WO2004055187A1 - Recombinant viral-based malaria vaccines - Google Patents
Recombinant viral-based malaria vaccines Download PDFInfo
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- WO2004055187A1 WO2004055187A1 PCT/EP2003/051019 EP0351019W WO2004055187A1 WO 2004055187 A1 WO2004055187 A1 WO 2004055187A1 EP 0351019 W EP0351019 W EP 0351019W WO 2004055187 A1 WO2004055187 A1 WO 2004055187A1
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Definitions
- the invention relates to the field of medicine. More in particular, the invention relates to the use of a recombinantly produced viral vector as a carrier of an antigenic determinant selected from a group of malaria pathogens for the development of a vaccine against malaria infections .
- Malaria currently represents one of the most prevalent infections in tropical and subtropical areas throughout the world. Per year, malaria infections lead to severe illnesses in hundreds of million individuals worldwide, while it kills 1 to 3 million people in developing and emerging countries every year. The widespread occurrence and elevated incidence of malaria are a consequence of the increasing numbers of drug- resistant parasites and insecticide-resistant parasite vectors . Other factors include environmental and climatic changes, civil disturbances and increased mobility of populations . Malaria is caused by the mosquito-borne hematoprotozoan parasites belonging to the genus Plas odium. Four species of Plasmodium protozoa (P. falciparum, P. vivax, P.
- ovale and P.malariae are responsible for the disease in man; many others cause disease in animals, such as P.yoelii and P.berghei in mice.
- P.-alciparum accounts for the majority of infections and is the most lethal type (“tropical malaria") .
- Malaria parasites have a life cycle consisting of several stages . Each one of these stages 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' form of the malaria parasite into the mammalian bloodstream. Sporozoites remain for few minutes in the circulation before invading hepatocytes .
- 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.
- CS circumsporozoite'
- the parasites replicate and develop into so-called schizonts' . These schizonts occur in a ratio of up to 20,000 per infected cell.
- T-lymphocytes especially CD8+ T-lymphocytes (Romero et al. 1998) .
- 'merozoites' After about one week of liver infection, thousands of so-called 'merozoites' are released into the bloodstream and enter red blood cells, becoming targets of antibody-mediated immune response and T-cell secreted cytokines. After invading erythrocytes, the merozoites undergo several stages of replication and transform into so-called 'trophozoites' and into schizonts and merozoites, which can infect new red blood cells. This stage is associated with overt clinical disease. A limited amount of trophozoites may evolve into 'gametocytes', which is 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 formation and subsequent transformation into ookinetes, 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.
- Current approaches to malaria vaccine development can be classified according to the different stages in which the parasite can exist, as described above. Three types of possible vaccines can be distinguished: - Pre-erythrocytic vaccines, which are directed against sporozoites and/or schizont-infected cells . These types of vaccines are mostly CS-based and should ideally confer sterile immunity, mediated by humoral and cellular immune response, preventing malaria infection. - Asexual blood stage vaccines, which are 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 .
- - Transmission-blocking vaccines which are designed to hamper the parasite development in the mosquito host. This type of vaccine should favor the reduction of population-wide malaria infection rates .
- the CS protein is the only P. falciparum antigen demonstrated to consistently prevent malaria when used as the basis of active immunization in humans against mosquito-borne infection, albeit it at levels that is often insufficient.
- Theoretical analysis has indicated that the vaccine coverage as well as the vaccine efficiency should be above 85%, or otherwise mutants that are more virulent may escape (Gandon et al. 2001) .
- One way of inducing an immune response in a mammal is by administering an infectious carrier, which harbors the antigenic determinant in its genome.
- an infectious carrier which harbors the antigenic determinant in its genome.
- One such carrier is a recombinant adenovirus, which has been replication- defective by removal of regions within the genome that are normally essential for replication, such as the El region.
- examples of recombinant adenoviruses that comprise genes encoding antigens are known in the art (WO 96/39178) , for instance HIV-derived antigenic components have been demonstrated to yield an immune response if delivered by recombinant adenoviruses (WO 01/02607; WO 02/22080) .
- recombinant adenovirus-based vaccines have been developed.
- Figure 1 shows the newly synthesized clone 02-148 (pCR- script.Pf), which is based on a range of known Plasmodium falciparum genes, and which encodes the novel circumsporozoite protein (A) (SEQ ID NO:3) , plus the codon-optimized nucleic acid sequence (B) (SEQ ID NO:1) .
- SEQ ID NO: 2 is the translated protein product translated from the Coding Sequence of SEQ ID N0:1 as generated by Patentln 3.1.
- SEQ ID NO:2 is identical to SEQ ID NO: 3 in content.
- Figure 2 shows the amino acid sequence (A) (SEQ ID NO: 6) and nucleic acid sequence (B) (SEQ ID NO: 4) of synthetic clone named 02-659 (pf-aa-sub) , which is the circumsporozoite gene of the P.falciparu-n strain 3D7, lacking the C-terminal 14 amino acids.
- SEQ ID NO: 5 is the translated protein product translated from the Coding Sequence of SEQ ID NO: as generated by Patentln 3.1.
- SEQ ID NO: 5 is identical to SEQ ID NO: 6 in content.
- Figure 3 shows the amino acid sequence (A) (SEQ ID NO: 9) and nucleic acid sequence (B) (SEQ ID NO: 7) of the codon- optimized circumsporozoite gene of P.yoelii .
- SEQ ID NO: 8 is the translated protein product translated from the Coding Sequence of SEQ ID NO:7 as generated by Patentln 3.1.
- SEQ ID NO: 8 is identical to SEQ ID NO: 9 in content.
- Figure 4 shows the (A) cellular immune response and the (B) humoral immune response in mice upon immunization with Ad5- and Ad35-based vectors harboring the P.yoelii circumsporozoite gene, administered via two routes: intra muscular and subcutaneous in different doses .
- Figure 5 shows the inhibition in mice of a P.yoelii sporozoite challenge following immunization with Ad5- and Ad35-based vectors harboring the P.yoelii circumsporozoite gene, administered in different doses, depicted in percentage of inhibition (A) , and in the presence of a parasite specific RNA molecules in the liver (B) .
- Figure 6 shows the cellular immune response raised by immunization with an Ad5-based vector harboring the full length P.falciparu circumsporozoite gene, and two deletion mutants, administered in different doses.
- the present invention relates to different kinds of replication-defective recombinant viral vectors comprising a heterologous nucleic acid encoding an antigenic determinant of several Plasmodium protozoa.
- viral vectors that comprise nucleic acids encoding the circumsporozoite (CS) protein of P.falciparum and P.yoelii.
- said viral vector is an adenovirus, preferably based on a serotype that is efficient in delivering the gene of interest, that encounters low numbers of neutralizing antibodies in the host and that binds to the relevant immune cells in an efficient manner.
- the CS protein is generated such that it will give rise to a potent immune response in mammals, preferably humans.
- the expression of the protein is elevated due to codon-optimization and thus altering the codon-usage such that it fits the host of interest.
- the novel CS proteins of the present invention are depicted in figure 1A (SEQ ID NO:3), 2A (SEQ ID N0:6) and 3A (SEQ ID NO:9), while the codon-optimized genes encoding said proteins are depicted in figure IB (SEQ ID NO:l), 2B (SEQ ID NO: 4) and 3B (SEQ ID NO:7) respectively.
- the invention also relates to vaccine compositions comprising a replication-defective recombinant viral vector according to the invention, and a pharmaceutically acceptable carrier, further comprising preferably an adjuvant. Furthermore, the invention relates to the use of a vaccine composition according to the invention in the therapeutic, prophylactic or diagnostic treatment of malaria. DETAILED DESCRIPTION OF THE INVENTION
- the present invention relates to the use of recombinant viruses as carriers of certain specific antigenic determinants selected from a group of malaria antigens. It is the goal of the present invention to provide a solution to at least a part of the problems outlined above for existing vaccines against malaria.
