Note: Descriptions are shown in the official language in which they were submitted.
CA 02584894 2007-04-13
DEMANDES OU BREVETS VOLUMINEUX
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COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE _2
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Brevets.
JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02584894 2007-04-13
Using a Reverse Genetic Engineering Platform to Produce Protein
Vaccines and Protein Vaccine of Avian Influenza Virus
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a method for preparing a functional
protein or vaccine, particularly a method for preparing a protein subunit
vaccine or a vaccinal virus strain capable of preventing or inhibiting
epidemic disease.
2. Description of Related Art
The recent breakthroughs in the research of molecular biology, the
development of various high-throughput instruments, and advanced
approaches enable researchers to obtain gene sequences of various species
readily. Through decoding these gene sequences which code life, scientists
are able to perform research that may improve human health. Among them,
one of the most important aspects of research is the R&D of functional
protein and vaccines.
The conventional developing procedures of a vaccine against epidemic
diseases are complicated. Moreover, these procedures are also inefficient
for developing vaccines against various novel epidemic diseases. Generally
speaking, the R&D work for developing a new vaccine must be conducted
under a series of protection measures and precautions. The subsequent
identification, classification and drug-resistance analysis must be
performed after sampling and culturing of wild type virus strain from the
affected subject is achieved. However, if the highly contagious strains are
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wrongly manipulated, they will be spread out from laboratories very easily.
The possible spreading actually poses great threats not only to the
researchers but also to the society as a whole.
To prevent the epidemic disease from further spreading, developing
vaccines is the one of the most appropriate directions in which to proceed.
However, it still requires a long period of time to tame and attenuate the
virus into a vaccinal strain in time for research. For a disease that has the
potential to spread so rapidly to cause a global pandemic, time is a great
challenge to human beings. Likewise, due to various limitations from
highly hazardous novel pathogens, the aggressive attempts to fight against
these diseases are somewhat thwarted. During an outbreak, medical
personnel in the affected areas are flooded by the problems of the patients,
and desperately await vaccines and drugs. Hence, only limited time is left, if
any, for developing vaccines and drugs. Furthermore, the people in the
non-affected areas, who are afraid to contact the pathogens, can only offer
and afford limited aid.
To prevent the crisis of a global outbreak of a new type Flu, developing
a safe and effective vaccine is required. For safety consideration of
mass-production, the newly developed reverse genetics of Flu virus strain is
promising. Reverse genetics Flu virus strains are based on conventional
viral strains and clone all genes into 8 vectors containing HINI PA, PB1,
PB2, NP, M, NS2, HA, and NA, respectively. If they are co-transformed
into a host cell, it is able to produce H1N1 virus bodies to enable massive
production of HIN1 vaccine under appropriate culturing conditions. In
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2002, Hoffmann E. used recombination approaches to recombine vectors
containing genes encoding structural proteins HA and NA of H3N2, H6N 1,
and H9N2, and vectors containing genes encoding PA, PB1, PB2, NP, and
M to generate a novel vaccinal strain. Though the method seems to be
practical, the recombination of the whole genome is potentially dangerous
since it could artificially induce harmful viral strains.
According to the past studies in immunology, antigens are able to
stimulate the lymphocytes carrying specific antigen receptors, induce
amplification and immunity, and subsequently eliminate these antigens
themselves. This is attributable to the antigen specificity of the immune
system. The sites on the antigens for recognition and binding on antibodies
are called "antigen determinant", or "epitope". The epitopes on an antigen
usually comprise 6-8 amino acids, which can be a structure with a
three-dimensional conformation.
The epitopes recognized by a T cell are epitope peptides consisting of a
series of amino acids as mentioned above. They are in conjunction with
MHC (major histocompatibility complex) class I/II and bind TCR (T T-cell
receptors) on the cell surface of T cells when they are functioning. Each
antigen typically has several epitopes, and the number of epitopes increases
with the complexity of the structure, and the molecular weight of the
antigens. Thus, whether the amino acid sequences of epitope peptides can
be obtained from the publication, and proceeded with in vitro transcription
to epitope peptides is critical to the research.
Meanwhile, to rigorously express the epitope peptides in the
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amplification system of the host cells, whether the host cells are able to
recognize the codons encoding the recognized epitope peptides is also
crucial. In addition to prior treatment, subsequent steps are necessary to
proceed with mass production, purification, and isolation.
Besides epitope peptides, antigenic neutralizing zones are also the key
to successful development of a vaccine. A vaccine able to induce a high
neutralizing titer can effectively inhibit infection and proliferation of the
virus. In addition to the above considerations, it is also important for the
vaccinal compositions to be safe and innocuous, and to cause no immune
disorders, immunotoxicity or allergy.
SUMMARY OF THE INVENTION
In view of the aforementioned problems, the present invention
provides a technological platform. The platform of the present invention
includes steps of the most recent generation of genetic engineering
technology, nucleic acid synthesis techniques, protein engineering and
reverse genetics, and results in a so-called "reverse genetic engineering
platform".
Using this platform, one can design and accomplish a vaccinal antigen
or a modified DNA sequence of any gene encoding a target protein through
the published data on the Internet, obviating the needs for contagious and
hazardous biological materials. The sequence can be ligated to a DNA
plasmid comprising a translocation system (for example, a pseudomonas
exotoxin) or a highly antigenic sequence (e.g. the KDEL family) so as to
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form a fusion gene.
