# Patent

## Title
Immunization against flavivirus

## Abstract

A method of inducing an immune response in a subject against a flavivirus. The method involves administering to the subject with a fusion polypeptide including a signal peptide and a part of an envelope protein of the flavivirus, or with an expression vector containing a nucleic acid encoding the fusion polypeptide. Also disclosed are a polypeptide, an expression vector, and vaccines containing the polypeptide or expression vector.

## Background
BACKGROUND
Flavivirus is a family of arthropod-borne viruses, including dengue virus, yellow fever virus, tick borne encephalitis virus, West Nile encephalitis virus, and Japanese encephalitis virus (JEV). All members of this family can cause diseases in human. In particular, infection of JEV is a leading cause of morbidity and mortality in tropical and sub-tropical regions of Asia. Immunization can be used to protect against JEV. Both inactivated and live-attenuated JEV viruses have been used as vaccines. However, their success has been limited due to lack of long-term immunity and adverse effects such as allergic reactions. Therefore, there is a need for effective and safe vaccines and immunization methods for protection flaviviruses, such as JEV.
SUMMARY
In one aspect, the present invention features a method of inducing an immune response in a subject, such as a human or a non-human animal, against a flavivirus, e.g., JEV, Dengue virus, tick borne encephalitis virus, or West Nile encephalitis virus. The method includes administering to the subject a fusion polypeptide including a signal peptide and a part of an envelope protein (E protein) of the flavivirus, or an expression vector containing a nucleic acid encoding the fusion polypeptide. The signal peptide contains the sequence of VVFTILLLLVAPAYS (SEQ ID NO: 5) and allows the fusion polypeptide to be soluble and correctly folded in a cell. The part of the E protein can be an amino part or a carboxyl part of the E protein. The induced immune response, i.e., the production of antibodies specifically against the flavivirus, can be determined by the assay described in Examples 1–4 or any analogous assays.
To induce an immune response against JEV, a subject can be administered with a first expression vector containing a nucleic acid encoding a fusion polypeptide of the sequence SEQ ID NO: 3, i.e., 1-VVFTILLLLVAPAYSFNCLGMGNRDFIEGASGATWVDLVLEGDSC LTIMANDKPTLDVRMINIEASQLAEVRSYCYHASVTDISTVARCPTTGEAHNEKRADSSYVCKQGFTD RGWGNGCGLFGKGSIDTCAKFSCTSKAIGRTIQSENIKYEVGIFVHGTTTSENHGNYSAQVGASQAAK FTVTPNAPSITLKLGDYGEVTLDCEPRSGLNTEAFYVMTVGSKSFLVHREWFHDLALPWTPPSSTAWR NRELLMEFEEAHATKQSVVALGSQEGGLHQALAGAIVVEYSSSVKLTSGHLKCRLKMDKLALKGTT-315. This 315-amino acid fusion polypeptide, named M15EA, includes a 15-amino acid fragment (SEQ ID NO: 5, the underlined segment of SEQ ID NO: 3 shown above) of the carboxyl-terminal part of the JEV M protein and a 300-amino acid fragment EA (SEQ ID NO: 1, the non-underlined segment of SEQ ID NO: 3) of the amino-terminal part of the JEV E protein. One can also administer to a subject a first expression vector containing a nucleic acid encoding another fusion polypeptide M15EB to induce an immune response. The sequence of M15EB is shown below: 1-VFTILLLLVAPAYSDKLALKGTTYGMCTEKFSFAKNPADTGHGTVVIELSY SGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRG DKQINHHWYKAGSTLGKAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGKAVHQVFG GAFRT-175 (SEQ ID NO: 11). This fusion polypeptide contains the just-mentioned 15-amino acid fragment (underlined) and a fragment EB (SEQ ID NO: 2; the non-underlined segment of SEQ ID NO: 11), which contains a 160-amino acid carboxyl-terminal part of the JEV E protein.
In one example, the above-mentioned first expression vector is administered to prime a subject. In general, priming with a vaccine does not induce satisfactory immune response. However, a primed subject can generate strong and long-term immune response after being administered with another vaccine, i.e., boosted. Here, after priming with the first expression vector, the subject can be boosted with the polypeptide EA (SEQ ID NO: 1, shown above) or EB (SEQ ID NO: 2shown above). Alternatively, the subject is boosted with a second expression vector containing a nucleic acid encoding EA or EB. In one embodiment, the subject is primed with a first expression vector containing a nucleic acid encoding M15EA and boosted with the polypeptide EA. In another embodiment, the subject is primed with a first expression vector containing a nucleic acid encoding M15EB and boosted with EB.
In another example, a first expression vector encoding M15EA or M15EB is administered to boost a subject. Prior to the boosting, the subject is primed with EA or EB, or a second expression vector containing a nucleic acid encoding EA or EB. In one embodiment, the subject is primed and boosted respectively with EA and a first expression vector containing a nucleic acid encoding M15EA.
In yet another example, a subject is primed or boosted with a mixture of a first expression vector and a second expression vector containing nucleic acids encoding M15rEA and EB respectively.
Instead of an expression vector encoding M15EA or M15EB, the M15EA or M15EB polypeptide itself can also be administered to a subject to induce an immune response against JEV. The subject can be further administered with EA, EB, or an expression vector containing a nucleic acid encoding EA or EB.
The present invention also features a method of inducing an immune response in a subject against a JEV by administering the subject with an expression vector containing a nucleic acid encoding EB. Among the expression vector and the polypeptide EB, one of them can be used to prime and the other to boost the subject.
In another aspect, the present invention features an isolated polypeptide having the sequence of SEQ ID NO: 3 (e.g., M15EA) or SEQ ID NO: 11 (e.g., M15EB) and an expression vector containing a nucleic acid encoding such a polypeptide. In the expression vector, a nucleic acid encoding the polypeptide is operably linked to an expression control sequence containing a promoter and, optionally, additional elements such as enhancers. Such an expression vector can be used to transfect cells to thereby produce a polypeptide encoded by the nucleic acid.
In yet another aspect, the present invention features a vaccine for protecting a subject against JEV. The vaccine contains an expression vector having a nucleic acid that encodes a polypeptide of the sequence SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 11 and is operably linked to an expression control sequence, and a pharmaceutically acceptable carrier. The present invention also features a vaccine containing a polypeptide of the sequence of SEQ ID NO: 3 or SEQ ID NO: 11, and a pharmaceutically acceptable carrier.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.


