vaccination

Abstract

This invention relates to vaccines comprising a fraction of registered vaccines and an adjuvant comprising carbohydrate monosulphate fatty acid ester (CMS) and the simple, one-step preparation method thereof by mixing a single dose of a vaccine with multiple doses of a ready-to-use novel CMS-based adjuvant delivering multiple doses of a non-inferior and often even improved vaccine. The mixing is preferably done by the end-user (vaccinator, general practitioner) briefly prior to vaccination, which offers a series of advantages including improved affordability, availability, and accessibility of vaccine products.

Description

[0001] Title: Vaccination

[0002] Introduction

[0003] Vaccination is one of the most cost-effective means to prevent and to control infectious diseases in both human and animal health. Vaccines contain either modified live antigens such as bacteria, viruses, parasites, recombinant (micro-)organisms, or inactivated (nonreplicating) antigens such as inactivated viruses, inactivated bacteria, inactivated parasites, subunits of infectious (micro-)organisms, proteins, polysaccharides, peptides, glycoproteins, polysaccharide-protein conjugates, peptide-protein- conjugates, and the like.

[0004] Often, the immune response against nonreplicating antigens and in some cases also to replicating (live) antigens is too low to establish sufficient level and / or duration of protection. Adjuvants are incorporated in these antigen formulations to augment the immune response. Adjuvants such as alum, emulsions, virus-like particles, etc. function as a vehicle for the antigen and present the antigen to the immune system in a way that the final result is to obtain an increase in the immune responses, which includes higher levels of antibodies, higher number of T cells, higher degree of immune protection, and the like. Vaccines are complex mixtures of biological materials and often contain many different immunogens that induce antigen-specific immune responses.

[0005] An example of such complex mixture is seasonal influenza virus vaccine that contain immunogens from three or four virus strains and may contain five or more strains in the future.

[0006] Another example of such complex mixture is pneumococcal conjugate vaccine (PCV) containing polysaccharide immunogens from 7, 13, 20, 21, or 31 serotypes and maybe contain even more of the 100 serotypes known in the future. The complexity of PCVs is the result of the complicated manufacturing process which includes many different steps for each serotype and subsequent mixing of the immunogens of each serotype to obtain the final vaccine product. The complexity of the manufacturing process of PCV is unambiguously illustrated by manufacturer’s information that production of PCV13 (PCV with 13 serotypes) involves 580 manufacturing steps, 1700 employees, 678 quality tests, 400 different raw materials, and 2.5 years. These numbers will be 50 or 100% higher for PCV with 20, 21, 31 or more serotypes.

[0007] Vaccine development includes various stages of preclinical evaluation of vaccine candidates in animal models and clinical evaluation in humans. An important step is that of dose-finding to identify the optimal dose of vaccine. The optimal dose is characterized by the highest benefit / risk-ratio, the highest benefit / cost-ratio, the highest efficacy / toxicity- ratio or any other trade-off of advantages towards disadvantages. The benefits of vaccines relate to the induction of sufficient degree and persistency of protective immunity in target population. Risks, costs, and toxicity relate to risks of local and systemic toxicity, costs of vaccination, stability of vaccines, etc.

[0008] For licensed PC VI 3 and other pneumococcal conjugate vaccines the optimal benefit / cost or benefit / risk is brought about by doses of between 2.2 and 4.4 jig of polysaccharide from each serotype and 34 pg of CRM197 as carrier protein plus alum as adjuvant.

[0009] A drawback of the complex manufacturing process of vaccines is the relatively high costs of goods and the relatively high end-user price as compared to other vaccines or other generic products. One way to solve the problem is to reduce the number of serotypes, which was done recently by Serum Institute India that recently launched Pneumosil® with 10 serotypes. A drawback of fewer serotypes, however, is that the vaccine is not effective against the serotypes that are not included in the vaccine. Another way to solve the problem is to use lower doses of immunogens, but this will confer lower, suboptimal immunological protection. A drawback of complex vaccines is their high costs of manufacturing, which hampers access to these products of parts of the target populations (LMICs). High vaccine end-user prices limit the potential impact of vaccines on global health.

[0010] The present invention provides enhanced efficiency of vaccine use. Optimized doses of existing vaccines may be divided in several parts, and combined with a CMS emulsion as defined herein, to provide multiple portions of vaccine without compromising vaccine efficacy or safety.

[0011] WO 2016 / 013938 discloses disaccharide fatty acid monosulphate esters combined with submicron squalane-in-water (herein referred to as CMS) and their use as vaccine adjuvant in newly developed vaccines.

[0012] Figures

[0013] Figure 1: Serum HI antibody titres (GMTs) against four vaccine influenza strains (Victoria, Austria, Darwin and Phuket) at different time intervals (days) after vaccination with a standard dose of VaxigripTetra (15 jig HA) without adjuvant (black line), 1 / 5 dose of VaxigripTetra (3 pg HA) plus 2 mg CMS (dark grey line) and 1 / 5 dose of VaxigripTetra (3 pg HA) plus 0.5 mg of CMS (light grey line). Subjects were vaccinated intramuscularly at Day 1 and antibody titers in serum were measured at Day 1, Day 8, Day 29 and Day 180 by hemagglutination inhibition assay.

[0014] Figure 2: Rabbit antibody levels after the first immunization with PCV13 or diluted PCV13 with CMS (see example 2) were determined by a fluorescent- beat-based multiplex immunoassay. Antibody concentrations or mean fluorescence intensity (MFI) signals specific for the 13 polysaccharides included in the vaccine were measured and the log-transformed geometric mean titers GMT (lOlog base) ± SD were calculated and plotted in histograms (Figure 2a). Factor of increase in antibody titers by CMS of the full and 1 / 5TH dose is presented in Figure 2b. Figure 3: Rabbit antibody levels after the second immunization with PCV13 or diluted PCV13 with CMS (see example 2) were determined by a fluorescent-beat-based multiplex immunoassay. Antibody concentrations or mean fluorescence intensity (MFI) signals specific for the 13 polysaccharides included in the vaccine were measured and the log-transformed geometric mean titers GMT (lOlog base) ± SD were calculated and plotted in histograms (Figure 3a). Factor of increase in antibody titers by CMS of the full and 1 / 5TH dose is presented in Figure 3b.

[0015] Detailed description

[0016] The invention provides a composition suitable for injection comprising 1) a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), and 2) a vaccine comprising one or more antigens other than an influenza whole or split virion antigen, a Clostridioides difficile toxoid A or toxoid B antigen, a recombinant Plasmodium falciparum glutamate-rich protein produced in Lactococcus lactis (such as a RO. IOC malaria antigen), a SARS-CoV-2 receptor-binding domain (RBD) nanoparticle antigen produced in Expi293F cells, or a VLP- based human epidermal growth factor receptor-2 (HER2) antigen produced in Drosophila melanogaster S2 insect cells.

[0017] In preferred embodiments, the vaccine comprises one or more antigens other than an influenza antigen, preferably a whole or a split virion influenza antigen, a Clostridioides difficile toxoid A or toxoid B antigen, a recombinant Plasmodium falciparum protein (such as a RO. IOC malaria antigen), a SARS-CoV-2 receptor -bin ding domain (RBD) nanoparticle antigen, or a (preferably VLP-based) human epidermal growth factor receptor-2 (HER2) antigen.

[0018] In further preferred embodiments, the vaccine comprises one or more antigens other than an influenza antigen, a Clostridioides difficile antigen, a malaria RO. IOC antigen, a SARS-CoV-2 antigen or a human epidermal growth factor receptor-2 (HER2) antigen. In some embodiments, the antigen further does not comprise a gonadotropin-releasing hormone peptide, such as for example coupled to keyhole limpet haemocyanin. The composition is suitable for vaccination by injection, preferably for vaccination of a human by injection.

[0019] The invention provides the advantage that the CMS adjuvant is a strong activator of the immune system for the antigens described herein, so that with a relatively low quantity of antigen, good efficacy can be obtained, with minor or no side effects. Hence, non-inferior immunogenicity, efficacy and safety can be obtained.

[0020] As such, one dose (a “standard” dose) of a vaccine can provide multiple injectable portions of the composition of the invention (a CMS- enhanced vaccine), said multiple portions having at least similar (noninferior), and generally even more, immunogenic activity as the original single “standard” dose of the vaccine. Thus, the efficiency of use of a vaccine antigen can be increased, without compromising vaccine efficacy. The combination of the vaccine with the CMS-emulsion is preferably achieved briefly prior to vaccination, and can be done by the end-user (vaccinator, general practitioner). Thereby, affordability, accessibility, and availability of vaccines can be enhanced.

[0021] One “standard” dose of vaccine (as for example defined in the market authorization of the vaccine) provides multiple doses (“portions”) of a CMS-adjuvanted vaccine, using a lower-than-normal quantity of antigen, but with similar (non-inferior) efficacy and tolerability as other vaccines which apply higher quantities of antigen. The present composition will be referred to as a “CMS-enhanced vaccine”. A single portion of CMS-enhanced vaccine is at least as effective for immunization, as the single dose of vaccine which it was prepared from. The vaccine

[0022] A vaccine, in the present context, is an immunogenic composition. The immunogenic composition comprises one or more antigens. The immunogenic composition is preferably a liquid immunogenic composition comprising one or more antigens. The immunogenic composition is preferably suitable for injection, such as suitable for vaccination of a human by injection.

[0023] The immunogenic composition may comprise a solvent, for example water for injection (WFI) or a buffer, preferably saline, phosphate buffered saline, or citrate buffered saline, more preferably an isotonic saline, an isotonic phosphate buffered saline, or an isotonic citrate buffered saline. The immunogenic composition may further comprise one or more cosolvents. The immunogenic composition may further comprise excipients and / or additives.

[0024] The vaccine is preferably a commercially-available, vaccine product with market authorization; a vaccine with market authorization may be referred to as a registered vaccine. Market authorization (or registration) means that the distributor of the vaccine has received authorization to market the vaccine product, generally from local authorities responsible for the authorization of pharmaceutical products. Generally, market authorization is granted after an application procedure, in which safety and efficacy of the vaccine is affirmed by technical experts in view of local legal requirements for market authorization.

[0025] Because requirements for market authorization may vary per jurisdiction, market authorization (or registration) as used herein is intended to mean market authorization in any jurisdiction. For example, a vaccine may be registered by the FDA (US), the EMA (Europe), the NMPA (CN), the CDSCO (IN) or another local authority, for a relevant jurisdiction. Market authorization, as described herein, refers to market authorization as of the present date. A registered vaccine comprises one or more antigens, as described in the market authorization, and furthermore comprises all excipients and additives described in the market authorization, in the quantities described in the market authorization. A vaccine in the present context is an injectable vaccine, that is, a vaccine suitable for injection. The vaccine, as defined herein, is the vaccine in a ready-to-use form, that is, in the form and quantity in which it is to be injected to the person requiring vaccination. The vaccine in the present context thus refers to a liquid formulation. The vaccine is in the form which can be used for vaccination according to the marketing authorization.

[0026] Market authorization of a vaccine is described in a public document (which in some jurisdictions may be called a Summary of Product Characteristics or SmPC, but which herein will be referred to as the market authorization), in which the composition of the vaccine for which authorization is granted, is described in detail.

[0027] Thus, a vaccine is defined by at least a registered volume for injection and / or a registered antigen quantity for injection, said registered volume for injection and said registered antigen quantity for injection together defining a single dose of the vaccine. The volume for injection and the antigen quantity for injection for a vaccine are defined in (derivable from; published in) the market authorization, and are referred to herein as the registered volume for injection and / or the registered antigen quantity for injection. Thus, the registered volume for injection and the registered antigen quantity for injection refers to the volume which can be injected according to the market authorization, and to the antigen quantity which is to be injected according to the market authorization. Together, the volume for injection and the antigen quantity for injection according to the market authorization represent a single dose of the vaccine (which may be called the registered dose), which is also derivable from the market authorization. The word “registered” in the present context thus refers to quantities (such as volume for injection, antigen quantity for injection, dose, concentrations) as published in the market authorization of the vaccine in question. This quantity can also be referred to as the quantity of antigen as found optimal in clinical trials. That is, the registered antigen quantity for injection refers to the quantity of antigen which elicits an immune response appropriate for immunization, while having the least side effects.

[0028] A vaccine in the present context is thus defined by the public document(s) on the basis of which market authorization has been granted. A vaccine is defined by the (registered) volume for injection and / or the (registered) antigen quantity for injection as derivable from the market authorization, as well as by any further compositional details provided in the market authorization.

[0029] The vaccine, as defined herein, is an injectable vaccine, preferably in the final form in which it is to be injected according to the market authorization. The vaccine is preferably injected by intramuscular, intradermal or subcutaneous injection.

[0030] That is, the vaccine is a liquid suitable for injection, generally having a volume of 0.1 - 2.5, preferably 0.2 - 1.0 ml, and comprising the registered antigen quantity. The (registered) volume for injection may be referred to as Vref, and the (registered) antigen quantity may be referred to as Mref.

[0031] In preferred embodiments, the vaccine comprises as antigen (where multiple antigens of the same type are present, the indicated quantities represent the quantity per antigen):

[0032] • an influenza antigen, preferably in a quantity of 1 - 60 pg hemagglutinin (HA) per dose;

[0033] • a pneumococcal polysaccharide, preferably in a quantity of 1 - 8.0 pg polysaccharide per dose; • a zoonotic influenza antigen, preferably in a quantity of 1 - 15 pg per dose;

[0034] • a meningococcal antigen, preferably in a quantity of 5 - 100 pg per dose;

[0035] • a smallpox and / or monkey pox antigen, preferably in a quantity of at least 5xl07infectious units per dose;

[0036] • a diphtheria toxoid, preferably in a quantity of not less than 20 IU per dose

[0037] • a tetanus toxoid, preferably in a quantity of not less than 40 IU per dose;

[0038] • a Bordetella pertussis antigen, preferably in a quantity of 3 - 50 pg per dose;

[0039] • a polio antigen, preferably in a quantity of at least 7 D-antigen units;

[0040] • a hepatitis B surface antigen, preferably in a quantity of 5 - 30 pg per dose;

[0041] • a Hemophilus (Haemophilus) influenza polysaccharide, preferably in a quantity of at least 3 pg per dose;

[0042] • a respiratory syncytial virus (RSV) antigen, preferably in a quantity of 50 - 150 pg per dose;

[0043] • a human papillomavirus antigen, preferably in a quantity of 10 - 60 pg per dose.

