WO2019068876A1 - Chimeric flavivirus vaccines - Google Patents

Abstract

The invention relates to chimeric live infectious attenuated flavivirusese of a first and a second flavivirus for use in the prevention of an infection by the first flavivirus, wherein at least one of the structural proteins of the first flavivirus is replaced by the 5 corresponding at least one of the structural proteins of the second flavivirus.

Description

Chimeric flavivirus vaccines Description

In spite of its spread worldwide, fiaviviruses are mostly restricted to their defined endemic geographical areas. However, recent trends suggest that flavivirus infections are not only escalating in magnitudes, but also are spreading to new areas and, therefore, fiaviviruses are classified as emerging and reemerging viruses [Ishikawa et a/. (2014) Vaccine. 32, 1326-1337; Mackenzie et a/. (2004) Nat Med. 10(12S), S98- 109; Petersen et a/. (2016) N Engl J Med. 374( 16), 1552-1563; Wilder-Smith & Monath (2016) Lancet Infect Dis. 22. 1473-3099(16), 30588-6] . To date, no approved/licensed antiviral therapy as well as vaccine (excluding YFV and JEV and Dengue) is available against any flavivirus. YFV vaccine (Stamari®/YFVax: YFV 17D and 17DD) is a life-attenuated vaccine (LAV) and is describe as the best available vaccine human kind ever had. Despite of a very efficient YFV vaccine, every year ~0.2 million YFV infections with 30,000 case-fatality are reported worldwide [Staples et a/. (2010) MMWR Recomm Rep. 59(RR-7), 1-27] . Recent YFV Angola outbreak and shortage of YF vaccine supply revealed serious concerns over our unpreparedness towards the disease management [Barrett (2016) N Engl J Med. 375, 301-303] and due to the presence of its vector Aedes mosquitoes, its spreading to Asia may aggravate the issue [Wasserman (2016) Int J Infect Dis. 48, 98- 103; Chen (2016) Em erg Microbes Infect. 5, e69] . There are also two approved vaccines ( 1) Ixiaro (inactivated vaccine) and Imojev (YFV 17D based-chimeric live-attenuated vaccine; c-LAV) against JEV [Li et al. (2014) Hum Vaccin Immunother. 10, 3579- 3593; Scott (2016) Drugs 76, 1301-1312] . Another YFV 17D based tetravalent chimeric-LAV against Dengue (Dengvaxia®) is also in clinical trials [Monath et al. (2015) Vaccine 33, 62-72] . However, there are severe concerns in the use of this vaccine, as it may induce antibody dependent enhancement, upon a subsequent infection with Dengue.

A c-LAV is defined as a Chimeric infectious Live-Attenuated Virus (e.g. YFV 17D), in which the nucleotide sequence of a prM-E protein is replaced with other fiaviviruses (e.g. Japanese encephalitis virus, dengue, Zika etc. ), so that functional prM-E protein, which is expressed in chimera does not belong to parental flavivirus. In spite of several safety issues, LAVs are most efficient vaccines as it provides both humoral and cellular immunity [Monath et al. (2015) Vaccine 33, 62-72; Minor (2015) Virol. 479-480, 379-392] . It is widely accepted in the art that all the humoral responses against flaviviruses are mainly because of domain 3 loop of its envelope protein, which results in the genesis of neutralizing antibodies [Li et al. (2014) Hum Vaccin Immunother. 10, 3579-3593]. Recent data suggest that Flavivirus infections are not only escalating in magnitudes but are also spreading to new areas and, therefore, Flaviviruses are classified as emerging and reemerging viruses [Ishikawa et al. (2014) Vaccine. 32, 1326-1337; Mackenzie et al. (2004) Nat Med. 10(12S), S98-109; Petersen et al. (2016) N Engl J Med. 374( 16), 1552-1563;Wilder-Smith & Monath (2016) Lancet Infect Dis. 22. 1473-3099(16), 30588-6.] .

In spite of safety concerns in immunocompromised individuals, live-attenuated vaccines (LAVs) are most efficient because they induce life-long humoral as well as cellular immunity [Monath et al. (2015) Vaccine 33, 62-72; Minor (2015) Virol. 479- 480, 379-392] . To date antiviral therapies are missing, and effective flavivirus vaccines are only available for TBEV, YFV and JEV). All currently licenced YFV vaccines (tradenames e.g. Stamaril®, YF-Vax®, others) are derived from the live-attenuated 17D strain (YFV 17D) that is considered as one of the best available vaccines ever made. Despite this efficient vaccine, ~0.2 million YFV infections and 30,000 fatal cases occur annually worldwide. The recent YFV outbreak in Angola and shortage of the YFV vaccine supply revealed serious unpreparedness [Barrett (2016) N Engl J Med. 375, 301-303.] . In addition, the presence and global spread of the yellow fever mosquitoes (Aedes sp.) poses a huge danger of YFV spread to Asia and other areas from which YFV is currently absent [Wasserman (2016) Int J Infect Dis. 48, 98-103; Chen (2016) Emerg Microbes Infect. 5, e69].

To date, the vaccine field broadly relies on the strategy to mount protective immunity against structural proteins of a virus, in case of Flaviviruses in particular neutralizing antibodies (nAb) elicited against the viral surface proteins (WHO Expert Committee on Biological Standardisation, WHO Technical Report Series 979, 2011, Annex 2 : Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines (live, attenuated); WHO Expert Committee on Biological Standardisation, Recommendations to assure the quality, safety and efficacy of Japanese encephalitis vaccines (live, attenuated) for human use, Technical Report Series 980, 2012 ). For instance, two vaccines against JEV are approved, namely Ixiaro®_ (an inactivated vaccine) and Imojev® (a YFV 17D-based chimeric live-attenuated vaccine comprising prM protein and envelope E protein of Japanese encephalitis virus; c-LAV) [Li et al. (2014) Hum Vaccin Immunother. 10, 3579-3593; Scott (2016) Drugs 76, 1301-1312] based on their ability to induce sufficiently high serum titers of JEV- specific nAb that are widely accepted as immune correlates of protection.

Chimeric vaccines have been developed wherein the prME genes of the envisaged pathogen are inserted into the backbone of another flavivirus.

Mathenge et al. (2004) J. Gen. Virol. 85, 2503-2513 and Yang (2017) Protein Cell 8, 225-229 disclose dual vaccination with chimeric viruses with a Japanese encephalitis virus backbone and Dengue Prm-E proteins.

The Imojev Vaccine against Japanese encephalitis (JE), is a yellow fever virus wherein the YF prME genes of the well-established YF17D yellow fever vaccine are replaced by those of the JE virus. The safety of the YFD vaccine has been the reason to consider this construct as a backbone for chimeric viruses. Unintentionally, the chimeric vaccine contains fragments of geographically distinct regions. Thus the unmodified yellow fever vaccine for vaccination of yellow fever is used in other geographic regions (Africa and south America) than the chimeric vaccine for Japanese encephalitis (Asia).

Alternatives for YF 17D vaccines have been suggested e.g. by Bassi et al. (2016) PLoS Negl Trop Dis 10, e0004464, disclose that a single immunization with an Ad- vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D.

Summary of the invention

The present invention is based on the surprise finding that the C protein and the non- structural proteins of a chimeric virus are sufficient to act as a vaccine, such that a chimeric vaccines not only function against the virus from which the prME insert originates, but also against the virus from which the remaining backbone portion originates.

The need to vaccinate a person against both JE and YF is limited to Asians travelling to Africa and vice-versa, or for persons outside these regions intending to visit both Africa and Asia.

Separate vaccines for these separate regions and viruses have been used up to now. Indeed, product information of the supplier of the Imojev vaccine states that in adults, IMOJEV may be administered at the same time as yellow fever vaccine using separate syringes, and into separate limbs. Individuals who received both vaccines could not have been aware that the use of the chimeric vaccine would have been sufficient to provide protection against both viruses.

Even the individual who received the chimeric construct as vaccine against JE, and who visited a risk area for Yellow Fever and did not attract YF, would be classified as a person who was not infected by YF, rather than a person who was protected against YF because of the antibodies originating from the backbone of the vaccine construct. The chimer vaccine against Dengue (Dengvaxia), containing the backbone of the YF 17D vaccine is up to know only approved in Mexico, where yellow fever does not occur. This chimeric vaccine is not yet routinely used in South American countries where both Dengue and Yellow fever occur.

The invention thus relates to chimeric live-attenuated Flaviviruses of the general genetic structure A-B-A and their use in vaccination against at least A, wherein A is particular virus A originally used as vector backbone (vector) and (ii) B representing the genetic material of another virus B and encoding for antigenic surface proteins of B (vaccine target) that has originally been introduced into A for the purpose of inducing protection from said virus B infection following vaccination. Surprisingly such A-B-A vaccines [e.g. the existing Imojev® and Dengvaxia® that are introduced in the yellow fever virus (YFV) strain 17D] can also been used to efficiently immunize against and fully protect from virus A (e.g. YFV in the case of Imojev® and Dengvaxia®) that was originally considered as antigenically irrelevant and employed only as vector component. The underlying mechanism conferring protection from A is specific for A, yet unrelated to that of the original A-B-A vaccine as it does not involve neutralizing antibodies.

Nonstructural proteins, especially NS1 due to antigen presentation and secretory nature can also be important in cellular and humoral responses. Therefore, it appears that humoral and cellular responses against both envelope and nonstructural proteins may form the basis of vaccination [Pierson et al. (2008) Cell Host Microbe. 4, 229- 238; Putnak et al. ( 1990) J Gen Virol. 71(Pt 8), 1697-1702; Schlesinger et al. ( 1993) Virology. 92(1), 132-141 ; Amorim et al. (2016) Virology 487, 41-49; Rastogi et al. (2016) Virol J. 13, 131; Watte rson et a/. (2016) Antiviral Res. 130, 7-18.] . c-LAV flavivirus vaccines (e.g. Imojev or Dengvaxia®) contain a structural part from one virus (e.g. JEV or Dengue) and a nonstructural part from other virus (e.g. YFV) and therefore, they have a potential to be developed as a dual vaccine against two flaviviruses.

The present invention demonstrates that in a chimeric virus, the presence of the nonstructural parental antigens, even in the absence of the parental PrME gene, is sufficient to be used as a vaccine against the parental virus.

The present invention further has the advantages that vaccination can be performed against a virus infection associated with antibody dependent enhancement, such as is the case with Dengue and Zika.