DETAILED DESCRIPTION
The present invention relates to vaccines and methods to induce an immune response in a subject against a flavivirus, such as JEV.
A flavivirus has a single stranded positive sense RNA genome. For example, the genome of JEV encodes a polyprotein that is proteolytically cleaved into at least 10 proteins in infected cells. These proteins include three structural proteins: an envelope protein (E), a membrane protein (M or precursor M (PrM)), and a capsid protein, and at least seven nonstructural proteins NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5. The nucleotide sequence corresponding to JEV genorne bp 933–2447 (SEQ ID NO: 21) is shown below.









GTGGTATTCACCATCCTCCTGCTGTTGGTCGCTCCGGCTTATAGTTTCAACTGTCTGGGA
992



 


ATGGGCAATCGTGACTTCATAGAAGGAGCCAGTGGAGCCACTTGGGTGGACTTGGTGCTA
1052


 


GAAGGAGACAGCTGCTTGACAATCATGGCAAACGACAAACCAACATTGGACGTCCGCATG
1112


 


ATCAACATCGAAGCTAGCCAACTTGCTGAGGTCAGAAGTTACTGCTATCATGCTTCAGTC
1172


 


ACTGACATCTCGACGGTGGCTCGGTGCCCCACGACTGGAGAAGCCCACAACGAGAAGCGA
1232


 


GCTGATAGTAGCTATGTGTGCAAACAAGGCTTCACTGATCGTGGGTGGGGCAACGGATGT
1292


 


GGACTTTTCGGGAAGGGAAGTATTGACACATGTGCAAAATTCTCCTGCACCAGCAAAGCG
1352


 


ATTGGAAGAACAATCCAGTCAGAAAACATCAAATACGAAGTTGGCATTTTTGTGCATGGA
1412


 


ACCACCACTTCGGAAAACCATGGGAATTATTCAGCGCAAGTTGGGGCGTCCCAGGCGGCA
1472


 


AAGTTTACAGTAACACCTAATGCTCCTTCGATAACCCTCAAACTTGGTGACTACGGAGAA
1532


 


GTCACACTGGACTGTGAGCCAAGGAGTGGACTAAACACTGAAGCGTTTTACGTCATGACC
1592


 


GTGGGGTCAAAGTCATTTTTGGTCCATAGGGAATGGTTTCATGACCTCGCTCTCCCTTGG
1652


 


ACGCCCCCTTCGAGCACAGCGTGGAGAAACAGAGAACTCCTCATGGAATTTGAAGAGGCG
1712


 


CACGCCACAAAACAGTCCGTTGTTGCTCTTGGGTCACAGGAAGGAGGCCTCCATCAGGCG
1772


 


TTGGCAGGAGCCATCGTGGTGGAGTACTCAAGCTCAGTGAAGTTAACATCAGGCCACCTA
1832


 


AAATGCAGGCTGAAAATGGACAAACTGGCTCTGAAAGGCACAACCTATGGTATGTGCACA
1892


 


GAAAAATTCTCGTTCGCGAAAAATCCGGCGGACACTGGTCACGGAACAGTTGTCATTGAA
1952


 


