Vaccine

Abstract

The invention provides a vaccine composition comprising a Yellow Fever Virus vaccine, for use in vaccinating an individual against infection by a Flavivirus; wherein the Flavivirus is not Yellow Fever Virus. The invention also provides a vaccine composition comprising a Yellow Fever Virus vaccine and one or more additional vaccine against a Flavivirus, for use in vaccinating an individual against infection by the Flavivirus; wherein the Flavivirus is not Yellow Fever Virus.

Claims

1. A vaccine composition comprising a Yellow Fever Virus vaccine, for use in vaccinating an individual against infection by a Flavivirus; wherein the Flavivirus is not Yellow Fever Virus, West Nile Virus or Dengue; and wherein the Yellow Fever Virus vaccine generates a cross-reactive immune response to the Flavivirus.

2. A vaccine composition for use according to claim 1 wherein the individual has not previously been infected with the Flavivirus and/or vaccinated against the Flavivirus.

5. A vaccine composition for use according to claim 1 wherein the individual has not previously been infected with Yellow Fever Virus and/or vaccinated with the Yellow Fever Virus vaccine.

6. A vaccine composition for use according to claim 1, wherein the individual has previously been infected with the Flavivirus and/or vaccinated against the Flavivirus.

7. A vaccine composition for use according to claim 1, wherein the individual has previously been infected with Yellow Fever Virus and/or vaccinated with the Yellow Fever Virus vaccine.

8. A vaccine composition for use according to claim 1, wherein the Flavivirus is one or more Flavivirus selected from the group consisting of: Zika Virus; Tick-Borne Encephalitis Virus; Japanese Encephalitis Virus; West Nile Virus; Saint Louis Encephalitis Virus; Omsk Haemorrhagic Fever Virus.

9. A vaccine composition according to claim 1, wherein vaccinating the individual against infection by the Flavivirus prevents and/or reduces a subsequent infection by the Flavivirus and/or reduces one or more disorder and/or condition associated with infection by the Flavivirus.

10. A vaccine composition according to claim 9 wherein the prevention and/or reduction is effective for a period of up to 1 year or 5 years or 10 years.

11. A vaccine composition according to claim 9 wherein the reduction is by 100%, or by 90%, or by 80%, or by 70%, or by 60%, or by 50%.

12. The vaccine according to claim 1, wherein vaccinating the individual against infection by the Flavivirus generates and/or increases immunity in the individual against the Flavivirus.

13. A vaccine according to claim 12, wherein immunity comprises: cellular immunity and/or adaptive immunity in the individual against the Flavivirus; or wherein cellular immunity comprises T cell activity in the individual against the Flavivirus; or wherein the T cell activity comprises CD4+ T cell activity and/or CDS+ T cell activity.

14. A vaccine composition for use, or a use, or a method, according to claim 13 wherein adaptive immunity comprises B cell activity and/or antibody activity in the individual against the Flavivirus.

15. The vaccine of claim 1 wherein the antibodies bind to SEQ ID NO: 32, SEQ ID No: 32 SEQ ID No: 33, and/or SEQ ID No: 34

16. A vaccine composition comprising a Yellow Fever Virus vaccine and one or more additional vaccine against a Flavivirus, for use in vaccinating an individual against infection by the Flavivirus; wherein the Flavivirus is not Yellow Fever Virus, West Nile Virus or Dengue and wherein the Yellow Fever Virus vaccine and the one or more additional vaccine against a Flavivirus generate a cross-reactive immune response to the Flavivirus

17. A vaccine composition according to claim 16, wherein the one or more additional vaccine is selected from the group consisting of: a Zika Virus vaccine; a Tick-Borne Encephalitis Virus vaccine; a Japanese Encephalitis Virus vaccine; a West Nile Virus vaccine; a Saint Louis Encephalitis Virus vaccine; an Omsk Haemorrhagic Fever Virus vaccine.

18. A vaccine according to claim 17 wherein the one or more vaccine is intended to treat the Flavivirus of the one or more vaccine.

19. A vaccine composition for according to claim 16, wherein the individual has not previously been infected with the Flavivirus and/or vaccinated against the Flavivirus.

20. A vaccine composition for according to claim 16, wherein the individual has not previously been infected with Yellow Fever Virus and/or vaccinated with the Yellow Fever Virus vaccine.

21. A vaccine composition for according to claim 16, wherein the individual has previously been infected with the Flavivirus and/or vaccinated against the Flavivirus.

22. A vaccine composition for according to claim 16, wherein the individual has previously been infected with Yellow Fever Virus and/or vaccinated with the Yellow Fever Virus vaccine.

23. A vaccine composition for according to claim 16, wherein the Flavivirus is one or more Flavivirus selected from the group consisting of: Zika Virus; Tick-Borne Encephalitis Virus; Japanese Encephalitis Virus; West Nile Virus; Saint Louis Encephalitis Virus; Omsk Haemorrhagic Fever Virus.

24. A vaccine composition for according to claim 16, wherein vaccinating the individual against infection by the Flavivirus prevents and/or reduces a subsequent infection by the Flavivirus and/or prevents and/or reduces one or more disorder and/or condition associated with infection by the Flavivirus.

25. A vaccine composition for use, or a use, or a method, according to claim 24, wherein the prevention and/or reduction is effective for a period of up to 1 year; or 5 years; or 10 years.

26. A vaccine composition for use, or a use, or a method, according to claim 24, wherein the reduction by 100%, or by 90%, or by 80%, or by 70%, or by 60%, or by 50%.

27. A vaccine composition for use, or a use, or a method, according to claim 16 wherein vaccinating the individual against infection by the Flavivirus generates and/or increases immunity in the individual against the Flavivirus.

