Human cytomegalovirus immunogenic composition
11524069 · 2022-12-13
Assignee
Inventors
- Pascal Chaux (Bully, FR)
- Rafaela Dumas (Porquerolles, FR)
- Jean Haensler (Grezieu la Varenne, FR)
- Sylvie Pichon (Brindas, FR)
- Fabienne Piras-Douce (Fleurieux sur l'Arbresle, FR)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
C12N2710/16122
CHEMISTRY; METALLURGY
C12N2710/16134
CHEMISTRY; METALLURGY
A61K2039/55555
HUMAN NECESSITIES
C12N2710/16111
CHEMISTRY; METALLURGY
International classification
A61K39/39
HUMAN NECESSITIES
C12N7/00
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
Abstract
The invention relates to an immunogenic composition comprising an HCMV gB antigen, an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen and a Th1-inducing adjuvant. If further relates to the immunogenic composition for use as an HCMV vaccine.
Claims
1. A subunit vaccine comprising an immunogenic composition consisting of: a purified protein HCMV gB antigen; a purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and a Th1-inducing adjuvant, wherein said Th1-inducing adjuvant comprises a TLR4 agonist or a linear or branched polyacrylic acid polymer salt with a weight average molecular weight Mw in the range of 350 to 650 kDa.
2. The subunit vaccine according to claim 1, wherein said Th1-inducing adjuvant induces in mice a lower IgG1:IgG2a,c ratio, and/or a higher INF-y level, and/or a lower IL-5 level than MF59 in a composition comprising the same purified protein HCMV gB antigen and the same purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen.
3. The subunit vaccine according to claim 1, wherein said purified protein HCMV gB antigen comprises one or several mutations at the endoproteolytic cleavage site.
4. The subunit vaccine according to claim 1, wherein said purified protein HCMV gB antigen is a full length gB polypeptide, a full length gB polypeptide lacking at least a portion of a transmembrane domain of the gB polypeptide, a full length gB polypeptide lacking at least 80% of the amino acid sequence of the transmembrane domain, a full length gB polypeptide lacking at least a portion of an intracellular domain of the gB polypeptide, a full length gB polypeptide lacking at least 80% of the amino acid sequence of the intracellular domain, or a full length gB polypeptide lacking at least 80% of the amino acid sequences of both the transmembrane domain and the intracellular domain.
5. The subunit vaccine according to claim 1, wherein said purified protein HCMV gB antigen is gBdTm.
6. The subunit vaccine according to claim 1, wherein in the said purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen, the gH antigen lacks at least a portion of a transmembrane domain of the full length gH.
7. The subunit vaccine according to claim 6, wherein said gH antigen comprises an ectodomain of the full length gH encoded by UL75 gene.
8. The subunit vaccine according to claim 1, wherein the purified protein HCMV gB and the purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex are the sole HCMV protein antigens.
9. The subunit vaccine according to claim 1, wherein said vaccine increases neutralizing antibody levels and/or persistence.
10. The subunit vaccine according to claim 6, wherein in the purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen, the gH antigen lacks at least 80% of the amino acid sequence corresponding to the transmembrane domain.
11. A subunit vaccine comprising an immunogenic composition consisting of: a purified protein HCMV gB antigen; a purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and a TLR4 agonist.
12. The subunit vaccine according to claim 11, wherein said TLR4 agonist is in combination with a delivery system.
13. The subunit vaccine according to claim 12, wherein the delivery system is selected from the group consisting of aqueous nanosuspension, calcium phosphate, liposomes, virosomes, ISCOMs, micro- and nanoparticles, and emulsions.
14. The subunit vaccine according to claim 11, wherein said TLR-4 agonist is selected from the group consisting of: a lipopolysaccharide, a monophosphoryl lipid A (MPL), a 3-de-O-acylated monophosphoryl lipid A (3D-MPL), a glucopyranosyl lipid adjuvant (GLA), a second-generation Lipid Adjuvant (SLA), a phospholipid dimer connected by a noncarbohydrate backbone and an aminoalkyl glucosaminide phosphate, or a derivative thereof.
15. The subunit vaccine according to claim 12, wherein TLR-4 agonist in combination with a delivery system is AS01 or AS02.
16. The subunit vaccine according to claim 11, wherein said TLR-4 agonist is GLA (CAS Number 1246298-63-4) TLR-4 agonist.
17. A subunit vaccine comprising an immunogenic composition consisting of: a purified protein HCMV gB antigen; a purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and a linear or branched polyacrylic acid polymer salt with a weight average molecular weight Mw in the range of 350 to 650 kDa.
18. The subunit vaccine according to claim 17, wherein said linear or branched polyacrylic acid polymer salt is PAA225000.
19. A method of preventing a disease associated with HCMV infection in a patient in need thereof, comprising the administration of an immunologically effective amount of a subunit vaccine comprising an immunogenic composition consisting of: a purified protein HCMV gB antigen; a purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and a TLR4 agonist or a linear or branched polyacrylic acid polymer salt with a weight average molecular weight Mw in the range of 350 to 650 kDa, thereby preventing the disease associated with HMCV infection in the patient.
20. A method of producing a subunit vaccine comprising an immunogenic composition, the method comprising: providing purified protein antigens consisting of a purified protein HCMV gB antigen and a purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and combining the purified protein antigens with a TLR4 agonist, to thereby produce an immunogenic composition.