CTTTCATACTCTGGGAGTGATGGCCCCTGCAAGATTCCGATTGTCTCCGTTGCTAGCCTC
2012


 


AATGACATGACCCCCGTCGGGCGGCTGGTGACGGTGAACCCCTTCGTCGCGACTTCCAGC
2072


 


GCCAACTCAAAGGTGCTGGTCGAGATGGAACCCCCCTTCGGAGACTCCTACATCGTAGTT
2132


 


GGAAGGGGAGACAAGCAGATTAACCACCATTGGTACAAGGCTGGAAGCACGCTGGGCAAA
2192


 


GCCTTTTCAACGACTTTGAAGGGAGCTCAAAGACTGGCAGCGTTGGGCGACACAGCCTGG
2252


 


GACTTTGGCTCTATTGGAGGGGTCTTCAACTCCATAGGGAAAGCTGTTCACCAAGTGTTT
2312


 


GGTGGTGCCTTCAGAACACTCTTTGGGGGAATGTCTTGGATCACACAAGGGCTAATGGGG
2372


 


GCCCTACTACTTTGGATGGGCATCAACGCACGAGACCGATCAATTGCTTTGGCCTTCTTA
2432


 


GCCACAGGAGGTGTGCTCGTGTTCTTAGCTACCAATGTGCATGCT
2477






The sequence bp 978–2477 (SEQ ID NO: 9) of the JEV genome encodes the full length E protein (SEQ ID NO: 4). The sequences bp 978–1877 (SEQ ID NO: 6) and bp 1851–2330 (SEQ ID NO: 7) encode EA and EB, respectively. Attempts have been made to used these two fragments expressed and prepared from E coli or mammalian cells to induce immune response in a subject against JEV. EA is not immunogenic as it is insoluble and does not properly fold when expressed in host cells. EB is a better immunogen than EA, but still cannot induce a satisfactory immune response in a subject.
The present invention discloses methods using EA or EB to induce satisfactory immune response against JEV. The methods are based in part on the unexpected features of a fusion polypeptide M15EA (SEQ ID NO: 3) encoded by bp 933–1877 of the JEV genome (SEQ ID NO: 8), and another fusion polypeptide M15EB (SEQ ID NO: 11) encoded by the fusion of bp 933–977 and bp 1851–2330 of the JEV genome (SEQ ID NO: 12). Both fusion polypeptides contain the carboxyl-terminal 15 amino acid fragment of the JEV M protein. This 15 amino acid fragment, encoded by bp 933–977 (SEQ ID NO: 10) of the JEV genome functions as a signal peptide, rendering the fusion polypeptides soluble. Also, as the fusion polypeptides can properly fold in host cells, they can be prepared by recombinant methods.
To prepare a fusion polypeptide described above, a nucleic acid encoding the polypeptide, e.g., SEQ ID NO: 8 or SEQ ID NO: 12, can be ligated into a recombinant expression vector, where the nucleic acid is operatively linked to a regulatory sequence suitable for a host cell for expressing the polypeptide. Examples of such a regulatory sequence include promoters, enhancers and other expression control elements that are known to those skilled in the art. Such an expression vector can be used to transfect host cells to thereby produce a protein or polypeptide of the invention. The recombinant expression vector of the invention can be designed for expression of polypeptides in prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, mammalian cells, or other suitable host cells. See Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. The expressed fusion polypeptide can be purified from corresponding host cells by methods well known in the art, including ammonium sulfate precipitation and fractionation column chromatography (e.g., ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.). The purified polypeptide can then be used in a vaccine to induce immune response in a subject against JEV in the manner described in Examples 1–4 below. The above-described expression vector can also be used to induce immune response in a subject against JEV.
The vaccine of this invention can contain pharmaceutically suitable carriers, diluents or excipient, such as sterile water, physiological saline, or the like. The vaccine can also contain adjuvant materials, such Freund's Complete Adjuvant (FCA) or Freund's Incomplete Adjuvant (ICA) to enhance an immune response.
To achieve a satisfactory immune response, the above-mentioned expression vectors or polypeptides are introduced into the subject at different times and, optionally, via different routes. Both of the expression vectors and the polypeptides can be used to prime or boost the immune response as described in Examples 2 and 3 below. The expression vectors can be introduced into the subject via intramuscular injection or the gene gun as described in Chen H W et al., J. Virol. 73: 10137–10145, 1999. The fusion polypeptide can be introduced into the subject in the manner described in Example 2 below. After introducing the expression vectors or polypeptides, the immune response in the subject can be evaluated following the procedures described in Example 4 below.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
EXAMPLE 1
1. Animals. Female C3H/HeN mice (6–7 weeks old) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). The care of the animals was provided in accordance with guidelines approved by the animal committee of the Institute of Biomedical Sciences, Academia Sinica.
2. Virus. The JEV strain Beijing-1 was maintained in suckling mouse brain and prepared as described in Chia S C et al., Microbial. Patho. 31: 9–19, 2001. To determine the 50% lethal dose (LD50), groups of mice (10–12 weeks old) were infected with a 10-fold serial dilution of JEV Beijing-1 followed by an intracerebral inoculation of 30 μl of phosphate buffered saline (PBS) (sham inoculation) to increase the susceptibility of infection of the central nervous system. A formalin-inactivated mouse-brain-derived JEV reference vaccine (Beijing strain, Tanabe Seiyaku Co., Osaka, Japan) was used as a positive control antigen. The LD50 dose for C3H/HeN mice was found to be 1×104 pfu/mouse.
3. Plasmids for DNA immunizations. Fragments of the gene encoding the E protein of JEV Beijing-1 were prepared from the plasmid p15ME (Chen H W et al., J. Virol. 73: 10137–10145, 1999) by PCR. This plasmid contains a cDNA segment encoding the full-length E protein and the carboxyl-terminal 15 amino acid of the membrane protein. This cDNA segment corresponds to nucleotide bases 933–2477 of the JEV genome and was constructed into the pcDNA3 vector (Invitrogen, San Diego). The double-stranded subfragments of JEV E gene were obtained by PCR using appropriate primer sets shown below:










