AU2014201356A1 - Combined measles-malaria vaccine - Google Patents

Abstract

The present invention relates to a combined measles-malaria vaccine containing different attenuated recombinant measles-malaria 5 vectors comprising a heterologous nucleic acid encoding several Plasmodium falciparum antigens. Preferably, it relates to viral vectors that comprise nucleic acids encoding the circumsporozoite (CS) protein of P. falciparum, the merozoite surface protein 1 (MSP 1) of P. falciparum, and its derivatives (p-42; p-83-30-38) in its 10 glycosylated and secreted forms, and apical membrane antigen 1 (AMA1) of P. falciparum, in its anchored or secreted form. The viral vector stems from an attenuated measles virus, based on a strain that is used as a vaccine and is efficient in delivering the gene of interest and that binds to and infects the relevant immune cells 15 efficiently. In a preferred embodiment, the CS, the MSP1 and the AMA1 proteins are generated from the virus such that they will give rise to a potent immune response in mammals, preferably humans; the expression of the proteins is elevated due to human codon optimisation. Furthermore, the invention relates to the use of the 20 recombinant vaccine in the prophylactic treatment of malaria. 5190235_1 (GHMatters) P88715.AU.1 11-Mar-14

Description

COMBINED MEASLES-MALARIA VACCINE The entire disclosure in the complete specification of our Australian Patent Application No. 2010245645 is by this cross reference incorporated into the present specification. 5 FIELD OF THE INVENTION The present invention relates to a combined measles-malaria vaccine containing different attenuated recombinant measles-malaria vectors comprising a heterologous nucleic acid encoding several LO Plasmodium falciparum antigens. Preferably, it relates to viral vectors that comprise nucleic acids encoding the circumsporozoite (CS) protein of P. falciparum, the merozoite surface protein 1 (MSP 1) of P. falciparum, and its derivatives (p-42; p-83-30-38) in its glycosylated and secreted forms, and apical membrane antigen (CAA1) L5 of P. falciparum, in its anchored or secreted form. The viral vector stems from an attenuated measles virus, based on a strain that is used as a vaccine and is efficient in delivering the gene of interest and that binds to and infects the relevant immune cells efficiently. In a preferred embodiment, the CS, the MSP1 and the !0 AMAl proteins are generated from the virus such that they will give rise to a potent immune response in mammals, preferably humans; the expression of the proteins is elevated due to human codon optimisation. Furthermore, the invention relates to the use of the recombinant vaccine in the prophylactic treatment of malaria. 25 BACKGROUND INFORMATION Measles Virus The invention relates to a vaccine containing recombinant attenuated measles viruses expressing antigens of Plasmodium 30 falciparum (Pf) and to their use for the preparation of recombinant measles-malaria vaccine which will confer immunity against both Measles and Malaria antigens. Measles virus (MV) is a member of the order Mononegavirales, i.e. viruses with a non-segmented negative-strand RNA genome. The 35 non segmented genome of MV has an antimessage polarity; thus, the genomic RNA is not translated either in vivo or in vitro. Furthermore, it is biologically active only when it is very 1 5490235.1(GHMuners) PSI715AU.1 1tMar-14 specifically associated with three viral proteins in the form of a ribonucleoprotein (RNP) complex (see below). Transcription and replication of non-segmented (-) strand RNA viruses and their assembly as virus particles have been reviewed extensively (1). 5 Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and additionally two-non structural tO proteins derived from the P gene, C and V, involved in counteracting the constitutive immune responses and in regulation of transcription/replication. The gene order is 3' N, P (including C and V), M, F, H, and L 51. In addition, from the 3'-terminal region a short leader RNA of about 50 nucleotides is transcribed. The cited L5 genes respectively encode the proteins of the ribonucleocapsid (RNP) of the virus, i.e., the nucleoprotein (N), the phosphoprotein (P), 1a 5190235_1 (GHMauers) P8S71.AU I 11-Mar-14 and the large polymerase/replicase protein (L), which very tightly associate with the genorne RNA, forming the RNP. The other genes encode the proteins of the viral envelope including the hemagglutinin (H), the fusion (F) and the matrix (M) proteins. The transcription of the MV genes follows a decreasing gradient: when the polymerase 5 operates on the genomic template it synthesizes more RNA made from upstream genes than from downstream genes. In this discontinuous transcription mode the mRNAs are capped and polyadenylated. Conversely, in the replication mode, the L protein produces full length antigenomic and genomic RNA which are immediately covered with N, P and L proteins to form infectious progeny RNPs. 10 The measles virus has been isolated in 1954: Enders and Peebles inoculated primary human kidney cells with the blood of David Edmoston, a child affected by measles, and the resulting Edmoston strain of MV (2) was subsequently adapted to growth in a variety of cell lines. Adaptation to chicken embryos, chick embryo fibroblasts (CEF), and/or dog kidney cells and human diploid cells produced the 15 attenuated Edmonston A and B (3), Zagreb (EZ) and AIK-C seeds. Edmonston B was licensed in 1963 as the first MV vaccine. Further passages of Edmonston A and B on CEF produced the more attenuated Schwarz and Moraten viruses (3) whose sequences have recently been shown to be identical (4; 5). Because Edmonston B vaccine was reactogenic, it was abandoned in 1975 and replaced by the 20 Schwarz/Moraten vaccine. Several other vaccine strains are also used: AIK-C, Schwarz F88, CAM70, TD97 in Japan, Leningrad-16 in Russia, and Edmonston Zagreb. The CAM70 and TD97 Chinese strains were not derived from Edmonston. Schwarz/Moraten and AIK-C vaccines are produced on CEF. Zagreb vaccine is produced on human diploid cells (WI-38). Today, the Schwarz/Moraten, AIK-C and EZ 25 vaccines are commonly used (6), but in principle, any one of these attenuated vaccine strains, which are all of the one unique MV serotype, proven to be safe and to induce long-lasting immune responses, can be used for the purposes of the invention. MV vaccines induce life-long immunity after a single or two low-dose injections. Protection against measles is mediated both by antibodies and by CD4 and 30 CD8 T cells. Persistence of MV-specific antibodies and CD8 cells has been shown for as long as 25 years after vaccination (7). MV vaccine is easy to produce on a large scale in most countries and can be distributed at low cost. Because the attenuation of MV genome results from an advantageous combination of numerous mutations, the vaccine is very stable and 35 reversion to pathogenicity has never been observed (6). Regarding safety, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration into host DNA. These characteristics make live attenuated MV vaccine an attractive candidate to be used as a multivalent vaccination vector. Such a vaccine may prove as efficient in eliciting long-lasting immune protection 40 against other pathogenic agents as against the vector virus itself. 2 Martin Billeter and colleagues cloned cDNA corresponding to the antigenome of Edmonston MV, and established an original and efficient reverse genetics procedure to rescue the virus (8), as described in International Patent Application WO 97/06270. The recombinant measles virus is recovered from the helper cell line 293 5 3-46, stably transfected and expressing MV N an P proteins as well as bacteriophage T7 RNA polymerase. For rescue of any variant or recombinant MV the helper cell line is then transiently transfected with an expression plasmid encoding L protein, and most importantly with any antigenomic plasmid appropriately constructed to yield any mutated or recombinant antigenomic RNA compatible to give rise to progeny MV. 10 The transient transfection step leads first to the transcription, preferably by the resident T7 RNA polymerase. The resulting antigenomic RNA is immediately (in statu nascendi) covered by the viral N, P and L proteins, to yield antigenonic RNP from which genomic RNP is produced. Second, the genomic RNP is transcribed by the attached L, to yield all viral mRNAs and the respective proteins. Finally, both genomic 15 and antigenomic RNPs are amplified by replication. In a slight variation of this procedure, rather than using stably transfected 293-3-46 helper cells, commercially available 293T cells have been transiently transfected, using simultaneously all 5 plasmids detailed in the original patent description, those encoding N, P and T7 polymerase (previously used to create the 20 helper cell line) as well as the plasmid encoding L and the antigenomic plasmid. Note that in the "fully transient transfection' procedure it is possible to use also variant expression plasmids and to avoid the use of T7 RNA polymerase altogether, utilizing instead the resident RNA polymerase II to express also the L protein and the antigenome (9). 25 To rescue individual recombinant MVs the antigenomic plasmids utilized comprise the cDNA encoding the full length antigenomic (+)RNA of the measles virus recombined with nucleotide sequences encoding the heterologous antigen of interest (heterologous nucleotide sequence), flanked by MV-specific transcription start and termination sequences, thus forming additional transcription units (ATUs). This MV 30 Edmonston strain vector has been developed by the original MV rescue inventors for the expression of foreign genes (10), demonstrating its large capacity of insertion (as much as 5 kb) and the high stability in the expression of transgenes (11; 12), such as Hepatitis B virus surface antigen, simian or human immunodeficiency viruses (SIV or HIV), mumps virus, and human IL-12. In particular, early on, recombinant measles 35 virus expressing Hepatitis B virus surface and core antigens either individually or in combination have been produced and shown to induce humoral immune responses in genetically modified mice. From the observation that the properties of the measles virus and especially its ability to elicit high titers of neutralizing antibodies in vivo and its property to be a 40 potent inducer of long lasting cellular immune response, the inventors have proposed 3 that it may be a good candidate for the production of recombinant viruses expressing antigens from P. falciparum, to induce neutralizing antibodies against said Malaria parasite which preferably could be suitable to achieve at least some degree of protection in animals and more preferably in human hosts. 5 Especially, MV strains and in particular vaccine strains have been elected in the present invention as candidate vectors to induce immunity against both measles virus and P. falciparum parasite whose constituent is expressed in the designed recombinant MV, in exposed infant populations because they are having no MV immunity. 