Description
FIGURES
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EXAMPLES
(14) TABLE-US-00008 TABLE 2 List of abbreviations Acronym/Abbreviation Designation/Description CMV Cytomegalovirus D Day ELISA Enzyme Linked Immuno Sorbent Assay EU ELISA Unit IFNg Gamma interferon Ig Immunoglobulin IL Interleukin IM Intra-muscular IP Intra-peritoneal M Month N/A Not Applicable SN Seroneutralization W Week
Example 1
(15) Material and Methods
(16) Material
(17) Product(s) Tested in the Examples
(18) Products are described in Table 3. Female 7-weeks-old C57BL/6J mice were immunized by the intra-muscular (IM) route (hind leg, quadriceps) under a volume of 50 μl on D0 and D20 and D227.
(19) TABLE-US-00009 TABLE 3 Product Name Concentration Source or composition Pentamer CMV 1 mg/ml The Native Antigen company, Oxford UK gB (CMV) 685.5 μg/ml gBdTM obtained as described in U.S. Pat. No. 6,100,064, which is a 806 AA long polypeptide PAA225000 8 mg/ml Polysciences Europe GmbH, Hirschberg (PAA) PAA 225000 an der Bergstrasse, Germany MF59 4% squalene Quantity per mL: Squalene 39.0 mg Polysorbate 80 4.7 mg Sorban trioleate 4.7 mg Sodium citrate, dehydrate 2.65 mg Citric acid, monohydrate 0.17 mg Water for injections q.s.p. 1 mL AF04 5% squalene, Obtained according to the process 0.04 mg/ml E6020 described in WO 2007/080308. GLA-SQEM 4% squalene, GLA from Avanti Polar Lipids Inc., 0.1 mg/ml GLA Alabaster, USA GLA-SQEM is obtained according to the process described above in the description of the invention
Methods
Group Definition
(20) Study groups are described in the following Table 4. Mice were randomly allocated to one of the following 7 groups. Each group is differentiated in 3 subgroups, i.e. A=>A1, A2 and A3 depending on the time-point analysis requiring mouse euthanasia to collect the spleens (days 34, 208 and 257), as summarized in the study schedule on
(21) TABLE-US-00010 TABLE 4 Sub- Groups (number Products under test of Active substance Adjuvant Adm. Groups mouse) Name dose Name Dose route A A1 (5) PBS — — — IM (15) A2 (5) 50 μl at D0, D21 A3 (5) IM 50 μl at D0, D21, M7 B B1 (5) gB + 2 μg — — IM (20) B2 (5) Pentamer 50 μl at D0, D21 B3 (10) 2 μg IM 50 μl at D0, D21, M7 C C1 (10) gB + 2 μg MF59 2% IM (35) C2 (10) Pentamer squalene 50 μl at D0, D21 C3 (15) 2 μg IM 50 μl at D0, D21, M7 D D1 (10) gB + 2 μg PAA 200 μg IM (35) D2 (10) Pentamer 50 μl at D0, D21 D3 (15) 2 μg IM 50 μl at D0, D21, M7 E E1 (10) gB + 2 μg AF04 1 μg IM (35) E2 (10) Pentamer 2 μg E6020, 50 μl at 2.5% D0, D21 E3 (15) squalene IM 50 μl at D0, D21, M7 F F1 (10) gB + 2 μg GLA- 2.5 μg IM (35) F2 (10) Pentamer SQEM GLA, 50 μl at 2% D0, D21 F3 (15) 2 μg squalene IM 50 μl at D0, D21, M7
(22) Biological Sampling and Analytical Tests
(23) Biological Sampling
(24) Blood samples were collected from all the animals under anesthesia. The anesthesia was performed by Imalgene® (1.6 mg of Ketamine) and Rompun (0.32 mg of Xylazine) administered in a volume of 200 μl via the intraperitoneal route. Around 1 mL of blood was collected in vials containing clot activator and serum separator (BD Vacutainer SST ref 367783). After a night at +4° C., blood was centrifuged at 3000 rpm during 20 minutes and serum was collected and stored at −20° C. until analysis.
(25) For cellular response assays, spleens were collected in sterile conditions and splenocytes were isolated as soon as possible after spleen sampling.
(26) Analytical Tests
(27) Seroneutralization Assays
(28) This technique is used to titrate the functional neutralizing antibodies present in the sera of CMV-gB+pentamer+adjuvant immunized animals. Based on the ability of the Cytomegalovirus to infect MRC5 fibroblasts and ARPE-19 cells (human epithelial cells), a serum containing specific functional antibodies against CMV-gB and/or CMV-pentamer can inhibit the viral infection of the cells.
(29) Briefly, 2.5×104 MRC5 fibroblasts or ARPE-19 cells were dispensed in 96-well dark plates the day before the microneutralization (MN) assay. On D0, sera were heat-inactivated at 56° C. for 30 min. Serum samples were serially two-fold diluted in DMEM/F12 1% FBS, starting from 1/10 to 1/10240 in a 96-deep-well plate and incubated with 4.2 log FFU/ml of the BADrUL131-Y4 CMV virus strain (provided by Thomas Shenk, as described in Wang et al., J. Virol., 2005, 79(16):10330-10338), titrating 4.89 or 4.71 log FFU/ml on ARPE-19 or MRC5 cells, respectively) for 60 min at 37° C. in a 5% CO2 cell culture incubator. The serum/virus mixtures were then transferred onto the MRC5 or the ARPE-19 cells and incubated at 37° C. in a 5% CO.sub.2 cell culture incubator. The incubation was performed on 3 days for the MRC5 cells and on 4 days for the ARPE cells.
