbits, and this fluid Immunity is reactive with 5ERA derived from blood stage parasitic bacteria, and the glycodylation of 5ERA is conjugation does not impair the immune response to this protein.

Example 9 - NF34 by PCH to insert AMA-1 into HA site The complete AMA-1 gene was isolated from the /3D7 clone. Amplified PCR fragment The piece was cloned into the vector pMP I3H to transform AMA-1 into cotton cininia I3. was placed under the control of the L promoter to form p731AMA-1. Complete AMA- The 1 nucleotide sequence has been determined and shown previously (see figure 'M4).

44 vector fragment. The resulting plasmid was p544AMA-1 , which directs the insertion of 13L/AMA-1 into the HA site. .

NYVAC (7) by recombination using the p544AMA-1 donor plasmid. I3L/AMA-1 was inserted into the HA site. The obtained vaccinia recombinant was It was named P1018.

Example 11 - Expression of AMA-1 by vP1018 vP101g infected cell table The expression of AMA-1 on the surface was determined by immunofluorescence using a pool of human antimalarial Igs. Shown by optical analysis. Additionally, this reagent was applied to vP1018-infected MRC-5 cells. The derived cell-associated protein of approximately 83 kD was immunoprecipitated. Interestingly, infection Approximately 90 kD AMA-1 peptide was released from the cells.

Example 12 - Formation of ABRA-containing vaccinia recombinant vP947 (Example 2 The ABRA-containing vaccinia recombinant named NYVAC (+KIL) inserted into the ATI site of and promoted to vaccinia H6 from FCR3 isolate. Contains the ABRA cDNA.

Using the pABRA-8 donor plasmid (see Example 2), recombinant Anyori, NY H6/5ERA was inserted into the ATI site of VAC. Obtained vaccinia recombinant was named vP1052.

by immunofluorescence using ABRA-specific mAb 3D5 (provided by WRAIR). showed ABRA expression in cells infected with vP947 and vP1052. . However, no product was detected in immunoprecipitation using this antibody. N Transient expression derived from pABRA-8 donor plasmid in YVAC-infected cells According to the analysis, it was detected by immunofluorescence analysis and immunoprecipitation using mAb 3D5. ABRA is shown to be expressed by the donor plasmid, as shown in be suggested.

Plasmid pNF 4. 1B (Klaslov et al, 19 88) The Pfs25 gene derived from NF34/3D7 in Pfs25-specific Primer JAT61 (SEQ ID No: 66) (5’-TAATCA TGAATAAAAACTTTACAGTTTG-3'), JAT62 (SE Q ID NO: 67) (5’-GGATCCTCGAGCTGCAGATC TATAAAAATTACATTATATAAAAGCATAC-3'), and amplified by PCR using plasmid pNF4.13 as a template. 5 The amplified fragment of approximately 650 bp with double-stranded ends was digested with ``upaI'' and /Pstl into the pMPI3H vector fragment. profit The generated plasmid pPfs25゜1 was linked to the vaccinia 13L promoter. Contains the Pfsi25 coding sequence. Sequence analysis was performed and T It was confirmed that no aq polymerase error was introduced.

I3L/Pfs25 cassette from pPfs25.1 into a 750 bp double strand/ psD5, which had been isolated as a ``Eng II fragment and digested with Smal/BgllI. 50 vector fragments. The obtained donor plasmid pSD25゜2 directs the insertion of I3L/Pfs25 into the I4L site.

Example 16 - Pfs25-containing vaccinia recombinant pPfs25.2 donor plastic I3L/Pfs25 was recombined into the I4L site of NYVAC using Sumid. inserted. The obtained vaccinia recombinant was named vP1085.

