GB2600468A - Adjuvant composition - Google Patents

Adjuvant composition Download PDF

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GB2600468A
GB2600468A GB2017282.1A GB202017282A GB2600468A GB 2600468 A GB2600468 A GB 2600468A GB 202017282 A GB202017282 A GB 202017282A GB 2600468 A GB2600468 A GB 2600468A
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antigen
adjuvant
immunogenic composition
formulation
composition
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GB202017282D0 (en
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Laing Peter
Wagner Andreas
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Excivion Ltd
Excivion Ltd
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Excivion Ltd
Excivion Ltd
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Priority to PCT/EP2021/080285 priority patent/WO2022090550A1/en
Publication of GB2600468A publication Critical patent/GB2600468A/en
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Abstract

An adjuvant composition comprising liposomes comprising a synthetic or natural Monophosphoryl Lipid A (MPLA) in the range of 70 to 100 µg/ml, a sterol in the range of 1.23 to 1.95 mg/ml, a phosphatidylcholine (PC) in the range of 2.81 to 4.69 mg/ml, a phosphatidylglycerol (PG) in the range of 0.36 to 0.60 mg/ml, a disaccharide at weight per unit volume in the range of 7% to 12% w/v, and a saponin in the range of 30 to 50 µg/ml is provided. The MPLA is preferably a synthetic derivative, the phosphatidylcholine is preferably dimyristoyl phosphatidylcholine, the phosphatidylglycerol is preferably dimyristoyl phosphatidylglycerol, the disaccharide is preferably sucrose and the saponin is preferably QS21. An immunogenic composition comprising the adjuvant and one or more antigens is provided. The antigen is preferably a flavivirus antigen selected from a Zika antigen and a dengue antigen. A method of manufacturing an immunogenic composition is provided. The immunogenic composition is preferably lyophilised and may be reconstituted prior to use in sterile water. The dried immunogenic composition is preferably stable on storage for at least one month at 40 °C.

Description

Intellectual Property Office Application No G132017282.1 RTM Date 12 May 2021 The following terms are registered trade marks and should be read as such wherever they occur in this document: Superdex Polymun GeneArt Gene Wiz Alhydrogel CytoFLEX Tween Triton Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo Adjuvant Composition
Technical Field
The invention relates to liposomal compositions for use as adjuvants in immunogenic compositions containing antigen, to enhance the cell-mediated immune response and antibody response to antigens to confer protective immunity.
Background Art
Vaccine safety and efficacy are of increasing concern, such that there is a desire to create new prophylactic and therapeutic vaccines (e.g., for pandemic preparedness or response, or for cancer therapy, or allergy) which will be effective without causing serious side effects. To this end, subunit vaccines based on recombinant subunit antigens, have an important role to play because of their favourable safety profile, e.g., being incapable of causing infections in subjects with compromised immunity (which are potentially fatal when live attenuated vaccines are unwittingly administered to such subjects). Although hi storically and generally subunit vaccines have had the disadvantage of lesser efficacy than live attenuated vaccines, requiring multiple doses in order to be efficacious, recent developments in adjuvant technology have permitted subunit vaccines that are very highly efficacious even after a single dose (e.g., Cervarix', a papillomavirus vaccine for prevention of cervical cancer), or which have outperformed comparable live-attenuated strategies: e.g., Shingrie (a herpes zoster vaccine for shingles prevention), which is 89% efficacious in persons over eighty years of age, compared to Zostavax® (a live attenuated zoster vaccine) which is only 51% efficacious in persons over sixty years of age'.
Obtaining optimal adjuvant performance in a vaccine formulation is challenging because there is a need to avoid unacceptable systemic reactions (e.g., pyrexia, shivering), which limits the dose of adjuvant that can be administered. A common feature of adjuvant materials used in subunit vaccines is that they can be detected as danger signals by receptors of the innate immune system', present in abundance on and in (in the membranes of endosomal vesicles) antigen presenting cells, such as macrophages and dendritic cells. Lipopolysaccharides (LPS, or endotoxin), comprising the outer membrane lipids of Gram-negative bacteria, are a potent danger signal to the immune system, signifying the presence of a potential infectious threat.
While some vaccines intentionally contain LPS (e.g. group-A meningococcal vaccine), this is achieved safely in the form of membrane vesicles in order to attenuate the toxicity of the LPS, but nevertheless LPS-adjuvanted vaccines and whole-cell pertussis vaccines (containing LPS) are among the most reactogenic of marketed vaccines'. In bacterial infections LPS is a major cause of toxic shock and cytokine release syndrome. In recent years however, much progress has been made in effectively managing the toxicity of LPS5, and several vaccines contain an attenuated version of LPS such as natural monophosphoryl lipid-A (MPLA), based on the core lipid-A' structure of LPS. MPLA has the remarkable property of being able to stimulate TLR4 (toll-like receptor-4, an important receptor of the innate immune system) in a manner that avoids the potentially fatal toxicity of LPS, and is surprisingly non-toxic even in rather high dosages, well in excess of those needed to obtain maximal adjuvant effect 6. Indeed, in mice, prior administration of MPLA, rather than mimicking the toxic effects of endotoxin, pre-empts toxic responses to subsequently administered endotoxing.
Plant saponins are another class of vaccine adjuvant material that have been widely used in veterinary and latterly in human vaccines. Kensil purified a fraction (QS21', from the soap-bark tree Ouillaja soponaria Molina) which comprises a mixture of triterpene glycosides that are particularly potent as vaccine adjuvants'''. QS21 has a differing and complementary mechanism of action to that of MPLA which is, as yet, incompletely understood, although it is known to involve recruitment of dendritic cells and action via the NLRP3 inflammasome, which is not activated by IMPLA. Initially the adjuvant activity of QS21 was thought to have been dependent upon its cell-lytic properties but it was discovered that QS21 can be incorporated into particles (e.g., ISCON4s, liposomes) which effectively attenuate its lytic toxicity while preserving its adjuvant activity'''. Particulate formulation strategies (ISCOM, emulsion, liposomal) also serve to direct adjuvant materials to antigen presenting cells, increasing their effectiveness as vaccine adjuvants, provided that the particles retain a nanoparticulate size distribution, which maximises access to these cells in the lymphoid tissue draining the injection site.
While formulations incorporating MPLA and/or QS21 have found great utility as vaccine adjuvants, especially when these are rendered in particulate form, generally these have required a refrigerated formulation that cannot be frozen without detrimentally affecting the particle structure by aggregation of the nanoparticles to form microparticulates, potentially reducing the adjuvant effect. Since accidental freezing is a major cause of vaccine spoilage in transit", the vulnerability of such formulations to freeze-spoilage is a significant problem in vaccine storage and distribution. Also, particulate vaccine formulations containing MPLA and QS21 are generally not stable to heat either, requiring refrigeration for long term storage and for field distribution, which is difficult to achieve in limited-resource settings.
Aqueous formulations containing QS21 and/or or MPLA are typically required to be stored at mild-acid pH as a precaution against hydrolytic degradation of QS21 and MPLA derivatives which, by simulating endosomal pH, is prone to trigger undesirable and irreversible conformational changes in certain vaccine proteins, particularly viral coat proteins which are important targets for direct-neutralising or complement-fixing antibodies. Also, many vaccine antigens are isoelectric in the mildly acidic range, making them prone to isoelectric precipitation in such conditions upon long term storage.
Comparable emulsion-based systems (as distinct from liposomes) have been developed that effectively harness the adjuvant activities of MPLA and QS21, however in the development of Mosquirixm, in side-by-side comparison of emulsion and liposome forms, the initial choice of an emulsion form (AS02) was reconsidered when it was discovered in clinical studies that the liposome form (AS01) was more effective in clinical trials of Mosquirix (Didierlaurentu) Furthermore, to the best of our knowledge, lyophilisable or heat stable forms of emulsion-based adjuvant systems, though notionally possible, have not so far been described.
Maintaining chemical stability, particle size stability (as nanoparticles), and antigenic stability of a liposomal vaccine formulation containing both MPLA and QS21 poses certain challenges. The use of saturated liposomes is desirable since these are fundamentally more stable both physically and chemically than are unsaturated liposomes such as the liposomes of AS01'2'1' (the adjuvant used in Shingrix® and Mosquirix®). Indeed, in general, the lipid bilayers of saturated liposomes are solid at the storage temperature of 2-8°C making them more stable to undesirable fusion and hydrolytic reactions The nature of some of these difficulties can be summarized as follows.
The addition of QS21 (a saponin) to saturated nanoparticulate NIPLA-containing liposomes results in formation of extremely large liposomes of up to 30 jun ('ALFQ") m. However, nanoparticulate size distribution is highly desirable for vaccine adjuvants in order to maximise their efficacy's The effect of aggregation is to dramatically reduce the 'molarity' of the adjuvant, (considering a single liposome as a molecule), that is, to increase the size of the particules but decrease their number, thereby reducing the ability of the larger liposome microparticulates to access draining lymph nodes and generally limiting their bioavailability to antigen-presenting cells. In contrast, the unsaturated liposomes of AS01 do not aggregate upon addition of QS21 (Vandepapeliere, P., 2011; US Patent Application No, 13/020,045, Publication No. US2011/0206758). However, AS01 liposomes, and vaccines that use them, have to be both stored and distributed in the refrigerated state due to the instability of their liposomes to freezing and to high temperatures. Recently, an adapted lyophilisable form of AS01 has been developed (see below) but, requiring a pH of 6.1, it has the disadvantage that it is not applicable to protein antigens that are not stable at mildly acid pH. Moreover the lyophilisable AS01 formulation uses potassium phosphate buffer and likely exhibits further pH excursion (due to precipitation of the buffering phosphate ion) into the acidic region below pH 6.1, upon the freezing step of lyophilisation, which is undesirable.
Accordingly there is a need for adjuvant formulations that minimise systemic toxicity, are stable on storage in lyophilised form and do not require refrigerated storage or transport, which can be readily rehydrated for administration while maintaining a uniform nanoparticulate size distribution and which can be used with antigens that are acid-labile.
Statements of Invention The invention provides:
1. An adjuvant composition comprising liposomes comprising: a synthetic or natural Monophosphotyl Lipid A (MPLA), preferably a saturated synthetic MPLA, at a concentration in the range of from 70 to 100 pg/ml, preferably at a concentration of about 80 g/m1; a sterol at a concentration in the range of from 1.23 to 1.95 mg/ml, preferably at a concentration of about 1.64 mg/ml; a phosphatidylcholine (PC) at a concentration in the range of from 2.81 to 4.69 mg/ml, preferably at a concentration of about 3.75 mg/mi.; a phosphatidylglycerol (PG) at a concentration in the range of from 0.36 to 0.60 mg/ml, preferably at a concentration of about 0.47 mg/ml; a disaccharide at weight per unit volume in the range of from 7 % to 12 (?,() w/v, preferably about 8 % w/v and a saponin at a concentration in the range of from 30 to 50 pg/ml, preferably at a concentration of about 40 jig/ml.
2. An adjuvant composition according to clause 1, wherein the N1PLA is a synthetic MPLA selected from 3D (6-acyl) PHADO, 3D-PHADO and PHADO; and / or the sterol is cholesterol; and / or the phosphatidylcholine (PC) is selected from the group consisting of dimyristoyl phosphatidylcholine (DNIPC), dipalmitoyl phosphatidylcholine (DPPC) and distearyl phosphatidylcholine (DSPC); and / or the phosphatidylglycerol (PG) is selected from dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG) and distearyl phosphatidylglycerol (DSPG); and / or the disaccharide is sucrose or trehalose; and / or the saponin is selected from QS21, QS7 and QS18.
3. An adjuvant composition according to clause I or clause 2 wherein the MPLA is 3D (6-acyl) PHAD®, the sterol is cholesterol, the phosphatidylcholine (PC) is dimyristoyl phosphatidylcholine (DMPC), the phosphatidylglycerol (PG) is dimyristoyl phosphatidylglycerol (DMPG), the disaccharide is sucrose and the saponin is QS21 4. An adjuvant composition according to any one of the preceding clauses further comprising a physiologically-acceptable buffer at pH in the range of from 7.0-8.5 5. An adjuvant composition according to clause 4 wherein the buffer is selected from FIEPES (lEPES-Na0H, HEPES KOH), PIPES, ACES, MOPSO, BIS TRIS propane, BPS, DIPSO, Tris, Tricine, Gly-Gly, EPPS(HEPPS), Bicine, TAPS, and AMPD at pH in the range of from 7.0-8.5.
6. An adjuvant composition according to any one of the preceding clauses comprising PBS and / or NaC1 at physiological osmolarity wherein the PBS and / or saline element comprises less than 7.5% (v/v) by volume of the composition.
7. An immunogenic composition comprising an adjuvant composition of any one of clauses 1 15 to 6 and one or more antigens 8 An immunogenic composition according to clause 7, wherein the concentration of antigen in the composition is in the range of from 10 ps / ml to 600 jig / ml, preferably about 60 pg/ml.
9. An immunogenic composition according to clause 7 or clause 8, wherein the one or more antigen is selected from a viral antigen, a protozoal antigen, bacterial antigen, a fungal antigen, a cancer antigen or an allergen.