M15EA upstream:
5′-CGCGGATCCAAATGGTGGTATTCACCATC-3′
(SEQ ID NO: 13)



 


M15EA downstream:
5′-TTTTCTAGAGCTTAGGTTGTGCCTTTCAG-3′
(SEQ ID NO: 14)


 


EA upstream:
5′-CGCGGATCCAAATGTTCAACTGTCTGGGA-3′
(SEQ ID NO: 15)


 


EA downstream:
5′-TTTTCTAGAGCTTAGGTTGTGCCTTTCAG-3′
(SEQ ID NO: 16)


 


M15EB upstream:
5′-CGCGGATCCAAATGGTGGTATTCACCATC-3′
(SEQ ID NO: 17)


 


M15EB downstream:
5′-TTTTCTAGACGTTATGTTCTGAAGGCACC-3′
(SEQ ID NO: 18)


 


EB upstream:
5′-GCTGCATATGGACAAACTGGCTCTG-3′
(SEQ ID NO: 19)


 


EB downstream:
5′-TTTTCTAGACGTTATGTTCTGAAGGCACC-3′
(SEQ ID NO: 20)






The upstream primers contain a BamHI site and an ATG start codon, and the downstream primer contains a stop codon and an EcoRI site to facilitate cloning. The PCR products were ligated into a PCR-Blunt vector (Invitrogen, San Diego) for enzyme mapping and sequencing. The identified plasmids were then digested with appropriate restriction enzymes to release DNA fragments containing bp 933–1877, 978–1877, and 1851–2330 of the JEV genome. The first fragment encodes a 15 amino acid signal peptide preceding the E protein and the amino-terminal part of the E protein (EA). The latter two fragments encode EA and EB protein fragments and make up 90% of the E protein, excluding the transmembrane domain and cytoplasmic tail (49 amino acids). These fragments were inserted into the pcDNA3 vector to produce pM15EA, pM15EB, pEA, and pEB, respectively. M15 indicates the nucleic acid 933 to 978 encoding the 15-amino acid signal peptide. These eukaryotic expression vectors contain the cytomegalovirus early promoter-enhancer sequence and the polyadenylation and the 3′-splicing signals from bovine growth hormone. Plasmid DNA was purified from transformed Escherichia coli DH5α by Qiagen Plasmid Giga Kits in accordance with the manufacturer's instructions and stored at −70° C. as pellets. The DNA was reconstituted in sterile saline at a concentration of 1 mg/ml before use.
4. Cloning and expression of JEV E protein fragment in E. coli. The cloning and expressing of EA and EB in E. coli were conducted in the same manner as described in Chia S C et al., Microbial. Patho. 31: 9–19, 2001. Briefly, the PCR products containing DNA encoding EA (bp 978–1877) and EB (bp 1851–2330) were inserted into the pET-28A vector (Novagen, Madison, Wis.) to obtain pET-EA and pET-EB. These vectors were transformed into E. coli JM109/DE3 respectively. The expressions of the proteins in the transformed E. coli were induced by culturing the E. coli in the presence of isopropyl thio-β-D-galactopyranoside (IPTG) for 6 hours at 30° C. The expressed proteins were purified by Nickel columns, and their amino acid sequences confirmed by the MALDI-TOF (Matrix assisted laser desorption—time of flight, Applied Biosystems, Foster City, Calif.) analysis. The recombinant proteins thus prepared were named as rEA and rEB to differentiate from plasmids pEA and pEB.
5. Immunization. The above-mentioned female C3H/HeN mice were immunized intramuscularly (im) with the purified proteins rEA or rEB according to the method described in Chia S C et al., Microbial. Patho. 31: 9–19, 2001. The mice were divided into 6 groups. Mice in Groups 1–3 were injected into each quadriceps muscle with rEA at a dose of 25 μg/injection for once, twice, and three times, respectively. Mice in Groups 4–6 were injected rEB in a similar manner. For the first injections and the following injections, the polypeptides were mixed with CFA and IFA, respectively.
Two weeks after the last injection, the immunized mice were challenged with lethal doses of JEV according to the method described in Chia S C et al., Microbial. Patho. 31: 9–19, 2001. Briefly, each of the mice was challenged intraperitonealy (ip) with JEV Beijing-I at a dose of 50 times of the LD50. The JEV-challenged mice were observed daily for symptoms of viral encephalitis as described in McMinn P J. of general virol. 78:2711–2722, 1991 for 30 days. The percentage of mice that survived in each group (i.e., the survival rate or level of protection) was determined.