- the present invention relates to a replication- defective recombinant viral vector comprising a heterologous nucleic acid encoding an antigenic determinant of Plasmodium falciparum.
- said viral vector is an adenovirus, an alphavirus or a vaccinia virus .
- said viral vector is an adenovirus, wherein said adenovirus is preferably derived from a serotype selected from the group consisting of: Ad5, Adll, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.
- the replication-defective recombinant viral vector according to the invention comprises an antigenic determinant that is the circumsporozoite (CS) protein, or an immunogenic part thereof.
- said heterologous nucleic acid is codon-optimized for elevated expression in a mammal, preferably a human. Codon- optimization is based on the required amino acid content, the general optimal codon usage in the mammal of interest and a number of provisions of aspects that should be avoided to ensure proper expression.
- Such aspects may be splice donor or -acceptor sites, stop codons, Chi-sites, poly (A) stretches, GC- and AT-rich sequences, internal TATA boxes, etcetera.
- the invention relates to a replication-defective recombinant viral vector according to the invention, wherein the adenine plus thymine content in said heterologous nucleic acid, as compared to the cytosine plus guanine content, is less than 87%, preferably less than 80%, more preferably less than 59% and most preferably equal to approximately 45%.
- the invention provides in one embodiment a replication- defective recombinant viral vector, wherein said circumsporozoite protein is the circumsporozoite protein as depicted in figure 1A and in another embodiment a codon-optimized heterologous nucleic acid as depicted in figure IB.
- the proteins can be in a purified form, but also expressed in vivo from nucleic acid delivery vehicles such as the recombinant viral vectors of the present invention. In a purified form, such proteins can be applied in other types of vaccines, wherein the protein is for instance enclosed in liposomes or other carriers used in the art.
- the nucleic acid can be cloned into other vectors than as disclosed herein, but also be applied as naked DNA in other vaccine-settings.
- the invention relates to a replication-defective recombinant viral vector according to the invention, wherein the circumsporozoite protein, or the immunogenic part thereof, is lacking a functional GPI anchor sequence.
- the invention also discloses novel genes that may be used in humans as well as in other mammals. Therefore, the invention also relates to a replication-defective recombinant viral vector comprising a heterologous nucleic acid encoding the circumsporozoite protein of Plasmodium yoelii, wherein said nucleic acid is codon-optimized for elevated expression in a mammal.
- said viral vector is an adenovirus, an alphavirus or a vaccinia virus, and it is even more preferred to use a recombinant adenovirus, which is preferably selected from the group consisting of: Ad5, Adll, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.
- a recombinant adenovirus which is preferably selected from the group consisting of: Ad5, Adll, Ad26, Ad34, Ad35, Ad48, Ad49 and Ad50.
- the adenine plus thymine content in said nucleic acid is less than 87%, preferably less than 80%, more preferably less than 59% and most preferably equal to approximately 45%.
- the invention provides in one embodiment a replication-defective recombinant viral vector according to the invention, wherein said circumsporozoite protein is the circumsporozoite protein as depicted in figure 3A, while in another embodiment, a replication-defective recombinant viral vector is provided, wherein said nucleic acid is the nucleic acid as depicted in figure 3B.
- the circumsporozoite protein, or the immunogenic part thereof is lacking a functional GPI anchor sequence.
- the invention further relates to an isolated nucleic acid encoding a circumsporozoite protein of Plasmodium falciparum as depicted in figure IB, wherein said nucleic acid is codon-optimized, and to an isolated nucleic acid encoding a circumsporozoite protein of Plasmodium falciparum strain 3D7, as depicted in figure 2B, wherein said nucleic acid is codon-optimized.
- Such isolated nucleic acids can be applied in subcloning procedures for the generation of other types of viral-based vaccines, apart for the types as disclosed herein.
- isolated nucleic acids can be used for naked DNA vaccines or in cloning procedures to generate vectors for in vitro production of the encoded protein, which, in itself can be further used for vaccination purposes and the like.
- the production can be in all kinds of systems, such as bacteria, yeasts or mammalian cells known in the art.
- an isolated nucleic acid encoding a circumsporozoite protein of Plasmodium yoelii as depicted in figure 3B is provided, wherein said nucleic acid is codon-optimized.
- a vaccine composition comprising a replication-defective recombinant viral vector according to the invention, and a pharmaceutically acceptable carrier is provided.
- Pharmaceutically acceptable carriers are well known in the art and used extensively in a wide range of therapeutic products.
- carriers are applied that work well in vaccines .
- More preferred are vaccines, further comprising an adjuvant.
- Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant.
- the invention also relates to the use of a vaccine composition according to the invention in the therapeutic, prophylactic or diagnostic treatment of malaria.
- Another embodiment of the present invention relates to a method of treating a mammal for a malaria infection or preventing a malaria infection in a mammal, said method comprising (in either order, or simultaneously) the steps of administering a vaccine composition according to the invention, and administering a vaccine composition comprising at least one purified malaria- derived protein or peptide.
- the invention also relates to a method of treating a mammal for a malaria infection or preventing a malaria infection in a mammal, said method comprising (in either order, or simultaneously) the steps of administering a vaccine composition comprising a replication-defective recombinant viral vector comprising a malaria circumsporozoite antigen according to the invention; and administering a vaccine composition comprising a replication-defective recombinant viral vector comprising another antigen, such as LSA-1 or SA-3 according to the invention.
- the advantages of the present invention are multifold.
- recombinant viruses such as recombinant adenoviruses can be produced to very high titers using cells that are considered safe, and that can grow in suspension to very high volumes, using medium that does not contain any animal- or human derived components
- the present invention combines these features with a vector harboring the circumsporozoite gene of Plasmodium falciparum.
- P. falciparum is the parasite that causes tropical malaria.
- the gene has been codon-optimized to give an expression level that is suitable for giving a proper immune response in humans .
- the present invention provides a vaccine against malaria infections, making use of for instance adenoviruses that do not encounter high titers of neutralizing antibodies.
- adenoviruses are serotype 11 and 35 (Adll and Ad35, see WO 00/70071 and WO 02/40665) .
- the nucleic acid content between the malaria-causing pathogen, such as P. falciparum and the host of interest, such as Homo sapiens is very different.
- the invention now provides a solution to some of the disadvantages of vaccines known in the art, such as expression levels that are too low to elicit a significant immune response in the host of interest, preferably humans.
- Recombinant viral vectors have been used in vaccine set-ups . This has been demonstrated for vaccinia-based vaccines and for adenovirus-based vaccines.
- a platform based on alphaviruses is being developed for vaccines as well.
- the invention relates to the use of recombinant adenoviruses that are replication defective through removal of at least part of the El region in the adenoviral genome, since the El region is required for replication-, transcription-, translation- and packaging processes of newly made adenoviruses.
- El deleted vectors are generally produced on cell lines that complement for the deleted El functions . Such cell lines and the use thereof for the production of recombinant viruses have been described extensively and are well known in the art.
- PER.C6TM cells as represented by the cells deposited under ECACC no.
- 96022940 at the European Collection of Animal Cell Cultures (ECACC) at the Centre for Applied Microbiology and Research (CAMR, UK) , are being used to prevent the production of replication competent adenoviruses (rca) .
- cells are being applied that support the growth of recombinant adenoviruses other than those derived of adenovirus serotype 5 (Ad5) .
- Ad5 adenovirus serotype 5
- Adenoviral-based vectors that have been used in the art mainly involved the use of Ad5 vectors.