10. An immunogenic composition according to clause 9, wherein the one or more viral antigen is selected from a flavivirus antigen, influenza antigen, an RSV antigen, a Varicella zoster antigen, a herpes simplex virus antigen, a cytomegalovirus antigen, an Ebola virus antigen and a coronavirus antigen 11. An immunogenic composition according to clause 9 or clause 10, wherein the one or more viral antigen is selected from a Zika antigen and a dengue antigen.
12. An adjuvant composition or immunogenic composition of any one of clauses 1 to 11 aqueous suspension haying a pH of 7.0 to 8.0, preferably pH 7.5, or in dry (lyophilized) form.
13. An adjuvant composition or immunogenic composition of any one of clauses 1 to 12, wherein the composition is in dry (lyophilized) form and on reconstitution in aqueous solution (e.g. in water) the liposomes have a predominantly nanoparticulate particle size distribution.
14. A adjuvant composition or immunogenic composition of any one of clauses 1 to 13, wherein the composition is in dry (lyophilized) form and on reconstitution of the adjuvant composition or immunogenic composition in aqueous solution (e.g. in water) >80%, preferably >90%, more preferably >95%, of the particles in suspension are in the nanoparticle size range (less than 1 micrometre diameter), as determined by area-under-the-curve measurement of intensity in DLS.
15. An immunogenic composition according to any one of clauses 7 to 14 formulated for intramuscular (IM), subcutaneous (SC), nasal or oral administration 16. An immunogenic composition according to any one of clauses 7 to 15, wherein a unit dose is in the range of from 0.25 to 1.5m1, preferably in the range of from 0.5 to 1.0 ml.
17. An immunogenic composition according to any one of clauses 7 to 16 for use in administration to a subject to stimulate an immune response.
18. A method for manufacture of an immunogenic composition according to any one of clauses 7 to 17, comprising the steps of: (a) providing an adjuvant composition of any one of clauses 1 to 6 (b) addition of one or more antigen to the adjuvant composition to form an immunogenic 20 composition, (c) optionally lyophilizing the immunogenic composition 19. A method for preparation of an immunogenic composition comprising reconstituting a lyophilized immunogenic composition of any one of clauses 7 to 17 in an aqueous solution, preferably in sterile water.
20. An immunogenic composition according to any one of clauses 7 to 17, wherein (a) the composition is stable on storage at 40 °C in the dry, lyophilized state for at least one month, and / or (b) the composition is stable at 2 to 8 °C for at least 9 months, and / or, (c) the dry, lyophilized composition is stable following freezing, stability being assessed following reconstitution with water, wherein stability is assessed by one or method selected from HPLC analysis of lipid components, immunoassay of protein antigen components, SDS electrophoresis of protein antigen components and measurement of liposomes by dynamic light scattering.
21. An immunogenic composition according to any one of clauses 7 to 17, wherein the lyophilized composition reconstituted in water is stable at 25 °C for at least 6 hours.
22 An adjuvant or immunogenic composition according to any one of clauses 1 to 17 comprising a composition as shown in Table D herein.
23. An adjuvant or immunogenic composition as described herein with reference to the
description, clauses and / or figures.
Advantages of the Invention Here we describe a novel formulation of a liposom al adjuvant preferably containing a synthetic saturated form of MPLA and QS21 which can be made resistant (by lyophilisation) to freeze-spoilage and moreover, in the lyophilised state, is sufficiently stable at 40 degrees Celsius to allow field distribution without refrigeration. In GLP toxicology studies, using a recombinantly-derived Zika exodomain dimeric antigen, we demonstrate that the formulation is easily rehydrated while maintaining a uniform nanoparticulate size distribution, avoids clinically evident systemic toxicity (pyrexia) in rabbits, and is well tolerated by intramuscular injection.
The adjuvant system, unlike several comparable systems, can be lyophilised, allowing better stability on refrigerated storage than liquid formulations and the more rapid development of novel vaccine products by conferring heat stability to the antigen and adjuvant materials in the dry state. Unlike other comparable systems it spares vaccine constituents from exposure to non-physiological pH values, which may trigger inappropriate irreversible conformational changes in an antigen, particularly viral antigens e.g., triggering of non-protective fusion conformations. The formulation comprises a liposome in which all lipid ingredients are saturated, and containing a synthetic derivative of monophosphoryl lipid-A (MPLA), plus the saponin QS21. The formulation is reconstituted rapidly and retains a uniform nanoparticulate character after lyophilisation, important to maximise adjuvant function after reconstitution from the lyophilized state, and is non-toxic.
A further advantage of the novel formulation is that it avoids the necessity to store aqueous formulations containing QS21 and/or or MPLA at mild-acid pH (a necessary precaution in aqueous formulations against hydrolytic degradation of QS21 and MPLA derivatives). This latter property of the compositions of the invention (i.e., maintenance of pH in the mildly alkaline range) markedly extends the utility of vaccine adjuvants based on MPLA and QS21 to an important subset of proteins which are acid-labile.
We have developed a novel saturated liposome adjuvant Formulation, based on known MPLA liposomes which (unlike ALFQ, defined above) substantially retains a nanoparticulate size distribution upon addition and insertion of QS21 and after lyophilisation and reconstitution in a manner which preserves the antigenic structure and immunogenicity of antigen co-present in the formulation. The new adjuvant formulation also allows the full expression of the functional properties of the adjuvant materials MPLA and Q S21, as well as maintaining their chemical stability and the stability of excipient lipids. The new formulation may comprise two components: a lyophilised vial with all vaccine solutes (Including the liposomes and the antigen) plus sterile water for reconstitution of the lyophilised material for injection. As such it is remarkably stable and resistant to accidental freezing in transit, and moreover does not require refrigerated distribution. Lyophilised immunogenic formulations of antigen in the novel liposome adjuvant have minimal activity in the MAT test for LPS and non-LPS pyrogens, which uses human monocytes. Moreover, a Zika subunit vaccine formulation studied in GLP toxicology in the rabbit at the full anticipated human dose was found to be non-toxic after four doses, exhibiting transient injection site reactions typical of an adjuvanted vaccine, with zero incidence of pyrexia. In these toxicology studies of the new formulation, expected elevations of CRP were seen after each dose, with minor escalation only after the third and fourth dose. CRP was measured frequently around the first and fourth dose, dropping back to normal levels following dosing, e.g., at two days after the fourth dose. CRP elevations were very similar to those seen in published toxicology studies of Shingrix0 in the rabbit (Giordano') indicating that the new adjuvant composition will enable safe and effective immunogenic vaccine compositions for human use, with the added advantage of stability. Likewise, neutrophil blood counts were only transiently elevated (as reported for Shingrix0)16, and there was no elevation of white blood cells generally or of monocytes (which were reduced somewhat after dosing). Except for local injection-site reactions commensurate with expectation for an adjuvanted vaccine product, there were no histological changes in non-injected tissues or systemic toxicological findings.
Another advantage of the new adjuvant formulation is that it enables the faster development of new vaccine products: i.e., its amenability to lyophilisation means that a vaccine developer can be confident at an earlier stage of product development that the formulation will transpire to be stable, upon lengthier stability studies. For example, in liquid storage, proteins and/or liposomes can exhibit slow aggregation behaviour, or a protein antigen may have a minor contamination with a proteolytic enzyme that becomes apparent (by antigen destruction) only after many months of storage. A lyophilised formulation avoids such risks, because these phenomena can only happen in the liquid state. This is a major advantage of the present adjuvant system, enabling more-rapid development of new subunit vaccines, or vaccines based on virus-like particles or inactivated viruses. Liposomal adjuvant compositions of the invention for use in immunogenic compositions containing viral antigen, enhance the cell-mediated immune response and antibody response to the viral antigens to confer protective immunity. The cell-mediated Th 1 profile of the response, including complement-fixing antibody subclasses, will also be advantageous for vaccines against parasitic infections, certain bacterial infections, cancer and allergy.
List of Figures Figure 1: in vitro antigen-stimulated T-cell responses in mice immunised with monomeric subunit and VLP forms of Zika exodomain antigen in various adjuvant formulations. Female BALB/c mice in groups of 8 were immunised on three occasions at 14-day intervals with four different molecular forms of Zika antigen and four different liposomal adjuvant compositions. At sacrifice (day 42), spleens were removed and cells cryopreserved for later measurement of antigen-stimulated IFN-y responses of individual cells from cell suspensions of individual animals by ELISPOT. Molecular forms were His-tagged Zika MC from HER cells, His tagged Aalto wild-type Zika, Strep-tag-II-tagged Zika HX from Tni cells (CR068) and wild-type Zika VLP from ELK cells, displayed in columns of panels (a-d respectively; e-h respectively, etc.). Adjuvant compositions are displayed as rows of panels, from top to bottom PHAD+Alum (a-d); 3D-6-Acyl+Alum (e-h); 3D-6-Acyl alone (i-1); 3D-6-Acyl+QS21 (m-p). Antigen re-stimulation in vitro used CR028 -a His-tagged Zika FIX monomeric exodomain made from Tni cells. A = raw data; B = statistical analysis as follows. Fold change from mean of unstimulated to antigen restimulated (Ag-restimulated), by adjuvant and shaded by molecular form, with 95% confidence intervals. Letters indicate homogeneous groups at the adjusted 5% levels, where different letters indicate significant differences: lower case letters are to compare between adjuvants within a molecular form (i.e. within a shade of grey-and uppercase letters are to compare between molecular forms within an adjuvant). A dotted line at a fold change of 1 illustrates the significance line for the lower confidence limit Figure 2: Direct neutralising antibody responses to various molecular forms and adjuvant formulations of Zika exodomain antigen. Sera taken at day 42 from the mice described in Fig. 1 were tested for anti-Zika neutralising antibodies by micro neutralisation assay following immunisation with the following monomeric antigens: (a) His-tagged ZikaHX from HEK; (I)) His-tagged Aalto wild-type Zika exodomain; Strep-lagged Zika-HX from Tni; (d) Zika VLP from FMK; (e) vehicle (PBS containing Alhydrogel, PHAD and QS21).
Figure 3: Assessment of Thl/Th2 polarisation of antibody response to the formulations of Fig. I by measurement of IgG2a and IgGI. Sera taken at day 42 from the mice described in Fig. I were subject to IgG isotype specific ELISA tests: a = His-tagged Zika HX from HEK PHAD + Alum; b = His-tagged Zika 1-1X from HEK 3D6Acyl+QS21; c = His-tagged Aa1to WT Zika PHAD + Alum; d = Strep-tag-II tagged Zika HX from Tni 3D6Acyl-Q S21; e = Zika VLP from HEK PHAD + Alum; N Zika VLP from HEK 3D6Acyl + Alum; g = Zika VLP from HEK 3D6Acyl only; h = Zika VLP from HEK 3D6Acyl+Q521. Controls: i = Zika VLP from HEK 3D6Acyl+QS21; j = murine monoclonal IgG2a antibody 4G2 starting at 1 microgram/ml; j = Purified 18G I coated direct to solid phase; k = Purified IgG2a coated direct to solid phase; m = comparison of curves from j and k.
Figure 4: Live challenge of mice immunised with Zika-HX dimer and Zika HX oligomer.
The Zika His-tagged antigens were formulated in 3D-6-Acyl-P1-IAD (40 jig per dose) plus QS21 (20 ps per dose), at a 30 jig protein dose, wherein the QS21 was added to Polymun liposomes according to the formulation process described under "Measurement of antigen-specific T-cell responses to Zika antigens and formulations". Statistical significance of differences from the vehicle control at day 3 post-challenge are indicated Error bars represent standard deviations.
Figure 5: Recombinant expression, cloning and purification of tag-free Zika-HX dimer in Drosophila S2 cells. Recombinant ZHX protein was produced in a cloned Drosophila S2 cell line and secreted into cell culture medium. Protein was harvested from supernatant by centrifugation, concentrated and buffer was exchanged using a tangential flow filtration system using 10-turnover volumes. Contaminating proteins were precipitated out with ammonium sulfate solution discarding the precipitate by filtration and the target protein was captured using hydrophobic interaction chromatography (HIC, Sartobind Phenyl) followed by ion-exchange chromatography (IEX, Sartobind-Q). Elution fractions were pooled and concentrated using Amicon Ultra spin concentrators and further purified by preparative size exclusion chromatography (SEC) using a Superdex 200 column (GE), eluted in 1xPBS. Fractions containing the target protein were pooled, concentrated and sterile filtered, yielding a solution of the target protein at 2.75 mg/ml, and stored frozen at -80 °C. The purified protein was analysed by SDS-PAGE with Coomassie blue staining and was found by densitometry to be 92.9% pure. The protein behaved as expected, having dimeric and monomeric bands of the appropriate molecular weight and converting from a dimer to a monomer when dithiothreitol was present in the SDS electrophoresis sample buffer. In the gel shown, lanes were as follows: 1-See blue plus-II molecular weight markers (ThermoFisher); 2 -BSA 0.1 mg (reduced); 3 -the purified protein (reduced) at 1 mg load; 4 -the purified protein (reduced) at 10 pig load; 5 -the purified protein (non-reduced) at 1 tig load; 6 -the purified protein (non-reduced) at 10 Fig load.