The results indicated that the injection of rEB for once, twice, and three times led to 20%, 27%, 58% levels of protection, respectively. The injection of rEA, on the other hand, showed no protection even after three injections. These results were comparable to those described in Chia S C et al., Microbial. Patho. 31: 9–19, 2001, suggesting that rEA, an insoluble protein, is less antigenic and less immunogenic, while rEB, a soluble protein, is antigenic and immunogenic.
Female C3H/HeN mice were also immunized im with the plasmids: pcDNA3, pM15EA, pEA, or pEB One week before the immunization, each mouse was injected with 200 μl of 10 μM cardiotoxin (Sigma, St. Louis) in the quadricep muscles on two of its legs (100 ul each). These mice were divided into 12 groups (Groups 1–12), anesthetized, and given different immunization regimens as indicated below in Table 1, column 2. The mice in Groups 1 and 2 were injected once with a control vaccine (Tanabe Seiyaku Co., Osaka, Japan) and saline at a dose of 100 ul/injection, respectively. The mice in Groups 3, 4, and 9 were injected once, respectively, with pcDNA3, pEA, and pEB at a dose of 50 μg/injection. The mice in Groups 5–8 and 10–11 were injected, respectively, with pcDNA3, pM15EA, pEA, and pEB at a dose of 50 μg/injection for twice or three times at 2 week-intervals. The mice in Group 12 were injected with a mixture of p15MEA and pEB at a dose of 50 μg/injection for three times at 2 week-intervals.
Two weeks after the last injection, the mice in each group were challenged with lethal doses of JEV and the survival rates determined in the same manner described above. The results were summarized in Table 1.







TABLE 1







Effects of Immunization by vectors containing JEV envelope (E) gene











Immunization
Number of mice surviving




regimens (number
after a JEV challenge/
Survival


Group No.
of injection(s))
Number of mice challenged
rate (%)













1
Control Vaccine
24/24
100.00**


2
Saline
 4/20
20.00


3
PcDNA3
 5/25
20.00


4
pEA (1)
 2/10
20.00


5
pEA (2)
 3/11
27.27


6
pEA (3)
 5/25
20.00


7
pM15EA (2)
 3/10
30.00


8
pM15EA (3)
13/24
54.17*


9
pEB (1)
 2/10
20.00


10
pEB (2)
 2/9
22.22


11
pEB (3)
12/30
40.00*


12
pM15EA + pEB (3)
14/19
73.68**





*p < 0.01,


**p < 0.001 by Fisher's exact test compared to value for Group 2.






As shown in Table 1, the injections of pEA for once, twice, and three times (Groups 4, 5, and 6) led to 20%, 27.27% and 30% levels of protection, which are not significantly different from that by the injection of pcDNA3. In contrast, the injection of pM15EA (3 times) led to a 54% level of protection (Group 8), which is significantly greater than that by the injection of pcDNA3. Since pM15EA encodes a polypeptide containing a 15-amino acid signal peptide (rM15EA) while pEA encodes a polypeptide without the signal peptide, these results suggest the importance of this signal peptide for rM15EA to be immunogenic.
Also as shown in Table 1, the injection of pEB protected mice from the lethal JEV challenge only after three injections (Group 11). Interestingly, after being immunized with pM15EA and pEB, most (73.6%) of the mice in Group 12 were protected against the lethal virus challenge.
EXAMPLE 2
Mice were immunized by a DNA priming-protein boosting immunization protocol, and challenged with lethal doses of JEV.
Mice were pretreated with cardiotoxin in the same manner described above in Example 1. One week later, the mice were divided into 10 groups. The mice in Groups 1, 2, and 3 were injected with a control vaccine, pcDNAs, and saline, respectively in the same manner described above in Example 1. The mice in Groups 4 and 7 were respectively primed with pM15EA and pEB twice at a dose of 50 μg/injection. The mice in Groups 5 and 6 were primed with pM15EA (50 μg/injection) twice, and two weeks later, boosted with rEA (25 μg/injection) once and twice, respectively. The mice in Groups 8 and 9 were primed with pEB (50 μg/injection) twice, and boosted with rEB (25 μg/injection) once and twice, respectively. Finally, the mice in Group 10 were primed with pM15EB twice (50 μg/injection) and boosted with rEB twice (25 μg/injection).
Two weeks after the last injection, the mice in each group were challenged with lethal doses of JEV and the survival rates determined in the same manner described above in Example 1. The results were summarized in Table 2.