Indeed, whereas a first infection with Dengue generally results in a mild disease, a second infection generally provokes a more severe disease pattern . Several publications warn for the possibility that the chimeric Dengue vaccine acts as a first infection without symptoms and that it upon a subsequent genuine infection, a severe disease symptoms occur. Vaccination with such vaccine is thus only recommended for persons who already had an earlier dengue infection .

The fact that subsequent infections are more severe can be attributed to the development of Antibody Dependent Enhancement (ADE). The antibodies responsible for this phenomenon are raised against the Envelope protein, and especially against the prM protein.

The methods of the present invention allow to vaccinate against Dengue using a chimeric vaccine wherein a Dengue backbone is used wherein the prM gene and typically also the E gene have been replaced by those of another virus (apart from Zika).

The same consideration applies to Zika virus wherein a chimeric vaccine with a Dengue backbone is used wherein the prM gene and typically also the E gene have been replaced by those of another virus (apart from Dengue).

This has the advantage that a Dengue or Zika vaccine can be used which does not elicit antibodies against the prM and E gene. The methods of the present invention also allow to use a single vaccine for serotypes 1, 2 and 3, of Dengue, possibly even against all 4 serotypes. Indeed, at present 4 different chimeric vaccines, each containing the prM-E genes of the 4 different serotypes are used as vaccine. A chimeric vaccine comprising the common backbone of Dengue and the prM E genes of another virus, (e.g . Yellow Fever flavivirus, preferably not Zika virus) overcomes the need for 4 different vaccines. Upon use of a chimeric flavivirus vaccine, antibodies are raised against the Capsid proteins and the Nonstructural proteins of the backbone and against the membrane protein and the envelope protein. It is generally accepted in the field that the antibodies against the membrane and/or envelope protein are essential to provide protection against the pathogen.

Although the skilled person recognized that the Non-structural proteins encoded by the backbone will also generate antibodies, this response is not believed to be sufficient to protect an individual from a viral infection. As a consequence chimeric vectors comprise the prM and E genes of the envisaged pathogen, which may lead to antibody-dependent enhancement (ADE), especially in the case of Dengue or Zika. It an aspect of the invention to provide a vaccination where the involvement of neutralizing antibodies originating from the envelope protein or the membrane protein is circumvented. The invention is further summarized in the following statements:

1. A chimeric flavivirus of a first and a second flavivirus wherein in the genome of the first flavivirus at least one of the genes encoding the structural proteins is replaced by the corresponding part of the genome of the second flavivirus for use as a vaccine in the prevention of an infection by said first flavivirus.

2. The chimeric virus in accordance with statement 1, for use in the prevention according to statement 1, wherein the prM-E genes of the first flavivirus are replaced by the prM-E genes of the second flavivirus. Preferably the second virus is not Dengue virus or Zika virus. Optionally also the signal peptide of the capsid protein of the first virus is replaced by the one of the second virus.

3. The chimeric virus according to statement 1 or 2, for use as a vaccine in the prevention of a infection by both said first and second flavivirus.

4. The chimeric virus in accordance with statement 1 or 2, for use in the prevention according to any one of statements 1 to 3, wherein said first flavivirus is not Yellow Fever virus.

5. The chimeric virus in accordance with statement 1 or 2, for use in the prevention according to any one of statements 1 to 4, wherein said second flavivirus is Yellow Fever flavivirus.

6. The chimeric virus in accordance with statement 1 or 2, for use in the prevention of an infection by Dengue virus, wherein the first flavivirus is Dengue virus and wherein the prM-E gene of Dengue virus is replaced by the prM-E gene of a second flavivirus, preferably other than zika. 7. The chimeric virus in accordance with statement 1 or 2, for use in the prevention

of an infection by Zika virus, wherein the first flavivirus is Zika virus and wherein the prM-E gene of Zika virus is replaced by the prM-E gene of a second flavivirus, preferably other than Dengue.

8. The chimeric virus in accordance with statement 6 or 7, for use in the prevention

of an infection by respectively Dengue or Zika virus where said second flavivirus is Yellow Fever virus.

9. The chimeric virus according to any one previous claims, for use in accordance with any one of the previous claims wherein the genome encoding the chimeric flavivirus is cloned in a Bacterial Artificial Chromosome comprising an inducible origin.

The invention equally relates to a method of preventing a flavivirus infection by vaccination, comprising the step of administering a chimeric flavivirus of a first and a second flavivirus wherein in the genome of the first flavivirus at least one of the genes encoding the structural proteins is replaced by the corresponding part of the genome of the second flavivirus, wherein the method aims to prevent a flavivirus infection by said first flavivirus.

This method can also be used to prevent a flavivirus infection by both said first flavivirus and said second flavivirus using the same above mentioned chimeric flavivirus.

Statements 1 to 9 as mentioned above are equally applicable to this method of prevention.

10. A chimeric live infectious attenuated flavivirus of a first and a second flavivirus for use in the prevention of an infection by said first flavivirus, wherein at least one of the structural proteins of the first flavivirus is replaced by the corresponding at least one of the structural proteins of the second flavivirus, and wherein the second flavivirus is not Dengue or Zika virus,

11. The chimeric flavivirus for use in the prevention according to claim 9, wherein the prM-E proteins of the first flavivirus are replaced by the prM-E proteins of the second flavivirus.

12. The chimeric flavivirus for use in the prevention according to claim 10, wherein in addition the signal peptide of the C terminal part of the capsid protein of the first flavivirus is replaced by the signal peptide of the C terminal part capsid protein of the second flavivirus.

13. The chimeric flavivirus for use in the prevention according to claim 9 or 10, wherein the signal peptide of the C terminal part of the capsid protein of the first flavivirus is not replaced.

14. The chimeric flavivirus according to any one of claims 9 to 12, for use in the prevention of a infection by both said first and second flavivirus.

15. The chimeric flavivirus according to any one of claims 9 to 13, for use in the prevention according to any one of claims 9 to, wherein said first flavivirus is not Yellow Fever virus.

16. The chimeric flavivirus according to any one of claims 9 to 14, for use in the prevention according to any one of claims 9 to 14, wherein said second flavivirus is Yellow Fever flavivirus.

17. The chimeric flavivirus according to any one of claims 9 to 14, for use in the prevention according to any one of claims 9 to 14, wherein said second flavivirus is

Japanese encephalitis.

18. The chimeric flavivirus according to any one of claims 9 to 16, for use in the prevention of an infection by Dengue virus, wherein the first flavivirus is Dengue virus and wherein the prM-E proteins of Dengue virus are replaced by the prM-E proteins of a second flavivirus other than Zika virus.

19. The chimeric flavivirus according to claim 17, for use in the prevention of an infection by Dengue where said second flavivirus is Yellow Fever virus.

20. The chimeric flavivirus according to claim 17, for use in the prevention of an infection by Dengue where said second flavivirus is Japanese encephalitis virus 21. The chimeric flavivirus according to any one of claims 9 to 16, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and wherein the prM-E proteins of Zika virus are replaced by the prM-E proteins of a second flavivirus other than Dengue.

22. The chimeric flavivirus according to claim 20, for use in the prevention of an infection by Zika virus where said second flavivirus is Yellow Fever virus.

23. The chimeric flavivirus according to claim 20, for use in the prevention of an infection by Zika virus where said second flavivirus is Japanese encephalitis virus.

24. The chimeric flavivirus according to claim 22, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and the second virus is Japanese encephalitis virus, and wherein the signal peptide of the C terminal part of the C protein is from Zika virus. 25. A polynucleotide comprising a sequence encoding a live infectious attenuated chimeric flavivirus of a first and a second flavivirus for use in the prevention of an infection by said first flavivirus, wherein the sequence encoding at least one of the structural proteins of the first flavivirus is replaced by the sequence of encoding at least one of the structural proteins the second flavivirus, and wherein the second flavivirus is not Dengue or Zika virus.

26. The polynucleotide for use in the prevention according to claim 25, wherein the sequence encoding the prM-E protein of the first flavivirus are replaced by the sequence encoding the prM-E proteins of the second flavivirus.

27. The polynucleotide according to claim 25, for use in the prevention according to claim 16, wherein further the sequence encoding the signal peptide of of the C terminal part of the capsid protein of the first flavivirus is replaced by the sequence encoding the signal peptide of the capsid protein of the second virus.

28. The polynucleotide according to claim 25 for use in the prevention according to claim 24 or 25, wherein the signal peptide of the C terminal part of the capsid protein of the first flavivirus is not replaced.

29. The polynucleotide according to any one of claims 24 to 27, for use in the prevention of an infection by both said first and second flavivirus.

30. The polynucleotide according to any one of claims 24 to 27, for use in the prevention according to any one of claims 24 to 27, wherein said first flavivirus is not

Yellow Fever virus.

31. The polynucleotide according to any one of claims 20 to 25, for use in the prevention according to any one of claims 20 to 25, wherein said second flavivirus is Yellow Fever flavivirus.

32. The polynucleotide according to any one of claims 24 to 28, for use in the prevention according to any one of claims 16 to 20, wherein said second flavivirus is Japanese encephalitis virus.

33. The polynucleotide according to any one of claims 24 to 31, for use in the prevention of an infection by Dengue virus, wherein the first flavivirus is Dengue virus and wherein the sequence encoding the prM-E proteins of Dengue virus are replaced by the sequence encoding the prM-E proteins of a second flavivirus other than Zika virus.

34. The polynucleotide according to claim 32, for use in the prevention of an infection by Dengue or Zika virus, wherein said second flavivirus is Yellow Fever virus.

35. The polynucleotide according to claim 32, for use in the prevention of an infection by Dengue or Zika virus, wherein said second flavivirus is Japanese encephalitis virus. 36. The polynucleotide according to claim 24 to 31, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and wherein the sequence encosing the prM-E proteins of Zika virus are replaced by the sequence encoding the prM-E proteins of a second flavivirus other than Dengue.

37. The polynucleotide according to claim 35, for use in the prevention of an infection by Zika virus where said second flavivirus is Yellow Fever virus.

38. The polynucleotide according to claim 38, for use in the prevention of an infection by Zika virus where said second flavivirus is Japanese encephalitis virus.

39. The chimeric flavivirus according to claim 38, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and the second virus is Japanese encephalitis virus, and wherein the signal peptide of the C terminal part of the C protein is from Zika virus.

40. The polynucleotide according to any one of claims 24 to 38, for use in the prevention according to any one of claim 24 to 39, wherein said polynucleotide is a Bacterial Artificial Chromosome comprising an inducible origin.

