MERS coronavirus vaccine

Abstract

The present invention relates to mRNAs suitable for use as mRNA-based vaccines against infections with MERS coronaviruses. Additionally, the present invention relates to a composition comprising the mRNAs and the use of the mRNAs or the composition for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the prophylaxis or treatment of MERS coronavirus infections. The present invention further describes a method of treatment or prophylaxis of infections with MERS coronavirus using the mRNA sequences.

Claims

1. A pharmaceutical composition comprising an isolated mRNA comprising at least one coding region, encoding a spike protein (S) of a Middle East respiratory (MERS) coronavirus, said coding region being at least 90% identical to a nucleic acid sequence of SEQ ID NO: 2361, wherein the mRNA is associated with lipid nanoparticles (LNPs).

2. The pharmaceutical composition according to claim 1, wherein the mRNA further comprises at least one histone stem-loop.

3. The pharmaceutical composition according to claim 1, wherein the mRNA comprises, in 5 to 3 direction, the following elements: a) a 5-cap structure, b) the at least one coding region, and c) a poly(A) sequence.

4. A method of treatment or prophylaxis of MERS coronavirus infections comprising the steps: a) providing the pharmaceutical composition of claim 1; and b) administering an effective amount of the pharmaceutical composition to an organism.

5. The method according to claim 4, wherein the pharmaceutical composition is administered to the organism by subcutaneous, intramuscular or intradermal injection.

6. The method according to claim 5, wherein the injection is carried out by using conventional needle injection or jet injection.

7. The pharmaceutical composition according to claim 3, wherein the mRNA is associated with LNPs comprising: (i) a cationic lipid, (ii) an aggregation reducing agent, (iii) optionally, a non-cationic lipid, and (iv) optionally, a sterol.

8. The pharmaceutical composition according to claim 7, wherein the aggregation reducing agent comprises a PEG-modified lipid.

9. The pharmaceutical composition according to claim 8, wherein the mRNA is associated with LNPs comprising: (i) the cationic lipid, (ii) the PEG-modified lipid, (iii) the non-cationic lipid, and (iv) the sterol.

10. The pharmaceutical composition according to claim 9, wherein the coding region of the mRNA is at least 95% identical to SEQ ID NO: 2361.

11. The pharmaceutical composition according to claim 9, wherein the coding region of the mRNA is identical to SEQ ID NO: 2361.

12. The pharmaceutical composition according to claim 9, wherein the mRNA further comprises a poly(A) sequence of 10 to 200 nucleotides.

13. The pharmaceutical composition according to claim 12, wherein the mRNA further comprises a 5 untranslated region (UTR) and/or a 3 UTR.

14. The pharmaceutical composition according to claim 13, wherein the mRNA further comprises a 5 UTR and a 3 UTR.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in cells after transfection of the mRNA. Shown are the results of a FACS analysis. Panels A and C show cell surface staining; Panels B and D show intracellular staining. Further details are provided in Example 2.

(2) FIG. 2: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in cells after transfection of the mRNA. Shown are the results of a western blot analysis. Further details are provided in Example 3.

(3) FIG. 3: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins are expressed in vivo and that humoral immune responses are induced in mice. Shown are the results of an ELISA analysis. Panel A shows IgG1 endpoint titers; Panel B shows IgG2 endpoint titers. Further details are provided in Example 4.

(4) FIG. 4: shows that mRNA encoding MERS coronavirus/MERS-CoV antigenic proteins induces specific humoral immune responses after immunization in mice. Further details are provided in Example 5.

EXAMPLES

(5) The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of mRNA Constructs for In Vitro and In Vivo Experiments

(6) For the present examples, DNA sequences encoding MERS coronavirus/MERS-CoV antigenic proteins were prepared and used for subsequent RNA in vitro transcription reactions. DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silico algorithms that increase the GC content of the respective coding sequence. Moreover, sequences were introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3-terminal end (poly-A-tail) (indicated as mRNA design 1 in Table X1). Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L45-UTR ribosomal 5TOP UTR and 3-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3-terminal end (poly-A-tail) (indicated as mRNA design 2 in Table X1). The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

(7) RNA in vitro transcription on linearized pDNA:

(8) The DNA plasmids prepared according to paragraph 1 were enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. RNA production is performed under current good manufacturing practice according to WO2016180430. The obtained mRNAs were purified using PureMessenger (CureVac, Tbingen, Germany; WO2008077592) and used for in vitro and in vivo experiments.