The fusion gene on the plasmid can produce functional proteins or a
subunit vaccine in a host cell; in addition, "reverse genetics preparing novel
novel vaccine RNA virus body" techniques can also be derived, leading to a
safe, immuno-effective and novel Flu vaccinal virus strain. The strain is
generated by inserting the determinants having antibody neutralization
titration epitope in the genes of a highly contagious and hazardous virus
into the corresponding loci of the original Flu H1N1 vaccine strain, which
are subsequently cloned into the plasmids having similar genetic
characteristics in the eight-plasmid-system used for producing vaccines,
then are co-transformed with seven other plasmids containing vaccinal
genes into host cells or embryos, so that these composite plasmids can
synthesize novel influenza vaccine strain in host cells.
As to pathogenic and harmful diseases, a vaccine that is effective in
inducing antibodies and is also safe can be rapidly developed by the
above-mentioned "reverse genetic engineering platform", allowing more
researchers to devote themselves to the R&D of various novel vaccines
against infectious diseases so as to prevent the diseases from spreading and
provide efficient and workable vaccine development techniques.
The key points of the present invention are as follow:
a. First, converting the target amino acid sequence to the corresponding
nucleotide codons in order to deduce a target nucleic sequence. Because one
amino acid sequence corresponds to multiple nucleotide sequences, those
suitable for expression in s E.coli systems should be selected from literature
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(for example, http://www.kazusa.or.jp/codon/), and those not easily
recognized and expressed by E.coli should be avoided. Likewise, if the
sequence is to be expressed in yeasts, sequences suitable for expression in
yeast systems (i.g. Saccharomyces, or Pichia spp.) should be selected.
b. Analyzing the restriction enzyme map of the target nucleic acid sequence
by software (such as DNA strider). Then designing and linking restriction
sites for restriction digestion to both termini of the target nucleic acid
sequence is achieved. The sequences of the restriction sites not supposed to
reside in the target sequence should be replaced by different codons
encoding the same amino acids, so that those restriction sites which can be
removed.
c. The regions that are possibly toxic, causing immune disorders, leading to
immune toxicity or allergy in the target protein according to literatures
should be modified through point mutation or deletion of its amino acid
sequence if possible. For example, ultra virulence caused by basic amino
acids existing in the structural proteins of the influenza virus can be
attenuated by mutating them to other non-basic amino acids. By modifying
the target protein in any given site, it is possible to provide design of a
protein vaccine capable of inhibiting an influenza virus.
d. Of course, the modified version of the target protein should be inspected
and converted to the corresponding restriction of the target gene. If any new
restriction site appears in the modified target gene and causes difficulties
in
cloning the gene into a plasmid, it can be removed by substituting the
condon with another one encoding the same amino acid.
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The aforementioned points and steps are further clarified by examples
given as follows. Avian flu virus belongs to a subtype of Influenza A virus,
generally has a diameter about 0.08-0.12pm, and is an RNA virus usually
classified by types of HA/hemagglutinin and NA/neuraminidase on its
surface. There are 15 HA subtypes and 9 NA subtypes. The human
influenza viruses are usually H1N1 or H3N2, which have been pervasive
for many years, so most people are immune-resistant to them. Generally,
avian flu viruses are genetically distinct from human flu viruses, but cases
of transmission from animal to human have been reported, such as H9, H7
and H5.
The three proteins of the capsule of A type flu virus are HA
(hemmagglutinin), NA(neuraminidase) and M2, which are the major targets
of recognition by antibodies of the host or anti-virus drugs. HA
glycoprotein forms spikes on the surface of the virus, controls adhesion to
sialoside receptors of the host cell and then enters the cell by fusion to the
cell membrane. NA forms spherical spikes and catalyzes the release of
viruses from infected cells, so as to spread the viruses. M2 is a membrane
protein that is responsible to form an ion channel, allowing genes of the
virus to be released and expressed.
A/H5N1 avian flu virus is also called "H5N1" virus, which is a novel
type-A influenza virus subtype existing mainly among birds. Bird flu virus
transmits continuously among birds and mutates very easily. Furthermore,
the habitation areas of birds, livestock and human beings overlap
considerably, so the cross-species transmission occurs easily. Pigs or
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humans can acquire different virus genes; for example, patients could be
infected by human flu virus and bird flu virus at the same time, resulting in
a "hybrid channel" of viruses and even new virus strains due to
recombination. When a virus is able to infect a person and cause serious
diseases, it possesses the characteristics of an outbreak-inducing flu virus
strain. The looming crisis is that these kinds of viruses are prone to undergo
gene flow, which could evolve to a pandemic of human-to-human
transmission. Before any antibody is available to be induced in human
bodies, a severe pandemic can be expected.
The present invention can derive development and application of two
kinds of vaccines. The first one utilizes eight-plasmid flu system and
reverse genetic engineering. First, HA structural protein is modified to a HA
plasmid in which the neutralization titration regions are replaceable. Then
the target sequences of the neutralization titration regions of H5N1 or other
virulent novel flu virus are generated by the nucleic acid synthesis method.
Finally, the synthesized fragments are inserted into replaceable HA
plasmids of the H1N1 vaccine strain. The antigen composition does not
include a full-length HA of the novel highly pathogenic strain H5NI, and
only neutralization titration regions H5N1-HA are substituted. Thus, the
properties of the virus are very similar to the H1N1 vaccine strain, so it is
less probable to evolve to a highly pathogenic virus strain, endowing more
safety to the manufacturing process of the vaccine. Besides, the novel flu
vaccine can generate antibodies having neutralization titration after
administration. Utilizing the above-mentioned method, that is, to insert the
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=
target antigen gene of the H5N1 -HA neutralization titration regions into the
homologous loci of the similar genes, then to employ reverse genetic
engineering and eight-plasmid flu system, and to generate a vaccine strain
against novel pathogens, is the best strategy for humans to fight against
novel infectious diseases.