41. A method of inducing a neutralizing antibody response against a first flavivirus in a subject, thereby preventing an infection by said first flavivirus, comprising the step of administering to said subject a chimeric live infectious attenuated flavivirus of a first and a second flavivirus in accordance with any one of claims 1 to 15 or administering to said subject a nucleotide sequence encoding chimeric live infectious attenuated flavivirus of a first and a second flavivirus in accordance with any one of claims 16 to 40.

Detailed description

Figure 1: Determination of lethal dose of JE-CVax in AG129 mice. Six-weeks (6W) old AG129 mice (n=5) were infected lO^3"5 PFUs of JE-CVax as well as PBS. Sixteen weeks (16W) old AG129 mice were also infected with 10⁵ PFUs of JE-CVax. Animals were observed for morbidity and mortality for next 28 dpi. We observed 100% mortality (p-value; 0.002 compared to PBS in one-way ANOVA) only in six- weeks old AG129 mice infected with 10⁵ PFUs while no negative impact was experienced in any of the other infected groups.

Figure 2: Protection of AG129 mice against YFV 17D lethal challenge after vaccination with chimeric JE-CVax and PLLAV-JE-CVax. AG129 mice were first vaccinated intraperitoneally (i.p.) with PBS (— : black solid line [1] ; n=34), 10⁴ PFUs JE-CVax ( : black broken line [2] ; n=36) or ΙΟμς PLLAV-JE-CVax (— : solid line

[3] ; n= 13) or transdermal^ with ΙΟμς PLLAV-JE-CVax ( : broken line [4] n=9) and 28 days post vaccination were challenged with 1000-LDioo YFV 17D i.p.. Animals were observed for morbidity (weight loss) and mortality for further 5 weeks. Log- rank (Mantel-Cox) survival analysis test was performed for statistical significance. Mean days to death for PBS vaccinated mice was 14.4±2.7 days. ****p-value <0.0001.

Figure 3: Evaluation of window for the vaccination with JE-CVax against YFV

17D. AG129 mice were first vaccinated with JE-CVax and challenged with YFV 17D on 0 (- - -) [1], day 4 (· . ·)[2], day 7 (· . ·)[3], day 14, day 21 and day 28 (— ) [4] days post vaccination. Mice were also either only vaccinated with JE-CVax (— ) [5] or only challenged with YFV 17D (— ) [6] and observed for morbidity and mortality for next 28 days.

Figure 4: Evaluation of protection of AG129 mice against ZIKV lethal challenge after vaccination with JE-CVax and YFV 17D challege. AG129 mice were first vaccinated with JE-CVax and 4 weeks later challenged with YFV 17D (i.p. ; N=6). These animals were challenged with 10⁴ pfu ZIKV MR776 (i.p.). Animals (N=7) neither vaccinated with JE-CVax nor challenged with YFV 17D of same age group were also challenged with 10⁴ pfu ZIKV MR776. All the animals were observed for morbidity and mortality for further 7 weeks. Log-rank (Mantel-Cox) survival analysis test was performed for statistical significance. Mean days to death for non-vaccinated and vaccinated mice were 23.5 ± 5.4 and 17.4±8.8 days.

Figure 5: Serological analysis for cross-reactive antibodies against JE-CVax and YFV 17D in YFV 17D-infected or JE-CVax-vaccinated AG129 mice. Prior to vaccination, serum was obtained (A). Then, AG129 mice were either infected IP with 10³ LDioo YFV 17D (B) or vaccinated with 10⁴ PFUs JE-CVax (C). Upon first signs of disease for YFV 17D or 28-days post vaccination (JE-CVax), serum was collected. Serum prior to vaccination (A), serum post infection with YFV (B) or serum from JE- CVax vaccinated mice (C) were analyzed for reactivity to YFV- or JEV-infected cells in indirect immunofocus assay (UFA).

Figure 6: Serological analysis for neutralizing antibodies against JE-CVax and YFV 17D in (PLLAV) JE-CVax vaccinated mice. AG129 mice were first vaccinated IP with 10⁴ PFUs JE-CVax (n=34). Four weeks later, animals were challenged with 10³ LDioo YFV 17D IP. Serum prior to vaccination, post vaccination or post challenge was harvested and CPENT assays were performed to determine neutralizing antibodies. \JEV and 'YFV represents data of neutralization assays performed against JE-CVax or YFV 17D, respectively. 'dO', 'd28' and 'end' represents CPENT assays performed on serum prior to vaccination, day 28 post-vaccination (prior to challenge) and at end of the study (35 days after the challenge), respectively. Our starting dilution was 1 : 20. Therefore non-reactive samples were assigned a value of 1.3010 (Iogio20) and set as limit of detection (LOD).

Figure 7: Characterization of serum for animals vaccinated with JE-CVax, YFV 17D and ZIKV MR776 for reactivity towards anti-NSl YFV 17D antibodies. (A) AG129 mice were first vaccinated with either PBS or lO^3"5 PFUs JE- CVax or infected with 10⁴ PFUs YFV 17D or 10⁵ PFUs ZIKV MR776. Serum was collected either on the humane end-point or 28 days post manipulation. Graph showing results of ELISA determining the level of antibodies reactive against YFV 17D NS 1 in serum quantified relative to anti-NSl YFV 17D monoclonal antibody clone 1A5. (B) Hek-293 cells were transfected with an NS1 and GFP expressing plasmid (top) or infected with YF17D-mCherry reporter virus (bottom). Either 48 h after transfection or 72 h after infection, cells were stained intracellularly with serum from naive, non-vaccinated mice (left), from mice that were vaccinated with JE-CVax (center) or anti-YFV NS l-specific monoclonal antibody (mAb, rig ht). Graph showing flow cytometric analysis of GFP or mCherry fluorescence, marking transfected or infected cells, respectively, and visualization of antibod ies in the mouse serum that bind YFV NS1 via addition of rabbit anti-mouse IgG secondary antibody conjugated to PE-Cy7.

Figure 8: Evaluation of protective effect of passive JE-CVax mice hyperimmune-serum transfer in non-vaccinated AG129 mice. Non-vaccinated AG129 mice were i . p. injected with 250 μΙ JE-CVax hyperimmune mouse serum two [ 1 ] to three [2] times i.e. either on day -01 and day 01 (A) or on day -01, 04, and 08 (B and C) and challenged with 10³ (A), 10² (B) or 10¹ (C) lethal dose-50 of YFV 17D. N = 5 and 10 animals were used in study A/C and B, respectively.

Figure 9: Depletion or blockage of innate effector functions in JE-CVax- vaccinated mice. AG 129 mice were vaccinated with JE-CVax and, four weeks later, challenged with 10³ PFU YFV 17D, a dose that is 100% lethal in non-vaccinated mice. Starting one day prior to infection challenge, innate effector functions were blocked in some mice. Monoclonal antibodies (mAbs) directed against Ly6G or Ly6C/G were inoculated one day prior to challenge to deplete neutrophils or monocytes/neutrophils, respectively; mAbs directed against Fc receptor CD16/32 were inoculated to block antibody binding ; or cobra venom factor (CVF) was inoculated to prevent complement-activation .

Figure 10: Protective efficacy of ZIK-JEprM/E in AG129 mice. (A) Weig ht loss of sham-vaccinated (n= 10) or vaccinated (n = 5 per group) mice following intraperitoneal challenge with 1x104 PFU of ZIKV MR766. (B) Survival curve of sham- vaccinated and vaccinated mice.

Figure 11: Protective efficacy of YF-ZIKprM/E in AG129 mice. (A) Schematic presentation of the vaccine efficacy protocol in AG129 mice. Weight loss (B) and survival (C) of sham-vaccinated (n= 10) and vaccinated (n= 15) mice following intraperitoneal challenge with 1x103 PFU of YFV-17D. Viremia (D) and virus dissemination (E) in sham vaccinated (n= 10) and vaccinated (n=5 or 10) following virus YFV-17D challenge. Log-rank (Mantel-Cox) test was used to measure statistical differences in survival rates between sham-vaccinated and vaccinated mice. Mann- Whitney two-tail test was used for comparisons between two groups in viremia and virus dissemination studies. P values >0.5 was considered statistically significant. *P=0.05, **P=0.01, ***P=0.001, ****P=0.0001.

Flaviviruses belong to the viral family of Flaviviridae and comprise many medically important viruses, including Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Tick-borne encephalitis virus (TBEV), Yellow fever virus (YFV) and Zika virus (ZIKV), Japanese Encephalitis virus (JEV), Murray Valley Encephalitis virus (MVE), St. Louis Encephalitis virus (SLE), Tick-borne Encephalitis (TBE) virus, Russian Spring-Summer Encephalitis virus (RSSE), Kunjin virus, Powassan virus, Kyasanur Forest Disease virus, Usutu virus, Wesselsbron and Omsk Hemorrhagic Fever virus. Flaviviruses are enveloped with a ~ 10-11 kb long (+)ssRNA genome encoding for 3 structural proteins (core, C; premembrane, prM; and envelope, E), which are incorporated in the virions, and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).

The signal peptide at the C terminus of the C protein (C-signal peptide; also called C-anchor domain ("canch") regulates flavivirus packaging through coordination of sequential cleavages at the N terminus (by viral NS2B/NS3 protease in the cytoplasm) and C terminus (by host signalase in the endoplasmic reticulum [ER] lumen) of the signal peptide sequence.

Generally, a c-LAV (Chimeric Life Attenuated Vaccine) of the prior art is defined as an infectious live-attenuated vaccine virus in which the nucleotide sequences of the structural prM and E proteins of e.g. YFV 17D are replaced with those of another Flavivirus (e.g. JEV, DENV, ZIKV etc.). The generic structure of such c-LAV can be described as A-B-A with (i) building blocks A comprise parts of a particular virus A originally used as vector backbone [typically the encoding the C protein and the Ns proteins] (vector) and (ii) building blocks B representing the genetic material of another virus B and encoding for the antigenic surface proteins of B (vaccine target) that has originally been introduced into A for the purpose of inducing protection from said virus B infection following vaccination. Along these lines, a YFV 17D-based tetravalent c-LAV against Dengue virus serotypes 1-4 (Dengvaxia®) has been developed and recently approved in some countries in Central and South America as well as South-East Asia. Here, the genes encoding prM and E proteins were switched into the backbone of non-structural genes of YFV 17D (general structure A-B-A; A from YFV 17D, B from representatives of all four DENV serotypes).