10 Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention results in a boost of anti-MV antibodies (13). The invention relates in particular to the preparation of recombinant measles 15 viruses bearing heterologous genes from P.falciparum parasites. The advantageous immunological properties of the recombinant measles viruses according to the invention can be shown in an animal model which is chosen among animals susceptible to measles viruses, and wherein the humoral and/or cellular immune response against the heterologous antigen and/or against the 20 measles virus is determined. Among such animals suitable to be used as model for the characterization of the immune response, the skilled person can especially use transgenic mice expressing CD46, one of the specific receptors for MV. The most promising recombinants can then be tested in monkeys. The recombinant measles virus nucleotide sequence must comprise a total 25 number of nucleotides which is a mutiple of six. Adherence to this so-called 'rule of six" is an absolute requirement not only for MV, but for all viruses belonging to the subfamily Paramyxouirinae. Apparently, the N protein molecules, each of which contacts six nucleotides, must cover the genomic and antigenomic RNAs precisely from the 5' to the 3' end. 30 It is of note that the location of the ATUs can vaxy along the antigenomic cDNA. Thus, taking advantage of the natural expression gradient of the mRNAs of MV mentioned above, the level of expression of inserted ATUs can be varied to appropriate levels. Preferred locations of ATUs are upstream of the L-gene, upstream from the M gene and upstream of the N gene, resulting in low, medium and strong expression, 35 respectively, of heterologous proteins. Malaria parasite Malaria currently represents one of the most prevalent infectious diseases in the world, especially in tropical and subtropical areas. Per year, malaria infections 40 lead to severe illnesses in hundreds of million individuals worldwide, killing between 1 and 3 million, primarily young infants in developing and emerging countries. The 4 widespread occurrence and elevated incidence of malaria are a consequence of the widespread ban of DDT and the increasing numbers of drug-resistant parasites as well as insecticide-resistant parasite vectors. Other factors include environmental and climatic changes, civil disturbances, and increased mobility of populations. 5 Malaria is caused by the mosquito-borne hematoprotozoan parasites belonging to the genus Plasmodium from the phylum Apicomplexa. Four species of Plasmodium genus infect humans: P. malariae, responsible for Malaria quartana, P. vivax and P. ovale, both of which cause Malaria tertiana, and P. falcparum, the pathogen of Malaria tropica and responsible for almost all fatal infections. Many 10 others cause disease in animals, such as P. yoelii and P. berghei in mice. Malaria parasites have a life cycle consisting of several stages. Each stage is able to induce specific immune. responses directed against the corresponding occurring stage-specific antigens. Malaria parasites are transmitted to man by several species of female Anopheles mosquitoes. Infected mosquitoes inject the "sporozoite" 15 form of the malaria parasite into the mammalian bloodstream. Sporozoites remain for a few minutes in the circulation before invading hepatocytes. At this stage, the parasite is located in the extra-cellular environment and is exposed to antibody attack, mainly directed to the "circumsporozoite" (CS) protein, a major component of the sporozoite surface. Once in the liver, the parasites replicate and develop into so 20 called "schizonts." These schizonts occur in a ratio of up to 20,000 per infected cell. During this intra-cellular stage of the parasite, main players of the host immune response are T-lymphocytes, especially CDS+ T-lymphocytes. After about one week of liver infection, thousands of so-called "merozoites" are released into the bloodstream. Apical membrane antigen 1 (AMA 1) and merozoite surface protein 1 (MSP1) are both 25 present on merozoites that emerge from infected liver cells: they are essential components of the asexual blood-stage merozoite, responsible for invasion of erythrocytes. Once they enter red blood cells, they become targets of antibody mediated immune response and T-cell secreted cytokines. After invading erythrocytes, the merozoites undergo several stages of replication, giving rise to so-called 30 "trophozoites" and to schizonts and merozoites, which can infect new red blood cells. A limited amount of trophozoites may evolve into "gametocytes," which constitute the parasite's -sexual stage. When susceptible mosquitoes ingest erythrocytes, gametocytes are released from the erythrocytes, resulting in several male gametocytes and one female gametocyte. The fertilization of these gametes leads to zygote 35 formation and subsequent transformation into cokinetes, then into oocysts, and finally into salivary gland sporozoites. Targeting antibodies against gametocyte stage specific surface antigens can block this cycle within the mosquito mid gut. Such antibodies will not protect the mammalian host but will reduce malaria transmission by decreasing the number of infected mosquitoes and their parasite load. 5 The MSP-1 is synthesised as 190-200 kDa (d-190) precursor which is proteolytically processed into fragments of 83, 30, 38 and 42 kDa (d-42) during schizogony (14). At the time of erythrocytic invasion the 42-kDa is further cleaved to yield a 33 kDa fragment which is shed with the rest of the complex, and a 19 kDa 5 fragment, which contains two epidermal growth factor (EGF)-like domains, that remains associated with the merozoite membrane during invasion. This secondary cleavage is a pre-requisite for successfully erythrocyte invasion (15). MSP-1 is an essentially dimorphic protein exhibiting high conservation within the dimorphic alleles characterised by the KI and MAD20 prototypes. 10 AMA-1 (16) is a structurally conserved type I integral membrane protein, comprising 622 aa in P. falciparum (PfAMA- 1), organised in a cytosolic region (50 aa), a tratismembrane region, and an ectodomain, which folds as an a N-terminal pro sequence and three domains (DI, DII, DIII) Expression of the protein is maximal in late schizogony: the precursor of AMA-1 (83 kDa) is processed proteolytically, to 15 cleave away the pro-sequence, converting the protein into a 66 kDa form, which allows the merozoite relocalisation. Antibodies recognise mainly DI and DII, and appear to react equally well with several allelic variants. Antibody responses to DliI are generally low, levels increasing in adults (17, 18). PfAMA-1 contains 64 polymorphic positions (9 in the pro-sequence, 52 in the 20 ectodomain, 3 in the cytosolic region), most of them are dimorphic, which are important epitopes for host immune responses. To develop PfAMA-1-based vaccines it should be important to cover the polymorphisms: Diversity Covering (DiCo 1, 2 and 3) PfAMA-1 are artificial sequences representing, to the greatest extent possible, the naturally occurring polymorphism of the PfAMA 1 ectodomain. It has been shown that 25 they induce immune responses which are functional against a range of parasites carrying diverse PfAMA 1 alleles. This approach may offer a means by which vaccines targeting PfAMAI can be produced such that a strong and a functional protection against the broad range of naturally occurring PJAMAI alleles can be induced. (19). The CS protein (CSP) has about 420 aa and a molecular weight of 58 kDa. It 30 represents the major surface protein of sporozoites: its function is fundamental for the maturation of sporozoites from occystis and for the invasion of hepatocytes, which is mediated from a conserved motif of positively charged aminoacids. CSP is organised into two non-repetitive regions at 5' and 3' ends, and a variable species-specific central region, consisting of multiple repeats of four-residues-long motifs, which 35 represents the main epitope within the CSP. Since CSP continues to be detectable for at least the first 3 days of schizogony, it is considered an attractive vaccine target for both antibody-mediated immuno response, directed against extracellular sporozoites, and cell-mediated immuno responses, directed against schizonts (20). Current approaches to malaria vaccine development can be classified 40 according to the different stages in which the parasite can exist, as described above. 6 Three types of possible vaccines can be distinguished: i) pre-erythrocytic vaccines, which are directed against sporozoites and/or schizont-infected cells. These types of vaccines are primarily CS-based, and should ideally confer sterile immunity, mediated by humoral and cellular immune responses, preventing malaria infection; ii) 5 asexual blood-stage vaccines, which are directed against merozoites- infected cells: MSPI and AMAL are leading malaria vaccine candidates, designed to minimize clinical severity. These vaccines should reduce morbidity and mortality and are meant to prevent the parasite from entering and/or developing in the erythrocytes; iii) transmission-blocking vaccines, which are designed to hamper the parasite 10 development in the mosquito host. This type of vaccine should favour the reduction of population-wide malaria infection rates. Next to these vaccines, the feasibility of developing malaria vaccines that target multiple stages of the parasite life cycle is being pursued in so-called multi-component and/or multi-stage vaccines. Today's global malaria vaccine portfolio looks promising with 47 new vaccine 15 candidates, 31 in preclinical development, narrowing down to 16 in clinical trials. One of these, the RTS,S vaccine, being developed by GSK Biologicals and PATH-MVI, should enter final phase Ill clinical trials in 2008 (21). Other interesting vaccine candidates are those based on live recombinant viruses used as vector, such as Modified Vaccinia Ankara (MVA), as described in International Patent Application 20 US2006127413, poxvirus (US6214353, AU7060294, AU1668197, W09428930, and US5756101), adenovirus (US2007071726, US2005265974, US2007088156 and CA2507915), cold-adapted attenuated influenza virus, or based on yeasts, such as Pichia pastoris and Sacchammyces spp., or on bacterial expression systems, such as Salmonella spp. (US5112749) and Escherchia coli (EB0191748) (22). 