Example 17 - Expression of Pfs25 by vP1085 Pfs25-specific m Immunofluorescence analysis using Ab 4B7 revealed that P on vP1085-infected cells Expression of fs25 was demonstrated. This surface expression is due to the hydrophobic transmembrane in Pfs25. This is consistent with the existence of a bran region. As detected by immunoprecipitation with 4B7 , two Pfs25 peptides of 25kD and 28kD in vP1085-infected cells. derived from the expressed NF34 sequence (Moelans et al., 1991a). Pfs16-specific oligonucleotide CO40 (SEQ IDN0:68) (5'-TAATCATGAATATTCGAAAGTTC-3') and C041(SEQ　IDN0:69)(5'-GCGAATTCATAAAA ATTAAGAATCATCTCCTTC-3') as the primer, P, by PCH using Lucipalum NF34 clone 3D7 genomic DNA as a template. The complete Pfs16 gene was formed. Approximately 5oobp with a 5' duplex end The amplified fragment of was digested with RI and RI. It was cloned into the pMPI 3H vector fragment. Amplified NF34/3D7 The Pfs16 sequence is the published NF34 sequence (Moelans et al., Formation of donor plasmid for the same as 1991a) The I3L/Pfs16 cassette was converted into a 600 bp double-stranded terminal fragment (and Saturday dIII/ and isolated from pPfs16.1 as (oRI digestion followed by Klenow embedding) Cloned into the pSD542 vector fragment that had been digested with IC1. did. The resulting donor plasmid pPfs16.2 is a TK of 13L/Pfs16. It directs insertion into the site.

NYVAC(7)T by recombination using pPfs16.2 donor plasmid. Inside the K part! , j3L/Pfs16 was inserted. The purified recombinant was isolated and H3x xl, H3xx2, H3xx3, and H3xx4.

Example 21 - Expression analysis of Pfs16-containing recombinants According to sedimentation analysis, human anti-inflammatory Pfs16 expression was detected in H3xx4-infected cells in the Laria 1gs pool. There wasn't. Also sensitive to H3xxl, H3xx2, H3xx3, and H3xx4. Pfs16 expression was also detected in this serum by immunoprecipitation analysis of stained cells. There wasn't. This human serum is MSA-1,5ERA expressed by vaccinia.

and AMA-1, but not against Pfs16. It may not contain antibodies.

Example 22 - Cloning of CS gene Isolation of NF34 of P. falciparum Things (WRAIR glue.

C8 derived from 307 clone (provided by Dr. D. Lanar) The construct lacks 9 repeat units (repeat #20-28), at position 1091. The difference between C and T changes the amino acid from Set to Phe. The expressed CS sequence of NF34 (Caspers et al., 1989) and are different. in plasmid pcOPcs-6H-CS containing this construct. In , C8 is linked to the vaccinia J6 bromotor.

Changes in vaccinia early transcription termination signals. This C8 sequence is at the 5' end of the coding sequence. It contained a vaccinia early transcription termination signal (TgNT) located near the end. P Using CR, this termination signal was altered without changing the amino acids.

Using pcOPcs-6H-C8 as a template, primer H6,5 (SEQ ID NO. Near 0) (5’-GAAAGCTTCTTTATTCTATAC-3’) and C8,5 (SEQ ID NO Near 1) (5'-CCTCAACAAAATA GGAAGGAAG-3') was used to amplify a fragment of approximately teobp. this The fragment extends from the 5' end of the H6 bromotor (including the Hindl11 site for cloning). The HaeIII site located 3' of the transcription termination signal has been reached. , modified nucleotides that remove this signal without changing the amino acid sequence It has an array. After digestion with HindlII, this HLndlII/H aeI II fragment 1,058b containing the remainder of the CS coding sequence p's earth 2 I I I / 5 l dan I fragment to, and kodan dIII / 5 tan n! digest it with p I B I 25-C5 vector array (International Blotechnologies, Inc., New Raven, C T). The resulting plasmid was named plBI25-C8, Contains the full length C8 gene linked to the H6 bromotor.

Formation of donor plasmid for insertion of C5 into vaccinia. CS codein pIBI25 containing the 3' end of the H6 bromotor linked to the sequence - cloned into a 1.100 bp vector fragment from C5. The donation thus obtained The body plasmid pcOPcs-5H contains a regenerating H6 bromotor linked to C3. and directs insertion into the C6L-KIL deletion site.

Example 23 - Modification of CS Coding Sequence Derivation of Lead Minus C8 Construct . Inducing a C8 construct lacking the N-terminal lead, the predicted intracellular trafficking alterations were observed in cs We investigated whether this would affect the induction of immune responses to.