[0044] • a hepatitis A antigen, preferably in a quantity of 1 - 100 pg per dose;

[0045] • a SARS-CoV-2 (COVID-19) antigen, preferably in a quantity of 1 - 50 pg per dose;

[0046] • an ebola antigen, preferably in a quantity of at least 72x 106plaque forming units;

[0047] • a dengue antigen, preferably in a quantity of 2.0 - 9.0 logio of the 50% cell culture infectious dose;

[0048] • an anthrax antigen,

[0049] • a cholera antigen • a rabies antigen;

[0050] • a rubella antigen.

[0051] In more preferred embodiments, the vaccine comprises

[0052] • a pneumococcal polysaccharide, preferably in a quantity of 1 - 8.0 pg per dose;

[0053] • a zoonotic influenza antigen, preferably in a quantity of 1 - 15 pg per dose;

[0054] • a meningococcal antigen, preferably in a quantity of 5 - 100 pg per dose;

[0055] • a smallpox and / or monkey pox antigen, preferably in a quantity of at least 5xl07infectious units per dose;

[0056] • a diphtheria toxoid, preferably in a quantity of not less than 20 IU per dose

[0057] • a tetanus toxoid, preferably in a quantity of not less than 40 IU per dose;

[0058] • a Bordetella pertussis antigen, preferably in a quantity of 3 - 50 pg per dose;

[0059] • a polio antigen, preferably in a quantity of at least 7 D-antigen units;

[0060] • a hepatitis B surface antigen, preferably in a quantity of 5 - 30 pg per dose;

[0061] • a Hemophilus (Haemophilus) influenza polysaccharide, preferably in a quantity of at least 3 pg per dose;

[0062] • a respiratory syncytial virus (RSV) antigen, preferably in a quantity of 50 - 150 pg per dose;

[0063] • a human papillomavirus (HPV) antigen, preferably in a quantity of 10 - 60 pg per dose.

[0064] • a hepatitis A antigen, preferably in a quantity of 1 - 100 pg per dose;

[0065] • a SARS-CoV-2 (COVID-19) antigen, preferably in a quantity of 1 - 50 pg per dose; • an ebola antigen, preferably in a quantity of at least 72x 106plaque forming units;

[0066] • a dengue antigen, preferably in a quantity of 2.0 - 9.0 logio of the 50% cell culture infectious dose;

[0067] • an anthrax antigen,

[0068] • a cholera antigen

[0069] • a rabies antigen;

[0070] • a rubella antigen.

[0071] In even more preferred embodiments, the vaccine comprises one or more pneumococcal polysaccharide antigens, preferably in a quantity of 1 - 8.0 pg per dose (per antigen); one or more meningococcal antigens, preferably in a quantity of 5 - 100 pg per dose (per antigen), one or more respiratory syncytial virus (RSV) recombinant antigens, preferably in a quantity of 50 - 150 pg per dose (per antigen), one or more human papillomavirus (HPV) antigens, preferably in a quantity of 10 - 60 pg per dose (per antigen), a dengue antigen, preferably in a quantity of 2.0 - 9.0 logio of the 50% cell culture infectious dose (per antigen), said dengue antigen preferably being a chimeric dengue antigen, or a rabies antigen, preferably in a quantity of more than 2.5 IE.

[0072] In most preferred embodiments, the vaccine comprises as antigen a pneumococcal polysaccharide, for example a pneumococcal polysaccharide conjugate antigen, preferably in a quantity of 1 - 8.0 pg per dose.

[0073] In some embodiments, the vaccine comprises an antigen not being an influenza antigen. In some preferred embodiments, the vaccine comprises an antigen not being an influenza (preferably a seasonal influenza) whole or split virion antigen. In other preferred embodiments, the vaccine comprises an antigen not being an avian influenza antigen, preferably an avian influenza whole or split virion antigen. In other preferred embodiments, the vaccine comprises an antigen not being a SARS- CoV-2 receptor-binding domain (RBD) nanoparticle antigen. In other preferred embodiments, the vaccine comprises an antigen not being a pneumococcal polysaccharide conjugate antigen (such as elsewhere defined). In some embodiments, the vaccine comprises an antigen not being a recombinant Plasmodium falciparum glutamate -rich protein produced in Lactococcus lactis antigen, preferably an RO. IOC malaria antigen, or a VLP- based human epidermal growth factor receptor-2 (HER2) antigen, such as produced in Drosophila melanogaster S2 insect cells.

[0074] The CMS-emulsion

[0075] The CMS-emulsion is an emulsion suitable for injection comprising an oil phase, an aqueous phase, and a carbohydrate monosulphate fatty acid ester (CMS).

[0076] CMS is preferably present in the oil phase. In preferred embodiments, the oil phase further comprises CMS. In preferred embodiments, at least 90 % of the CMS in the composition is present in the oil phase, more preferably at least 99 %.

[0077] The CMS is an immunostimulant. It has been found that CMS activates the immune system, so that less antigen is required to provoke an immune response. The immunostimulatory power of CMS allows for “dilution” of a registered vaccine so as to provide multiple single doses (herein called “portions”), of CMS-enhanced vaccine from one single dose of a vaccine according to the market authorization, without loss of immunization power / vaccine efficacy, and without additional side effects.

[0078] CMS is a compound which comprises a carbohydrate scaffold, said carbohydrate scaffold being substituted with one sulphate group, and one or more fatty acid groups. The carbohydrate can also be called a saccharide.

[0079] The saccharide can be a monosaccharide, a disaccharide, a trisaccharide or a (linear or cyclic) polysaccharide with 4 - 10 monosaccharide units, or a mixture thereof. Preferably, the saccharide is monosaccharide, a disaccharide, a trisaccharide or a cyclodextrin, even more preferably a disaccharide and / or a monosaccharide, and most preferably the carbohydrate is a disaccharide.

[0080] If the saccharide is a monosaccharide, the saccharide can be selected from the group of pentoses with the general formula C5H10O5 and from hexoses with the general formula C6H12O6. The monosaccharide is preferably selected from the group of allose, altrose, glucose, gulose, mannose, sorbose, iodose, talose, gentiobiose, fructose, galactose, inositol, ribose, arabinose, xylose and lysose. More preferably, the monosaccharide is one or more selected from fructose, galactose and glucose.

[0081] If the saccharide is a disaccharide, the saccharide can be a sugar with the general formula C12H22O11 The disaccharide is preferably selected from the group of sucrose, maltose, lactose, lactulose, cellobiose, trehalose, gentiobiose, turanose, isomaltulose and melibiose. More preferably, a disaccharide is one or more selected from lactose, maltose and sucrose. Most preferably, a disaccharide is maltose and / or lactose.

[0082] If the saccharide is a trisaccharide, the saccharide can be a sugar with the general formula C18H32O16. A trisaccharide is preferably selected from the group of raffinose, melezitose, maltotriose, isomaltotriose, raffinose, kestose and negerotriose, more preferably the group of isomaltotriose, maltotriose and raffinose, and most preferably selected from raffinose and / or maltotriose.

[0083] If the saccharide is a polysaccharide, the saccharide consists of 4 - 10 monosaccharide units, which may be arranged linearly or cyclic. Preferably, the polysaccharide is a cyclic polysaccharide, preferably consisting of glucose units, i.e. a cyclodextrin. In much preferred embodiments, the polysaccharide is an alpha-, beta- or gamma- cyclodextrin, that is, a cyclic polyglucose having six, seven or eight glucose units, respectively. In the CMS, the saccharide as defined above is substituted with one sulphate group, and one or more fatty acid groups. Said substitution is on the hydroxyl groups of the saccharide. Substitution means that other groups are attached to the carbohydrate through the hydroxyl- groups of the carbohydrate via a covalent (ester) bond, so that the substituted carbohydrates in the present context are carbohydrate esters. Substitution is with a sulphate group through a sulphate ester bond, and with at least one fatty acid through an ester bond. Thus, sulphate groups are covalently bound to the carbohydrate through an ester group on a carbohydrate hydroxyl group, and similarly, fatty acids are covalently bound to the carbohydrate through an ester group on a carbohydrate hydroxyl group.

[0084] In CMS, one hydroxy group per saccharide molecule is substituted with a sulphate group (-SO3 ). Said sulphate group can be substituted with a monovalent cation, said monovalent cation preferably being selected from H+, Na+, K+, Li+, NHA, NRH3+, NR2H2+, NR3H+, with R being selected, independently, on each occurrence, from a Cl - C6 saturated or unsaturated alkyl group.

[0085] CMS is a monosulphate, that is, CMS is substituted with a single sulphate group as defined above. Preferably, CMS is substantially pure monosulphate. That is, CMS preferably comprises less than 10 mol%, preferably less than 5 mol%, more preferably less than 1 mol% of poly sulphate.

[0086] CMS is furthermore substituted with at least one fatty acid. A fatty acid, in the present context, is defined as a C6 - C22 linear or branched, saturated or unsaturated fatty acid. In preferred embodiments, the fatty acid is a linear fatty acid having 6 - 18 carbon atoms, preferably 8 - 12 carbon atoms. Fatty acids may be saturated or unsaturated, and may be branched, but are preferably linear.

[0087] Generally, a fatty acid can be a saturated fatty acid according to general formula -O-(C=O)-(CH2)X-CH3 wherein x is between 4 and 16. Furthermore, a fatty acid can be an unsaturated fatty acid according to one of the formulas -O-(C=O)-(CH2)x-CH=CH-(CH2)y-CH3 wherein x+y is between 4 and 14, or -O-(C=O)-(CH2)x-CH=CH-(CH2)y-CH=CH-(CH2)z-CH3wherein x+y+z is between 2 and 12.

[0088] Preferably, fatty acids may be of the general structure of -O- (C=O)-(CH2)X-CH3 wherein x may be 4 (hexanoic acid), 6 (octanoic acid), 8 (decanoic acid), 10 (dodecanoic acid; also referred to herein as lauric acid), 12 (tetradecanoic acid also known as myristic acid) or 14 (hexadecanoic acid also known as palmitic acid). Further preferred are fatty acids with the general structure of -O-(C=O)-(CH2)x-CH=CH-(CH2)y-CH3 wherein x+y may be 14 (for example oleic acid). Further preferred are fatty acids with the general structure of -O-(C=O)-(CH2)x-CH=CH-(CH2)y-CH=CH-(CH2)z-CH3wherein x+y+z is 12 (for example lin oleic acid).

[0089] Preferred fatty acids include hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, oleic acid and linoleic acid. Most preferably, the fatty acid is decanoic acid or dodecanoic acid.

[0090] When a carbohydrate is substituted with more than 1 fatty acid, any combination of fatty acids as described above is possible. Preferably however, the carbohydrate is substituted with a single type of fatty acid, on one or more hydroxy groups. In preferred embodiments, the carbohydrate is substituted on at least 50 %, preferably at least 75 %, more preferably at least 80 %, more preferably at least 90 % of the hydroxyl groups with a fatty acid as defined above.

[0091] In preferred embodiments, the saccharide comprises said one sulphate group, and is further substantially saturated with fatty acids. For pentose monosaccharides, this means, on average, preferably 2 - 3 fatty acids per molecule, more preferably 3. For hexose monosaccharides, this means, on average, preferably 2 - 4 fatty acids per molecule, preferably 3 or 4, more preferably 4. For disaccharides, this means, on average, preferably at least five fatty acids per molecule, more preferably at least 6, most preferably about 7 fatty acids per molecule. For trisaccharides and polysaccharides, reference is made to the above, where the proportion of hydroxyl groups substituted with a fatty acid is provided.

[0092] The CMS present in the emulsion may be obtained following the procedures described in WO 2016 / 013938.

[0093] Preferred CMS comprises a scaffold selected from maltose, galactose or sucrose, which is substituted with one sulphate group and further substantially saturated with C8 - C14 linear fatty acids, preferably octanoic acid, decanoic acid, dodecanoic acid or tetradecanoic acid, most preferably decanoic acid or dodecanoic acid.

[0094] In particular, CMS can be selected from the group of monosulphate heptadecyl maltose, monosulphate heptadodecyl maltose, monosulphate tetradodecyl galactose, monosulphate heptadodecyl sucrose, and monosulphate heptadecyl sucrose, preferably with a tri (Cl - C6) alkyl amine counterion, such as triethylamine. Most preferably, CMS is monosulphate hepta(C8-C14)alkyl sucrose, preferably monosulphate heptadecyl or heptadodecyl sucrose.

[0095] The CMS is preferably present in the CMS-emulsion in a quantity so as to provide 0.01 - 10 mg CMS per injectable portion of the composition, preferably 0.1 - 5 mg, more preferably 0.5 - 2.5 mg.

[0096] The CMS is preferably present in the CMS-emulsion in a concentration of 0.02 - 20 mg / mL, more preferably 0.2 - 10 mg / mL, even more preferably 1.0 - 5.0 mg / mL.

[0097] The CMS-emulsion furthermore comprises an oil phase. The oil phase comprises one or more oils, preferably one oil, selected from the group of squalane, squalene, hexadecane, a vegetable oil or a mineral oil. Appropriate vegetable oils and mineral oils for injection are known in the art. Preferably, the oil phase comprises squalane. The CMS emulsion comprises oil in a quantity defined by the weight ratio CMS : oil, of 1 : 10 - 5 : 1, preferably 1 : 5 - 2 : 1, more preferably 1 : 1 - 1 : 3.