(9) RNA constructs used in the present Examples are provided in Table X1. Therein, the respective antigen name is indicated, the corresponding protein SEQ ID NO and the mRNA SEQ ID NO. Three different Spike constructs were tested (S=full length; S(V1060P,K1061P)=mutant comprising two consecutive proline residues at the beginning of the central helix for retaining S protein in the prototypical prefusion conformation; S1) comprising either the endogenous signal peptide (R1-R3) or a heterologous signal peptide (HsIgE; R4-R6).

(10) TABLE-US-00011 TABLE X1 mRNA constructs of the Example section (Example 1): SEQ ID NO of SEQ ID NO: encoded mRNA Name antigen design 2 RNA ID S 16 2377 R1 S(V1060P, K1061P) 2357 2373 R2 S1 1463 2378 R3 HsIgE(1-18)_S(18-1353) 2358 2374 R4 HsIgE(1-18)_S(18-1353, 2359 2375 R5 V1060P, K1061P) HsIgE(1-18)_S(18-747) 2360 2376 R6

Example 2: Expression of MERS Coronavirus/MERS-CoV Proteins in HeLa Cells and Analysis by FACS

(11) The results of the present Example show that mRNA encoding MERS coronavirus/MERS-CoV proteins are translated in cells after transfection of the mRNA.

(12) To determine in vitro protein expression of the mRNA constructs, HeLa cells were transiently transfected with mRNA encoding MERS coronavirus/MERS-CoV antigens and stained using a suitable anti-S protein antibodies (raised in mouse), counterstained with a FITC-coupled secondary antibody (F5262 from Sigma). HeLa cells were seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. HeLa cells were transfected with 1 g and 2 g unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs prepared according to Example 1 and listed in Table X1 were used in the experiment (SEQ ID NOs: 2373-2378; RNA ID: R1-R6), including a negative control (water for injection). 24 hours post transfection, HeLa cells are stained with suitable anti anti-NiV or anti-HeV antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.). The results are shown in FIG. 1.

(13) Results:

(14) The results show that the used constructs led to a detectable protein expression at the cell surface for full length S construct (R1 and R4) and the stabilized S construct (R2 and R5). Moreover, a detectable intracellular protein expression for full length S construct (R1 and R4), the stabilized S construct (R2 and R5), and the S1 construct (R3 and R6) was shown. The results exemplify that the inventive mRNA encoding S proteins is translated in cells and that alternative RNA constructs as described in the present invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 3: Expression Analysis of MERS Coronavirus/MERS-CoV Proteins Using Western Blot

(15) The results of the present Example shows that mRNA encoding Nipah virus G protein and Hendra virus G protein are expressed in HeLa cells after transfection.

(16) For the analysis of MERS coronavirus/MERS-CoV, HeLa cells were transfected with unformulated mRNA (wfi as negative control) using Lipofectamine as the transfection agent. The mRNA constructs prepared according to Example 1 and listed in Table X1 were used in the experiment (SEQ ID NOs: 2373-2378; RNA ID: R1-R6), including a negative control (water for injection) and a positive control (S protein). Post transfection, HeLa cells were detached by trypsin-free/EDTA buffer, harvested, and cell lysates were prepared. Cell lysates were subjected to SDS-PAGE followed by western blot detection. Western Blot analysis was performed using an anti-S protein antibody used in combination with a suitable secondary antibody. The presence of -tubulin was analyzed (-tubulin; Cell Signalling Technology; 1:1000 diluted). MERS S protein was used as a positive control.

(17) Results:

(18) As shown in FIG. 2, the mRNA encoding MERS coronavirus/MERS-CoV S proteins is expressed in HeLa cell lysates.

(19) The results exemplify that the inventive mRNA encoding MERS coronavirus/MERS-CoV S proteins is translated in cells and that alternative mRNA as described in the present invention may also be translated in cells, which is a prerequisite for an mRNA-based vaccine.

Example 4: Vaccination of Mice with mRNA Encoding MERS Coronavirus/MERS-CoV Antigens and ELISA Analysis

(20) The results of the present Example shows that mRNA encoding MERS coronavirus/MERS-CoV antigens are expressed in mice after intradermal injection. In addition, the expressed antigens provided by the inventive mRNA induces humoral immune responses after immunization in mice.