- administration of Ad5 and efficient delivery to the target cells of interest, responsible for sufficient immunogenic responses is hampered by the presence of high titres of neutralizing antibodies circulating in the bloodstream if a subject previously encountered an Ad5 infection.
- serotypes are better suited for therapeutic use, and it turned out that a limited number of serotypes encountered neutralizing antibodies in only a small percentage of individuals in the human population.
- the invention relates to the use of adenovirus serotype 11, 26, 34, 35, 48 and 50, and more preferably to Adll and Ad35, since these serotypes encountered no neutralizing antibodies in the vast majority of tested samples.
- Adll and Ad35 adenovirus serotype 11 26, 34, 35, 48 and 50
- Adll and Ad35 adenovirus serotype 11 26, 34, 35, 48 and 50
- Adll and Ad35 preferably to Adll and Ad35
- certain adenovirus serotypes such as Adl6, Ad35 and Ad50, carry capsid proteins that specifically bind to certain receptors present on dendritic cells (WO 02/24730) .
- Ad5 is a serotype that is mainly homing to the liver, which may be a disadvantage if sufficient numbers of viral particles should infect cells of the immune system. It was found that at least in in vitro experiments some of the serotypes, different from Ad5 could infect dendritic cells multi-fold better than Ad5, suggesting that also in vivo the delivery to such cells is more efficient. It still remains to be seen whether this in vitro to in vivo translation holds up, and if serotypes other than Ad5 will give rise to the required protection level.
- Ad50 was mistakenly named Ad51.
- the Ad51 serotype that was referred to in the mentioned publications is the same as serotype Ad50 in a publication by De Jong et al. (1999) , wherein it was denoted as a B-group adenovirus.
- Ad50 as used herein, is the B-group Ad50 serotype as mentioned by De Jong et al. (1999) .
- the invention further relates to methods for vaccinating mammalian individuals in which a priming vaccine composition comprises a replication-defective recombinant adenovirus of a first serotype, while in a boosting vaccine composition a replication-defective recombinant adenovirus of a second serotype are used.
- adenoviral serotypes that encounter low pre-existing immunities in subjects that are to be treated.
- Preferred examples of such serotypes are the recombinant mentioned, wherein Ad5 is not excluded for individuals that have never experienced an Ad5 infection.
- the settings described and claimed in the applications mentioned above relate to the use of adenoviral vectors carrying transgenes such as those from measles, or gag from HIV (for treatment of humans) or SIV (for treatment and studies in monkeys) .
- a prime-boost set-up towards Malaria is a setting in which, next to different adenovirus serotypes, also different antigenic determinants may be used.
- an antigen different from CS is the Liver Specific Antigen 1 (LSA-1, Kurtis et al. 2001) .
- LSA-1 Liver Specific Antigen 1
- Such set-ups are at least for one reason useful, namely that the CS antigen is expressed mainly during the blood-stage of the parasite, while its expression goes down in the liver-stage.
- LSA-1 this situation is more or less the opposite; it is expressed to low levels during the blood-stage, but is highly expressed during the liver-stage.
- one could use both antigens in subsequent administrations it may also be used at the same time to provide protection against the parasite at the blood-stage as well as at the liver-stage.
- both antigens may be delivered by one adenovirus serotype (either cloned together in the same vector, or separately in separate vectors of the same serotype) .
- both antigens are delivered by different serotypes that may be delivered at the same time or separately in time, for instance in a prime-boost setting.
- the vaccines of the present invention may also be used in settings in which prime- boosts are being used in combination with naked DNA or other delivery means, unrelated to the replication- defective viral vectors of the present invention, such as purified proteins or peptides.
- proteins that may be used in prime-boosts (Ad/protein; protein/Ad; protein/Ad/ d; Ad/protein/Ad; Ad/ d/protein, etc) are CS, LSA-1, LSA-3, MSP-1, MSP-119, MSP-142 (see below), or the hepatitis B particles-containing and CS-derived vaccine composition known as RTS,S (see Gordon et al. (1995; US 6,306,625 and WO 93/10152).
- poxviruses vaccinia viruses, such as MVA
- flaviviruses such as alphaviruses .
- alphaviruses Non-limiting examples of alphaviruses that may be applied for delivering the immunogenic Plasmodium components of the present invention are: Ndumu virus, Buggy Creek virus, Highland J.
- Virus Fort Morgan virus, Babanki virus, Kyzylagach virus, Una virus, Aura virus, Whataroa virus, Bebaru virus, South African Arbovirus No. 86, Mayaro virus, Sagiyama virus, Getah virus, Ross River virus, Barmah Forest virus, Chikungunya virus, O'nyong-nyong virus, Western Equine Encephalitis virus (WEE) , Middelburg virus, Everglades virus, Eastern Encephalitis virus (EEE) , Mucambo virus and Pixuna virus.
- an alphavirus is the virus of choice, Semliki Forest Virus, Sindbis virus or Venezuelan Equine Encephalitis virus are applied.
- a sequence is 'derived' as used herein if a nucleic acid can be obtained through direct cloning from wild- type sequences obtained from wild-type viruses, while they can for instance also be obtained through PCR by using different pieces of DNA as a template. This means also that such sequences may be in the wild-type form as well as in altered form. Another option for reaching the same result is through combining synthetic DNA. It is to be understood that 'derived' does not exclusively mean a direct cloning of the wild type DNA. A person skilled in the art will also be aware of the possibilities of molecular biology to obtain mutant forms of a certain piece of nucleic acid. The terms 'functional part, derivative and/or analogue thereof are to be understood as equivalents of the nucleic acid they are related to.
- Such alterations are for instance deletions in the viral backbone to enable the cloning of larger pieces of heterologous nucleic acids .
- Examples of such mutations are for instance E3 deletions or deletions and/or alterations in the regions coding for the E2 and/or E4 proteins of adenovirus .
- Such changes applied to the adenoviral backbone are known in the art and often applied, since space is a limiting factor for adenovirus to be packaged; this is a major reason to delete certain parts of the adenoviral genome.
- E2, E3 and/or E4 regions of the genome may be related to stability or integrity of the adenoviral vector, as for instance described in international patent applications PCT/NL02/00280, PCT/EP03/50125, PCT/NL02/00281, PCT/EP03/50126 (non published) .
- These applications relate amongst others to the use of an E4orf6 gene from a serotype from one subgroup in the backbone of an adenovirus from another subgroup, to ensure compatibility between the E4orf6 activity and the E1B-55K activity during replication and packaging in a packaging cell line.
- 'Replication defective' as used herein means that the viral vectors do not replicate in non-complementing cells. In complementing cells, the functions required for replication, and thus production of the viral vector, are provided by the complementing cell.
- the replication defective viral vectors of the present invention do not harbor all elements enabling replication in a host cell other than a complementing cell.
- 'Heterologous' as used herein in conjunction with nucleic acids means that the nucleic acid is not found in wild type versions of the viral vectors in which the heterologous nucleic acid is cloned.
- the heterologous nucleic acid that is cloned in the replication defective adenoviral vector is not an adenoviral nucleic acid.
- 'Antigenic determinant' as used herein means any antigen derived from a pathogenic source that elicits an immune response in a host towards which the determinant is delivered (administered) .
- antigenic determinants of Plasmodium that can be delivered by using the replication defective recombinant viruses of the present invention are the circumsporozoite protein, the SE36 polypeptide, the merezoite surface protein 119 kDa C-terminal polypeptide (MSP-119), MSP-1, MSP-142, Liver Stage Antigen 1 or 3 (LSA-1 or -3) , or a fragment of any of the aforementioned.
- the invention relates to the circumsporozoite (CS) protein from P. falciparum.
- 'Codon-optimized' as used herein means that the nucleic acid content has been altered to reach sufficiently high expression levels of the protein of interest in a host of interest to which the gene encoding said protein is delivered.