Figure 6: Preservation of nanoparticulate size distribution in a lyophilised vaccine formulation following addition of QS21 and antigen to saturated MPLA liposomes and following subsequent lyophilisation and reconstitution. A sample of Polymun stock PHAD liposomes were rendered into 9% w/v trehalose 20 mM HEPES pH 7.5 by diafiltration. QS2 I was then added either to sucrose-based or to trehalose-based liposomes, as described for Table 3, except that monomeric antigen CR068 (SEQ ID NO: 2) was subsequently added to each formulation at a concentration of 0.8 mg/ml in PBS. The mixtures were diluted with 9% w/v of the appropriate sugar containing 20 mM HEPES-NaOH pH 7.5 to create formulations conforming to the Specification of Table 3 (except for the use of monomeric Zika-EXCR068 in place of the dimeric Zika-EX, and except that one of these formulations contained trehalose in place of sucrose). Graphs depict size-distribution (diameter) of liposomes measured by DLS: trehalose -left three panels; sucrose right three panels. Process stages are as follows: fresh formulation containing all ingredients including antigen (top panels); after lyophilisation and immediate reconstitution (middle panels); and after one week's storage in the dry state at 2-8 C (lower panels). Trehalose (a,b,c), sucrose (d,e,f). Fresh -a, d; reconstituted immediately b, e; reconstituted after 1 week, c, Reconstitution used deionised water.
Figure 7: GLP toxicology studies of a Zika liposomal vaccine formulation containing dimeric tag-free Zika-HX antigen in the rabbit. Male and female NZW rabbits were dosed four times with the vaccine formulation of Table 3 at 0.5 ml per dose i.m., at 14 day intervals, as described in Materials and Methods. Measurements of CRP (a), neutrophil counts (b), monocyte counts (c) and white blood cell counts (d) were taken at intervals as measures of systemic inflammation. Histogram bars are in clusters of four as follows: i) saline-control, male; ii) saline-control control, female; iii) lyophilised Zika vaccine formulation, male; iv) Zika vaccine formulation, female.
Figure 8: Development of anti-Zika IgG responses in pooled sera from the toxicology study of Fig. 7 after successive doses of lyophilised Zika vaccine formulation or saline control. Class-switched (IgG) antibody responses were measured by ELTSA in rabbit sera (counting the first day of injection as day-1 as distinct from day-zero in previous figures) The first cluster of each paired set of data points is the saline control, the second the vaccine formulation.
Examples
Materials and Methods Ethical Statement
All animal procedures were conducted according to local or national regulations for the welfare of animals in scientific research, as regulated by the Home Office in the UK.
Recombinant Zika antigen expression cloning and purification All recombinant proteins used were generated as DNA by GeneArt (Life Technologies) or GeneWiz using appropriate species-appropriate codon optimisation. Zika E-protein 'FIX' exodomain antigen having a mutated fusion-loop containing an introduced N-linked glycosylation sequon at residue 100 (i.e. Nl'HT, in place of the G1'WG of natural Zika exodomain fusion-loop sequences) and a hexahistidine tag at the C-terminal end of the soluble exodomain sequence was recombinantly expressed by transient transfection with linear polyethyleneimine in human embryonic kidney cells as described in PCT/US2017/033882 (SEQ ID NO: 1). The recombinant protein was purified using immobilized metal chelate affinity chromatography (IMAC) using cobalt chelate. The same sequence was also expressed via baculovirus expression in Tni cells as described (for Sf9 cells) by Hitchmae, and purified by INIAC using a Nickel chelate affinity chromatography medium. A variant of the fusionloop-glycosylated 'IIX' exodomain protein was also expressed from Tni cells with a Strep-tagII at its C-terminal end (SEQ ID NO: 2). Fusion-loop glycosylated dimeric Zika FIX antigens (having an inter-subunit disulfide bond) were also produced (Zika-HX dimeric antigen) in His-tagged SEQ ID NO: 3 and tag-free forms SEQ ID No: 4 (the latter from a cloned line of Drosophila S2 cells). The tag-free protein was purified from cultures of 52 cells by ammonium sulfate precipitation, to precipitate contaminating proteins which were filtered out. The clarified supernatant was then purified by successive rounds of hydrophobic interaction, anion-exchange and gel-permeation chromatography, selecting the dimeric species.
Measurement of antigen-specific T-cell responses to Zika antigens and jOrmulations Groups of eight BALB/c female mice 6-8 weeks old were immunised with various liposomal formulations (with or without Alhydrogel) containing 5 micrograms (a non-saturating dose) of various monomeric Zika antigens (SEQ IDs below) in a 60 R1 volume by intramuscular injection. Dosing was conducted on days 0, 14 and 28. Spleens were removed at sacrifice on day 42 and spleen-cell suspensions were ciyopreseryed in liquid nitrogen to allow subsequent measurement of antigen-specific IFN-gamma-secreting T-cell responses by ELISPOT. Mice were bled from the tail vein on days 14, 28 and 42 for antibody measurements. Antigens were monomeric Zika exodomains as follows: His-tagged Zika HX from HEK cells (pCR028, SEQ ID NO: 1); His-tagged wild-type Zika (Aalto AZ6312, lot 3903); Strep-tagged Zika HX from Tni cells (CR068, SEQ ID No: 2). A Zika VLP from HEK cells was also used (The Native Antigen Company ZIKV-VLP 17061910). Strep-tag-II-tagged Zika -HX from Tni was further filter-sterilised using a 0.22 um PVDF membrane (Merck SLGV013SL). Other antigens were provided sterile. Sterile adjuvant formulations were all based on two types of liposomes obtained from Polymun Scientific of Austria, in iso-osmolar sucrose as follows: liposomal PHADR); liposomal 3D-6-acyl-PHADTh. Further experimental adjuvant formulations were created (where indicated) by adding additional adjuvant materials to these liposomes: liposomal PHAD plus Alhydrogel; liposomal 3D-6-acyl-PHAD plus Alhydrogel; and 3D-6-Acyl-liposomes plus QS21. Alhydrogel (2%) was obtained from Brenntag Biosector (Denmark). QS21 was obtained from Desert King International (USA). QS21 was dissolved in sterile deionised water at 1 mg/ml at room temperature with occasional agitation for one hour to create a uniform micellar solution before addition to liposome formulations. Zika antigen stock solutions were stored in aliquots at -80 degrees C. Formulations were prepared at physiological osmolarity fresh for each dosing occasion. Formulations including Alhydrogel (Brenntag Biosector 2% Alhydrogel 843061, batch 5369) first involved a conditioning step where the Alhydrogel colloidal suspension (provided sterile in water) was mixed at room temperature with 10x PBS pH 7.4 to render the suspension isotonic and to form phosphate groups on the surface of the colloidal particles Formulations that did not use Alhydrogel used an equivalent volume of PBS. For Alhydrogel-containing formulations, the various protein antigens were rendered to equivalent protein concentrations by dilution with PBS, and added to conditioned Alhydrogel. This was followed by addition of liposome suspension. Liposomal formulations containing Q521 were prepared by adding QS21 solution (at 1 mg/ml in deionised water) to liposome suspensions at room temperature, followed by mixing, and then addition of Zika antigen and further mixing. This order of addition was designed to avoid exposure of the antigen to the surfactant activity of QS21 which is quenched by the liposomes. Prior to i.m. injection, formulations were gently vortexed, avoiding frothing. A control formulation lacking antigen was prepared at each timepoint by mixing 90 jd Alhydrogel, 10 pl 10x PBS, 233 RI PHAD liposomes, 100 pl Q521 and 168 p1 lx PBS (in order of additions), to provide 600 pl of formulation, sufficient to inject 8 mice with 20% overage. PBS solutions were 10x PBS -Gibco, 70011; lx PBS -Gibco, 10010. Q521, once dissolved, was stored frozen in aliquots at -70 C and thawed freshly for each dosing point. Alhydrogel was stored at 2-8 degrees C. PHAD and 3D-6-Acyl PHAD were from Avanti Polar Lipids (product numbers 699800 and 699855, respectively). Polymun liposomes were made as described in European Patent EP 1 337 322 "Method and Device for Producing Lipid Vesicles". The composition of the two types of Polymun liposomes (containing PHAD or 3D6-Acyl-PHAD) are described in the tables below (Table A and Table B respectively). Polymun liposomes were stored and transported at 2-8 °C. (Absolute concentrations vary from batch to batch due to process factors, but relative amounts of the ingredients are constant for successive batches of each formulation).
Table A: Polymun PHAD liposomes (stock solution) MPLA content (as PHAD) 0.86 mg/ml DMPC content 32.1 mg/ml Cholesterol content 14.8 mg/ml DMPG content 4.5 mg/ml Particle size (z-average) 78 um Polydispersity index 0.10 Zeta Potential -39.7 mV pH 6.6 Osmolality 337 mOsmol/kg Table B: Polymun 3D-6-Acyl-PHAD liposomes (stock solution) 3D 6-acyl MPLA content 0.83 mg/ml DMPC content 39.0 mg/ml Cholesterol content 16.8 mg/m1 DMPG content 4.7 mg/ml Particle size (z-average) 74 nm Polydispersity index 0.11 Zeta Potential -39.6 mV pH 6.7 Osmolality 336 mOsmol/kg The composition of the four antigen-containing formulations used for immunisation for the measurement of antigen-specific T-cell responses to Zika antigens in female BALB/c mice, with respect to their content of adjuvant actives is summarized in Table C below:-Table C: Final composition of vaccine formulations used for measurement of antigen-specific T-cell responses with respect to adjuvant and anfgen active ingredients per dose Formulation MPLA as PHAD QS21 per dose Alhydrogel (Mum) per dose Antigen per dose Volume per i. m. dose or 3D-6-Acyl PHAD per dose PHAD+Alum 20 Mg 0 Mg ISO Mg 5 itg 60 id 3D-6-Acyl+Alum 20 Mg 0 Mg 180 n 5 itg 60 til 3D-6-Acyl alone 20 Mg 0 Mg 0 Mg 5 pg 60 til 3D-6-Acyl+QS21 20 Mg 10 jig 0 Mg 5 pg 60 til Table D: Final composition of adjuvant formulations.
Ingredient Final Final Molecular mass Molarity concentration concentration rig/ pg / ml dose (0.5ml dose) 3D 6-acyl PHAD 80 40 1747 469 4.578E-05 DMPC 3750 1875 677.25 0.0055371 Cholesterol 1640 820 386,66 0,00424145 DN1PG 470 235 688.85 0.0006823 HEPES free acid 2109 1054,5 238.3 0.00885019 HEPES sodium 2304 1152 260.29 0.00885167 salt NaC1 612.5 306.25 58.44 0.01048084 orthophosphate 84.1 42.05 94.971 0.00088553 sucrose 79650 39825 342.3 0.23269062 QS21 40 20 1990.13 2.0099E-05 Assay of antigen-specific T-cell responses in cryopreserved spleen cells by ELISPOT After sacrifice, spleens were removed from each mouse and transferred to 5 ml of cold, serum free, RPMI 1640 medium (Sigma, R0883) supplemented with mixture of 100U penicillin/100m streptomycin (Sigma, P0781). Each spleen was processed individually. Spleens were manually disassociated using 70 nm cell strainers (Falcon®, 352350) and serum free RPMI 1640 medium prepared as described above (SFM RPMI). 40 ml of cell suspension were produced from each spleen. Cell suspensions were spun down 200 x g, 10 min at 10 °C. Cell pellets were reconstituted in 3 ml/pellet of ACK lysis buffer (Lonza, 10-548E) and incubated for 5 min at room temperature to lyse red blood cells. 30 ml of cold SFM RPN1E medium were added. Suspensions were spun down 200 x g, 10 min at 10 °C. Pellets were reconstituted using 10 ml /pellet SFM RPMI medium. Suspensions were passed through 70 pm cell strainers. 50 p1 of each suspension were added to 96-well plate and stained with 50 pl of master mix containing 50 tig/m1 propidium iodide (PI) (Biolegend, 421301) and 5 pg/ml anti-mouse CD45 FITC antibody (Biolegend, 103107). PI and antibody were diluted in 1% BSA (Fisher Scientific, BP9705) in D-PBS (Gibco, 14190094). Staining was carried out for 10 min at room temperature in the dark. At the end of incubation 150 M1 of 1% BSA/D-PBS were added to each well. Live CD45+ cells were counted using Beckman Coulter flow cytometer (CytoFLEX S/N AW50407). Using these methods, viability was determined to be 87.8% (mean) ± 7.7% (SD). Cell suspensions were spun at 400 x g, 10 mm at room temperature. For cryopreservation, each pellet was reconstituted in a volume of freezing solution producing a 5 x 106 cells/ml suspension. Freezing solution (10% v/v DNISO (Sigma, D2650) in heat inactivated FBS (Biosera, FB-1001)) was added to each pellet in two equal volumes. Splenocyte suspensions were aliquoted 1 ml/cryovial and stored at -70 °C in CoolCells (BioCision, USA). After 24-72 hrs cryovials were placed in vapour phase LN2 storage. Cellular response was evaluated using IFN-gamma ELISPOT end-point. Four testing occasions were conducted (4 ELISpot plates/occasion). Occasion L groups 1 -4; Occasion 2: groups 5 -8; Occasion 3: groups 9 -12; Occasion 4: groups 13-16; Each group of 8 animals was tested on the same ELI SPOTplate. Spleen cells of Group 17 (which were treated with vehicle control lacking antigen) was distributed equally throughout the 4 testing occasions. Each occasion incorporated one animal from group 16 with predicted high level of cellular responses. Each ELISPOT plate included the test system controls described in the protocol.