[0098] Oil is preferably present in the CMS emulsion in a concentration of 0.5 - 100 mg / mL, more preferably 0.8 - 50 mg / mL, even more preferably 1.0 - 25 mg / mL, more preferably 1.0 - 10.0 mg / mL, most preferably 2.0 - 8.0 mg / mL.

[0099] The CMS-emulsion furthermore comprises an aqueous phase. The aqueous phase in this context may be pure water suitable for injection, or it may be a buffered water solution, or an isotonic solution, which may be ionic or non-ionic. The water is preferably water for injection (WFI), saline, phosphate buffered saline or citrate buffered saline, more preferably an isotonic saline, an isotonic phosphate buffered saline, or an isotonic citrate buffered saline.

[0100] The quantity of aqueous phase in the CMS-emulsion is preferably between 500 and 995 mg / mL, more preferably between 616 and 984 mg / mL.

[0101] In preferred embodiments, the CMS-emulsion comprises an emulsifier. The emulsifier can be any emulsifier, or combination of emulsifiers, which is suitable for injection, as generally known in the art. Preferred emulsifiers include a polysorbate, a sorbitan ester, polyoxyethylene sorbitan ester, or polyethylene glycol ester, most preferably a polysorbate emulsifier, such as for example Polysorbate 80.

[0102] The emulsifier is preferably present in a quantity so as to provide a stable emulsion, both before and after combination with the vaccine. Appropriate quantities depend on the type of emulsifier, and are generally known in the art. Exemplary quantities of emulsifier in the CMS-emulsion are 0.5 - 100 mg / mL, preferably 0.8 - 50 mg / mL, even more preferably 1.0 - 25 mg / mL, more preferably 1.0 - 10.0 mg / mL, most preferably 2.0 - 8.0 mg / mL.

[0103] A particularly preferred CMS-emulsion comprises an aqueous phase and between 1.0 and 10.0, preferably 1.5 - 5.0 mg / mL CMS, between 2.0 and 20.0, preferably between 3.0 and 10.0 mg / mL oil and preferably between 2.0 and 20.0, more preferably between 3.0 and 10.0 mg / mL emulsifier, with the aqueous phase, the CMS, the oil and the emulsifier being as defined above.

[0104] A particularly preferred CMS-emulsion comprises CMS, oil and emulsifier at weight ratio of CMS : oil phase : emulsifier of 1 : 0.5 - 10 : 0.5 - 5, more preferably in a weight ratio of 1 : 5 - 1 : 0.75 - 2.5, with the aqueous phase, the CMS, the oil and the emulsifier as defined above. In preferred embodiments, the CMS emulsion comprises CMS, squalane and Polysorbate 80 in a weight ratio of 1:2:1, in an injectable aqueous phase.

[0105] The CMS emulsion can be obtained by mixing all components using appropriate emulsification equipment to obtain an injectable emulsion, as is known in the art. One example is high pressure emulsification, for example by using a Microfluidizer® (Microfluidics International Corporation, Westwood, USA) or similar equipment, at pressure range of 200 to 1500 bar.

[0106] The CMS emulsion is preferably sterile. The CMS-emulsion is preferably an oil-in-water emulsion. The CMS-emulsion is preferably a submicron emulsion, characterized by an average droplet size of less 500 nm, preferably less than 200 nm and most preferably less than 80 nm.

[0107] The composition of the invention

[0108] The composition of the invention comprises the immunogenic composition (such as a vaccine) defined above and the CMS-emulsion defined above. The composition may be referred to as a CMS-enhanced vaccine. Alternatively, the composition can be referred to as an immunostimulated vaccine. Immunostimulation, in this context, means that the immune response of the individual which receives the composition is stronger, as compared to the situation where the same quantity of antigen would be injected, without the CMS emulsion.

[0109] The CMS-enhanced vaccine can be used for vaccination of individuals in need of vaccination by administration of a single portion of the CMS-enhanced vaccine. A portion of CMS-enhanced vaccine generally has a volume of 0.3 - 1.5 ml, said volume comprising CMS in a quantity of 0.2 - 3.0 mg. A portion of CMS-enhanced vaccine can also be defined by reference to the market authorization of the vaccine from which the CMS- enhanced vaccine was prepared.

[0110] In preferred embodiments, a portion of the CMS-enhanced vaccine has a volume which is comparable to the registered volume of injection of the vaccine it was prepared from. Comparable, in this regard, means that the volume of one portion of the CMS-enhanced vaccine is 50 - 150 vol.%, preferably 80 - 120 vol.%, more preferably 90 - 110 vol.%, most preferably 100 ± 5 vol.% of the registered volume of injection of the vaccine that the CMS-enhanced vaccine was prepared from. In further embodiments, the volume of one portion of the CMS-enhanced vaccine can be 25 - 300 vol.%, preferably 30 - 250 vol.%, more preferably 40 - 200 vol.% of the registered volume of injection of the vaccine that the CMS-enhanced vaccine was prepared from. Also, the mode of administration of the CMS-enhanced vaccine can be described by reference to the market authorization of the vaccine from which the CMS-enhanced vaccine was prepared. That is, if the market authorization refers to a particular mode of injection (e.g. intramuscular or intradermal injection), then the CMS-enhanced vaccine is preferably also injected through that same mode of injection.

[0111] The composition comprises the antigen(s), preferably as defined in a market authorization of the vaccine, and the immunostimulant in the form of the CMS-emulsion. The composition can be obtained by combining the vaccine and the CMS-emulsion, for example by adding the vaccine to a container comprising the CMS-emulsion, preferably followed by mixing, for example through gently swirling of the container. The addition may for example be achieved through the use of a syringe.

[0112] The container can be any suitable container, such as for example a vial. The container is preferably sterile. The container is preferably provided with a puncturable closing, suitable for receiving a syringe. The container is preferably pre-filled with the CMS-emulsion, so that the vaccine can be added through the puncturable closing using a syringe, thereby obtaining the composition.

[0113] The composition preferably comprises, relative to the registered volume for injection of the vaccine, 1) the vaccine in a volume of 5 - 100 vol.% of the registered volume for injection, and 2) the CMS-emulsion in 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents. A volumetric equivalent, as defined herein, is a parameter which takes a volume of a first liquid as a base value, so as to measure a volume of a second liquid in terms of the number of volumes of the first liquid. A volumetric equivalent thus means an equal volume of the measured (second) liquid, relative to the volume of the first liquid.

[0114] That is, the composition of the invention preferably comprises a quantity of the vaccine which is 5 - 100 vol.% of the registered volume for injection of the vaccine, preferably 50 - 100 vol.% of the registered volume for injection of the vaccine. The CMS-emulsion is present in the composition of the invention in a quantity, expressed relative to the volume of the vaccine, of 1 - 25 times the volume of the vaccine, preferably 2 - 20 times the volume of the vaccine, more preferably 3 - 10 times.

[0115] In further preferred embodiments, one portion of the composition of the invention comprises, expressed as vol.% of the composition, 1) the vaccine in a volume of 5 - 50 vol.% of the registered volume for injection, and 2) the CMS-emulsion in a volume of 50 - 95 vol.% of the registered volume for injection of the vaccine, preferably 80 - 90 vol.%.

[0116] Thus, if the vaccine is characterized by a registered volume for injection Vref(which volume Vrefcomprises the registered antigen quantity Mref), and if the actual volume of the vaccine in the composition is v (v measured in number of doses), then the composition of the invention preferably comprises a volume of CMS-emulsion of (1 to 25)xvxVref, preferably (2 to 20)xyxVref, more preferably (3 to 10)xvxVref, with v being a number larger than 0, preferably 0.001 - 100, more preferably 0.1 - 10, most preferably 1.

[0117] In preferred embodiments, when one registered dose of vaccine is used to prepare multiple portions of CMS-enhanced vaccine, then v = 1. However, it is also possible to combine a part of a registered dose of the vaccine with an appropriate quantity of CMS-emulsion, in which case v < 1. It is furthermore possible to combine multiple registered doses of vaccine with an appropriate quantity of CMS-emulsion, in which case v > 1. For both situations where v 1, the appropriate quantity of CMS-emulsion follows from the above formulae.

[0118] Alternatively, the composition of the invention comprises a volume of the vaccine (Vv) and a volume of the CMS-emulsion (Ve), such that the volumetric ratio Vv : Ve is 1 : (1 - 25), preferably 1 : (2 - 20), more preferably 1 : (3 - 10).

[0119] Further alternatively, the vaccine can be defined by a registered antigen quantity for injection. Using this definition, the composition of the invention comprises 1) the vaccine in a volume so as to provide 5 - 100 wt.% of the registered antigen quantity for injection, and 2) the CMS-emulsion in 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents. The composition of the invention can be obtained by dilution of the vaccine with the CMS-emulsion. Because the antigen quantity for injection has been optimized using dose finding studies (so that the registered antigen quantity for injection must be considered the optimal dose), and given the dilution of the vaccine using the CMS-emulsion to prepare from one dose of vaccine multiple portions of CMS-enhanced vaccine, this means that the composition of the invention comprises the antigen in a sub-optimal quantity. A sub-optimal quantity, in this context, means less than the optimal quantity, that is, less than the registered quantity for injection. The composition of the invention is therefore, per portion, characterized by an antigen quantity for injection which is less than the registered antigen quantity for injection. One portion of the composition of the invention preferably contains less than 90 % of the registered antigen quantity for injection, more preferably less than 75 %, more preferably less than 50 %, more preferably less than 35 %. Preferably, one portion of the composition of the invention provides 1 - 90 % of the registered antigen quantity for injection, more preferably 5 - 75 %, more preferably 8 - 50 %. In much preferred embodiments, the composition of the invention provides per portion 10 - 30 % of the registered antigen quantity for injection.

[0120] In much preferred embodiments, one dose of vaccine (as defined in the market authorization by for example volume and / or antigen quantity) provides 2 - 20 portions of CMS-enhanced vaccine, more preferably 3 - 10 portions.

[0121] The skilled person appreciates that it may be dependent on the type of vaccine how many portions of CMS-enhanced vaccine can be obtained from a single dose of registered vaccine. In order to determine how many portions of CMS-enhanced vaccine can be obtained from a single dose of (registered) vaccine, the skilled person can routinely create a dilution series, for example in volumetric proportions of vaccine : CMS-emulsion of 1 : 1 - 1 : 10, and test the immune response using an immune response test appropriate for the vaccine in question. An appropriate test can generally be derived from the market authorization documents, or would otherwise be generally known in the art. Examples are a fluorescent-bead-based multiplex immunoassay (MIA) adapted from Stenger et al. (Clin Vaccine Immunol 2011;18(4):595-603.) for the measuring of a pneumococcal immune response, or a hemagglutination inhibition assay for the measuring of an influenza immune response.

[0122] Dilutions where the immune response upon vaccination with the CMS-enhanced vaccine, after a specific time frame, is at least as high as the immune response following vaccination with the full dose of vaccine without CMS (after the same time frame), are considered appropriate dilutions to practice the invention. Dilutions where the immune response following vaccination with the CMS-enhanced vaccine is higher than the immune response following vaccination with the full dose of vaccine without CMS, are preferred.

[0123] One portion of CMS-enhanced vaccine provides an at least as good immune response (without additional side effects) as the single dose of vaccine that the CMS-enhanced vaccine was prepared from. One portion of CMS-enhanced preferably has a volume as described above: 50 - 150 vol.%, preferably 80 - 120 vol.%, more preferably 90 - 110 vol.%, most preferably 100 ± 5 vol.% of the registered volume of injection of the vaccine that the CMS-enhanced vaccine was prepared from. One portion of CMS-enhanced vaccine preferably has a volume of between 0.1 and 2.0 mL preferably 0.2 - 1.0 mL.

[0124] One portion of CMS-enhanced vaccine preferably comprises 0.1 - 10 mg of CMS, more preferably 0.5 - 5.0 mg, most preferably 0.8 - 2.5 mg of CMS. One portion of CMS-enhanced vaccine further comprises at least one antigen, said antigen being contributed by the vaccine the CMS-enhanced vaccine was prepared from. The quantity of antigen in the CMS-enhanced vaccine is about 5 - 90 % of the registered antigen quantity for injection (as defined in the market authorization), preferably 10 - 50 %, more preferably 15 - 35 %. One portion of CMS-enhanced vaccine furthermore comprises oil, which stems from the CMS-emulsion as well as from (optional) oil which may be present in the vaccine according to the market authorization.

[0125] The CMS-enhanced vaccine may furthermore comprise an emulsifier, stemming from the CMS-emulsion and / or from the vaccine, and any other excipient as defined in the market authorization, in concentrations which are below the registered concentration.

[0126] In preferred embodiments, a portion of the CMS-enhanced vaccine can be obtained, for example, by providing a container which provides CMS- emulsion in a quantity so as to provide 0.5 - 25 mg of CMS, preferably 1.0 - 10.0 mg of CMS, in a volume of 0.5 - 20.0 mL CMS-emulsion, preferably 1.0 - 10.0 mL, more preferably 1.5 - 5.0 mL; subsequently adding to said container one dose of the vaccine (as defined in the market authorization), and subsequently extracting multiple single portions of the CMS-enhanced vaccine, preferably 2 - 20 portions, more preferably 3 - 10 portions, of the CMS-enhanced vaccine.

[0127] In alternative embodiments, a portion of the CMS-enhanced vaccine can be obtained, for example, by providing a container comprising CMS-emulsion which provides a quantity of CMS sufficient to enhance the immune response in one individual, such as for example 0.1 - 10 mg of CMS, more preferably 0.5 - 5.0 mg, most preferably 0.8 - 2.5 mg of CMS. To said container there is added part of a single dose of the vaccine, such as for example 5 - 90 vol.% of the registered volume for injection of the vaccine, more preferably 10 - 50 vol.% of the registered volume, even more preferably 15 - 35 vol.% of the registered volume of the vaccine, thereby providing a single portion of CMS-enhanced vaccine.