In addition to the neutralization titration regions, ELISA using
non-neutralization titration regions of H5N1-HA is still able to distinguish
species-specific antibodies arising from a natural infection. Thus, the
ELISA system using reaction to this specific antibody after administration
of the vaccine will not interfere with current detection systems in
surveillance of real time situations regarding the spreading of a novel flu.
Another method of vaccine preparation of the present invention is the
method to prepare a target subunit vaccine, comprising: (a) providing an
amino acid sequence of at least one epitope peptide of a target antigen
protein, and converting the amino acid sequence to a corresponding wild
type nucleic acid sequence; (b) modifying the wild type nucleic acid
sequence of the epitope to a modified nucleic acid sequence which is
recognizable to a host cell and encodes the epitope peptide; (c)
synthesizing primers of the modified nucleic acid sequence, wherein the
primers are nucleic sequences having 5-200 nucleic acids, the primers are
identical or complementary to portions of the modified nucleic acid
sequence, and the 3' ends of the forward primers and the 3' ends of the
reverse primers among the primers comprise sequences of 5-20 nucleic
acids that are complementary to each other; (d) synthesizing the modified
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nucleic acid in vitro using the primers; (e) linking the synthesized nucleic
acid fragments to a nucleic acid sequence having the functions of binding
and translocation and a plasmid having carboxyl terminal moiety to produce
a modified plasmid; (f) transforming the modified plasmid into a host cell,
so as to produce the epitope peptide encoded by the modified nucleic acid;
and (g) collecting and purifying the epitope peptide.
The length of the primers used in the present method are not limited.
Preferably, the primers are of 5-200 nucleic acids. More preferably, the
primers are of 5-80 nucleic acids.
In the method of the present invention, the target antigen genes can also
be inserted into the homologous loci of the similar genes in the vaccine
strain, and then reverse genetic engineering and eight-plasmid flu system
are employed so as to generate a vaccine strain against novel pathogens.
In prior arts of the related field, it is known that a short peptide sequence
can be used to prepare antibodies against the peptide itself, and the
antibodies are able to recognize the original whole protein. Therefore, the
method for preparing a subunit vaccine disclosed in the present invention is
to select a segment on the epitope sequence to serve as a target synthesized
peptide, without using a full-length sequence. Thus, there is no particular
limitation to the sequences suitable for this method; the sequences can be
mainly functional fragments, such as epitopes stimulating B-cell or T-cell
immunity, or the choice of desired fragments can be based on
hydrophobicity of the structure. The hydrophilic regions are more reactive
to intracellular components, so it is preferable that the epitope peptides are
CA 02584894 2007-04-13
derived from hydrophilic regions of the target antigen protein structure. The
fragments used are not limited. Several fragments of choices can be joined
together to form a large fragment of a peptide, a single fragment can be
selected from an epitope sequence, or epitopes stimulating B-cell or T-cell
immunity can be fused to form a fusion protein.
The protein structures of sequences of the present invention are not
directly isolated from natural bacteria or virus, so it is necessary to
synthesize and produce target proteins by using host cells. There is no
particular limitation to suitable host cells, but the host cells are
preferable to
be microbe cells, plant cells or animal cells, and more preferable to be
E.coli or yeasts. Besides, the target peptide (having a synthesized nucleic
sequence) synthesized by host cells must encode the same target epitope
peptide as in a wild type nucleic acid sequence to achieve the effects of
specificity of the antigens prepared by the method disclosed in the present
invention. Itis advantageous for preparing an antigen vaccine having great
safety and for achieving the same specificity as wild type virus antigens.
Although proteins of some antigens have immune-toxicity or could cause
immune disorders, it is possible to modify them to endow safety and
immune protection.
There is no particular limitation to the method to synthesize the modified
nucleic acid of the present invention in accordance with an epitope of a wild
type target protein. The method is preferable to be in vitro synthesis by PCR.
There is no particular limitation to the source of the nucleic acid sequence
of the carboxyl terminal moiety comprised by the synthesized nucleic acid
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of the present invention, but it is preferable to be derived from a portion of
pseudomonas exotoxin, and more preferable to be amino acid sequence
comprising KDEL or its corresponding sequence.
The method for preparing a target type subunit vaccine of the present
invention comprises a subunit vaccine, which is able to induce protection
titers and effectively inhibit infection by an Avian Influenza virus. The
structure of the protein vaccine comprises: an epitope of; a peptide having
the functions of binding and translocation; and a carboxyl terminal KDEL
peptide.
In the present invention, the epitope peptide encoding an avian flu viral
protein encoding is artificially synthesized, instead of isolating and
preparing from a natural Avian Influenza virus, and thus obviates the need
to contact pathogens that transgress human bodies, so as to improve the
safety of the working environment of researchers and to accelerate research
speed of vaccines and drugs.
The avain flu virus in the present invention is orthomyxoviridae H5N1.
The nucleic acid sequences of suitable epitopes of bird flu virus proteins
inust be modified so that the encoding epitope peptides are identical to
those of naturally occurring virus strains, while at the saine time achieving
high-level expression in desired host cells.
Meanwhile, the synthesized target antigen used in the target type subunit
vaccine are preferable to be generated by converting a full-length epitope
peptide of a wild type viral protein to nucleotide codons, selecting a portion
of the nucleotide sequence that suits the desired functions, or combining
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several fragments of the nucleotide sequence, and producing the
corresponding epitope peptides (synthesized) of the viral protein by
microbes.
The synthesized peptides prepared in accordance with the present
invention have the effects of inducing antibodies in vivo, while infection
during immunization of the subjects is prevented, so they can serve as
relative safe vaccines of antibody compositions.