28. A vaccine composition for use, or a use, or a method, according to claim 27, wherein immunity comprises cellular immunity and/or adaptive immunity in the individual against the Flavivirus or wherein cellular immunity comprises T cell activity in the individual against the Flavivirus.

29. A vaccine composition, according to claim 28, wherein the T cell activity comprises CD4+ T cell activity and/or CDS+ T cell activity.

30. A vaccine composition according to claim 29 wherein adaptive immunity comprises B cell activity and/or antibody activity in the individual against the Flavivirus.

31. The vaccine of claim 30 wherein the antibodies bind to SEQ ID NO: 32, SEQ ID No: 32 SEQ ID No: 33, and/or SEQ ID No: 34

32. The use of a Yellow Fever Vaccine as an adjuvant.

33. A use according to claim 32, wherein the Yellow Fever Vaccine is used as an adjuvant for a vaccine, for example an inactivated vaccine.

34. A vaccine composition according to claim 16 wherein the Yellow Fever Virus vaccine is a live vaccine, and is preferably a live attenuated vaccine.

35. A vaccine composition according to claim 1, wherein the Yellow Fever Virus vaccine comprises a Yellow Fever Virus non-structural protein, or a fragment, variant or derivative thereof.

36. A vaccine composition wherein the Yellow Fever Virus vaccine comprises a polynucleotide sequence encoding a Yellow Fever Virus non-structural protein, or a fragment, variant or derivative thereof.

37. A vaccine composition for use, or a use, or a method, according to claim 36 wherein the Yellow Fever Virus non-structural protein comprises the NS5 protein, or a fragment, variant or derivative thereof.

38. The vaccine of claim 37 wherein the NS5 protein or fragment is selected from SEQ ID No: 1, SEQ ID No:39, SEQ ID No: 40, SEQ ID No: 41, SEQ ID No: 42, SEQ ID No:43, SEQ ID No: 44, SEQ ID No: 45, SEQ ID No: 46, SEQ ID No: 47, SEQ ID No:48, SEQ ID No: 49, SEQ ID No:50, SEQ ID No: 51, SEQ ID No:52, SEQ ID No: 53, SEQ ID No: 54, SEQ ID No: 55, SEQ ID No: 56, SEQ ID No: 57, SEQ ID No: 58, SEQ ID No:59, SEQ ID No:60, SEQ ID No:61, SEQ ID No:62, SEQ ID No: 63, SEQ ID No: 64, SEQ ID No: 65, SEQ ID No: 66, SEQ ID No: 67, SEQ ID No: 68 and/or SEQ ID No:69.

39. The vaccine of claim 1 wherein the vaccine generates antibodies which bind to E protein and NS1.

40. The vaccine of claim 16 wherein the vaccine generates antibodies which bind to E protein and NS1.

41. The vaccine composition of claim 1 further comprising a pharmaceutically-acceptable excipient or diluent.

42. The vaccine composition of claim 16 further comprising a pharmaceutically-acceptable excipient or diluent.

43. The vaccine of claim 1 wherein the vaccine is an Asibi strain, 17-DD, 17D204 or TBEV NEudorfl, TBEV Sojifin, TBEV Fe-205, TBEV Senzhang, JEV Nakayama, JEV SA-14-14-2, ZIKV Polyesian Strain 2013.

44. The vaccine of claim 1 which binds e protein or NS1 with 100, 1000 or 1000 times affinity of any other molecule in the patient.

45. The vaccine of claim 16 wherein the vaccine is an Asibi strain, 17-DD, 17D204 or TBEV NEudorfl, TBEV Sojifin, TBEV Fe-205, TBEV Senzhang, JEV Nakayama, JEV SA-14-14-2, ZIKV Polyesian Strain 2013.

46. The vaccine of claim 16 which binds e protein or NS1 with 100, 1000 or 1000 times affinity of any other molecule in the patient.

47. A method for vaccinating an individual against infection by a Flavivirus, the method comprising the step of administering to the individual a vaccine composition comprising a Yellow Fever Virus vaccine; wherein the Flavivirus is not Yellow Fever Virus; and wherein the Yellow Fever Virus vaccine generates a cross-reactive immune response to the Flavivirus.

48. The method of claim 47 further comprising administering at least additional vaccine against a Flavivirus; wherein the Flavivirus is not Yellow Fever Virus.

49. The method according to claim 48, wherein the Yellow Fever Virus vaccine and the at least one additional vaccine against a Flavivirus are administered simultaneously to the individual.

50. The method according to claim 48 wherein the Yellow Fever Virus vaccine and the one or more additional vaccine against a Flavivirus are administered sequentially to the individual.

Description

[0218] The invention includes a kit substantially as claimed herein with reference to the accompanying claims, description, examples and figures.

[0219] FIG. 1. Flavivirus RNA encodes for 10 proteins.

[0220] FIG. 2. Schematic figure of the NetCTL prediction score sites.

[0221] FIG. 3. Appearance of short-lived antibody-secreting plasmablasts at day 0, 7 and 15 or 28 (28 after TBE vaccine) after vaccinations. (A) Flow plot showing the plasmablasts in one alpha donor before and at day 7 and 15 after vaccination. (B) Plots showing the plasmablasts after administration with either YFV alone (blue), TBE vaccine alone (black), and YFV and TBE vaccines administered at the same time (red). Plots show plasmablasts in percent of total B cells.

[0222] FIG. 4. Levels of TBEV neutralizing antibodies after yellow fever vaccination. TBEV neutralizing antibodies in sera from three donors vaccinated with YFV vaccine. Alpha T0 received concomitant YFV and TBE prime vaccines, and thereafter two additional doses of TBE vaccine. Sampling time point was three year after primary immunizations, and levels of neutralizing antibodies measured to 80%. Beta Y1 received several TBE vaccines previously, with the last booster 4 months prior YFV vaccine and levels of neutralizing antibodies increase from 20% (before YFV vaccine) to 40% (80 days after YFV vaccine). Beta Y2 is TBE vaccine negative, and received YFV vaccine and no TBEV neutralizing antibodies were detected (data not shown).