25 Currently, no commercially available vaccine against malaria is available, although the development of vaccines against malaria has already been initiated more than 30 years ago. Many factors make malaria vaccine development difficult and challenging. First, the size and genetic complexity of the parasite mean that each infection presents thousands of antigens to the human immune system. Understanding which 30 of these can be a useful target for vaccine development has been complicated, and to date at least 40 different promising antigens have been identified. Second, the parasite changes through several life stages even while in the human host, presenting, at each stage of the life cycle, a different subset of molecules to the immune system. Third, the parasite has evolved a series of strategies that allow it to 35 confuse, hide, and misdirect the human immune system. Finally, it is possible to have multiple malaria infections of not only different species but also of different strains at the same time. Hence the present invention fulfil the long felt need of prior art by providing combined measles-mnaria vaccine containing different attenuated recombinant measles 7 malaria vectors comprising a heterologous nucleic acid encoding several Plasmodium falciparum antigens. It is to be understood that, if any prior art publication is 5 referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. SUMMARY OF THE INVENTION LO in one embodiment of the present invention provides a combined measles-malaria vaccine comprises a recombinant measles vaccine virus which express malaria antigens capable of eliciting immune response and protection both against measles and malaria. L5 In another embodiment, the present invention provides the recombinant measles vaccine virus having nucleotide sequence which expresses MSP1 malaria antigen. In preferred embodiment, recombinant measles vaccine virus having nucleotide sequence which expresses malaria antigen d190 or d83-30-38 or d42 in both anchored 20 and secreted forms from 3D7 strain and the FCB1 strain. In yet another embodiment, the present invention provides the recombinant measles vaccine virus having nucleotide sequence which expresses Diversity Covering (DiCo) AMA1 malaria antigen. 25 In yet another embodiment, the present invention provides the recombinant measles vaccine virus having nucleotide sequence which expresses CS malaria antigen. 30 DESCRIPTION OF THE FIGURES Fig. 1: Schematic representation of the antigenomic DNA p(+)MV-EZ of measles virus. p(+)MV-EZ is a plasmid derived from pBluescript containing the complete sequence of the measles virus (Edmoston Zagreb), under the control of the T7 RNA polymerase promoter (T7), 35 containing three ATU respectively in position 1 (before the N gene of the measles virus), 2 (between the P and the M genes of the measles virus) and 3 (between the H and the L genes of the measles virus), and exactly terminated by the hepatitis delta ribozyme and 8 5190235..1 (GHMalers) P88715 AU.1 11-Mar-14 T7 RNA polymerase terminator (6 T7t). The size of the plasmid is 18941 bp. Fig. 2: Representation of the MSP-1 synthetic gene (d-190) from 3D7 5 strain. The coding nucleotides on the flanking regions of the d-190 gene fragments (d-83-30-38 and d-42) and the corresponding amminoacids are shown. Unique restriction sites added for cloning procedures are in colours; SP: signal peptide; GPI: glycosyl phosphatidil-inositol sequence coded for membrane-anchored region. LO Fig. 3: Representation of the MSP-1 synthetic gene (d-190) from FCB1 strain. The coding nucleotides on the flanking regions of the d-190 gene fragments (d-83-30-38 and d-42) and the corresponding amminoacids are shown. Unique restriction sites added for cloning .5 procedures are in colours; SP: signal peptide; GPI: glycosyl phosphatidil-inositol sequence coded for membrane-anchored region. SP and GPI regions are from 3D7 strain. Sa 5190235_1 (GHMauers) PB9716.AU.1 11-Mar-14 Fig. 4. Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 -EZ-d190-3D7. It is a plasmid derived from p(+)MV-EZ containing d-190 malaria gene (317 strain), 5253 bp, coding the GPI-anchored form of the protein, cloned in position two of the measles genome by SgrAl-BssHII digestion. The size of 5 the recombinant plasmid is 24323 bp. Fig. 5: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 EZ-d19O*-3D7. It is a plasmid derived from p(+)MV-EZ containing d-190* malaria gene (3D7 strain), 5160 bp, coding the secreted form of the protein, cloned in position two of the measles genome by SgrAl-BssHIIl digestion. The size of the 10 recombinant plasmnid is 24227 bp. Fig. 6: Schematic representation of the recombinant measles-malaria plasmid, p(+)MVa-EZ-d190-3D7 or p(+)MV3-EZ-dl90*-3D7. It is a plasmid derived from p(+)MV EZ containing the d- 190 malaria gene (3D7 strain), 5253 bp, coding the OPI-anchored form of the protein, or the d-190* malaria gene (3D7 strain), 5160 bp, coding the 15 secreted form of the protein, cloned in position three of the measles genome by SgrAl BssHII digestion. The recombinant plasmid p(+)MV-EZ-d190 is 24323 bp, and p(+)MVa-EZ-d 190* is 24227 bp Fig. 7: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 -EZ-d83-30-8-3D7. It is a plasmid derived from p(+)MV-EZ containing d-83 20 30-38 malaria gene (3D7 strain), 4122 bp, coding the GPI-anchored form of the protein, cloned in position two of the measles genome by SgrAl-BssHII digestion. The size of the recombinant plasmid is 23195 bp. Fig. 8: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV- 2 EZd83-30-38*-3D7. It is a plasmid derived from p(+)MV-EZ containing d-83 25 30-38* malaria gene (307 strain), 4029 bp, coding the secreted form of the protein, cloned in position two of the measles genome by SgrAl-BssHIH digestion. The size of the recombinant plasmid is 23105 bp. Fig. 9: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV-EZ-d83-30-38-3D7 or p(+)MVr-EZ-d83-30-38*-3D7. It is a plasmid derived 30 from p(+)MV-EZ containing d-83-30-38 malaria gene (3D7 strain), 4122 bp, coding the GPI-anchored form of the protein, or the d-83-30-38* gene (3D7 strain), 4029 bp, coding the secreted form of the protein, cloned in position -three of the measles genome by SgrAI-BssHII digestion. The recombinant plasnid p(+)MV-EZ-d83-30-38 is 23195 bp, p(+)MV-EZ-d83-30-38* is 23105 bp. 35 Fig. 10: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 -EZ-d42-3D7. It is a plasmid derived from p(+)MV-EZ containing d-42 malaria gene (307 strain), 1347 bp, coding the GPI-anchored form of the protein, cloned in 9 position two of the measles genome by SgrAl-BssHl1 digestion. The size of the recombinant plasmid is 20417 bp. Fig. 11: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 EZ-d42*-3D7. It is a plasmid derived from p(+)MV-EZ containing d-42* 5 malaria gene (3D7 strain), 1254 bp, coding the secreted form of the protein, cloned in position two of the measles genome by SgrAI-BssHII digestion. The size of the recombinant plasmid is 20345 bp. Fig. 12: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV-EZ-d42-3D7 or p(+)MVa-EZ-d42*-3D7. It is a plasmid derived from p(+)MV-EZ 10 containing d-42 malaria gene (3D7 strain), 1347 bp, coding the GPI-anchored form of the protein, or the d-42* malaria gene (307 strain), 1254 bp, coding the secreted form of the protein, cloned in position three of the measles genome by SgrAI-BssHlI digestion. The recombinant p(+)MV-EZ-d42 is 20417 bp, the p(+)MVa-EZ-d42* is 20345 bp. 15 Fig.13: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV 2 -EZ-d190-FCBI. It is a plasmid derived from p(+)MV-EZ containing d-190 malaria gene (FCB1 strain), 5013 bp, coding the GPI-anchored form of the protein, cloned in position two of the measles genome by SgrAI-BssHIlI digestion. The size of the recombinant plasmid is 24083 bp. 20 Fig. 14: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV-EZ-d190-FCB1. It is a plasmid derived from p(+)MV-EZ containing the d-190 malaria gene (FCB1 strain), 5013 bp, coding the GPI-anchored form of the protein, cloned in position three of the measles genome by SgrAl-BssHI digestion. The recombinant plasmid p(+)MVa-EZ-d 190 is 24083 bp. 25 Fig. 15: Representation of the CS synthetic gene. The coding nucleotides on the flanking regions of the CS gene and the corresponding amminoacids are shown. Unique restriction sites added for cloning procedures are in colours. Fig. 16: Schematic representation of the recombinant measles-malaria plasmid, p(+}MV 2 -EZ-CS. It is a plasmid derived from p(+)MV-EZ containing CS gene, 1119 bp, 30 cloned in position two of the measles genome by SgrAI-BssHII digestion. The size of the recombinant plasmid is 20219 bp. Fig. 17: Schematic representation of the recombinant measles-malaria plasmid, p(+)MVa-EZ-CS, It is a plasmid derived from p(+)MV-EZ containing CS gene, 1119 bp, cloned in position three of the measles genome by SgrAI-BssHII digestion. The size of 35 the recombinant plasmid is 20219 bp. Fig. 18: Representation of the DiCo-1 complete synthetic gene. The coding nucleotides on the flanking regions of the DiCol complete gene domains (ecto and trans-cyto) and the corresponding amminoacids are shown. Unique restriction sites 10 added for cloning procedures are in colours; SP: signal peptide human codon optinilsed. Fig. 19: Representation of the DiCo- 1 ecto synthetic gene. The coding nucleotides on the flanking regions of the DiCol ecto domain and the corresponding amminoacids 5 are shown. Unique restriction sites added for cloning procedures are in colours; SP: signal peptide (human codon optimised. Fig. 20: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV2-EZ-DiCo 1-complete. It is a plasmid derived from p(+)MV-EZ containing DiCol complete gene, 1689 bp, coding the transmembrane form of the protein, cloned in 10 position two of the measles genome by SgrAl-BssHlI digestion. The size of the recombinant plasmid is 20753 bp. Fig. 21: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV-EZ-DiCol-complete. It is a plasmid derived from p(+)MV-EZ containing DiCol complete gene, 1689 bp, coding the transmembrane form of the protein, cloned in 15 position three of the measles genome by SgrAI-Bss!'HI digestion. The size of the recombinant plasmid is 20753 bp. Fig. 22: Schematic representation of the recombinant measles-malaria plasmid, p(+)MV2-EZ-DiCol-ecto. It is a plasmid derived from p(+)MV-EZ containing DiCol ecto gene, 1458 bp, coding the secreted form of the protein, cloned in position two of the 20 measles genome by SgrAl-BssHlI digestion. The size of the recombinant plasmid is 20525 bp. Fig. 23: Schematic representation of the recombinant measles-malaria, plasmid, p(+)MV-EZ-DiCol-ecto. It is a plasmid derived from p(+)MV-EZ containing DiCol ecto gene, 1458 bp, coding the secreted form of the protein, cloned in position three of the 25 measles genome by SgrAl-BssHl digestion. The size of the recombinant plasmid is 20525 bp. Fig. 24: Complete nucleotide sequence of p(+)MV 2 EZ-GFP. The sequence can be described as follows with reference to the position of the nucleotides: - 592-608 T7 promoter 30 - 609-17354 MV Edmoston Zagreb antigenomne - 4049-4054 Mlul restriction site - 4060-4067 SgrAI restriction site - 4079-4084 BssHll restriction site - 4085-4801 Green Fluorescent Protein (GFP) ORP 35 - 4805-4810 BssHlI restriction site - 4817-4822 Aal restriction site - 17355-17580 HDV ribozyme and T7 terminator 11 Fig. 25: Complete nucleotide sequence of p(+)MVaEZ-GFP. The sequence can be described as follows with reference to the position of the nucleotides: - 592-608 T7 promoter - 609-17359 MV Edmoston Zagreb antigenome 5 - 9851-9856 Mlul restriction site - 9862-9869 SgrAI restriction site - 9886-9891 BssIlI restriction site - 9892-10608 Green Fluorescent Protein (GFP) ORF - 10612-10617 BssHI restriction site 10 - 10624-10629 Aatll restriction site - 17360-17585 HDV ribozyme and T7 terminator Fig. 26; ANlOITE: this is the MSP1 d-190 3D7 sequence ORF cloned by the inventors. The sequence can be described as follows with reference to the position of 15 the nucleotides: - 1-3 Start codon - 4-99 d- 1903D7 signal peptide - 100-105 BamHil restriction site - 4014-4020 BstEll restriction site 20 - 5152-5157 AcI! restriction site - 5158-5250 GPI sequence - 5251-5253 STOP codon Fig. 27: AN102TE: this is the MSPL d-190* 3D7sequence ORF cloned by the 25 inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon - 4-99 d- 190*3D7 signal peptide - 100-105 BamHl restriction site 30 - 4014-4020 BstEll restriction site - 5152-5157 AcEI restriction site - 5158-5160 STOP codon Fig. 28: AN103TE: this is the MSP1 d-83-30-38 3D7 sequence ORF cloned by the 35 inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon - 4-99 d-83-30-38 3D7 signal peptide - 100-105 BamH] restriction site 40 - 4014-4020 BstEII restriction site 12 - 4021-4026 Ac! restriction site - 4027-4119 GPI sequence - 4120-4122 STOP codon 5 Fig. 29: AN 104TE: this is the MSP1 d-83-30-38* 3D7 sequence ORF cloned by the inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon - 4-99 d-83-30-38* 3D7signal peptide 10 - 100-105 BamHI restriction site - 4014-4020 BstEll restriction site - 4027-4029 STOP codon Fig. 30: AN 105TE: this is the MSP1 d-42 3Dl7sequence ORF cloned by the inventors. 