There is no particular limitation to the epitope peptides of avian flu viral
proteins suitable to the target type subunit vaccines covered by the present
invention but they are preferably selected from one of the group consisting
of: H5N 1-S 1, H5N1-NP, H5N 1-HA neutralization titration regions,
H5N1-M2, and H5N1-NA enzymatically active sites.
The target subunit vaccines in the present invention, in addition to
reacting to antigens, are also functionally related to a vaccine delivery
system and have the functions of binding and translocation to antigen
presenting cells. There is no particular limitation to the sources of the
nucleic acid sequences having the functions of binding and translocation,
but they are preferably derived from domain I and domain II of
pseudomonas exotoxin. Domain I of pseudomonas exotoxin is a ligand
which functions to bind the receptors of a target cell. The suitable target
cells can be any one known in the art but they are preferably selected from
at least one of the group consisting: T cells, B cells, dendritic cells,
monocytes and macrophages. The suitable receptors are selected from at
least one of the group consisting of: TGF receptors, IL2 receptors, IL4
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receptors, IL6 receptors, 1GF1 receptors, CD4 receptors, IL18 receptors,
IL12 receptors, EGF receptors, LDL receptors,a 2 macroglobulin receptors,
and heat shock proteins.
In the target subunit vaccines in the present invention, the target
antigen genes are inserted into the homologous loci of the similar genes,
and then reverse genetic engineering and eight-plasmid flu system are
employed so as to generate a vaccine strain against novel pathogens.
The design idea of the fusion protein in the present invention is to
develop vaccines having a conserved common immunogen, e.g. vaccines
having antigens like M2. In other words, immune reactions induced from
the vaccine comprising the fusion proteins of the present invention can
response for various types of influenza viruses, e.g. H5N1, H5N2, HINl,
and so forth, even though the virus mutates vary rapidly. Therefore, the
vaccines of the present invention comprising the conserved common
immunogen can be used for treating disease infected by the virus without
the drawbacks of the conventional vaccines, i.e. changing into or
developing new vaccines every year. Hence, this design idea of the
vaccines of the present invention is a future trend for the development of an
influenza vaccine. The design idea illustrated above is unlike a
conventional method, which only focuses on enhancing the protection of
neutralizing antibody titer. In fact, treatment for a disease induced from a
virus with the conventional vaccine frequently becomes useless if a new
mutant virus of the same type virus appears next year. Nevertheless, the
vaccine comprising the fusion protein of the present invention can be
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efficient for various types of viruses even though new viruses appear
through mutation quickly.
Other objects, advantages, and novel features of the invention will
become more apparent from the following detailed description when taken
in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is the electrophoresis photo of PCR-sythesized H5N 1-NS 1;
Fig. 2a-2d is the electrophoresis photo of PCR-sythesized H5N1-NP,
wherein 2a refers to the fragment H5N 1-NP-a (256bp), 2b refers to the
fragment H5N 1-NP-b (365bp), 2c refers to the fragment H5N1-NP-c
(464bp), and 2d refers to the fragment H5N1-NP-d (2488p);
Fig.3 is the electrophoresis photo of PCR-sythesized H5N 1 -HA(4 86bp);
Fig.4 is the electrophoresis photo of PCR-sythesized H5N1-NA(501bp);.
Fig.5 is the vector scheme of H5N1-NS1;
Fig.6 is the vector scheme of H5N 1-NP, wherein 6a refers to the fragment
H5N 1-NP-a, 6b refers to the fragment H5N 1-NP-b, 6c refers to the
fragment H5N 1-NP-c, and 6d refers to the fragment H5N 1-NP-d;
Fig.7 is the vector scheme of H5N1-HA;
Fig.8 is the vector scheme of H5N1-eM2;
Fig.9 is the vector scheme of H5N1-NA;
Fig.10 shows the results of protein expression of vector
PE-H5Nl-NP-a-K3-PE-H5N1-NP-d-K3, wherein the results of a-d are
illustrated;
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Fig.ll shows the results of protein expression of vector
PE-H5N1-HA-K3;
Fig.l2 shows the results of protein expression of vector
PE-H5N1-eM2-K3;
Fig.13 shows the results of protein expression of vector
PE-H5N 1-NA-K3 ;
Fig.14 illustrates the titers of M2 antibody after immunization of mice by
different levels of PE-H5N 1-eM2-K3 in Example 6
Fig.15 illustrates changing titers of IgY antibody after immunization of
leghorn chicken in Example 7;
Fig. 16 shows pathological sections of lungs of the ICR mice after 14 days
of challenged with H5N2 type virus in Example 8;
Fig. 17 shows the death rate of chickens during the period of
immunization in Example 9;
Fig. 18 shows the egg production of chickens during the period of
immunization in Example 9;
Fig. 19 illustrates the titer of the antibodies got from the eggs produced by
chickens during the period of immunization in Example 9;
Fig.20 illustrates the titer of the 500-times dilution of the antibodies got
from the eggs produced by chickens, against H5N1-M2 during the period of
immunization in Example 9; and
Fig.21 illustrates the titer of the 500-times dilution of the antibodies got
from the eggs produced by chickens, against H5N 1-HA during the period of
immunization in Example 9.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The technical platform for preparing a targeting subunit vaccine of the
present invention makes it possible to use a peptide sequence having
functions of binding and translocation, and a plasmid of carboxy-terminal
KDEL type peptide to construct a plasmid capable of producing a target
protein in vitro, after the sequence of the target protein is obtained,
codon-converted and modified.
The following embodiments utilize several peptides of avian influenza
virus H5N1 as the target antigens.
