MULTIPLE MOSQUITO-BORNE FLAVIVIRUS VACCINE AND USE THEREOF IN INDUCING NEUTRALIZING ANTIBODIES

Abstract

The present invention relates to a peptide immunogen, comprising at least one copy of amino acid sequence RCPTTGE (called JBP). The peptide immunogen of present invention can induce an effective immune response to two or more mosquito-borne flavivirus. The present invention also provides mono/multivalent virus vaccines to protect animal against multiple mosquito-borne flavivirus infection, including diseases caused by Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV) infections.

Claims

1. A peptide immunogen, comprising at least one copy of amino acid sequence RCPTTGE (SEQ ID No.1), wherein the peptide induces an antibody in response to two or more mosquito-borne flavivirus.

2. The peptide immunogen of claim 1, wherein the peptide induces neutralizing antibodies against multiple mosquito-borne flavivirus in immunized animal.

3. The peptide immunogen of claim 2, wherein the mosquito-borne flavivirus comprises at least one flavivirus selected from Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV).

4. An isolated nucleotide sequence, coding for the peptide immunogen of claim 1.

5. A multiple mosquito-borne flavivirus vaccine, comprising the peptide immunogen of claim 1.

6. The multiple mosquito-borne flavivirus vaccine of claim 5, which induces an effective neutralizing antibody response to at least one flavivirus selected from Japanese encephalitis virus (JEV), West Nile virus (WNV), Dengue Virus (DENV), and Zika virus (ZIKV).

7. The multiple mosquito-borne flavivirus vaccine of claim 5, which induces protecting antibodies against JEV, DENVs and ZIKV.

8. A method for protecting animal against mosquito-borne flavivirus infection, which comprises immunizing the host in need with the peptide immunogen of claim 1.

9. The method of claim 8, the peptide immunogen comprises an amino acid sequence of RCPTTGERCPTTGERCPTTGERCPTTGERCPTTGE (SEQ ID No.2).

10. A method for controlling mosquito-borne flavivirus infection, which comprises administration of antibodies induced by the peptide immunogen of claim 1.

Description

BREIF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 demonstrates the conserved region between residues 73 to 79 in E protein domain II.

[0017] FIG. 2. shows the effects of the JBP in neutralizing antibody response against JEV, DENVs and ZIKV.

[0018] FIG. 3. shows the protective effect of JBP induced antibodies against JEV challenge.

[0019] FIG. 4. shows the protective effect of JBP induced antibodies against DENV-1 and DENV-2 challenge.

[0020] FIG. 5. illustrates the inhibition of viremia after challenge with DENV-4 and ZIKV.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Other features and advantages of the present invention will be further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

[0022] As used herein, the term animal has its ordinary meaning and includes, but not limited to, a bird, a fish and a mammal. In some embodiments, the mammal comprises, but not limited to, dogs, cats. horses, sheep, rodents and primates, including human.

EXAMPLE 1

Production of Neutralizing Antibodies Against Multiple Tick-Borne Flaviviruses After JBP Vaccination

[0023] The neutralizing capacity of the antibodies induced by JBP vaccination is evaluated. BALB/c mice are divided into three groups (6 mice per group) and immunized with 30 g 1 JBP, 5 JBP or PBS by the subcutaneous injection respectively, and then all the mice are boosted two times with a two-week interval during immunization. Sera from individual BALB/c mice were collected at the sixth week after first vaccination. Sera were pooled and serially diluted from 1:8 to 1:512 with PBS. The neutralizing titer of the immunized mice sera against JEV, four serotypes of DENVs, and ZIKV is assessed by focus reduction neutralization tests (FRNT.sub.50).

[0024] The results indicate that 5 JBP-immunized sera exhibits higher FRNT.sub.50 titers ranging from 64 to 256 and 1 JBP-immunized sera shows moderate FRNT.sub.50 titers ranging from 32 to 128, and both are significantly higher than those of the control PBS-immunized sera (FIG. 2), which indicates that JBP can induce the neutralizing antibodies against JEV, DENVs and ZIKV.