Following the same A-B-A blueprint, other DENV c-LAVs are currently in development, yet using attenuated DENV strains (Torresi et a/. (2017) Hum Vaccin Immunother.

13, 1059-1072) or ZIKV (Xie et al (2017) MBio. 8, e02134-16) as vector backbone. Similar c-LAV candidates has been developed for a variety of Flaviviruses each time targeting for neutraling antibody (nAb) responses by the envelope and membrane protein (Lai et al. (2003) Adv Virus Res. 61, 469-509) including DENV4/TBEV (Langatvirus) chimeras to protect from TBEV by nAb neutralizing TBEV (Pletnev & Men (1998) Proc Natl Acad Sci USA. 95, 1746-1751). However, nAbs play a dual role in the pathogenesis of DENV infection. Due to the existence of four antigenically related DENV serotypes, pre-existing cross-reactive nAbs against a particular serotype may not only confer cross-protection from infection by a heterologous serotype, yet may also pose the risk of antibody- dependent enhancement (ADE) : During ADE pre-existing antibodies that are directed against viral surface proteins and were generated for instance following a first infection with one serotype or a suboptimal vaccination can lead to increased disease severity during infection with another heterologous DENV serotype (Guzman and Harris (2015) Lancet Rev 385, 453-465). In line, clinical studies suggested that administration of Dengvaxia® may under certain circumstances rather increases the risk for dengue-related hospitalization in young children (Cameron & Simmons (2015) N Engl J Med 373, 1263-1264; Guy & Jackson (2016) Nature Rev. Microbiol.

14, 45-54). Hence, in particular in the case of DENV and the risk of ADE, vaccines that do not induce nAb may be favorable, and this in contrast with best practice of the skilled person and the advice of key experts (WHO Expert Committee on Biological Standardisation, WHO Technical Report Series 979, 2011, Annex 2 : Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines (live, attenuated); WHO Expert Committee on Biological Standardisation, Recommendations to assure the quality, safety and efficacy of Japanese encephalitis vaccines (live, attenuated) for human use, Technical Report Series 980, 2012).

Prior vaccine strategies focused on the induction of nAb responses that protect from Flavivirus infections and are mostly directed against the respective envelope protein [Li et a/. (2014) Hum Vaccin Immunother. 10, 3579-3593] . However, in addition to this widely accepted correlate of vaccine-mediated protection by nAb, historical as well as recently published evidence suggested that, in addition, nonstructural proteins and, due to its antigenic and secreted nature, in particular NS1 may contribute to some extent to vaccine efficacy via protective cellular and humoral immune responses [Pierson et al. (2008) Cell Host Microbe. 4, 229-238; Putnak et al. (1990) J Gen Virol. 71(Pt 8), 1697-1702; Schlesinger et al. (1993) Virology 92(1), 132-141 ; Amorim et a/. (2016) Virology 487, 41-49; Rastogi et a/. (2016) Virol J. 13, 131 ; Watterson et a/. (2016) Antiviral Res. 130, 7-18] . We here show that, unexpectedly, c-LAV Flavivirus vaccines (e.g. Imojev or Dengvaxia® that are approved to protect against JEV or DENV, respectively) have the potential to fully protect from massively lethal vaccine challenge fully without induction of any relevant nAb and may therefore be developed as dual vaccines, also protecting against a second Flavivirus, namely against that virus which provided the backbone of non- structural proteins (e.g. of YFV 17D) for replication of the c-LAV. In other words, c- LAVs of the generic structure A-B-A protect against infection with two viruses b and A because these chimeric vaccines contain the structural proteins of one virus B (e.g. JEV or Dengue) and the nonstructural proteins of a second virus A (e.g. YFV). This principle extends to the already approved chimeric Flavivirus vaccines (e.g. Imojev or Dengvaxia®), as well as to chimeras between viruses from different virus families.

A BAC as referred to in the present application comprises:

- an inducible bacterial ori sequence for amplification of said BAC to more than 10 copies per bacterial cell, and

- a viral expression cassette comprising a cDNA of an the RNA virus genome and comprising cis-regulatory elements for transcription of said viral cDNA in mammalian cells and for processing of the transcribed RNA into infectious RNA virus.

As is the case in the present invention the RNA virus genome is a chimeric viral cDNA construct of two RNA virus genomes.

In these BACS, the viral expression cassette comprises a cDNA of a positive-strand

RNA virus genome, an typically a RNA polymerase driven promoter preceding the 5' end of said cDNA for initiating the transcription of said cDNA, and

an element for RNA self-cleaving following the 3' end of said cDNA for cleaving the RNA transcript of said viral cDNA at a set position.

The BAC may further comprise a yeast autonomously replicating sequence for shuttling to and maintaining said bacterial artificial chromosome in yeast. An example of a yeast ori sequence is the 2μ plasmid origin or the ARS1 (autonomously replicating sequence 1) or functionally homologous derivatives thereof.

The RNA polymerase driven promoter of this first aspect of the invention can be an RNA polymerase II promoter, such as Cytomegalovirus Immediate Early (CMV-IE) promoter, or the Simian virus 40 promoter or functionally homologous derivatives thereof.

The RNA polymerase driven promoter can equally be an RNA polymerase I or III promoter.

The BAC may also comprise an element for RNA self-cleaving such as the cDNA of the genomic ribozyme of hepatitis delta virus or functionally homologous RNA elements.

The formulation of DNA into a vaccine preparation is known in the art and is described in detail in for example chapter 6 to 10 of "DNA Vaccines" Methods in Molecular Medicine Vol 127, (2006) Springer Saltzman, Shen and Brandsma (Eds.) Humana Press. Totoma, NJ. and in chapter 61 Alternative vaccine delivery methods, Pages 1200-1231, of Vaccines (6th Edition) (2013) (Plotkin et al. Eds.). Details on acceptable carrier, diluents, excipient and adjuvant suitable in the preparation of DNA vaccines can also be found in WO2005042014, as indicated below.

"Acceptable carrier, diluent or excipient" refers to an additional substance that is acceptable for use in human and/or veterinary medicine, with particular regard to immunotherapy.

By way of example, an acceptable carrier, diluent or excipient may be a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic or topic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate and carbonates, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulphates, organic acids such as acetates, propionates and malonates and pyrogen-free water. A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N. J. USA, 1991) which is incorporated herein by reference.

Any safe route of administration may be employed for providing a patient with the DNA vaccine. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection may be appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines. It is also contemplated that micro particle bombardment or electroporation may be particularly useful for delivery of nucleic acid vaccines.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

DNA vaccines suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of plasmid DNA, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the DNA plasmids with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is effective. The dose administered to a patient, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent (s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. Furthermore DNA vaccine may be delivered by bacterial transduction as using live- attenuated strain of Salmonella transformed with said DNA plasmids as exemplified by Darji et al. (2000) FEMS Immunol Med Microbiol 27, 341-349 and Cicin-Sain et al. (2003) J. Virol. 77, 8249-8255 given as reference.

Typically the DNA vaccines are used for prophylactic or therapeutic immunisation of humans, but can for certain viruses also be applied on vertebrate animals (typically mammals, birds and fish) including domestic animals such as livestock and companion animals. The vaccination is envisaged of animals which are a live reservoir of viruses (zoonosis) such as monkeys, mice, rats, birds and bats.

In certain embodiments vaccines may include an adjuvant, i.e. one or more substances that enhances the immunogenicity and/or efficacy of a vaccine composition However, life vaccines may eventually be harmed by adjuvants that may stimulate innate immune response independent of viral replication. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween-80; Quill A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1 ; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOMt) and ISCOMATRIX (B) adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran or with aluminium phosphate; carboxypolymethylene such as Carbopol'EMA; acrylic copolymer emulsions such as Neocryl A640; vaccinia or animal poxvirus proteins; sub-viral particle adjuvants such as cholera toxin, or mixtures thereof.

As illustrated in the below example using Imojev® (JE-CVax) that is a c-LAV which has been clinically developed and licensed as a prophylactic vaccine to protect against JEV infection, expresses the envelope proteins of JEV and hence does not induce nAbs against YFV, we unrevealed that such c-LAV has a surprisingly high protective efficacy, even from an exceedingly aggressive challenge dose in a very stringent vaccine challenge model of YFV infection. Also peculiarly, protection is conferred within an unexpectedly short period of time following vaccination and after only a single dose. Both latter findings argue against a rather unspecific partial cross- protection that is due to a marginally cross- reactive and hence rather weak cross- immunity of JEV and YFV. By contrast JE-CVax turned out to be a very potent and specific YFV vaccine in which non-nAb responses targeting cell bound YFV NS1 may play a crucial role as exemplified by ELISA and passive serum transfer experiments. In fact, heterologous protection from YFV by JE-CVax appeared so strong that even (partial) depletion of several effector principles prior to or during vaccine challenge hardly could affect infection outcomes: no effect of depletion of complement, T cells nor neutrophils, yet macrophages.

Previous studies had demonstrated that AG 129 mice are highly susceptible to several Flavivirus infections including DENV [Calvert et a/. (2006) J Gen Virol. 87(Pt 2), 339- 346], JEV [Calvert et a/. (2014) Vaccine. 32, 258-264], WNV [Calvert et a/. (2011) Virology. 410, 30-37], YFV [Thibodeaux et a/. (2012) Vaccine 30, 3180-3187] and ZIKV [Zmurko et al. (2016) PLoS Negl Trop Dis. 10, e0004695] . While literature teaches that only low amounts of YFV are required to sicken AG 129 [Thibodeaux et al. (2012) Vaccine. 30, 3180-3187], till now no similar study have been available about Imojev®/JE-CVax. In fact, in the lethal AG 129 i. p. YFV 17D infection model only 1 pfu of YFV 17D is sufficient to induce lethal infection in AG129 mice. A similar high susceptibility of AG 129 mice has been reported for the JEV SA 14-14-2 vaccine strain [Calvert et al. (2014) Vaccine 32, 258-264] and ZIKV MR776 [Zmurko et al. (2016) PLoS Negl Trop Dis. 10, e0004695] . Using this sensitive AG 129 mouse model and i. p. infection with tissue-culture derived viruses, the attenuation of JE-CVax is compared to YFV 17D in vivo. This analysis revealed that Imojev®/JE-CVax is 10⁵- fold more attenuated compared to Stamaril®/YFV 17D in vivo (Fig-1 ). This high degree of attenuation allows to test JE-CVax vaccine efficacy against lethal YFV challenge in the stringent AG129 mouse model using YFV 17D as surrogate for wild- type YFV.