For ELISPOT assays, cryopreserved cells were thawed and viability determined as above. One cryovial per animal was removed from vapour phase LN2 storage. Splenocytes were thawed in water bath at 37°C 3 min). Cryovials were thawed in batches consisting of cryovials from 1 animal group. 500)11 of thawing medium were added to each vial. Thawing medium was prepared fresh by adding benzonase (Sigma, E1014) to complete medium to final concentration of 10 U/ ml Complete medium: 900 ml of RPMI 1640 (Sigma, R0883), 100 ml of heat inactivated FBS (Biosera, FB-1001), 10 ml of L-glutamine (Sigma, G7513), 10 ml Penicillin/Streptomycin (Sigma, P0781). Contents of cryovials were transferred to 16 ml of thawing medium. Suspension was spun 330 x g, 10 mm at room temperature. Supernatants were discarded and pellets were resuspended in 10 ml of thawing medium. Suspensions were spun again 330 x g, 10 mm at room temperature and pellets were resuspended in 10 ml of complete medium. 50 pl of each cell suspension were stained as above. The mean and SD values of viability of thawed cells on the four occasions were (respectively): 70.9+5.9; 80.3+9.9; 85.0+5.1; 81.9+4.3.
ELISPOT procedures were as follows. Cell suspensions were spun down 330 x g, 10 mm at room temperature and pellets were reconstituted in assay medium to produce suspensions at 2.5 x 106 live cells/ml. Assay medium is complete medium with 0.05 mM 2-ME. 2-ME (Sigma, M3148) was pre-diluted in PBS (Gibco, 10010) to 5 mM. ELISPOTplates (R&D Systems, XEL485) were blocked at room temperature with 200 p1/well complete medium for minimum 20 min. Medium was removed before stimulant and cell addition. His tagged Zika HX antigen (CR028, 1.3 mg/ml) was diluted with assay medium to 2x final concentration (50 pg/m1).
Antigen was prepared fresh for each assay occasion. Concanavalin A (ConA, Sigma, C5275) was reconstituted in sterile water to produce stock at I mg/ml. Stock was aliquoted and stored at -20 °C. ConA (a non-antigen specific stimulant) stock solution was diluted with assay medium to 2x final concentration (10 pg/m1) -fresh dilution was prepared for each assay.
Addition of stimulants and cells to the blocked ELISpot plates: Wells Al:D9 100 p1/well antigen at 50 pg/ml. Wells El:H9 100 pl/well assay medium.
Wells Al:H9 100 MI/well cell suspension containing 2 x 105 cells (see plate layouts on slide 12).
Well All:D11 100 Ml/well mouse recombinant IFN-gamma control (R&D Systems, XEL485, reconstituted in 500 R1 of assay medium).
Well El 1:H11 100 p1/well antigen at 50 mg/m1 + 100 Rl/well of cell suspension at 2.5 x 106 cells/ ml.
Well Al2:D12 200 RI/well assay medium.
Well E12:H12 100 gl/well ConA at 10 Rg/ml + 100 RI/well of cell suspension at 2.5 x 106 cells/ml, Plates were agitated horizontally and vertically to assure equal distribution of cells. Plates were incubated at 37°C, 5% CO2 for 17 -21 hours. Following incubation plates were removed from the incubator. 160 R1 of supernatant was removed from each well and transferred to V-bottom 96-well plate (Corning, 3894) which were sealed and stored at -70 °C for use in MSD cytokine profiling. Wash buffer concentrate (10x) (from ELISPOT kit) was diluted with sterile water to produce lx working solution. Detection antibody concentrate (I 20x) was diluted with Dilution buffer 1 to produce lx working solution. ELISPOT plates were washed with wash buffer. Washes were conducted using autowasher (3 x at 300 RI/well). Plates were blotted against paper towel. 100 pl of diluted detection antibody were added per well except for wells El I H11 (kit negative control) where 100 M1 of PBS (Gibco, 10010015) were added instead. Plates were placed in 2-8 °C for overnight incubation, then washed and blotted as above. Streptavidin-AP concentrate (120x) was diluted using dilution buffer 2 to produce lx working solution. 100 RI of Streptavidin-AP working solution were added / well of the ELISPOT plates. Plates were incubated for 2 hours at room temperature and washed and blotted as before. 100 RI of chromogenic substrate (BCIP/NBT) were added / well of the ELISPOT plates. Plates were incubated for 1 hours at room temperature in the dark, washed with water, blotted against paper towels and left to diy at room temperature for minimum of 16 hours. Plates were analysed using AID ELISPOT instrument ELRO3 (software version 3.5 build 2550). Spot definitions were set as follows: Min. intensity 10; Min. size: 15; No gradient setting; Emphasis: small; ELISpot v.3.2x algorithm. The same settings were used to read all 16 plates. The same exposure setting was applied to each well within the 96-well plate. The count data (IFN-y-secreting cells / 106 cryopreserved spleen cells) were analysed by generalised linear mixed models using a Poisson distribution on log link function. Analysis was performed using R v3,4,3 statistical software with libraries lmer, multcomp and ggplot2.
Multiplex ELISA assay of cytokine profile of stimulated spleen cell supernatants Multiple cytokines were measured simultaneously in the supernatants of spleen cell ELISPOT assays using the Mesosca1e Discovery technology (MSD V-plex mouse kit, Rockville, MD, USA). Detection antibody was prepared by adding 210 pl of Blocker D-B (10%) to 2.73 mL of Diluent 100 and 60 gl of detection antibody mix. 25 Ml/well of detection antibody were added to the plate. Sealed plate was incubated at room temperature for 17 hrs and 19 min. Wash buffer was prepared by mixing 250 pl of Tween 20 and 500 ml of PBS (Gibco, 10010015).
Read buffer was prepared by mixing 12 5 mL of 4x read buffer with 12.5 mL of deionised water. Plate was washed 3x 250 ttl of 0.05% Tween 20 in PBS using automated plate washer. Wash buffer was removed and 150 ttL of read buffer added per well. Plate was read immediately using SECTOR 6000 MSD instrument (S/N 1200070820371). Raw data was transferred to a Microsoft Excel spreadsheet and imported to SoftMax Pro GxP 6.4 software (Molecular devices) for analysis. Four-parameter logistic curves were fitted to standard curve datasets for each cytokine using 1/Y2 weighting function (approach suggested by MSD kit supplier). Sample concentration (pg/ml) was interpolated from the appropriate standard curves.
Measurement of murine IgG isotype-specific antibody responses (IgGI, IgG2q) Nunc NIaxiSorpTIVI flat-bottom ELISA plates (ThermoFisher 44-2404-21) were coated with '°°R1 per well Zika VLPs (The Native Antigen Company ZlICV-VLP-100; Batch: 17061910) at 0.5pg/m1 in carbonate-bicarbonate buffer (pH 9.3 -9.9) 'Coating Buffer', made from Merck/Sigma-Aldrich Carbonate-Bicarbonate Buffer Capsules C3041. For reference purposes additional plates were coated with either purified murine monoclonal IgGrx or IgG2aw immunoglobulins (BioLegend MG145, MG2a-53) in a 1/3 serial dilution from 0.5pg/ml. All plates were sealed and incubated for 2hours at room temperature. Serial dilutions of the reference antibodies for coating purposes were made in quadruplicate.
VLP-coated Maxisorp plates and antibody-coated plates were washed 5x with 0.05% v/v Tween-20 in PBS (PBS-Tween) for 5 mins on a shaker, and were blocked with 200 pl/ well 2% w/v neutral bovine serum albumin (Sigma, A7906100g; Lot: SLBW1273; Heat shock fraction, pH7, >98%) in PBS (BSA Blocking Buffer) and incubated a further 30 mins at room temperature. Plates were emptied and washed a further 4x with PBS-Tween.
In separate round-bottom 96-well polystyrene plates (Dilution Plates'), murine test sera from immunised animals and controls, and a reference IgG2a antibody against the fusion loop of dengue-serotype-2 E protein 4G2 (Millipore, MAB10216, Lot 2786064) were diluted in BSA Blocking Buffer, in fivefold serial dilution, in duplicate. Sera were diluted in six steps starting from 1/100; 4G2 starting from 1 jig/ml. The eighth row of each plate ('H') received BSA Blocking Buffer only. 100 pl from each well of the Dilution Plate was transferred to the corresponding well of the VLP-coated ELISA test plate, starting at row-H. Assay plates were incubated at 37 C for two hours. Plates were emptied and washed 4x with PBS-Tween as above.
Secondary antibodies (at stock concentrations of 0.5 mg/ml) were then introduced into appropriate wells of the assay plates at 100 p1/well at 1/2000 dilution in BSA Blocking Buffer as follows: biotin-labelled rat-anti-mouse IgG1 (Biolegend 406603, Lot: B208212) and biotin-labelled rat-anti-mouse-anti-IgG2a (Biolegend 407103, Lot: B204551). Plates were incubated at 37 C for one hour, and washed 4x as above.
Assay plates were then incubated with 100 p1/well streptavidin-horseradish-peroxidase conjugate (Biolegend, 405210, Lot: B255152) at a dilution of 1/2000 in BSA Blocking Buffer, and incubated for 30 mins at room temperature in the dark. Plates were washed 5x as above, and 100 lit of Sigma TIVIE substrate (Life Technologies TMB Single Solution 002023, Lot 06176181-7). Plates were stopped with 100 ttl of IN FI2SO4 after 7 mins and read at 450 nm in an ELISA reader.
Direct antibody micro-neutralisation asscg, Heat-inactivated mouse or rabbit serum samples (56 C, 30 mins) were diluted initially ten-fold (or in some cases initially five-fold) and then 3-fold serially in Vero-cell culture medium (Life Technologies M199 31150-022) containing 2% foetal bovine serum (Life Technologies FBS 61870-010) containing penicillin and streptomycin, mixed with a constant concentration of Zika virus MP1751 (Uganda 1962) (Public Health England) to allow a multiplicity of infection of 0.5, and incubated for I h at 37 C. The antibody-virus mixtures (70 pl samples) so created were then transferred to monolayers of Vero cells in 96-well plates and incubated for a further 2 h at 37 °C to allow infection. The supernatant was washed off with PBS and cells were incubated for a further 48 hours to allow virus proliferation. For fluorescent staining, cell monolayers were fixed with fixed with 4% formaldehyde (Life Technologies 10010-015) for 30 min in the dark, quenched for 20 min with 50 mM ammonium chloride in PBS at room temperature, washed with PBS, permeabilised for 10 mins with 0.1% v/v Triton-X100 in 0.5% BSA. Monolayers were then incubated with antibodies in in PBS 0.5% BSA (Sigma, A7906-100g; LOT: SLBW1273) (washing buffer) on a laboratory shaker at medium speed: first, a flavivinis cross-reactive murine anti-dengue-2 envelope-protein antibody (402, Millipore MAB003, R&D) recognising the fusion loop of Zika virus at a dilution of 1/500 in wash buffer.
The 402-containing medium was then washed off and a secondary goat-anti-mouse Alexa488-labelled antibody (Life Technologies al 1001) was then added to the cells at a concentration of 5 jig/m1 in wash buffer for 1 hour in the dark. Hoechst fluorochrome (Sigma bis-Benzimide H 33258; B2883 lot B2883) was used as a nuclear stain at a final concentration of 5 pg/m1 in the Alexa-labelled antibody solution. Finally the cells were washed in PBS to remove unbound antibodies, and stored in PBS until ready to read. Immunostained fluorescent cells were enumerated using a Perkin Elmer Opera Phenix high-content imaging system, using Columbus software. Fluorescent staining was controlled for by use of an isotype-matched IgG2a control antibody (R&D Systems, MAB003, clone 20102) of irrelevant specificity. Uninfected cells and no-antibody samples were used as additional controls, at 4 wells each per plate.
Live challenge studies of Zika subunit vaccine formulations Recombinant His-tagged Zika-HX antigen from Drosophila S2 cells was purified by serial nickel chelate affinity chromatography and gel permeation chromatography yielding three fractions: an oligomer (eluting near the void volume of the gel permeation column) containing covalently linked dimer; a pure covalently linked dimer; and a pure monomeric subunit.
BALB/c female mice were immunised (30 lig per dose of oligomeric or dimeric antigen) with a formulation comprising 3D-6-Acyl-PHAD (40 lig per dose) plus QS21 (20 pg per dose), in Polymun liposomes with formulation process as described under "Measurement of antigen-specific T-cell responses to Zika antigens and formulations". A control group received PBS in place of vaccine formulation. Doses of 120 pl volume in PBS/isotonic sucrose (the latter from the liposomes) were administered subcutaneously on three occasions, day 0, day 14 and day 28. In order to render mice susceptible to Zika virus infection with PRVABC59 strain (Zeptometrix, USA 0810525CF) two doses of a blocking antibody against the murine interferon alpha receptor IFNAR-(Leinco, USA 'Platinum' grade Cat. No. 11 188 clone MARI5A3) were administered (1 mg i.p. in 200 pl PBS) on days 44 and 46, immediately before and after challenge with 250 PFU/mouse of PRVABC59 virus, administered i.v., on day 45. Viraemia was monitored by reverse-transcriptase RT-PCR with real time monitoring using Sybr-green according to Lanciotti et. al.' -on days 48 (day 3 post-challenge) and 51 (day 6 post-challenge). RNAs were purified from 100u1 of mouse serum samples by extraction with Qiagen RNeasy 96 Kit (74181) and eluted in 60p.I of elution buffer provided by the kit. SYBR Green based RT-qPCR was conducted using primers previously described by Lanciotti et. al. RNA quantification was evaluated from the standard curve derived from seven 10-fold serially diluted ZIKV FSS13205 RNA starting at 4.03E+08 RNA copies.