(30) On D3 or D4, after removal of culture supernatant, cells were fixed with 100 μl of 1% formol in PBS for 1 hour at room temperature. The plates were then washed three times with PBS and air-dried at room temperature before analysis on the Microvision fluorescent plate reader to count infected cells in each well.
(31) As control, two wells of cell control (without virus) and six wells with cells infected with half of the viral dilution containing the 4.2 log FFU/mL were present on each plate. The mean of these six wells defined the threshold of seroneutralization, determined as 50% of the specific-signal value. Neutralizing end-point titers were defined as the reciprocal of the last dilution that fell below the calculated 50% specific-signal value. Neutralizing titers (pPRNT50) were defined for each individual serum as the last dilution that induced 50% reduction of infected cells, i.e. the last dilution that induced lower infected cells than the calculated 50% specific-signal value. Geometric mean neutralizing antibody titers were calculated for each group.
(32) ELISA Assay
(33) Serum IgG1 and IgG2c antibodies directed against CMV-gB antigen or against CMV-pentamer antigen were titrated by a robot ELISA assay according to the following procedure.
(34) Dynex 96-well microplates were coated overnight at 4° C. with 1 μg/well of CMV-gB or CMV-pentamer, in 0.05 M carbonate/bicarbonate buffer, pH 9.6 (Sigma). Plates were then blocked at least 1 hour at 37° C. with 150 μL/well of PBS Tween-milk (PBS pH7.1, 0.05% Tween 20, 1% (w/v) powdered skim milk (DIFCO)). All next incubations were carried out in a final volume of 100 μL, followed by 3 washings with PBS pH 7.1, 0.05% Tween 20. Serial two-fold dilution of serum samples were performed in PBS-Tween-milk (starting from 1/1000 or 1/10000) and were added to the wells. Plates were incubated for 90 min at 37° C. After washings, goat anti-mouse IgG1 or IgG2c peroxydase conjugate antibodies (Southern Biotech) diluted in PBS-Tween-milk at 1/2000 were added to the wells and plates were incubated for 90 min at 37° C. Plates were further washed and incubated in the dark for 30 min at 20° C. with 100 μL/well of a ready-to-use Tetra Methyl Benzidine (TMB) substrate solution (TEBU). The reaction was stopped with 100 μL/well of HCl 1M (Prolabo).
(35) Optical density (OD) was measured at 450 nm-650 nm with a plate reader (VersaMax—Molecular Devices). The IgG1 or IgG2c antibodies titers were calculated using the CodUnit software, for the OD value range of 0.2 to 3.0 from the titration curve (reference mouse hyper immune serum put on each plate). The IgG1 or IgG2c titer of this reference, expressed in arbitrary ELISA Units (EU) corresponds to the log 10 of the reciprocal dilution giving an OD of 1.0. The threshold of antibody detection was 10 ELISA units (1.0 log 10).
(36) All final titers were expressed in log 10 (Log).
(37) IgG1/IgG2c ratios were calculated using the individual arithmetic values and the geometric mean of individual IgG1/IgG2c ratios was calculated for each group.
(38) Fluorospot
(39) The fluorescent-linked immunospot (FLUOROSPOT) is used for detecting and enumerating individual cells secreting the IFN-γ and IL-5 cytokines.
(40) On D0, the membrane of the 96-well IPFL-bottomed microplates (Multiscreen) was pre-wetted for 1 minute with 25 μL of 35% ethanol. Ethanol was then removed and each well was washed twice with 200 μL of PBS 1×. Microplates were then coated with a rat anti-mouse IFN-γ or rat anti-mouse IL-5 antibodies (10 μg/ml, Pharmingen) diluted at 1/100 and 1/50 respectively and were incubated overnight at 4° C.
(41) On D1, plates were washed with PBS and then blocked at least 2 h at 37° C. with RPMI 10% FBS. After plates washing, 5×10.sup.5 freshly isolated splenocytes/well were incubated overnight with the CMV-gB antigen (0.1 μg/ml), CMV-pentamer (0.1 μg/ml) or concanavalin A (Con A, 2.5 μg/mL) as a positive control, in presence of murine IL-2 (10 U/ml).
(42) On D2, the plates were washed 6 times with PBS 1×-BSA 0.1% (200 μL/well). After the washing step, 100 μL/well of the biotinylated anti-mouse IFN-γ or anti-mouse IL5 antibodies were added at 1 μg/mL in PBS1×-BSA 0.1% for 2 hours at room temperature, in the dark. The plates were washed again 3 times with PBS 1×-BSA 0.1% (200 μL/well). Then, 100 μL/well of streptavidin-PE at 1 μg/mL in PBS 1×-BSA 0.1% was incubated for 1 hour at room temperature, in the dark.
(43) The plates were further washed 6 times with PBS 1×-BSA 0.1% (200 μL/well). The plates were stored at 5° C.±3° C. in the dark until reading.
