Patent Grant
8623381
The invention relates to viral strains derived from the first generation vaccinia virus Lister smallpox vaccine and particularly a viral clone of this traditional vaccine called “Lister VACV-107” and to pharmaceutical composition containing thereof.
Smallpox was eradicated through a worldwide effort coordinated by the World Health Organization (WHO) global vaccination campaign in the second half of the last century (Fenner F. et Al., 1988). This disease was “the most dreadful scourge of the human species” (Fenner F., 1984) and claimed hundreds of millions of victims for centuries (Fenner F. et AL., 1988). Variola virus (VARV), its causative agent, spreads easily and exclusively from human to human by the respiratory route. It causes fever, severe rash and in about 30% of cases, death (Fenner F., et Al., 1984).
The fear of the release of VARV through bioterrorism has generated renewed interest in the prevention of smallpox because of the high proportion of unimmunised people in the global population and because vaccination is the only currently effective means to curtail a smallpox epidemic. Live vaccinia virus (VACV) is the active ingredient of the smallpox vaccine administered by scarification. VACV and VARV belong to the Orthopoxvirus genus within the family Poxyiridae and both these viruses display considerable serological cross-reactivity, allowing VACV to provide protection from VARV infection, the accepted basis of its use as a smallpox vaccine. In view of the smallpox threat, a number of countries have maintained stockpiles of the first-generation smallpox vaccine. Inevitably, there will be a need to replace or to increase the stockpiles, but the historical manufacturing process, in the skin of live animals is no longer acceptable. This has stimulated interest in developing second-generation vaccines made of live, replicative, vaccinia virus, but manufactured by virus replication in cell cultures.
Several new second-generation smallpox vaccines have been developed using tissue culture-adapted virus: one such vaccine (ACAM2000™, a live vaccinia virus smallpox vaccine) is derived from a New York City Board of Health (NYCBH) strain first-generation vaccine through cloning and propagation in MRC-5 and Vero cell cultures (Monath T P, et Al., 2004) (Weltzin R, et Al., 2003), and others are derived from a Lister/Elstree first-generation vaccine without cloning.
Second-generation vaccines have the advantage over first-generation vaccines of being produced and controlled according to Good Manufacturing Practices (GMP), thus being more standardised and free of adventitious agents. Nevertheless, these second-generation vaccines are unsatisfactory because they may still induce the same vaccine complications as those induced by the first generation vaccines.
H. Mahnel and colleagues passaged the vaccinia virus Ankara strain (CVA) more than 500 times in chicken embryo cells and isolated a highly attenuated vaccine named MVA (Mahnel, H. and Mayr, A., 1994). During the multiple passages of the CVA virus in tissue culture that ultimately led to the MVA virus, 6 main regions of the viral genome were deleted and numerous point mutations and smaller deletions occurred (Antoine, G., F. et Al., 1998 and Meyer, H., et Al., 1991; Meisinger-Henschel et AL., 2007)
The MVA strain has been extensively characterised and has been found to be efficacious in protecting animals from challenge infections mimicking smallpox and to display a very promising profile as a smallpox vaccine in clinical trials. Nevertheless, potential drawbacks of the MVA smallpox vaccine lie in the fact that it must be employed at very high doses (108 PFU/injection intramuscularly or intradermally, a dose more than 100 fold higher than the dose used with the first generation smallpox vaccine) because it does not replicate in human cells and a booster vaccination is recommended to achieve long-lasting immunity. Furthermore, the MVA vaccine produces no visual take at the site of inoculation as produced by the traditional smallpox vaccine.
Therefore, there is still an important need for new viral strains derived from the vaccinia virus which have better vaccine potency at a lower dose than the MVA strain dose.
A first object of the invention relates to a viral strain derived from the vaccinia virus Lister VACV-107 wherein strain contains in its genomic sequence (SEQ ID N°1) at least one deletion selected from the group consisting of: deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22) and deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23).
Another object of the invention relates to a viral strain according to the invention comprising at least one homologous and/or heterologous nucleic acid sequence.
Another object of the invention relates to a pharmaceutical composition comprising the viral strain according to the invention.