15 The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon - 4-99 d-42 3D7 signal peptide - 100-105 BamlI! restriction site 20 - 108-114 BstElI restriction site - 1246-1251 Ac restriction sites - 1252-1344 GPI sequence - 1345-1347 STOP codon 25 Fig. 31: AN106TE: this is the MSPI d-42* 3D7 sequence ORF cloned by the inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon - 4-99 d-42* 3D7 signal peptide 30 - 100-105 BamH restriction site - 108-114 BstBIl restriction site - 1246-1251 Ac! restriction sites - 1252-1254 STOP codon 35 Fig. 32: AN107TE: this is the MSPI d-190 FCBl sequence ORF cloned by the inventors. The sequence can be described as follows with reference.to the position of the nucleotides: - 1-3 Start codon - 4-99 d-190 PCB1 signal peptide 40 - 100-105 BamIft restriction site 13 - 146-151 HindIll restriction site - 3825-3831 BstEll restriction site - 4912-4917 Ac restriction sites - 4918-5010 GPI sequence 5 - 5011-5013 STOP codon Fig. 33: AN108TE: this is the CS -sequence ORP cloned by the inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon 10 - 4-1116 CS sequence - 1117-1119 STOP codon Fig. 34: AN109TE: this is the DiCo 1 complete sequence ORF cloned by the inventors. The sequence can be described as follows with reference to the position of the 15 nucleotides: - 1-3 Start codon - 4-99 DiCol complete signal peptide - 100-105 BamHI restriction site - 106-1686 DiCo 1 complete sequence ORP 20 - 1687-1689 STOP codon Fig. 35: AN 11 OTE: this is the DiCo 1 ecto sequence ORP cloned by the inventors. The sequence can be described as follows with reference to the position of the nucleotides: - 1-3 Start codon 25 - 4-99 DiCol ecto signal peptide - 100-105 BamHI restriction site - 106-1455 DiCo 1 ecto sequence ORF - 1456-1458 STOP codon 30 Fig. 36: Comparable cytopathic effects produced on Vero cells after infection with the recombinant Measles-p-42 Malaria virus MV virus vaccine. Fig. 37: Expression of the d-42 3D7 transgene inserted into position three of the Measles vector (MV 3 EZ-d-42 SgrAl). Cell lysates from passage 1, 5 and 10 analysed by 35 Western Blot against empty Measles vector (MVEZ) and a negative control (MV 3 L1, a recombinant MV-Papilloma virus). Fig. 38: Expression of the d-42 3D7 transgene inserted into position three of the Measles vector (MV3EZ-d-42 SgrAl) analysed by immunofluorescence, compared with 40 empty Measles vector (MVEZ) and a negative control (MV2EZLI, a recombinant MV 14 Papilloma virus). Arrows point to the same syncythia as they looked using an optical microscope before and after immunostaining. Fig. 39: Growth kinetics curve of the recombinant Measles-p-42 S Malaria virus compared with that of the MV virus vaccine. Fig. 40: Expression of the d-190 FCBl transgene inserted into position two and three of the Measles vector (MV 2 -3EZ-d-190 SgrAI FCB1). Cell lysates analysed by Western Blot against empty Measles LO vector (MVEZ) and a negative control (MV2EZL1, a recombinant MV Papilloma virus). Fig. 41: Growth kinetics curve of the recombinant Measles-p-190-FCB1 Malaria virus compared with that of the MV virus vaccine. L5 DETAILED DESCRIPTION OF THE INVENTION The object of the invention is the production of a combined measles-malaria vaccine from a recombinant Measles vectors capable of containing stably integrated DNA sequences which code for CS, i0 MSP-1 or partial sections of it and AMA-1 or partial sections, in the secreted or surface anchored forms, of P. falciparum. The invention shall also include the rescue of recombinant MV Malaria viruses which are capable of infection, replication and expression of PfCS, PfMSP-1 and PfAMA-1 antigens in susceptible 25 transgenic mice, monkeys and human host. Furthermore, the invention intends to include the construction of multivalent recombinant measles-malaria vectors, in which two different antigens are simultaneously cloned and expressed in the same vector, conferring immunity against both of them. 30 Moreover, the invention relates to the combination of three different recombinant measles-malaria viruses, each carrying a different gene and expressing different antigens, in a manner to elicit immuno response in the host, directed against the different stages of the parasite's life-cycle. 35 In addition, the invention includes a process to produce recombinant measles-malaria viruses which are avoided of defective interfering particles (DIs). The DIs are known to significantly 15 5190235_1 (GHMatilers) P8871$.AU 1 11-Mar-14 inhibit the growth of virus in any production system and to successfully suppress immune response in human individuals. Furthermore, the invention comprises a method to produce a vaccine containing such recombinant viruses. 5 The examples below describe the preferred mode of carrying out the invention. It should be understood that these examples are provided for illustration and should not be construed as limiting the scope of the invention in any way. In the claims which follow and in the description of the LO invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in 'S various embodiments of the invention. 15a 51902351 (GHMuians) P88715SAU- 111Mr-14 Example 1: construction of recombinant MV-PfMSP-1 plasmids All cloning procedures were done as per the techniques described in Sambrook et at (1989). All the restriction enzymes were from New England BioLabs; the 5 oligonucleotides PCR primers and DNA polylinkers were from Invitrogen. PfMSP1 and its fragments (d-83-30-38 and d-42) either in the secreted and anchored form, have been chemically synthesized and human codon optimised. They have been cloned into the pZE21MV intermediate vector and have been slightly modified by adding SgrAl cloning site at the 5' end followed by an optimised Kozak sequence 10 (TCATCA). These modifications have been checked by sequencing at MWG Biotech. List of the recombinant MV-PMSP-1 plasmids, GPI-anchored and secreted (* forms, from 3D7 strain, which belongs to the MAD20 prototype, and from FCB1 strain, which belongs to the KI prototype: p(+)MV2EZ-d-190-SgrAI (3D7) 15 p(+)MVaEZ-d- 190-SgrAI (3D7) p(+)MV2EZ-d-83-30-38-SgrAl (3D71 p(+)MVaEZ-d-83-30-38-SgrAl (3D7) p(+)MV2EZ-d-42-SgrAl (3D7) p(+)MVaEZ-d-42-SgrA (3D7) 20 p(+)MV 2 EZ-d-190*-SgrAI (3D7) p(+)MVaEZ-d-190*-SgrAI (3D7) p(+)MV2EZ-d-83-30-38*-SgrAl (3D7) p(+)MVaEZ-d-83-30-38*-SgrAI (3D7) p(+)MV2EZ-d-42*-SgrA (3D7) 25 p(+)MVaEZ-d-42*-SgrA (3D7) p(+)MV 2 EZ-d-190-SgrAI (FCB1) p(+)MVaEZ-d- 190-SgrAl (FCB1) la) Construction of p(+)MV2EZ-d-190-SgrAl (3D7, 24323 bp) and p(+)MVaEZ-d-190 30 SgrAI (3D7, 24323 bp). lpg of MV plasmid DNA containing the green fluorescent protein (GFP) (p(+)MV 2 -aEZ GFP Berna strain, 19774 bp: figure 24 and 25) was digested with one unit of both SgrAI and BssHII restriction enzymes, for two hours at their optimal temperature, in 50p1 final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 35 Volt for about 2 hours. Then, the proper band (19048 bp) was excised from the gel, purified by QIAEX gel purification and the DNA concentration was calculated by absorbance at 260 nm and adjusted to 1 sg/ml. 16 1pg of d-190 gene, inserted into an intermediate plasmid (pZE21MV-d-190 SgrAl, 7564 bpJ was taken out by SgrAl-BssHll digestion (one unit of each enzyme), for two hours at their optimal temperature, in 50LI final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. Then, the proper band 5 (5275 bp) was excised from the gel, purified by QIAEX gel purification kit and the DNA concentration was calculated by absorbance at 260 nim and adjusted to 1 pg/ml. Thus, the vector (MV DNA: figure 1) and the insert (d-190 DNA: figure 2), were ligated in an equimolar ratio overnight at 16'C, using one unit of T4 DNA Ligase and its own reaction buffer in 100pl final volume. 10 XL1O Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et at. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maid kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences, aligned 15 with the assumed ones using a DNA Strider software, showed 100% identity. The d-190-3D7 gene, inserted into position 2 of the MV vector (SgrAl, pos. 4060, and BssHlI, pos. 9335) is represented in figure 4 and its Open Reading Frame (ORP) is listed in figure 26. The d- 190- 3D7 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, and 20 BssHlI, pos. 15137) is represented in figure 6. The genome's length (starting at ACC, pos. 609, to GGT, pos. 21884) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of the recombinant MV 2 .a-d-190-3D7 viruses. 25 1b) Construction of p(+)MV2EZ-d-83-30-38-SgrAI (3D7, 23195 bp) and p(+)MVaEZ-d 83-30-38-SgrA (3D7, 23195 bp). The measles vectors were prepared as detailed described in example 3a. The pZE2 1MV-d- 190 SgrAl was digested BstEll-AclI to cut out the d-42 fragment;. a polylinker, with cohesive BstElI and Acl ends, had been ligated to obtain the 30 intermediate plasmid pZE21MV-d-83-30-38-SgrAl (6436 bp). The sequence of the polylinker was: 5'-CGTCACCAGCGGCCGCAA-3'. lpg of pZE21MV-d-83-30-38 SgrAI was digested SgrAI-BssHIl (one unit of each enzyme), for two hours at their optimal temperature, in 50 1 pl final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. 35 Then, the proper band (4147bp) was excised from the gel, purified by QlAEX gel purification kit and the DNA concentration was calculated by absorbance at 260 nm and adjusted to 1Dpg/ml. 17 Thus, the vector (MV DNA: figure 1) and the insert (d-83-30-38 DNA: figure 2), were ligated in an equimolar ratio overnight at 16*C, using one unit of T4 DNA Ligase and - its own reaction buffer in 100pl final volume. XL10 Gold chemical competent cell were then transformed with all ligation volume, 5 following a standard transformation protocol (Sambrook et aL 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maid kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences were then aligned with the assumed ones using a DNA Strider software. 