(44) Each spot, corresponding to an IFN-γ or IL5 secreting cell (IFN-γ SC or IL5 SC), was enumerated with an automatic FLUOROSPOT plate reader (Microvision). Results were expressed as number of IFN-γ or IL-5 secreting cell per 10.sup.6 splenocytes.
(45) IgG, IgG1 and IgG2c FLUOROSPOT Assay
(46) The fluorescent-linked immunospot (FLUOROSPOT) is used for detecting and enumerating individual B cells secreting antibodies irrespective of antigen specificity (IgG1, IgG2c or total IgG).
(47) On D0, the membrane of the 96-well IPFL-bottomed microplates (Multiscreen) was pre-wetted for 1 minute with 25 μL of 35% ethanol. Ethanol was then removed and each well was washed twice with 200 μL of PBS 1×. Microplates were then coated with CMV-gB antigen (10 μg/ml, Sanofi), CMV-pentamer (10 μg/ml, NAC) or total IgG antibody (10 μg/ml, KPL) diluted at 1/68, 1/100 and 1/100 respectively and were incubated overnight at 4° C.
(48) On D1, plates were washed with PBS and then blocked at least 2 h at 37° C. with RPMI 10% FBS.
(49) After plates washing, 5×10.sup.5 freshly isolated splenocytes/well for CMV-gB antigen or CMV-pentamer and 2.5.10.sup.5 freshly isolated splenocytes/well for total IgG antibody were incubated 5 hours.
(50) After 5 hours, the plates were washed 3 times with PBS 1× and stored à 4° C. for the night.
(51) On D2, the plates were washed 6 times with PBS 1×-BSA 0.1% (200 μL/well). After the washing step, 100 μL/well of the anti-mouse IgG1 PE or anti-mouse IgG2c FITC or anti-mouse total IgG antibodies were added respectively at 4, 2 or 0.5 μg/mL in PBS1×-BSA 0.1% for 2 hours at room temperature, in the dark. The plates were washed again 6 times with PBS 1×-BSA 0.1% (200 μL/well). The plates were stored at 5° C.±3° C. in the dark until reading.
(52) Each spot, corresponding to an antibody secreting cell (ASC) (IgG1 ASC, IgG2c ASC or total IgG ACS), was enumerated with an automatic FLUOROSPOT plate reader (Microvision). Results were expressed as number of antibody secreting cell per 10.sup.6 splenocytes.
(53) Results
(54) Humoral Response
(55) Longitudinal Analysis of the Neutralizing Antibody Response on ARPE-19 Epithelial Cells Between Day 20 and 257
(56) The neutralizing activity against the BADrUL131-Y4 CMV virus strain on epithelial cells (ARPE-19) was monitored by seroneutralization assays in individual intermediate serum samples collected monthly from all animals from subgroups 3 from day 20 to day 257 (i.e. at days 20, 34, 62, 90, 118, 153, 187, 226 and 257). The seroneutralization technique is detailed in the material and methods section and raw data are shown in Tables 5 a-b.
(57) Tables 5 a-b
(58) TABLE-US-00011 ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE Group Serum D20 + C M1 + C M2 + C M3 + C M4 + C M5 + C M6 + C M7 + C M8 + C A PBS GMT 24 16 25 29 23 21 17 22 16 B Pentamer: GMT 33 133 212 183 191 136 164 90 1026 2 μg gB: 2 μg C Pentamer: GMT 220 3625 3516 3748 3562 2034 2973 1058 6792 2 μg gB: 2 μg MF59: 2.3% squalene D Pentamer: GMT 879 15990 11598 10962 10724 10266 8681 5792 37166 2 μg gB: 2 μg PAA: 200 μg E Pentamer: GMT 383 12648 9288 7833 9048 5653 4494 3559 17936 2 μg gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: GMT 976 30755 20844 15957 17068 11231 11156 8505 35897 2 μg gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene a-Sub Group 3-intermediary-Seroneutralization ARPE with complement
(59) TABLE-US-00012 ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE Group Serum D20 − C M1 − C M2 − C M3 − C M4 − C M5 − C M6 − C M7 + C M8 + C A PBS GMT 30 16 18 13 71 22 12 12 13 B Pentamer: GMT 32 94 138 170 109 122 92 65 815 2 μg gB: 2 μg C Pentamer: GMT 74 2020 1973 1928 1264 1174 1028 655 5449 2 μg gB: 2 μg MF59: 2.3% squalene D Pentamer: GMT 83 3297 4890 3768 3589 3289 3580 2201 28657 2 μg gB: 2 μg PAA: 200 μg E Pentamer: GMT 91 3734 3354 3918 2728 2344 2359 1274 11910 2 μg gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: GMT 106 8048 7446 7774 5392 4812 3459 2883 9150 2 μg gB: 2 μg GLA- SQEM: 2.5 μg GLA, 2% squalene b-Sub Group 3-intermediary-Seroneutralization ARPE without complement
(60) Geometric mean titers (GMT) as well as individual neutralizing titers are depicted in
(61) M1=D34, M2=D62, M3=D90, M4=D118, M5=D153, M6=D187, M7=D226, M8=D257.
