BIVALENT DENGUE/HEPATITIS B VACCINES

Abstract

The present invention relates to the construction of, and immunization with viral vaccines. In particular, bivalent vaccines that are capable of providing simultaneous virus infection protection for two or more different viruses. Furthermore, the bivalent vaccines contemplated herein are contemplated as being effective in a neonatal mammal. One such bivalent viral vaccine comprises two antigenic epitopes against the dengue viruses and at least one antigenic epitope against hepatitis B virus. Immunization cross-reactivity may also provide infection protection against other viruses as well.

Claims

1. A bivalent vaccine peptide comprising a first dengue antigenic epitope, a spacer peptide and a second dengue antigenic epitope, wherein said spacer peptide is attached to said first dengue antigenic epitope and said second dengue antigenic epitope.

2. The bivalent vaccine peptide of claim 1, wherein said spacer peptide comprises a plurality of glycine residues.

3. The bivalent vaccine peptide of claim 2, wherein said plurality of glycine residues comprises at least six glycine residues.

4. The bivalent vaccine peptide of claim 1, further comprising a crosslinker.

5. The bivalent vaccine peptide of claim 4, further comprising a hepatitis B virus epitope conjugated to said second dengue antigenic epitope with said crosslinker.

6. The bivalent vaccine peptide of claim 4, further comprising a hepatitis B virus epitope attached to said first dengue antigenic epitope with said crosslinker.

7. The bivalent vaccine peptide of claim 4, further comprising a first hepatitis B virus epitope conjugated to said first dengue antigenic epitope and a second hepatitis B virus epitope conjugated to said second dengue antigenic epitope with said crosslinker.

8. The bivalent vaccine peptide of claim 1, wherein said first dengue antigenic epitope is SEQ ID NO:1.

9. The bivalent vaccine peptide of claim 1, wherein said second dengue antigenic epitope is SEQ ID NO:2.

10. The bivalent vaccine peptide of claim 1, wherein said hepatitis B virus epitope is rHepBsAg.

11. The bivalent vaccine peptide of claim 1, wherein said bivalent vaccine peptide is SEQ ID NO:4.

12. The bivalent vaccine peptide of claim 4, wherein said crosslinker comprises an amine reactive group.

13. The bivalent vaccine peptide of claim 4, wherein said crosslinker comprises a carboxyl reactive group.

14. The bivalent vaccine peptide of claim 4, wherein crosslinker comprises a heterobifunctional crosslinker.

15. The bivalent vaccine peptide of claim 14, wherein said heterobifunctional crosslinker comprises an amine reactive group and a carboxyl reactive group.

16-36. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0047] FIG. 1 presents exemplary illustrations of predicted 3D structures (models 1-5) of the peptide GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:3). (bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/).

[0048] FIG. 2 presents exemplary illustrations of experimental structures of

[0049] FIG. 2A: DENV2

[0050] FIG. 2B: DENV3 envelope proteins. (cgLucsfedu/chimera).

[0051] FIG. 3 presents one embodiment of a method for the mouse vaccination.

[0052] FIG. 4 presents exemplary data of serum anti-DENVs 1-4 neutralizing antibody curves in female Balb/c mice vaccinated with a bivalent dengue vaccine peptide (e.g., rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE; SEQ ID NO:4) using a protocol shown above in FIG. 3. Anti-DENV's 1-4 antibody neutralization assays were performed utilizing U937/DC-SIGN cells. de Alwis et al., “Measuring antibody neutralization of dengue virus (DENV) using a flow cytometry-based technique” Methods Mol Biol. (2014) 1138:27-39. Data points for each mouse were performed in duplicate. Sigmoid neutralization curves were fit utilizing SigmaPlot version 11.0.

[0053] FIG. 4A: DENV1 West Pacific 74

[0054] FIG. 4B: DENV216681

[0055] FIG. 4C: DENV3 H87

[0056] FIG. 4D: DENV4 TVP360.