In accordance with the aforementioned mechanisms of viral infection,
the present invention employs highly conserved regions of some key
immunogenic proteins among them (i.e. epitope) for testing, to elicit
immunity in vivo without occurrence of viral infection in the course of
research and administration of vaccines. The target proteins used in the
following examples are: H5N1-NS1, H5N1-NP, H5N1-HA, H5N1-M2, and
H5N 1-NA.
The target type subunit plasmid discovered in the experiments shows
poor efficiency in the induction of protein synthesis in the host E. coli
cells,
possibly attributable to the toxicity of the protein itself. Therefore, the
hydrophobic regions of M2 are removed, and the hydrophilic regions of the
protein remain. The modified protein is dubbed H5N1-eM2. Through this
modification, H5N1-eM2 could be expressed in large scale in E.coli. The
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encoded amino acids are not influenced and the regions of high
immunogenecity are reserved according to the result of the sequence
comparison. Thus, the H5N1-M2 related antigens are mainly represented
by H5N1-eM2 in the present invention.
Example 1. The selection of target sequence fragments
The DNA sequences and the correspondent amino acid sequences of
H5N1-NS1, H5N1-NP, H5N1-HA, H5N1-M2, and H5N1-NA were
retrieved from the National Center of Biotechnology Information (NCBI,
USA) database.
According to the theories of vaccine preparation, not all of the peptide
sequences of antigen proteins were able to induce the antibodies reactive to
a whole antigen protein. The sequence must at least be located on the
surface of the antigen and be able to contact water. Thus, the
above-mentioned DNA sequences were each entered into the software used
for evaluating the hydrophobicity (i.e. DNA strider V1.0), then the desired
segments to be synthesized were selected according to the resulting
evaluation plot. In this Example, hydrophilic regions were selected to
proceed with the preparation of the synthesized peptides. Note that this
Example is not set to limit the selection of desired regions, and other
regions able to induce similar effects are covered by the claims of the
present invention.
According to the above results of evaluation by the software, several
hydrophilic segments were selected from each target protein of the Example.
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The amino acid sequences include: one from H5N1-NS 1( SEQ. ID. NO.1)
four from H5N1-NP (SEQ. ID. NO.2, SEQ. ID. NO.3, SEQ. ID. NO.4, SEQ.
ID. NO.5), one from H5N1-HA (SEQ. ID. NO.6), one from H5N1-eM2
(SEQ. ID. NO.7 ), and H5N1-NA ( SEQ. ID. NO.8 ).
The resulting target must undergo restriction digestion, so it was
preferable that the target DNA sequences have no restriction site. Thus,
software must be employed to evaluate the fact whether these sites reside in
the DNA sequences or not. If these sites reside in the DNA sequences, they
must be replaced with other condons encoding the same amino acids. The
software can also check the existence of the designed restriction sites at
both termini of the DNA, which makes following cloning procedures
possible.
Example 2. In vitro synthesis of the target sequences
The nucleic acid sequence encoding the wildtype protein was modified to
make the protein be expressed in large scale in E. coli; the key point of the
modification was to modify each single nucleotide without affecting the
originally expressed amino acids, and at the same time to express them
effectively in E. coli. The nucleic acids of the modified nucleic acid
sequence were synthesized by polymerase chain reaction. The primers were
numbered as shown in Table 1.
Table 1
Target antigen Number of Seq. ID. No. Number of Seq. ID. No.
forward rimers forward primers
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NSI 6 9-14 6 15-20
NP-a 4 21-24 4 25-28
NP-b 6 29-34 6 35-40
NP-c 8 41-48 8 49-56
NP-d 8 57-64 8 65-72
HA 8 73-80 8 81-88
eM2 4 89-92 4 93-96
NA 8 97-104 8 105-112
First, non-DNA-template PCRs were used by using forward and
reverse primers to proceed with enzyme-catalyzed annealing of nucleotide
fragments, wherein the 3' ends of each primers have 10-15 bases that were
complementary to each other. A PCR DNA product was then generated
through reading and complementation of polymerase.
After the first round PCR, 0.01-141 of the product was used as the DNA
template of the second round PCR. The second primer pair was added at a
suitable amount together with dNTPs, reagents and Pfu polymerase, and
then the second round PCR was perforined. Subsequently, different primer
pairs were added each time in this manner, so that modified nucleotide
sequences were respectively synthesized.
All synthesized nucleotide fragments were analyzed by electrophoresis
to check if the produced fragments were of the expected sizes. H5N1-NS
(396bp), as shown in Fig. 1; H5N1-NP (four fragments a, b, c and d were
used, with 256 bp of fragment a, 365 bp of fragment b, 464 bp of fragment c,
and 488bp of fragment d), as shown in Fig. 2a-2d; H5N1-HA (486bp), as
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shown in Fig.3; H5N1-eM2; and H5N1-HA (501bp), as shown in Fig.4.
Example 3. Constructing the plasmid comprising the target sequence
Eight of the aforementioned synthesized fragments (including NP-a-d)
were digested by EcoRI and Xhol and then ligated to a peptide sequence
having the functions of binding and translocation and an E. coli plasmid
containing a carboxyl terminal moiety. The plasmids constructed were:
1. pPE-H5N 1-NS l-K3 plasmid:
The construct was built in a pET vector system having an ampicilin
resistance fragment, which was able to express H5N 1-NS 1 fusion protein.
(The vector scheme is illustrated in Fig. 5.)
2. pPE-H5NI -NP, a, b, c, d-K3 plasmid:
This constructed pET 15 vector system is able to express
H5N 1-NP 1 A-H5N 1-NP 1 D fusion proteins. (The vector scheme is
illustrated in Figs. 6a-6d.)