[0128] Using the above teachings, the skilled person is capable to provide the composition of the invention on a larger scale (preferably more than 10, more preferably more than 20, even more preferably more than 30 portions of CMS-enhanced vaccine) in a single step, by combining multiple doses of vaccine with an appropriate quantity of CMS-emulsion, to provide the composition of the invention in larger volume, from which larger volume a multitude of single portions of CMS-enhanced vaccine can be obtained, for example for use in mass vaccination.

[0129] In all of the below specific embodiments 1 - 7, one portion of CMS- enhanced vaccine obtained using the vaccines mentioned below, may comprise 0.2 - 3.0 mg CMS, preferably 0.5 - 2.5 mg, more preferably 0.8 - 2.0 mg CMS. One portion preferably has a volume of 0.3 - 1.5 ml, preferably 0.4 - 1.2 ml. Said portion is defined as a single portion which is suitable for injection while providing an immune response which is at least as good as the immune response provoked by the vaccine the CMS-enhanced vaccine was obtained from (as defined elsewhere).

[0130] 1) If the vaccine comprises a pneumococcal polysaccharide antigen, then the registered volume for injection can be 0.5 ml (e.g. SmPC for Prevenar 20, or Prevenar 13). Said registered volume for injection contains a range of different pneumococcal polysaccharide antigens, in different registered quantities.

[0131] Pneumococcal polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F all have a registered quantity for injection of 2.2 pg (in said registered volume for injection of 0.5 ml). Pneumococcal polysaccharide serotype 6B has a registered antigen quantity for injection of 4.4 pg.

[0132] A composition of the invention, when prepared using a pneumococcal vaccine such as Prevenar 13 or Prevenar 20, preferably contains per portion of CMS-enhanced vaccine, the pneumococcal polysaccharide serotypes 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg of the pneumococcal polysaccharide, per portion (per antigen serotype). For serotype 6B, the quantity per portion of CMS-enhanced vaccine is preferably 0.04 - 2.2 pg, more preferably 0.2 - 2.2 pg, more preferably 0.3 - 1.5 pg. Furthermore, pneumococcal polysaccharide antigens serotypes 2, 6C, 9N, 15A, 16F, 17F, 20, 24F, and / or 35B may also be present, in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg of the pneumococcal polysaccharide, per portion (per antigen serotype).

[0133] That is, the immunogenic composition may comprise one or more antigens selected from the group of pneumococcal polysaccharide serotypes

[0134] 1, 2, 3, 4, 5, 6A, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg per antigen; and / or a pneumococcal polysaccharide serotype 6B antigen in a quantity of 0.04 - 2.2 pg, more preferably 0.2 - 2.2 pg, more preferably 0.3 - 1.5 pg.

[0135] 2) Alternatively, if the vaccine comprises a chimeric yellow fever dengue virus antigen, such as chimeric yellow fever dengue virus serotype 1,

[0136] 2, 3 and / or 4, the registered antigen quantity for injection is 4.5 - 6.0 logio CCID50 (CCID50 = the 50% cell culture infectious dose) per registered volume for injection of 0.5 ml (see e.g. the Dengvaxia SmPC).

[0137] A composition of the invention, when prepared using a dengue vaccine such as Dengvaxia, preferably contains per portion of CMS- enhanced vaccine, a quantity of chimeric yellow fever dengue virus serotype 1, 2, 3 and / or 4, of less than 3.9 logio CCID50, preferably 0.5 - 3.5 logio CCID50, more preferably 1.0 - 3.0 logio CCID50.

[0138] 3) Further alternatively, if the vaccine comprises as antigen a human papillomavirus (HPV) Type 6 LI, 18 LI, 31 LI, 33 LI, 45 LI, 52 LI or 58 LI, such as a HPV vaccine, then the registered quantity for injection may be 20 pg per dose (registered volume for injection = 0.5 ml). The antigens human papillomavirus Type 11 LI, 16L1 and 18L1 may have a registered quantity for injection of 40 pg per dose (see, e.g. the SmPC for the HPV vaccine Gardasil or Gardasil 9). A composition of the invention, when prepared using a HPV vaccine such as Gardasil, preferably contains per portion of CMS-enhanced vaccine, a quantity less than 15 pg, preferably 1 - 15, more preferably 2 - 10 pg of human papillomavirus Type 6 LI, 18 LI, 31 LI, 33 LI, 45 LI, 52 LI and / or 58 LI. Alternatively or additionally, a composition of the invention prepared using a HPV vaccine may contain per portion of CMS-enhanced vaccine, a quantity less than 20 pg, preferably 2 - 20 pg, more preferably 4 - 15 pg of human papillomavirus Type 11 LI, 16L1 and 18L1.

[0139] 4) Further alternatively, if the vaccine comprises an inactivated rabies antigen, such as a rabies vaccine, then the registered antigen quantity for injection can be > 2.5 IE (per dose of registered volume of 1.0 ml). See, e.g., the SmPC of Rabipur.

[0140] A composition of the invention, when prepared using a rabies vaccine such as Rabipur, preferably contains per portion of CMS-enhanced vaccine, a quantity of inactivated rabies virus of 0.1 - 2.0 IE, more preferably 0.2 - 1.0 IE.

[0141] 5) Further alternatively, if the vaccine comprises a meningococcal antigen, such as Neisseria meningitidis group A, C, Y and / or W polysaccharide, such as in a meningococcal vaccine, then the registered antigen quantity for injection may be 10 pg per dose (registered volume for injection 0.5 ml). See, e.g., the SmPC for Menquadfi. For meningococcal group B antigens, (e.g. Neisseria meningitidis group B NHBA fusion protein, group B Nada protein or group B fusion protein, the registered antigen quantity for injection may be 50 or 60 pg per dose (registered volume for injection 0.5 ml). See, e.g., the SmPC for Bexsero or Trumenba).

[0142] 5a) A composition of the invention, when prepared using a meningococcal vaccine comprising antigens of groups A, C, Y and / or W such as Menquadfi, preferably contains per portion of CMS-enhanced vaccine, a quantity of Neisseria meningitidis group A, C, Y and / or W antigen, of 0.5 - 5.0 pg, preferably 1.0 - 3.5 pg. 5b) A composition of the invention, when prepared using a meningococcal group B vaccine such as Bexsero or Trumenba, preferably comprises per portion of CMS-enhanced vaccine, a quantity of meningococcal group B antigens, (e.g. Neisseria meningitidis group B NHBA fusion protein, group B Nada protein or group B fHbp fusion protein) of 2.5 - 25.0 pg, preferably 3.0 - 20 pg, more preferably 5.0 - 15 pg (per group B protein type).

[0143] 6) Further alternatively, if the vaccine comprises an RSV (respiratory syncytial virus) antigen, such as in an RSV vaccine, the registered antigen quantity for injection may be 50 mg per dose (registered volume for injection 0.5 ml). See, e.g., the SmPC for Beyfortus. Alternatively, for RSV recombinant antigens such as RSVPreF3 antigen, or RSV subgroup A and / or B glycoprotein F antigen, the registered antigen quantity for injection may be 60 or 120 pg per dose (registered volume for injection 0.5 ml). See, e.g., the SmPC for Arexvy or Abrysvo.

[0144] 6a) A composition of the invention, when prepared using an RSV vaccine comprising the RSV antigen , preferably contains per portion of CMS-enhanced vaccine 2.5 - 25 mg, preferably 5 - 20 mg of RSV antigen.

[0145] 6b) A composition of the invention, when prepared using an RSV vaccine comprising RSV recombinant antigens, preferably contains per portion of CMS-enhanced vaccine 3.0 - 50.0 pg, more preferably 5.0 - 40 pg, even more preferably 8 - 30 pg of RSV recombinant antigens.

[0146] 7) Further alternatively, if the vaccine comprises an influenza antigen, such as from a Victoria, Austria, Darwin or Phuket strain, such as in a influenza vaccine, then the registered antigen quantity may be 15 pg of each of the HA antigens (e.g. A / Victoria / 4897 / 2022 (H1N1), A / Darwin / 9 / 2021 (H3N2), B / Austria / 1359417 / 2021 and B / Phuket / 3073 / 2013) in a single 0.5 ml dose. See. E.g., the SmPC of VaxiGrip Tetra.

[0147] A composition of the invention, when prepared using one or more influenza antigens, preferably comprises per portion of the CMS-enhanced vaccine, 1 - 10 pg, preferably 1.5 - 8 pg, more preferably 2.0 - 5.0 pg of influenza antigen.

[0148] Uses of the composition of the invention

[0149] The composition of the invention is for use in vaccination. Preferably, said use is use in humans or animals, more preferably a human or animal in need of vaccination. An animal, in this regard, is preferably a mammal or a bird. Further preferably, said use for vaccination is in a mammal, including humans, or a bird. Most preferably, use is for vaccination of a human by injection.

[0150] Use for vaccination, in the present context, preferably comprises a) providing a vaccine suitable for injection as a single dose, or part thereof; b) mixing said vaccine with said CMS-emulsion to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine; c) administration of a single portion of said CMS-enhanced vaccine to a human or animal for which vaccination is desired.

[0151] In one embodiment, the CMS-enhanced vaccine is administered to a human desiring (or in need of) vaccination. In another embodiment, the CMS-enhanced vaccine is administered to an animal in need of vaccination.

[0152] The vaccine is defined above, and may refer to a vaccine with market authorization, in the form in which it is to be injected. The market authorization defines what constitutes a single dose of the vaccine: an antigen quantity for injection, which has been optimized through one or more clinical trials and / or dose-finding studies, and a volume for injection, which comprises said antigen quantity, as well any other excipients and ingredients as outlined in the market authorization.

[0153] The vaccine may be provided in the form of a single dose, as prescribed in the market authorization, or as part thereof. If in part, then the (partial) dose is 5 - 90 vol.% of the registered volume for injection of the vaccine, more preferably 10 - 50 vol.% of the registered volume, even more preferably 15 - 35 vol.% of the registered volume of the vaccine, as described above.

[0154] The single dose of vaccine (or part thereof) can subsequently be mixed with the CMS-emulsion defined above to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine. The number of portions of CMS-enhanced vaccine which may be obtained from a single dose of vaccine is preferably 2 - 20 portions, more preferably 3 - 10 portions.

[0155] In one embodiment, said use may comprise combining part of a dose of the vaccine with a quantity of CMS-emulsion, so as to provide a single portion or multiple portions of CMS-enhanced vaccine, preferably a single portion.

[0156] In another embodiment, said use may comprise combining a single dose of the vaccine to a quantity of CMS-emulsion so as to provide multiple portions of CMS-enhanced vaccine. A portion of the CMS- enhancedvaccine has been defined above, and refers to an injection volume which is 50 - 150 vol.%, preferably 80 - 120 vol.%, more preferably 90 - 110 vol.%, most preferably 100 ± 5 vol.% of the registered volume of injection of the vaccine that the CMS-enhanced vaccine was prepared from, which comprises the quantities of CMS, antigen(s), oil, aqueous phase and further optional components as defined above.

[0157] In preferred embodiments, administration of a single portion of said CMS-enhanced vaccine to a human or animal for which vaccination is desired is by intramuscular, intradermal, or subcutaneous injection. In much preferred embodiments, the administration is achieved in the way prescribed in the market authorization, taking into account any further guiding prescribed in the market authorization.

[0158] In preferred embodiments, the invention provides a composition for use in vaccination of a human by injection, said composition comprising 1) a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), and 2) a vaccine comprising one or more antigens, said composition being in the form of an emulsion, and said composition representing a single portion having a volume of 0.3 - 1.5 ml, said volume comprising CMS in a quantity of 0.1 - 10 mg, preferably 0.2 - 3.0 mg, more preferably 0.5 - 2.5 mg, most preferably 0.8 - 2.0 mg, wherein said one or more antigens are as defined under a-m: a. one or more influenza antigens in a quantity of 1 - 10 pg, preferably 1.5 - 8 pg, more preferably 2.0 - 5.0 pg b. one or more antigens selected from the group of pneumococcal polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg per antigen; and / or a pneumococcal polysaccharide serotypes 6B antigen in a quantity of 0.04 - 2.2 pg, preferably 0.2 - 2.2 pg, more preferably 0.3 - 1.5 pg; one or more of yellow fever dengue virus serotype 1, 2, 3 and 4 antigens, preferably in a quantity of 0.5 - 3.5 loglO CCID50, more preferably 1.0 - 3.0 loglO CCID50 per antigen; c. one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 6 LI, 18 LI, 31 LI, 33 LI, 45 LI, 52 LI and 58 LI, preferably in a quantity of 0.5 - 15, more preferably 2 - 10 pg per antigen; and / or one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 11L, 16 LI and 18 LI, preferably in a quantity of 1 - 20 pg, preferably 2 - 20 pg, more preferably 4 - 15 pg per antigen; d. an inactivated rabies antigen, preferably in a quantity of 0.1 - 2.0 IE, more preferably 0.2 - 1.0 IE; e. one or more meningococcal antigens of groups A, C, Y and / or W, preferably in a quantity of 0.5 - 5.0 pg, more preferably 1.0 - 3.5 pg per antigen; f. one or more meningococcal antigens of group B, preferably in a quantity of 2.5 - 25.0 pg, more preferably 3.0 - 20 pg, even more preferably 5.0 - 15 pg, per antigen; g. one or more respiratory syncytial virus (RSV) recombinant antigens, preferably in a quantity of 3.0 - 50.0 pg, more preferably 5.0 - 40 pg, even more preferably 8 - 30 pg of RSV per antigen; h. a smallpox and / or monkey pox antigen, preferably in a quantity of up to 4 x 107infectious units; i. a diphtheria toxoid, preferably in a quantity of up to 15 infectious units; j. a tetanus toxoid, preferably in a quantity of up to 40 infectious units; k. a Bordetella pertussis antigen, preferably in a quantity of up to 18 pg l. a polio antigen, preferably in a quantity of up to 25 D antigen units for type 1, and / or up to 6 D antigen units for type 2, and / or up to 25 32 D antigen units for type 3; m. a hepatitis B surface antigen, preferably in a quantity of up to 8 pg-

[0159] The invention furthermore provides a composition comprising a CMS-emulsion for use in vaccination, said CMS-emulsion being as defined above, wherein said use comprises a) providing a vaccine suitable for injection as a single dose, or part thereof; b) combining said vaccine with said CMS-emulsion to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine; and c) administration of a single portion of said CMS-enhanced vaccine to a human or animal for which vaccination is desired, all aspects being as defined above.