[0057] FIG. 5 presents exemplary data showing serum anti-HepBsAg lgG levels in female Balb/c mice vaccinated with the bivalent dengue vaccine peptide (e.g., rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE; SEQ ID NO:4), using an immunization protocol shown in FIG. 3. Anti-HepBsAg lgG was measured by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates to the construction of, and immunization of mammals with, viral vaccines. In particular, bivalent vaccines that are capable of providing simultaneous virus infection protection for two or more different viruses simultaneously. Furthermore, the bivalent vaccines contemplated herein are contemplated as being effective in a neonatal mammal. One such bivalent viral vaccine comprises two antigenic epitopes against the dengue viruses and at least one antigenic epitope against the hepatitis B virus. Immunization cross-reactivity may also provide infection protection against other viruses as well.

[0059] In one embodiment, the present invention contemplates a method comprising immunizing a mammal with a 23mer synthetic peptide that contains two different conserved DENV envelope protein fusion epitopes recognized by broadly neutralizing human monoclonal antibodies. Although hapten conjugation to a hepatitis B vaccine has been previously studied in order to improve the immunogenicity of some infectious disease vaccines, this approach has not been suggested or considered in regards to a dengue vaccine. A potential long-term advantage of this proposed strategy is that a single vaccine product could provide protection against hepatitis B virus infection and dengue virus infection simultaneously. Furthermore, a vaccination series could begin at birth, providing protection against dengue virus and hepatitis virus infections starting in infancy (e.g., immunization of a neonatal mammal). This type of vaccine and immunization protocol provides significant advantages over the dengue vaccine products currently licensed or in pre-clinical and clinical trials.

I. Dengue

[0060] DENV infections produce a wide spectrum of clinical illnesses. Symptoms range from from: i) being asymptomatic; ii) an expression of a mild illness; iii) an exhibition of classic dengue fever (DF); or iv) a severe and potentially life threatening disease of dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS) (severe dengue).

[0061] Protective immunity to the homotypic DENV serotype is life-long; however, serial heterologous DENV infections can occur and are a risk factor for developing severe dengue. Developing a DENY vaccine is, therefore, a preventive goal for effective disease control. In order to prevent serial infections with more than one DENV serotype, vaccination strategies have been examining products directed against all 4 DENV serotypes simultaneously. A tetravalent chimeric dengue vaccine, where the envelope proteins of DENVs 1-4 have been inserted into a recombinant yellow fever (YF)-17D backbone, has been developed by Sanofi Pasteur and is licensed in a few countries. However, the results of Phase IIb and Phase III clinical trials of this vaccine presented a mixed picture. Capeding et al., “Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial” Lancet (2014) Epub 2014/07/16; Villar et al., “Efficacy of a tetravalent dengue vaccine in children in Latin America” N Engl 1 Med. (2015) 372(2):113-23; and Sabchareon et al., “Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial” Lancet (2012) 380(9853):1559-67. In addition, the yellow fever/dengue vaccine protocol requires three (3) injections over a period of twelve (12) months and is only approved for older children. Another tetravalent live attenuated dengue vaccine product (e.g., by a deletion in the DENV 3′ UTR region) is beginning Phase III clinical trials. Other dengue vaccine products that are still in pre-clinical phases include an adjuvanted recombinant envelope protein vaccine, an adjuvanted purified inactivated virus vaccine, and a plasmid DNA vaccine.

II. Bivalent Epitope Dengue Vaccine Compositions

[0062] In one embodiment, the present invention contemplates a bivalent dengue vaccine configured to induce high and long-lasting levels of broadly neutralizing antibodies against DENVs 1-4 with low anti-DENV antibody enhancing activity. In one embodiment, the vaccine comprises a synthetic peptide that contains two (2) conserved envelope protein conformational epitopes adjacent to, and in, the fusion loop. In one embodiment, the dengue vaccine is linked to the hepatitis B virus surface antigen (e.g., rHepBsAg) virus-like particles (VLPs) (the hepatitis B vaccine) as a carrier. In one embodiment, the hepatitis B virus surface antigen VLP is a yeast cell-derived recombinant hepatitis B virus surface antigen VLP. Although it is not necessary to understand the mechanism of an invention, it is believed that a synthetic peptide that contains two (2) conserved envelope protein conformational epitopes adjacent to, and in, the fusion loop, and recognized by anti-DENVs 1-4 broadly neutralizing antibodies, can be linked to yeast cell-derived recombinant hepatitis B virus surface antigen (rHepBsAg) virus-like particles (VLPs) (the hepatitis B vaccine) as the carrier.