We constructed a DNA infectious clone of JE-CVax/Imojev® : PLLAV-JE-CVax. Recent studies revealed that immune deficient AG 129 mice could be used as model to evaluate vaccine efficacy [Sumathy et al. (2017) Sci Rep. 7, 46375] . Combining JE- CVax vaccination and YFV 17D challenge, we evaluated the protective efficiency of JE-CVax (and of its genuine PLLAV-JE-CVax version), that the present invention illustrates that both the vaccines were able to provide almost complete protection against YFV infection in immunodeficient AG 129 mice (Fig-2). Vaccination with 10⁴ PFUs JE-CVax or 10. ug PLLAV-JE-CVax confers 97% (n = 35/36) and 89-100% protection, respectively. This protection appears to be very specific to YFV as animals vaccinated with JE-CVax and subsequently challenged with YFV failed to survive another lethal ZIKV challenge without any significant difference with placebo (Fig-4). Our data reveals that the safety window between complete protection against YFV 17D lethal challenge ( 10⁴ PFUs) vs. absolute lethality of JE-CVax inoculation ( 10^s PFUs) in young 6-8 weeks old AG 129 mice is very narrow. If older animals were vaccinated ( 16 weeks old) this window could be increased. Experimentation with kinetics of protection post vaccination against YFV revealed that there was 60% and 100% protection already seven days and 14 days post vaccination (Fig-3). This is quite comparable to what we see in human population following YFV 17D vaccination, where vaccine-mediate immunity is raised within 10 days [Xie et al. (2017) MBio. 8(1), e02134-16] . It has previously been reported that non-structural proteins may also provide (partial) protection against Flaviviruses [Schlesinger et al. (1993) Virology 92(1), 132-141 ; Amorim et a/. (2016) Virology. 487, 41-49; Rastogi et al. (2016) Virol J. 13, 131 ; Watterson et al. (2016) Antiviral Res. 130, 7-18; Campbell et al. (2011) Bull World Health Organ. 89( 10), 766-774, 774A-774E] . However, the present invention provides evidence for a viable LAV vaccination platform based on nonstructural proteins, as well as chimeric vaccine-mediated protection against its parental target. The dual protection conferred by vaccination with c-LAVs like Imojev® or Dengvaxia® against YFV as well as JEV/DENV will be advantageous in (i) firstly in reducing number of vaccinations in vaccination regimens and (ii) secondly will provide alternative options for vaccination in case of YFV vaccine shortage [Gershman et al. (2017) MMWR Morb Mortal Wkly Rep. 66, 457-459] . Another advantage with chimeric vaccinations will be that spillage of one Flavivirus to other areas with the prevalence of other Flavivirus and common vector may be controlled. Likewise, using c-LAVs constructed in DENV backbone [Halstead & Russell (2016) Vaccine 34, 1643-1647] may also help to overcome with the problem of antibody dependent enhancement of dengue vaccines [Barrett et al. (2009) Curr Opin Immunol. 21, 308-313] . Similar will apply to c-LAV for ZIKV using a ZIKV backbone. Our UFA based serological analysis with only JE-CVax vaccinated or only YEV challenged animals revealed that there is serological cross-reactivity between both JEV and YFV infected serum (Fig-5). It is well established that any Flavivirus infection or vaccination generally results in the induction of a certain amount of broadly Flavivirus cross-reactive serology with non-significant cross-neutralization [Stiasny et al. (2006) J Virol. 80, 9557-9568] . In line, we failed to detect any neutralizing antibodies against YFV in only JE-CVax vaccinated animals in a CPENT assay (Fig-6). All the animals post JE-CVax vaccination had neutralizing antibodies against JEV ( log ioCPENTso: 2.4478 ± 0.2890; 1.7161-3.0392) but none against YFV. This was further reconfirmed in JE-CVax vaccinated and twice boosted animals. A recent vaccination study by Nasveld et. al. comparing vaccination outcomes with JE-CVax and YFV 17D revealed that these finding also apply in humans which also that there is no cross-neutralization of JEV or YFV after YFV 17D or JE-CVax vaccination, respectively [Nasveld et a/. (2010) Hum Vaccin. 6, 906-914] . All the animals except one survived post YFV challenge, however, there were variation in YFV neutralizing antibody titers post YFV challenge (log ioCPENTso: 1.7307 ± 0.3123; non-detectable at 1 : 20 dilution; 1.3010-2.4384). Some animals did not reveal any neutralizing antibodies against YFV post challenge, which also may be indicative of protection mediated by non-neutralizing antibodies against NS 1 and E [Stiasny et al. (2006) J Virol. 80, 9557-9568] and/or by immune cells [Mladinich et al. (2012) Immunogenetics 64, 1 1 1- 121 ] . Previous studies had demonstrated that NS1 based vaccination can provide protection against lethal YFV challenge in mice and monkeys [Putnak et al. ( 1990) J Gen Virol. 71(Pt 8), 1697- 1702; Schlesinger et al. ( 1993) Virology 92, 132- 141] . Gershman et al. (2017) MMWR Morb Mortal Wkly Rep. 66, 457-459] and Schlesinger et al. [cited above] also reported presence of non- neutralizing antibodies in monkeys serum post NS1 vaccination and absolute protection post lethal YFV challenge. Similarly, our ELISA analysis also revealed that in YFV 17D challenged and JE-CVax animals there are comparable levels of anti-YFV NS1 antibodies and these are absent in serum of placebo or ZIKA challenged animals alone [Fig-7] . There seems to be some correlation data between anti-NS l antibody level and JE-CVax dose needed for protection. Conversely, our passive adoptive hyperimmune-serum transfer data indicate that there is a partially protective effect of anti-NSl antibody levels against YFV 17D (due to the stringency of the fatal model seen only at low viral inoculum). This may be attributed to the fact that as YFV 17D replicates very efficiently in the AG 129 mouse YFV lethal challenge model and minimum antibody concentrations required for protection could not been consistently reached . The definite mechanism of action of JE-CVax-mediated protection against lethal YFV challenge may depend on the synergy of several effector principles, however protection is strangely not correlated with nAb.

To conclude, c-LAV such as I mojev®/ JE-CVax can be medically used in another manner than originally indicated, namely against YFV, which in case is not mediated by neutralizing antibodies and suggest that Imojev® could be used off-label against YFV and also as dual vaccine against JEV and YFV both. Therefore, development of c-LAVs may reduce number of vaccinations required in vaccination regimen, minimize the spillage concerns of viral infection in newer areas and provide sustainable alternative vaccine strategy in case of shortage of parental vaccine. A similar principle applies to all current c-LAV Flavivirus vaccine (such as Dengvaxia®) as well as c-LAV currently under development. Likewise, c-LAV can be generated against DENV and other viruses at risk of ADE by switching their antigenic surface proteins for those of antigenically distinct and distantly related viruses or serotype avoiding the induction of potentially harmful nAb responses. Examples

Example 1. Methodology

Cells and medium

BH K-21J (baby hamster kidney cells; ATCC CCL-10) and Vero E6 (Vero C1008; ATCC CRL- 1586) cells were maintained in seeding medium containing MEM Rega-3 medium (Gibco, Belgium) supplemented with 10% fetal calf serum (FCS, Gibco, Belgium), 1% sodium bicarbonate (Gibco, Belgium) and 1% glutamine (Gibco, Belgium). Virus culture and cytopathic effect based virus neutralization assays (CPENT) were performed in the assay medium, which is the seeding medium supplemented with only 2% FCS. All cultures were maintained at 37°C in an atmosphere of 5% C02 and 95%-99% humidity.

Virus, Vaccine and construct

The examples of the present application are based on the earlier published PCT application WO2014/174078 using inducible bacterial artificial chromosomes, in which we combine a reverse genetics approach with LAVs. Using this technology we re-engineered the c-LAV (Chimerivax-JE, JE-CVax) present in Imojev® (Arroyo et a/. (2001) Virol. 75, 934-942; US7459160) as a biological replicate as represented by its genomic nucleotide sequence.

Stamaril™ (lot G5400) were bought from Sanofi Pasteur (France), passaged two- times in vero E6 cells (YFV 17D-G5400P2), aliquoted and stored at (-80°C). YFV 17D- G5400P2 was used throughout the study to challenge AG129 mice and is referred as YFV 17D. Three DNA fragments encoding the prM and envelope proteins of JEV (as in the Imojev® c-lav) were custom synthetized as gene block and assembled in- house into the YFV 17D backbone of the inducible BAC construct disclosed in WO2014174078 to generate YFV 17D based Japanese encephalitis c-PLLAV (PLLAV- JE-CVax). BHK 21J cells were the transfected with PLLAV-JE-CVax plasmid using TransIT®-LTl transfection reagent (Mirrus Bio LLC, Belgium) following manufacturer's instructions. Upon onset of cytopathic effect (CPE) JE-CVax virus was harvested, centrifuged at 4000 rpm at 4°C for 10 minutes, aliquoted and stored in (- 80°C). JE-CVax virus was subsequently passaged on BHK 21J to generate virus stocks. As an alternative challenge virus, ZIKV strain MR766 was used in the present study [Zmurko et al. (2016) PLoS Negl Trop Dis. 10, e0004695] . Virus titers were determined on BHK 21J cells by plaque assays (plaque forming unit/ml) and CPE- based assays (TCIDso/ml) as described below. Animals, vaccination and infection