[0160] The invention furthermore provides a composition comprising a vaccine for use in vaccination, said vaccine being as defined above, wherein said use comprises a) providing a CMS-emulsion suitable for injection; b) combining said vaccine with said CMS-emulsion to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine; and c) administration of a single portion of said CMS-enhanced vaccine to a human or animal for which vaccination is desired, all aspects being as defined above.

[0161] Said use of the composition of the invention for vaccination, in the form of a single portion as defined elsewhere, provides an immunological response which is at least as good as the immunological response obtained from administration of the vaccine without CMS. In preferred embodiments, administration of a single portion of the CMS-enhanced vaccine provides an immune response which is even improved, relative to administration of the vaccine without CMS. Improvement, in this regard, can be defined as an immune response obtained from administration of a single portion of the CMS-enhanced vaccine which is at least 110 %, preferably at least 150%, more preferably at least 200 %, of the immune response obtained after administration of one single (full) dose of the vaccine (without CMS).

[0162] The immune response can be measured by methods generally known in the art. Said method may vary with the type of antigen(s) in the vaccine. Preferably, the directions for measurement of an immune response can be found in the market authorization documents, such as an SmPC, for any particular vaccine, or is available as common general knowledge.

[0163] The invention furthermore provides use of a CMS-emulsion (defined elsewhere) comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS) for the preparation of a CMS-enhanced vaccine (defined elsewhere) comprising one or more antigens other than an influenza antigen, a Clostridioides difficile antigen, a malaria antigen, or a SARS-CoV-2 antigen (as defined in more detail elsewhere).

[0164] The invention also provides use of a CMS-emulsion (defined elsewhere) comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS) for the manufacture of a medicament for vaccination, said medicament being a CMS-enhanced vaccine comprising one or more antigens (as defined elsewhere).

[0165] Methods to obtain CMS-enhanced vaccines, and kits for achieving the same.

[0166] The invention furthermore provides a method for the preparation of a (CMS-enhanced) vaccine. This method is a method for vaccine distribution, which method enhances efficiency of vaccine use, while maintaining or increasing (preferably increasing) vaccine efficacy, as measured by the immune response, without additional side effects.

[0167] Said method comprises a) providing a vaccine suitable for injection (as defined elsewhere) as a single dose, or part thereof; b) combining said vaccine with a CMS-emulsion (as defined elsewhere); thereby providing one or more portions of a CMS-enhanced vaccine. All aspects of the method have been elaborated upon above.

[0168] In preferred embodiments, the vaccine is provided in a volume of volume of 0.05 - 2.5 mL, preferably 0.1 - 1.5 mL, more preferably 0.15 - 1.0 mL, or any other volume as defined elsewhere. More preferably, the vaccine is provided as a single (registered) dose (as defined in the market authorization), at a volume of preferably 0.1 - 1.0 ml. The combination of the vaccine with the CMS-emulsion occurs as described elsewhere: the volume of the CMS-emulsion represents 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents. This provides, if the vaccine is provided as a single dose, multiple portions of the CMS-enhanced vaccine are obtained. Alternatively, if the vaccine is provided as part of a single dose, the CMS-enhanced vaccine can be obtained as a single portion.

[0169] The invention also provides a method for vaccination, comprising a) providing a vaccine (as defined elsewhere); b) providing a CMS-emulsion (as defined elsewhere) suitable for injection; c) combining said vaccine and said CMS-emulsion to provide at least one portion of a CMS-enhanced vaccine (as defined elsewhere); d) administering one portion of CMS-enhanced vaccine to a human or animal for which vaccination is desired (as defined elsewhere).

[0170] The invention also provides a container comprising CMS-emulsion (as defined elsewhere), said container being adapted to receive a volume of a vaccine (as defined elsewhere) so as to provide a CMS-enhanced vaccine. Said container comprising CMS-emulsion comprises air space sufficient to receive a vaccine, preferably a single dose of a vaccine, as defined in the market authorization. Thus, preferably, the container comprises 0.2 - 10 mL air space, more preferably 0.5 - 5.0 mL. Further preferably, the container comprises 1.0 - 10.0 ml CMS-emulsion, more preferably 1.5 - 5.0 mL.

[0171] If the container is adapted to receive a single dose of a vaccine, or part thereof, then preferably, the container comprises a quantity of CMS emulsion of 1 - 10 volumetric equivalents of said single dose of vaccine, or part thereof, and / or air space sufficient to allow for 1 - 5 volumetric equivalents of said single dose of vaccine, or part thereof. Appropriate volumes have been defined elsewhere. Said container is further preferably equipped with a puncturable closing. Said container is preferably a vial. The container is further defined elsewhere.

[0172] The invention furthermore provides kits which provide essential aspects of the invention separately, thereby allowing to practice the invention. Thus, the invention also provides a kit of parts, comprising

[0173] 1) at least one container comprising a CMS-emulsion (as defined elsewhere); and

[0174] 2) one or more of a) a vaccine (as defined elsewhere), b) one or more syringes, and c) instructions for use of the kit; wherein the kit is adapted to combine a volume of the vaccine with a volume of the emulsion such that after combination, a CMS-enhanced vaccine is obtained.

[0175] A container, in this context, can be any container which is suitable to contain an injectable formulation. The container is preferably sterile. Further preferably, the container is provided with a puncturable closing, adapted to reversibly receive a syringe.

[0176] Preferably, the container is a vial, defined as a glass container, preferably having a cylindrical shape, said vial being provided with a cap, which cap is preferably puncturable by a syringe. The glass may be colored or without color, and is preferably without color. The glass is preferably transparent.

[0177] In the present case, the container has a size at least sufficient to hold one or more portions of CMS-enhanced vaccine. In preferred embodiments, the container has additional free space (“air space”) to receive the vaccine.

[0178] The kit may furthermore comprise one or more syringes. A syringe, in this regard, is a medical tool suitable for injection of a liquid into a human or animal, as herein defined. Syringes which are suitable for this purpose are known in the art.

[0179] The kit may furthermore comprise instructions for use of the kit. Such instructions may come in any form known in the art, such as on paper, for example by textual or graphical instructions. Alternatively, instructions may take the form of a printed reference to a website or QR-code, which links to the instructions for use online.

[0180] The kit may take any appropriate form. The kit may for example further comprise a box, which provides appropriate, preferably sterile, packaging for each of the parts.

[0181] In one embodiment, the kit comprises a container which comprises CMS-emulsion in a quantity so as to provide multiple portions of CMS-enhanced vaccine. In this embodiment, the container comprising CMS- emulsion has sufficient air space to allow for introduction of one or more doses of the vaccine. In this embodiment, the kit furthermore comprises one or more of a) one or more doses of the vaccine; b) one or more syringes, and c) instructions for use of the kit.

[0182] In another embodiment, the kit comprises a container which is adapted to receive a quantity of CMS-emulsion so as to provide multiple portions CMS-enhanced vaccine. In this embodiment, the container is preferably an empty container with sufficient air space to allow for introduction of one or more doses of the vaccine and sufficient CMS- emulsion to provide multiple doses of CMS-enhanced vaccine. In this embodiment, the kit furthermore comprises a container with CMS-emulsion, and one or more of a) one or more doses of the vaccine; b) one or more syringes, and c) instructions for use of the kit.

[0183] In another embodiment, the kit comprises at least one, and preferably 2 - 10, more preferably 3 - 8 containers, each container containing CMS-emulsion in a quantity so as to provide CMS in a quantity sufficient to enhance an immune response in a single subject (defined elsewhere; for example 0.1 - 10 mg of CMS, more preferably 0.5 - 5.0 mg, most preferably 0.8 - 2.5 mg of CMS). In this embodiment, each container is adapted to receive part of a dose of a vaccine, such as 5 - 90 vol.% of a single dose of the vaccine, preferably 10 - 50 vol.% (see elsewhere), such that each container provides a single portion of the CMS-enhanced vaccine.

[0184] Variations of the above ways of composing kits to practice the invention are easily conceived by the skilled person, and considered encompassed in the invention.

[0185] Thus, the kit may comprise a container comprising or adapted to receive the CMS-emulsion in multiple volumetric equivalents of the one or more doses of the vaccine, preferably 1 - 25, more preferably 2 - 20, more preferably 3 - 10 volumetric equivalents. The container may further be adapted to receive one or more doses of the vaccine, so that the container provides multiple portions of the CMS-enhanced vaccine per dose of the vaccine.

[0186] Alternatively, the kit may comprise a plurality of containers comprising or adapted to receive a volume of the CMS-emulsion, each container of said plurality of containers further adapted to receive a part of the dose of the vaccine, such that after combination of the volume of the CMS-emulsion with said part of the dose of the vaccine, a container provides at least one portion of CMS-enhanced vaccine. Preferably, in this case, the kit comprises 1 - 25, more preferably 2 - 20, more preferably 3 - 10 containers comprising or adapted to receive the CMS-emulsion, for combination with 1 - 50 vol.%, preferably 2 - 40 vol.%, more preferably 3 - 30 vol.%, more preferably 4 - 25 vol.%, most preferably 5 - 25 vol.%, of a single dose of vaccine (as defined in the market authorization).

[0187] The kit may comprise the vaccine in a liquid or a solid form. If liquid, the vaccine is preferably in a ready-to-use form. If solid, the vaccine is preferably reconstituted following the guidance in the market authorization, to provide the liquid vaccine in a ready-to-use form, prior to combination with the CMS-emulsion.

[0188] Clauses pertaining to the invention

[0189] 1. A composition suitable for injection comprising 1) a vaccine and 2) a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS).

[0190] 2. A composition according to clause 1, wherein the vaccine is a registered vaccine, defined by a registered volume for injection and a registered antigen quantity for injection, said registered volume for injection and said registered antigen quantity for injection together defining a single dose of the vaccine.

[0191] 3. A composition according to clause 1 or 2, wherein the vaccine is defined by a registered volume for injection, and wherein the composition comprises, relative to the registered volume for injection of the vaccine, 1) the vaccine in a volume of 5 - 100 vol.% of the registered volume for injection, and 2) the CMS-emulsion in 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents.

[0192] 4. A composition according to any of clauses 2 - 3, wherein the composition comprises, relative to the registered volume for injection of the vaccine, 1) the vaccine in a volume of 5 - 50 vol.% of the registered volume for injection, and 2) the CMS-emulsion in a volume of 50 - 95 vol.% of the registered volume for injection of the vaccine, preferably 80 - 90 vol.%.

[0193] 5. A composition according to clause 1 or 2, wherein the vaccine is defined by a registered antigen quantity for injection, and wherein the composition comprises, relative to the registered antigen quantity for injection of the vaccine, 1) the vaccine in a volume so as to provide 5 - 100 wt.% of the registered antigen quantity for injection, and 2) the CMS- emulsion in 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents.

[0194] 6. A composition according to any of clauses 1 - 5, wherein the CMS- emulsion comprises CMS, defined as a carbohydrate monosulphate fatty acid ester, wherein

[0195] • the carbohydrate is a monosaccharide, a disaccharide, a trisaccharide or a polysaccharide comprising 4 - 10 saccharide units, preferably a monosaccharide selected from allose, altrose, glucose, gulose, mannose, sorbose, iodose, talose, gentiobiose, fructose, galactose, inositol, ribose, arabinose, xylose and lysose, a disaccharide selected from sucrose, maltose, lactose, lactulose, cellobiose, trehalose, gentiobiose, turanose, isomaltulose and melibiose, a trisaccharide selected from raffinose, melezitose, maltotriose, isomaltotriose, raffinose, kestose and negerotriose, or a polysaccharide selected from an alpha-, beta- or gamma-cyclodextrin; and / or

[0196] • the carbohydrate is substituted on one hydroxyl group with a sulphate group (-SO3 ), said sulphate group optionally being substituted with a monovalent cation, said monovalent cation preferably being selected from H+, Na+, K+, Li+, NHA, NRH3+, NR2H2+, NR3H+, with R being selected, independently, on each occurrence, from a Cl - C6 saturated or unsaturated alkyl group; and / or

[0197] • the carbohydrate is substituted on at least 50 %, preferably at least 75 %, more preferably at least 80 % of the hydroxyl groups with a fatty acid, said fatty acid being defined as a C6 - C22 linear or branched, saturated or unsaturated fatty acid, preferably a linear fatty acid having 6 - 18 carbon atoms.