To investigate if a vaccinia virus strain with fewer alterations than those found in the MVA strain may display satisfactory attenuation while maintaining total vaccine efficacy, the inventors have now constructed a series of new strains derived from the Lister VACV 107 strain (a viral clone selected from the vaccinia virus Lister first generation smallpox vaccine) which are multiply deleted in the six major regions deleted in the MVA strain so as to create all of the possible combinations of the six major deletions. This led to the creation of 17 distinct viral mutants which multiplied well in tissue culture cells. The inventors show that deletion of several regions of the Lister genome does not entail a significant loss of vaccine potency when compared to the parental Lister VACV-107 strain. In fact, for most of the deletion mutants, vaccination against a challenge infection that mimics smallpox (intranasal infection of mice with cowpox virus) is as efficient as vaccination with the parental Lister strain. The inventors show that most of the deletion mutants were more attenuated than the parental Lister strain as demonstrated by experimental infection of immunocompromised mice (athymic Nude mice) concomitantly with unaltered vaccine potency. Thus, the inventors have now produced viral strains derived from the vaccinia virus Lister VACV-107 which provide good protection against cowpox virus infection (a model infection for smallpox) and which can be used as efficient vaccines at a low viral dose. Moreover, the usefulness of the new virus strains as new vectors for heterologous vaccination is pointed out.
As used herein the term “Smallpox” denotes an infectious disease unique to humans, caused by either Variola major or Variola minor.
As used herein the term “vaccinia virus” or “VACV” denotes a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome approximately 190 kbp in length, and which encodes approximately 200 proteins. The dimensions of the virion are roughly 360×270×250 nm.
As used herein the terms “vaccinia virus Lister VACV-107” or “Lister VACV-107” denote a strain of vaccinia virus. As used in the invention, this vaccinia virus Lister VACV-107 has been cloned from the original live vaccinia virus Lister strain (production lot X5533 obtained from the Sanofi-Pasteur Company). The GenBank/EMBL/DDBJ accession number for its nucleic acid sequence is DQ121394 (SEQ ID N: °1).
The deletions obtained in the genomic sequence ID NO°1 are described in the table A below.
So a first object of the invention relates to a viral strain derived from the vaccinia virus Lister VACV-107 wherein strain contains in its genomic sequence (SEQ ID N°1) at least one deletion selected from the group consisting of: deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), deletion of the nucleotides acid 6118 to 9677 in the sequence ID NO°1 (Δ22), deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23).
The inventors have demonstrated that particular viral strains of this kind:
In a preferred embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) at least two deletions selected from the group defined above.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18) and the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22). According to the invention, said viral strain is named: VACV-107Δ18/22 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18) and the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20). According to the invention said viral strain is named: VACV-107Δ18/20 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20) and the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22). According to the invention said viral strain is named VACV-107Δ20/22 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ21/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20) and the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22). According to the invention said viral strain is named: VACV-107Δ18/20/22 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ18/21/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ21/22/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ20/21/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ18/21/22/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ18/20//21/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ20/21/22/23 deletion mutant.
In another embodiment, the viral strain according to the invention contains in its genomic sequence (SEQ ID N°1) the deletion of the nucleotides 19758 to 28309 in the sequence ID NO°1 (Δ18), the deletion of the nucleotides 161293 to 164811 in the sequence ID NO°1 (Δ20), the deletion of the nucleotides 181231 to 183304 in the sequence ID NO°1 (Δ21), the deletion of the nucleotides 6118 to 9677 in the sequence ID NO°1 (Δ22) and the deletion of the nucleotides 1833 to 3574 and 185848 to 187589 in the sequence ID NO°1 (Δ23). According to the invention said viral strain is named: VACV-107Δ18/20/21/22/23 deletion mutant.
In another embodiment, the invention relates to the genomic sequence SEQ ID NO°1 with at least one deletion as defined above.
In a further embodiment of the invention, the viral strain according to the invention may comprise at least one heterologous nucleic acid sequence.
As used herein, the term “heterologous” denotes any combination of nucleic acid sequences that is not normally found intimately associated with the viral strain in nature, such viral strain is also called “recombinant viral strain”. Furthermore, the genomic sequence of the virus with the heterologous nucleic acid sequence is called “recombinant genomic sequence”.
Accordingly, recombinant viral strains according to the invention may be used as vectors for introducing a homologous and/or heterologous nucleic acid sequence into a host cell.
The term “vector” means the vehicle by which a nucleic acid sequence can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
For example, the introduction of such a heterologous nucleic acid sequence into a target cell may be used to produce in vitro heterologous peptides or polypeptides and/or complete viruses encoded by said sequence. This method comprises the infection of a host cell with the recombinant viral strain, cultivation of the infected host cell under suitable conditions, and isolation and/or enrichment of the peptide, protein and/or virus produced by said host cell.
So the invention relates to a host cell infected with the viral strains according to the present invention.
According to another embodiment, the recombinant viral strain can be used in gene therapy. Indeed, the viral strain may be used to insert in cells or animal (including humans) any nucleic acid sequence of therapeutic interest.