[0063] A. Development and Clinical Strategy

[0064] There are several advantages to utilizing rHepBsAg as a carrier protein linked to a dengue vaccine. For example, a yeast cell-derived rHepBsAg (the hepatitis B vaccine) has been clinically very effective as an immunogenic vaccine that can be given starting in the neonatal period. Further, a hepatitis B vaccine's immunogenicity likely derives from an ability of rHepBsAg to self-assemble into VLPs, and the presence of yeast-derived lipids serve as an adjuvant in the vaccine, thereby providing additive efficacy. The hepatitis B vaccine has been incorporated into the World Health Organization (WHO) Expanded Programme on Immunization (EPI) throughout the world. Consequently, a synthetic dengue peptide vaccine that induces broadly neutralizing antibodies against all 4 DENY serotypes with low anti-DENVs 1-4 antibody enhancing activity, and conjugated to the hepatitis B vaccine, would take advantage of the hepatitis B vaccine's immunogenicity and allow a single vaccine series to protect against both hepatitis B and dengue starting at birth.

[0065] Although it is not necessary to understand the mechanism of an invention, it is believed that a balance between anti-DENV neutralizing antibodies and anti-DENV enhancing antibody activities depends on several factors: i) the targeted epitope(s); ii) the antibody titer; iii) the mix of immature and mature virus particles; and iv) the infecting cell type. It is further believed that essentially all anti-DENY antibodies with neutralizing activity have also been found to have varying degrees of activity that enhance DENY infection in Fcy receptor-bearing cells, and usually at lower antibody titers.

[0066] In some embodiments, the present invention contemplates a vaccination method having a high likelihood of generating antibody responses with high neutralizing capability (e.g., viral-infection protection) and low enhancing activities (e.g., clinically ineffective infection enhancement) to DENVs 1-4. In one embodiment, the method comprises a synthetic bivalent dengue peptide vaccine generating a neutralizing antibody titer to more than one of the conserved conformational epitopes involved in the fusion process of the DENY envelope glycoprotein.

[0067] It has been recently reported that a passive immunization with a broadly neutralizing human monoclonal antibody directed against the DENY envelope protein fusion loop protected against viremia in DENV2-challenged rhesus macaques. Simmons et al., “Antibody Prophylaxis Against Dengue Virus 2 Infection in Non-Human Primates” Am J Trop Med Hyg. (2016) 95(5):1148-56. In one embodiment, conjugation of a synthetic dengue peptide vaccine linked to a rHepBsAg VLP (the hepatitis B vaccine) induces high and long-lasting antibody titers, and thereby minimizes the production of potential antibodies that enhance infection activity. In one embodiment, a synthetic dengue peptide vaccine lacks DENV prM protein epitopes (e.g., derived from immature virus particles), which also contributes to the minimization of potential antibody enhancing activity.

[0068] In one embodiment, the present invention contemplates a method comprising: i) immunizing a mammal with a bivalent dengue vaccine peptide conjugated to a hepatitis B vaccine; ii) immunizing a mammal with a hepatitis B vaccine, and iii) immunizing a mammal with the dengue vaccine peptide. Although it is not necessary to understand the mechanism of an invention, it is believed that differences between these two immunization vaccines will demonstrate that the conjugated vaccine has high anti-DENV's 1-4 neutralizing antibody activity, low anti-DENV's 1-4 enhancing antibody activity, and an equivalent anti-HepBsAg antibody titer.

[0069] B. Vaccine Construction

[0070] Adjacent to a fusion loop of a DENV envelope protein, an epitope in the bc loop was found to be recognized by a potent broadly neutralizing human monoclonal antibody (e.g., mAb 1C19). Smith et al., “The potent and broadly neutralizing human dengue virus-specific monoclonal antibody 1C19 reveals a unique cross-reactive epitope on the bc loop of domain II of the envelope protein” MBio. (2013) 4(6):e00873-13. This seven (7) amino acid peptide sequence (RCPTQGE: SEQ ID NO:1) resides in the bc loop and was found to be conserved in 5,495 DENV envelope protein sequences encompassing all four (4) DENV serotypes (ViPR database, viprbrc.org). Similarly, a conserved ten (10) amino acid peptide sequence (GWGNGCGLFG: SEQ ID NO:2) was located in a fusion loop of DENV envelope proteins. This ten amino acid sequence was found to contain active binding residues for several broadly neutralizing human monoclonal antibodies. Costin et al., “Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop” J Virol. 2013) 87(1):52-66. This ten amino acid sequence peptide was found to be conserved in 10,029 DENV envelope protein sequences encompassing all four (4) DENV serotypes (ViPR database, viprbrc.org). Neither SEQ ID NO:1 nor SEQ ID NO:2 are present in a HepBsAg protein (e.g., the adw subtype).