TABLE 2







Effects of Immunization by DNA priming - protein boosting












Number of mice surviving




Immunization
after a virus


Group
regimens (number of
challenge/Number of mice
Survival


No.
injection(s))
challenged
rate (%)













1
Vaccine
10/10
100.00**


2
pcDNA3
 4/13
30.70


3
Saline
2/8
25.00


4
pM15EA (2)
 3/12
30.00


5
pM15EA (2) + rEA (1)
 5/10
50.00*


6
pM15EA (2) + rEA (2)
 9/10
90.00**


7
pEB (2)
2/9
22.20


8
pEB (2) + rEB (1)
 4/10
40.00*


9
pEB (2) + rEB (2)
13/18
72.20**


10
pM15EB (2) + rEB (2)
18/18
100.00**





*p < 0.01,


**p < 0.001 by Fisher's exact test compared to value of Group 2.






As shown in Table 2, the injections (twice) of pM15EA or pEB alone provided no protection against the JEV challenge. Unexpectedly, priming with pM15EA twice followed by one or two boostings with non-immunogenic rEA (Groups 5 and 6) led to levels of protection (50% and 90%) significant higher than that by pcDNA3 or saline. The level of protection in Group 6 was almost equal to that the control vaccine. These results indicate that, if the priming is sufficient, the non-immunogenic rEA can serve as an effective immunogen.
Priming with pEB twice followed by one or two boostings with rEB (Groups 8 and 9) led to significant levels of protection (40% and 72.2%). These levels of protection are much higher than those resulted from injection of pEB (twice) or rEB (twice, see Table 1) alone, indicating DNA priming-protein boosting regimen is more effective DNA only or protein only regimen. Finally, it is unexpected that all mice were protected against from JEV if they had been primed twice with pM15EB and boosted twice with rEB (Group 10). These results suggest that the M15 signal peptide could increase the immunogenicity of EB.
EXAMPLE 3
Mice were immunized by a protein priming-DNA boosting immunization protocol, and challenged with lethal doses of JEV.
Mice were divided into 6 groups. Those in Groups 1, 2, and 3 were injected once with a control vaccine, pcDNAs, and saline, respectively, in the same manner described above in Example 1. The mice in Groups 4, 5, and 6 were primed by intramuscular injection of 25 μg rEA or rEB protein (prepared in CFA), and, two weeks later, were primed again by 25 μg rEA or rEB protein prepared in IFA. One week later, the mice in these three groups were treated with 100 μl of 10 μM cardiotoxin in each quadricep muscle. One and three weeks later, the mice in these three groups were, respectively, boosted with pM15EA, pEA, and pEB at a dose of 25 μg/boosting. The mice in each group were challenged with lethal doses of JEV and the survival rates determined in the same manner described above in Example 1. Two independent experiments were performed for each group. The results were summarized in Table 3.







TABLE 3







Effects of Immunization by Protein priming - DNA boosting.











Immunization
Number of mice surviving



Group
regimens (number of
after a virus challenge/
Survival


No.
injection(s))
Number of mice challenged
rate (%)