According to a further embodiment, the heterologous nucleic acid sequences may encode for antigenic epitopes so that the recombinant strains may be used for vaccine purpose against a microorganism (bacteria, virus . . . ).
For example, said antigenic epitope can be selected from another poxviral or a vaccinia source.
Alternatively, the heterologous nucleic acid sequences may encode for an antigenic epitope, which may be selected from any non-vaccinia source. For example, said recombinant viral strain may express one or more antigenic epitopes from Plasmodium falciparum, Mycobacteria, Influenza virus, or from viruses selected from the family of Flaviviruses, Paramyxoviruses, Hepatitis viruses, Human immunodeficiency viruses or from viruses causing hemorrhagic fever such as Hantaviruses or Filoviruses, i.e., Ebola or Marburg virus.
According to a further preferred embodiment of the invention, the expression of heterologous nucleic acid sequence is preferably, but not exclusively, under the transcriptional control of a poxvirus promoter, more preferably of a vaccinia virus promoter.
According to still a further embodiment the insertion of heterologous nucleic acid sequence is preferably into a non-essential region of the virus genome.
Viral strains according to the invention may be obtained by methods well known to the person skilled in the art. Such a method is described in Example here below.
Viral strains according to the invention (recombinant or not) may be used for the preparation of a pharmaceutical composition.
Hence, the present invention also provides a pharmaceutical composition comprising a viral strain according to the invention (recombinant or not). The pharmaceutical composition may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. This pharmaceutical composition can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration.
As used herein, the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
Since the viral strains according to the invention are highly attenuated as demonstrated by experimental infection of immunocompromised animals, they are ideal to immunize or treat a wide range of mammals including humans.
Hence, in a particular embodiment, pharmaceutical compositions according to the invention can be used as vaccine.
For example, the pharmaceutical composition according to the invention can be used as vaccine against smallpox.
Indeed, the pharmaceutical composition according to the invention may induce vaccinia virus neutralizing antibodies or vaccinia specific T cell responses of a similar magnitude to those induced by the standard smallpox vaccine.
In still a particular embodiment, the pharmaceutical composition according to the invention can be used as a vaccine against other infectious diseases (the infectious diseases can be but are not limited to malaria, tuberculosis, hepatitis), cancer or other non-infectious diseases.
Alternatively, pharmaceutical compositions according to the invention may be used for gene therapy.
For vaccination or therapy, pharmaceutical compositions according to the invention may be administered either systemically or locally, i.e. by parenterally, intramuscularly, subcutaneous or any other path of administration know to the skilled practitioner. The mode of administration, the dose and the number of administrations can be optimized by those skilled in the art in a known manner.
The invention will be further illustrated by the following figures and examples.
a and 1b: Multiplication of Selected Deletion Mutants in Human HeLa Cells and Human MRC5 Fibroblasts
HeLa cells grown in monolayers were infected with approximately 0.01 PFU/cell for one hour. Unadsobed virus was then removed and fresh medium was added. Samples of infected cells were harvested 3 hours or 48 hours post-infection and the virus titers in the samples were determined by titration on BHK21 cells. The X axis indicates the viruses examined and the Y axis the titer of each sample. MRC5 cells grown in monolayers were infected with approximately 0.01 PFU per cell for one hour. Unadsorbed virus was removed and fresh medium was added. Samples of infected cells were harvested 1 hour or 31 hours post-infection and the virus titers in the samples were determined by titration on Vero cells. The X axis indicates the viruses examined and the Y axis the log of the ratio of the titer of the sample taken at 31 hours over that taken at 1 hour.
Groups of six Nude mice (athymic immunocompromized) were infected by tail scarification with 105 PFU of the VACV deletion mutants in two separate experiments and the percentage in weight change was followed over the course of time.
Groups of six Nude mice were infected by tail scarification with 105 PFU of the VACV deletion mutants and the percentage in weight change was followed over the course of time.
a: Induction of Neutralizing Antibodies in Mice Vaccinated with Lister VACV-107 Deletion Mutants
BALB/C mice were vaccinated by scarification at the base of the tail with 105 PFU (six animals per group). Four weeks later the mice were bled and vaccinia virus neutralizing antibodies in the serum of each animal were titrated. The graph depicts the reciprocal of the serum dilution which led to a 50% reduction in the VACV plaque count compared with a negative control and the mean of this dilution for each deletion mutant is indicated at the top of each series. Neutralization titers in serum samples from uninfected animals were less than 10, the threshold of neutralization (a 1/10 dilution of the serum resulted in no neutralization).
b: Induction of VACV-specific CD4+ and CD8+ Lymphocytes in Mice Vaccinated with VACV Lister Deletion Mutants
BALB/C mice were vaccinated by scarification at the base of the tail with 105 PFU (six animals per group). Four weeks later the mice were bled, spleens recovered and the percentage of CD4 (top panel) or CD8 T lymphocytes (bottom panel) able to secrete interferon γ in the presence of dendritic cells presenting VACV antigens was determined. The mean values for 6 animals in each group and the corresponding standard deviations are plotted. Asterisks are positioned above values that were significantly different from those obtained after infection with the VACV-107 virus (students T test p≦05).