Immunodeficient interferon (IFN)-a/ and -γ receptor knockout mice (AG129; B&K Universal, Marshall Bio resources, UK) were bred in house. AG129 mice have been shown to be highly susceptible to lethal YFV 17D infection serving as a well established surrogate rodent challenge model for wild type YFV infection (Meier KC, Gardner et al (2009) PLoS Pathog. 5, el000614; Thibodeaux et al. (2012) Vaccine 30, 3180-3187). Six to eight weeks old male AG129 mice were used for all experiments after random assignment to different groups. Animals were kept in ventilated type-2 filter top cages on a 12 hour day/night cycle with water and food ad libitum. Housing of animals and procedures involving animal experimentation were conducted in accordance with institutional guidelines approved by the Ethical Committee of the KU Leuven, Belgium on licenses P168/2012 and P103/2015. Throughout the study, animals were vaccinated intraperitoneally (i.p.) with 10⁴ plaque forming units (PFUs) of JE-CVax or 10μg PLLAV-JE-CVax. Four weeks later, vaccinated animals were challenged i.p. with 10³ PFUs of YFV 17D (corresponding to 10³-times a 100% lethal dose), if not stated otherwise. An additional four weeks post YFV 17D challenge, some animals were rechalleged i.p. with 10⁴ PFUs ZIKV MR776. All transdermal PLLAV JE-CVax vaccinations were performed via jet injection (Injex®, United Kingdom) using transfection reagent polyethylenimine (PEI, in wVo-jetPEI®, Polyplus, France) as per manufacturer's instruction. Similarly, all needle inoculations of PLLAV JE-CVax via the i. p. route were performed also using PEI. Animals were observed for morbidity and mortality (weight loss and humane end-point) once daily. The humane end point is defined as paresis/difficulty in walking, paralysis (hind legs/soured eyes), moribundity / ataxia / tremors / difficulty breathing, 20% weight loss or quick weight loss (15% within 1 or 2 days) and animals were immediately euthanized once reaching the humane end-point. To evaluate the role of antibody-mediated protection against lethal YFV challenge by JE-CVax, we inoculated non-vaccinated AG129 mice passively with serum that, based on our results, does not contain neutralizing antibodies against YFV. For adoptive serum transfers, hyperimmune serum was prepared by vaccinating AG129 mice with 10⁴ PFUs JE-CVax, followed by two boosts with 10⁵ PFUs in 14-day intervals. Another 14 days after the second booster, animals were bled once to twice per week for the following four weeks. All serum batches then were pooled and CPENT assays were performed before serum transfers. Non-vaccinated animals were administered with 250 μΙ of serum one day prior to challenge with either 10¹, 10² or 10³ PFU. Animals were re-administered with 250 μΙ of serum on day 1 or day 4 and on day 8; and observed for morbidity and mortality for 4 weeks.

Depletion of serum complement, T cells, neutrophils and macrophages prior to challenge

Innate cell depletion and Fc receptor blockage in vivo

Four weeks after vaccination of AG129 mice with JE-CVax, different innate effector functions were depleted or blocks to determine the mode of protection against YFV challenge. Therefore, 500 μg of anti-Ly6G mAb (1A8, BioXCell) were inoculated IP one day prior to challenge to deplete neutrophils; 200 μg of anti-Ly6C/G (Grl) mAb (RB6-8C5, BioXCell) were inoculated IP one day prior to challenge to deplete neutrophils and monocytes; 500 μg of non-depleting anti-Fc receptor CD16/32 mAb (2.4G2, BioXCell) were inoculated IP one day prior and one day post challenge to block Fc receptor-mediated antibody effector function; or 20 μg cobra venom factor (CVF) was inoculated IP one day prior to challenge to deplete complement-activating functions.

Indirect Immunofluorescence assay (UFA)

To determine the seroconversion of animals, all JEV, YFV and ZIKV IgG-IIFAs were performed as per manufacturer's instruction (Euroimmune, Germany) except for the use of labelled secondary antibody and mounting agent glycerine, which were replaced by Alexa Fluor 488 goat anti-mouse Ig (A-11029, ThermoFisher, Germany) and DAPI (ThermoFisher, Germany), respectively. Serum from non-vaccinated animals served as naive, negative control. Slides were visualized using a fluorescent microscope (FLoid Cell Imaging Station, ThermoFisher, Germany).

Plaque assay Viral titers of YFV 17D or JE-CVax preparations were determined using plaque assays on BHK-21J cells overlaid with agarose [Lindenbach et a/. (1999) J Virol. 73, 4611- 4621] with some modifications. In brief, 106 BHK-21J cells per well were plated in 6- well plates and cultured overnight in seeding medium. Cells were washed with PBS and inoculated with virus of different dilutions prepared in the assay medium for one hour at room temperature (RT). Culture supernatants of uninfected cells were used as negative controls. Cells were thoroughly washed with the assay medium and overlaid with 2x MEM (Gibco, Belgium) supplemented with 20% FCS and 1% sodium bicarbonate containing 0.5% low melting agarose (Invitrogen, USA). The overlay was allowed to solidify at RT, cells were then cultured for 7 days at 37 °C, fixed with 8% formaldehyde and stained by methylene blue. Plaques were manually counted and plaque titer was determined as PFUs/ml.

Cell based cytopathic assay (CPE) and CPE based virus neutralization assay (CPENT)

Viral titers for culture derived YFV 17D or JE-CVax (TCID₅₀) and 50% neutralizing antibody titers (logioCPENT₅o) were determined using cytopathic effect (CPE)-based cell assays and CPE-based virus neutralization assays (CPENT), respectively, on BHK- 21J cells [Jochmans et al. (2012) J Virol Methods. 183, 176-179] with some modifications. In brief, 20,000 BHK-21J cells/well were plated in 96-well plates overnight in seeding medium. The medium then was replaced with assay medium containing different virus dilutions and cultured for 5 days at 37°C. Later, assays were first manually scored for CPE by eyes and then medium was replaced with 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-(4-sulfophenyl)-2H- tetrazolium / phenazinemethosulfate (MTS/PMS, Promega, Netherlands) solution. After incubation for 1.5 hours at 37°C in the dark, the optical density (OD) at 498 nm was determined for each well. All assays were performed in six replicates and TCID50 was determined using the Reed and Munch method [Reed & Muench (1938) Am. J. Hyg. 27, 493-497] .

CPENT assay was performed in a similar way as the CPE assays for viral titration with some modifications. Briefly, an additional step was added, where different serum dilutions made in the assay medium were first inoculated with 100 TCID50 YFV 17D or JE-CVax virus for 1 h at 37 °C and then added to the cells. All sera were assayed in triplicate in serial dilutions 1 : 20, 1;66, 1 :200, 1 :660, 1 : 2000 and 1 : 6600. CPE neutralization was calculated with the following formula : % neutralization activity = % CPE reduction = (OD us+serum - OD_vc) * 100 / (OD_cc - OD_Vc) and 50% neutralization titers (CPENT50) were calculated using the Reed and Munch method [Reed & Muench (1938) Am. J. Hyg. 27, 493-497]. Culture-derived YFV 17D or JE- CVax were used as positive virus controls, while culture supernatants of uninfected cells were used as negative cell control CPENT50 value for each sample represent geometric mean of three independent CPENT50 values.

Enzyme-linked immunosorbent assay (ELISA)

Serum antibodies recognizing YFV NSl were detected by indirect ELISA as previously described [43,44] . In brief, ELISA plates (Nunc MaxiSorp, Thermo Fisher Scientific cat. no. 44-2404-21) were coated with 1 μg/ml recombinant YFV NSl (Biorad, cat. no. PIP052A) in 50 mM carbonate buffer (pH 9.6) overnight at 4°C. After three washes with PBS-T (PBS with 0,05% Tween 80), plates were blocked with 2% BSA in PBS-T for 1 h at 37°C, or alternatively overnight at 4°C. After three washes with PBS-T, wells were treated with serial dilutions of test sera (2-fold serial dilution in PBST) for 2 h at room temperature. Serial dilutions of the YFV NSl-specific mouse IgG2a monoclonal antibody (clone 1A5) starting at 10 μg/mL served as standard. After four washes with PBS, plates were incubated with horseradish peroxidase- labelled goat anti-mouse IgG antibody (Sigma-Aldrich, USA, diluted 1 : 3000 in PBS- T). After another four washes with PBS, bound antibodies were detected via conversion of added TMB (SureBlue TMB Microwell Peroxidase; KPL, USA). The reaction was stopped after 10 minutes by adding equal quantities of 1 M HCI solution, and results were red at 450nm (OD450). After background subtraction, relative anti- YFV NSl titers were determined by comparison to the standard curve generated for mAb 1A5. In general, equivalence dilutions were derived at serum dilutions at which the respective OD450 equaled 1. Only values that exceeded three time the background signal were considered positive.

Intracellular staining of NSl protein in HEK-293 cells

HEK-293 cells were detached with trypsin-EDTA (0,05%), centrifuged (at 2500 rpm and 4°C for 5 minutes in a tube) and the supernatant was removed. The cells were then suspended in FACS-B and not more than 5 x 10⁶ cells per well were seeded in a round-bottom 96-well plate (200 μΙ/well). The plate was centrifuged and the supernatant was removed. Dead cells were stained in vitro with ZombieAqua, 1 : 500 diluted in DPBS, to exclude from final results. The cells were washed with FACS-B, the plate was centrifuged and the supernatant was removed. Next, all cells were fixed with 2% PFA in FACS-B followed by another washing step. FACS-B with 0,1% saponin, 1% NMS and streptavidin was added to the cells to permeabilize the cells and to block endogenous biotin, and the cells were stained with a fourfold concentrated anti-NSl primary antibody solution, or JE-ChimeriVax mouse serum, in permeabilizing and blocking solution, followed by a washing step. FACS-B with 0,1% saponin, 1% NMS and biotin was added to permeabilize the cells and to block residual streptavidin. Then, the primary antibody was stained with a fourfold concentrated biotinylated goat anti-mouse IgG secondary antibody solution (1 : 200 dilution), followed by a washing step. The biotinylated secondary antibody was stained with streptavidin-PE-Cy7 (1 : 200 dilution). After washing the cells, they were resuspended in FACS-B and filtered through 100 μιτι nylon meshes for analysis.

Statistical analysis

Graph Pad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA) was used or all statistical evaluations. Quantitative data were represented as mean ± standard deviation or geometric mean and obtained from at least three independent experiments. Statistical significance was determined using survival analysis with log- rank (Mantel-Cox) test and one-way ANOVA analysis. Values were considered statistically significantly different at p-values < 0.05. Example 2. Comparative evaluation of attenuation of JE-CVax in AG129 mice compared to YFV 17D

Chimeric Japanese encephalitis vaccine (JE-CVax/Imojev) has pre-membrane and envelope genes from Japanese encephalitis virus in YFV 17D backbone. Comparative evaluation of attenuation of JE-CVax in AG129 mice compared to YFV 17D : In plaque assays, we show that the chimeric JE-CVax virus seems to be highly attenuated compared to the parental YFV 17D, as the former generates smaller plaques (data not shown). No previous study is available comparing the pathogenicity of JE-CVax in susceptible AG129 mice. Therefore, we determined the lethal dose of JE-CVax in highly susceptible mice, in which even 1 PFU of YFV 17D causes 100% lethal disease. Six-weeks old AG129 mice (n= 5) were infected with different amounts of JE-CVax virus or YFV 17D and observed for morbidity and mortality for four-weeks. Animals (n= 5) injected with PBS were used as negative control. Additionally, we infected sixteen-weeks old animals (n=4) with 10⁵ PFUs JE-CVax or 10³ PFUs YFV 17D also. Irrespective of age, we observed 100% mortality in AG129 mice even if animal is infected with 1 PFU of YFV 17D (data not shown), whereas we experienced 100% mortality in six-weeks old AG129 mice only when infected with 10⁵ PFUs JE- CVax (Fig. 1). No negative impact of JE-CVax was observed in groups infected with lower PFUs than 10⁴ of JE-CVax or sixteen-week old animals. Therefore, we concluded that JE-CVax is ~ 10⁵-folds attenuated compared to YFV 17D in AG129 mice.