Prior to removal of the leader sequence, p542MLF-C3 (pSD542 H6/CS cassette cloned as Mimiko II/ll±II fragment in one site. g) H6/CS cassette 11±I/day! plBI24 ( International Biotechnologies, Inc. New Raven, CT) and pMLF-C5,24 was formed. pMLF-C5,24 then approximately 110 ml where the leader sequence is located. The leader sequence was deleted by removing the bp Nrul/BstXI fragment. , this sequence was converted into a homologous NruI/BstXI fragment with a precise leading sequence deletion. Replaced with. The fragment with this “deletion” is the oligonucleotide NruMLFCS (SEQ ID No: 72) (5’-GATTATCGCGATAT CCGTTAAGTTTGTATCGTAATGCAGGAATAACCAGTG CTATGGAAGTTCGTCAAAC-3') and NruMFCSR ( SEQ ID NO Near 3) (5’-GTTTGACGAACTTCCATA GCACTGGTATTCCTGCATTACGATACAAAACTTAACG GATATCGCGATAATC-3') and its subsequent (Nr Obtained by digestion with ul and BstXI. The resulting plasmid pMLF C8,2°24 contains a lead minus cs gene linked to H6.

of the donor plasmid for inserting the lead minus the C8 gene into vaccinia. Formation. 3′ of the H6 bromotor linked to the lead minus cs coding sequence pMI containing ends, FCS, 2.24 to 1.040 bp NruI/Kp The nl fragment was isolated. This fragment was prepared using pc that had been digested with Nrul/Goritan I. Cloned into the OPcs-51 vector fragment. Donor plasmid thus obtained pMLF-C8,3 contains the regenerating H6 promoter linked to the leader minus C8. and directs insertion into the C6L-KIL deletion site.

Example 24 - Formation of C5-containing vaccinia recombinant C8-containing donor plus as described above Insert CS into the C6L-KIL deletion site in vP668 by recombination using I entered.

vP868(H6/C3) was purified using the pcOPcs-CS donor plasmid. Form vP1056 (H6/leading part minus C3) using MLF-CS, 3 did.

C8 expression by vP868 was demonstrated by both immunofluorescence and immunoprecipitation. rabbit Immunofluorescence analysis using anti-C8 repeat and anti-repeat cs sera showed that C8 Expressed on the surface of 868-infected vero cells. Anti-repeat serum showed VP868 sensitivity. Two C8s of 60kD and 50kD were isolated by immunoprecipitation of stained Ver□ cell lysates. Protein is detected. The expression of the duplex is similar to other studies in which C8 was expressed from vaccinia. (Cheng et al., 198B). Also, vP Duplexes were also detected by immunoprecipitation of 1056 infected cell lysates.

However, the molecular weight of these peptides is similar to that expressed from vP868E. The body weight is also slightly smaller (5g, 5kD and 54.5 compared to 80kD and 56kD). kD). These results suggest that the C8 leading sequence (19 amino acids) (predicted to have a molecular weight of 2.1 kD) is a vaccinia infection. It may not be fully degraded in the cells. This difference is not significant and is expressed This does not affect the utility of the recombinant product or its expression source.

To examine the immunogenicity of C5 expressed by vaccinia, two rabbits were incubated at 10 ’　Circically immunized with PFU of vP868 and at 3.6 and 9 weeks post-inoculation. The same dose of stimulation was given. for specific sequences in the repetitive region and the non-repetitive regions on either side. ELIsA titers against the corresponding NF34/3D7-derived CS peptides were measured. vP Immunization of rabbits with 868 produced antibodies in both the repeat and flank regions. body is triggered, but the response to the lateral regions is not as strong as that to the repetitive regions. It was.

VP868 was injected into mice and a peptide corresponding to amino acids 368-390 of C8 was injected into mice. The primary T cell response was examined by analyzing in vitro amplification of cells. of inoculation A significant T cell amplifying response was detected in pancreatic cells collected after 7 days.

For humans immunized with irradiated sporozoites and protected from sporozoite counter-doses. Experiments conducted show that cells infected with vP868 in vitro. stimulates C5-specific cytotoxic T cells and targets these CTLs. It can act as such.

and 5ERA to form a donor plasmid containing both P126. 18 (mentioned above) to 42/5 ERA cassette a, ooobp mutual 52 H1/ p542 isolated as a fragment and digested with BamHI/XhoI MLF-C8 (described above) was cloned into the vector fragment. The donor plate obtained in this way Sumido p126/C3-TK2 to 42/5ERA and H6/CS (reverse direction It contains a promoter arranged “head-to-head” with a transcriptional orientation of Direct insertion into the senior recombinant position.