Mice were vaccinated with either 104, 103 or 102 PFU by tail scarification and challenged 28 days later with cowpox virus. The results are displayed as the number of surviving mice (Y axis) out of a total of 6 mice per group for each vaccine dose employed (X axis). The protective dose, theoretical dose able to protect 50% of the animals (PD50) was calculated and is shown for each virus at the right of the graphs. Note that all unvaccinated mice succumbed to the challenge infection (not shown).
Mice were vaccinated with either 104, 103 or 102 PFU by tail scarification and challenged 28 days later with cowpox virus. The results are displayed as the number of surviving mice (Y axis) out of a total of 6 mice per group for each vaccine dose employed (X axis). The protective dose, theoretical dose able to protect 50% of the animals (PD50) was calculated and is shown for each virus at the right of the graphs. Note that all unvaccinated mice succumbed to the challenge infection (not shown).
The weight loss for mice vaccinated with 104 PFU by tail scarification and challenged 28 days later with cowpox virus, as reported in
Plasmids used to create deletions are listed in the left column. The size of the corresponding deletions in the MVA strain relative to the VACV Copenhagen strain (Antoine et AL. 1998) is listed in the second column. The oligonucleotide primers used to amplify the fragments by PCR which flank the targeted regions are listed in the third and fourth columns.
The plasmid used to create deletion Δ18 is listed in the left column and the corresponding deletion in the MVA strain as defined by Antoine et AL. 1998 is listed in the second column. The oligonucleotide primers used to amplify PCR fragments at the left end or right end of the targeted deletions are listed in the third and fourth columns.
Deletions were checked by PCR analysis of total DNA extracted from infected cells using the 3 oligonucleotide primers A, A′ and B as listed in table 2. Primers A and B together were designed to flank the areas deleted and generate fragments of approximately 500 bp. Primer A′ was chosen so that it lies within the sequence expected to be deleted so that together with B it enables amplification of a fragment in the parental virus. Virus stocks containing mixtures of the two types of viruses generated the two PCR products listed in the fourth column whereas virus stocks containing only virus deleted in the targeted region generated only an approximately 500 bp fragment as listed in the fourth column except in the case of Δ23 which generated a 1000 bp fragment.
General Outline of the Method Used to Construct New Viral Strains by Deletion of Selected Regions of a Clonal Isolate of the Vaccinia Virus (VACV) Lister-107 Strain:
Prior to this invention, biological clones of the virus contained within the smallpox Lister vaccine (production lot X5533 obtained from the Sanofi-Pasteur Company) were produced by standard virus cloning procedures on the human fibroblast cell line MRC-5. One clone, designated VACV-107, was selected for further study because it displayed similar properties to the parental virus population in that it protected mice from a lethal challenge infection with cowpox virus (an animal model of smallpox in man) as efficiently as the parental Lister strain and it displayed a similar level of pathogenicity upon intracerebral injection into newborn mice (Garcel et AL. 2009). Analysis of the genomic sequence of VACV-107 (≈190 Kbp) demonstrated that its genotype is closely related to other isolates of VACV albeit with several distinctive features. For instance VACV-107 has a series of unique open reading frames from ORF 194 to ORF 196 as compared to a number of other VACV strains (Garcel et AL. 2007).
We then employed a strategy that was previously developed to create deletions in the VACV genome (Falkner et AL., 1990) and which operates in a similar manner to the strategy we had previously employed to delete unwanted selection markers from the fowlpox virus genome (Spehner et al., 1990). In a first step, we amplified by PCR of the VACV-107 genome, two approximately 500 bp fragments on each side of the regions to be deleted. The two amplified fragments surrounding each region to be deleted were then assembled together on one bacterial plasmid so as to replicate the desired deletions on the plasmids. We then added adjacent to the rightmost fragment on each plasmid both a gene encoding the green fluorescent protein (GFP) and a gene encoding the enzyme guanine phosphoribosyl-transferase (GPT) which confers resistance to mycophenolic acid (MPA). Both the GFP and GPT genes were positioned behind specific VACV promoters so as to ensure their expression.