1
Vaccine
9/10
90.00**


2
Saline
2/10
20.00


3
PcDNA3
2/10
20.00


4
rEA (2) + pM15EA (2)
8/8 
100.00**


5
rEA (2) + pEA (2)
1/8 
12.50


6
rEB (2) + pEB (2)
5/7 
71.43**





*p < 0.01,


**p < 0.001 by Fisher's exact test compared to value of Group 2.






As shown in Table 3, 100% and 71.43% of the mice in Groups 4 and 5 were protected from the lethal JEV challenges. These levels of protection were highly significant and comparable to those in the groups of mice that were DNA-primed and protein-boosted as described above in Example 1 above (see Table 2, Groups 6 and 9). In contrast, only 12.5% of the mice in Group 5 were protected, indicating the importance of the 15-amino acid signal peptide in the immunogenic activity of M15EA.
Examples 2 and 3 described above have indicated unexpected results: rEA by itself is non-immunogenic or non-protective, but it acts as an excellent immunogen in either priming or boosting. One reason for these results is that rEA contains certain minor epitopes. Although rEA is not immunogenic by itself, it can induce a full-fledge secondary immune response if a priming or the boosting agent is immunogenic (e. g., pM15EA).
EXAMPLE 4
Survived mice from the groups described above in Examples 1–3 were tested for the presence anti-JEV E protein antibodies according to the ELISA method described in Chia et al., Microbial. Patho. 31: 9–19, 2001.
It is known that different immunization approaches (e.g., different antigen delivery routes) can affect the antibody isotypes and T helper cell types in an immune response. For example, intramuscular injection of DNA generates almost exclusively IgG2a antibody due to Th1-cell activation. In contrast, gene gun delivery of plasmid DNA mostly leads IgG1 antibody production, which is enhanced by Th2-cell activation. Further, Th2-cell activation suppresses IgG2a production.
To test the presence of anti-JEV E protein antibodies, serum samples were collected from 4–5 mice in each group subjected to above-mentioned immunization regimens at 2 days before (prechallenge) and 2 weeks after (postchallenge) the virus challenges. Each sample was added to a well on a microtiter plate that was coated with JEV prepared from tissue culture. Anti-JEV E protein antibodies in the sample would bind to the JEV.
To detect IgG in the sample, the plate was then incubated with an HRP-conjugated goat anti-mouse IgG Fc (1:1000, ICN/Cappel, Aurora, Ohio). After color was developed by incubating with ABTS, the absorbance at 405 nm was measured on an ELISA reader. The resultant reading indicated the level of IgG in the sample.
To detect IgG1 or IgG2a isotype in the sample, the plate were incubated with biotin-conjugated rat anti-mouse IgG1 (1:1000; PharMingen, San Diego, Calif.) or rat anti-mouse IgG2a (1:1000; PharMingen). The results were visualized by Avidin-HRP (1:2000; PharMingen) and ABTS, and the absorbance at 405 nm measured in the same manner as described above.
The results indicated that, in general, the OD405 readings of IgG2a were greater than those of IgG1 irrespective of the immunization regimens. At the prechallenge stage, the IgG2a and IgG1 OD405 levels did not vary very much from group to group except in the samples from mice subjected to the DNA priming—protein boosting or protein priming—DNA boosting regimen. In such mice, the IgG2a levels were significantly higher than those of IgG1. At the postchallenge stage, the IgG2a level in each group increased significantly, while the IgG1 level only increased slightly. Also, the level of IgG2a was much greater than those of IgG1 (p ranging from <0.01 to <0.001). Further, EA antigen tended to lead to higher IgG2a level than any other antigens, while pcDNA3 led to low levels of both IgG1 and IgG2a.
To examine the ability of antisera collected from immunized mice to neutralize JEV virus, a plaque reduction neutralization test (PRNT) was conducted in the same manner as the “50% plaque reduction assay with BHK-21 cells” described in Johnson M P et al., p. 189–192. In H. Ginsberg, F. Brown, R. A. Lerner, and R. M. Chanock (ed.), Vaccines 88. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Serum samples were collected from the mice in the groups described above in Example 1–3 at 2 days before (prechallenge) and 2 weeks after (postchallenge) the virus challenges. The samples were serially diluted in 5% BCS-PBS and incubated at 56° C. for 30 minutes to inactivate complement. Each sample was then mixed with an equal volume of minimum essential medium (MEM)-BCS containing JEV to yield a mixture containing approximately 1000 pfu of virus/ml. The mixture was incubated at 4° C. for 18–21 hours and then added to a well in a six-well plate (in triplicates) containing confluent monolayers of BHK-21 cells. The plate was then incubated at 37° C. for 1 hour and gently rocked every 15 minutes. The wells of the plate were then overlaid with 2 ml of 1% methyl cellulose prepared in MEM supplemented with 5% BCS and incubated at 37° C. in 5% CO2 for 4 days.
After plaques appeared in the wells, they were stained with naphthol blue black and counted under a microscope. The neutralization antibody titer was calculated as the reciprocal of the highest dilution resulting in 50% reduction of plaques as compared to that of a control JEV virus in the absence of any antiserum. At least three serum samples from each group were determined for the PRNT titer. Results were shown in Table 4.







TABLE 4







Effects of immunizations on neutralizing antibody titres and


on survival rates













Survival



Immunization
Plaque neutralization titers
rate











Groups
regimens
Prechallenge
Postchallenge
(%)
















1
Vaccine
 1:80
>1:1280
100% 



2
Saline
<1:5 
1:40 
20%



3
pcDNA3
1:5
1:40 
20%



4
pM15EA
1:5
1:160
54%



5
pEA
<1:5 
1:40 
20%



6
pEB
1:5
1:40 
40%



7
pM15EA + pEB
1:5
1:640
74%



8
pM15EA + rEA
 1:40
1:640
90%



9
pEB + rEB
 1:10
1:640
72%



10
rEA + pEA
<1:5 
1:40 
13%



11
rEA + pM15EA
 1:20
1:640
100% 



12
rEB + pEB
1:5
1:320
71%







Numbers underlined: only one determination was done due to limited amount of serum available.