10 The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. The d-83-30-38-3D7 gene, inserted into position 2 of the MV vector (SgrAl, pos. 4060, and BssHIll, pos. 8207) is represented in figure 7 and its Open Reading Frame (ORF) is listed in figure 28. 15 The d-83-30-38-3D7 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, and BssHlI, pos, 14006) is represented in figure 9. The genome's length (starting at ACC, pos. 609, to GOT, pos. 20756) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of the recombinant MV 2 .3-d-83-30-38-3D7 viruses. 20 1c) Construction of p(+)MV 2 EZ-d-42-SgrAl (3D7, 20417bp) and p(+)MVaEZ-d-42-SgrAl (3D7, 20417 bp). The measles vectors were prepared as detailed described in example 3a. lpg of d-42gene, inserted into an intermediate plasmid (pZE21MV-d-42 SgrAJ, 3658 25 bp) was taken out by SgrAI-BssHll digestion (one unit of each enzyme), for two hours at their optimal temperature, in 50pl final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. Then, the proper band (1369 bp) was excised from the gel, purified by QIAEX gel purification kit and the DNA concentration was calculated by absorbance at 260 nm and adjusted to lOpg/ml. 30 Thus, the vector (MV DNA: figure 1) and the insert (d-42 DNA: figure 2), were ligated in an equimolar ratio overnight at 160C, using one unit of T4 DNA Ligase and its own reaction buffer in 100sl final volume. XLIO Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et a. 1989), plated and 35 selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes 18 digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. The d-42-3D7 gene, inserted into position 2 of the MV vector (SgrAI, pos. 4060, and BssHII, pos. 5429) is represented in figure 10 and its Open Reading Frame (ORF) is 5 listed in figure 30. The d-42-3D7 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, and BssHII, pos. 11231) is represented in figure 12. The genome's length (starting at ACC, pos. 609, to GOT, pos. 17978) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of 10 the recombinant MV 2 4 -d-42-3D7 viruses. The recombinant Measles-p-42 Malaria viruses and MV vaccine induced similar cytopathic effect (figure 36). The transgene is rather stably expressed: its expression was completely maintained in all analysed progeny clones derived from single original rescued clones after ten serial virus passages in human diploid cell MRCS (figure 37 15 38). The growth curves of recombinant MV-Malaria virus and MV vaccine showed the same kinetics (figure 39). id) Construction of p(+)MV 2 EZ-d- 190*-SgrAl (3D7, 24227 bp) and p(+)MV 3 EZ-d- 190* SgrAI (3D7, 24227 bp). 20 The measles vectors were prepared as detailed described in example 3a. Using the intermediate vector pZE21MVd-190-SgrAI as template, a PCR reaction has been performed to delete the GPI anchor region, which is located between AcIl (pos. 5434) and Clal (pos. 5536) sites. PCR amplifications were carried out using the proofreading Pfu DNA polymerase 25 (Stratagene). DNA sequences of the synthetic oligonucleotides primers are given in lower case for the MV nucleotides and in upper case for non MV nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. The following oligonucleotides primers have been used: For-Clal, 5' CCAATAAACrGT'TAAT AGatceattacgcgcgctctagc -3', and Rev-Avdil, 5' 30 gcctttgagtgagctgatacc-3'. For-Clal is homologous to the template at the level of the Clal and BssHI sites and contains an overhang (in upper case) with two stop codons (TAATAG), the Acd site (AACGTT), and a 6 bp long-protection site for Aci! (CCAATA). In the so-called PCR-GPI and in the final construct d-190*, Ac! will become close to Clal. 35 Rev-AvrIl is homologous to the template (from pos. 5704 to 5724). PCR product was 207 bp-long: its digestion with Acf+Avtil and ligation with the pre digested Acf+AvrHl intermediate vector pZE21MVd-190-SgrAI has produced pZE21MVd-190*-SgrAL. 19 In detail, the digestion of the vector with Acfl+Avrll has produced two bands of 7318 bp and 246 bp (containing the GPI region to delete): the 7.3 kb-fragment was purified from agarose gel by using QIAEX If purification kit (Qiagen) and was ligated to the digested Acil-AvrH PCR (insert) to obtain pZE21MVd-190*-SgrAl. 5 To screen for positive clones, Nool digestion has be done, producing a single band of 7 kb from the d- 190* intermediate vector, and two bands of 1.3 and 5.7 kb from the original GPI-anchor construct. To construct the definitive recombinant p(+)MeV 2 EZ-dl9O* and p(+)MeV3EZ-d190* (figure 5 and figure 6), according to the "rule of six", MeV vectors and intermediate 10 plasmid were digested with SgrAl+BssHII and afterwards ligated each other. In detail, pZE21MVd-190*-SgrAI digested SgrAI+BssHI has produced three bands, 5.2 kb + 1.3 kb + 900 bp. D-190* sequence was contained in the 5.2 kb fragment, that has been cut, purified and ligated with MeV2EZ and MeV 3 EZ vectors SgrAI+BssHlI digested (19 Kb in length), in an equimolar ratio overnight at 16*C, 15 using one unit of T4 DNA Ligase. XLiO Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et at 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kitj and restriction enzymes 20 digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. The d- 190*- 3D7 gene, inserted into position 2 of the MV vector (SgrAl, pos. 4060, and BssHII, pos. 9239) is represented in figure 5 and its Open Reading Frame (ORF) is listed in figure 27, 25 The d- 190*- 3D7 gene, inserted into position 3 of the MV vector (SgrA[, pos. 9862, and BssHI, pos. 15041) is represented in figure 6. The genome's length (starting at ACC, pos. 609, to GGT, pos. 21788) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of the recombinant M 3V2--d-190*-3D7 viruses. 30 le) Construction of p(+)MV2EZ-d-83-30-38*-SgrAI (3D7, 23105 bp) and p(+)MVaEZ-d 83-30-38*-SgrAI (3D7, 23105 bp). The measles vectors were prepared as detailed described in example 3a. The intermediate vector pZE21MVd-190-SgrAl was digested BstElI-Clal to cut out the 35 d-42 fragment and the GPI region, which is located between Acil (pos. 5434) and Cial (pos. 5536) sites; a polylinker, with cohesive BstEll and Clal ends, had been ligated to obtain the intermediate plasmid pZE21MV-d-83-30-38*-SgrAI (6346 bp). The sequence of the polylinker was: 5'-GTCACCGGGGAATAATAGCGCAT-3'. 20 DNA sequence of the synthetic oligonucleotide polylinker is given in upper case for non MV nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. Polylinker contains the BstElI (GTCACC) and Clal (AT) sticky ends, two stop codons 5 (TAATAG), and a triplet (GCG) to keep the rule of six. 1pg of pZE21MV-d-83-30-38* SgrAl was digested SgrAl-BssHll (one unit of each enzyme), for two hours at their optimal temperature, in 50p final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. Then, the proper band (4057bp) was excised from the gel, purified by Q!AEX gel 10 purification kit and the DNA concentration was calculated by absorbance at 260 nm and adjusted to lopg/mi. Thus, the vector (MV DNA: fig. 1) and the insert (d-83-30-38* DNA: fig. 2), were ligated in an equimolar ratio overnight at 16"C, using one unit of T4 DNA Ligase and its own reaction buffer in 100pI final volume. 15 XL10 Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et al. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences were then 20 aligned with the assumed ones using a DNA Strider software. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. The d-83-30-38*- 3D7 gene, inserted into position 2 of the MV vector (SgrAl, pos. 4060, and BssHII, pos. 8117) is represented in figure 8 and its Open Reading Frame 25 (ORF) is listed in figure 29. The d-83-30-38*- 3D7 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, and BssHHl, pos. 13919) is represented in figure 9. The genome's length (starting at ACC, pos. 609, to GGT, pos. 20666) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of 30 the recombinant MV2.s-d-83-30-38*-3D7 viruses. 1f) Construction of p(+)MV2EZ-d-42*-SgrAl (3D7, 20345bp) and p(+)MVsEZ-d-42* SgrA (3D7, 20345 bp). The measles vectors were prepared as detailed described in example 3a. 35 Using the intermediate vector pZE21MVd-42-SgrAl (3658 bp) as template, a PCR reaction has been performed to delete the GPI anchor region, which is located between Ac! (pos. 1528) and Clal (pos. 1630) sites. 21 PCR amplifications were carried out using the proofreading Pfu DNA polymerase (Stratagene). DNA sequences of the synthetic oligonucleotides primers are given in lower case for the MV nucleotides and in upper case for non MV nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. 5 The following oligonucleotides primers have been used: For-Clt, 5' CCAATAAACGTrTAAT AGatcattacgacctctagc -3', and Rev-Auri1, 5' gcctttgagtgagctgatacc-3'. For-Clad is homologous to the template at the level of the Clad (pos. 1630) and BssHIU (pos. 1639) sites and contains an overhang (in upper case) with two stop codons 10 (TAATAG), the Acil site (AACGTT), and a 6 bp long-protection site for AclH (CCAATA). In the so-called PCR-GPI and in the final construct d-42*, Acl will become close to Cad. Rev-AvrII is homologous to the template (from pos. 1798 to 1818). PCR product was 207 bp-long: its digestion with Acl[+Avdl and ligation with the pre digested Acil+Aurif intermediate vector pZE21MVd-42-SgrAI has produced 15 pZE21MVd-42*-SgrAL. In detail, the digestion of the vector with Acl[+Audl has produced two bands of 3412 bp and 246 bp (containing the GPI region to delete): the 3.4 kb-fragment was purified from agarose gel by using QIAEX II purification kit (Qiagen) and was ligated to the digested Acil-Avil PCR (insert) to obtain pZE2 1 MVd-42*-SgrAI. 20 To screen for positive clones, Ncol digestion has be done, producing-a single band of 3.4 kb from the d-42* intermediate vector, and two bands of 1.3 and 2.3 kb from the original GPI-anchor construct. To construct the definitive recombinant p(+)MeV 2 EZ-d42* and p(+)MeVaEZ-d42*, according to the "rule of six", MeV vectors and intermediate plasmid were digested 25 with SgrAI+BssHiI and afterwards ligated each other. In detail, pZE21 MVd-42*-SgrAl digested SgrA1+BssHI1+Spei has produced four bands, 1.3 kb + 936 bp + 800 bp + 400 bp. D-42* sequence was contained in the 1.3 kb fragment, that has been cut, purified and ligated with MeV 2 EZ and MeVaEZ vectors SgrAI+BssHil digested (19 Kb in length), in an equimolar ratio overnight at 16'C, 30 using one unit of T4 DNA Ligase. XLIO Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et at. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes 35 digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. The d-42*-3D7 gene, inserted into position 2 of the MV vector (SgrAi, pos. 4060, and BssHIIl, pos. 5357) is represented in figure 11 and its Open Reading Frame (ORF)~is listed in figure 31. -22 The d-42*-3D7 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, and BssHII, pos. 11159) is represented in figure 12. The genome's length (starting at ACC, pos. 609, to GGT, pos. 17906) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of 5 the recombinant MV 2