[0198] 7. A composition according to any of clauses 1 - 6, wherein the oil phase in the CMS-emulsion comprises squalane, squalene, hexadecane, a vegetable oil or a mineral oil, preferably squalane. 8. A composition according to any of clauses 1 - 7, wherein the CMS- emulsion comprises CMS in a concentration of 0.5 - 25 mg / mL, more preferably 0.8 - 10 mg / mL, even more preferably 1.0 - 5.0 mg / mL and / or wherein the CMS-emulsion comprises oil in a concentration of 0.5 - 100 mg / mL, more preferably 0.8 - 50 mg / mL, even more preferably 1.0 - 25 mg / mL, more preferably 1.0 - 10.0 mg / mL, most preferably 2.0 - 8.0 mg / mL

[0199] 9. A composition according to any of clauses 1 - 8, wherein the vaccine comprises

[0200] • an influenza antigen, preferably in a quantity of 1 - 60 pg per dose;

[0201] • a pneumococcal polysaccharide, preferably in a quantity of 1 - 8.0 pg per dose;

[0202] • a zoonotic influenza antigen, preferably in a quantity of 1 - 15 pg per dose;

[0203] • a meningococcal antigen, preferably in a quantity of 5 - 100 pg per dose;

[0204] • a smallpox and / or monkey pox antigen, preferably in a quantity of at least 5xl07infectious units per dose;

[0205] • a diphtheria toxoid, preferably in a quantity of not less than 20 IU per dose;

[0206] • a tetanus toxoid, preferably in a quantity of not less than 40 IU per dose;

[0207] • a Bordetella pertussis antigen, preferably in a quantity of 3 - 50 pg per dose;

[0208] • a polio antigen, preferably in a quantity of at least 7 D-antigen units;

[0209] • a hepatitis B surface antigen, preferably in a quantity of 5 - 30 pg per dose;

[0210] • a Haemophilus influenza polysaccharide, preferably in a quantity of at least 3 pg per dose;

[0211] • a Respiratory Syncytial Virus (RSV) antigen, preferably in a quantity of 50 - 150 pg per dose; • a human papillomavirus (HPV) antigen, preferably in a quantity of 10 - 60 pg per dose.

[0212] • a hepatitis A antigen, preferably in a quantity of 1 - 100 pg per dose;

[0213] • a SARS-Cov-2 (COVID- 19) antigen, preferably in a quantity of 1 - 50 pg per dose;

[0214] • an ebola antigen, preferably in a quantity of at least 72x 106plaque forming units;

[0215] • a dengue antigen, preferably in a quantity of 2.0 - 9.0 logio of the 50% cell culture infectious dose;

[0216] • an anthrax antigen,

[0217] • a cholera antigen

[0218] • a rabies antigen;

[0219] • a rubella antigen.

[0220] 10. A composition according to any of clauses 2 - 9, said composition being in the form of an emulsion, and said composition representing a single portion having a volume of 50 - 150 vol.%, preferably 80 - 120 vol.%, more preferably 90 - 110 vol.%, most preferably 100 ± 5 vol.% of the registered volume of injection of the vaccine, said composition comprising:

[0221] • 0.1 - 10 mg of CMS, preferably 0.5 - 5.0 mg, most preferably 0.8 - 2.5 mg; and

[0222] • at least one antigen, in a quantity of 5 - 90 % of the registered antigen quantity for injection of the vaccine, preferably 10 - 50 %, more preferably 15 - 35 %; and

[0223] • at least one oil, said oil preferably comprising squalane; and

[0224] • an aqueous phase.

[0225] 11. A composition according to any of clauses 1 - 9, said composition being in the form of an emulsion, and said composition representing a single portion having a volume of 0.3 - 1.5 ml, said volume comprising CMS in a quantity of 0.2 - 3.0 mg, preferably 0.5 - 2.5 mg, more preferably 0.8 - 2.0 mg, and further comprising • one or more antigens selected from the group of pneumococcal polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg per antigen; and / or a pneumococcal polysaccharide serotypes 6B antigen in a quantity of 0.04 - 2.2 pg, more preferably 0.2 - 2.2 pg, more preferably 0.3 - 1.5 pg; or

[0226] • one or more yellow fever dengue virus serotype 1, 2, 3 and 4 antigens, in a quantity of 0.5 - 3.5 logio CCID50, more preferably 1.0 - 3.0 logio CCID50 per antigen; or

[0227] • one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 6 LI, 18 LI, 31 LI, 33 LI, 45 LI, 52 LI and 58 LI in a quantity of 0.5 - 15, more preferably 2 - 10 pg per antigen; and / or one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 11L, 16 LI and 18 LI in a quantity of 1 - 20 pg, preferably 2 - 20 pg, more preferably 4 - 15 pg per antigen; or

[0228] • an inactivated rabies antigen in a quantity of 0.1 - 2.0 IE, preferably 0.2 - 1.0 IE; or

[0229] • one or more meningococcal antigens of groups A, C, Y and / or W in a quantity of 0.5 - 5.0 pg, preferably 1.0 - 3.5 pg per antigen; or

[0230] • one or more meningococcal antigens of group B in a quantity of 2.5 - 25.0 pg, preferably 3.0 - 20 pg, more preferably 5.0 - 15 pg, per antigen; or

[0231] • one or more respiratory syncytial virus (RSV) recombinant antigens in a quantity of 3.0 - 50.0 pg, more preferably 5.0 - 40 pg, even more preferably 8 - 30 pg of RSV per antigen; or

[0232] • One or more influenza antigens in a quantity of 1 - 10 pg, preferably 1.5 - 8 pg, more preferably 2.0 - 5.0 pg, wherein said influenza antigen can be a seasonal influenza antigen or a pandemic influenza antigen.

[0233] 12. The composition of any of clauses 1 - 11 for use in vaccination.

[0234] 13. The composition for use according to clause 12, wherein said use comprises a) providing a vaccine suitable for injection as a single dose, or part thereof; b) mixing said vaccine with said CMS-emulsion to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine; c) administration of a single portion of said CMS-enhanced vaccine to a human or animal for which vaccination is desired.

[0235] 14. A composition for use according to clause 12 or 13, wherein said use comprises administration by intramuscular, intradermal or subcutaneous injection.

[0236] 15. A method for the preparation of a vaccine, comprising a) providing a vaccine suitable for injection as a single dose, or part thereof; b) mixing said vaccine with an emulsion suitable for injection comprising an oil phase, an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), thereby providing one or more portions of a CMS-enhanced vaccine.

[0237] 16. A method according to clause 15, wherein the vaccine suitable for injection has a volume of 0.05 - 2.5 mL, preferably 0.1 - 1.5 mL, more preferably 0.15 - 1.0 mL.

[0238] 17. A method according to clause 15 or 16, wherein the volume of the emulsion represents 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents.

[0239] 18. A method according to any of clauses 15 - 17, wherein if the vaccine is provided as a single dose, multiple portions of the CMS-enhanced vaccine are obtained, and / or if the vaccine is provided as part of a single dose, the CMS-enhanced vaccine is obtained as a single portion or multiple portions.

[0240] 19. A container comprising a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), said container being adapted to receive a volume of a vaccine.

[0241] 20. A container according to clause 19, wherein said container comprises air space in a volume sufficient to receive the vaccine.

[0242] 21. A kit of parts, comprising 1) at least one container comprising a CMS-emulsion suitable for injection comprising an oil phase, aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), and 2) one or more of a) a vaccine; b) one or more syringes, and c) instructions for use of the kit; wherein the kit is adapted to combine a volume of the vaccine with a volume of the emulsion such that after combination, a CMS-enhanced vaccine is obtained.

[0243] 22. A kit of parts according to clause 21, wherein the vaccine is a registered vaccine, defined as a vaccine with market authorization.

[0244] 23. A kit of parts according to clause 21 or 22, wherein either a) the kit comprises a container comprising or adapted to receive the emulsion in multiple volumetric equivalents of the one or more doses of the vaccine, and further adapted to receive said one or more doses of the vaccine, so that the container provides multiple portions of the CMS- enhanced vaccine per dose of the vaccine; or b) the kit comprises a plurality of containers comprising or adapted to receive a volume of the emulsion, each container of said plurality of containers further adapted to receive a part of the dose of the vaccine, such that after combination of the volume of the emulsion with said part of the dose of the vaccine, a container provides at least one portion of CMS- enhanced vaccine. EXAMPLES general

[0245] CMS in the below examples was monosulphate heptadecyl sucrose with a triethyl amine counterion, unless otherwise mentioned.

[0246] CMS was isolated, and subsequently stored as a CMS-emulsion using a saline aqueous phase and a squalane oil phase. A stock of CMS emulsion was obtained by combining 20 mg / ml CMS, 40 mg / ml squalane, and 20 mg / ml Polysorbate 80 in the saline aqueous phase, applying high pressure emulsification to obtain a stable emulsion The said stock CMS- emulsion was diluted with saline to obtain the desired concentration of the CMS-emulsion, unless otherwise indicated.

[0247] To obtain CMS-enhanced vaccines with 1 / 5THdose of a registered vaccine and 0.5 mg of CMS per dose for injection in humans or animals, 0.125 mL of the said stock of CMS-emulsion and 1.875 mL of saline were introduced into vials, to which there was added a single dose of a registered (authorized) vaccine, in the quantity indicated in the authorization documents (0.5 ml in all cases). This provided 2.5 mL of the composition of the invention, representing 5 portions of CMS-enhanced vaccine. Alternatively, 0.25 mL of the said stock of CMS-emulsion and 3.75 mL of saline were introduced into vials, to which there was added two doses of a registered (authorized) vaccine, in the quantity indicated in the authorization documents (0.5 ml in all cases). This provided 5.0 mL of the composition of the invention, representing 10 portions of CMS-enhanced vaccine.

[0248] To obtain CMS-enhanced vaccines with 1 / 5THdose of a registered vaccine and 1 mg of CMS per dose for injection in humans or animals, 0.25 mL of the said stock of CMS-emulsion and 1.75 mL of saline were introduced into vials, to which there was added a single dose of a registered (authorized) vaccine, in the quantity indicated in the authorization documents (0.5 mL in all cases). This provided 2.5 mL of the composition of the invention, representing 5 portions of CMS-enhanced vaccine.

[0249] To obtain CMS-enhanced vaccines with 1 / 5THdose of a registered vaccine and 2 mg of CMS per dose for injection in humans or animals, 0.50 mL of the said stock of CMS-emulsion and 1.50 mL of saline were introduced into vials, to which there was added a single dose of a registered (authorized) vaccine, in the quantity indicated in the authorization documents (0.5 ml in all cases). This provided 2.5 ml of the composition of the invention, representing 5 portions of CMS-enhanced vaccine.

[0250] In preclinical and clinical studies, the quantity of CMS in the final CMS-enhanced vaccine was varied, and the type of CMS was varied. Monosulphate heptadecyl maltose, monosulphate heptadodecyl maltose, monosulphate tetradodecyl galactose, monosulphate heptadodecyl sucrose, and monosulphate heptadecyl sucrose were all found to provide comparable results, at comparable quantities, as described below for monosulphate heptadecyl sucrose.

[0251] Example 1

[0252] CMS-emulsions were prepared as above, comprising 1 mg / ml CMS, 2 mg / ml squalane, and 1 mg / ml Polysorbate 80 (CMS-1), or 4 mg / ml CMS, 8 mg / ml squalane, and 4 mg / ml Polysorbate 80 (CMS-2).

[0253] A vial of CMS-emulsion (CMS-1 or CMS-2; 2.0 ml) was combined with one registered dose (0.5 ml) of an influenza vaccine (VaxiGrip Tetra), providing 5 portions of a CMS-enhanced influenza vaccine.

[0254] VaxiGrip Tetra contains 15 pg HA of each of the antigens A / Victoria / 4897 / 2022 (H1N1), A / Darwin / 9 / 2021 (H3N2), B / Austria / 1359417 / 2021 and B / Phuket / 3073 / 2013 in a single 0.5 ml dose. One portion of the CMS-1 and CMS-2 enhanced influenza vaccines thus contains 3 pg of each of these antigens, and 0.5 mg (CMS-1) or 2 mg (CMS-2) of CMS.

[0255] In a clinical trial (20 humans per group), efficacy and side effects of a single 0.5 ml portion of the CMS-1 and CMS-2 enhanced vaccine was evaluated, using a full (0.5 ml) dose of VaxiGrip Tetra as control.

[0256] After intramuscular vaccination at day 1, serum hemagglutination inhibition (HI) antibody titers (GMT) were determined at day 1 (prior to injection), and days 8, 29 and 180 using a hemagglutination inhibition assay, for the antigens Victoria, Austria, Darwin and Phuket. It was found that the CMS-1 and CMS-2 enhanced influenza vaccines provided an at least equal immune response, as compared to the full dose of VaxiGrip Tetra. After 180 days, the GMT antibody titers were better after using 3 pg of antigen in CMS-1 or CMS-2 enhanced vaccine, than after using 15 pg of antigen using the regular (non-CMS-enhanced) vaccine. See Figures la - Id. There was no significant difference in side effects, relative to the control.

[0257] Example 2

[0258] The stock CMS-emulsion was used, providing 1.0 mg CMS (monosulphate heptadecyl sucrose ) per portion of CMS-enhanced vaccine, in combination with the registered vaccine Prevenar 13 (PCV-13), a pneumococcal vaccine (registered dose 0.5 ml, containing pneumococcal polysaccharide antigens of serotype 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23 F in a quantity of 2.2 pg, and pneumococcal polysaccharide antigens of serotype 6B in a quantity of 4.4 pg).

[0259] New Zealand White Rabbits, 3-4 months of age, were divided in four groups of 6 animals (each 3 males and 3 females). Animals were healthy and were acclimatized for 7 days before treatment. The animal facility conditions were a day / night cycle (12h / 12h) with a monitored temperature of 21 to 23 °C and a relative humidity of 65-67%. Animals were randomly assigned to treatment groups using a generalized randomized block design on the base of body weight. Animals were checked daily for overt signs of disease and treatment-related adverse events.

[0260] The following 4 vaccines were investigated:

[0261] Group 1, Vaccine 1: Full dose of PCV13 without CMS

[0262] Group 2, Vaccine 2: Full dose of PCV13 with 1 mg of CMS

[0263] Group 3, Vaccine 3: 1 / 5THdose of PCV13 without CMS Group 4, Vaccine 4: 1 / 5THdose of PCV13 with 1 mg of CMS The injected doses in group 2 were prepared by combining full single doses of PCV (0.5 ml) with one volumetric equivalent (0.5 ml) of a CMS-emulsion providing 1 mg of CMS.