The plasmids thus constructed for each region to be deleted were then transfected into cells that had been infected with VACV-107. Two days later, virus derived from this initial infection/transfection was used to infect fresh cells in the presence of MPA so as to select for virus clones that had integrated the entire plasmid. Observation of the fluorescence of plaque isolates at this stage confirmed that the entire plasmid was integrated into the viral genome. The strategy used implies an initial recombination event between virus DNA and plasmid DNA that generates virus recombinants that bear the region to be deleted as well as the entire plasmid DNA. After the initial isolation of an MPA resistant virus, further cloning steps are carried out in the presence of MPA to ensure that the virus population contains mostly virus harbouring the entire plasmid. The virus enriched in this manner is then plated on cells in the absence of MPA so as to allow detection of a second recombination event which can generate either virus deleted of the region targeted or parental virus. Both parental viruses and viruses that have deleted the GPT gene can be recognized because of their lack of GFP fluorescence and such viruses are picked and amplified on fresh cell monolayers. To distinguish viruses deleted in the regions targeted from parental virus, DNA extracted from cells infected with each virus isolate is analysed by PCR using specific oligonucleotides. Once a deleted virus is recognized it is cloned several times again to ensure its purity and the deletion is confirmed by PCR with selected oligonucleotides. In this way we isolated viruses containing deletions in 5 distinct regions of VACV-107 which correspond to 6 major regions deleted in the MVA strain. Two regions deleted in the MVA strain that are very close together (D4.8 and D2.8) were combined in one deletion in our study. Furthermore, we created viruses harboring combined deletions of each of the 5 deletions initially created by repeating the operations outlined above on previously deleted viruses. In addition, because it has previously been shown that inactivation of the VACV thymidine kinase gene (TK) leads to virus attenuation without loss of the vaccine potency (Lee et AL. 1992) we also deleted a portion of the TK gene and subsequently used this virus as a comparator in the evaluation of newly constructed deletion mutants. The pEM25 plasmid contains the deletion in the TK gene used to construct VACV-107Δ25.
Methods Used for PCR and Molecular Cloning:
PCR amplification of viral DNA and molecular cloning in plasmids was carried out according to the methods previously described (Sambrook et Russell, 2001). Approximately 500 bp DNA fragments of VACV-107 were amplified on both sides of the regions to be deleted using the oligonucleotides listed in table 1a and 1b. This table provides the name of the plasmid used to create the deletions in VACV-107, the designation of the corresponding deletion in the MVA virus (according to Antoine et AL., 1998) and the sequence of the oligonucleotides used to amplify the leftmost and rightmost regions surrounding each region targeted. In the case of plasmids pEM 23, pEM22, pEM20 and pEM21, DNA fragments were amplified using the indicated oligonucleotides, cut with the restriction enzymes positioned at each end and inserted into the appropriate plasmid as described in more detail in the results section for one plasmid. In the case of pEM18, each region was amplified by PCR with two different sets of oligonucleotide primers chosen so as to be able to generate the desired restriction sites at their ends upon denaturation of the fragments and reannealing of the two fragments together. The fragments surrounding each targeted deletion were then joined together two by two in a single bacterial plasmid so as to reproduce the deletions on the plasmid.
Virus Propagation and Cell Culture Methods:
The VACV Lister strain derived from the first generation smallpox vaccine was initially amplified and cloned in the human diploid fibroblast cell line MRC5 (obtained from bioMérieux) cultivated in RPMI 1640 Glutamax medium supplemented with 10% fetal calf serum (obtained from South America) and 40 μg/ml gentamycine. Lister clone VACV-107 and deletion mutants derived from it were amplified and cloned in the chicken embryonic stem cell line EbxR (obtained from Vivalis) cultivated in DMEM/F12 medium supplemented with 10% fetal calf serum, non essential amino acids, 1 mM sodium pyruvate and 40 μg/ml gentamycine. Baby hamster kidney cells (BHK21) were cultivated in BHK21 medium supplemented with 1.5 g/ml bacto-tryptose phosphate, gentamycine and 10% fetal calf serum. The BHK21 cell line was used to determine virus titers after infection of the majority of the cell lines examined but was never used to passage virus or produce virus stocks. Vero cells were cultivated in M199 medium supplemented with 5% fetal calf serum, 100 international units (IU) penicillin and 100 IU streptomycin. Human HeLa cells were cultivated in DMEM with 10% fetal calf serum and 40 μg/ml gentamycine. The titers obtained after infection of MRC5 cells were determined on Vero cells.