Formulation development of a lyophilised vaccine Prinulation containing 3D-6-Acyl-PHAD liposomes and QS2 I for preclitticd toxicology A modified adjuvant formulation was developed for lyophilisation that had a similar composition to the non-lyophilised formulation used for mouse live challenge studies (above), although having a lesser PBS content (contributed by antigen solution) relative to the sucrose excipient of the liposomes, in order to minimise saline and phosphate content of the final formulation. As a first step in the creation of this new formulation, Polymun 3D-6-Acyl-PHAD liposomes (defined above, in phosphate buffered sucrose pH 6.6) were diafiltered into a medium comprising 9% w/v sucrose in 20 mIVI HEPES-NaOH buffer pH 7.5. A micellar solution of QS21 was created by dissolving Q521 at 1 mg/ml in 1 mM ammonium bicarbonate solution, according to manufacturer's instructions (Desert King, USA). The Q521 solution was then added to the liposome suspension which was gently mixed to allow insertion of QS21 into the liposome membrane to create [3D-6-Acyl-PHAD + Q521] liposomes. Having quenched the surfactant activity of the QS21 with the liposomes, antigen (Zika-HX tag-free (SEQ ID NO: 4) dimer antigen from Drosophila S2 cells) at 2.75 mg/ml of protein in PBS was then added with further gentle mixing. Further 9% sucrose in 20 mM HEPES-NaOH at pH 7.5 was added to make up the volume. The formulation was then distributed into 2 ml vials at 0.7 ml per vial and lyophilised, for later reconstitution with 0.7 ml of water-for-injection (WFI). Lyophilisation was conducted at a drying temperature of-IS C in a Christ Epsilon 2-4 LSCplus freeze dryer. The resulting composition of the formulation is shown in Table 3 under Specification'. NB this formulation contained 9% sucrose w/v as excipient which was not analysed chemically in stability studies although is apparent in the osmolality measurements (the isotonic PBS saline contribution to osmolality from the antigen stock solution having a minor contribution thereto, of 10% or less).
For stability studies, SDS polyacrylamide gel electrophoresis (PAGE) was conducted in 4-12% polyacrylamide Bis-Tris gels (Bolt, Thermo Fisher) according to manufacturer's instructions, employing a molecular mass standard mixture (BioRad 161-0375) Gels were stained with Coomassie blue using Imperial Stain (Thermo Fisher). HPLC was reverse phase HPLC with charged aerosol detection (CAD). Dynamic light scattering (DLS) and Zeta potential were determined using a Malvern Nano ZS (Zeta Sizer) instrument (Malvern Panalytica1 Ltd, UK) Stability studies of the Zika vaccine liposomal Ibrmulation In order to characterise the stability of the novel lyophilised nanoparticulate liposome formulation (described in Table 3), studies were conducted at three temperatures: 2-8 °C, 25 °C and 40 °C. Also, an in-use stability study was conducted on reconstituted formulation at 25 °C for six hours, in order to establish working stability for the day of injection.
Repeated dose GLP toxicology study by intramuscular administration of lyophilised Zika vaccine in rabbits A study was conducted in accordance with Executive Order No. 1245 of 12 December 2005 on GLP for Medicinal Products as required by the Danish Medicines Agency. The study complied with OECD Principles of Good Laboratory Practice (as revised in 1997). These Principles are in conformity with other international GLP regulations. The associated immunogenicity study was not performed to GLP. The objective of this study, conducted at Charles River Laboratories Copenhagen NS, was to assess the repeated dose toxicity of a lyophilised Zika subunit vaccine formulation in rabbits. The vaccine was administered by intramuscular injection 4 times over a period of 43 days. The injections were performed with an interval of 14 days between each treatment. The study was performed with 40 albino rabbits (20 males and 20 females) of the New Zealand White strain. Group 1 animals received 0.9 % NaC1 whereas Group 2 received the lyophilised Zika subunit vaccine formulation of Table 3 at: 30 ttg antigen/dose (Zika-HX tag-free dimer); 40 ug 3-D-6Acyl-PHAD/dose and 20 1..tg Q521/dose, in a volume of 0.5 ml/dose.
Half of the animals (main animals) in each group were terminated three days after the last injection (Day 46) whereas the remainder of the animals (recovery animals) were terminated 21 days after the last injection (Day 64). During the study, clinical signs, injection site reactions, body weight, food consumption, body temperature and ophthalmoscopy were evaluated Furthermore, haematology, clinical chemistry (including C-Reactive Protein), immunogenicity (IgG) and organ weight analyses were also performed. In addition, macroscopic and microscopic examinations were undertaken.
Lyophilised vaccine in 2 ml vials was reconstituted with 0.7 ml of water-for-injection (WEI) per vial in order to allow extraction of 0.5 ml dose volume for injection. The vial was reconstituted once removed from the refrigerator. Reconstitution took a few seconds. All formulations were prepared in glass containers. Lyophilised vials were stored in the dark, and reconstituted formulation was pooled in dark glass vials for injection.
Measurement of rabbit IgG antibodies in sera from toxicology studies ELISA plates, Coating Buffer, BSA Blocking Buffer, washing procedures, diluent (PBSTween), and horseradish peroxidase substrate were the same as above for 'murine IgG i sotypespecific antibody responses' above. ELISA plates were coated with a monomeric C-terminally His-tagged baculovirus-expressed Zika exodomain CR028' with a glycan in the fiision loop, as per the dimeric antigen of the toxicology test formulation, wherein the natural residues in the region of W101 (i.e. GWG) had been replaced by the residues NHT to programme insertion of an N-linked glycan at residue 100 (SEQ ID NO: 1). ELISA plates were coated with 100 4/well CR028 at 10 pg/m1 in Coating Buffer for 2 h at room temperature and then at 4C for 16 h. Plates were washed then blocked with 300 0 Starting Block blocking solution (ThermoFisher 37542 for 30 min at room temperature). Blocking solution was removed and plates were washed 5x as above. Test sera were diluted in fivefold serial dilution in BSA Blocking Buffer starting at 1/100. As a standard GeneTex 133314 affinity-purified rabbit antiZika antibody (1.07 mg/m1) was used, also at 5-fold serial dilution. Diluted sera at 100 p1/well were incubated at 37C for 2 h in the CR028-coated plate, washed 5x, and plates were incubated in the presence of 100 p1/well goat-anti-rabbit IgG H&L horseradish peroxidase conjugate (Abcam Ab6721) at a dilution of 1/40,000 in BSA Blocking Buffer for lh at 37C. Plates were washed 5x and incubated with 100 p1/well Sigma TN1111 substrate as above. Substrate was incubated for 8 mm and stopped as above. Absorbance data were background-subtracted for non-specific binding of conjugate (determined from wells that received no serum) and a 4-parameter curve-fit (GraphPad Prism v5) was used to determine the antigen-specific IgG content of the test sera, by interpolation of the standard curve generated using the GeneTex antibody.
Results, Discussion and Conclusions
Figure 1 demonstrates that the glycoprotein form of the antigen influenced the T-cell response.
Thus, as judged by ELISPOT assay, in the Alum-free formulations most-permissive of Thl expression, the HX-form Zika exodomain subunit antigens, which have two glycans, were stronger Thl immunogens than wild-type subunit antigen or VLP antigens, and moreover these responses were highest in formulations containing QS21. The higher Thl response to the HX antigens may reflect better uptake by antigen-presenting cells via lectin receptors (e.g, surface receptors, or indirectly via mannose-binding protein). The enhanced generation of Th 1 - polarised T-cells (judged by antigen-stimulated IFN-7 release in ELISPOT), by insect-expressed FIX antigens mediated by addition of QS21 to 3D-6-Acyl-PHAD liposomes (exceeding that of VLP-form antigen) was deemed a desirable feature of the formulation, according to our hypothesis.
Fig. 2 shows direct (complement independent) neutralising antibody responses to the various formulations of Fig. 1, demonstrating that neutralising responses asymptoting to 100% were generated by all formulations (except for the wild-type Zika exodomain), particularly by Zika VLP formulations. However, the productivity of recombinantly-expressed flavivirus VLPs in culture is rather low making these antigens difficult to manufacture, and moreover they exist in various stages of maturity (as do flavivirus virions during natural infection and in live attenuated or in inactivated-virus flavivirus vaccines) with significant expression of fusion-loop epitope (FLE). As such, VLP antigens, like live attenuated viruses, and like natural and inactivated flaviviruses, are liable (in dengue naive subjects) to give rise to infection-enhancing antibodies which may enhance dengue severity, which is highly undesirable.
Figure 3 further demonstrates that the subclass distribution of the antibody response to the various formulations (between IgG2a and IgG1 indicative of Thl and Th2 responses, respectively) is radically affected by formulation composition, and differs by antigen type (VLP or exodomain subunit). First, Alhydrogel-containing formulations (which also contain PHAD or 3D-6Acyl-PHAD liposomes) gave rise to a balanced production of IgG2a and igGl, indicating expression of both Thl and Th2 profiles of the PHADs and the Alhydrogel respectively when formulated together, at the level of antibody production. Notably, the performance of 3D-6-Acyl PHAD was equivalent to PHAD. However, in the case of the VLP antigen formulations, omission of Alhydrogel gave rise both to markedly increased IgG2a production and a marked decrease in IgG1 production, indicating that Alhydrogel detracts from the full expression of the Th 1 profile of 3D-6-Acyl-PHAD when these adjuvants are used together. This may be significant for vaccine formulations intended to prevent or treat viral infections since the effect of IgG1 would be to reduce the ability of IgG2a to fix complement on viral surfaces or upon cells infected with virus (IgG2a is a complement fixing subclass).
In the case of Zika-HX exodomain, subunit antigens made from HEK or Baculovirus/Tni cells, with the exception of the wild-type Zika exodomain antigen which (by comparison) was not strongly immunogenic, addition of QS21 to liposomes containing 3D-6-Acyl-PHAD, resulted in a further increase in IgG2a production. These observations frilly confirm and extend the results of the ELISPOT analyses (above) and cytokine assays (below) which demonstrate a very clear effect of the QS21 element of the formulation, at the levels of both cellular and humoral immunity. The findings reported in Figs. 1-3 demonstrate that liposomal [3D-6-AcylPHAD plus QS2 I] is an effective vehicle for harnessing the Thl adjuvant effect of Q521 (and 3D-6-Acyl-PHAD) for Zika-FIX antigens, generating strong antigen-specific T-cell responses, and that its Thl character is best achieved in the absence of Alhydrogel.
Table 1 describes antigen-stimulated cytokine responses to the various formulations of Fig. 1 measured in the supernatants of antigen-restimulated spleen cells in vitro, following immunisation with the sixteen formulation permutations of Figure 2. There was a very strong antigen-stimulated 1L2 response, indicative of T-cell activity, when QS2 I was present in the immunising formulation, particularly for the subunit antigen forms. The striking prominence of IL2 in the cytokine response profile is reflective of human studies of Shingrix1.20, increasing our confidence that these observations (of Thl profile of the novel adjuvant) will translate to human studies. Several other Thl and Th2 cytokines were also elevated by immunisation with the various adjuvant formulations, upon antigen-restimulation in vitro. TNF-a, an inflammatory Thl cytokine, was also elevated, although modestly so by comparison to IL2. Unlike the antigen-specific IL2 response, which was specifically attributable to QS21, other elevations were not specific to any particular antigen or adjuvant formulation. Taken together these observations are indicative of immunity more so than inflammation (IL2 represents a T-cell growth factor signal stimulated in an antigen-specific manner by the presence of antigen-specific T-cells). On the basis of our hypothesis above, and these observations on the effects of QS21, liposomes containing the synthetic IVIPLA form 3D-6-Acyl P1-TAD' plus QS21 were selected for further development. N)
Table 1: Cytokine concentrations in supernatants of antigen-stimulated spleen cells from EL1SPOT analysis by MSD multiplex immunoassay.
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Next, we sought to achieve higher recombinant expression of the Zika-HX exodomain antigen in order to facilitate manufacturing and began expressing the His-tagged Zika-HX antigen in Drosophila S2 cells, where we made a dimeric form of Zika-HX (SEQ ID NO: 3), having an inter-subunit disulfide bond. We then explored the capability of the chosen formulation (liposomes containing 3D-6-Acyl-PHAD and Q52 I) with the 52-expressed antigen to protect mice against live challenge with Zika virus. In order to render the wild-type BALB/c mice more susceptible to infection with Zika virus, two injections with an anti-interferon-alpha receptor antibody were used, before and during the challenge phase. Fig. 4 shows that following live challenge, control BALB/c mice (that received the full liposomal adjuvant formulation minus antigen) experienced a prominent viraemia at day three post-inoculation with the Zika challenge virus, which resolved at day six. However, mice immunised with the full formulation containing fusion-loop-glycosylated Zika-HX (SEQ ID NO: 3) dimer exodomain antigen were protected against infection. Likewise, a higher molecular mass oligomeric species of the Zika-HX (SEQ ID NO: 3) (a side product of purification of the dimer by GPC), estimated by GPC to be a trimer, also gave rise to protection. The absence of protection in the adjuvant-only ('vehicle') formulation demonstrated protection to be dependent upon the presence of either dimeric or oligomeric Zika-FIX (SEQ ID NO: 3) antigen in the full formulation.