Formation of C3/5ERA duplex recombinants. p126/C3-TK2 donor plasmid 42/5ERA and H6/CS by recombination using NYVAC+7) It was inserted into the TK site. The vaccinia recombinant obtained in this way was specifically identified as vP1007 and Life A. VP10 by immunoprecipitation with different rabbit sera and anti-unrepeated C8 serum. 5ERA and C8 in 07-infected cells are shown, respectively, and this result can be compared to the appropriate monomer. It was equivalent to that observed in the recombinant.

Immunization of rabbits with VP1007. 2 rabbits 10” PFU vP10 The mice were immunized subcutaneously at 07, and the same dose of booster was administered at 3.6 and 9 weeks after inoculation. It was. Serum was collected before immunization and weekly thereafter from week 2 to week 12. inoculation The previous serum contained no antibodies specific for 5ERA or C8, whereas the Post-seed serum contains antibodies.

Therefore, vP1007 can be used to stimulate an immune response, e.g. as a vaccine. or useful for the expression (production) of 5ERA and C8 in vitro. Ru.

6/C5-TK2 donor plasmid and recombination to 42/5ERA and and H6/CS were inserted into the TK site of vP924. vP924 is vaccinia P, falci promoted by H6 and inserted into the ATI site of NYVAC. palm MSA-1 gene. CS/5ERA/MSA thus obtained The vaccinia recombinant containing -1 was named vP967.

Expression of C8,5ERA and MSA-1 by vP967. appropriate serum reagent CS, 5ERA, and MSA in vP967-infected cells were determined by immunoprecipitation using -1 expression was detected and was comparable to that observed in the appropriate single recombinant. .

Immunization of rabbits with vP967. Two rabbits were treated with 10”ppu of vP868. The mice were immunized subcutaneously, and booster doses of the same amount were given at 3, 6, and 9 weeks after inoculation.

Serum was collected before immunization and weekly thereafter from week 2 to week 12. Blood before vaccination whereas the supernatant does not contain antibodies specific for C55SERA or MSA-1. However, the serum after vaccination contains antibodies. Therefore, vP967 is e.g. CS, 5ER or in vitro to stimulate the immune response as a It is useful for the expression (production) of A and MSA-1.

Example 29 - Multiple P, falsinoculum gene C5°5ERA, MSA-1, and A.M.A. -1 vaccinia recombinant containing Formation of C5/5ERA/MSA-1/AMA-1 heavy recombinants. p544AMA- vP967 (CS in NYVAC) was recombined using a donor plasmid. I3L/AMA-1 was inserted into the HA site of /5ERA/MSA-1).

NYVAC recombinant VP1108 obtained by isolation and purification of the recombinant is C55S Contains ERA, MSA-1, and AMA-1 genes and is suitable for immunoprecipitation experiments. Therefore, its expression is indicated.

ALVAC recombinant containing Formation of donor plasmid for insertion of C8 into the 05 site. CS coding arrangement 1 from pcOPcs-CS containing the 3' end of the H6 bromotor linked to the column. , 100 bl) was isolated. This fragment, ordinary craftsman ul/ Cloned into the pNVQH6C5SP-18 vector fragment that had been digested with Kpnl. It has become an online version. The donor plasmid pMLF-CS, 4 thus obtained, is linked to CS. It contains a regenerated H6 bromotor that directs insertion into the C5 site. Ru.

Formation of CS-containing ALVAC recombinants. pMLF-CS, using 4 donor plasmids , by recombination, ALVAC (canarypox CPpp with reduced national power) 0 H6/C8 was inserted at 5 sites. Isolate ALVAC recombinant (ALVAC-C8) When purified, the gene is found to be present.

The donor plasmid for inserting Pfs25 into the 05 site is 750 bp B. a m HI / B g±1! Separate the fragments and digest with BamHI. was cloned into the pNcLsP-5 vector fragment. The thus obtained plasmid p Pfs25.3 directs the insertion of 13L/Pfs25 into the 05 site. Ru.

Formation of Pfs25-containing ALVAC recombinants. pPfs25゜3 donor plasmid 13L/Pfs25 was inserted into the 05 site of ALVAC by recombination using . Isolation and purification of the ALVAC recombinant (ALVAC-Pf 525) resulted in the production of residues. It was confirmed that the gene was present.

was isolated as a Do211/Kou9 fragment and digested with Mimi mHI/XhoI. It was cloned into the pVQCP3L vector fragment. The donor plate obtained in this way Sumido p126, C3 goes to C31! 5th place 42 K/S E RA (Djl This is for attaching the input f direction.