Assay for the Determination of Vaccinia Virus Neutralizing Antibodies and Vaccinia Virus T Lymphocyte Responses
Mice were anesthetized with pentobarbital and bled by intracardiac puncture. Vaccinia virus neutralizing antibodies were then titrated according to the method described by Leparc-Goffart et AL. (2005). Serum samples were first incubated at 56° C. for 30 minutes then submitted to serial two-fold dilutions in PBS. The samples were then mixed with an equal volume of vaccinia virus containing 15-55 PFU/0.1 ml for one hour at 37° C. and added to Vero cell monolayers. Two days later virus plaques were counted. The neutralization titer was expressed as the reciprocal serum dilution that led to a 50% reduction in the number of plaques as compared to the control. The threshold of the titration was 10.
To measure T lymphocyte responses the percentage of CD4+ and CD8+ lymphocytes expressing intracellular IFN-γ was measured by flow cytometry as previously described by Ferrier-Rembert et al, (2007). Briefly, mature bone marrow dendritic cells from uninfected Balb/c mice were infected with VACV then incubated with spleen cell suspensions from infected animals for six hours at a ratio of one mature dendritic cell for two splenocytes. Brefeldin A (5 μg/ml) was added for the last four hours to block cytokine secretion. Cells were stained with FITC-coupled anti-CD8b2 and APC-couple anti-CD4 mAbs, fixed and permeabilised. Intracellular IFN-γ was stained with a PE-coupled anti-IFN-γ mAb and cells were fixed in 2% formaldehyde diluted in PBS. Flow cytometry was performed on a Beckman Coulter FC500 and the data analyzed with the CYTOMICS RXP® data analysis software (Beckman Coulter).
Methods Used to Obtain Virus Deletions: Transfection, Selection and Screening:
In order to isolate deletion mutants, EBxR cells were plated at approximately 300 000 cells per 35 mm2 Petri dish and incubated overnight at 37° C. in a cell culture incubator containing 5% CO2. The cells were then infected with VACV-107 or one of its derivatives in which a deletion was to be introduced with approximately 0.1 PFU/cell for one hour. After this period of time the suspension containing unadsorbed virus was removed, the cells were washed twice with serum free medium and the appropriate plasmid was transfected into the cells using Lipofectamine (Invitrogen). About 1 μg of purified plasmid DNA in 10 μl DNA buffer was mixed with 100 μl EBxR culture medium without serum and 5 μl Lipofectamine were mixed with 100 μl of the EBxR culture medium without serum. The DNA mixture was then added to the Lipofectamine mixture and left to stand at room temperature for 15 minutes. Culture medium without serum (0.8 ml) and the DNA/Lipofectamine mix (0.2 ml) were then added to the previously infected cells. The cells were put into a 37° C. incubator under 5% CO2. One day later cells were observed under the microscope by UV illumination to visualize expression of GFP, a marker of successful transfection and the cells were frozen down at −20° C. On the same day or several days later, the infected cells were thawed and then submitted to several additional rounds of freezing thawing to lyse all cells and the virus in the cell lysates was used to infect fresh EBxR cells in the presence of a selective pressure for the expression of the GPT gene. Selection was carried out under cell culture medium containing 1.2% low melting temperature agarose, 0.025 μg/ml mycophenolic acid, 0.25 μg/ml xanthine and 15 μg/ml hypoxanthine. One day later viral plaques formed in the presence of the selective medium were visualized by microscopy under UV light and fluorescent plaques were picked with a pipette and placed in 0.5 ml EBxR culture medium. The virus from each plaque was amplified by infection of EBxR cells in the presence of selective pressure but without agarose. This procedure of cloning the virus under selective pressure and amplification was repeated several times. Two to three independent virus clones able to form fluorescent plaques were then used to infect fresh EBxR cells after limiting dilution and in the absence of selective pressure but under an agarose overlay. This time only non fluorescent plaques were picked after two days infection and the virus was amplified on EBxR cells in the absence of selective pressure. A sample of the amplified virus stock was used to extract total DNA and the DNA was submitted to PCR using one of the appropriate pairs of primers indicated in table 2. The primers were designed so as to be able to detect the presence of the deleted viral DNA as well as undeleted viral DNA or a mixture of the two. The clones which contained only deleted viral DNA were submitted to two additional cloning steps in the absence of selective pressure, picking and amplification on EBxR cells. The viral deletions were again confirmed in another round of PCR analysis of DNA from the more thoroughly cloned virus mutants. Finally virus stocks were made by amplification on EBxR cells.
Animal Experiments:
Animal experiments were carried out using mice in accordance with French regulations on laboratory animals and received permission from the ethics committee where they were performed (CRSSA).