EXAMPLE 2

Induction of Prospective Antibodies Against Japanese Encephalitis Virus and Dengue Virus in Animal Model

[0025] The protective effect of 1 and 5 JBP-immunized sera against JEV is examined. The IgG purified from pre-immunized sera, 1 or 5 JBP-immunized sera is respectively adoptive transferred to ICR mice and the mice are challenged with 110.sup.5 PFU of JEV by the intraperitoneal plus intracardiac route on day 1 post transfer.

[0026] As showed in FIG. 3, the 1 and 5 JBP-immunized sera IgG-transferred mice exhibit the higher survival rates (37.5%) after 30 days post infection; however, all the pre-immune sera IgG-transferred mice are dead within 12 days post infection. The results suggest that JBP vaccination can induce the production of the IgG with the protective effect against JEV challenge.

[0027] Furthermore, the protective effect of JBP-immunized sera against JEV is examined as well. Six-week-old AG129 mice are intraperitoneally (intraperitoneal injection) transferred with 50 g , 5 JBP-immunized sera IgG or 50 g pre-immune sera IgG as a control. The IgG transferred mice are challenged with 2.6510.sup.8 FFU of DENV-2 or 110.sup.7 FFU of DENV-1 by intraperitoneal injection on day 1 post transfer. The mice are monitored for mortality for 60 days.

[0028] Likewise, mice transferred with 1 or 5 JBP-immunized sera IgG possess complete protective effect against DENV-2 challenge (100% survival rate). In contrast, mice transferred with pre-immunized sera IgG have lower survival rate (FIG. 4A). On the other hand, the mice transferred with 5 JBP-immunized sera IgG also can defense DENV-1 infection with 50% survival rate when the mice of control group are all dead (FIG. 4B). Overall, the results indicate that 5 JBP-immunized sera IgG provide the protective effect against DENV-1 and DENV-2 challenge.

EXAMPLE 3

The inhibition of Type IV Dengue Virus and Zika Virus Viremia by JBP-Induced Antibodies

[0029] The protective effect against DENV-4 and ZIKV of the sera IgG induced by 5 JBP immunization is examined. Six-week-old AG129 mice are transferred with 1 g, 10 g, or 50 g 5 JBP-immunized sera IgG or 50 g pre-immunized sera IgG by intraperitoneal injection. The IgG transferred mice are challenged with 210.sup.7FFU of DENV-4 or 10 FFU ZIKV by the intraperitoneal route on day 1 post transfer. The survival rates of these mice were monitored daily. Viral load in the sera after DENV-4 or ZIKV challenge is determined by focus forming assay on day 3 post challenge.

[0030] As shown in FIG. 5, comparing with the mice treated with 50 g pre-immunized sera IgG, the viremia levels of the mice treated with 5 JBP-immunized sera IgG are significantly decreased after DENV-4 or ZIKV challenge (FIGS. 5A and 5C). The inhibition effect of 5 JBP-immunized sera IgG is dose-dependent; however, the dose of 10 g and 50 g 5 JBP-immunized sera IgG shows the similar inhibition effect. Therefore, the dose of 5 JBP-immunized sera IgG in further survival test is chosen as 10 g 5 JBP-immunized sera IgG.

[0031] In the further survival test, no mice survive in any group after DENV-4 or ZIKV challenge, but the mice treated with 10 g 5 JBP-immunized sera IgG live longer than the mice treated with 10 g pre-immunized sera IgG (FIGS. 5B and 5D). The results indicate that 5 JBP immunization induce the production of protective antibodies against DENV-4 and ZIKV challenge to reduce viremia levels.

[0032] In summary, the present invention first discloses an amino acid sequence conserved in the E protein domain II of mosquito-borne flavivirus, and designs a neutralizing epitope RCPTTGE (named JBP) to be developed as the efficacious multivalent mosquito-borne flaviviruses vaccine and as the efficacious antigen to product anti-virus antibody.