[0223] FIG. 5. Cross reactive CD4 T cells against TBEV and ZIKV arise after concomitant administration of YFV and TBE vaccines. (A) PBMC from before and at days 15 and 22 after concomitant vaccinations were activated with ZIKV or TBEV overlapping NS5 libraries in one donor (alpha T2). (B) PBMC from before and at day 15 after vaccinations were activated with TBEV overlapping NS5 libraries in one donor (alpha T1). (C) PBMCs activated with TBEV overlapping NS5 libraries in one donor from day 28 after 2:nd and 3:rd dose of FMSE.

[0224] FIG. 6. Cross-reactive CD8 T cells against TBEV and ZIKV arise after administration with YFV and TBE vaccines. (A) Plots show IFN-γ and TNF double positive CD8 T cells after activation with either nothing (−cntr), YFV-NS4B covering library (+cntr), TBEV NS5 covering peptide library or ZIKV NS5 covering library in one donor vaccinated with YFV only (beta Y1). (B) Plots show IFN-γ and TNF double positive CD8 T cells at day 0 (top), 15 (middle) and 22 (bottom) after vaccination. Cells were activated with either YFV-NS4B covering library (+cntr), or ZIKV NS5 covering peptide library in one donor (alpha T2) receiving concomitant YFV and TBE vaccinations. (C) Plots show IFN-γ and TNF double positive CD8 T cells over time after activation with ZIKV NS5 covering peptide library in one donor (alpha T1) receiving concomitant YFV and TBE vaccinations. (D) Plots show PBMC from a TBE single vaccine, 28 days after administration after second and third TBE booster dose, activated with TBEV NS5 peptide library.

[0225] FIG. 7. Cross reactive T cells against TBEV and ZIKV arise after vaccination with YFV vaccine alone. (A) PBMC from two YFV vaccinated donors (HLA-A1 positive) were expanded for 6 days with either nothing (negative control, left panel), or predicted TBEV peptide ETACLSKAY (SEQ ID NO: 1) (right panel), 15 days after vaccination with YFV. Specific cells are measured as proliferating (CFSE low) and IFN-γ positive. (B) T cells 1 day and 15 days after vaccination were activated for 6h with predicted ZIKV NS5 CD8 epitopes. Specific cells are measured by the simultaneous expression of CD107a (degranulation) and TNF.

[0226] FIG. 8. Zika virus-specific T cells arise after yellow fever vaccination. PBMCs from two individuals (donor 1 and donor 2), activated with a Zika virus-specific NS5 library, before (day 0) and at day 15 following YFV vaccination. CD4 and CD8 T cells responding to the Zika-NS5 library are identified by production of IFN-γ and TNF. Numbers in figure indicate percentage of CD4 T cells and CD8 T cells.

[0227] FIG. 9. Vaccination and blood draw schedule for concomitant YFV and TBEV. A booster TBEV vaccine will be given at day 28 with blood draw the same day.

[0228] FIG. 10. Viral strains derived from Asibi strains.

[0229] FIG. 11. JEV NS5 stimulations of CD8 T cells (12h) on frozen PBMCs from a healthy donor receiving YVF vaccine (donor By4), before (day 0) and at 15 days after vaccination. Production of TNF increases from 0.03% to 0.3% which is a 10-fold increase in response to the JEV NS5 peptide library.

EXAMPLE 1: YFV VACCINE INDUCES/ENHANCES IMMUNE RESPONSE AGAINST OTHER FLAVIVIRUSES

[0230] Flaviviruses belong to the family of Flaviviridae and comprise over 70 viruses that cause severe diseases. These viruses are responsible for hundreds of thousands of deaths annually and additional significant morbidity. There are currently commercially available vaccines against three flaviviruses; YFV, JEV and TBEV (Table 1). Of these vaccines, the live attenuated YFV vaccine is one of the most effective and uses vaccines on earth.

TABLE-US-00002 TABLE 1 Overview of flaviviruses, endemic area and available vaccines. Virus Vector Endemic area Vaccine available JEV Mosquito (Culex) Asia IXIARO ®-Inactivated ZIKV Mosquito (Aedes) Africa, None available Latin America YFV Stamaril ®/YF-VAX ®- Live attenuated TBE Tick (Ixodes) Eurasia FSME-IMMUN and Encepur- Inactivated

[0231] This Example investigates the cross-reactivity of the YFV vaccine to other Flaviviruses, and in particular to TBEV and 21 KV.

Methods

Peptide Libraries

[0232] Peptide libraries are used to display multiple, linear peptide fragments in parallel to deduce specific epitopes. We have designed peptide libraries covering TBEV and ZIKV NS5 proteins, designed to activate both CD4 and CD8 T cells, with overlapping peptide segments of the entire antigen. Binding is assessed by flow cytometry and this method has been especially useful for developing vaccines.

Prediction of CD8 T Cell Epitopes

[0233] To identify virus-specific CD8 T cells with flow cytometry, T cell epitopes in the virus have to be identified if they are not already known. Epitope identification requires a systematic screening of the antigen, which can be difficult when the antigen has multiple conformations or binding domains. Another method to identify T cell epitopes to a specific pathogen is to utilize online search engines, which consider HLA-type and possible peptide presentation by the MHC molecule on the cell surface. The traditional whole genome library have the advantage of being HLA-unbiased while predicative algorithms represent a more high throughput technology. We have generated ZIKV and TBEV CD8 pools of predicted epitopes in ZIKV (Table 2) and TBEV (Table 3) NS5 proteins to the most common HLA-types (HLA-A1, A2, A3, B7 and B8). We used the NetCTL search engine to predict the epitopes, which integrates prediction of peptide-MHC class I binding, proteasomal C terminal cleavage and TAP transport efficiency (FIG. 2).