[0264] The injected portions for group 4 were prepared by mixing one full dose (0.5 ml) of PCV with 5 volumetric equivalents of CMS-emulsion (2.5 ml), thereby providing 5 portions of 0.6 ml of CMS-enhanced pneumococcal vaccine (each containing 1 mg of CMS).

[0265] Each portion (0.6 ml) of CMS-enhanced vaccine as used in Group 4 contained pneumococcal polysaccharide antigens of serotype 1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F and 23 F in a quantity of 0.44 pg, and pneumococcal polysaccharide antigens of serotype 6B in a quantity of 0.88 dg-

[0266] Animals were injected with 0.6 mL of the vaccine, according to the distribution above. Blood samples were collected from overnight-fasted rabbits from the ear vein pre-immunization and 29 and 57 days after the first immunization. Blood samples were shortly kept at room temperature to allow clotting. Serum was isolated by centrifugation of the blood samples at 16.000g at 4 °C and stored / shipped at <-20 °C until analysis.

[0267] Antibody levels against all thirteen polysaccharides included in the PCV13 vaccine were measured using a fluorescent -bead-based multiplex immunoassay (MIA). The protocol was adapted from Stenger et al. (Clin Vaccine Immunol 2011;18(4):595-603.). Pneumococcal polysaccharide (PnPS) types 1, 3, 4, 5, 6B, 9V, 14, 19A and 23F were obtained from Merck and types 6A, 7F, 18C and 19F were obtained from Pfizer. Coupling of PnPS to 4-(4,6-dimethoxy[l,3,5]triazin-2- yl)-4-methylmorpholinium chloride (DMTMM) was performed as described by Schlottmann et al. (J. Immunol Methods 2006;309(l-2):75-85.). MagPlex® Microsphere beads for 13 different regions were obtained from Luminex. Beads were vortexed for 30 s at max speed and sonicated for 20 s before each step in the protocol.

[0268] 250 pL of each previously obtained PnPS-DMTMM solution was incubated with 500 pL of the corresponding bead solution, in a total volume of 750 jiL. Incubation was performed overnight, rotating at room temperature (RT). Beads were washed twice with PBS, before storage in bead storage buffer (1 % BSA 0,05 % NaN3 in PBS) at 4 °C. Prior to the MIA, beads were counted and a mix containing 2000 beads per region in 25 jiL PBS was prepared. 25 pL bead solution was mixed with 25 pL standard or test sample in dark, flat bottom 96-wells plates (Greiner). Serial dilutions of a pool of serum from PCV13-vaccinated rabbits were used as a standard.

[0269] Serum samples were diluted 1 / 500 in adsorption buffer (3% w / v BSA and 240 pg / mL multi cell wall polysaccharide (CWPS, Statens Serum Institute) in PBS). Plates with beads and samples were incubated for 45 min at RT, shaking at 600 rpm. Beads were washed three times with 0.05 % Tween 20 in PBS in a magnetic plate washer (Biotek, 405 TS). PE- conjugated Goat anti-Rabbit IgG, (Southern Biotech, origin, US, 4030-09) was diluted 1 / 200 in PBS. 50 jiL of the conjugate dilution was added to each well and the plates were incubated shaking for 20 min at RT. Washing was performed as described above and the beads were resuspended in 0.05 % Tween 20 in PBS. The median fluorescence intensity (MFI) was measured for 50 beads per region on a FLEXMAP 3D® (LUMINEX). Data was analysed using the BioPlex Manager software (Bio-Rad). Titres were listed for each animal and for each group the geometric mean antibody titer (GMT) of the loglO-value of individual titers, standard deviation (SD), antilog-value (10GMT), student T-test and factor of increase (FOI) were calculated in Excel.

[0270] Results

[0271] The four groups of rabbits were injected intramuscularly (IM) at Day 0 and Day 28 with a full (optimal) dose or 1 / 5TH(suboptimal) dose of PCV13 with or without 1 mg of CMS, as indicated.

[0272] Before (Day 0), four weeks after the first vaccination (Day 29) and four weeks after the second vaccination (Day 57) blood samples were collected to determine the antibody titers in serum against the 13 polysaccharides included in PCV13 by a fluorescent -beat -based multiplex immunoassay. Serum samples collected before immunization and prediluted 500-fold, demonstrated no or low antibody titers to the polysaccharides with exception of two animals that showed significant titers against serotype 14.

[0273] Post vaccination 1 (PV-1), the standard (full) dose of PCV13 plus CMS gave significantly higher responses to the 9 serotypes than without adjuvant and factor of increase (FOI) varied between 3 and 6.7 and similar responses to 4 serotypes 6B 19F, 19A and 23F with FOI of between 0.7 and 2.3. The responses to a suboptimal dose of 1 / 5TH of PCV13 without CMS were lower than those to the optimal, full dose of PCV13, while responses to suboptimal dose of 1 / 5TH of PCV13 with CMS were higher than those to the optimal, full dose of PCV13. Overall, 1 / 5 dose of PCV13 with CMS gave 2- fold higher responses than the optimal, full dose of PCV13 without CMS. See Figure 2.

[0274] Post vaccination 2 (PV-2), antibody responses were increased against most serotypes. The antibody responses to suboptimal dose of 1 / 5TH of PCV13 without CMS were lower than those to the full, optimal dose of PCV13 without CMS, while responses to suboptimal dose of 1 / 5TH of PCV13 with CMS were similar or higher than those to the full, optimal dose of PCV13 without CMS (Figure 3). Overall, the suboptimal dose of 1 / 5 of PCV13 plus CMS gave similar responses as the full, optimal dose of PCV13 without CMS. See Figure 3.

[0275] It is concluded that the immune response to a dose of l / 5th of the standard dose of PCV13 was lower than to a full, optimal dose, but that presence of CMS enhances the immune response so as to provide an at least as good immune response, as a single full dose without CMS. No significant effects on side effects were observed.

[0276] Example 3

[0277] Safety and efficacy of combinations of CMS-enhanced vaccines based on equal proportions of CMS-emulsion (also called “LVA”, LiteVax Adjuvant) and Gardasil (HPV vaccine) and Rabipur (rabies vaccine) was determined in rabbits. The standard (registered) dose of the vaccine was combined with CMS emulsion, so as to provide portions of CMS-enhanced vaccine containing 2 mg of CMS per portion and the standard dose of the registered quantity of antigen. This provided readily acceptable safety profiles. Efficacy of a portion of CMS-enhanced vaccine is at least as good as the efficacy of the vaccine without CMS.

[0278] Materials and methods

[0279] • Vaccine Product Gardasil-9: 9 valent human papillomavirus (HPV) vaccine adsorbed to alum, commercial vaccine, MSD.

[0280] • LiteVax Adjuvant (LVA): CMS emulsion in vials containing 20 mg / mL CMS, 20 mg / mL Polysorbate 80 and 40 mg / mL squalane in phosphate buffered saline (isotonic).

[0281] • Vaccine Product Rabipur: Inactivated rabies virus vaccine, non- adjuvanted commercial vaccine, Bavarian Nordic. • Saline (0.9% NaCl solution in water-for-inj ection) for vaccine preparation, Braun, 10 mL ampoules.

[0282] • Empty vials for vaccine preparation, 10 mL glass sterile vials plus stopper and cap. 1-mL syringes with 0.1 mL graduation, 26x / 2” G injection needles to take-up vaccine from the vial plus injection needles for the immunization.

[0283] New Zealand White Rabbits, males and females, 14 weeks of age, were checked for their health status and acclimatized for 7 days before treatment. The animal facility conditions were a day / night cycle (12h / 12h) with a monitored temperature of 21.0 to 22.5 °C and a relative humidity of 63-80%. Three females and three males were randomly assigned to treatment groups. Animals were checked daily for overt signs of disease and treatment-related adverse events. Weighing, (blood) sampling and rectal temperature measurements were performed without anaesthesia of the rabbits. Ethical approval was obtained.

[0284] Study Vaccines with l / 5th dose of Vaccine Product were prepared by mixing 0.10 mL of the Vaccine Product, 0.05 ml of LiteVax Adjuvant (LVA) and 0.35 mL of saline. The Study Vaccines were administered within 4 hours after preparation. A volume of 0.5 mL of the Study Vaccines or the Vaccine Product was administered slowly as intramuscular injection into quadriceps muscle of by using a suitably graduated disposable sterile syringe attached with a sterile needle.

[0285] The dose of antigen per injection was a full human dose of Gardasil 9 or Rabipur without LVA or l / 5thdose of Gardasil 9 or l / 5thdose of Rabipur with LVA. The dose of LVA was 1 mg CMS per injection. A full human dose of Gardasil 9 contains human papillomavirus (HPV) 30 jig of 6-Ll-protein, 40 pg of 11-L1 -protein, 60 jig of 16-L1 -protein, 40 jig of 18-L1 -protein, 20 jig of 31-L1- protein, 20 jig of 33-Ll-protein, 20 jig of type 45-Ll-protein, 20 jig of type 52-Ll-protein and 20 jig of 58-Ll-protein. A l / 5th-dose of Gardasil 9 contains human papillomavirus (HPV) 6 jig of 6-Ll-protein, 8 jig of 11-L1- protein, 12 jig of 16-Ll-protein, 8 jig of 18-L1 -protein, 4 jig of 31-L1- protein, 4 jig of 33-Ll-protein, 4 jig of type 45-Ll-protein, 4 jig of type 52-Ll- protein and 4 jig of 58-Ll-protein.

[0286] A full human dose of Rabipur contains 2.5 IE inactivated rabies virus strain Flury LEP. A l / 5th-dose of Rabipur contains 0.5 IE inactivated rabies virus strain Flury LEP.

[0287] Blood samples were collected from overnight-fasted rabbits from the ear vein before immunisation, and 21 days after immunisation. Blood samples were shortly kept at room temperature to allow clotting. Serum was isolated by centrifugation of the blood samples at 16.000g at 4 °C and stored / shipped at <-20 °C until analysis.

[0288] Virus neutralisation antibody levels against HPV were measured using a high-throughput pseudovirion-based neutralization assay (HT-PBNA) described by Sehr et al (Sehr P, Rubio I, Seitz H, Putzker K, Ribeiro -Muller L, Pawlita M, et al. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses. PloS One (2013) 8(10):e75677. doi:

[0289] 10.1371 / journal.pone.0075677). The HT-PBNA was applied using an assay cut-off based on the mean EC50 value measured in sera of non-immunized (Day -7) rabbits. Titres were listed for each animal and for each group the geometric mean antibody titer (GMT) of the loglO-values of individual titers and standard deviation (SD) were calculated. Statistical difference was analysed by using the Student T-test and P-values < 0.05 were considered statistically distinct. Serum or antibody preparations displaying EC50 > 100 were considered neutralizing positive.

[0290] Virus neutralisation antibody levels against rabies were measured using a Rapid Fluorescent Focus Inhibition Test (RFFIT). The results were expressed in International Units per mL (lU / mL), which reflects the level of virus-neutralizing antibodies in serum. Titres were listed for each animal and for each group the geometric mean antibody titer (GMT) of the log 10- values of individual titers and standard deviation (SD) were calculated. Statistical difference was analysed by using the Student T-test and P-values < 0.05 were considered statistically distinct. Serum or antibody preparations displaying EC50 > 100 were considered neutralizing positive.

[0291] Results

[0292] Immunogenicity Gardasil

[0293] Groups of 3 male and 3 female rabbits were injected IM at Day 0 with a full dose of Gardasil-9 HPV vaccine without LVA or a mixture of l / 5th-dose of vaccine with a quantity of LVA to provide 1 mg of CMS. Serum antibody titers against 6 HPV serotypes of the HPV-9 vaccine were measured by a serum neutralization assay. GMTs, SD and P-values are presented in the table below.

[0294] Table: Virus neutralizing antibody titers after a single immunization with a full dose of Gardasil-9™ or l / 5th-dose of Gardasil-9™ with LVA in rabbits, expressed as 10-log values.

[0295] Before immunization, no detectable antibodies against 5 out of the 6 serotypes tested were observed in the animals (data not shown). Low titers against serotype 18 were detected prior to immunization. After vaccination, antibody titers to the 6 serotypes tested were increased in both groups.

[0296] There were no statistical differences between the group that received the Vaccine Product and the group that received the Study Vaccine (P>0.05).

[0297] Immunogenicity Rabipur Groups of 3 male and 3 female rabbits were injected IM with a full dose Rabipur™ without LVA or with l / 5th-dose of the vaccine mixed with a quantity of LVA contributing 1 mg of CMS. The antibody titers were measured 21 days later by a RFFIT and GMTs, SD and P-values are presented in the table below.

[0298] Table: Virus neutralizing antibody titers after a single immunization with a full dose of Rabipur™ or l / 5th-dose of Rabipur with LVA in rabbits. Before immunization, no detectable antibodies against rabies were observed in the animals (data not shown). Three weeks after immunization, both groups exhibited significant RFFIT antibody titers that were not statistically distinct.

[0299] Safety and tolerability

[0300] IM injection of the Vaccine Products or the Study Vaccines did not result in any clinical signs, mortality, food consumption, or effects on body weights. Mild redness was observed at the injection sites in some animals, but the incidence and severity were comparable between the Vaccine Products and the Study Vaccines. No additional signs of adverse reactions were noted during the study.

[0301] Conclusion

[0302] Compared to the standard (full) dose of Vaccine Products Gardasil-9 and Rabipur, a fractional (l / 5th) doses of Gardasil-9 and Rabipur supplemented with CMS-emulsion gave similar antibody responses and similar safety profiles. Antigen-sparing by LVA offers obvious advantages, which might be exploited by mixing bulk of antigen and bulk of adjuvant aiming at a vaccine product with a fractional dose of antigen or by mixing a single dose of the Vaccine Product with multiple (e.g. 5) doses of LVA aiming at 5 doses of vaccine product with a fractional dose of antigen.