3. pPE-H5N 1-HA-K3 plasmid
This constructed pETl5 vector system is able to express H5N1-HA fusion
protein. (The vector scheme is illustrated in Fig. 7.)
4. pPE-H5N1-eM2-K3 plasmid
This constructed pET 15 vector system was able to express H5N 1-eM2
fusion protein. (The vector scheme is illustrated in Fig. 8.)
5 .pPE-H5N 1-NA-K3 plasmid
This constructed pET15 vector system is able to express H5N1-NA fusion
protein. (The vector scheme is illustrated in Fig. 9.)
21
CA 02584894 2007-04-13
76302-51
Finally, the above plasmids were transformed into cells of different E. coli
strains.
Example 4. Expressing and analyzing the target proteins
The E.coli strains, after being checked that 90% of the bacteria
populations have the above plasmids with desired genes, were stored at -70 C
in glycerol in 2-ml aliquots.
In a sterile environment, 2 ml of the stored clones was inoculated in a
500m1 flask containing 200m1 LB(+500pg /ml Amp), incubated in a 37 C
rotating incubator and shaken at 150rpm for 10-12 hours, and the bacteria
cultures were generated. OD600 of the cultures should attain 1.0 0.3.
In a sterile environment, 50m1 culture liquid was inoculated in each eight
sterilized 3000m1 flasks, each of them containing 1250m1 LB(+500pg /ml
Amp+50m1 10% glucose), incubated in a 37 c rotating incubator and shaken
at 150rpm for 2-3 hours, added with IPTG to a final concentration 50ppm,
and further incubated in a 37 C rotating incubator and shaken at 150rpm for 2
hours, so that the protein production was completed.
Subsequently, the antigen protein fragments in inclusion bodies were
resolved by 8M urea extraction method, such as PE-H5NI-NSI-K3,
PE-H5N1-NP-a-K3-PE-H5N 1-NP-d-K3 (Figl0),
PE-H5N 1-HA-K3 (Fig l 1) , PE-H5N 1-eM2-K3 (F ig l 2) , and
PE-H5N1-NA-K3(Fig13). Each of them was extracted with 10 liter bacteria
culture liquid, and 300-400mg antigen was obtained. Totally 3000-9000
22
CA 02584894 2007-04-13
injection doses can be obtained from 300 mg antigen.
Each antigen solution was quantified by Western-blotting, coomasie blue
staining, and SDS-PAGE electrophoresis with measurement of density of
the bands by a densitometer. 0.03 0.003mg of the above antigen protein
was used as the primary content of a high-dose injection, and 0.01
0.0001mg was used as the primary content of a low-dose injection.
Example 5. Preparation of Avian Influenza vaccines
In a class 100 laminar flow, each antigen solution was added with 8M
urea to a final volume 40m1, 40 ml was A 206 adjuvant was then added, and
the mixture was stirred at 50rpm for 10 minutes in a stirring bucket,
sterilized water was added and the stirring speed was increased to 100rpm
to further stir for one hour.
The stirring buckets were transferred to a dispensing room for dispensing,
capping and labeling with 1ml per dose in each sterilized injection bottle, so
that 100 doses of injection of Avian Influenza vaccine were obtained.
Example 6. The test of immunity of the vaccine using mice as a model
organism
Taking PE-H5N1-eM2-K3 as an example, 0.3 0.03mg was used as the
primary content of an ultra-high-dose injection(VH), 0.03 0.003mg was
used as the primary content of a median dose injection 0.01 0.01mg was
used as the primary content of a high-dose injection(H), and 0.01
0.0001mg was used as the primary content of a low-dose injection, which
23
CA 02584894 2007-04-13
were mixed with adjuvant of different doses (Spec was A 206) and used to
immunize Balc/C mice, each group having 12 mice; the mice were
immunized in two weeks and had to be immunized 3 to 4 times in total.
A blood sample was taken after immunization and assayed by ELISA.
For M2 anti-specific IgG, an antibody titer was detected after the second
round of immunization by anti-eM2 ELIZA through 1/10 dilution end-point
titration assay. Ultra-high-dose injection(VH)(0.1 0.01mg)and High-dose
injection(H)(0.3 0.03mg) induced similar titers; the titers peaked after the
third round immunization and reached a plateau after the fourth round. The
median dose of injection and the low dose of injection induce lower titers,
but both reached 10,0000 times after the fourth round immunization. The
results are shown in Fig. 14.
Example 7. Vaccinal immunization tests of Egg-laying Leghorn
chicken model
Taking PE-H5N 1-eM2-K3 as an example, 0.1 0.01 mg of the fusion
antigen was used as a dose of injection, mixed with appropriated adjuvants,
and administered to a Leghorn chicken at egg-laying stage. After three to
four times of immunization, high-titer anti-avian influenza antibodies were
accumulated in the yolks, with the titer of ELIZA 1/10 dilution end-point
titration assay higher than 10,0000. However, when the antigens used were
PE-H5N 1-eM2 and or eM2 subunit protein antigen, the IgY titers were very
low, only 10-100 times as high as those of the blank, non-immunized group.
24
CA 02584894 2007-04-13
Example 8. Immunization and virus challenge test of Al subunit
vaccines in ICR mice
The fusion proteins expressed in the present invention are
conserved common immunogens of the H5N1 type influenza virus.