TABLE-US-00003 TABLE 2 Predicted ZIKV epitopes. HLA NetCTL Nr ZIKV SEQ ID NO: Supertype Score 1 ETACLAKSY 1 A1 1.7742 2 RTWAYHGSY 2 A1 1.6323 3 ALNTFTNLV 3 A2 1.2792 4 YMWLGARFL 4 A2 1.1720 5 RTTWSIHGK 5 A3 1.3336 6 CVYNMMGKR 6 A3 1.2696 7 GLVRVPLSR 7 A3 1.1146 8 YTYQNKVVK 8 A3 0.9942 9 NMMGKREKK 9 A3 0.8719 10 GLGLQRLGY 10 A3 0.8095 11 VPCRHQDEL 11 B7 0.9698 12 GLQRLGYVL 12 B8, A2 1.1986 13 WLGARFLEF 13 B8 1.0892 14 LLYFHRRDL 14 B8, A2 1.0109 15 SGQVVTYAL 15 B8 1.0040

TABLE-US-00004 TABLE 3 Predicted TBEV epitopes. HLA NetCTL Nr TBEV SEQ ID NO: Supertype score 1 STLNGGLFY 16 A1 2.9180 2 ETACLSKAY 17 A1 2.0304 3 GVEGISLNY 18 A1 1.9546 4 VMEWRDVPY 19 A1 1.2679 5 VLAPYRPEV 20 A2 1.2973 6 YMWLGSRFL 21 A2, B8 1.1707 7 MLVSGDDCV 22 A2 0.8937 8 YALNTLTNI 23 A2 0.8088 9 TLTNIKVQL 24 A2 0.7921 10 CVYNMMGKR 25 A3 1.2472 11 KLGEFGVAK 26 A3 1.2261 12 AKVKSNAAL 27 B7 0.8632 13 VVTYALNTL 28 B7 0.8505 14 IAKVKSNAA 29 B8 1.4929 15 SGQVVTYAL 30 B8 1.0018

Study Design and Subjects

[0234] Peripheral blood and sera was collected before and at days 7 and 15 from four donors vaccinated with a primary dose of YFV vaccine (Beta Y donors), and before and at days 7, 15 and 22 from two donors that received concomitant vaccination with primary doses of YFV and TBE (alpha T donors). As a negative control group, PBMCs were collected at day 28 from the second and third dose from individuals vaccinated with TBE (FSME) only. PBMC were isolated from EDTA tubes (BD Biosciences, San Jose, Calif.). PBMC were either stained fresh or cryopreserved in 90% FCS and 10% DMSO for later analysis.

Antibodies for Flow Cytometry

[0235] Immune responses were assessed using multi-color flow cytometry, and the monoclonal antibodies (mAbs) used were: anti-CD107a FITC, anti-CD4 brilliant ultraviolet 395, anti-CD19 Brilliant ultra violet 395, anti-CD4 brilliant ultraviolet 737, anti-HLA-DR APC, anti-Ki67 Alexa Fluor 700, anti-MIP-1β Alexa Fluor 700, anti-CD14 BD horizon V500, anti-CD19 BD horizon V500 and anti-TNF PE-CF594 and were all from BD Biosciences (San Jose, Calif.). Anti-CD45RA APC-Cy7, anti-IFN-γ Brilliant Violet 421, anti-CD27 Brilliant Violet 650, anti-CD38 brilliant violet 785, anti-IgG PE, anti IgD PE-Cy7 were all from Biolegend (San Diego, Calif.). Anti-CD8 Qdot 605 and Aqua Live/Dead were all from Invitrogen (Carlsbad, Calif.). Anti-CD3 PE-Cy5 and anti-CD56 ECD were from Beckman Coulter (Brea, Calif.).

Flow Cytometry

[0236] For phenotypic analysis of cells, PBMCs were incubated for 30 minutes 4° C. in the dark, with surface mAbs, followed by washing with PBS. For the CD107a staining, the CD107a antibody was present during the 6 hours stimulation, and then additional CD107a antibody was added together with the surface mAbs for 30 minutes incubation at 4° C. in the dark. Cells were fixed and permeabilized with fix/perm (eBioscience) for 30 minutes at 4° C. in the dark. Cells were then washed and stained with intracellular mAbs. Samples were acquired on a BD LSRFortessa instrument (BD Biosciences) and analyzed using FlowJo software version 9.4 (Tree Star, Ashland, Oreg.). B cell plasmablasts were identified as lymphocytes (extended gate), single cells, live cells and CD14/CD123 negative, CD3 and CD4 negative, CD20 negative and CD19 positive, CD27 and CD38 double positive. T cells were identified as lymphocytes, singlets, dump negative (CD19, CD14 and aqua dead cell marker) and positive for CD3, CD4 or CD8 positive.

In Vitro Functional Assays

[0237] PBMCs were rested in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 1% penicillin and streptomycin (Invitrogen) overnight at 37° C. Cells were stimulated with 10 μg peptides for 12 or 6 hours in 96-well round bottom plates in the presence of Brefeldin A (Sigma-Aldrich, St. Louis, Mo.), monensin (BD Biosciences) and purified anti-CD28/CD49d (1 μl/ml) (BD Bioscienses). Staining, flow cytometry and analyses were performed as described above.