(62) Similar kinetics neutralizing antibody titer profiles were detected in presence and absence of complement in the epithelial-based neutralizing assay as depicted in
(63) For the group administered with unadjuvanted CMV-gB and pentamer, a low neutralizing antibody response was detected at day 20 (GMT=33 and 32, with or without complement, respectively) and then increased up to day 62 to reach a plateau with GMT ranging from 90 to 212 or from 65 to 170, in presence or absence of complement, between day 62 and day 226. The 3rd injection at day 226, boosted the neutralizing antibody titers as detected at days 257 with GMT=1026 or 815, in presence or absence of complement, respectively.
(64) For all the adjuvanted groups (MF59, PAA, AF04 and GLA-SQEM), neutralizing antibody titers were detected in presence or absence of complement on day 20 (i.e. 20 days after the first injection) with GMTs at 220 or 74, respectively, for group C3 administered with CMV-gB and pentamer adjuvanted with MF59 and with GMTs≥383 and ≥83 for groups D3 to F3 administered with the other adjuvant formulations. At day 34 (i.e. 14 days after the 2nd injection), all the adjuvanted groups presented the peak of the response after 2 injections with GMTs ranging from 3 625 to 30 755 or from 2 020 to 8 048, in presence or absence of complement, respectively.
(65) Over the 6 month period (between day 34 and 226), the epithelial-based neutralizing antibody titers slightly decreased down to titers ranging from 1 058 to 8 505 or from 655 to 2883, in presence or absence of complement, respectively. Similarly the 3rd injection at day 226, boosted the neutralizing antibody titers as detected at days 257 with GMTs ranging 6 792 (i.e. for MF59-) to 37 166 (i.e. for PAA-) or from 5 449 (i.e. for MF59-) to 28 657 (i.e. for -PAA adjuvanted group), in presence or absence of complement, respectively.
(66) To compare the different adjuvanted groups i.e. SPA09, AF04 and GLA-SQEM to the MF59 reference, a statistical mixed model with 2 fixed factors (group and time) was performed on repeated neutralizing antibody titers between days 34 to 226.
(67) With respect to the group comparison as presented in Table 6, in presence of complement, the neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with MF59 were not significantly superior to the neutralizing antibody titers obtained in mice administered with unadjuvanted CMV-gB and pentamer whereas all the other adjuvanted groups (i.e. PAA, AF04 and GLA-SQEM) were significantly superior to the neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with MF59 (all p-values <0.001).
(68) In absence of complement, the neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with MF59 were not significantly superior to the neutralizing antibody titers obtained in mice administered with unadjuvanted CMV-gB and pentamer. The neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with AF04 were not significantly superior to the neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with MF59, whereas all the other adjuvanted groups (i.e. PAA and GLA-SQEM) were significantly superior to the neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with MF59 (all p_values 0.009). The neutralizing antibody titers obtained in mice administered with CMV-gB and pentamer adjuvanted with AF04 were significantly superior to the neutralizing antibody titers obtained in mice administered with unadjuvanted CMV-gB and pentamer (all p-values <0.001).
(69) TABLE-US-00013 TABLE 6 In presence of In absence of ARPE-19 neutralizing assay Complement Complement Comparison P-value* P-value* (B3) unadjuvant vs (C3) MF59 1.000 (NS) 1.000 (NS) (D3) PAA vs (C3) MF59 <0.001 (S) 0.009 (S) x4.2 x2.4 (E3) AF04 vs (C3) MF59 <0.001 (S) 0.077 (NS) x2.8 (F3) GLA-SQEM vs (C3) MF59 <0.001 (S) <0.001 (S) x6.6 x4.0 Statistical comparison of the different groups within estimated repeated neutralizing antibody titers between days 34 to 226 (Superiority test, *p-values with Dunnett adjustment, NS: not significant or S: significant superiority, when significant the fold increase is indicated in italic).
(70) Detailed Neutralizing Antibody Response on Epithelial Cells (ARPE-19) and Fibroblasts (MRC-5) at Days 34 (M1), 208 (M7) and 257 (M8)
(71) The neutralizing activity against the BADrUL131-Y4 CMV virus strain on epithelial cells (ARPE-19) and fibroblasts (MRC-5) was monitored by seroneutralization assays in individual serum samples collected from all animals from subgroups 1, 2 and 3 at, respectively, days 34 (2 weeks after the second immunization), 208 (7 months after the primary vaccination series) and 257 (1 month after the booster injection at M7). The seroneutralization technique is detailed in the material and methods section and raw data are shown in Tables 7 a-f.