This illustrates that the AG129 mice is an excellent model to demonstrate the protective effect of a compound or treatment against the YFV 17D induced death.

Example 3. Evaluation of protective effect of chimeric JE-CVax vaccination against YFV 17D lethal challenge

JE-CVax has the pre-membrane and envelope gene from Japanese encephalitis virus (JEV) but the remaining backbone contains core and nonstructural (NS1-NS5) genes from YFV 17D. TThe protective effect of JE-CVax virus and PLLAV-JE-CVax BAC against yellow fever virus vaccine strain 17D (YFV 17D) was evaluated in the above described AG129-YFV 17D lethal challenge model. When we challenged animals (n = 5) with 1000 LDioo of YFV 17D 28 days later after vaccinating with 10³ or 10⁴ PFUs JE-CVax, we observed 80% and 100% survival, respectively. Therefore, throughout the study we vaccinated animals with 10⁴ PFUs. Our data presented in figure 2 indicate that all the JE-CVax vaccinated animals (n=35/36) except one survived YFV 17D challenged without any negative outcome, whereas all the PBC vaccinated animals (placebo; n=34) died due to YFV infection (mean days to death; 14.4±2.7 days, p-value; >0.0001). Therefore, we concluded that vaccination with JE-CVax can provide absolute protection against YFV in AG129 mice. Similarly to our analysis with JE-CVax virus, all the PLLAV-JE-CVax BAC + PEI (N = 13/13) intraperitoneal and almost all the PLLAV-JE-CVax BAC+ PEI (N=8/9) transdermal vaccinated animals survived YFV 17D challenged without any negative outcome. Therefore, we concluded that apart from JE-CVax virus, vaccination with PLLAV-JE- CVax BAC also provides protection against YFV in AG129 mice.

To further evaluate the time required for protection against lethal challenge of YFV 17D after vaccination with JE-CVax virus in AG129 mice, animals (n= 5) were first vaccinated with JE-CVax virus and were after 0, 4, 7, 14, 21 and 28 days post vaccination (dpv), challenged with YFV 17D. Controls animals were also infected with YFV 17D and JE-CVax alone. All the animals were observed for morbidity and mortality for a further five weeks. We observed 100% mortality in 0 dpv (MDD: 11.0 ± 1.7 days), 4 dpv (MDD: 11.6 ± 1.9 days) and YFV 17D alone (MDD: 14.2 ± 1.6 days) groups (Figure 3). 60% animals were protected against YFV 17D challenge after 7 dpv (MDD: 12.0 ± 0.0) and all the animals were protected from YFV 17D lethal challenge from 14dpv onwards (Figure 3). Therefore, we concluded that within two weeks of vaccination JE-CVax virus provides absolute protection against lethal YFV 17D challenge. Example 4. Evaluation of cross-protection against ZIKV in dual vaccinated (JE-CVax and YFV 17D) AG129 mice

To evaluate whether the cross protection against YFV against Fiaviviruses in general or specific against YFV 17D, JE-CVax vaccinated and YFV challenged AG129 mice were re-challenged with 10⁴ PFUs ZIKV MR776 and animals were observed for morbidity and mortality. Non-vaccinated animals of same aged group were also challenged with ZIKV. We did not observe any protection against ZIKV after JE-CVax vaccination and YFV 17D challenge (Figure 4). This revealed that protection against YFV by JE-CVax is specific and for Fiaviviruses in general. The experiment further demonstrates that dual vaccination with JE-CVax and YFV 17D did not provide any protection against ZIKV.

Example 5. Serological analysis of JE-CVax/PLLAV-JE-CVax vaccinated and YFV 17D challenged animals

To evaluate the mechanism behind the JE-CVax-mediated protection against YFV, we analyzed serum of pre-vaccinated (day 0), YFV 17D infected alone, JE-CVax/PLLAV- JE-CVax vaccinated (day 28) and YFV 17D post-challenge serum (day 35) using either UFA or CPENT assay for seroconversion/cross-reactive antibodies and neutralizing antibodies, respectively. As expected, we were failed to detect any reactivity against JEV or YFV in pre-serum samples (Figure 5) but observed cross-reactivity for JEV and YFV in YFV 17D infected and JE-CVax vaccinated serum samples, respectively. In align to the UFA data we were not able to detect any YFV 17D or JE-CVax neutralizing activity in pre-serum of infected or vaccinated individuals. We also failed to detect any neutralizing antibodies against JE-CVax in YFV 17F infected serum (data not shown), confirming limited cross-neutralisation. When animals were vaccinated with JE-CVax or PLLA-JE-CVax, we consistently observed only high levels of nAb for JE- CVax (logioCPENTso: 2.4478 ± 0.2890; 1.7161-3.0392) prior to YFV challenge, but detected varying levels of neutralizing antibodies against YFV 17D (logioCPENTso: 1.7307 ± 0.3123; non-detectable at 1 : 20 dilution; 1.3010-2.4384) post YFV 17D challenge (Figure 6). Serological analysis of serum from JE-CVax hyperimmune animals also revealed that animals do not have neutralizing antibodies against YFV but do have neutralizing antibodies for JE-CVax (logi₀CPENT₅₀: 2.9279 ± 0.2018; n = 13).

Similar findings were obtained with PLLAV-JE-CVax (data not shown). Therefore, we can conclude that protection provided by JE-CVax is not dependent on neutralizing antibodies but some other mechanism is involved in protection against YFV. However, YFV challenge induced seroconversion against YFV, serving as marker for exposure. Similar data were obtained from JE-CVax hyperimmune animals, which were vaccinated trice by JE-CVax every two weeks and bled two weeks post final vaccination. All the animals (n= 13) revealed neutralizing antibodies against JEV (logioCPENT₅o: 2.9279 ± 0.2018) but fail to reveal any neutralizing antibodies against YFV.

Example 6. Determination of anti-NSl YFV antibody levels in JE-CVax vaccinated animals

From our serological analysis, we concluded that JE-CVax vaccinated serum contained antibodies that did not neutralize but bound to components of YFV-infected cells. This reactivity may be attributed to NS1, which is a secreted protein that can induce protective immunity. To evaluate the presence of anti-NSl YFV antibodies in JE-CVax-vaccinated animals, ELISA was performed using serum from mice inoculated with PBS (sham) or vaccinated with 10³-10⁵ PFUs JE-CVax, 10⁴ PFUs YFV 17D or 10⁵ PFUs ZIKV MR339. Serum was collected either at onset of disease (YFV 17D, 10⁵ PFUs JE-CVax and ZIKV) or 28 days post vaccination. ELISA data revealed (Figure 7A) that equivalent amounts of anti-NSl YFV antibodies in JE-CVax-vaccinated animals (10³-10⁵ PFUs: 12.2 ± 4.9, 21.3 ± 3.4 and 20.1 ± 8.1 ng/ml, p-value; 0.535, 0.705 and 0.774, respectively) or YFV 17D-infected animals (17.2 ± 5.6 ng/ml). However, no anti-NSl YFV antibodies could be detected in sham administered animals (p-value; 0.0004) and only very low level of antibodies cross-reacting with YFV-NS1 in ZIKV infected serum sample (1.7 ± 0.5 ng/ml, p-value; 0.0022). No significant difference in serum anti-NSl YFV antibodies levels existed between JE- CVax vaccinated and YFV 17D infected mice (p-value > 0.05). Along the same lines, flow cytometry revealed that antibodies in the serum of JE-CVax-vaccinated mice bound to overexpressed NS1 as well as to cells infected with YFV 17D (Figure 7B). The observed staining pattern of serum JE-CVax-vaccinated mice was similar to that of a monoclonal antibody (mAb) specifically detecting YFV NS1. Therefore, we conclude that most YFV-reactive antibodies in the serum of JE-CVax most likely recognize YFV NS1. Example 7. Evaluation of passive protection mediated by adoptive serum transfer

To evaluate whether the YFV-reactive antibodies mediate the JE-CVax-induced protection against YFV, a passive transfer of JE-CVax hyperimmune serum was performed into non-vaccinated AG129 mice. The hyperimmune serum was first evaluated for the presence of neutralizing antibodies against YFV 17D in CPENT assays. We did not observe any YFV 17D neutralizing antibodies in the hyperimmune serum but observed ~4-fold higher neutralizing titers against JE-CVax (logio CPENT50 hyperimmune = 3.03 ± 0.17 vs. serum from single-vaccinated mice 2.45 ± 0.29). In non-vaccinated AG129 mice (n > 5), we transferred 250 μΙ of the hyper-immune serum two to three times i.e. either on days (-01) and 01 or on days (-01), 04 and 08 post infection with 10³ LD₅₀ or Ιθ θ² LD₅₀ of YFV 17D [1] . We did not observe any significant effect on survival of mice after challenge with 10³ LD50 YFV post 2X- serum transfer. However, we did observe that when the frequency of serum transfer was increased from 2X (Figure 8A) to 3X (Fig 8B and 8C) and viral titer of infection was decreased from 10³ LD₅₀ (Fig-8A) to 10² LD₅₀ (Fig-8B) and 10¹ LD₅₀ (Fig-8C) [2], respectively, a marked delay or even protection against YFV 17D challenge could be observed. Therefore, we conclude that passive transfer of JE-CVax-immune serum can provide some protection against lethal YFV challenge, likely being mediated via antibodies targeting YFV NSl.