Plaque Reduction Neutralization Test (PRNT)

[0238] Sera from vaccinated individuals were prepared according to standard clinical diagnostic protocol at Swedish Institute for infectious disease control.sup.22. Sera, including positive and negative controls, were inactivated and diluted two-fold in Hanks' basal salt solution with 2% inactivated FCS, 2% 1 M HEPES. Equal amounts of serum dilutions and virus at approximately 50 PFU: 100m l were mixed and tubes were incubated at 37° C. and 5% CO2 for 1 h. Following incubation, 200 μl of the serum-virus mixture was added to the plates with Vero cell monolayers. After incubation at 37° C. and 5% CO2 for 1 h, the wells were overlaid with 2 ml mixture of one part 1% agarose and one part 2 basal Eagle's medium supplemented with 8% FCS, 2% 1 M HEPES. The plates were incubated at 37° C. and 5% CO2 for 6 days, when a second overlay containing 3.3% neutral red was added at 2 ml/well. Plates were returned to the incubator and plaques were enumerated the following day. The test was accepted if the virus dose was in the range 30-70 PFU. Neutralizing antibody titres were calculated as the reciprocal of the serum dilution that gave an 80% reduction of the number of plaques, as compared to the virus control.

Results

High Cross-Homology in Flavivirus NS5 Protein

[0239] NS5 is a multifunctional, conserved protein in flaviviruses and constitutes the viral polymerase. We compared the sequences the NS5 protein in YFV, TBEV JEV and ZIKV and found that, YFV NS5 protein has over 60% homology with the NS5 proteins of TBEV, JEV and ZIKV (Table 4).

TABLE-US-00005 TABLE 4 Results after protein BLAST of flavivirus protein sequences. Complete protein NS5 protein Virus and strain (vaccine homology to YFV- homology to YFV- name) 17D204 (%) 17D204 (%) YFV 17D204 (Stamaril ®) 100 100 JEV-SA14-14-2 (IXIARO ®) 45 63 TBEV Neudeurfl (FSME ®) 43 61 ZIKV-polynesian isolate 46 64 West Nile virus-human isolate 46 63 St. Louis encephalitis 47 65 virus-2015 Omsk hemorrhagic fever virus 43 61 Murray valley encephalitis No virus sequence — virus available Complete protein NS3 protein homology to YFV homology to YFV Virus 17D204 (%) 17D204 (%) YFV 17D204 100 100 JE-SA14-14-2 45 55 TBEV Neudeurfl 43 48 ZIKV-polynesian 46 56 WNV-human isolate 46 57 SLEV-2015 47 58 OHFV 43 47 MVEV ND ND

[0240] We isolated blood from individuals vaccinated with either YFV alone, or individuals that received concomitant vaccinations with YFV and TBE vaccines.

Concomitant YFV and TBE Vaccination Generates a Higher Amount of Antibody Producing B Cells than the YFV Vaccine Alone

[0241] Immune responses after vaccination can be measured in multiple ways. One of the earliest responding immune cells in infection and vaccination are B cells. Germinal centers are transiently developed after antigen/vaccine exposure and are critical sites for selecting and differentiating B cells to become short-lived antibody-producing plasmablasts and memory B cells. Plasmablast responses have been shown to be a predictive measure of antibody levels induced by vaccination and can therefore aid in early evaluation of the efficacy of a vaccine.sup.23. Plasmablasts can be detected by flow cytometry in the acute-stage of infection.sup.24, however it is a bit more challenging to detect after primary vaccination, due to low amounts generated. Several booster doses are usually required to detect them after vaccination.

[0242] To assess the appearance of plasmablasts after vaccinations, we stained freshly isolated PBMCs before and at day 7 and 15 after administration with either YFV alone (Beta Y donors), or after concomitant administration of YFV and TBE (alpha T donors) (FIGS. 3A and B). Plasmablasts are visible in both cohorts at day 7 after vaccination, however the alpha donors have a peak with numbers as high as 10-30% of total B cells at day 15 (FIGS. 3A and B). These are levels measurable with acute dengue infection.sup.24.

[0243] To measure the neutralizing effect of the antibodies generated, we performed a plaque reduction neutralization test (PRNT) for TBEV22 on three donors vaccinated with the YFV vaccine. One donor was previously negative for both YFV and TBE vaccines (Beta Y2), one donor received several TBE vaccines previously, with the last booster 4 months prior YFV vaccine (Beta Y1), and one donor received concomitant YFV and TBE prime vaccines, and had thereafter followed a full TBE vaccine program (two additional doses of TBE vaccine, with the last booster dose four months prior sampling time point (Alpha T0).

[0244] Beta Y1 showed a 100% increase (from 20-40% neutralization) before and after administration of YFV vaccine (FIG. 4). Furthermore, Alpha T0 measured highest levels of neutralizing antibodies against TBEV (80% neutralization). Together this indicates that 1; YFV vaccine does not generate cross neutralizing antibodies against TBEV (measured by this assay); 2; YFV vaccine boost already existing TBEV Nab and 3; a high degree of TBEV Nab is generated if prime YFV and TBE vaccines are given concomitantly.

YFV Vaccine Generates Protective Cross-Reactive CD4 T Cells to TBEV and ZIKV

[0245] CD4 T cells responding to antigens have critical functions in activating and regulating immune responses via the production and release of various cytokines and play an important role in the protective immunity against viruses, primed by infection or by vaccination. CD4 T cells can promote contact of CD8 T cells with DCs in secondary lymphoid tissue.sup.25,26, as well as recruit lymphoid cells into draining lymph nodes 27, and recruit innate or antigen-specific effectors to the site of viral replication.sup.28,29. CD4 T cells can be divided into distinct subsets depending on their cytokine profile. In a simplified view, Th1 subsets activate cellular immune responses via the production of IFN-γ, TNF and IL-2, and Th2 subsets mainly produce cytokines supporting B cell activation 30.