According to the knowledge of the influenza virus, a person having the
ordinary skill in the art understands that H1N1 type virus has N1 type
characteristics of H5N1 type virus, and H5N2 type virus has H5 type
characteristics of H5N1 type virus. Hence, vaccines comprising the fusion
proteins, which are conserved common immunogens of the H5N1 type
influenza virus, expressed in the present invention can protect a host against
both of H1N1 type and H5N2 type virus. However, because H5N1 type
influenza virus is extremely harmful to human bodies, it is unsuitable to
perform experiments by using this type virus directly. Through being
challenged respectively with H1N1 type and H5N2 type influenza virus, the
vaccines of the present invention can be proved that they are really efficient
to H5N1 type virus due to the H 1N 1 type and H5N2 type virus respectively
having N1 type and H5 type characteristics of H5N1 type virus.
A. Challenged with H IN 1 type human influenza virus
First, ICR mice are separated into five groups, and each group
possesses six ICR mice. Each of the different fusion proteins is taken with a
determined dose of injection, and then is mixed with an appropriate dose of
an adjuvant. They are as follows: ICR mice of Group I are immunized with
PE-H5N 1-eM2-K3 (H, 0.1 0. 01 mg); ICR mice of Group II are
CA 02584894 2007-04-13
immunized with PE-H5N 1-NP-(a+b+c+d)-K3 (H, 0.1 0. 01 m g); ICR
mice of Group III are immunized with PE-H5N1-HA-K3 (H, 0.1 0.01
m g); ICR mice of Group IV are immunized with PE-H5N 1-NS 1-K3 (L,
0.01 0.001 mg); and ICR mice of Group V are a blank group immunized
with nothing.
After three to four times of immunization, the immunized ICR mice
of Groups I-V are challenged with H1N1 virus. After four days of
post-challenged, the salvia of each ICR mouse in each group is tested for
checking the existence of virus excretion. Besides, the healthy condition of
every mouse in each group is also observed and recorded. The results are
shown in Table 2.
Table 2
number of number of number of
Group Fusion protein immunized mice excreting ill mice at 4
mice virus at 4 D c*
Dpc*
p
I PE-H5N 1-eM2-K3 (H) 6 2 1
II PE-H5N 1-NP-(a+b+c+d)-K3 6 2 0
(H)
III PE-H5N 1-HA-K3 (H) 6 5 3
IV PE-H5N 1-NS 1-K3 (L) 6 4 4
Blank 6 5 5
group
* Dpc: days of post-challenged.
According to Table 2, after four days of post-challenged, the ICR
mice immunized with the fusion proteins of the present invention have
fewer mice with virus excretion in saliva than those of the blank group.
Furthermore, the PE-H5N1-eM2-K3 and PE-H5N1-NP-(a+b+c+d)-K3
26
CA 02584894 2007-04-13
fusion proteins in the high dose exhibit the best vaccinal effect, and they
can
decrease the number of mice with virus excretion in saliva. Moreover,
although other fusion proteins do not exhibit the same effect of
PE-H5N 1 -eM2-K3 and PE-H5N 1 -NP-(a+b+c+d)-K3, they still have better
effect against avian influenza than the blank group does.
B. Challenged with H1N1 type human influenza virus
First, ICR mice are separated into six groups, and each group
possesses five ICR mice. Each of the different fusion proteins is taken with
a determined dose of injection, and then is mixed with an appropriate dose
of an adjuvant. They are as follows: ICR mice of Group I are immunized
with PE-H5N 1-eM2-K3 (H, 0.1 0. 01 mg); ICR mice of Group II are
immunized with PE-H5N 1-NP-(a+b+c+d)-K3 (H, 0.1 0. 01 m g); ICR
mice of Group III are immunized with PE-H5N 1-HA-K3 (L, 0.01 0. 0 01
m g); ICR mice of Group IV are immunized with PE-H5N 1-NS 1-K3 (L,
0.01 0.001 m g); ICR mice of Group V are immunized with
PE-H5N1-NA-K3 (H, 0.1 0.01 mg); and ICR mice of Group VI are a
blank group immunized with nothing.
After three to four times of immunization, the immunized ICR mice
of Groups I-VI are challenged with H1N1 virus. After four days of
post-challenged, the salvia of each ICR mouse in each group is tested for
checking the existence of virus excretion. Besides, the healthy condition of
every mouse in each group is also observed and recorded. The results are
shown in Table 3.
27
CA 02584894 2007-04-13
Table 3
number of number of number of
Group Fusion protein immunized mice excreting ill mice at 4
mice virus at 4 D c*
Dpc* p
I PE-H5N1-eM2-K3 (H) 5 2 1
II PE-H5N 1-NP-(a+b+c+d)-K3 5 2 0
(H)
III PE-H5N 1-HA-K3 (H) 5 2 0
IV PE-H5N1-NS1-K3 (L) 5 2 0
V PE-H5N1-NA-K3 (H) 5 2 0
Blank --- 5 4 2
group
* Dpc: days of post-challenged.
According to Table 3, after four days of post-challenged, the ICR
mice immunized with the fusion proteins of the present invention have
fewer mice with virus excretion in saliva than those of the blank group do.
Further, all of the fusion proteins in the present invention have better
effect
against avian influenza than the blank group does.
Additionally, after 14 days of post-challenged, ICR mice
immunized with PE-H5N 1-eM2-K3 are anatomized and their lungs are
taken to process pathological sections. As compared with the pathological
lung sections of the unimmunized and unchallenged ICR mice and
unimmunized and challenged ICR mice, severity levels of interstitial
pneumonia are determined and recorded. The results are shown in Table 4
and FIG. 16.