Formation of 5ERA-containing ALVAC recombinants. p126. By recombination using C3, /5ERA was inserted at 42 into the C3 site of ALVAC. ALVAC recombinant (A When LVAC-8ERA) was isolated and purified, it was confirmed that it contained the gene. I can't stand it.

Formation of a donor plasmid for insertion of Pfs16 into the 03 site opPfs16 ．． The 13L/Pfs16 cassette from 2 was isolated as an XhoI/BamHI fragment. , cloned into the pVQCP3L vector that had been digested with Xhol/BamHI. It became. The thus obtained plasmid pPfs16°C3 has I3L/ Direct the insertion of Pfs 16.

Formation of Pfs16-containing ALVAC recombinants. For recombination using pPfs16゜03 Therefore, ■3L/Pfs16 was inserted into the 03 site of ALVAC. ALvAC group When the transgenic (ALVAC-Pfs16) was isolated and purified, it was found that it contained the gene. It is permitted to do so.

Formation of a donor plasmid for insertion of AMA-1 into the 06 site. p731AM A-1 to I3L/AMA-1 cassette was converted into a 2.000 bp double-stranded terminal fragment ( HindIII digestion followed by Klenow embedding and SmaI digestion) and cloned into pC6LC6L pecta which had been digested with SmaI. Ta. The plasmid thus obtained was named pC5AMA-1, and the I3 to C6 site was This directs the insertion of L/AMA-1.

Formation of ANA-containing ALVAC recombinants using pC6AMA-1 donor plasmid , 1. within the 06 site of ALVAC by recombination. :I3L/AMA-14 inserted . When the ALvAC recombinant (ALVAC-AMAI) was isolated and purified, the gene is recognized to be included.

Although preferred embodiments of the present invention have been described in detail above, The present invention should not be limited to the above detailed description, and the spirit of the invention should not be construed. It is understood that many obvious changes are possible without departing from the Should.

References x3. Clswell, D, B,, J, Bacteriol, AE0. 667-676 (1972).

Is, C1yda, D, F,, Am, J, Trop, Mad, H yg, 24.397-4 (11 (1975).

30,) Iattori, M, and 5akaki, Y, Ana I, Biocham, Engineering 52°2 Co. 2-237 (1986).

32, Higgins, o+, McConnall, o,, 5harp, p, , Nature 340°604 (1989).

45, Laammli, υ, ni,, Nature 22f, 680-685 (1970).

49, Mason, P., Virology Engineering G9.354-364 (1 989).

65, Riackmann, X, H,, Trans, Roy, Soc, Trop, ad, HY9-611.258-259 (1974).

69, Schmitt, ff, F,, and Stunnenberg, H, G, J, Virol, 62°1889-'1897 (1988) .

■!　Li VXLA Roro Ll’l Roro Ii Bozo Kakudai 985 Hiroshi Nu88 area 8ro 8 Hiroshi 8″ Ma = 3 police = Tatami ≧ CC’d N + FJ xg to ω Approximately = octopus :ff ::ffLl"l Roro Hi V Piroro Good Lolo good mouth P/’l++he-ro~Jr e’JPz%j-hay- FIXI Koh to NFL+l ζ-i- ψ to h to ψ Φh88 枳 43g 6 $28 ρ 88 Kyou $sa ON’l + @P/% NJr N”if Jtu LI’LLI’l u:lLI’l Nu’l 11014) International search report Continuation of front page (51) Int, C1,5 identification symbol Agency M! Number A61K 39/285 ADY 9284-4CC12N 15/31 (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), AU, CA, JP, KRI (72) Inventor: Tine, John A. New York, United States of America 12 302 Scotia Broad Street

Claims (26)