[0246] To study whether YFV and TBEV specific, as well ZIKV cross reactive CD4 T cells arise after vaccination, we cultured PBMC with TBEV or ZIKV NS5 covering libraries from day 0 and 15 (FIG. 5) from donors vaccinated with YFV and TBE. In donor alpha T2, specific cells against both TBEV and ZIKV NS5 libraries could be detected. ZIKV specific cells also appeared at day 15 in the double vaccinated alpha T1 donor (FIG. 5B). However, TBE vaccine alone appear not to induce CD4 specific T cells after the second of third dose of TBE vaccine (FIG. 5C).

YFV Vaccine Enhances Production of TBEV Specific T Cells after Concomitant Vaccination with TBEV and Generates Cross-Reactive T Cells Against ZIKV

[0247] CD8 T cells have a spectrum of functions to control viral infections. Here, we assessed cytokine expression (IFN-γ and TNF) and degranulation (CD107a) of after activation with TBE/ZIKV NS5 libraries (FIG. 6) or with predicted ZIKV/TBEV CD8 T cell pools (FIG. 6) from before (day 0) and at days 15 and 22 after vaccinations. YFV alone generates a small amount of cross-reactive CD8 T cells against both TBEV NS5 and ZIKV NS5 at day 15 in donor beta Y1 (FIG. 6A). ZIKV specific CD8 T cells are absent before, but detectable at 15 days after concomitant administration with YFV and TBEV vaccines (FIGS. 6B and C). These cells are completely absent after multiple second and third dose of the TBE vaccine (FIG. 6D), demonstrating that TBEV vaccine alone may not generate specific CD8 T cells. Together, this indicates that TBEV CD8 T cells are generated after one single dose of the TBE vaccine if given simultaneously as the YFV vaccine.

[0248] To assess the existence of CD8 T cell responses against single TBEV peptide sequences (Table 3), we CFSE-labeled PBMCs from 2 HLA-A1 positive donors recently vaccinated with YFV, and cells were cultured with the corresponding synthetic peptides for 6 d. By the end of day 6, these cultures were re-stimulated for 12 h in the presence of peptide, and responses were determined by intracellular staining for IFN-γ (FIG. 7A). Responding cells were identified as double positive for CFSE dilution, indicative of proliferation during culture with peptide, and IFN-γ production. One peptide, predicted to be presented in HLA-A1 positive donors induced responses in both donors, with up to 1.9% IFN-γ positive cells. Next, we activated T cells with the predicted CD8 ZIKV pool (Table 2) in one donor previously activated with the YFV vaccine from before and at 15 days after vaccination. ZIKV specific T cells, producing CD107a and TNF arise at day 15 after YFV administration.

Conclusion

[0249] With these data, we show that antibody-producing plasmablasts arise and peak at day 15 after administration of YFV and YFV together with TBE vaccine. The levels of plasmablasts were higher in individuals receiving concomitant vaccinations as compared to individuals receiving the YFV alone (FIG. 3). This may indicate that there is a synergy-adjuvant effect generated when receiving concomitant vaccinations of TBEV and YFV.

[0250] The YFV alone generate small but detectable protective cross-reactive CD8 T cells against ZIKV, JEV and TBEV (FIGS. 6 and 7). However in the individuals receiving concomitant vaccinations with YFV and TBE, a better CD4 T cell response against TBEV NS5 peptides was detected, as compared to individuals that receive the TBE vaccine alone (FIG. 5). Individuals receiving concomitant vaccinations with YFV and TBE show a strong CD8 T cell response against ZIKV NS5 library (FIG. 5), indicating cross-species protective effects of flavivirus vaccines.

Discussion

[0251] We have for several years established and studied a cohort of healthy volunteers that were vaccinated with the YFV with a focus on how NK and T cell responses evolve over time. Vaccines take years to develop, which can be detrimental to global health with emerging pathogens. If we had a better understanding of how flavivirus vaccines work and the responses they induce, we could better design vaccines towards emerging viruses like ZIKV and/or improve the TBEV or JEV vaccines or immunization schedules. There is no commercial vaccine for ZIKV yet, but cross-reactivity in immunity gained from other flavivirus vaccinations could potentially aid in protecting those in high-risk areas from infection. Furthermore, if future ZIKV vaccines prove not to be entirely effective, co-vaccination with the YFV vaccine may yield improved vaccination.

[0252] The TBE and JE vaccines are relatively weak that require multiple booster doses, and with the number of vaccine failures reported in recent years, there is great need for improvement. If simultaneous immunizations lead to more robust immune responses, better protection against the infection could be acquired and less vaccine failures would occur. The results would also lead to more cost effective vaccination regimens as fewer boosters are required, and those vaccinated would sustain sufficient protection for a longer period. The vaccinations could be made more economically available for people living in TBEV or JEV endemic areas, ultimately reducing the number of infections and thereby fewer deaths or life long complications associated with the diseases.

[0253] Investigating the cross-reactivity of vaccines to related pathogens is important for understanding how immune responses to vaccines develop, and the mechanisms involved. The data generated can give significant insight into the immune mechanisms behind these vaccinations, which could lead to improved flavivirus vaccination strategies as well as provide potential protection against Zika virus while its vaccine is still in development. The data generated herein contributes to our understanding of flavivirus vaccines and leads to a potential novel vaccination method.