As shown in Table 4, at the prechallenge stage, the mice in most of the groups had low PRNT titers raging from 1:5 to 1:10, except those had been immunized with a control vaccine (Group 1), pM15EA +rEA (Group 8), and rEA +pM15EA (Group 11). The mice in these 3 groups had PRNT titers ranging from 1:20 to 1:80. At the postchallenge stage, the PRNT titers of mice in all groups increased considerably and correlated positively with the survival rate of mice in respective groups. In general, it appeared that a PRNT titer of 1:40 was enough to protect against the virus infection.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

## Claims

1. A method of inducing an immune response in a subject against a flavivirus, the method comprising administering to the subject a fusion polypeptide including a signal peptide and a part of an envelope protein of the flavivirus, or an expression vector containing a nucleic acid encoding the fusion polypeptide, wherein the signal peptide contains SEQ ID NO: 5 and SEQ ID NO: 5 is located at the amino terminus of the fusion polypeptide.

2. The method of claim 1, wherein the flavivirus is a Japanese encephalitis virus.

3. The method of claim 2, wherein the part of the envelope protein is an amino terminal part or a carboxyl terminal part of the envelope protein.

4. The method of claim 4, wherein the subject is administered a first expression vector containing a nucleic acid encoding a fusion polypeptide of the sequence SEQ ID NO: 3 or 11.

5. The method of claim 5, wherein the first expression vector is administered to prime the subject.

6. The method of claim 5, wherein, after performing the administering step, the subject is boosted by administering a polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 2, or a second expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

7. The method of claim 6, wherein the subject is primed with the first expression vector containing the nucleic acid encoding the fusion polypeptide of the sequence of SEQ ID NO: 3 and boosted with the polypeptide of the sequence SEQ ID NO: 1.

8. The method of claim 6, wherein the subject is primed with the first expression vector containing the nucleic acid encoding the fusion polypeptide of the sequence of SEQ ID NO: 11 and boosted with the polypeptide of the sequence SEQ ID NO: 2.

9. The method of claim 4, wherein first expression vector is administered to boost the subject.

10. The method of claim 9, wherein, before performing the administering step, the subject is primed by administering a polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 2 or a second expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 1 or SEQ ID NO: 2.

11. The method of claim 10, wherein the subject is primed with the polypeptide of the sequence SEQ ID NO: 1 and is boosted with the first expression vector containing the nucleic acid encoding the fusion polypeptide of the sequence of SEQ ID NO: 3.

12. The method of claim 4, wherein the subject is further administered a second expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 2.

13. The method of claim 3, wherein the subject is administered a fusion polypeptide of the sequence SEQ ID NO: 3 or 11.

14. The method of claim 13, wherein the subject is further administered a polypeptide of the sequence SEQ ID NO: 1 or an expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 1.

15. The method of claim 13, wherein the subject is further administered a polypeptide of the sequence SEQ ID NO: 2 or an expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 2.

16. The method of claim 1, wherein the flavivirus is a Dengue virus, a tick borne encephalitis virus, or a West Nile encephalitis virus.

17. The method of claim 1, wherein the part of the envelope protein is an amino terminal part or a carboxyl terminal part of the envelope protein.

18. A method of inducing an immune response in a subject against a Japanese encephalitis virus, the method comprising administering to the subject an expression vector containing a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 2.

19. The method of claim 18, wherein the expression vector and a polypeptide having the seguence as set forth in SEQ ID NO: 2 are administered to prime and boost the subject, respectively.

20. The method of claim 18, wherein a polypeptide having the sequence as set forth in SEQ ID NO: 2 and the expression vector are administered to prime and boost the subject, respectively.

21. An isolated polypeptide comprising the sequence SEQ ID NO: 3 or 11.

22. An expression vector comprising a nucleic acid encoding a polypeptide of the sequence SEQ ID NO: 3 or 11, wherein the nucleic acid is operably linked to an expression control sequence.

23. An immunogenic agent comprising an expression vector containing a nucleic acid that encodes a polypeptide of the sequence SEQ ID NO: 2, 3, or 11 and is operably linked to an expression control sequence, and a pharmaceutically acceptable carrier.

24. An immunogenic agent comprising a polypeptide of the sequence of SEQ ID NO: 3 or 11, and a pharmaceutically acceptable carrier.

25. An immunogenic agent comprising a nucleic acid that encodes a polypeptide of SEQ ID NO: 2 or 3, and a pharmaceutically acceptable carrier.