[Claims] 1. Plasmodium-derived DNA is included in the essential region of the box virus genome. Recombinant box virus with. 2. The DNA encodes the Plasmodium gene of Plasmodium falciparum. The recombinant boxwidth according to claim 1, characterized in that the recombinant boxwidth is Luz. 3. The Plasmodium falciparum genes are SERA, ABRA, Pfhs p70, AMA1, Pfs25, Pfs16, CS and MSA1 and The second claim is selected from the group consisting of a combination of Recombinant box virus as described in Section. 4. The DNA is expressed in the host by the generation of a Plasmodium coding sequence. The recombinant poxwirl according to claim 1, characterized in that Luz. 5. The coding sequence is SERA, ABRA, Pfhsp70, AMA1, Consisting of Pfs25, Pfs16, CS and MSA1 and combinations thereof The recombinant box according to claim 4, characterized in that the recombinant box is selected from the group Kusuvirus. 6. The claim characterized in that the poxvirus is vaccinia virus. Recombinant poxvirus according to scope 1. 7. The vaccinia virus is characterized in that it has reduced bacterial vigor. The recombinant poxvirus according to claim 6. 8. The vaccinia virus is NYVAC vaccinia virus. The recombinant box virus according to claim 7. 9. Claims characterized in that said poxvirus is canarypox virus. Recombinant box virus according to scope 1. 10. The canarypox virus is characterized in that it has reduced bacterial vigor. The recombinant box virus according to claim 9. 11. Particularly, the canarypox virus is ALVAC canarypox virus. 11. The recombinant poxvirus according to claim 10, wherein the recombinant poxvirus has the following characteristics. 12. vP870, vP947, vP905, vP1039, vP1040, v P1023, vP1018, vP1052, vP1085, H3xx1, H3x x2, H3xx3, H3xx4, v868, vP1056, vP1006, vP 967, vP924, vP1108, ALVAC-CS, ALVAC-Pfs2 5. ALVAC-SERA, ALVAC-Pfs16 or ALVAC-AM The recombinant poxvirus according to claim 1, which is A1. 13. A vaccine that induces an immune response in an inoculated host animal, the vaccine The tube consists of a carrier and a recombinant pod containing Plasmodium-derived DNA in the essential region. A vaccine characterized by consisting of a virus. 14. The DNA co-copies the Plasmodium gene of Plasmodium falciparum. 14. The vaccine according to claim 13, characterized in that the vaccine is coded with: 15. The Plasmodium falciparum genes are SERA, ABRA, Pfh sp70, AMA1, Pfs25, Pfs16, CS and MSA1 and their Claim No. 1 selected from the group consisting of a combination of these Vaccine according to item 14. 16. Claim characterized in that the poxvirus is vaccinia virus. The vaccine according to item 13. 17. The vaccinia virus is characterized in that it has reduced bacterial vigor. The vaccine according to claim 16. 18. Particularly, the vaccinia virus is NYVAC vaccinia virus. 18. The vaccine according to claim 17, wherein the vaccine has the following characteristics: 19. Claim characterized in that said poxvirus is canarypox virus. The vaccine according to item 13. 20. The canarypox virus is characterized in that it has reduced bacterial vigor. The vaccine according to claim 19. 21. Particularly, the canarypox virus is ALVAC canarypox virus. 21. The vaccine according to claim 20, wherein the vaccine has the following characteristics: 22. The box viruses are vP870, vP947, vP905, and vP10. 39, vP1040, vP1023, vP1018, vP1052, vP108 5, H3xx1, H3xx2, H3xx3, H3xx4, v868, vP105 6, vP1006, vP967, vP924, vP1108, ALVAC-CS , ALVAC-Pfs25, ALVAC-SERA, ALVAC-Pfs16 also or ALVAC-AMA1 according to claim 13. vaccine. 23. SERA, ABRA, Pfhsp70, AMA1, Pfs25, Pfs1 6, plus selected from the group consisting of CS and MSA1 and combinations thereof; A method of producing a Modium falciparum immunogen, the method comprising: In vitro, the DNA encoding or expressing the immunogen is contained in the essential region. A method comprising infecting a recombinant poxvirus with a recombinant poxvirus. 24. Claim characterized in that the poxvirus is vaccinia virus. The method according to item 23. 25. Claim characterized in that said poxvirus is canarypox virus. The method according to item 23. 26. The poxviruses include vP870, vP947, vP905, and vP10. 39, vP1040, vP102-3, vP1018, vP1052, vP10 85, H3xx1, H3xx2, H3xx3, H3xx4, v868, vP10 56, vP1006, vP967, vP924, vP1108, ALVAC-C S, ALVAC-Pfs25, ALVAC-SERA, ALVAC-Pfs16 or ALVAC-AMA1, as described in claim 23. the method of.