- Sufficiently high expression levels in this context means that the protein levels should be high enough to elicit an immune response in the host in order to give protection to a malaria-inducing parasite that may enter the treated host before or after treatment.
- some vaccines give an immune response in humans, through which approximately 60% of the vaccinated individuals is protected against illnesses induced by subsequent challenges with the pathogen (e.g., sporozoites). Therefore the expression levels are considered to be sufficient if 60% or more of the treated individuals is protected against subsequent infections.
- nucleic acids disclosed in the present invention are codon-optimized for expression in humans. According to Narum et al. (2001), the content of adenine plus thymine (A+T) in DNA of Homo sapiens is approximately 59%, as compared to the percentage cytosine plus guanine (C+G) . The adenine plus thymine content in P. falciparum is approximately 80%.
- the adenine plus thymine content in the CS gene of P. falciparum is approximately 87%. To obtain sufficient protection it is believed to be necessary to improve production levels in the host.
- One way to achieve this is to optimize codon usage by altering the nucleic acid content of the antigenic determinant in the viral-based vector, without altering the amino acid sequence thereof.
- the replication-defective recombinant viral vectors according to the invention have an adenine plus thymine content in the heterologous nucleic acids of the present invention of less than 87%, preferably less than 80%, and more preferably less than or equal to approximately 59%. Based on codon-usage in human and the amino acid content of the CS genes of P.
- the percentages of the codon-optimized genes were even lower, reaching approximately 45% for the amino acid content as disclosed by the present invention. Therefore, as far as the CS genes are concerned it is preferred to have an adenine plus thymine content of approximately 45%. It is to be understood, that if another species than humans is to be treated, which may have a different adenine plus thymine concentration (less or more than 59%) , and/or a different codon usage, that the genes encoding the CS proteins of the present invention may be adjusted to fit the required content and give rise to suitable expression levels for that particular host.
- Example 1 Assembly of the Plasmadlum falciparum circumsporozoite synthetic gene.
- CS complete or partial sequences were used in order to identify variation between the different geographical areas and identified lab-strains .
- the final amino acid consensus sequence determined was thoroughly examined.
- the inventors of the present invention subsequently adjusted this consensus sequence and to have a new CS gene synthesized (Fig. 1) .
- the novel amino acid sequence is shown in Fig. 1A.
- the new CS protein harbors the aspects listed below (from N-terminus to C-terminus) :
- N-terminal signal sequence which would direct the protein to the endoplasmic reticulum, is left unchanged.
- HLA binding peptide amino acid (31-40) as well as region 1 (predominant B-cell epitope) are conserved, therefore these sequences are left unchanged.
- the Th2R region and the immunodominant CD8 epitope (Lockyer et al. 1989; Zevering et al. 1994) : a single consensus sequence that differs in some respects from that of the known, and frequently used lab-strain 3D7 sequence was determined. This sequence is sometimes referred to as the 'universal epitope' in literature (Nardin et al. 2001) .
- the TH3R region which is considered to be a less important CD8 epitope, is used in the form of a consensus sequence, since only point mutations were found.
- the gene encoding the newly designed CS protein was codon-optimized in order to improve its expression in mammalian cells, taking care of the following aspects to avoid cis-acting sequences: no premature pol (A) sites and internal TATA boxes should be present; Chi-sites, ribosomal entry sites and AT-rich sequence clusters should be avoided; no (cryptic) splice acceptor and - donor sites should be present; repetitive sequence stretches should be avoided as much as possible; and GC- rich sequences should also be avoided.
- the final codon- optimized gene is shown in Fig. IB.
- CS consensus sequence was synthesized and cloned into pCR-script (Stratagene) by GeneArt (Regensburg, Germany) , using methodology known to persons skilled in the art of synthetic DNA generation, giving rise to a clone named 02-148 (pCR-script.Pf) (SEQ ID NO:l) .
- Example 2 Codon-optimization of the circumsporozoite gene of the rodent-specific malaria parasite Plasmodium yoelii .
- Example 3 Generation of recombinant adenoviral vectors based on Ad5.
- RCA-free recombinant adenoviruses can be generated very efficiently using adapter plasmids, such as pAdApt, and adenovirus plasmid backbones, such as pWE/Ad.Aflll- rlTRsp.
- adapter plasmids such as pAdApt
- adenovirus plasmid backbones such as pWE/Ad.Aflll- rlTRsp.
- the adapter plasmid containing the transgene of interest in the desired expression cassette is digested with suitable enzymes to free the recombinant adenovirus sequences from the plasmid vector backbone.
- the adenoviral complementation plasmid pWE/Ad.Aflll-rlTRsp is digested with suitable enzymes to free the adenovirus sequences from the vector plasmid DNA.
- Plasmid 02-149 (GeneArt, see above) containing the codon optimized CS gene was digested with Hindlll and BamHI restriction enzymes.
- the 1.1 Kb fragment corresponding to the P.yoelii CS gene was isolated from agarose gel and ligated to Hindlll and BamHI-diges ed pIPspAdaptl vector (described in WO 99/64582) .
- the resulting plasmid was named pAdapt .CS .Pyoel and contains the CS gene under the transcriptional control of full length human immediate-early (IE) cytomegalovirus (CMV) promoter and a downstream SV40 poly (A) signal .
- IE human immediate-early
- CMV cytomegalovirus
- A downstream SV40 poly
- Plasmid 02-148 pCR-script.Pf (see above) containing the codon optimized CS gene was digested with Hindlll and BamHI restriction enzymes .
- the 1.2 kb fragment corresponding to the CS gene was isolated from agarose gel and ligated to Hindlll and BamHI- digested pIPspAdaptl vector.
- the resulting plasmid was named pAdapt .CS. Pfale and contains the CS gene under the transcriptional control of full length human immediate- early (IE) cytomegalovirus (CMV) promoter and the downstream SV40 poly (A) signal.
- IE immediate- early
- CMV cytomegalovirus
- the enzyme Pwo DNA polymerase (Inno-train Diagnostic) was used, while the following program was applied: 1 cycle of 5 min at 94°C, 1 min at 50°C, 2 min 30 sec at 72°C; 5 cycles of 1 min at 94°C, 1 min at 50°C, 2 min 50 sec at 72°C; 20 cycles of 1 min at 94°C, 1 min at 54°C, 2 min 50 sec at 72°C; and 1 cycle of 1 min at 94°C, 1 min at 54°C, followed by 10 min at 72°C.
- Pwo DNA polymerase Inno-train Diagnostic
- the amplified PCR product was digested with the restriction enzymes Hindlll and BamHI and then cloned into pIPspAdaptl which was also digested with Hindlll and BamHI .
- the resulting plasmid was designated pAdapt.
- CS.Pfale (-28) and contains the CS gene under the transcriptional control of full length human immediate- early (IE) cytomegalovirus (CMV) promoter and the downstream SV40 poly (A) signal.
- the enzyme Pwo DNA polymerase (Inno-train Diagnostic) was used, while the following program was applied: 1 cycle of 5 min at 94°C, 1 min at 50°C, 2 min 30 sec at 72°C; 5 cycles of 1 min at 94°C, 1 min at 50°C, 2 min 50 sec at 72°C; 20 cycles of 1 min at 94°C, 1 min at 54°C, 2 min 50 sec at 72°C; and 1 cycle of 1 min at 94°C, 1 min at 54°C, followed by 10 min at 72°C.
- Pwo DNA polymerase Inno-train Diagnostic
- the amplified PCR product was digested with the restriction enzymes Hindlll and BamHI and then cloned into pIPspAdaptl also digested with Hindlll and BamHI .