(72) Tables 7 a-f
(73) TABLE-US-00014 ARPE ARPE Group Serum J34 +C J34 −C A PBS GMT 12 12 B Pentamer: 2 μg GMT 46 43 gB: 2 μg C Pentamer: 2 μg GMT 4168 1934 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 14698 3862 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 7179 2919 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg gB: 2 μg GMT 13302 3146 GLA-SQEM: 2.5 μg GLA, 2% squalene a.- Sub Group 2- Seroneutralization ARPE D234 (D208)
(74) TABLE-US-00015 ARPE ARPE Group Serum M7 +C M7 −C A PBS GMT 14 17 B Pentamer: 2 μg GMT 46 51 gB: 2 μg C Pentamer: 2 μg GMT 3739 1834 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 4484 2230 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 6101 2718 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 7719 1758 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene b- Sub Group 2- Seroneutralization ARPE M7 (D208)
(75) TABLE-US-00016 ARPE ARPE Group Serum M8 +C M8 −C A PBS GMT 16 13 B Pentamer: 2 μg GMT 1026 815 gB: 2 μg C Pentamer: 2 μg GMT 6792 5449 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 37166 28657 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 17936 11910 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 8505 9150 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene c- Sub Group 3- Seroneutralization ARPE M8 (D257)
(76) TABLE-US-00017 MRC5 MRC5 Group Serum D34 +C D34 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 8 5 gB: 2 μg C Pentamer: 2 μg GMT 636 71 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 2699 84 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 1477 153 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 3131 146 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene d- Sub Group 1- Seroneutralization MRC5 - D34
(77) TABLE-US-00018 MRC5 MRC5 Group Serum M7 +C M7 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 5 5 gB: 2 μg C Pentamer: 2 μg GMT 147 27 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 500 39 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 451 48 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 913 14 gB: 2 μg GLA-SQEM: 2.5μg GLA, 2% squalene e- Sub Group 2- Seroneutralization MRC5 M7 (D208)
(78) TABLE-US-00019 MRC5 MRC5 Group Serum M8 +C M8 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 29 15 gB: 2 μg C Pentamer: 2 μg GMT 695 151 gB: 2 μg MF59: 2.3% squalene D Pentamer: 2 μg GMT 6645 536 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 1977 438 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 6152 427 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene f- Sub Group 3- Seroneutralization MRC5 M8 (D257)
(79) Geometric mean titers (GMT) as well as individual neutralizing titers are depicted in
(80) Similar neutralizing antibody profiles were observed on both epithelial- and fibroblast-based neutralizing assays, with higher neutralizing titers monitored in the epithelial-based neutralizing assay with at least 5-fold or 11-fold higher GMTs in presence or absence of complement, respectively.
(81) At day 34, i.e. 14 days after the 2nd injection, no or low neutralizing antibody titers were detected in mice immunized with unadjuvanted CMV-gB and pentamer (GMT 8 on MRC-5 and 46 on ARPE-19 cells, respectively). For all the CMV-gB and pentamer adjuvanted groups, a marked adjuvant effect was observed with a 14- up to 337-fold increase of the SN titers on MRC-5 and 44- to 319-fold increase on ARPE-19 cells, irrespective of the presence or absence of complement, compared to the unadjuvanted group.
(82) With respect to the neutralizing antibody titers on ARPE-19 epithelial cells in presence of complement (
(83) At the opposite, with respect to the neutralizing antibody titers on ARPE-19 cells in absence of complement (
(84) With respect to the neutralizing antibody titers on MRC-5 fibroblasts in presence of complement (
(85) Lastly, with respect to the neutralizing antibody titers on MRC-5 fibroblasts in absence of complement (
(86) With respect to the neutralizing antibody titers on ARPE-19 epithelial cells in presence or absence of complement (
(87) Lastly, with respect to the neutralizing antibody titers on MRC-5 fibroblasts in absence of complement (
(88) At day 257 (
(89) In all adjuvanted sub-groups 3 except MF59, the neutralizing antibody titers detected at day 257 after the 3rd injection were significantly higher than those detected at day 34 after the 2nd injection, whatever the cell type and whatever the presence or absence of complement (all p_values ≤0.002).
(90) At day 257, with respect to the adjuvant comparison to the MF59 reference, all the adjuvants (i.e. PAA and AF04) except GLA-SQEM induced higher neutralizing antibody titers than MF59, whatever the cell type and whatever the presence or absence of complement (test of superiority, unilateral Dunnet adjustment, all p-values ≤0.05). Regarding GLA-SQEM the induced complement dependent neutralizing antibody titers were significantly higher to those induce by MF59 (5.3- or 8.9-fold higher in ARPE-19 or MRC-5 cells respectively, test of superiority, unilateral Dunnet adjustment, all p_values <0.001), whereas in absence of complement the induced neutralizing were not significantly different, whatever the cell type.
(91) At D208, i.e. up to 7 months after the 2.sup.nd injection the composition comprising gB+Pentamer+AF04 or PAA or GLA-SQEM give higher neutralization antibody levels than a composition comprising gB+Pentamer+MF59, showing a better persistence of the functionality of the antibodies. At D257, 1 month after the boost, the measured neutralizing antibody increase reflects the memory response and shows higher titers for composition comprising gB+Pentamer+AF04 or PAA or GLA-SQEM than a composition comprising gB+Pentamer+MF59.
(92) All these results show that the immunogenic composition comprising gB+Pentamer+AF04 or PAA or GLA-SQEM give higher neutralization antibody levels and persistence than a composition comprising gB+Pentamer+MF59.
(93) IgG1 and IgG2c Antibody Responses
(94) CMV gB-specific and pentamer-specific IgG1 and IgG2c antibody responses elicited by the CMV gB and pentamer antigens administrated without or with different adjuvants were measured by ELISA in individual serum samples collected from all animals from subgroups 1, 2 and 3 at, respectively, days 34 (2 weeks after the second immunization), 208 (7 months after the primary vaccination series) and 257 (1 month after the booster injection at M7). Mean ELISA antibody titers (log 10 EU) are depicted in
(95) With respect to the IgG1 and IgG2c antibody responses similar profiles were obtained irrespective of the CMV-antigen specificity either gB or pentamer, whatever the analyzed time-point.