Protection of Mice Against a Lethal Intranasal Cowpox Virus Infection after Vaccination:
The ability of the deleted viruses to confer protective immunity after vaccination was assayed in 4 week old female BALB/c mice. Groups of 6 animals were tested and treated in the same manner for each condition studied. Before vaccination, mice were anesthetised by intraperitoneal injection of ketamine (75 mg/kg) and atropine (1.5 mg/kg). The mice were then scarified at the base of the tail with the tip of a 23 gauge needle. Three μl of virus at various concentrations was deposited at the scarification site. Twenty eight days after vaccination, the mice were anesthetised as described above and challenged by intranasal instillation with 2×106 50% tissue culture infectious doses 50 (TCID50) of cowpox virus (Brighton strain VR302 obtained from the ATCC and amplified in Vero cells) which corresponds to a dose of virus 30 fold higher than the dose that kills 50% of mice (Ferrier-Rembert et AL., 2007b). For the challenge, 50 μl of virus or 50 μl of NaCl 9°/°° were deposited in both nostrils using a micropipette with a sterile tip. Clinical signs of disease (weight loss, ruffling of the fur, and reduced mobility) and mortality were noted for the following three weeks. The animals were euthanized at the end of the experiment by cervical dislocation.
Assessment of Attenuation in Nude Mice Deficient in T Lymphocytes
Five week old Swiss Nude mice (Charles Rivers Laboratories) were anesthetised (100 mg/kg ketamine et 1.5 mg/kg atropine) and challenged with Lister VACV-107 or deletion mutants derived from it by tail scarification as described above using 5.0 log PFU of each virus. Clinical signs of disease and mortality were followed over an 8 week period.
Results
Construction of Plasmids Used to Isolate Deletion Mutants of VACV-107:
The construction of the plasmids used to delete targeted regions of the VACV-107 genome was carried out in the same manner for each of the deletions. First, the leftmost fragment of the targeted region in VACV-107, corresponding to the 3.5 Kb deletion in MVA, was first amplified by PCR using primers 1 and 2 designed for this deletion and indicated in table 1a. The DNA fragment obtained by PCR and separated by electrophoresis on an agarose gel was purified then cut with Xho I and Bam HI. The fragment was then inserted by ligation into a small bacterial plasmid, pEM8, to generate pEM9. The rightmost fragment of the targeted region (deletion 3.5 Kb in MVA) was then amplified by PCR using primers 3 and 4 indicated in table 1a and in the same manner as for the leftmost fragment was cut with Bam HI and Sac I then inserted by ligation into pEM9 to generate pEM10. We also constructed the pEM7 plasmid so as to contain side by side the GFP gene (gene encoding enhanced green fluorescent protein from BD Biosciences Clontech) under the control of a synthetic early promoter (Davison, A. J. and Moss, B., 1990; Howley et AL., 1996) and the GPT gene under the control of the p7.5 kd VACV promoter by modification of the pG08 plasmid (Sutter et AL., 1994). The juxtaposed GFP and GPT genes were flanked by an Eco RI site at the left end and a Pst I site at the right end. Thus a DNA fragment containing the GFP and GPT genes was cut out from pEM7 using Eco RI and Pst I and inserted by ligation into pEM10 previously cut with Sac I and Pst I. A single stranded oligonucleotide linker (5′ gagctaattc3′) was added to the ligation reaction to enable ligation of the Eco RI end of the fragment to the Sac I end of the pEM10 plasmid. This procedure generated plasmid pEM20 which was used in the transfection experiments described below. All of the other plasmids were constructed in the same manner except pEM18 in which case the oligonucleotides used for PCR amplification were designed to create the Xho I, Bam HI and Sac I sites after annealing together two PCR fragments for both the leftmost and rightmost regions targeted.
Isolation of Virus Deletion Mutants:
In a first step, mutants deleted in a single region were isolated after transfection of one of the plasmids into chicken EBxR cells infected with VACV-107 as described in detail in the methods section. Deletion mutants were designated according to the name of the plasmid from which they were isolated. For instance the use of pEM20 generated a virus named VACV-107Δ20 and for convenience virus names are sometimes shortened to the include only the deletion considered (for example VACV-107Δ20 may be shortened to Δ20). To isolate viruses deleted in two regions simultaneously, EBxR cells were infected with a virus deleted in one region and transfected with another plasmid required to delete another region. For instance virus Δ18/22 was isolated from cells infected with virus Δ18 and transfected with plasmid pEM22. The deletions were all confirmed by PCR analysis of DNA extracted from infected cells. Deletion Δ23 which is in an area of the genome that is repeated at both ends was initially isolated as a virus having only one end deleted. After cloning this virus on EBxR cells and analysis of several more virus clones we readily isolated a Δ23 virus with both ends deleted.