- the resulting plasmid was designated pAdapt.CS.Pfalc (-14) and contains the CS gene under the transcriptional control of full length human immediate- early (IE) cytomegalovirus (CMV) promoter and the downstream SV40 poly (A) signal.
- IE immediate- early
- CMV cytomegalovirus
- Plasmid 02-659 pf-aa-sub (see above) containing the codon-optimized CS gene is digested with Hindlll and BamHI restriction enzymes.
- the 1.1 kb fragment corresponding to the CS gene is ligated to Hindlll and BamHI-digested pIPspAdaptl vector.
- the resulting plasmid is designated pAdapt.CS.Pfalc (pf-aa- sub) and contains the CS gene under the transcriptional control of full length human immediate-early (IE) cytomegalovirus (CMV) promoter and the downstream SV40 poly (A) signal .
- IE human immediate-early
- CMV cytomegalovirus
- A downstream SV40 poly
- CS . yoel The generation of the recombinant virus named Ad5 ⁇ E3.
- CS . yoel was performed as follows.
- pAdapt .CS . Pyoel was digested by Pad restriction enzyme to release the left-end portion of the Ad genome.
- Plasmid pWE/Ad.Aflll- rlTRsp containing the remaining right-end part of the Ad genome has a deletion of 1878 bp in the E3 region (Xbal deletion). This construct was also digested with Pad.
- Ad5-based vectors were separately transfected with Pad digested pWE.Ad.Aflll-rlTRsp into PER-E1B55K producer cells (cells have been described in WO 02/40665) using lipofectamine transfection reagent (Invitrogen) using methods known in the art and as described in WO 00/70071. Homologous recombination between overlapping sequences led to generation of the recombinant virus named Ad5 ⁇ E3.CS. Pyoel. It is to be understtod that Ad5-based vectors can also be produced on PER.C ⁇ TM cells, which cells are represented by the cells deposited under ECACC no. 96022940 (see above) .
- the adenoviral vector, in crude lysates, resulting from this transfection were plaque purified using methods known to persons skilled in the art. Single plaques were analyzed for the presence of ' he CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cmVflask) . Upon amplification cells are harvested at full CPE and the virus is purified by a two-step Cesium Chloride (CsCl) purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- CsCl Cesium Chloride
- Ad5 ⁇ E3.CS .Pfale The generation of the recombinant virus named Ad5 ⁇ E3.CS .Pfale was performed as follows. pAdapt .CS . Pfale was digested by Pad restriction enzyme to release the left-end portion of the Ad genome. Plasmid pWE/Ad.Aflll- rlTRsp containing the remaining right-end part of the Ad genome has a deletion of 1878 bp in the E3 region (Xbal deletion) . This construct was also digested with Pad. pAdapt.CS.Pfalc was transfected with Pad digested pWE.Ad.Aflll-rlTRsp into PER-E1B55K producer cells using lipofectamine transfection reagent.
- Ad5 ⁇ E3.CS. Pfale The adenoviral vector, in crude l sates, resulting from this transfection was plaque purified using methods known to persons skilled in the art. Single plaques were analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cm 2 /flask) . Cells were harvested at full CPE and the virus was purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- Ad5 ⁇ E3.CS.Pfalc (-28) and Ad5 ⁇ E3.CS.Pfalc (-14) was performed as follows.
- pAdapt.CS.Pfalc (-28) and pAdapt.CS.Pfalc (-14) were separately digested by Pad restriction enzyme to release the left-end portion of the Ad genome.
- Plasmid pWE/Ad.Aflll-rlTRsp containing the remaining right-end part of the Ad genome has a deletion of 1878 bp in the E3 region (Xbal deletion) . This construct was also digested with Pad.
- pAdapt.CS.Pfale (- 28) and pAdapt.CS.Pfale (-14) were separately transfected with Pad digested pWE.Ad.Aflll-rlTRsp into PER-E1B55K producer cells using lipofectamine transfection reagent. Homologous recombination between overlapping sequences led to generation of recombinant viruses named respectively Ad5 ⁇ E3.CS.Pfalc(-28) and Ad5 ⁇ E3.CS.Pfale (- 14) .
- Adenoviral vectors in crude lysates resulting from these transfections are plaque purified using methods known to persons skilled in the art.
- Single plaques are analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cmVflask) .
- Cells are harvested at full CPE and the virus is purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- Ad5 ⁇ E3.CS.Pfale The generation of the recombinant virus named Ad5 ⁇ E3.CS.Pfale (pf-aa-sub) is performed as follows. pAdapt.CS.Pfale (pf-aa-sub) is digested by Pad restriction enzyme to release the left-end portion of the Ad genome. Plasmid pWE/Ad.Aflll-rlTRsp containing the remaining right-end part of the Ad genome is also digested with Pad.
- pAdapt.CS.Pfale (pf-aa-sub) is transfected with Pad digested pWE.Ad.AflII-rITRsp ⁇ E3 into PER.C6TM or PER-E1B55K producer cells using lipofectamine transfection reagent, or by other means such as electroporation or other transfection methods known to persons skilled in the art. Homologous recombination between overlapping sequences leads to generation of the recombinant virus named Ad5 ⁇ E3.CS .Pfale (pf-aa-sub) .
- the adenoviral vector, in crude lysates, resulting from this transfection is plaque purified using methods known to persons skilled in the art.
- Single plaques are analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cmVflask) .
- Cells are harvested at full CPE and the virus is purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- a first 101 bp PCR fragment containing the Ad5 pIX promoter (nucleotides 1509-1610) was generated with the primers SV40for (5'-CAA TGT ATC TTA TCA TGT CTA G-3') (SEQ ID NO: 16) and pIX5Rmfe (5'-CTC TCT CAA TTG CAG ATA CAA AAC TAC ATA AGA CC-3' ) (SEQ ID N0:17) .
- the reaction was done with Pwo DNA polymerase according to manufacturers instructions but with 3% DMSO in the final mix.
- pAdApt was used as a template.
- the program was set as follows: 2 min at 9 °C; 30 cycles of: 30 sec at 9 °C, 30 sec at 52°C and 30 sec at 72°C; followed by 8 min at 72°C.
- the resulting PCR fragment contains the 3' end of the SV40 poly (A) signal from pAdApt and the Ad5-pIX promotor region as present in Genbank Accession number M73260 from nucleotide 3511 to nucleotide 3586 and an Mfel site at the 3' end.
- a second PCR fragment was generated as described above but with primers ⁇ IX35Fmfe (5'-CTC TCT CAA TTG TCT GTC TTG CAG CTG TCA TG-3') (SEQ ID NO: 18) and 35R4 (for reference to the sequence of the 35R4 primer, see WO 00/70071) .
- pAdApt35IPl (described in WO 00/70071) was used as a template, the annealing was set at 58°C for 30 sec and the elongation of the PCR program was set at 72°C for 90 sec.
- This PCR procedure amplifies Ad35 sequences from nucleotide 3467 to nucleotide 4669 (sequence numbering as in WO 00/70071) and adds an Mfel site to the 5' end. Both PCR fragments were digested with Mfel and purified using the Qiagen PCR purification kit (Qiagen) . Approximate equimolar amounts of the two fragments were used in a ligation reaction. Following an incubation of two hours with ligase enzyme in the correct buffers, at room temperature, the mixture was loaded on an agarose gel and the DNA fragments of 1.4 kb length were isolated with the Geneclean II kit (BI0101, Inc) .
- the purified DNA was used in a PCR amplification reaction with primers SV40for and 35R4.
- the PCR was done as described above with an annealing temperature of 52°C and an elongation time of 90 sec.
- the resulting product was isolated from gel using the Qiagen gel extraction kit and digested with Agel and Bglll.