[0071] In one embodiment, the present invention contemplates a 23mer synthetic peptide containing SEQ ID NO:1 and SEQ ID NO:2 are attached with a spacer protein that may comprise a plurality of glycine residues. In one embodiment, the 23mer synthetic peptide comprises six (6) glycine residue. In one embodiment, the 23mer synthetic peptide has the amino acid sequence of GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:3; the first and second conserved DENV epitopes are italicized for emphasis). In one embodiment, SEQ ID NO:1 is derived from a DENV envelope protein. In one embodiment, SEQ ID NO:2 is derived from a DENV envelope protein. Although it is not necessary to understand the mechanism of an invention, it is believed that a glycine spacer peptide placed between the two conserved DENV epitopes allows the steric flexibility for different antibodies/B-cell receptors to bind to each of the respective epitopes simultaneously. In fact, predicted 3D structures of this synthetic peptide show open loops encompassing both neutralizing antibody epitopes. See, FIG. 1. Furthermore, structures of the DENV2&3 envelope proteins demonstrate that these two conserved epitopes, recognized by anti-DENVs 1-4 broadly neutralizing antibodies, also form open loops. ViPR database, viprbrc.org; and FIG. 2. In one embodiment, the synthetic bivalent dengue vaccine peptide GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:3) comprises at least two cysteine residues, for example, at amino acid residues six (6) and eighteen (18). Although it is not necessary to understand the mechanism of an invention, it is believed that these two cysteine residues do not form a disulfide bond. Ensemble-based Disulfide Bonding Connectivity Pattern Prediction Server, biomedical.ctust.edu.tw/edbcp.

[0072] Although it is not necessary to understand the mechanism of an invention, it is believed that a conjugation of GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:3) with rHepBsAg maximizes the probability that a carboxyl reactive end of a crosslinker will attach to carboxyl groups of a rHepBsAg protein An et al., “Preparation and testing of a Haemophilus influenzae Type b/Hepatitis B surface antigen conjugate vaccine” Vaccine (2015) 33(13):1614-9. and that an amine reactive end of a crosslinker will attach to an N-terminus of SEQ ID NO:3. Other combinations may occur, but preliminary studies suggest that SEQ ID NO:3 is the most likely fusion protein combination. In the event a different approach is required, the peptide GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:3) could be attached to a crosslinker using a carboxyl group on, for example, a C-terminal glutamate residue or by using a sulthydryl group on a cysteine residue. Alternatively, sulfhydryl groups are available on reduced cysteine residues on the HepBsAg protein could also be involved in crosslinker conjugation.

[0073] In one embodiment, the present invention contemplates a bivalent dengue vaccine comprising at least two conserved epitopes recognized by anti-DENVs 1-4 broadly neutralizing antibodies and a crosslinker attaching the bivalent dengue vaccine to a yeast-cell derived rHepBsAg. In one embodiment, at least two conserved epitopes are conformational epitopes. In one embodiment, the bivalent dengue vaccine is GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO: 3). In one embodiment, the yeast-cell derived rHepBsAg is the adw subtype. In one embodiment, the crosslinker is non-immunogenic. In one embodiment, the crosslinker is a heterobifunctional crosslinker. In one embodiment, the crosslinker comprises an amine reactive group. In one embodiment, the crosslinker comprises a carboxyl reactive group. In one embodiment, the crosslinker comprises an amine reactive group and a carboxyl reactive group. In one embodiment, the bivalent dengue/rHepBsAg vaccine is rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:4). Although it is not necessary to understand the mechanism of an invention, it is believed that a crosslinker attaches a synthetic dengue vaccine peptide to a rHepBsAg in such a configuration to allow anti-HepBsAg antibodies to develop. If a spacer is required in order to generate anti HepBsAg IgG antibodies, alternative strategies can use a homobifunctional crosslinker containing 9 repeating units of polyethylene glycol ((PEG)9) (spacer arm 35.8 angstroms) with either two amine reactive ends or two sulfhydryl reactive ends (for conjugation to reduced cysteine residues in the rHepBsAg protein and the 23mer synthetic peptide).