Having selected a mode of expression (in Drosophila S2 cells) and a formulation for the antigen, we then made a tag-free version of the Zika-HX exodomain in S2 cells (SEQ ID NO: 4). In order to maximise expression, stable cell-lines were made and clones of S2 cells stably expressing high levels of tag-free Zika-HX antigen were selected by iterative subcloning for further development. The dimeric antigen was purified by conventional chromatographic methods (including hydrophobic interaction, anion-exchange and gel permeation) resulting in a product with 92.5% purity (Fig. 5).
In order to reduce the risk that the antigen or its novel formulation composition might be unstable in solution (something that might take two years to discover), and in order to create a formulation that would be useful in limited-resource settings (such as tropical countries, where refrigerated distribution might be difficult) we sought to develop a heat-stable product which could avoid the requirement for cold-chain distribution. We therefore explored the lyophilisability of the formulation. Although there are licensed vaccine products on the market using liposomal adjuvants (Shingrix®, Mosquirix0) these products require cold-chain distribution because the liposomes are in solution (suspension) and are prone to fuse at higher temperatures and, moreover, are also vulnerable to freeze-spoilage since freezing can also cause liposomal fusion. We therefore embarked on a programme of research to develop a formulation that could be distributed without refrigeration; i.e., a cold-chain free formulation in which all solutes were lyophilised, including the liposomes, requiring only water for injection.
Table 2 describes the effect of QS21 addition and insertion into Polymun liposomes containing PHAD. These pilot studies did not use antigen in the formulation. They demonstrate that such formulations can be created without transformation into a microparticulate size-distribution, unlike ALFQ, and furthermore demonstrate that this can be achieved with either trehalose or sucrose. However, we found later that these two sugar excipients (which are largely believed to be equivalent in performance for this purpose) behaved differently when antigen was added to the formulation when we began to make antigen-containing formulations using model 'tagged' antigens, described below.
Fig. 6 describes the initial development of the novel lyophili sable liposomal Zika vaccine formulation containing liposomal MPLA (as 3D-6-Acyl-PHAD) and QS21 which was done with model antigen CR068, a tagged monomeric Zika-HX exodomain antigen having Streptag-II at its C-terminal end (SEQ ID NO: 2). After an initial screen of various excipients in formulations lacking antigen we had identified sucrose and trehalose as the best candidates to create a lyophilised formulation. Fig. 6 represents a comparison of the use of these two sugars as excipients in an antigen-containing formulation upon lyophilisation and reconstitution. We had expected trehalose (which is a natural preservative against dehydration, exploited by desert plants) to be better than sucrose, but found that sucrose gave rise to better retention of nanoparticulate size distribution upon lyophilisation and reconstitution. Based on the earlier results of Table 2 (which used P1-TAD liposomes as distinct from 3D-6-Acyl-P1-IAD), in antigen-free formulations, wherein phosphate-buffered sucrose was less effective than HEPES-buffered sucrose at the same pH at retaining a small particle size and limited polydispersity, we consider this difference in behaviour between sucrose and trehalose excipients in the present antigen-containing formulation (ie. giving rise to differing particle size distribution profiles in antigen-containing formulations) as most-likely being attributable to the saline constituents (phosphate buffer, NaCl) added in with the antigen (which in the case of CR068, was in the form of a 0.8 mg/ml stock protein concentration in PBS). In any event, these observations favour sucrose as the chosen excipient material, in order to maintain the desired nanoparticulate size and size-distribution profile (polydispersity).
Table 2: The effect of QS21 on particle size and polydispersity of PHAD liposomes before and after lyophilisation in iso-osmotic sucrose and trehalose-containing formulations in various buffers at pH 7.5. (No antigen was used in these model formulations).
Size Pdl (nm) before QS21 addition 77.41 0.098 EXC/080519/1 in 20 mIVI HEPES Sucrose pH 7.5 after QS21 insertion 74.89 0.098 75.15 0.110 after lyophilizat on 118.9 0.190 before QS21 addition 77.80 0.101 EXC/080519/2 in 20mM HEPES Trehalose pH 7.5 after QS21 insertion 74.85 0.104 75.23 0.091 after lyophilization 114.6 0.188 before QS21 addition 71.53 0.108 EXC/080519/5 in 10 mk1 phosphate-buffered-Sucrose pH 7.5 after QS21 insertion 73.58 0.101 81.24 0.138 after lyophilization 146.50 0.320 Vandepapeliere (EP2364721B1) describes a liposome formulation in which the human dose must contain 30 micrograms of both QS21 and of MPLA (eg. as MPLA from Salmonella minnesota) or less. Subsequently it has emerged that it is not necessary to have less than 30 micrograms each of the MPLA and QS21 elements per human dose, because Shingrix0, a licensed vaccine, uses 50 micrograms each of Salmonella minnesola MPLA and of QS21 per dose in the same liposomes described by Vandepapeliere. However, Shingrix0, while a very successful vaccine, nevertheless causes systemic toxicity (e.g. pyrexia) in 17% of human recipients (Shingrix EPAR), i.e. an acceptable but undesirably high rate of reactogenicity (provided, hypothetically speaking, that adequate efficacy of a vaccine could be achieved with lesser side-effects). It is also indicated from the published toxicology of ShinDix, where the full human dose was administered to rabbits'', that it is the AS01 adjuvant per se that is primarily responsible for toxicological findings of the vaccine. These observations are in accord with those made earlier by Destexhe in comparing various adjuvant strategies with A50121. Based on observations by Alving et al cited below, we recognized that a different ratio, and different amounts of MPLA and QS21 could be used per dose to make an effective vaccine, that might further give rise to a more tolerable side-effect profile than was seen for Shingrix0 Since QS21 is more toxic than is MPLA (which is remarkably non-toxic') in order to achieve a lesser reactogenicity profile we decided to elevate (over Vandepapeliere) the MPLA content per-dose of our formulation to 40 micrograms per dose (substantially exceeding the specification of Vandepapeliere but less than is used in Shingrix), while reducing the '30 ug' amount of QS21 specified by Vandepapeliere substantially, to 20 micrograms per dose, for toxicology studies of the lyophilised formulation in the rabbit at the full intended human dose. In so doing we expected to achieve a lesser reactogenicity profile, as QS21 is the more toxic of the two adjuvant materials (ie is more toxic than MPLAs). We reasoned that if (perchance) we saw inadequate immunogenicity with the new saturated formulation (with 40 micrograms of MPLA and 20 micrograms of QS21 per human-intended dose) in clinical studies, that we could always elevate the amount of liposomal adjuvant per dose (by increasing either its concentration or its injectate volume) to compensate, e.g., to 50 micrograms of QS21 per dose and 100 micrograms of MPLA (as 3D-6Acyl-PHAD), with minimal risk of MPLA toxicity which would still be at a safe dose6, provided there were appropriate toxicological data to support such elevation in dose.
Alving et. al. described a saturated liposome formulation containing an MPLA plus QS21 "ALFQ" (US 10,434,167 B2) which differs from the specification of Vandepapeliere, by using elevated amounts of both MPLA derivative (synthetic 3D-6-Acyl-PHAD) and of QS21 per dose, as well as a different mass ratio of these adjuvant active materials (2:1 respectively) compared to Vandepapeliere. However, ALFQ is defined by having unilamellar liposomes of median diameter in the micrometre range. The unique composition of ALFQ was achieved by employing saturated liposomes containing an elevated content of cholesterol (e.g. 55 moles per cent of the lipid composition), in order to accommodate a higher concentration of QS21 (which binds strongly to cholesterol in the liposomes). However, in the making of ALFQ, upon addition of QS21 to the MPLA-containing (3D-6-Acyl-PHAD containing) 'hi-cholesterol' saturated liposomes, the size and polydispersity of the liposomes increase markedly -i.e. are no longer uniformly nanoparticul ate, containing particles as large as 6 microns". While ALFQ is an effective and non-toxic adjuvant system, such a large increase in particle size is not necessarily desirable since the optimal effectiveness of liposomal or emulsion particulate adjuvant systems generally benefits greatly from their nanoparticulate size distribution which allows effective access to draining lymph nodes following injection of the formulation. Also, in some applications the higher viscosity associated with a larger particle size distribution could be a limiting factor.
In light of the observations of Singh and Alving (above of ALFQ), we were surprised therefore 5 that when a lesser amount of QS21 (representing half that of an MPLA derivative in mass terms) was added to the saturated Polymun liposomes of Table B (in Materials and Methods), containing the synthetic MPLA derivative (3D-6-Acyl-PHAD) that the liposomal particles remained nanoparticulate. Notably, the stock Polymun liposomes of Table B contain about 40 moles per cent of cholesterol as distinct from >50% moles per cent cholesterol used in ALFQ. 10 Moreover, to our further considerable surprise, after these liposomes had been made up to volume with further 9% w/v sucrose 25 mM HEPES pH 7.5 to meet the concentrations specified in Table 3, we found that even upon lyophilisation and reconstitution of the full formulation (containing Zika-HX (SEQ ID NO: 4) dimeric antigen from Drosophila S2 cells) we found only a modest increase in size and polydispersity (attributed to fusion of a subpopulation of liposomes) -from about 80 nm diameter (of the liposome stock) to about 130 mu (median diameter), with the vast majority of particles remaining in the nanoparticulate range (>95°,10 less than 1 um diameter).
Fig. 6, using monomeric Zika antigen CR068 (SEQ ID NO: 2), illustrates how the novel formulation of the present disclosure (whose specification is described in Table 3, for the tag-free dimer species SEQ ID NO: 4) maintains its nanoparticulate character throughout addition of QS21 and throughout lyophilisation and reconstitution under specified conditions.
Table 3: Stability at 2-8 C of sucrose-based Zika-HX tag-free dimer lyophilised formulation containing 3D-6-Acyl-PHAD and QS21 used for toxicology studies.
Test Method Specification 0 2 weeks 1 3 6 9
months month months months months Appearance before Visual inspection White, pass pass pass pass pass pass reconstitution homogeneous lyo cake Appearance after reconstitution Visual inspection White to light-beige N/A pass pass pass pass pass opalescent or milky pH pH meter 7.5 T 0.5 7.6 7.5 7.6 7.5 7.6 7.6 Osmolality mOsmol/kg Freezing-point depression Report result 317 315 316 310 295 313 MPLA content HPLC-CAD 80 mind +25% 98 88 88 80 79 71 QS21 content HPLC-CAD 40 jig/m1 +25% 44 41 42 43 47 41 DN1PC content HPLC-CAD 3.75 mg/m1 3.9 3.39 3.19 3.31 3.22 3.00 ±25% Cholesterol content HPLC-CAD 1.64 ing/m1 ± 1.70 1.49 1.40 1.42 1.41 1.32 25% DNfPG content HPLC-CAD 0.47 mg/ml ± 25% 0.54 0.41 0.43 0.44 0.42 0.38 Antigen integrity Reducing SDS-PAGE Single band pass pass pass pass pass pass (antigen present migrating between marker bands 37 and 50 kD at 60pg/ml, added from 2.75 mg/in! stock in PBS) Non-reducing SDS-PAGE Single band pass pass pass pass pass pass migrating between marker band 50 and 100 kD Particle size, Dynamic light.-scattering Report result 131 132 128 128 124 129 Zavg Polydispersity index Dynamic light-scattering Report result 0.339 0.350 0.323 0.331 0.330 0.339 Zeta potential Dynamic light-scattering Report result -57.3 -53.2 -54.5 -55.3 -56.5 -58.1 Endotoxin EP 2.6.30 <25.00 EEU/m1 2.95 Sterility EP 2.6.1 Sterile pass Table 4: Stability at 25 °C of the of sucrose-based Zika-HX tag-free dimer lyophilised formulation containing 3D-6-Acyl-PHAD and QS21 used for toxicology studies.
Test Method Specification 0 1
months month Appearance before Visual inspection White, homogeneous lyo cake pass pass reconstitution Appearance after Visual inspection White to light- beige N/A pass reconstitution opalescent or milky pH pH meter 7.5 + 0.5 7.6 7.6 Osmolality mOsmol/kg Freezing-point depression Report result 317 315 MPLA content HPLC-CAD 80 pg/ml +25% 98 86 QS21 content HPLC-CAD 40 pg/m1 +25% 44 38 DMPC content HPLC-CAD 3.75 mg/ml +25% 3.90 3.16 Cholesterol content HPLC-CAD 1.64 mg/ml ± 25% 1.70 1.38 DA/PG content HPLC-CAD 0.47 mg/ml ± 25% 0.54 0.43 Antigen integrity Reducing SDS-PAGE Single band migrating pass pass between marker bands 37 and 50 kD Non-reducing SDS-PAGE Single band migrating pass pass between marker band 50 and 100 kD Size of liposomes Dynamic light- Report result 131 126 scattering Polydispersity index Dynamic light- Report result 0.339 0.327 scattering Zeta potential Dynamic light-scattering Report result -57.3 -53.9 Table 5: Stability at 40 °C of sucrose-based Zika-I-DC tag-free dimer lyophilised formulation containing 3D-6-Acyl-PHAD and QS21 used for toxicology studies.