- the resulting 0.86 kb fragment containing the complete 100 nucleotide pIX promoter form Ad5 the Mfel site and the pIX ORF (fragment Mfel-Agel, including the ATG start site) from Ad35, but without a poly (A) sequence, was isolated from gel using the Geneclean II kit.
- RCA-free recombinant adenoviruses based on Ad35 can be generated very efficiently using adapter plasmids, such as pAdApt535 (described below) and adenovirus plasmid backbones, such as pWE/Ad35. ⁇ IX-rITR ⁇ E3
- pAdApt535 (described in WO 02/40665) .
- pAdApt35.Luc (described in WO 00/70071) was digested with Bglll and Agel and the resulting 5.8 kb vector was isolated from gel. This fragment was ligated with the isolated 0.86 kb Bglll-Agel fragment containing the Ad5- Ad35 chimeric pIX promotor described above, to result in a plasmid named ⁇ AdApt535.Luc, which was subsequently digested with Bglll and Apal. The resulting 1.2 kb insert was purified over gel.
- pAdApt35IPl was digested with Bglll and Apal and the 3.6-kb vector fragment was isolated over gel. Ligation of the 1.2 kb Bglll-Apal insert from pAdApt535.Luc and the 3.6 kb Bglll-Apal digested vector resulted in pAdApt535.
- Plasmid 02-149 containing the codon optimized P.yoelii CS gene was digested with the restriction enzymes Hindlll and BamHI.
- the 1.1 kb fragment corresponding to the CS gene was isolated over agarose gel and ligated to the Hindlll and BamHI digested pAdapt535 vector.
- the resulting plasmid was named pAdapt535-CS.Pyoel and contains the CS gene under the transcriptional control of the full length human CMV promoter and the downstream SV40 poly (A) signal.
- Plasmid 02-148 containing the codon optimized CS gene of P. falciparum was digested with the restriction enzymes Hindlll and BamHI.
- the 1.2 kb fragment corresponding to the CS gene was isolated over agarose gel and ligated to the Hindlll and BamHI digested pAdapt535 vector.
- the resulting plasmid was named pAdapt535-CS.Pfalc and contains the CS gene under the transcriptional control of the full length human CMV promoter and the downstream SV40 poly (A) signal.
- the cloning of the gene encoding the CS P. falciparum protein minus the GPI anchor sequence, thus with the deletion of the last 28 amino acids, into pAdapt535 is performed as follows.
- the 1.1 kb PCR fragment obtained as described above using primers Forw.Falc and Rev.Falc.CS- 28, is digested with the restriction enzymes Hindlll and BamHI and then cloned into pAdapt535 vector also digested with Hindlll and BamHI .
- the resulting plasmid is designated pAdapt535.CS.Pfale (-28) and contains the CS gene under the transcriptional control of the full length human CMV promoter and the downstream SV40 poly(A) signal.
- the cloning of the gene encoding the CS P. falciparum protein minus the GPI anchor sequence, now with the deletion of the last 14 amino acids, into ⁇ Adapt535 is performed as follows.
- the 1.1 kb PCR fragment obtained as described above using primers Forw.Falc and Rev.Falc.CS- 14, is digested with the restriction enzymes Hindlll and BamHI and then cloned into pAdapt535 vector also digested with Hindlll and BamHI .
- the resulting plasmid is designated pAda ⁇ t535.CS.Pfale (-14) and contains the CS gene under the transcriptional control of the full length human CMV promoter and the downstream SV40 poly (A) signal.
- Plasmid 02-659 pf-aa-sub (see above) containing the codon optimized CS gene is digested with Hindlll and BamHI restriction enzymes.
- the 1.1 kb fragment corresponding to the CS gene is ligated to Hindlll and BamHI digested pAda ⁇ t535 vector.
- the resulting plasmid is designated pAda ⁇ t535.CS.Pfale (pf-aa- sub) and contains the CS gene under the transcriptional control of the full length human CMV promoter and the downstream SV40 poly (A) signal.
- Ad35 ⁇ E3.CS.Pyoel The generation of the recombinant virus named Ad35 ⁇ E3.CS.Pyoel was performed as follows. pAdapt535.CS.Pyoel was digested by Pad restriction enzyme to release the left-end portion of the Ad genome. Plasmid ⁇ WE.Ad35. ⁇ IX-rITR ⁇ E3, containing the remaining right-end part of the Ad genome with a deletion of 2673 bp in the E3 region is digested with Notl. pAdapt535.CS.Pyoel was transfected with Notl digested pW ⁇ .Ad35.pIX-rITR ⁇ E3 into PER-E1B55K producer cells using lipofectamine transfection reagent.
- PER.C6TM cells were stably transfected with Seal linearised pIG35-55K DNA, carrying the E1B-55K gene of adenovirus serotype 35, after which a selection procedure with G418 yielded in 196 picked colonies. Further culturing of a limited number of well growing colonies resulted in stable cell lines that upon numerous subcultures stably expressed the Ad35 E1B-55K gene and supported the growth of recombinant Ad35 viruses, while the original PER.C6TM cell were very inefficient in supporting this .
- Ad35 ⁇ E3.CS.Pyoel The adenoviral vector, in crude lysates, resulting from this transfection was plaque purified using methods known to persons skilled in the art. Single plaques were analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cmVflask) . Upon amplification cells were harvested at full CPE and the virus was purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- Ad35 ⁇ E3.CS.Pfale The generation of the recombinant virus named Ad35 ⁇ E3.CS.Pfale was performed as follows. pAdapt535.CS.Pfale was digested by Pad restriction enzyme to release the left-end portion of the Ad genome. Plasmid pWE.Ad35.pIX-rITR ⁇ E3, containing the remaining right-end part of the Ad genome with a deletion of 2673 bp in the E3 region was digested with Notl. pAdapt535.CS.Pfale was transfected with Notl digested pWE.Ad35.pIX-rITR ⁇ E3 into PER- ⁇ 1B55K producer cells using lipofectamine transfection reagent.
- Ad35 ⁇ E3.CS.Pfale The adenoviral vector, in crude lysates, resulting from this transfection was plaque purified using methods known to persons skilled in the art. Single plaques were analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cm 2 /flask) . Upon amplification cells were harvested at full CPE and the virus was purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- Ad35 ⁇ E3.CS.Pfale (-28) and Ad35 ⁇ E3.CS.Pfalc (-14) are separately digested by Pad restriction enzyme to release the left-end portion of the Ad genome.
- Plasmid pWE.Ad35.pIX-rITR ⁇ E3 containing the remaining right-end part of the Ad genome is digested with Notl.
- pAdapt535.CS.Pfale (-28) and pAdapt535.CS.Pfale (-14) are separately transfected with Notl digested pWE.Ad35. ⁇ IX-rITR ⁇ E3 into PER-E1B55K producer. Homologous recombination between overlapping sequences leads to generation of recombinant viruses named respectively Ad35 ⁇ E3.CS.Pfale (-28) and Ad35 ⁇ E3. CS.Pfale (-14) .
- Adenoviral vectors in crude lysates resulting from these transfections are plaque purified using methods known to persons skilled in the art.
- Single plaques are analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cm 2 /flask) .
- Cells are harvested at full CPE and the virus is purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- the generation of the recombinant virus named Ad35 ⁇ E3.CS.Pfale (pf-aa-sub) is performed as follows.
- pAdapt535.CS.Pfale (pf-aa-sub) is digested by Pad restriction enzyme to release the left-end portion of the Ad genome.
- Plasmid pWE.Ad35.pIX-rITR ⁇ E3 containing the remaining right-end part of the Ad genome is digested with Notl.
- pAdapt535.CS.Pfale pf-aa-sub
- pf-aa-sub is transfected with Notl digested pWE.Ad35.pIX-rITR ⁇ E3 into PER-E1B55K producer cells using lipofectamine transfection reagent (Invitrogen) using methods known in the art and as described in WO 00/70071 or by electroporation or other transfection methods known to those skilled in the art.