Multiplication of Deletion Mutants in Selected Cell Lines.
The deletion mutants were routinely propagated on the chicken embryonic stem cell line EbxR and all of them were found to multiply to high levels. Titration of virus was performed on hamster BHK21 cells and all of the deletion mutants produced similar and clearly visible plaques after a two day incubation period.
HeLa cells were also infected to investigate whether these human cells were permissive for multiplication of the mutants. Initial experiments showed that all of the mutants with single deletions multiplied well in HeLa cells except mutant Δ22 which induced virus yields about 5-10 fold lower than the parental VACV 107 strain. The influence of the Δ22 deletion within the background of other deletions was then investigated. Virus titers in the infected cell cultures were assayed on BHK21 cells after several cycles of freezing and thawing samples either 3 hours after infection (to determine the amount of input virus) or 48 hours after infection (to determine the yields). The input 3 hours post infection titers varied up to two fold. Forty eight hours after infection all deletion mutants containing the Δ22 deletion multiplied to levels 4 to 5 fold lower than the VACV 107 strain (
Assessment of Attenuation of the Deletion Mutants in Nude Mice:
Traditional smallpox vaccination can not be performed in immunocompromised people suffering from active HIV infection, drug induced or innate immunodepression. It should generally be recommended that these people and even their families are excluded from vaccination campaigns even though some may escape notice. In case of extensive exclusion from vaccination, too many people could be unvaccinated. Thus, it appears essential to have a vaccine available that is without any danger in immunocompromised individuals. In order to assess the potential danger associated with the use of the deletion mutants described above their pathogenicity upon vaccination of Nude mice lacking a thymus was studied. Nude mice are deficient in the production of antigen specific lymphocytes and T cell dependent antibody responses and therefore highly susceptible to vaccinia virus infection unlike immunocompetent mice (Ramshaw et AL. 1987). The experiments in
Another experiment was performed to assess attenuation of one deletion mutant that had not been tested in the previous experiments (VACV-107Δ18/20) and others which appeared to be the most highly attenuated according to the experiments presented in
Assessment of Immunogenicity
The ability of the some of the most severely attenuated deletion mutants to induce vaccinia virus neutralizing antibodies in mice was assessed using a standardized assay. (
The cell mediated immune responses were also assayed after tail scarification of groups of 6 Balb/C mice with 105 PFU/animal (
Vaccination of Mice by Deletion Mutants and Challenge with Cowpox Virus:
In order to quantitatively assess the ability of the most highly attenuated VACV mutants harboring specific deletions, BALB/c mice were vaccinated by scarification at the base of the tail with a total of 104, 103 or 102 PFU per mouse. Each dose was administered to 6 mice then one month after vaccination the mice were challenged with cowpox virus and clinical signs of disease and mortality were noted. The survival of the animals after the challenge infection, performed in two independent experiments, is presented in
Another experiment was performed to assess vaccine efficacy of the most highly attenuated deletion mutants which were initially found to be as effective in vaccination as the parental VACV-107 strain. As previously, mice were vaccinated with either 104, 103 or 102 PFU per animal by tail scarification. They were then challenged one month later with cowpox virus and animal deaths were recorded. The survival data are presented in
In order to study the protection against disease provided by vaccination, animal weight loss observed after the challenge infection reported above was recorded. Data for the 104 vaccine dose are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 08305570 | Sep 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/062114 | 9/18/2009 | WO | 00 | 3/18/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/031837 | 3/25/2010 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050031643 | Szalay et al. | Feb 2005 | A1 |
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| Kidokoro et al.; “Geneticlally Stable and Fully Effective Smallpox Vaccine Strain Constructed from Highly Attenuated Vaccinia LC16m8”; Proceedings of the National Academy of Sciences of the United States of America, vol. 102, No. 11, Mar. 2005, pp. 4152-4157. |
| Garcel et al.; “Genomic Sequence of a Clonal Isolate of the Vaccinia Virus Lister Strain Employed for Smallpox Vaccination in France and it's Comparison to Other Orthopoxviruses”; Journal of General Virology, vol. 88, No. Part 7, Jul. 2007, pp. 1906-1916. |
| Meyer et al.; “Mapping of Deletions in the Genome of the Highly Attenuated Vaccinia Virus MVA and Their Influence on Virulence”; Journal of General Virology, vol. 72, Jan. 1991, pp. 1031-1038. |
| Number | Date | Country | |
|---|---|---|---|
| 20110171256 A1 | Jul 2011 | US |