Test Method Specification 0 n 1
months weeks month Appearance before White, Report result pass pass pass reconstitution homogeneous lyo cake Appearance after reconstitution White to light- Report result N/A pass pass beige opalescent or milky pH pH meter 7.5 +0.5 7.6 7.5 7.6 Osmolality mOsmol/kg Freezing-point depression Report result 317 311 314 MPLA content HPLC-CAD 80 Fig/m1 +25% 98 92 75 QS21 content HPLC-CAD 40 pg/m1 +25% 44 42 39 DMPC content HPLC-CAD 3.75 mg/ml +25% 3.90 3.71 3.17 Cholesterol content HPLC-CAD 1.64 mg/ml + 25% 1.70 1.63 1.39 DMPG content HPLC-CAD 0.47 mg/ml + 25% 0.54 0.45 0.43 Antigen integrity Reducing SDS- Single band migrating pass pass pass PAGE between marker bands 37 and 50 kD Non-reducing SDS-PAGE Single band migrating between marker band 50 and 100 kD pass pass pass Size of liposomes Dynamic light- Report result 131 152 134 scattering Polydispersity index Dynamic light- Report result 0.339 0.376 0.283 scattering Zeta potential Dynamic light- Report result -57.3 -56.9 -55.3 scattering Table 6: Working stability of the reconstituted sucrose-based Zika-HX tag-free dimer lyophilised formulation containing 3D-6-Acyl-PHAD and QS21 used for toxicology studies.
Test Method Specification 0 2 6 hours at 25 °C after months weeks reconstitution Appearance before White, Report result pass pass pass reconstitution homogeneous lyo cake Appearance after White to light-beige opalescent or milky Report result N/A pass pass reconstitution pH pH meter 7.5 + 0.5 7.6 7.5 7.5 Osmolality Freezing-point depression Report result 317 315 315 mOsmol/kg MPLA content HPLC-CAD 80 [tWml +25% 98 88 90 QS21 content HPLC-CAD 40 mg/m1 +25% 44 41 38 DMPC content HPLC-CAD 3.75 mg/ml +25% 3.90 3.39 3.45 Cholesterol content HPLC-CAD 1.64 mg/ml ± 1.70 1.49 1.52 25% DMPG content HPLC-CAD 0.47 mg/ml +25% 0.54 0.41 0.42 Antigen integrity Reducing SDS-PAGE Single band pass pass pass migrating between marker bands 37 and 50 kD Non-reducing SDS-PAGE Single band pass pass pass migrating G between marker band 50 and 100 Id) Size of Dynamic light- Report result 131 132 134 liposomes scattering Liposomal size distribution Dynamic light- Report result 0.339 0.350 0.351 scattering Zeta potential Dynamic light- Report result -57.3 -53.2 -54.9 scattering Following these favourable results from pilot stability tests with model antigens, we went on to create a formulation based upon a dimeric species lacking a tag, i.e. tag-free Zika-HX (SEQ ID NO: 4) which was made in Drosophila S2 cells. Table 3 describes stability studies conducted at 2-8 C on the lyophilised formulation of the toxicology study which incorporated the tag-free Zika-FlX covalent dimer (SEQ ID NO: 4). These data demonstrate that all potentially labile components of the formulation were found to be stable within specified limits for nine months in the dry state at this temperature. Further studies at the elevated temperature of 25 °C demonstrate similar findings for a duration of 1 month (Table 4).
do Table 5 demonstrates that, remarkably, all components of the formulation are stable for one month at 40 degrees Celsius, which is adequate to allow field distribution without a cold-chain. Early confidence in formulation stability is a key advantage in the development of new vaccines, particularly those having protein or particulate components (e.g., liposomes) which are prone to commence aggregation at random intervals even after a period of stability. By having all components in the dry state, the opportunity for particle aggregation (of liposomes or protein) in the formulation is either obviated completely or minimised to what can happen upon lyophilisation. The negative charge of the liposomes is advantageous in this respect, as most proteins are acidic and negatively charged in the physiological pH range (near pH 7.4). At the formulation pH (pH 7.5) the electrostatic repulsion of the (DMIPG-containing) liposomes and the protein, serves to guard against physical association of the protein antigen with the liposomes, which could otherwise result in liposomal fusion and protein aggregation.
Table 6 demonstrates working stability of the formulation for the day of injection. A lyophilised formulation that had been stored for two weeks in the dry state at 2-8 °C was reconstituted, analysed immediately following rehydration, and after six hours incubation in the liquid state at 25 °C. All potentially labile ingredients were preserved within the specified limits following reconstitution, allowing sufficient time for the formulation to be administered in toxicology studies without deterioration.
Lyophilised, heat-stabilised liposomal vaccines containing MPLA and QS21 have recently been described by others. Wui22 described a DOTAP-containing formulation, but DOTAP is a xenobiotic entity which (though used experimentally in gene therapy and DNA vaccination) lacks an established history of safe use in vaccine products. Also, Fortpied" have adapted the AS01 formulation to make it lyophilisable, but this adapted formulation, at pH 6.1, is more acidic than the original AS01 (pH 6.6). As such the Fortepied formulation is expected to be prone to more substantial acidic pH excursions (upon precipitation of its phosphate buffer during freezing) simulating the acidic pH of endosomes -likely to trigger adverse conformational transitions in viral proteins, or protein precipitation of protein antigens with mildly acidic isolectric points. The stated reason for the lower pH used by Fortepied (as distinct from the earlier pH of 6.6 for regular AS01) was to protect the adjuvant-active MPLA and QS21 components of the formulation against hydrolysis on storage in their lyophilised formulation. Surprisingly, given these efforts of Fortepied to maintain adjuvant-active stability (of MPLA and QS21) upon lyophili sati on by using a distinctly acid pH of 6.1, these ingredients are very well preserved in the novel lyophilised formulation of the present disclosure (Table 3) at the higher, more protein-compatible, pH of 7.5, using a zwitterionic buffer (HUES) which is not prone to acidify upon freezing, and which is present in the final formulation in substantial excess of any phosphate that is added with an antigen solution.
We anticipate that our formulation would also be suitable for many antigens stored in aqueous liquid form at refrigerated temperatures (2-8 degrees Celsius), if using mildly (pH 6.6) or distinctly (pH 6 1) acidic buffers in the liquid form, at least for that subset of protein antigens which is stable under these conditions, but it is important to note that these acidic pH values are less versatile with respect to protein stability, and will not be suitable for all proteins. Notably the Zika-HX dimer used here has a predicted isoelectric point of 6.2 and might be prone to self-aggregate upon long-term storage in mildly acidic buffers, as would wild-type equivalent dimer lacking the glycan in the fusion loop, which will have similar isoelectric properties.
The present formulation uses synthetic adjuvants that are entirely natural from a toxicology point of view (though synthetic, containing no un-natural bonds or chemical entities of xenobiotic origin) and maintains the adjuvant system at all times at a more favourable pH for protein stability. The mild alkaline pH of the formulation does not (as may be feared by reading Fortepied who further acidified the AS01 formulation in order to develop a lyophilised form, in order to guard against hydrolysis of the MPLA and QS21) compromise the stability of any of the formulation ingredients, including adjuvant-actives and excipient lipids, which, though they may be labile at mild alkaline pH in the aqueous liquid state, we find are remarkably stable in the dry state when lyophilised from a HEPES containing formulation at pH 7.5.
Figure 7 describes results from GLP preclinical toxicology studies of the lyophilized liposomal Zika vaccine formulation in the rabbit. First, the formulation was tested for endotoxin-related and non-endotoxin pyrogens by the monocyte activation test (MAT test) which is sensitive to both endotoxin and non-endotoxin classes of pyrogen, in order to demonstrate human-acceptable levels of pyrogens in the formulation. Pyrogen levels were very low and within acceptable limits (well below specification) for rabbit studies at the full intended human dose (<<25 endotoxin-equivalent units/ml by MAT test using human monocytes, reported in Table 4). Groups of 20 New Zealand White rabbits (10 male, 10 female) were immunised i.m. four times at 14-day intervals. The findings of the toxicology study were as follows. No treatment-related adverse effects were observed clinically or ophthalmoscopically and no adverse effects were seen on body temperature, body weight or food consumption. Furthermore, no systemic changes were observed upon macroscopic or microscopic examinations. A slightly higher incidence and severity of injection site reactions, more specifically erythema, swelling and bruising, was observed in vaccine-treated animals (Group 2) compared to the control (Group] saline-injected) group. In some individual animals of Group 2, the reactions were observed up to 3 days post dose. Reactions were primarily observed after the 3rd (Day 29, counting first injection day as day 1) and 4th (Day 43) dose and were minimally more severe after the 4th dose. As was expected for an adjuvanted vaccine product candidate, treatment related effects were observed on neutrophils (transient elevation) and lymphocytes (slight transient reduction), on the day following each dosing in Group 2 compared to the control group, returning to normal in two days. Values were comparable between the groups for samples obtained in the pre-treatment period and just prior to dosing. In addition, treatment related effects were observed on C-reactive protein (CRP) where similar elevations were seen after doses one and two, and further, successive elevation of the CRP response (indicating an increasing response to antigen) was observed following doses three and four in Group 2 animals, at all times commensurate or lower than levels of CRP seen in the published toxicology of Shingrix in the rabbit (Giordano et. al.).
Anti-vaccine IgG antibody responses, indicative of a class-switched antibody response, were detected on Days 29 (14 days post 2nd dose), 43 (14 days post 3rd dose), 46(3 days post 4th dose) in all Group 2 rabbits following injection and at day 64 (21 days post 4th dose) in the late sacrifice group 2 rabbits, thus demonstrating antigenic function of the vaccine, and meaningful exposure of the treated animals to the antigen ingredient of the lyophilised vaccine formulation. For injection sites examined three days after a 4th repeated injection (Day 46), focal moderate to marked degeneration/necrosis associated with subacute inflammation was evident. Twenty-one days after the 4th injection (Day 64), the injection sites of five vaccine treated animals (3 males and 4 females) showed minimal to mild focal/multifocaI inflammatory cell infiltrate, focal degeneration/regeneration of myofibers and focal fibroplasia, indicating partial recovery. It was concluded that four intramuscular injections, with an interval of 14 days (a shorter interval than would be used in human studies), of lyophilised Zika subunit vaccine formulation containing: 30 jig Zika-HX dimeric antigen (SEQ ID NO: 4); 40 ps 3D6-Acyl-PHAD; 20 pg QS21 in Polymun liposomes /dose, in a 0.5 ml dose volume i.m.] to male and female New Zealand White albino rabbits, induced anti-vaccine IgG antibody production, but caused no findings considered to be of toxicological significance. A local subacute inflammatory response characterised by focal moderate to marked degeneration/necrosis was observed at the injection sites. The recovery process was complete or in progress at termination 21 days after the 4th dose.
The transient elevations of CRP that were observed following vaccine formulation dosing demonstrate that the innate immune-stimulation effects of the adjuvant active materials (3D-6-Acyl-PHAD and Q521) were harnessed effectively in the new formulation (Table 3) of this disclosure. Moreover, the fact that the degree of CRP-elevation observed was no greater, even at the highest level reached after four doses, than the peak level that was observed for Shingrix' (Giordano) indicates that the formulation will be acceptable at the tested dose, 0.5 ml of the liposomal formulation of Table 3, which contains 30 pg Zika-HX dimeric antigen; 40 pg 3D6-Acyl-PHAD; 20 pg Q S21 in Polymun liposomes, in man. Given that the studies of Giordano (above) and of Destexhe' demonstrate that it is the AS01 adjuvant per se (independently of the various antigens that were studied with AS01) that is responsible for toxicological findings in the rabbit, these observations on the new lyophilisable adjuvant formulation further indicate (based on clinical experience with Shingrix0 and Mosquirix0) that it will be safe and nontoxic for use in man with a variety of antigen actives. A further difference of the present vaccine formulation from previous non-lyophi li sable or lyophilised forms (which require acidic pH in order to be stable), is that it is rendered at mild-alkaline/near-physiological pH (pH 7.5, buffered with 20 mM HEPES). As such it is capable of preserving a wider variety of labile antigens, such as spike proteins and haemagglutinin proteins of viruses, or inactivated virions (such as influenza viruses) that may be acid-labile, even to mild-acid pH, without having to re-engineer the sequence of these proteins to prevent fusion, which is not a trivial undertaking, and moreover may delete potentially neutralising epitopes.
The findings of the present toxicology study with the lyophilised Zika vaccine formulation were also similar to those of Cawlfield et a124 with their malaria vaccine candidate that used ALFQ, having elevated levels per dose of both IVIPLA and QS21 (in hi-cholesterol liposomes, i.e. elevated compared to the specification of Vandepapeliere and to that of the Shingrix® formulation). However, it has not been demonstrated whether ALFQ formulations can be stabilised by lyophilisation. Also, in the present vaccine formulation, more economic use is made of the adjuvant-active-ingredients 3D-6-Acyl-PHAD and QS21 than in ALFQ, since the amounts of these adjuvant actives per dose is lower than ALFQ or AS01, while the toxicology findings demonstrate effective harnessing of their adjuvant functions.
Fig. 8 further demonstrates that, in the toxicology studies described above, of the novel vaccine formulation, very strong IgG antibody responses were generated in the full vaccine formulation group, but not in the saline controls, demonstrating effective (immunogenic) exposure of the vaccine in the experimental group(s). These responses represent a significant percentage of the total IgG content of the sera.