(21) Preparation of Protamine Complexed mRNA:

(22) Nipah virus mRNA construct (SEQ ID NO: 1353) was prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA was complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

(23) Immunization:

(24) Female BALB/c mice (6-8 weeks old) were injected intradermally (i.d.) with mRNA vaccine compositions with doses, application routes and vaccination schedules as indicated in Table X2. As a negative control, one group of mice was vaccinated with buffer (ringer lactate). All animals were vaccinated on day 0, 21 and 42. Blood samples were collected on day 21 (post prime) and 35 (post boost) for the determination of antibody titers (ELISA).

(25) TABLE-US-00012 TABLE X2 Vaccination regimen (Example 4): Group Composition Dose Route Volume 1 Full length S 80 g id.; 2 50 l SEQ ID NO: 2377; RNA ID: R1 back of the Protamine formulated animal 2 HsIgE(1-18)_S(18-747) 80 g i.d.; 2 50 l SEQ ID NO: 2360; RNA ID: R6 back of the Protamine formulated animal 3 100% RiLa id.; 2 50 l Control back of the animal

(26) Determination of IgG1 and IgG2 antibody titers using ELISA:

(27) Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the MERS coronavirus/MERS-CoV antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. IgG1 and IgG2 titers directed against MERS coronavirus/MERS-CoV antigens were measured by ELISA on day 21 (post prime vaccination) and 35 (post boost vaccination).

(28) Results:

(29) As shown in FIG. 3, the mRNA encoding MERS coronavirus/MERS-CoV antigens are expressed in mice after intradermal injection. In addition, the expressed antigens provided by the inventive mRNA induces humoral immune responses after immunization in mice. In detail, boost vaccination induces clear ELISA responses in 4/6 animals in R1 (full length S) and 6/6 animals in R6 ((HsIgE(1-18)_S(18-747)). The results exemplify that the inventive mRNA encoding MERS coronavirus/MERS-CoV S proteins is translated in vivo and induces humoral immune responses in mice. Notably, alternative mRNA constructs as described in the present invention may also be translated in vivo and induce humoral immune responses in mice, which is a prerequisite for an effective mRNA-based MERS coronavirus/MERS-CoV vaccine.

Example 5: Analysis of Antigen-Specific Humoral Immune Responses in Mice Using a Cell-Based Assay

(30) The results of the present Example shows that mRNA encoding MERS coronavirus/MERS-CoV antigens induces antigen-specific humoral immune responses after immunization in mice.

(31) Hela cells were transfected with 2 g of RNA ID: R1 (SEQ ID NO: 2377) using lipofectamine. The cells were harvested 20 h post transfection, and seeded at 110.sup.5 per well into an 96 well plate. The cells were incubated with sera of mice vaccinated with RNA ID: R1 (SEQ ID NO: 2377) (diluted 1:50; day 35) obtained from Example 4, followed by FITC-conjugated anti-mouse IgG antibody. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

(32) Results:

(33) As shown in FIG. 4, the mRNA encoding MERS coronavirus/MERS-CoV S1 protein (mRNA construct R1) is expressed in mice after i.d. administration. Moreover, as antigen-specific IgGs were detected in sera of immunized mice, the results also show that the applied mRNA vaccine is suitable to induce antigen-specific humoral immune responses. The results exemplify that the inventive mRNA-based vaccine works and that similar mRNA vaccines comprising alternative mRNA constructs according to the invention may also be suitably used.

Example 6: Preparation of MERS Coronavirus/MERS-CoV Vaccine Compositions

(34) The results of the previous Examples showed that the inventive MERS coronavirus/MERS-CoV mRNA constructs are expressed in vitro and in vivo, and that the mRNA vaccine (protamine formulated) induces specific humoral immune responses in mice after i.d. administration. For further in vivovaccination experiments, other mRNA vaccine compositions are prepared, preferably using constructs listed in Table X1. One composition comprises mRNA that is encapsulated in lipid nanoparticles (LNPs), and one composition comprises polymer-lipidoid complexed mRNA.