-

3 -d-42*-3D7 viruses. 1g) Construction of p(+)MV 2 EZ-d-190-SgrAI (FCBI, 24083 bp) and p(+)MVaEZ-d-190 SgrAl (FCB1, 24083 bp). First of all, the cloning of the synthetic gene for MSP- 1 of the FCB 1 strain into the 10 intermediate plas mid pZE2 1 MV-SgrAI has been performed, keeping the signal peptide and the GPI-anchor region from MSP-1 of 3D7 strain. D-190 gene (FCBi) was obtained stepwise from an intermediate vector, called pZE23f-GX- 190H, as follow: i). I pg of the plasmid pZE21MV-d-190-SgrAI (3D7) was digested with HindIll + Act! restriction enzymes, for two hours at their optimal temperature, in 50d final volume. 15 All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. Then, the proper band (2558 bp), corresponding to the vector, was excised from the gel, purified by QIAEX gel purification and the DNA concentration was calculated by absorbance at 260 rnm. U). a PCR reaction was performed, using the pZE23f-GX- 190H as template, in order to 20 amplify and recover the d-42 portion of the MSP-i/FCB1. PCR amplification was carried out using the proofreading Pfu DNA polymerase (Stratagene). DNA sequences of the synthetic oligonucleotides primers are given in lower case for the MV nucleotides and in upper case for non MV nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. 25 The following oligonucleotides primers have been used, designed on the pZE23f-GX 190H sequence: For-1 FCB1, 5'-CCCAAGCTccaggtggccggAgagctgtcactcc -3', and Rev-1 FCB1, 5'- GCCTGCaacgttGCTagagctggagcaGaaGatcccgtcg -3'. For-1 FCB1 is homologous to the template from pos. 4509 to pos. 4538, comprising the BstEII site (ggtcacc). The A (in upper case) was a t in the template, and it has been 30 modified to eliminate a SgrAl site. It contains an overhang (in upper case) with the HinduII site (AAGCTT), after its 3 bp long-protection site (CCC). Rev-1 FCB1 contains an Acl site (aacgtt), preceded by a 6-bp protection site (GCCTGC). It was introduced a triplet GCT, coding for a seine, to keep the rule of six; two a have been modified in G to avoid a poly(A) site. 35 The obtained PCR-HindIll-AcE (1.1 kb) has been digested Hindill + Ac and ligated, overnight at 16*C in an equimolar ratio, to the pre-digested pZE21MV-d-190-SgrAI with Hindll + Ac! (step i), obtaining the pZE21MV-d-42-SgrAI-FCB1 (3657 bp). XL10 Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et at 1989), plated and 23 selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and by restriction enzymes digestion with HindIll + Acil (expected fragments 2558 bp + 1099 bp). iii). the pZE21MV-d-42-SgrAI-PCBI, obtained as described in step ii, has been 5 digested Hindll + BstEll (HinduI, pos. 428, and BstEll, pos. 440), and the proper band (3645 bp), corresponding to the opened vector, was loaded on a 1% agarose gel, excised from the gel, purified by QIAEX gel purification and the DNA concentration was calculated by absorbance at 260 nm. iv). The pZE23f-GX- 190H was digested HindH + BstEll and the proper band of 3679 10 bp (insert), corresponding to the d-83-30-38/FCB1 fragment, was purified from the gel, as previously described. v). the Hindlll + BstEll digested fragment of 3657 bp (vector), obtained from pZE21MV-d-42-SgrAl-FCB1, has been ligated to the HindU!I + BstEll fragment of 3679 bp (insert), containing the d-83-30-38/FCB1 and obtained by digestion from pZE23f 15 GX-190H. Ligation was done in an equimolar ratio overnight at 16*C, using one unit of T4 DNA Ligase, obtaining the pZE2lMV-d- 190-SgrAl-FCB1 (7324 bp). Afterwards, XLIO Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et at 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA 20 plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes digestion. To construct the p(+)MV 2 EZ-d-190-SgrAl-FCB1 and p(+)MVaEZ-d-190-SgrAl-FCB1, the measles vectors were prepared as detailed described in example 3a. lpg of d-190/FCB1 gene, inserted into an intermediate plasmid (pZE21MV-d-190 25 SgrAI-FCBX, 7324 bp), was taken out by SgrAl-BssHil digestion (one unit of each enzyme), for two hours at their optimal temperature, in 5OV1 final volume. All the digested DNA was loaded onto a 1% agarose gel, run at 80 Volt for about 2 hours. Then, the proper band (5035 bp) was excised from the gel, purified by QIAEX gel purification kit and the DNA concentration was calculated by absorbance at 260 nm 30 and adjusted to 10pg/ml. Thus, the vector (MV DNA: figure 1) and the insert (d- 190/PCB 1 DNA: figure 3), were ligated in an equimolar ratio overnight at 16*C, using one unit of T4 DNA Ligase and its own reaction buffer in 100pL final volume. XL1O Gold chemical competent cell were then transformed with all ligation volume, 35 following a standard transformation protocol (Sambrook et al. 1989), plated and selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QZAGEN, mini- midi and maxi kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. 24 The d-190- FCB1 gene, inserted into position 2 of the MV vector (SgrAl, pos. 4060, and BssHlII, pos. 9095) is represented in figure 13 and its Open Reading Frame (ORF) is listed in figure 32. The d-190- FCB1 gene, inserted into position 3 of the MV vector (SgrAl, pos. 9862, 5 and BssHII, pos. 14897) is represented in figure 14. The genome's length (starting at ACC, pos. 609, to GQT, pos. 21884) of the recombinant Measles-Malaria plasmids was a multiple of six, allowing the rescue of the recombinant MV24-d-190-FCB1 viruses. The transgene is rather stably expressed: its expression was completely maintained in 10 all analysed progeny clones derived from single original rescued clones after ten serial virus passages in human diploid cell MRC5 (figure 40). The growth curves of recombinant MV-Malaria virus and MV vaccine showed the same kinetics (figure 41). Example 2: designing of DiCol nucleic acid sequence. 15 Starting from the aminoacidic DiCol sequence (ecto, trans and cytoplasmic domains: aa 97-622) and using the DNA Strider software, a correspondent nucleic acid sequence has been designed comparing the DiCo 1 DNA degenerate sequence to a selected PfAMAl gene (accession number AAO141.1), which represents the most similar sequence to the DiCol after BLAST alignment. 20 At the 5 end suitable unique restriction sites has been added (Mul and SgrAI) as cloning sites, followed by an optimal KOZAC sequence and a human optimised Signal Peptide (SP). At the 3' end, two stop codons and a BssHl cloning site have been added. Following this scheme, we designed two nucleotides sequences (respecting the 'rule of six" for the further expression into the measles vector), 25 encoding the anchored and the secreted forms of the DiCol protein: the first gene comprises the ectoplasmasmic, the transmembrane and cytoplasmic domains (Figure 18), while the second one corresponds to the ectodomain alone (Figure 19). The two sequences has been human codon optimised by GENEART, to reduce AT% content, to avoid poly(A) sequence and RNA instability motif. 30 DiCol complete ORF and DiCol ectodomain ORF are listed respectively in figure 34 and 35. Example 3: construction of recombinant MV-PFJAA- 1 plasmids All cloning procedures were done as per techniques described in Sambrook et at (1989). 35 PfAMA1, and in particular Diversity Covering sequences 1 (DiCol) either in the secreted and anchored form, have been chemically synthesized and human codon optimised. 25 The codon optimised DiCol secreted and anchored forms were digested SgrAI+BssHll and ligated, overnight at 16C in an equimolar ratio, to the pre-digested MeV 2 EZ and MeV 3 EZ vectors (19 Kb in length), using one unit of T4 DNA Ligase, obtaining the following recombinant MV- PAMA-1 plasmids: p(+)MV 2 EZ-DiCol 5 complete (Figure 20), P(+)MVaEZ-DiCol-complete (Figure 21), p(+)MV2EZ-DiCol-ecto (Figure 22), and p(+)MV3EZ-DiCo1-ecto (Figure 23). Example 4: construction of recombinant MV-PfCS plasmids fSW (20219 bol and pt+MVqEZ. CS-SwAI 120219 bal All cloning procedures were basically as described in Sambrook et al. (1989). 10 PJCSl, cloned into an intermediate vector pAdApt35Bsu.CS.Pfalc.aa-sub.gcc, has been amplified by PCR, and directly cloned into the definitive MV vectors, obtaining two recombinant MV-PfCS plasmids: p(+)MV 2 EZ-CS and p(+)MV 3 EZ-CS. In detail, a PCR reaction was performed, using the pAdApt35Bsu.CS.Pfalc.aa-sub.gcc as template, in order to amplify and recover the CS gene (figure 15). PCR amplification 15 was carried out using the proofreading Pfu DNA polymerase (Stratagene). DNA sequences of the synthetic oligonucleotides primers are given in lower case for the MV nucleotides and in upper case for non MV nucleotides; sequences of relevant restriction endonucleases recognition sites are underlined. The following oligonucleotides primers have been used, designed on the 20 pAdApt35Bsu.CS.Pflc.aa-sub.gcc sequence: For-SgrAl, 5' ACTTCTCACCGGTGTggaggettaccac catgat -3', and Rev-BssHll-CS 5'- TAGCCGCttagaggatccttatcage -3'. For-SgrAl is homologous to the template from pos. 1356 to pos. 1375, comprising the HindIll site (aagctt). It contains an overhang (in upper case) with SgrAl restriction site 25 (CACCOGTO), after 6-bp long-protection site (ACTrCT). Rev-BssHI-CS contains an overhang (in upper case) with BssHII restriction site (GCGCGC), which will be close to Xbal (tctaga) in the PCR-CS (1187 bp). The obtained PCR-CS has been digested SgrAl + BssHI and ligated, overnight at 16'C in an equimolar ratio, to the pre-digested MeV 2 EZ and MeVaEZ vectors SgrAI+BssHl 30 (19 Kb in length), using one unit of T4 DNA Ligase, obtaining, respectively, p(+)MV2EZ-CS-SgrAI (20219 bp, figure 16) and p(+)MV 3 EZ- CS-SgrAl (20219 bp, figure 17). The CS ORF is listed in figure 33. XL10 Gold chemical competent cell were then transformed with all ligation volume, following a standard transformation protocol (Sambrook et al. 1989), plated and 35 selected on LB-Agar plates for ampicillin resistance. Colonies were screened by DNA plasmid preparation (QIAGEN, mini- midi and maxi kit) and restriction enzymes digestion. The right clones were sent to MWG for sequencing: the sequences, aligned with the assumed ones using a DNA Strider software, showed 100% identity. 26 Example 5: Cells and viruses Cells were maintained as monolayers in Dulbecco's Modified Eagles Medium (DMEM), supplemented with 5% Foetal Calf Serum (FCS) for Vero cells (African green monkey kidney) and with 10% FCS and 1% penicillin/streptomycin (P/S) for 5 293T cells (human embryonic kidney); DMEM supplemented with Glutamax (F12) and 10% PCS for MRC-5 (human foetal fibroblast); DMEM supplemented with 10% FCS and 1.2 mg/ml of G 418 for 293-3-46. To grow MV virus stocks reaching titers of about 107 pfu/ml, recombinant viruses and the vaccine strain Edmoston Zagreb were propagated in MRC-5 cells: 10 plaque purification was carried out by transferring a syncythium to 35 mm MRC-5 cell culture which was expanded first to a 10 cm dish, and afterwards to a 175 cm flask. Virus stocks were made from 175cm 2 cultures when syncythia formation was about 90% pronounced. Medium corresponding to the so-called "free-cell virus fraction" was collected, freeze and thawed three times and spun down to avoid cell 15 debris. The medium was then stored at -800C. Cells, which correspond to the so called "cell-associated virus fraction", were scraped into 3 ml of OPTIMEM (Gibco BRL) followed by three rounds freezing and thawing, spun down and the cleared surnatant stored at -80*C. Example 6: Transfection of plasmids and rescue of MV viruses 20 293T cells were seeded into a 35mm well to reach - 50-70% confluence when being transfected. 4 h before transfection, the medium was replaced with 3 ml DMEM containing 10% FCS. All recombinant plasmids were prepared according to the QIAGEN plasmid preparation kit. The kit for the Ca 2 +phosphate coprecipitation of DNA was from Invitrogen. 25 Cells were co-transfected with the plasmids in the follows final concentration: pCA-L 0.5 pg, pCA-N 0.5 pg, pCA-P 0.1 pg, pCA T7 1 pg and the recombinant Measles-Malaria plasmid 4 pg. All five plasmids, diluted in H 2 0, were added in a Eppendorf tube containing 2M CaC2, the mix was added to another Eppendorf tube containing HEPES buffer under shaking conditions, and was incubated 30 min at 30 room temperature (RT). Thus, the co-precipitates were added dropwise to the culture and the transfection was carried out at 37'C and 5% C02 for about 18h. Then, the transfection medium was replaced with 3ml of DMEM containing 10% FCS. Another way to obtain recombinant measles-malaria vaccine viruses is described hereafter, using the 293-3-46 helper cell (human embryonic kidney cells), 35 stably expressing the measles N and P proteins as well as the T7 RNA polymerase. The viral RNA polymerase (large protein, L) was expressed by co-transfecting the cells with 15 ng of the plasmid peMCLa. To improve transfection efficiency 300 ng of pSC6 T7 Neo were added. Calcium-phosphate method was used for transfection. 