List of Abbreviations 3D-6-Acyl PHAD 4G2 A264C
ALF
ALFQ AS01 AS02
BALB/c
BC IP
Blocker D-B
BSA C-tag CD4 CD45 CD8 ConA CR028 CR068
CRP CTL DLS DN1PC DN1PG
DMSO
DOTAP
E P
EPAR
FBS
monophosphoryl hexa-acyl lipid A, 3-deacyl (synthetic) murine anti-dengue-2 fusion-loop antibody a mutation in a Zika exodomain at position 264 from alanine to cysteine Army Liposome Formulation Army Liposome Formulation containing QS21 GSK liposomal adjuvant formulation containing MPLA from Salmonella minnesota plus QS21 GSK emulsion-form adjuvant system defined mouse strain definition bromo-chloro-indolyl-phosphate proprietary blocking buffer of Mesoscale Discovery bovine serum albumin amino-acid sequence EPEA cluster of differentiation antigen 4 cluster of differentiation antigen 45 cluster of differentiation antigen 8 conconavalin A monomeric Zika exodomain having a glycan in the fusion loop and C-terminal H6 tag monomeric Zika exodomain having a glycan in the fusion loop and Cterminal Strep-tag-II C-reactive protein cytotoxic T-cell dynamic light scattering dimyristoyl phospatidyl choline dimyristoyl phosphatidyl glycerol dimethyl sulphoxide 1,2-Dioleoy1-3-trimethylammonium propane European Pharmacopeia European Public Assessment Report foetal bovine serum FITC flourescein isothiocyanate FLE fusion loop epitope GLP Good Laboratory Practice H2SO4 sulfuric acid HEK-293 human embryonic kidney cells-293 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid His-tagged hexa-histidine tag HPLC-CAD reverse phase high performance liquid chromatography using charged aerosol detection HX hyperglycosylated i.m. intra-muscular i.p. intra-peritoneal IFN-y ELISPOT interferon-gamma enzyme-linked immunospot (assay) IFNAR-1 murine interferon-alpha receptor IgG immunoglobulin G IL2 interleukin-2 1MAC immobilised metal chelate chromatography ISCOMs immuno-stimulatory complexes (containing QS21) LN2 liquid nitrogen LPS lipopolysaccharides MAT test monocyte activation test -in vitro human test for pyrogens using monocytes ME mercaptoethanol MPLA monophosphoryl lipid-A NaC1 sodium chloride NBT nitro-blue tetrazolium NLRP3 NLRP3 inflammasome (organelle) PBS phosphate buffered saline PFU plaque forming units PHAD monophosphoryl lipid A-504 PI propidium iodide PVDF polyvinylidine difluo de QS21 onillcna.suponaria Molina fraction 21 RH5 a protein of the malaria parasite Plasmodium lalciparzem RT-qPCR reverse-transcriptase quantitative polymerase chain reaction s.c sub-cutaneous S2 Drosophila Schneider-2 cells SD standard deviation SDS-PAGE sodium dodecyl sulfate -polyacrylamide gel electrophoresis SEM RPMI serum-free RPMI medium Strep-tag-II a peptide sequence 'WSHPQFEK' that binds streptavidin Streptavidin -AP streptavi din -alkaline-phosphatase conjugate Th T-helper-I subset of T-lymphocytes Th2 T-helper-2 subset of T-lymphocytes TLR4 toll-like receptor-4 TIVIB tetramethylbenzidine TNF-a tumour necrosis factor alpha Tni Trichophisict ni cells VLP virus-like-particle WEI water for injection Zavg particle size derived from intensity-based harmonic mean by differential light scattering References: I. Levin, NI. J. et at Th I memory differentiates recombinant from live herpes zoster vaccines-IC//n Invest 128, 4429-4440 (2018).
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13. Cawlfield, A. et at Safety, toxicity and immunogenicity of a malaria vaccine based on the circumsporozoite protein (FMP013) with the adjuvant army liposome formulation containing 20 Q521 (ALFQ). Vaccine 37,3793-3803 (2019) 14. Singh, P., Beck, Z., Matyas, G. R. & Alving, C. R. Saturated phospholipids are required for nano-to micron-size transformation of cholesterol-containing liposomes upon QS21 addition. JLiposorne Res 29, 1-16 (2018).
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Sequence Listing Information SEQ ID NO: 1: pCR028 (additional glycosylation site in bold) from HEK-293 cells IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAE VRSYCYEASISDNIASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRNHTNGCGLFGKG 5 SLVTCAKFACSKKNITGKSIQPENLEYRINILSVHGSQHSGMIVNDTGHETDENRAKV EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLP WHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEM DGAKGRLSSGHLKCRLKNIDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA GTDGPCKVPAQMAVDNIQTLTPVGRLITANPVITESTENSKININELELDPPFGDSYIVIG 10 VGEKKITITHWERSGSTGGSGGSGGSHETHHHII >SEQ ID NO: 2: CR068 (additional glycosylation site, and C-terminal >Strep-tag-II in bold) from Tni cells
IRCIGVSNRDF VEGM SGGTW VD VVL EHGGCVTVM AQ DK PTVDI ELVTTT VSNM A E VRS YCYEAS ISDMA SD SRCPTQGEA YLDKQSDTQY VCKRTL VDRN HTNGCGLFGKG
SLVTCAKFACSICKNITGKSIQPENLEYREVILSVHGSQHSGMIVNDTGLIETDENRAKV EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNICHWLVHICEWFLIDIPLP WHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAENI DGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA GTDGPCKVPAQMAVDNIQTLTPVGRLITANPVITESTENSKINIMLELDPPFGDSY IVIG
VGEKKITHHWHRSGSTGGSGGSGGSGGSAWSHPQFEK
SEQ ID NO: 3. ZHX C-terminal His-tag from 52 cells (additional glycosylation site and C substitution in bold)
IRCIGVSNRDEVEGMSGGTWVDVVLEHGGCVTVNIAQDKPTVDIELVTTTVSNMAE VRSYCYEASISDNIASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRNHTNGCGLFGKG
SLVTCAKFACSKKMTGKSIQPENLEYRIN4LSVHGSQHSGMIVNDTGHETDENRAKV EITPNSPRAEATLGGEGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLP WHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGCVHTALAGALEAEM DGAKGRLSSGHILKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA GTDGPCKVPAQMAVDIVIQTLTPVGRLITANPVITESTENSKIVINELELDPPFGDSYIVIG VGEKKITHI-1WHRSGSTGGSGGSGGSHHHHEIH SEQ ID NO: 4: ZHX tag-free from S2 cells (additional glycosylation site and C substitution in bold)
IRCIGVSNRDEVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAE VRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRNHTNGCGLFGKG
SLVTCAKFACSKKMTGKSIQPENLEYRINILSVHGSQHSGMIVNDTGHETDENRAKV EITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLP WHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGCVHTALAGALEAEM DGAKGRLSSGITLKCRLKNIDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYA GTDGPCKVPAQMAVDNIQTLTPVGRLITANPVITESTENSKNIIVILELDPPFGDSYIVIG
VGEKKITIRIVVERSGST

Claims (23)

  1. Claims 1. An adjuvant composition comprising liposomes comprising: a synthetic or natural Monophosphoryl Lipid A (MPLA), preferably a saturated synthetic MPLA, at a concentration in the range of from 70 to 100 jig/ml, preferably at a concentration of about 80 pg/m1; a sterol at a concentration in the range of from 1.23 to 1.95 mg/ml, preferably at a concentration of about 1.64 mg/ml, a phosphatidylcholine (PC) at a concentration in the range of from 2.81 to 4.69 mg/ml, preferably at a concentration of about 3.75 mg/m1; a phosphatidylglycerol (PG) at a concentration in the range of from 0.36 to 0.60 mg/ml, preferably at a concentration of about 0.47 mg/m1; a disaccharide at weight per unit volume in the range of from 7 % to 12 % w/v, preferably about 8 % w/v and a saponin at a concentration in the range of from 30 to 50 pg/ml, preferably at a concentration of about 40 pg/ml.
  2. 2. An adjuvant composition according to claim 1, wherein the MPLA is a synthetic MPLA selected from 3D (6-acyl) PHADO, 3D-PHADO and PHADO, and / or the sterol is cholesterol and / or the phosphatidylcholine (PC) is selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC) and distearyl phosphatidylcholine (DSPC) and / or the phosphatidylglycerol (PG) is selected from dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG) and distearyl phosphatidylglycerol (DSPG) and / or the disaccharide is sucrose or trehalose and / or the saponin is selected from QS21, QS7 and QS18.
  3. 3. An adjuvant composition according to claim 1 or claim 2 wherein the MPLA 3D (6-acyl) PHAD1), the sterol is cholesterol, the phosphatidylcholine (PC) is dimyristoyl phosphatidylcholine (DMPC), the phosphatidylglycerol (PG) is dimyristoyl phosphatidylglycerol (DMPG), the disaccharide is sucrose and the saponin is QS21,
  4. 4. An adjuvant composition according to any one of the preceding claims comprising a physiologically-acceptable buffer at pH in the range of from 7.0-8.5.
  5. 5. An adjuvant composition according to claim 4 wherein the buffer is selected from HEPES (HEPES-Na0H, HEPES KOH), PIPES, ACES, MOPSO, BIS_TRIS propane, BPS, DIPSO, 5 Tris, Tricine, Gly-Gly, EPPS(HEPPS), Bicine, TAPS, and 4WD at pH in the range of from 7.0-8.5.
  6. 6. An adjuvant composition according to any one of the preceding claims comprising PBS and / or NaC1 at physiological osmolarity wherein the PBS and / or saline element comprises less than 7.5% (v/v) by volume of the composition.
  7. 7. An immunogenic composition comprising an adjuvant composition of any one of claims 1 to 6 and one or more antigens.
  8. 8. An immunogenic composition according to claim 7, wherein the concentration of antigen in the composition is in the range of from 10 ug / ml to 600 ug / ml, preferably about 60 ug/ml.
  9. 9. An immunogenic composition according to claim 7 or claim 8, wherein the one or more antigen is selected from a viral antigen, a protozoal antigen, bacterial antigen, a fungal antigen, a cancer antigen and an allergen
  10. 10. An immunogenic composition according to claim 9, wherein the one or more viral antigen is selected from a flavivirus antigen, influenza antigen, an RSV antigen, a Varicella zoster antigen, a herpes simplex virus antigen, a cytomegalovirus antigen, an Ebola virus antigen and a coronavirus antigen.
  11. 11. An immunogenic composition according to claim 9 or claim 10, wherein the one or more viral antigen is selected from a Zika antigen and a dengue antigen.
  12. 12. An adjuvant composition or immunogenic composition of any one of claims 1 to 10 aqueous suspension having a pH of 7.0 to 8.0, preferably pH 7.5, or in dry (lyophilized) form.
  13. 13. An adjuvant composition or immunogenic composition of any one of claims 1 to 12, wherein the composition is in dry (lyophilized) form and on reconstitution in aqueous solution (e.g., water) the liposomes have a predominantly nanoparticulate particle size distribution.
  14. 14. A adjuvant composition or immunogenic composition of any one of claims 1 to 13, wherein the composition is in dry (lyophilized) form and on reconstitution of the adjuvant composition or immunogenic composition in aqueous solution (e.g., water) >80%, preferably >90%, more preferably >95%, of the particles in suspension are in the nanoparticle size range (less than 1 micrometre diameter), as determined by area-under-the-curve measurement of intensity in DLS
  15. 15. An immunogenic composition according to any one of claims 7 to 14 formulated for intramuscular (IM), subcutaneous (SC), nasal or oral administration.
  16. 16. An immunogenic composition according to any one of claims 7 to 15, wherein a unit dose is in the range of from 0.25 to 1.5m1, preferably in the range of from 0.5 to 1.0 ml.
  17. 17 An immunogenic composition according to any one of claims 7 to 16 for use in administration to a subject to stimulate an immune response.
  18. 18. A method for manufacture of an immunogenic composition according to any one of claims 7 to 17, comprising the steps of: (a) providing an adjuvant composition of any one of claims 1 to 6 (b) addition of one or more antigen to the adjuvant composition to form an immunogenic composition, (c) optionally lyophilizing the immunogenic composition.
  19. 19. A method for preparation of an immunogenic composition comprising reconstituting a lyophilized immunogenic composition of any one of claims 7 to 17 in an aqueous solution, preferably in sterile water.
  20. An immunogenic composition according to any one of claims 7 to 17, wherein (a) the composition is stable on storage at 40 °C in the dry, lyophilized state for at least one month, and / or (b) the composition is stable at 2 to 8 °C for at least 9 months, and / or, (c) the dry, lyophilized composition is stable following freezing, stability being assessed following reconstitution with water, wherein stability is assessed by one or method selected from HPLC analysis of lipid components, immunoassay of protein antigen components, SDS electrophoresis of protein antigen components and measurement of liposomes by dynamic light scattering.
  21. 21. An immunogenic composition according to any one of claims 7 to 17, wherein the lyophilized composition reconstituted in water is stable at 25 °C for at least 6 hours.
  22. 22. An adjuvant or immunogenic composition according to any one of claims I_ to 17 comprising a composition as shown in Table D herein.
  23. 23. An adjuvant or immunogenic composition as described herein with reference to thedescription, claims and / or figures.
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