(96) Regarding IgG1 antibody titers, all the tested adjuvants significantly increased the IgG1 antibody titers compared to the unadjuvanted group. No significant difference was shown for AF04 when compared to MF59, whatever the antigen and the time-point. An adjuvant effect with significantly lower IgG1 titers than MF59 was observed for PAA at day 34 and 208 (at least 2.4-fold decrease, all p-values ≤0.045, test of difference, unilateral Dunnet adjustment) but not at day 257 after the 3rd booster injection. Compared to the MF59 reference, GLA-SQEM induced significantly lower anti-gB IgG1 titers (at least 2.5-fold decrease, all p-values ≤0.033, test of difference, unilateral Dunnet adjustment) at all the tested time-point and lower anti-pentamer IgG1 titers (at least 2.7-fold decrease, all p-values ≤0.005, test of difference, unilateral Dunnet adjustment) at day 208 and 257. Regarding IgG2c antibody titers, all the tested adjuvants significantly increased the IgG2c antibody titers compared to the unadjuvanted group. An adjuvant effect with significantly higher IgG2c titers than MF59 was observed for all the tested adjuvants i.e. PAA, AF04 and GLA-SQEM (at-least 11-fold higher; all p-values <0.001, test of difference, unilateral Dunnet adjustment) either for IgG2c specific to gB or pentamer whatever the time-point.
(97) ELISA IgG1/IgG2c Ratio
(98) In order to evaluate the Th2/Th1 orientation, IgG1/IgG2c ratios were calculated for all the adjuvanted groups and are detailed in
(99) As shown in
(100) Cellular Response
(101) IL5 and IFN-γ Cytokine Secreting Cells Monitored by FLUOROSPOT
(102) The IL5 and IFN-γ secreting cell frequencies were measured by FLUOROSPOT on splenocytes collected from all animals from subgroups 1, 2 and 3 at, respectively, days 34 (2 weeks after the second immunization), 208 (7 months after the primary vaccination series) and 257 (1 month after the booster injection at M7). During the FLUOROSPOT assay, each splenocyte suspension was ex-vivo stimulated overnight with either 0.1 μg/ml of recombinant CMV-gB or CMV-pentamer.
(103) The FLUOROSPOT technique is detailed in the material and methods section.
(104) As shown in
(105) At the opposite, high cytokine secreting cell frequencies were detected upon CMV-pentamer stimulation (
(106) With respect to the IFN-γ secreting cells frequencies, all the tested adjuvants i.e. PAA, AF04 and GLA-SQEM a significant 8- up to 29-fold increase of IFN-γ production was recorded compared to MF59 (all p-values ≤0.001, test of difference, unilateral Dunnet adjustment).
(107) At days 208, as shown in
(108) At day 257, both IL-5 and IFN-γ responses upon CMV-gB and CMV-pentamer stimulation increased as compared to day 34, however the Th1/Th2 profiles were conserved. With respect to the IL-5 secreting cells, high IL-5 SC frequencies were detected in mice administered with MF59 (268 and 2284 IL-5 SC/10.sup.6 splenocytes upon CMV-gB or pentamer stimulation, respectively).
(109) IL-5 secretions detected in groups administered with PAA, AF04 and GLA-SQEM were significantly lower than those obtained with MF59 (p-values ≤0.003, test of difference, unilateral Dunnet adjustment).
(110) With respect to the IFN-γ secreting cells frequencies, all the tested adjuvants i.e. PAA, AF04 and GLA-SQEM a significant increase of IFN-γ SC frequencies was recorded compared to MF59 (all p-values ≤0.001, test of difference, unilateral Dunnet adjustment). Taking together, all the tested adjuvants induced a more Th-1 biased overall response profile than MF59 consistent with the trend indicated by IgG1/IgG2c ratio.
(111) Consistent with the trend indicated by IgG1/IgG2c ratio, taking together, all the tested adjuvants induced a Th-1 biased overall cellular response profile while MF59 induced a Th2-biased overall cellular response profile.
(112) IgG1 and IgG2c Antibody Secreting Plasmablasts Monitored by ELISPOT
(113) The IgG1 and IgG2c antibody secreting plasmablast frequencies were measured by ex-vivo FLUOROSPOT on splenocytes collected from all animals from subgroups 1, 2 and 3 at, respectively, days 34 (2 weeks after the second immunization), 208 (7 months after the primary vaccination series) and 257 (1 month after the booster injection at M7). During the ELISPOT assay, each splenocyte suspension was deposited in wells coated either with recombinant CMV-gB or CMV-pentamer to capture either IgG1 or IgG2c specific antibodies presented at the plasmablast cell surface. IgG1 and IgG2c CMV-gB and pentamer-specific Antibody Secreting cells are enumerated and reported according the total IgG secreting cells; percentage of either IgG1 or IgG2c on total IgG are calculated. The FLUOROSPOT technique is detailed in the material and methods section.
(114) As presented in
(115) As expected, with respect to the detected ASC at day 208, responses were low, indicating that 6 months after the primary vaccination series, low rates of circulating plasmablasts were detected in mouse spleens.