27 First syncytia appeared 3-4 days after transfection when the cells were still subconfluent. To allow syncytia formation to progress more easily, almost confluent cell monolayer of each 35mm well were then transferred to a 10 cm dish. Each syncytium was taken up in 300 pl of transfection medium and put in a sterile 5 Eppendorf tube containing 700 pl of OPTIMEM, freeze and thaw for three rounds, and stored at -80*C. Example 7: virus titration by plaque assay Serial 10-times dilutions of virus preparations were carried out using OPTIMEM to a final volume of 0.5 ml. Each dilution was added on 35 mm Vero cell 10 cultures. After I h of virus adsorption, the inoculum was removed and the infected cells were overlaid with 2m of DMEM containing 5% FCS and 1% low melting point agarose (LMP agarose). After 5 days of incubation at 37*C and 5% C0 2 , cultures were fixed with Iml of 10% TCA for 1 h, then UV cross-linked for 30 min. After removal of the agarose overlay, cell monolayers were stained with crystal violet dissolved in 4% 15 ethanol, washed with water and the plaques were counted under the inverted microscope. Example 8: MRC-5 virus serial passages of recombinant viruses Rescued viruses were serially passaged 10-times on MRC5 cells, seeded into 10 crn diameter plates, that were infected with the standard and the recombinant MV 20 viruses at MOI of 0.01 PFU/cells. After monolayer was full infected, 1% surnatant of each culture was used to infect the subsequent MRC5 cells monolayer. To test transgene expression and stability, viruses from passage 1, 5, and 10 were used for further characterisation of expression by Western blot and immunofluorescence. Example 9: Western blot, immunofluorescence 25 To analyse the expression either MV and Malaria, Western blot and immunofluorescence were carried out. For Western blot, Vero cells seeded on 35 mm dish (1-5x10 5 ) were monitored the next day for 90% confluence and infected with cleared virus suspension from cell associated virus fraction, using 0.1 MOI (Multiplicity Of Infection), including MVEZ as 30 control. When about 80% syncythia formation was observed, cells were first washed with PBS and then scraped in 1 ml PBS and collected in an Eppendorf tube, and centrifuge at 2000 RPM/4 min. Cells were then lysated 5 min/RT with 70 P of lysis buffer (1% NP-40, 50 mM Tris pH 8, 150 mM NaCl) supplemented with protease inhibitor cocktail (Complete Mini, Roche, 1 836 153). Surnatants were cleared by 35 centrifuge at 13000 RPM/5 min, and transferred into a new tube: 30 I! of 4x loading buffer (Invitrogen) were added; samples were mixed and boiled at 95*C/2 min, spun down and stored at -20*C. 28 An SDS-PAGE migration was performed, running a NuPAGE 12% Bis acrylamide gel in reducing conditions, using lx Running Buffer, for 50 min at 200V (start 100-125 mA, end 60-80 mA). Then, semi-dry method was used to transfer separated cell-proteins to 5 Nitrocellulose Membrane, at 14V/ 1h30. As first antibodies, rabbit. polyclonal against MSP1-p-83, diluted in PBST at least 1:30000, and against MSPI-p-42,' diluted at leastl:50000, were used. The second antibody was a swine anti-rabbit antibody coupled to horse-radish peroxidase allowing the visualization of the bands by the enhanced chemiluminescence kit 10 (ECLTM, Amershan LifeScience). For immunofluorescence, Vero cells were seeded on a 24mm x 24mm glass cover slips in 35 mm wells, cultured overnight and infected with rescued recombinant virus. 3 days after infection cells on coverslips were fixed with 3,7% paraformaldehyde in PBS, and permeabilized with 0.1% TX-100, washed with 15 blocking solution (PBS containing 1% BSA) for lh, and stained with the specific antibodies. Mouse hybridoma supernatant mAb 5.2, which recognises a EGF-like domain in the p-19 portion of p-42, was used in a dilution 1:100 followed by FITCH conjugated goat anti-mouse serum, diluted 1:250. Example 10: growth kinetics curve 20 MRC5 cells seeded on 35 mm dish (1-5x10 5 ) were monitored for 90% confluence and infected with cleared virus suspension from cell-associated virus fraction, using 0.1 MOI, including MVEZ as control. Samples, corresponding to the so-called "free-cell virus fraction" and to the so-called "cell-associated virus fraction", were collected daily for one week and titrated. 25 Example 11: Mice immunisation The immunogenic power of the rescued recombinant MV-Malaria viruses described was proven by immunisation tests performed on transgenic mice IFNAR/CD46, susceptible to MV infections. The animals were kept under optimal' hygienic conditions and were immunized at 6-8 weeks of age. Below is provided an 30 example of mice immunization with two recombinant Measles-Malaria virus: the MeV2EZ-d-p42-SgrAl (the GPI anchored form) and the MeV2EZ-d-p42* (the secreted form). Immunisation was performed intra-muscularly using 10s PFU of each recombinant MV-Malaria in three injections at 0, 4 and 8 weeks. Mice immunized with recombinant-empty Measles (rMVEZ13- Empty cloned) served as negative 35 control. UV inactivated rMV was used as a control to determine the effect of virus replication on activation of immune responses. The immune response of the MV vectored antigen was tested compared to the purified d-42 protein (0.5mg/ml): mice 29 were immunized sub cutaneously with 20 pg of protein in Incomplete Freund's Adjuvant. The presence of MV-specific antibodies in the sera from the immunised IFNAR/CD46 mice (6 per test group and 3 for control group) was determined by 5 ELISA using 96-microwell plates, coated with Measles virus EIA bulk (ATCC VR-24), for IgG antibody detection. Protein was diluted 0.6 pg/ml with 0.05 M carbonate buffer (pH 9.4), and 100 pl per well was added to 96-well-microtiter plates. The plates were incubated overnight at 4"C, washed with PBS/0.05% Tween 20 (PI) (ph 7.4), incubated with PT (0.1 ml/weUl)-10% BSA for 60 min at 37'C, and washed again with 10 PT. Serial 2-folds dilutions of the tested sera were added (100 pl/well), and the plates were incubated for 60 min at 37'C. The plates were washed with PT and were incubated with 100 pl of goat anti-mouse IgG HRP diluted 1:2000 in P' for 30 min at 37"C. The plates were washed with PT and incubated with 100 pl OPD (o Phenylendiamin, Fluka 78411). The reaction was stopped after 3-4 min. Plates were 15 read on a MicroElisa Reader at a wave length of 490 nm. Readings higher than three folds negative controls were scored as positive reaction. The presence of MV-Malaria-specific antibodies in the sera of immunised CD46 mice (at least 10 per test group) was determined by ELISA assay. Briefly, 96-microwell plates were coated 50 ng/ well MSP- 1-d42 3D7 strains, diluted with carbonate buffer 20 pH 9.4. The plates were incubated overnight at 4*C, washed with PBS/0.05% Tween 20 (PT). Subsequently, unspecific interaction were blocked with 10% defatted milk dissolved in PT for Ihour at 37*C and wells were washed again with PT. The plates were consecutively incubated with various dilutions of mouse sera (starting at 1:200, followed by serial two-fold dilutions), peroxidase-conjugate goat anti-mouse [go and 25 with OPD substrate. Optical density values were measured at 490 nm. Values above the cut-off background level (mean value of sera from MV immunised mice multiplied by a factor of 2.1) were considered positive. Titres were depicted as reciprocal end dilutions. The humoral immune responses against Measles are shown in Figure A. The humoral 30 immune responses against Malaria p42 are shown in Figure B. 35 30 Figure A: Humoral response against Measles 1077: W4 1mn':W5 T IONIC1Z077;OW6 $~ 10DODa . -P- 10 -as 10000 1000 i10 1000 P P42* UV-P42*protein rMV P42 PA UVEj42* W;n rv 1077: Wa MR7: W10 _ e_ 10000 * Ion" 10 1017:1W0 107:0i P42 P42* UV-P42"protein rMV P42 P42" UV-P42* protein rW rMeV2EZ-d-p42-SgrAI rMeV2EZ-d-p42* 5SgrAl 100 3 ICO slo* seee 10010 Wa I& lo-0 4 WB w Blood was taken regularly and tested for Meastes IgG Titers. 31 Figure B: Humoral response against Malaria W4 W6 100000 100000.42 1 -- 1000 100 100 0 10 ft 10eUin I I f I P42 PWr W-PWr protn rM P42 P42* IN2 r atafn dilV towo 110000. 0 *c 1o -. 4. . IWO. £ ic1oooc 100 10 S 2P4 2 a; w- protein M rMeV2EZ-d-p42-SgrAI rMeV2EZ-d-p42*-SgrAI I001000. g10100 10. 19.' - W4 ws we VAO 10 Blood was taken regularly and tested for Malaria IgO Titers 32 Example 12: Purification of recombinant measles virus expressing malaria antigens from Defecting Interfering Particles (Dis) by plaque purification. It is known from literature that after a certain number of passages with Paramyxoaviruses, and in particular with measles virus, an accumulation of defective 5 interfering particles (Dis) will occur (23, 24). It has been described that these Dis develop various defects: negative impact on vaccine safety, negative influence on virus yields in production, genome instability and suppression of immune reaction after vaccination. In order to avoid such Dis with our new recombinant viruses, we have applied the method of plaque purification as described in example 6 with the 10 exception that we use MRC5 cell instead of 293T cells. After the formation of clear, well defined syncytia we aspirated under the microscope with a micropipette such material for further passaging in a fresh MRC5 tissue culture. Example 13: Purification of recombinant measles virus expressing malaria antigens from Defecting Interfering Particles (Dls) by end point dilution. 15 The end point dilution technique was applied in microplates: in all wells a fresh monolayer of MRC5 cells had just developed. The virus suspension containing recombinant measles-malaria viruses was prepared in two fold dilutions. From the well of the latest monolayer where a syncytia was detected the supernatant was aspirated with a pipette. The supernatant was mixed with a suspension containing 20 MRC5 cells. This mixture was incubated at 4*C for 1 hour. Finally, it was transferred in a small Costar flask and incubated at 35*C/5%C 2 and harvested for purify recombinant measles-malaria virus after ten days. Example 14: Production of a combined measles-malaria vaccine The working seed of the described recombinant measles-malaria virus has been 25 incubated on MRC5 cell monolayer in 1750cm 2 roller bottles at 35 *C for ten days. The cells have been monitored every day for status of health and confluence. On day ten at highest level of syncytia formation, the supernatant was pumped in a steel cylinder for storage in liquid nitrogen, The same procedure was repeated two days later. After performing of all the tests (virus titer, genome stability, virus safety, cell 30 safety, chemical analysis, sterility and others), the harvests have been thawed up and mixed with stabilizer containing gelatine, sorbitol, amminoacids and other sugars to final dilution of 10s. With a automated filling machine small lyo bottles (F3) have been inoculated with 0.5ml each. A specially calculated lyophilisation program was used to guarantee maximal survival of the product during the freeze-drying process. 33 BIBLIOGRAPHY 1. Fields Virology, fifth edition (2007), eds.-in-chief Knipe, D.M. &. Howley, P. M. Lippincott Williams & Wilkins, Philadelphia PA 19106, USA. 2. Enders, J. F., and Peebles, T. C. (1954). Propagation in tissue cultures of 5 cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med., 86: 277-286. 3. Griffin, D. (2007) Measles virus. In: Fields Virology, fifth edition, eds.-in-chief Knipe, D.M. &. Howley, P. M. Lippincott Williams & Wilkins, Philadelphia PA 19106, USA. 10 4. Parks, C. L., Lerch, R. A., Walpita, P., Wang, H. P., Sidhu, M. S., and Udem, S. A. (2001). Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J. Virol., 75: 921-933. 5. Parks, C. L., Lerch, R. A., Walpita, P., Wang, H. P., Sidhu, M. S., and Udem, S. A. (2001). Comparison of predicted amino acid sequences of measles virus strains in the 15 Edmonston vaccine lineage. J. Virol., 75: 910-920. 6. Hilleman, M.R. (2002). Current overview of the pathogenesis and prophylaxis of measles with focus on practical implications. Vaccine, 20: 651-665. 7. Ovsyannikova IG., Reid, K.C., Jacobson, R.M., Oberg, A.L., Klee, G.G., Poland, G.A. (2003). Cytokine production patterns and antibody response to measles vaccine. 20 Vaccine, 21(25-26); 3946-53. 8. Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, K. D6tsch, G. Christiansen, and M. Billeter. (1995). Rescue of measles viruses from cloned DNA. EMBO Journal., 14: 5773-5784. 9. Martin, A., Stacheli, P. and Schneider, U. (2006). RNA polymerase Il-controlled 25 expression of antigenomic RNA enhances the rescue efficacies of two different members of the Mononegavirales independently of the site of viral genome replication. J. Virol., 80: 5708-5715. 10. Radecke, F., and M. Billeter. (1997). Reverse genetics meets the nonsegmented negative-strand RNA viruses. Rev. Med. Virol., 7: 49-63. 30 11. Singh M. R., Cattaneo, R., Billeter, M.A. (1999). A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice. J. Virol., 73: 4823-4828. 12. Wang, Z.L., Hangartner, L., Cornu, T.I., Martin, L. R., Zuniga, A., and Billeter, M. (2001). Recombinant measles viruses expressing Deterologous antigens of mumps 35 and simian immunodeficiency viruses. Vaccine, 19: 2329-2336. 84 13. Dilraj, A., Cutts, F.T., de Castro, J.F., Wheeler, J.G., Brown, D., Roth, C., Coovadia, H.M., Bennett, J.V. (2000). Response to different measles vaccine strains given by aerosol and subcutaneous routes to schoolchildren: a randomised trial. Lancet, 355(9206): 798-803. 5 14. Holder A.A. and Freeman, R.R. (1984). The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J. Exp. Med, 160(2): 624-9. 15. Blackman M.J., Whittle H., and Holder A.A. (199 1). Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton 10 secondary processing product which is shed prior to erythrocyte invasion. Mol. Biochem. Parasitol., 49(1): 35-44. 16. Remarque, E.J., Faber, B.W., Kocken, C.H.M., and Thomas, A.W. (2007). Apical membrane antigen 1: a malaria vaccine candidate in review. Trends Parasitol, 24: 74 83. 15 17. Polley, S.D., Mwangi, T., Kocken, C.H., Thomas, A.W., Dutta, S., Lanar, D.E., Remarque, E., Ross, A., Williams, T.N., Mwambingu, G., Lowe, B., Conway, D.J., and Marsh, K. (2004). Human antibodies to recombinant protein constructs of Plasmodiumfaliparum apical membrane antigen 1 (AMA 1) and their association with protection from malaria. Vaccine, 23: 718-728. 20 18. Cort6s, A., Mellombo, M., Masciantonio, R., Murphy, V.J., Reeder, J.C., and Anders, R.F. (2005). Allele specificity of naturally acquired antibody responses against Plasmodium falcipamum apical membrane antigen I. Infect. Immun., 73: 422 430. 19. Remarque, E.J., Faber, B.W., Kocken, C.H.M., and Thomas, A.W. (2008). A 25 diversity-covering approach to immunisation with Plasmodium falcparum AMAI induces broader allelic recognition and growth inhibition responses in rabbits. Infect. Immun. 20. Garcia, J.E., Puentes, A., and Patarroyo, M.E. (2006). Developmental biology of sporozoite-host interactions in Plasmodium faliparum malaria: implications for 30 vaccine design. Clin. Microbiol. Rev., 19(4): 686-707. 21. Ballou, W.R., and Cahill, C.P. (2007). Two Decades of Commitment to Malaria Vaccine Development: GlaxoSmithKline Biologicals. Am. J. Trop. Med. Hyg., 77(6..Suppl): 289-295. 22. Girard, M.P, Reed, Z.H., Friede, M., and Kieny, M.P. (2007). A review of human 35 vaccine research and development: Malaria. Vaccine, 25: 1567-1580. 35 23. Roux, L., Simon, A.E., Holland, J.J. (1991). Effects of Defective Interfering Viruses on virus replication and pathogenesis in vitro and in vivo. Adv. Virus Res., 40: 181 221. 24. Calain, P., and Roux, L (1988). Generation of measles virus defective interfering 5 particles and their presence in a preparation of attenuated live-virus vaccine . J. Virol., 62 (8):2859-2866. 36