EXAMPLE 2: ZIKA VIRUS-SPECIFIC T CELL RESPONSES GENERATED BY THE YELLOW FEVER VACCINE

[0254] On Feb. 1, 2016, the WHO declared that the reported clusters of microcephaly and other neurological disorders constituted a Public Health Emergency of International Concern (PHEIC). The Zika virus epidemic represents an unprecedented health crisis affecting significant parts of the world.sup.32. The epidemic is currently ongoing in Latin America and the Caribbean, and impacts of the infection are already seen in large populations in Brazil, Colombia, Mexico, Peru and beyond. The infection currently stands without specific treatment or a vaccine, although several vaccine candidates currently are under development.sup.33.

[0255] The Zika virus is a mosquito borne virus belonging to the group of flaviviruses (family Flaviviridae), closely related to, e.g., the yellow fever virus (YFV). In 1939, Max Theiler succeeded in attenuating the YFV. Soon thereafter, a vaccine towards the YFV was developed and distributed worldwide and, to date, over 300 million doses of the vaccine have been administered to humans.sup.34. Indeed, the YFV vaccine is still considered as one of the world's most efficient vaccines, where a single dose gives an at least 10-year (or most likely lifelong) immunity to the infection.sup.35. Herein, we report that the YFV vaccine generate cross reactive CD4 and CD8 T cell responses to Zika virus antigens. The responses detected are mounted towards the NS5-protein of Zika virus.

[0256] The NS5-protein is a multifunctional, conserved protein in flaviviruses that constitutes the viral polymerase. By comparative analysis of NS5 protein sequences of the currently used live attenuated YFV vaccine and Zika virus.sup.36, we found the yellow fever vaccine-derived NS5 protein to have 64% homology with Zika virus NS5. This led us to hypothesize that cross-reactive responses may occur between T cells obtained following YFV vaccination and Zika virus specific antigens. To test this, peripheral blood was collected from two Zika virus naïve donors before (day 0) and 15 days after (day 15) administration of the YFV vaccine. Cells were stimulated (according standard methods,.sup.37) with a Zika-NS5 overlapping peptide library (Zika virus French polynesia isolate, 18 aminoacids in length and 10 aminoacids overlapping; library from GenScript) for 12 hours. Zika virus-specific T cells were subsequently identified as lymphocytes, singlets, dump- (CD19, CD14 and dead cell marker), CD3+CD4+/CD3+CD8+ and IFNγ+TNF+ cells. CD4 T cells producing both IFN-γ and TNF in response to the Zika-NS5 library were clearly increased at 15 days after YFV vaccination. Likewise, CD8 T cells producing both IFN-γ and TNF in response to the Zika-NS5 library were clearly increased at 15 days after YFV vaccination (see FIG. 8). These results indicate that the homology between the YFV vaccine and Zika virus-NS5 is high enough to induce Zika-specific T cells of both CD4 and CD8 lineage.

[0257] Neutralizing antibodies produced by B cells, are critical for vaccine-mediated protection against viral diseases. Cross-reactive antibodies among flaviviruses have been reported previously, and it is currently debated whether these are protective or contribute to pathogenesis.sup.38,39. In contrast, cross-reactive T cell-mediated immunity amongst flaviviruses has been less well studied. It was recently demonstrated that the vaccine against Japanese encephalitis generates T cells that are cross-reactive with Dengue virus and West Nile virus.sup.40. The present findings indicate that the YFV vaccine generates cell-mediated immune responses against Zika virus.

EXAMPLE 3: SYNERGY IN RESPONSES INDUCED BY CONCOMITANT YFV AND TBEV VACCINATION

[0258] Methodology: Blood from an initial 20 individuals, immunized with YFV vaccine (Day 0) and TBE (Day 0+28) vaccines simultaneously, are collected before and at several time points after vaccination (FIG. 9). Individuals immunized with YFV and TBE vaccines alone are used as control groups. The immune responses are determined with several different approaches.

[0259] We have previously identified epitopes in both YFV and TBEV.sup.10,31, and these are utilized to (i) study the phenotype of the TBE and YFV specific T cells (including markers for CD3, CD8, CD4, CD28, CTLA-4, CCR5, CD127, T-bet, Eomes, CD45RA, Ki67, CD69, perforin, granzyme B and CD38) and (ii), study T cells functions induced by the vaccines including markers for degranulation (CD107a), as well as cytokines and chemokines (TNF, IFN-γ, MIP-1β and IL-2).

[0260] The kinetics and magnitude of plasmablast appearance after vaccination are studied with multicolor flow cytometry. Phenotypical assessment over time is performed with markers for CD20, intracellular IgG, Ki67, PD-1, HLA-DR and CD80. Our pilot experiments have shown that plasmablasts are readily detectable following booster immunization to TBEV, indicating the feasibility of this approach (FIG. 3).

[0261] Elispot is used to functionally verify that the plasmablasts detected are vaccine-specific. Such methods are currently established in our lab (see Jahnimatz et al, 2013, J Immunol Methods, 391(1-2): 50-9).

[0262] Neutralizing antibodies are evaluated using plaque reduction neutralization tests.sup.22 and correlated with vaccine specific memory B cell and plasmablast numbers.

[0263] Multiplex assay on sera from all time points is performed, and inflammatory cytokines together with innate and adaptive cytokines will be measured.

EXAMPLE 4: SYNERGY IN RESPONSES INDUCED BY CONCOMITANT YFV AND JEV VACCINATION

[0264] Methodology: Blood from 20 individuals, immunized with YFV (Day 0) and JEV (Day 0+28) vaccines simultaneously, is collected before and at several time points after vaccination (with the same collection schedule as in Example 3, FIG. 1). Individuals immunized with YFV and JE vaccines alone are used as control groups. The immune responses are then determined with the same approaches as in Example 3.

[0265] The YFV vaccine is given at day 0 simultaneously with JEV vaccine and a booster JEV vaccine at day 28. Early innate mechanism activation is evaluated with the same approaches as described in Example 3.