[0074] It was previously reported that a crosslinker facilitated the generation of anti-HepBsAg antibodies in immunized mice when conjugating a hapten to rHepBsAg. An et al., “Preparation and testing of a Haemophilus Influenzae Type b/Hepatitis B surface antigen conjugate vaccine” Vaccine (2015) 33(13):1614-9.

EXPERIMENTAL

Example I

Construction of a Bivalent Dengue/rHepBsAg Vaccine

[0075] To generate rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE

[0076] 1. Equilibrate all solutions to room temperature before using.

[0077] 2. Make a solution of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) 5 mg/ml in 100 mM MES (2-(N-morpholino)ethanesulfonic acid) activation buffer pH 5.5-5.7.

[0078] 3. Add 100 mM MES activation buffer, pH 5.5-5.7, to the HepBsAg vial (50 μg) (2.1×10-3 μmoles) to bring the final volume to 100 μl (2×10-5 μM).

[0079] 4. Add 1 μl of EDC solution to 100 μl HepBsAg solution.

[0080] 5. Make a solution of sulfo-NHS 15 mg/ml in 100 mM MES activation buffer pH 5.5-5.7

[0081] 6. Add 1 μl of the sulfo-NHS solution to the HepBsAg/EDC solution.

[0082] 7. Incubate (mix well)×15 minutes at room temp.

[0083] 8. Dialyze against PBS pH 7.2 (20 kDa membrane)×1 hour at room temp.

[0084] 9. Increase the pH of the dialysate to pH 8 with NaOH (do this after making the peptide solutions below).

[0085] 10. Add 25×10-3 μmoles of the 23mer synthetic peptide to the HepBsAg/EDC/sulfo-NHS (N-hydroxysulfosuccinimide) solution pH 8. (use solutions of the peptide in PBS pH 8 in order to do this).

[0086] 11. Mix well and incubate×2 hours at room temp.

[0087] 12. Dialyze against PBS pH 7.2 overnight at 4 deg C.

Example II

Confirmation of rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:4) VLP Generation

[0088] This example confirms the generation of rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:4) VLPs.

[0089] In order to remove any remaining excess free synthetic peptide, rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:4) VLPs were desalted, run on a SDS-PAGE gel, and the VLP band was excised for proteomic analysis. After chymotrypsin digestion, digest products from HepBsAg (adw subtype) and the synthetic peptide digest product GGGGGGGRCPTQGE (SEQ ID NO:5) were detected by MALDI-TOF mass spectrometry.

Example III

Immunization of a Mammal with rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:4) VLP

[0090] This example demonstrates the immunization of female Balb/c mice with rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:4). See, FIG. 3.

[0091] Serum anti-DENVs 1-4 neutralizing antibody titers were measured using a U937/DC-SIGN flow cytometry based neutralization assay and serum anti-HepBsAg lgG levels using an ELISA. de Alwis et al., “Measuring antibody neutralization of dengue virus (DENY) using a flow cytometry-based technique” Methods Mol Biol. (2014) 1138:27-39.

[0092] The data show that an active immunization with SEQ ID NO:4 produced a potent (e.g, demonstrated by a high avidity) anti-DENV 1-4 neutralizing antibodies in Balb/c mice. See, FIGS. 4A-4D. The Hill slopes of the sigmoid neutralization curves shown in FIG. 4 reflect the avidities of the vaccine-induced neutralizing antibodies. The EC.sub.50'vs reflect the serum neutralizing antibody titers. The EC.sub.50 is a function of an antigen dose used in an immunization protocol and an amount of virus used in the in vitro antibody neutralization assays. There are no pre-defined correlates of protective immunity for anti-DENV neutralizing antibodies in either humans or any animal model. The data also verify that anti-HepBsAg lgG antibodies were produced in the above described immunization. See, FIG. 5.