(35) Preparation of LNP Encapsulated mRNA:

(36) A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNA mixture is diluted to a total concentration of 0.05 mg/mL in 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol). The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles are filtered through a 0.2 m pore sterile filter. Lipid nanoparticle particle diameter size is determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

(37) Preparation of Polymer-Lipidoid Complexed mRNA:

(38) 20 mg peptide (CHHHHHHRRRRHHHHHHCNH2; SEQ ID NO: 1443) TFA salt is dissolved in 2 mL borate buffer pH 8.5 and stirred at room temperature for approximately 18 h. Then, 12.6 mg PEG-SH 5000 (Sunbright) dissolved in N-methylpyrrolidone is added to the peptide solution and filled up to 3 mL with borate buffer pH 8.5. After 18 h incubation at room temperature, the reaction mixture is purified and concentrated by centricon procedure (MWCO 10 kDa), washed against water, and lyophilized. The obtained lyophilisate is dissolved in ELGA water and the concentration of the polymer is adjusted to 10 mg/mL. The obtained polyethylene glycol/peptide polymers (HO-PEG 5000-S(SCHHHHHHRRRRHHHHHHCS-)7-S-PEG 5000-OH amino acid component: SEQ ID NO: 1443) are used for further formulation and are hereinafter referred to as PB83.

(39) Preparation of 3-C12-OH lipidoid: First, lipidoid 3-C12 was obtained by acylation of tris(2-aminoethyl)amine with an activated lauric (C12) acid derivative, followed by reduction of the amide. Alternatively, it may be prepared by reductive amination with the corresponding aldehyde. Lipidoid 3-C12-OH was prepared by addition of the terminal C12 alkyl epoxide with the same oligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5. Preparation of compositions with nanoparticles of polymer-lipidoid complexed mRNA: First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) or PBS buffer may be used), respective amounts of lipidoid, and respective amounts of a polymer (PB83) are mixed to prepare compositions comprising a lipidoid and a peptide or polymer. Then, the carrier compositions are used to assemble nanoparticles with the mRNA by mixing the mRNA with respective amounts of polymer-lipidoid carrier and allowing an incubation period of 10 minutes at room temperature such as to enable the formation of a complex between the lipidoid, polymer and mRNA. In order to characterize the integrity of the obtained polymer-lipidoid complexed mRNA particles, RNA agarose gel shift assays are performed. In addition, size measurements are performed (gel shift assay, Zetasizer) to evaluate whether the obtained nanoparticles have a uniform size profile.

Example 7: Vaccination of Mice and Evaluation of Specific Immune Response

(40) Female BALB/c mice are injected intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 6) with doses, application routes and vaccination schedules as indicated in Table X3. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below).

(41) TABLE-US-00013 TABLE X3 Vaccination regimen - Nipah virus experiment (Example 7) Number Route/ Vaccination of mice Vaccine composition Volume Schedule (day) 8 5 g RNA construct R1; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R1; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35 8 5 g RNA construct R2; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R2; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35 8 5 g RNA construct R3; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R3; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35 8 5 g RNA construct R4; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R4; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35 8 5 g RNA construct R5; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R5; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35 8 5 g RNA construct R6; LNP formulated i. m.; 2 25 l 0/21/35 8 20 g RNA construct R6; polymer-lipidoid complexed i. m.; 2 25 l 0/21/35

(42) Determination of IgG1 and IgG2 antibodies by ELISA:

(43) ELISA performed essentially as described in Example 4.

(44) Determination of antigen-specific humoral immune responses using a cell based assay:

(45) Cell based assay performed essentially as described in Example 5.

(46) Intracellular cytokine staining:

(47) Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (210.sup.6 cells per well). The cells are stimulated with a mixture of four Nipah virus protein specific peptide epitopes (5 g/ml of each peptide) in the presence of 2.5 g/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37 C. in the presence of a protein transport inhibitor. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFN-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fc-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

(48) Plaque reduction neutralization test (PRNT50):

(49) Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art.

(50) Briefly, obtained serum samples of vaccinated mice are incubated with MERS coronavirus/MERS-CoV. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 8: Clinical Development of a Nipah Virus and Hendra Virus mRNA Vaccine Composition

(51) To demonstrate safety and efficiency of the mRNA vaccine composition(s), a clinical trial (phase I) is initiated. In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times. In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis). The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.