- lipofectamine transfection reagent Invitrogen
- Homologous recombination between overlapping sequences leads to generation of the recombinant virus named Ad35 ⁇ E3.CS .
- Pfale pf-aa-sub
- the adenoviral vector, in crude lysates, resulting from this transfection is plaque purified using methods known to persons skilled in the art.
- Single plagues are analyzed for the presence of the CS transgene and amplified for large-scale production in triple-layer flasks (3x175 cmVflask) .
- Cells are harvested at full CPE and the virus is purified by a two-step CsCl purification procedure as routinely done by those skilled in the art and generally as described in WO 02/40665.
- Example 5 Inducing protection against P.yoelii malaria infection using recombinant adenoviral-based vaccines in vivo.
- Ad5 Adenovirus serotype 5 (Ad5) -based vectors genetically engineered to express the CS antigen of the rodent malaria P.yoelii have been shown capable to induce complete protection against P.yoelii infection (Rodrigues et al. 1997) .
- Ad5 and Ad35 vectors carrying the codon-optimized P.yoelii CS gene was designed to investigate the immune response that is induced, and to investigate their ability in raising protection against P.yoelii parasite infection in mice.
- mice 4A and B The experiments were performed on mice that were sacrificed two weeks after injection with the recombinant adenoviruses . Each of the bars represents the average of 5 mice. If mice were not sacrificed they were used for a challenge with live sporozoites, after which the rate of protection was determined (Fig. 5A and B) . Each of these bars represents the average of 5 mice.
- the experiments on humoral and cellular immune responses are performed with immunological assays well known to persons skilled in the art and as described for instance by Bruna-Romero et al. (2001a) . The immunization, challenge and read out are scheduled in Table II and III.
- Antibodies titers against sporozoites can be determined by an indirect immunofluorescence assay or with an ELISA.
- Figure 4B shows the results as calculated with an ELISA.
- Cellular immune responses were determined by ex-vivo ELISPOT assay measuring the relative number of CS- specific, IFN- ⁇ -secreting, CD8+ and CD4+ T cells.
- the immunization with Ad5- and Ad35-based vectors was performed as follows . Aliquots of recombinant adenoviruses that were stored at -70°C were gradually thawed on ice and diluted to 100 ⁇ l in the desired concentration in PBS with 1% heat-inactivated Normal Mouse Serum. Subsequently the samples were sonicated for 5 sec. Sub-cutaneous administration was performed at both sides of the tail base with a volume of 50 ⁇ l at each side. Intra muscular administration was performed in both thighs with a volume of 50 ⁇ l at each thigh.
- the Indirect Immunofluorescence Assay is performed according to Bru ⁇ a-Romero et al. (2001a) .
- infected mosquitoes are generated by initially having a native mouse infected with an infected mosquito by having the mouse bitten at three different sites. Blood is removed from the mouse after 8 days when parasitemia is 4-8% and diluted to 1%. Then, other na ⁇ ve mice are injected i.p. with the diluted blood sample. After 3 days the blood is taken which serves as a blood meal for starved mosquitoes . These are fed for 2 days .
- P.yoelii sporozoites are isolated from the blood-fed mosquitoes by anaesthetizing infected mosquitoes on ice and subsequently saturating them in 70% ethanol. Then, the mosquitoes are transferred to PBS pH 7.4 and the salivary glands are dissected. These are subsequently grinded on ice and the sporozoites are separated from the debris by centrifugation. Using this method, approximately 35,000 P.yoelii sporozoites can be obtained from 1 mosquito.
- ELISA plates (Immulon II, Dynatech) were coated with 2 ⁇ g/ml antigen in PBS by adding 100 ⁇ l per well of this solution and leaving it overnight at 4°C.
- the antigen that was used is a 3x6 amino acid repeat of the P.yoelii CS protein: QGPGAPQGPGAPQGPGAP (SEQ ID NO: 19).
- the plates were subsequently washed three times with washing buffer (lx PBS, 0.05% Tween), and 200 ⁇ l blocking buffer (10% FCS in washing solution) was added per well. Plates were incubated for 1-2 h at room temperature. Then, plates were washed three times again with washing buffer including 5% FCS.
- Dilutions of the sera were made as follows: 50 ⁇ l washing buffer plus 5% FCS was added to wells 2-12. Then 100 ⁇ l washing buffer plus 5% FCS is added to the first well and 1:2 serial dilutions are made by transferring 50 ⁇ l from well 1 to 2, then from 2 to 3, etc. Plates are incubated for 1 h at room temperature. Then the plates are washed three times with washing buffer and 100 ⁇ l of a 1:2000 diluted peroxidase-labeled Goat anti-Mouse IgG (anti Heavy and Light chain, human absorbed, Kirkegaard & Perry Laboratories, catalogue no. 074-1806) is added per well and incubated.
- Goat anti-Mouse IgG Anti Heavy and Light chain, human absorbed, Kirkegaard & Perry Laboratories, catalogue no. 074-1806
- ABTS substrate solution (ABTS 1- Component, Kirkegaard & Perry Laboratories, catalogue number 50-66-18) is added to each well. The reaction is terminated by the addition of 50 ⁇ l 1% SDS, and plates are read at 405 nm in an ELISA reader.
- the ELISPOT assay to determine the relative number of CS-specific IFN- ⁇ -secreting, CD8+ and CD4+ T cells in the spleen, and the reverse transcriptase PCR and real- time PCR to quantify the amount of parasite specific RNA present in the liver of the challenged mice were all performed as described by Bruf-a-Romero et al. (2001a and 2001b) except for the fact that the number of cycles in the real-time PCR was 45.
- Figure 4A shows that with an administration of 10 9 and 10 10 viral particles per mouse the Ad35-based vector is at least as effective in inducing a cellular immune response as the Ad5-based vector, if not superior. It can be concluded that with this set-up that there is no dramatic difference in cellular response as indicated by the number of IFN- ⁇ -secreting cells after intra muscular and subcutaneous delivery.
- Figure 4B shows the antibody titers in the same experiment and performed on the same sera using the indirect immuno-fluorescence experiment outlined above. If compared to the results shown in Figure 4A it is clear that at a dose of 10 9 viral particles, the Ad35 based vector induces a significant cellular immune response but does not give rise to very high titers of anti-sporozoite antibodies .
- mice that were immunized received s.c. 10 9 or 10 10 viral particles (vp) and were challenged after 14 days with the sporozoites and then sacrificed after 48 h.
- Negative controls were empty vectors without antigen and non-immunized mice.
- a high percentage of inhibition is obtained when using the Ad5-based vector as well as with the Ad35-based vector, applying the two doses, while no protection was found in the negative controls (Fig. 5A) .
- Fig. 5A Importantly, only a low number of parasite-specific 18S ribosomal RNA's could be determined in the liver of the immunized mice, while the mice that received no adenoviral vector or empty vectors contained large numbers of these RNA's (Fig. 5B) .
- Ad35-based vector like the Ad5-based vector can give rise to significant protection against the malaria parasite, even after a single round of immunization.
- Example 6 Inducing immunity against P.falciparum malaria infection using recombinant adenoviral-based vaccines in vivo.
- a side-by-side comparison between Adenovirus serotype 5 (Ad5) and Adenovirus serotype 35 (Ad35) vectors is designed to investigate the ability to induce humoral and cellular immune responses against the CS antigen of the P. falciparum parasite in mice.
- immunogenicities of Adenovirus vectors containing full length and GPI minus CS are compared.
- Immunogenicity of the Ad35-based vectors is expected to be superior or at least comparable to the immunogenicity triggered by Ad5-based vectors.