(116) Thirty days after the 3rd injection (at day 257), the ASC frequencies, either IgG1 or IgG2c specific to CMV-gB or CMV-pentamer, increased as compared to day 208. Again, the means of IgG1 ASC frequencies at day 257 were ranging between 3.1% and 9% without significant differences between all the tested adjuvants. Regarding the IgG2c ASC frequencies, low % were detected when mice were administered with CMV-gB and pentamer adjuvanted with MF59. At the opposite, CMV-gB and pentamer adjuvanted with PAA, AF04 and GLA-SQEM induced significantly higher % of IgG2c ASC than MF59 (all p_values <0.001, test of difference, unilateral Dunnet adjustment), whatever the antigen specificity either CMV-gB or CMV-pentamer.
(117) IgG1 and IgG2c Antibody Secreting B Memory Cells Monitored by FLUOROSPOT
(118) The IgG1 and IgG2c antibody secreting cells frequencies were measured by FLUOSPOT at day 34, 208 and 257 on activated and enriched B cell splenocyte cultured for 4 days upon in vitro stimulation with IL-2 and R848. The FLUOROSPOT technique is detailed in the material and methods section.
(119) As presented in
(120) With respect to the detected ASC at day 208, B memory cells were detected mainly for IgG1 ASC specific to CMV-pentamer with % ranging from 1.6% to 3.24% independently of the tested adjuvant. Regarding the IgG2c ASC frequencies, low % were detected when mice were administered with CMV-gB and pentamer adjuvanted with MF59. At the opposite, CMV-gB and pentamer adjuvanted with PAA, AF04 and GLA-SQEM induced significantly higher % of IgG2c ASC than MF59 (all p_values <0.001, test of difference, unilateral Dunnet adjustment).
(121) Thirty days after the 3rd injection (at day 257), the means of IgG1 ASC frequencies were ranging between 1.1% and 3.75% without significant differences between all the tested adjuvants.
(122) Regarding the IgG2c ASC frequencies, low % were detected when mice were administered with CMV-gB and pentamer adjuvanted with MF59. At the opposite, CMV-gB and pentamer adjuvanted with PAA and GLA-SQEM induced significantly higher % of IgG2c ASC than MF59 (all p_values <0.001, test of difference, unilateral Dunnet adjustment), whatever the antigen specificity either CMV-gB or CMV-pentamer.
(123) These results show a higher memory response level with a composition comprising gB+pentamer+PAA or AF04 or GLA-SQEM than a composition comprising gB+pentamer+MF59. It is clear also that this higher memory cell frequency, which is known to be the mediator of the protection persistence, keeps a predominant Th1-type response profile.
Example 2
(124) Complementary Effect of the Two Antigens
(125) In a design of experiment study the inventors studied the combined dose-ranging effect of the two antigens in presence of PAA adjuvant. For that purpose 11 groups of 10 female C57/BI6J mice received by intra-muscular route on days 0 and 22 doses ranging from 0 to 5 μg of CMV-gH/gL/UL128/UL130/UL131 pentamer with or without doses ranging from 1.2 to 5 μg of CMV-gB in presence of PAA adjuvant. The antibody response was assessed by ELISA specific to gB and gH/gL/UL128/UL130/UL131 pentamer (IgG1/IgG2c subclasses) and neutralization assays on D22 (with complement, on ARPE-19 epithelial cells) and D35 (with and without complement, on MRC5 fibroblasts and ARPE-19 epithelial cells). The cellular response was assessed on D35 by IFN-y ELISPOT upon in-vitro stimulation with gB and pentamer recombinant proteins and pentamer peptide pools.
(126) The neutralizing activities monitored either on epithelial cells ARPE-19 or fibroblasts MRC-5 presented similar profiles, with higher neutralizing titers recorded on epithelial cells than fibroblasts (2- to 5-fold higher titers in ARPE-19 than MRC-5 cells). On day 20, (
(127) In conclusion, in presence of complement, the addition of the gB on the top of pentamer allows to increase the SN titers on both epithelial and fibroblast cells.
(128) On day 35, i.e. 14 days after the 2nd administration, high neutralizing antibody titers inhibiting both epithelial cells and fibroblasts infection in presence of baby rabbit complement were detected whatever the administered doses of either gB or gH/gL/UL128/UL130/UL131 pentamer. The detected neutralizing activities were at a plateau with no significant dose effect for neither the pentamer nor the gB (all p-values ≥0.240) (
(129) On day 35, the complement independent neutralizing activities inhibiting both epithelial cells and fibroblasts infection in absence of baby rabbit complement was also monitored.
(130) As depicted in
(131) In conclusion, in absence of complement, the addition of the pentamer on the top of gB allows to increase the SN titers on both epithelial and fibroblast cells Thus, complementary effect of the two antigens was evidenced by their respective effect on the neutralizing antibody response quality. With respect to the analysis of the functional humoral responses, i.e. the complement dependent and independent neutralizing antibodies, it was demonstrated that the combination of the two antigens provided an extended mode of action for virus neutralization. CMV-gB allows increasing neutralizing antibody titers on epithelial cells and fibroblasts in presence of complement and CMV-gH/gL/UL128/UL130/UL131 pentamer allows achieving complement independent neutralizing antibody on epithelial cells and fibroblasts.
(132) Moreover, this broadening property of the CMV-gB and CMV-gH/gL/UL128/UL130/UL131 pentamer combination was also noticed on the induced cellular responses. As depicted in
(133) In conclusion, the addition of the pentamer on the top of gB allows to increase the IFN-γ cellular response by broadening the number of cellular epitopes.