Example IV

Comparative Analysis of Immunization of a Mammal with rHepBsAg-GWGNGCGLFGGGGGGGRCPTQGE (SEQ ID NO:4) VLP with rHepBsAg or GWGNGCGLFGGGGGGGRCPTQGE Immunization Alone

[0093] Inbred Balb/c mice will be divided into three groups (n=12 mice/group, n=6 female and n=6 male). One group will be immunized subcutaneously (s.q.) with 1 μg of the 23mer synthetic peptide GWGNGCGLFGGGGGGGRCPTQGE adsorbed to alum and boosted at 30 and 60 days (group 1). The second group will be immunized s.q. with 1 μg yeast cell derived rHepBsAg VLPs adsorbed to alum and boosted at 30 and 60 days (group 2). The third group will be immunized s.q. with 1 μg rHepBsAg GWGNGCGLFGGGGGGGRCPTQGE adsorbed to alum and boosted at 30 and 60 days (group 3). 21 days after the last booster vaccination, the mice will be sacrificed and sera collected for the measurement of anti-DENVs 1-4 antibody neutralizing activity and anti DENVs 1-4 antibody enhancing activity (groups 1&3), and anti-HepBsAg lgG levels (groups 2&3). The flowchart for the vaccination protocol is shown in FIG. 3. Balb/c is the inbred mouse strain commonly used to examine hepatitis B vaccine immunogenicity. There is no immunocompetent inbred mouse strain that provides an animal model for DENV infection. However, anti DENV neutralizing antibody activity can be measured in Balb/c mice.

[0094] Serum anti HepBsAg lgG levels will be measured by ELISA; serum anti DENVs 1-4 neutralizing antibody assays will be performed using U937/DC-SIGN cells in a flow cytometry based method; and, serum anti-DENVs 1-4 enhancing antibody assays will be performed using U937 cells in a flow cytometry based method. The flow cytometry based anti-DENV neutralizing antibody assay correlates with the current gold standard assay, the plaque reduction neutralization assay Kraus A A, Messer W, Haymore L B, de Silva A M. Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization. J Clin Microbiol. 2007; 45(11):3777-80. U937 is a human monocytic cell line. In previous studies, mouse antibodies (IgG2a subclass) have been able to enhance DENV infection in U937 cells. The following prototypical DENV strains will be used in the antibody neutralization and enhancement assays: DENV1 West Pacific 74, DENV2 16681, DENV3 H87, and DENV4 TVP360.

[0095] In preliminary studies, active immunization with the subunit dengue vaccine product produced potent (high avidity) anti DENVs 1-4 neutralizing antibodies and anti-HepBsAg IgG in two Balb/c mice. It is anticipated that active immunization with rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:4) in the Balb/c mice will produce high serum titers of anti-HepBsAg IgG (equivalent to rHepBsAg VLP (hepatitis B vaccine) immunization), along with high titers of potent anti DENVs 1-4 neutralizing antibodies and low anti DENVs 1-4 antibody enhancing activity. This example will demonstrate a conjugated bivalent vaccine product that elicits protection against dengue and hepatitis B viruses together, potentially starting at birth.

[0096] It is anticipated that active immunization with rHepBsAg-GWGNGCGLFG-GGGGGG-RCPTQGE (SEQ ID NO:4) in inbred Balb/c and outbred mice will produce high serum titers of anti-HepBsAg IgG (equivalent to rHepBsAg VLP (hepatitis B vaccine) immunization), along with high titers of potent anti-DENVs 1-4 neutralizing antibodies and low anti DENVs 1-4 antibody enhancing activity. Non-parametric statistical tests will be used to compare antibody titers between the three vaccination groups in the Balb/c mice (and within the male and female subgroups).

SUPPLEMENTAL REFERENCES

[0097] Brewoo et al., “Immunogenicity and efficacy of chimeric dengue vaccine (DENVax) formulations in interferon-deficient AG129 mice” Vaccine (2012) 30(8):1513-20. [0098] Diamond et al., “Infection of human cells by dengue virus is modulated by different cell types and viral strains” J Virol. (2000) 74(17):7814-23. [0099] Littaua et al., “Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection” J Immunol. (1990) 144(8):3183-6. [0100] Puerta-Guardo et al., “Antibody-dependent enhancement of dengue virus infection in U937 cells requires cholesterol-rich membrane microdomains” J Gen Virol. (2010) 91(Pt 2):394-403. [0101] Shouval et al., “Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-S1 and pre-S2 antigens as compared with conventional yeast-derived vaccines” Vaccine (1994) 12(15):1453-9.