Claims (42)

1. A combined measles-malaria vaccine comprising a recombinant measles vaccine virus which express malaria antigens capable of eliciting immune response and protection both against measles and malaria. 5

2. The combined measles-malaria vaccine as claimed in claim 1 wherein the recombinant measles vaccine virus expresses single or different malaria antigens.

3. The combined measles-malaria vaccine as claimed in claim 1 wherein the recombinant measles vaccine virus expresses MSP1 malaria antigen. 10

4. The combined measles-malaria vaccine as claimed in claim I wherein MSPL malaria antigen is 190 to 200KDa (d190).

5. The combined measles-malaria vaccine as claimed in claim 1 wherein MSPI malaria antigen is d83-30-38.

6. The combined measles-malaria vaccine as claimed in claim I wherein MSP I 15 malaria antigen is d42.

7. The combined measles-malaria vaccine as claimed in any preceding claims wherein the recombinant measles vaccine virus expresses MSP1 malaria antigen in both anchored and secreted forms.

8. The combined measles-malaria vaccine as claimed in any preceding claims 20 wherein the recombinant measles vaccine virus expresses MSP1 malaria' antigen in both anchored and secreted forms 3D7 strain and MAD 20 preferably the FCBI strain.

9. The combined measles-malaria vaccine as claimed in any preceding claims wherein the recombinant measles vaccine virus expresses MSP1 malaria 25 antigen in both anchored and secreted forms PCBl strain.

10. The combined measles-malaria vaccine as claimed in claim wherein the recombinant measles vaccine virus expresses Diversity Covering (DiCo) AMA 1 malaria antigen.

11. The combined measles-malaria vaccine as claimed in claims wherein the 30 recombinant measles vaccine virus expresses DiCo-1 of AMA1 malaria antigen

12. The combined measles-malaria vaccine as claimed in claim wherein the recombinant measles vaccine virus expresses DiCo-2 of AMAl malaria antigen 35

13. Tie combined measles-malaria vaccine as claimed in claims wherein the recombinant measles vaccine virus expresses DiCo-3 of AMA1 malaria antigen

14. The combined measles-malaria vaccine as claimed in claim wherein the recombinant measles vaccine virus expresses DiCo-1, DiCo-2 and DiCo-3 40 of AMAl malaria antigen 37

15. The combined measles-malaria vaccine as claimed in claim 10 to claim 14 wherein the recombinant measles vaccine virus expresses Diversity Covering (DiCo) of AMAI malaria antigen in trans membrane and secreted forms. 5

16. The combined measles-malaria vaccine as claimed in claim wherein the recombinant measles vaccine virus expresses CS malaria antigen

17. The combined measles-malaria vaccine as claimed in any preceding claims wherein the malaria antigen is cloned between P and M or H and L protein of recombinant measles vaccine virus. 10

18. A measles vaccine virus vector comprising the nucleotide sequence of antigen of malaria.

19. The vector as claimed in claim 18 wherein the nucleotide sequence is selected from figure 26 to figure 35.

20. The vector as claimed in claim 18 and claim 19 wherein the measles 15 vaccine virus vector further comprises nucleotide sequence selected from figure 24 to figure 25.

21. The vector as claimed in claim 18 wherein the nucleotide sequence encodes malaria antigens selected from d83-30-38 and d42 and d190 fragments of MSP1 or Diversity Covering (DiCo) AMA1 or CS protein. 20

22. A host comprising the vector of claim 18.

23. The host as claimed in claim 22 is selected from E.coli or mammalian cell line.

24. The combined measles-malaria vaccine as claimed in any preceding claims wherein the recombinant measles virus originating from a vaccine strain 25 derived from Edmoston Zagreb.

25. The combined measles-malaria vaccine as claimed in any preceding claims wherein recombinant measles vaccine virus which expresses at least one malaria antigens selected from d83-30-38 and d42 and d190 fragments of MSP1 or Diversity Covering (DiCo) AMA1 or CS protein. 30

26. The combined measles-malaria vaccine as claimed in any preceding claims wherein recombinant measles vaccine virus which expresses two or more malaria antigens selected from d83-30-38 and d42 and d190 fragments of MSP1 or Diversity Covering (DiCo) AMAI or CS protein or combination thereof. 35

27. The combined measles-malaria vaccine as claimed in any preceding claims wherein recombinant measles vaccine virus comprises the following sequences. MSP-1 d-190-3D7 AN1OTE MSP-l d-190*-3D7 AN102TE 40 MSP-1 d-83-30-38-3D7 AN103TE 38 MSP-l d-83-30-38*-3D7 AN 104TE MSP-1 d-42-3D7 AN105TE MSP-1 d-42*-3D7 AN106TE MSP-1 d-190-FCBJI AN107TE S CS ANI08TE DiCo l-complete AN 109TE DiCol-ecto ANI IOTE

28. The combined measles-malaria vaccine as claimed in any preceding claims 10 wherein recombinant measles vaccine virus encods in addition to the malaria antigens protein with adjuvantic properties.

29. The vaccine as claimed in any preceding claims comprising recombinant measles viruses encoding in addition to the malaria antigens an interleukin, preferably interleukin 2. 15

30. The vaccine as claimed in any preceding claims comprises one of the described recombinant measles malaria viruses or a mixture of two to several such viruses.

31. The vaccine as claimed in any preceding claims wherein the described recombinant measles malaria viruses or a mixture of two to several such 20 viruses devoid of defective interfering particles (Dis)

32. The vaccine as claimed in any preceding claims wherein the adventitiously arisen DI particles have been eliminated by plaque purification.

33. The vaccine as claimed in any preceding claims wherein the adventitiously arisen DI particles have been eliminated by end point dilution. 25

34. The vaccine as claimed in any preceding claims wherein the adventitiously arisen DI particles have been eliminated by physical methods such as differential centrifugation.

35. The vaccine as claimed in any preceding claims being a component of a combined vaccine where the other components are rubella, mumps, 30 varicella or another life attenuated vaccine virus, naturally attenuated or recombinant, alone or in combination.

36. The vaccine as claimed in any preceding claims comprises suitable stabilizer, such as gelatin and/or human serum albumin and sorbitol as main components, ideal for parenteral application. 35

37. The vaccine as claimed in any preceding claims comprises with a suitable stabilizer and/or adjuvant ideal for intranasal application.

38. The vaccine as claimed in any preceding claims comprises suitable stabilizer and/or adjuvant ideal for inhalation application.

39. The vaccine as claimed in any preceding claims comprises suitable 40 stabilizer and/or adjuvant ideal for oral application. 39

40. The vaccine as claimed in any preceding claims comprises suitable stabilizer and/or adjuvant ideal for trans dermal application.

41. The vaccine as claimed in any preceding claims comprises suitable stabilizer and/or adjuvant ideal for any suppository formulation. 5

42. A composition of combined measles-malaria vaccine comprising stabilizer and/or adjuvant. 10 15 20 25 30 40