Example 8. Depletion of innate effector cells or blockage of Fc receptors cannot abolish JE-CVax-mediated protection against YFV

We hypothesized that JE-CVax-induced antibodies may bind to NSl on the surface of YFV-infected cells and thus mediate protection via antibody-dependent cytotoxicity (ADCC). During this process, the Fc receptors of innate immune cells recognize antibody-opsonized, infected cells and kill them via secretion of cytotoxic granules. To test this hypothesis, we depleted neutrophils (inoculation of anti-Ly6G) or monocytes & neutrophils (anti-Ly6C/G, Grl), complement-activating components (inoculation of cobra venom factor, CVF) or blocked antibody binding to Fc receptors (anti-CD16/32) in AG129 mice, four weeks after vaccination with JE-CVax. Non- depleted but vaccinated mice served as controls. Even though flow cytometric analysis (data not shown) confirmed marked depletion of neutrophils and/or monocytes or complement, none of these depleting strategies led to abrogation of the JE-CVax-induced protection against YFV infection. We thus conclude that the cross protection in JE-CVax-vaccinated animals against YFV infection is an extremely robust phenotype that not only relies on anti-YFV NS1 antibodies but may also involve T cell responses directed against the NS proteins of YFV.

Example 9. Mice vaccinated with YF-ZIKprM/E vaccine virus are fully protected against Zika and YFV-17D challenge viruses.

Zika virus (ZIKV) strains BeH819015 (Widman et al. mBio 2017) was used as vector to generate a chimeric virus (ZIK/JEprM/E) wherein the structural proteins, prM and E including the C anchor sequence of ZIKV were replaced by those of the Japanese encephalitis virus (JEV).

To demonstrate the safety of ZIK-JEprM/E, AG129 mice were either sham-vaccinated or vaccinated with decreasing doses of virus, namely, 10⁶, 10⁵, 10⁴ and 10³ PFU and were observed for 28 days. No mortality was recorded following vaccination. To investigate vaccine efficacy, the aforementioned mice were challenged with 10⁴ PFU (10⁴ LDioo) (Zmurko et al. 2016) of a heterologous ZIKV strain, MR766. As expected, sham-vaccinated mice succumbed to virus-induced weight loss (Fig. 10A) and were subsequently euthanized (Fig. 10B), with mean days to euthanasia (MDE) of 14± 1. In contrast to sham-vaccinated mice with 100% mortality, 42% (8/19) of vaccinated mice succumbed to ZIKV infection with a significant delay in mortality (MDE = 25±3). The remaining 58% (11/19) survived the challenged and stayed healthy throughout the observed period of 28 days.

The protective efficacy and safety of YF-ZIKprM/E against ZIKV was shown in stringent AG129 mouse models in which doses as low as 10² PFU were sufficient to elicit robust neutralization antibody (nAb) titres as early as 7 days post vaccination (Kum et al 2018). The vaccine virus was also shown to elicit strong CD4⁺ and CD8⁺ T cell responses against the ZIKV structural and YFV-17D non-structural proteins (Kum et a/ 2018), raising the question as to whether the multi-functional (memory) T cell responses against the YFV-17D non-structural proteins may confer protection against a lethal YFV challenge. To address this, mice deficient in both interferon-α/β and -γ receptors (AG129) (known to be highly susceptible to flavivirus infections) were either sham-vaccinated with 2% FBS medium or vaccinated with 10⁴ PFU of YF- ZIKprM/E. Twenty-eight (28) days post infection, vaccinated and sham-vaccinated mice were challenged with lxlO³ PFU of YFV-17D (Fig. 11A) and monitored for signs of challenge virus infection such weight loss, hunch back, ruffled fur and mortality. As expected, all sham-vaccinated mice were moribund; progressively lost weight (Fig. 11B) and were euthanized between days 13 and 18 post challenge (Fig . 11C). Five (5) days post challenge, vaccinated and sham-vaccinated mice were bled to quantify challenge virus viremia by qRT-PCR. Both vaccinated and sham-vaccinated mice developed viremia following challenge but YF-ZIKprM/E vaccinated mice had a significantly reduces viral load compared to the sham-vaccinated mice (Fig. 11D). At euthanasia, organs of mice were harvested, and virus RNA was quantified by qRT- PCR. For every two symptomatic sham-vaccinated mice euthanized, one asymptomatic vaccinated mouse was euthanized for RNA extractions in organs. Vaccinated mice had a significant reduction in viral load in various organs like the brain, spleen and liver (Fig. HE). Subsequently, these mice were completely protected against persistent virus induced weight loss and survived throughout the observed period of 28 days (Fig. 11C). This was in sharp contrast to sham-vaccinated mice that progressively weight lost (Fig. 11B) and showed overt signs of infection like paralysis, lethargy and were subsequently euthanized (Fig. 11C).

Claims

Claims:

1. A chimeric live infectious attenuated flavivirus of a first and a second flavivirus for use in the prevention of an infection by said first flavivirus, wherein at least one of the structural proteins of the first flavivirus is replaced by the corresponding at least one of the structural proteins of the second flavivirus, and wherein the second flavivirus is not Dengue or Zika virus.

The chimeric flavivirus for use in the prevention according to claim 1, wherein the prM-E proteins of the first flavivirus are replaced by the prM-E proteins of the second flavivirus.

The chimeric flavivirus for use in the prevention according to claim 2, wherein in addition the signal peptide of the C terminal part of the capsid protein of the first flavivirus is replaced by the signal peptide of the C terminal part capsid protein of the second flavivirus.

The chimeric flavivirus for use in the prevention according to claim 1 or 2, wherein the signal peptide of the C terminal part of the capsid protein of the first flavivirus is not replaced.

The chimeric flavivirus according to any one of claims 1 to 4, for use in the prevention of a infection by both said first and second flavivirus.

The chimeric flavivirus according to any one of claims 1 to 5, for use in the prevention according to any one of claims 1 to 5, wherein said first flavivirus is not Yellow Fever virus.

7. The chimeric flavivirus according to any one of claims 1 to 6, for use in the prevention according to any one of claims 1 to 6, wherein said second flavivirus is Yellow Fever flavivirus.

8. The chimeric flavivirus according to any one of claims 1 to 6, for use in the prevention according to any one of claims 1 to 6, wherein said second flavivirus is Japanese encephalitis.

9. The chimeric flavivirus according to any one of claims 1 to 8, for use in the prevention of an infection by Dengue virus, wherein the first flavivirus is Dengue virus and wherein the prM-E proteins of Dengue virus are replaced by the prM-E proteins of a second flavivirus other than Zika virus.

10. The chimeric flavivirus according to claim 9, for use in the prevention of an infection by Dengue where said second flavivirus is Yellow Fever virus.

11. The chimeric flavivirus according to claim 9, for use in the prevention of an infection by Dengue where said second flavivirus is Japanese encephalitis virus.

12. The chimeric flavivirus according to any one of claims 1 to 8, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and wherein the prM-E proteins of Zika virus are replaced by the prM-E proteins of a second flavivirus other than Dengue.

13. The chimeric flavivirus according to claim 12, for use in the prevention of an infection by Zika virus where said second flavivirus is Yellow Fever virus.

14. The chimeric flavivirus according to claim 12, for use in the prevention of an infection by Zika virus where said second flavivirus is Japanese encephalitis virus. 15. The chimeric flavivirus according to claim 14, for use in the prevention of an infection by Zika virus. wherein the first flavivirus is Zika virus and the second virus is Japanese encephalitis virus, and wherein the signal peptide of the C terminal part of the C protein is from Zika virus, 16. A polynucleotide comprising a sequence encoding a live infectious attenuated chimeric flavivirus of a first and a second flavivirus for use in the prevention of an infection by said first flavivirus, wherein the sequence encoding at least one of the structural proteins of the first flavivirus is replaced by the sequence of encoding at least one of the structural proteins the second flavivirus, and wherein the second flavivirus is not Dengue or Zika virus. The polynucleotide for use in the prevention according to claim 17, wherein the sequence encoding the prM-E protein of the first flavivirus are replaced by the sequence encoding the prM-E proteins of the second flavivirus.

The polynucleotide according to claim 17, for use in the prevention according to claim 16, wherein further the sequence encoding the signal peptide of of the C terminal part of the capsid protein of the first flavivirus is replaced by the sequence encoding the signal peptide of the capsid protein of the second virus.

The polynucleotide according to claim 17 for use in the prevention according to claim 16 or 17, wherein the signal peptide of the C terminal part of the capsid protein of the first flavivirus is not replaced.

The polynucleotide according to any one of claims 16 to 19, for use prevention of an infection by both said first and second flavivirus.

The polynucleotide according to any one of claims 16 to 19, for use in the prevention according to any one of claims 16 to 19, wherein said first flavivirus is not Yellow Fever virus.

22. The polynucleotide according to any one of claims 12 to 17, for use in the prevention according to any one of claims 12 to 17, wherein said second flavivirus is Yellow Fever flavivirus.

23. The polynucleotide according to any one of claims 16 to 20, for use in the prevention according to any one of claims 16 to 20, wherein said second flavivirus is Japanese encephalitis virus.

The polynucleotide according to any one of claims 16 to 23, for use in the prevention of an infection by Dengue virus, wherein the first flavivirus is Dengue virus and wherein the sequence encoding the prM-E proteins of Dengue virus are replaced by the sequence encoding the prM-E proteins of a second flavivirus other than Zika virus. The polynucleotide according to claim 24, for use in the prevention of an infection by Dengue or Zika virus, wherein said second flavivirus is Yellow Fever virus.

The polynucleotide according to claim 24, for use in the prevention of an infection by Dengue or Zika virus, wherein said second flavivirus is Japanese encephalitis virus.

The polynucleotide according to claim 16 to 23, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and wherein the sequence encosing the prM-E proteins of Zika virus are replaced by the sequence encoding the prM-E proteins of a second flavivirus other than Dengue.

The polynucleotide according to claim 27, for use in the prevention of infection by Zika virus where said second flavivirus is Yellow Fever virus.

29. The polynucleotide according to claim 27, for use in the prevention of an infection by Zika virus where said second flavivirus is Japanese encephalitis virus.

The chimeric flavivirus according to claim 29, for use in the prevention of an infection by Zika virus, wherein the first flavivirus is Zika virus and the second virus is Japanese encephalitis virus, and wherein the signal peptide of the C terminal part of the C protein is from Zika virus.

The polynucleotide according to any one of claims 16 to 30, for use in the prevention according to any one of claim 16 to 30, wherein said polynucleotide is a Bacterial Artificial Chromosome comprising an inducible origin.

32. A method of inducing a neutralizing antibody response against a first flavivirus in a subject, thereby preventing an infection by said first flavivirus, comprising the step of administering to said subject a chimeric live infectious attenuated flavivirus of a first and a second flavivirus in accordance with any one of claims

1 to 15 or administering to said subject a nucleotide sequence encoding chimeric live infectious attenuated flavivirus of a first and a second flavivirus in accordance with any one of claims 12 to 31.