EXAMPLE 5: CROSS REACTIVITY OF YFV VACCINATION TO ZIKV

[0266] Methodology: We have collected a cohort of healthy volunteers that were vaccinated against yellow fever virus. PBMC and sera have been collected before and at days, 10, 15 and 90 after administration of the vaccine. To measure possible cross-reactive T cells, we have generated overlapping peptide libraries of the conserved NS5 region of ZIKV and also TBE viruses.

[0267] The overlapping peptide libraries of ZIKV and TBEV are used to activate T cells in the YFV vaccinated donors.

[0268] The responses induced by ZIKV and TBEV peptides in the YFV vaccinated individuals are studied by flow cytometry. We will have markers for CD4 (T helper) and CD8 (cytotoxic T cells) together with classical markers for T cell function (IFN-γ, MIP-1β, IL-2).

[0269] HLA class I tetramers are generated for selected epitopes to study the appearance, magnitude and phenotype of cross-reactive T cells.

TABLE-US-00006 NS5 sequences YFV asibi (SEQ ID NO: 36) VSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAAAQKEVSGVKGFT LGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSS VTEGERTVRVLDTVEKWLACGVDNFCVKVLAPYMPDVLEKLELLQRRFGG TVIRNPLSRNSTHEMYYVSGARTLEADVILPIGTRSVETDKGPLDKEATE ERVERIKSEYMTSWEYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKIL TYPWDRIEEVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVN RWLFRHLAREKNPRLCTKEEFIAKVRSHAAIGAYLEEQEQWKTANEAVQD PKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMW LGARYLEFEALGFLNEDHWASRENSGGGVEGIGLQYLGYVIRDLAAMDGG GFYADDTAGWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVV KVLRPAPGGKAYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEM VIHHQHVQDCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVVRPIDDRFG LALSHLNAMSKVRKDISEWQPSKGWNDWENVPFCSHHFHELQLKDGRRIV VPCREQDELIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLL SLAVSSAVPTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQ DKTMVKEWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIG QEKYTDYLTVMDRYSVDADLQ YFV 17DD (YF VAX) (SEQ ID NO: 37) VSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAAAQKEVSGVKGFT LGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSS ITEGERTVRVLDTVEKWLACGVDNFCVKVLAPYMPDVLEKLELLQRRFGG TVIRNPLSRNSTHEMYYVSGARTLEADVILPIGTRSVETDKGPLDKEATE ERVERIKSEYMTSWEYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKIL TYPWDRIEEVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVN RWLFRHLAREKSPRLCTKEEFIAKVRSHAAIGAYLEEQEQWKTANEAVQD PKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMW LGARYLEFEALGFLNEDHWASRENSGGGVEGIGLQYLGYVIRDLAAMDGG GFYADDTAGWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVV KVLRPAPGGKAYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEN VIHHQHVQDCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVVRPIDDRFG LALSHLNAMSKVRKDISEWQPSKGWNDWENVPFCSHRFHELQLKDGRRIV VPCREQDELIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLL SLAVSSAVPTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQ DKTMVKKWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIG QEKYTDYLTVMDRYSVDADLQ YFV 17D204 (Stamaril) (SEQ ID NO: 38) VSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAAAQKEVSGVKGFT LGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSS VTEGERTVRVLDTVEKWLACGVDNFCVKVLAPYMPDVLEKLELLQRRFGG TVIRNPLSRNSTHEMYYVSGARTLEADVILPIGTRSVETDKGPLDKEATE ERVERIKSEYMTSWEYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKIL TYPWDRIEEVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVN RWLFRHLAREKNPRLCTKEEFIAKVRSHAAIGAYLEEQEQWKTANEAVQD PKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMW LGARYLEFEALGELNEDHWASRENSGGGVEGIGLQYLGYVIRDLAAMDGG GFYADDTAGWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVV KVLRPAPGGKAYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEM VIHHQHVQDCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVVRPIDDRFG LALSHLNAMSKVRKDISEWQPSKGWNDWENVPFCSHHFHELQLKDGRRIV VPCREQDELIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLL SLAVSSAVPTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQ DKTMVKKWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIG QEKYTDYLTVMDRYSVDADLQ

EXAMPLE 6: CROSS REACTIVITY OF YFV VACCINATION TO JEV

Methodology:

[0270] For mapping of T cell epitopes, freshly isolated PBMCs from one previously JEV-negative donor receiving the YFV vaccine, were incubated with JEV NS5 peptide library (18mer peptides, 70% pure, overlapping the NS5 protein of JEV) for 12 hours in the presence of brefeldin A and monensin (Sigma-Aldrich).

[0271] For phenotypic analysis, cells were incubated for 30 min at 4° C. in the dark with surface mAbs. For intracellular staining of TNF and IFN-γ, cells were fixed and permeabilized with Fix/Perm (eBioscience) for 30 min at 4° C. in the dark.

[0272] The following mAbs were used in flow cytometry: anti-CD4 Brilliant ultraviolet 737, anti-CD8 Brilliant ultraviolet 395, anti-CD3 PE-Cy5, anti-TNF Pacific Blue, anti-IFN-γ Brilliant Violet 785, Near IR, anti CD19 V500, anti CD14 V500. Flow cytometry data were acquired on a BD LSRFortessa (BD Biosciences) and analysed.

Results:

[0273] FIG. 11 shows CD8 T cells from one donor receiving the YVF vaccine (donor By4), before (day 0) and at 15 days after vaccination. Production of TNF increases from 0.03% to 0.3% that is a 10 fold increase in response to the JEV NS5 peptide library.

Conclusions

[0274] FIG. 11 demonstrates that JEV-specific T cells, able to produce TNF in response to JEV peptides, arise when given the YFV vaccine alone.

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