Tab1e 4
group mice No. total scores
unimmunized and BK-1 7
challenged ICR mice BK-2 6
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CA 02584894 2007-04-13
BK-3 7
BK-4 5
ICR mice immunized 8-1 2
with PE-H5N1-eM2-K3 8-2 3
and challenged with 8-3 3
H5N2 type virus 8-4 4
CTL-1 1
unimmunized and CTL-2 1
unchallenged ICR mice CTL-3 1
CTL-4 1
*Score marker: minimal=l, mild=2, moderate=3, and severe=4; in addition,
multifocal=l, diffuse=2, and subacute=l; the most severe mouse is scored
as 7 points; and the mouse with little interstitial pneumonia is scored as 3
points.
With reference to FIG. 16 and Table 4, compared with the
unimmunized and challenged ICR mice, the ICR mice immunized with
PE-H5N1-eM2-K3 have few symptoms of interstitial pneumonia close to the
ICR mice without being challenged with H5N2 type virus.
Example 9. Field trial in a chicken farm infected with H5N2 type avian
influenza virus
A field trial in a chicken farm broken out with H5N2 type avian
influenza virus is performed through immunizing the chickens in the
chicken farm with a dose of a complex vaccine comprising 0.05 mg
PE-H5N 1-eM2-K3, 0.01 mg PE-H5N 1-NP-a-K3, 0.01 mg
PE-H5N 1-NP-b-K3, 0.01 mg PE-H5N 1-NP-c-K3, 0.01 mg
PE-H5N1-NP-d-K3, 0.05 mg PE-H5N1-HA-K3, 0.05 mg
PE-H5N1-NA-K3, and ISA206 of 10%. The complex vaccine is prepared
29
CA 02584894 2007-04-13
by the way illustrated in Example 9. Additionally, the immunized chickens
are immunized again every two weeks till four or five times of
immunization are achieved. By comparing the immunized chickens and the
chickens in a blank group without being immunized by the above complex
vaccine, as shown in FIG. 17, the death rate of the chickens immunized with
the above complex vaccine is decreased to under about 5%. However, the
death rate of the chickens in the black group without being immunized by
the above complex vaccine is raised to about 60% to 70% by time pass.
In addition, the egg production of the immunized chickens tends
upwards by times of immunization, and the results are shown in FIG 18.
The yolks got from eggs produced by the immunized chickens after three to
five times of immunization perform ten-fold serial end-point diffusion test
to check the titer of IgY antibodies. As shown in FIG. 19, the titer of the
IgY
antibodies against HA, NA, M2, PE, or E. coli is increased by the times of
immunization. Furthermore, the yolks got from eggs produced by the
immunized chickens after five times of immunization are also tested to
check the titer of IgY antibodies against H5N1-M2 or H5N1-HA. We
discover that 500 times dilution of the antibodies still has dramatic positive
reaction, and the results are shown in FIG 20 and FIG 21 respectively.
In conclusion, the method of vaccine preparation obviates the needs
to contact highly hazardous biological samples and viral materials. Instead,
the amino acid sequence is retrieved directly from the Internet, enabling
researchers to generate a safe and effective vaccine. The establishment of
this platform is essential for vaccinal preparation in countries that are
CA 02584894 2007-04-13
currently unaffected by but highly vulnerable to the infectious disease. The
design idea of the fusion protein in the present invention is to develop a
conserved common immunogen, e.g. vaccines having antigens like M2. In
other words, immune reactions induced from the vaccine comprising the
fusion proteins of the present invention can response for various types of
influenza viruses, e.g. H5N1, H5N2, HIN1, and so forth, even though the
virus mutates vary rapidly. Therefore, the vaccines of the present invention
comprising the conserved common immunogen can be used for treating
disease infected by the virus without the drawbacks of the conventional
vaccines, i.e. changing into or developing new vaccines every year. Hence,
this design idea of the vaccines of the present invention is a future trend
for
the development of an influenza vaccine. The design idea illustrated above
is unlike a conventional method, which only focuses on enhancing the
protection of neutralizing antibody titer. In fact, treatment for new mutant
virus with the conventional vaccine frequently becomes useless if a new
mutant virus of the same type virus appears next year. Nevertheless, the
vaccine comprising the fusion protein of the present invention can be
efficient for various type viruses even though new viruses appear through
mutation quickly.
Although the present invention has been explained in relation to its
preferred embodiment, it is to be understood that many other possible
modifications and variations can be made without departing from the scope
of the invention as hereinafter claimed.
31
rn
0
C-n
Plasmids depicted in Figs 5, 6a-6d, 7, 8 and 9 have been
deposited at DSMZ (Deutsche Sammiung von Mikroorganismen und
Zellkulturen GmbH) as follows:
aeo le N
cn
original
Identification Refei'-ence given by the Depositor Country Institution
Accession Number 1l7aterial deposit
~ pPE(dieltalll)-H5N1-NSI-H-K3/pET-15b Germany DSMZ DSM 19253 plasmid 20070329
pPE(deltalll)-NPIA-K3/pET-15b Gerniany DSMZ DSM 19254 plasrnid 20070329
pPE(de1ta1ll)-NPIB-K3/pET-15b Germany DSMZ DSM 19255 plasmid 20070329
pPE(deital)))-NPIC-K3/pET-15b Germany DSMZ DSM 19256 plasmid 20070329 W
pPE(deltal1l)-NPID-K3/pET-15b Germany DSMZ DSM 19257 plasmid 20070329
pPE(deltal(I)-eM2K3/pET-15b Gezmany DSMZ DSM 19258 plasmid 20070329
pPE(deltalll)-H5N1-NSI-HA-K3/pET-15b Germany DSMZ DSM 19259 plasmid 20070329
pPE(deltalll)-H5N1-NSI-NA-K3/pET-15b Germany DSMZ DSM 19260 plasmid 20070329
CA 02584894 2007-04-13
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