- Figure 6 shows the results that were obtained by using the Ad5- based vector containing the full length gene encoding the P. falciparum CS protein, the gene encoding the protein with the 14 amino acid deletion and the gene encoding the protein with the 28 amino acid deletion. The results indicate that all three (Ad5-based) vectors are able to induce a cellular immune response as measured by the number of CS-specific IFN- ⁇ -secreting cells in a population of splenocytes, determined by the ex-vivo ELISPOT assay described above, and generally as in Brufla- Romero et al. (2001a) .
- Example 7 Inducing a long-lasting protection against P.yoelii malaria infection by prime-boost regimens with different adenovirus serotype-based vaccines.
- Recombinant Adenovirus serotype 5 expressing a CS antigen of P.yoelii was shown to elicit protection when used in prime-boost regimen in combination with a recombinant vaccinia virus carrying the same antigen (Bruna-Romero et al. 2001a) .
- An experiment to investigate the capability of prime/boost regimens based on adenovirus vectors carrying codon-optimized CS and derived from two different serotypes to induce long- lasting protection against the P. yoelii CS antigen was designed. This study enrolls Balb/C mice distributed in experimental groups of 12 mice each.
- Animals are immunized by intra-muscular injection of an optimal dose of Ad5 ⁇ E3 or Ad35 ⁇ E3 viral vectors carrying either the P.yoelii CS gene (Ad5 ⁇ E3-CS.Pyoel and Ad35 ⁇ E3-CS. Pyoel) or no transgene (Ad5 ⁇ E3-empty and Ad35 ⁇ E3-empty) .
- Ad5 ⁇ E3-CS.Pyoel and Ad35 ⁇ E3-CS. Pyoel no transgene
- mice Another group of mice is primed at week 0 with Ad35 ⁇ E3-CS.Pyoel and boosted at week 8 with Ad5 ⁇ E3-CS.Pyoel.
- Other groups of mice are primed at week 0 with Ad35 ⁇ E3-CS.Pyoel or Ad5 ⁇ E3-CS.Pyoel and boosted at week 8 with the same vector.
- a control group of mice is primed at week 0 with Ad5 ⁇ E3-empty and boosted at week 8 with
- Ad35 ⁇ E3-empty At week 2 post-boost, 6 mice of each group are sacrificed to allow evaluation and characterization of humoral and cellular immune responses with immunological assays well known to persons skilled in the art, the remaining 6 mice from each group are challenged with live sporozoites . The immunization, challenge and read out are scheduled in Table VI. Protection against malaria infection will be monitored and measured using assays well known to people skilled in the art as described above. Vaccine regimens based on Ad35 alone or Ad5/Ad35 combinations are expected to be superior or at least comparable in efficacy as compared to regimens based solely on Ad5.
- Example 8 Inducing a long-lasting immunity against P.falciparum malaria infection by prime-boost regimens with different adenovirus serotype-based vaccines.
- An experiment to investigate the ability of prime/boost regimens based on adenovirus vectors derived from two different serotypes to induce long-lasting immunity against the P. falciparum CS antigen was designed. The study enrolls B10.BR mice distributed in experimental groups of 24 mice each.
- Animals are immunized by intra-muscular injection of an optimal dose of adenoviral vectors carrying either the full length CS gene (Ad5 ⁇ 3-CS.Pfalc and Ad35 ⁇ E3-CS.Pfalc) or the GPI- anchor sequence minus CS gene (Ad5 ⁇ E3-CS.Pfale (-28) / (-14) and Ad35 ⁇ E3-CS.Pfalc (-28) / (-14) ) or no transgene (Ad5 ⁇ E3- empty and Ad35 ⁇ E3-empty) .
- adenoviral vectors carrying either the full length CS gene (Ad5 ⁇ 3-CS.Pfalc and Ad35 ⁇ E3-CS.Pfalc) or the GPI- anchor sequence minus CS gene (Ad5 ⁇ E3-CS.Pfale (-28) / (-14) and Ad35 ⁇ E3-CS.Pfalc (-28) / (-14) ) or no transgene (
- mice One group of animals is primed at week 0 with Ad5 ⁇ E3-CS.Pfale or Ad5 ⁇ E3-CS.Pfale (-28) / (- 14) and boosted at week 8 with Ad35 ⁇ E3-CS.Pfalc or Ad35 ⁇ E3-CS.Pfalc(-28) /(-14) .
- Another group of mice is primed at week 0 with Ad35 ⁇ E3-CS.Pfale or Ad35 ⁇ E3-
- mice are primed at week 0 with Ad35 ⁇ E3-CS.Pfalc or Ad35 ⁇ E3-CS.Pfalc(-28)/(-14) and boosted at week 8 with the same vector.
- a control group of mice is primed at week 0 with Ad5 ⁇ E3-em ⁇ ty and boosted at week 8 with Ad35 ⁇ E3-empty.
- mice are sacrificed at each time point and cellular and humoral responses are monitored with immunological assays well known to persons skilled in the art and as described above. The immunization, challenge and read out are scheduled in Table VII.
- Vaccine regimens based on Ad35 alone or Ad5/Ad35 combinations are expected to be superior or at least comparable in efficacy as compared to regimens based solely on Ad5.
- Example 9 Inducing an immune response against the P. falciparum CS antigen by prime/boost regimens using different Adenovirus serotype-based vaccines in non-human primates .
- Rhesus monkeys are vaccinated with adenoviral vectors carrying either the full-length CS gene (Ad5 ⁇ E3- CS.Pfalc or Ad35 ⁇ E3-CS.Pfale) or the GPI-anchor sequence minus CS gene (Ad5 ⁇ E3-CS.Pfale (pf-aa-sub) or Ad35 ⁇ E3- CS.Pfalc (pf-aa-sub) ) .
- Prime/boost regimens (Ad5 followed by Ad35 or Ad35 followed by Ad5) are compared to generally applied prime/boost regimens (Ad5 followed by Ad5 or Ad35 followed by Ad35) .
- Humoral and cellular immune responses are monitored using immunological assays well known to persons skilled in the art. Serum of immunized monkeys is tested by ELISA assay to determine the nature and magnitude of the antibody response against the repeat region of CS. Cellular immune response is measured by ELISPOT assay to determine the amount of antigen-specific IFN- ⁇ secreting cells.
- Plasmodium falciparum the case for an LSA-1 vaccine. Trends in Parasitology 17:219-223
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CA2507915A1 (en) | 2004-07-01 |
EP1573012A1 (en) | 2005-09-14 |
US20100172928A1 (en) | 2010-07-08 |
US7521229B2 (en) | 2009-04-21 |
US7387894B2 (en) | 2008-06-17 |
US20070071726A1 (en) | 2007-03-29 |
AU2003298361B2 (en) | 2009-05-14 |
US8097453B2 (en) | 2012-01-17 |
NZ539813A (en) | 2008-04-30 |
US7300657B2 (en) | 2007-11-27 |
US20120082694A1 (en) | 2012-04-05 |
US7524947B2 (en) | 2009-04-28 |
SG156535A1 (en) | 2009-11-26 |
US7867764B2 (en) | 2011-01-11 |
US20110150930A1 (en) | 2011-06-23 |
US8361478B2 (en) | 2013-01-29 |
US20070088156A1 (en) | 2007-04-19 |
EP2258850B1 (en) | 2013-07-17 |
CY1112634T1 (en) | 2016-02-10 |
US20050265974A1 (en) | 2005-12-01 |
AU2003298361A1 (en) | 2004-07-09 |
CA2507915C (en) | 2013-07-02 |
EP1573012B1 (en) | 2011-11-30 |
US20080220014A1 (en) | 2008-09-11 |
EP2258850A1 (en) | 2010-12-08 |
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