Claims

Claims1. A composition suitable for injection comprising 1) a CMS- emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), and 2) a vaccine comprising one or more antigens other than an influenza whole or split virion antigen, a Clostridioides difficile toxoid A or toxoid B antigen, a recombinant Plasmodium falciparum glutamate-rich protein produced in Lactococcus lactis, a SARS-CoV-2 receptor-binding domain (RBD) nanoparticle antigen produced in Expi293F cells, or a VLP- based human epidermal growth factor receptor-2 (HER2) antigen produced in Drosophila melanogaster S2 insect cells.

2. A composition according to claim 1, wherein the composition is in the form of an emulsion, said composition representing a single portion having a volume of 0.3 - 1.5 mL, said volume comprising CMS in a quantity of 0.1 - 10 mg, preferably 0.2 - 3.0 mg, more preferably 0.5 - 2.5 mg, most preferably 0.8 - 2.0 mg.

3. A composition according to claim 2, wherein the antigen is as defined in one or more of: a. one or more antigens selected from the group of pneumococcal polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B in a quantity of 0.02 - 2.0 pg, preferably 0.1 - 1.1, more preferably 0.15 - 0.80 pg per antigen; and / or a pneumococcal polysaccharide serotypes 6B antigen in a quantity of 0.04 - 2.2 pg, preferably 0.2 - 2.2 pg, more preferably 0.3 - 1.5 pg; b. one or more of yellow fever dengue virus serotype 1, 2, 3 and 4 antigens, preferably in a quantity of 0.5 - 3.5 loglO CCID50, more preferably 1.0 - 3.0 loglO CCID50 per antigen;c. one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 6 LI, 18 LI, 31 LI, 33 LI, 45 LI, 52 LI and 58 LI, preferably in a quantity of 0.5 - 15, more preferably 2 - 10 pg per antigen; and / or one or more HPV antigens selected from the group of human papillomavirus (HPV) Type 11L, 16 LI and 18 LI, preferably in a quantity of 1- 20 pg, preferably 2 - 20 pg, more preferably 4 - 15 pg per antigen; d. an inactivated rabies antigen, preferably in a quantity of 0.1 - 2.0 IE, more preferably 0.2 - 1.0 IE; e. one or more meningococcal antigens of groups A, C, Y and / or W, preferably in a quantity of 0.5 - 5.0 pg, more preferably 1.0- 3.5 pg per antigen; f. one or more meningococcal antigens of group B, preferably in a quantity of 2.5 - 25.0 pg, more preferably 3.0 - 20 pg, even more preferably 5.0 - 15 pg, per antigen; g. one or more respiratory syncytial virus (RSV) recombinant antigens, preferably in a quantity of 3.0 - 50.0 pg, more preferably 5.0 - 40 pg, even more preferably 8 - 30 pg of RSV per antigen; h. a smallpox and / or monkey pox virus antigen, preferably in a quantity of up to 4 x 107infectious units; i. a diphtheria toxoid, preferably in a quantity of up to 15 infectious units; j. a tetanus toxoid, preferably in a quantity of up to 40 infectious units; k. a Bordetella pertussis antigen, preferably in a quantity of up to 18 pg l. a polio virus antigen, preferably in a quantity of up to 25 D antigen units for type 1, and / or up to 6 D antigen units for type 2, and / or up to 25 32 D antigen units for type 3;m. a hepatitis B virus surface antigen, preferably in a quantity of up to 8 pg;4. The composition according to any of claims 1 - 3 for use in vaccination.

5. The composition for use according to claim 4, wherein said use comprises vaccination by injection, preferably by intramuscular, intradermal or subcutaneous route of administration.

6. The composition for use according to claim 4 or 5, wherein said vaccination is vaccination of a human.

7. A composition for use in vaccination of a human by injection comprising 1) a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), and 2) a vaccine comprising one or more antigens, said composition being in the form of an emulsion, and said composition representing a single portion having a volume of 0.3 - 1.5 mL, said volume comprising CMS in a quantity of 0.2 - 3.0 mg, preferably 0.5 - 2.5 mg, more preferably 0.8 - 2.0 mg, wherein said one or more antigens comprise one or more influenza antigens in a quantity of 1 - 10 pg, preferably 1.5 - 8 pg, more preferably 2.0 - 5.0 pg.

8. The composition according to any of claims 1 - 3 or the composition for use according any of claims 4 - 7, wherein the oil phase in the CMS-emulsion comprises squalane, squalene, hexadecane, a vegetable oil or a mineral oil, preferably squalane.

9. The composition according to any of claims 1 - 3 or the composition for use according any of claims 4 - 7, wherein CMS is defined as a carbohydrate monosulphate fatty acid ester, wherein a) the carbohydrate is a monosaccharide, a disaccharide, a trisaccharide or a polysaccharide comprising 4 - 10 saccharide units, preferably a monosaccharide selected from allose, altrose, glucose, gulose, mannose, sorbose, iodose, talose, gentiobiose, fructose, galactose, inositol, ribose, arabinose, xylose and lysose, a disaccharide selected from sucrose, maltose,lactose, lactulose, cellobiose, trehalose, gentiobiose, turanose, isomaltulose and melibiose, a trisaccharide selected from raffinose, melezitose, maltotriose, isomaltotriose, raffinose, kestose and negerotriose, or a polysaccharide selected from an alpha-, beta- or gamma-cyclodextrin; and / or b) the carbohydrate is substituted on one hydroxyl group with a sulphate group (-SO3 ), said sulphate group optionally being substituted with a monovalent cation, said monovalent cation preferably being selected from H+, Na+, K+, Li+, NH4+, NRH3+, NR2H2+, NR3H+, with R being selected, independently, on each occurrence, from a Cl - C6 saturated or unsaturated alkyl group; and / or c) the carbohydrate is substituted on at least 50 %, preferably at least 75 %, more preferably at least 80 % of the hydroxyl groups with a fatty acid, said fatty acid being defined as a C6 - C22 linear or branched, saturated or unsaturated fatty acid, preferably a linear fatty acid having 6 - 18 carbon atoms.

10. The composition according to any of claims 1 - 3 or the composition for use according any of claims 4 - 7, wherein the vaccine optionally comprises quantities of excipients and / or additives.

11. The composition for use according to any of claims 6 - 10, wherein said use comprises a) providing a vaccine suitable for injection as a single dose, or part thereof; b) mixing said vaccine with said CMS-emulsion to provide multiple portions of a CMS-enhanced vaccine per dose of vaccine; c) administration of a single portion of said CMS-enhanced vaccine to a human for which vaccination is desired.

12. A method for the preparation of a vaccine, comprising combining 1) a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS) with 2) a vaccine comprising one or more antigens other than an influenza whole or split virion antigen, a Clostridioides difficile toxoid A or toxoid B antigen, a recombinant Plasmodium falciparumglutamate-rich protein produced in Lactococcus lactis, a SARS-CoV-2 receptor-binding domain (RBD) nanoparticle antigen produced in Expi293F cells, or a VLP -based human epidermal growth factor receptor-2 (HER2) antigen produced in Drosophila melanogaster S2 insect cells.

13. A method according to claim 12, comprising a) providing a vaccine suitable for injection as a single dose, or part thereof, comprising one or more antigens; b) mixing said vaccine with an emulsion suitable for injection comprising an oil phase, an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), thereby providing one or more portions of a CMS-enhanced vaccine.

14. A method according to claim 12, wherein the vaccine suitable for injection has a volume of 0.05 - 2.5 mL, preferably 0.1 - 1.5 mL, more preferably 0.15 - 1.0 mL.

15. A method according to claim 12 or 13, wherein the volume of the emulsion represents 1 - 25 volumetric equivalents of the volume of the vaccine, preferably 2 - 20 volumetric equivalents, more preferably 3 - 10 volumetric equivalents.

16. A method according to any of claims 12 - 14, wherein if the vaccine is provided as a single dose, multiple portions of the CMS-enhanced vaccine are obtained, and / or if the vaccine is provided as part of a single dose, the CMS-enhanced vaccine is obtained as a single portion or multiple portions.

17. A container comprising a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS), said container being adapted to receive a volume of a vaccine.

18. A container according to claim 17, wherein said container comprises air space in a volume sufficient to receive the vaccine.

19. A kit of parts, comprising 1) at least one container comprising a CMS-emulsion suitable for injection comprising an oil phase, aqueous phaseand a carbohydrate monosulphate fatty acid ester (CMS), and 2) one or more of a) a vaccine; b) one or more syringes, and c) instructions for use of the kit; wherein the kit is adapted to combine a volume of the vaccine with a volume of the emulsion such that after combination, a CMS-enhanced vaccine is obtained.

20. The kit of parts according to claim 19, wherein either a) the kit comprises a container comprising or adapted to receive the emulsion in multiple volumetric equivalents of the one or more doses of the vaccine, and further adapted to receive said one or more doses of the vaccine, so that the container provides multiple portions of the CMS- enhanced vaccine per dose of the vaccine; or b) the kit comprises a plurality of containers comprising or adapted to receive a volume of the emulsion, each container of said plurality of containers further adapted to receive a part of the dose of the vaccine, such that after combination of the volume of the emulsion with said part of the dose of the vaccine, a container provides at least one portion of CMS- enhanced vaccine.

21. Use of a CMS-emulsion comprising an oil phase comprising at least one oil, and further comprising an aqueous phase and a carbohydrate monosulphate fatty acid ester (CMS) for the preparation of a CMS-enhanced vaccine comprising one or more antigens other than an influenza whole or split virion antigen, a Clostridioides difficile toxoid A or toxoid B antigen, a recombinant Plasmodium falciparum glutamate -rich protein produced in Lactococcus lactis, a SARS-CoV-2 receptor-binding domain (RBD) nanoparticle antigen produced in Expi293F cells, or a VLP -based human epidermal growth factor receptor-2 (HER2) antigen produced in Drosophila melanogaster S2 insect cells.

22. Use according to claim 21, wherein the CMS emulsion comprises a. oil in a quantity of 0.5 - 100 mg / mL, more preferably 0.8 - 50 mg / mL, even more preferably 1.0 - 25 mg / mL, more preferably 1.0 - 10.0 mg / mL, most preferably 2.0 - 8.0 mg / mL, said oil phasecomprising one or more oils, preferably one oil, selected from the group of squalane, squalene, hexadecane, a vegetable oil or a mineral oil, most preferably squalane; and b. between 500 and 995 mg / mL, preferably between 616 and 984 mg / mL, of the aqueous phase, said aqueous phase preferably being water for injection (WFI), saline, phosphate buffered saline or citrate buffered saline; and c. 0.02 - 20 mg / mL, preferably 0.2 - 10 mg / mL, even more preferably 1.0 - 5.0 mg / mL of CMS, CMS being defined as a carbohydrate monosulphate fatty acid ester, wherein a) the carbohydrate is a monosaccharide, a disaccharide, a trisaccharide or a polysaccharide comprising 4 - 10 saccharide units, preferably a monosaccharide selected from allose, altrose, glucose, gulose, mannose, sorbose, iodose, talose, gentiobiose, fructose, galactose, inositol, ribose, arabinose, xylose and lysose, a disaccharide selected from sucrose, maltose, lactose, lactulose, cellobiose, trehalose, gentiobiose, turanose, isomaltulose and melibiose, a trisaccharide selected from raffinose, melezitose, maltotriose, isomaltotriose, raffinose, kestose and negerotriose, or a polysaccharide selected from an alpha-, beta- or gammacyclodextrin; and / or b) the carbohydrate is substituted on one hydroxyl group with a sulphate group (-SO3 ), said sulphate group optionally being substituted with a monovalent cation, said monovalent cation preferably being selected from H+, Na+, K+, Li+, NH4L NRH3L NR2H2L NR3H+, with R being selected, independently, on each occurrence, from a Cl - C6 saturated or unsaturated alkyl group; and / or the carbohydrate is substituted on at least 50 %, preferably at least 75 %, more preferably at least 80 % of the hydroxyl groups with a fatty acid, said fatty acid being defined as a C6 - C22linear or branched, saturated or unsaturated fatty acid, preferably a linear fatty acid having 6 - 18 carbon atoms; d. optionally 0.5 - 100 mg / mL, preferably 0.8 - 50 mg / mL, even more preferably 1.0 - 25 mg / mL, more preferably 1.0 - 10.0 mg / mL, most preferably 2.0 - 8.0 mg / mL of an emulsifier, said emulsifier preferably comprising polysorbate, a sorbitan ester, polyoxyethylene sorbitan ester, or polyethyleneglycol ester.

23. Use according to claim 22, wherein the CMS emulsion comprises an aqueous phase and between 1.0 and 10.0, preferably 1.5 - 5.0 mg / mL CMS, between 2.0 and 20.0, preferably between 3.0 and 10.0 mg / mL oil and preferably between 2.0 and 20.0, more preferably between 3.0 and 10.0 mg / mL emulsifier.

24. Use according to any of claims 21 - 23, wherein the CMS emulsion comprises oil in a quantity defined by the weight ratio CMS : oil, of 1 : 10 - 5 : 1, preferably 1 : 5 - 2 : 1, more preferably 1 : 1 - 1 : 3.

25. Use according to any of claims 21 - 24, wherein the vaccine comprises: a. a pneumococcal polysaccharide, preferably in a quantity of 1 - 8.0 pg polysaccharide per dose; b. a meningococcal antigen, preferably in a quantity of 5 - 100 pg per dose; c. a smallpox and / or monkey pox antigen, preferably in a quantity of at least 5xl07infectious units per dose; d. a diphtheria toxoid, preferably in a quantity of not less than 20 IU per dose; e. a tetanus toxoid, preferably in a quantity of not less than 40 IU per dose; f. a Bordetella pertussis antigen, preferably in a quantity of 3 - 50 pg per dose; g. a polio antigen, preferably in a quantity of at least 7 D-antigen units;h. a hepatitis B surface antigen, preferably in a quantity of 5 - 30 pg per dose; i. a Respiratory Syncytial Virus (RSV) antigen, preferably in a quantity of 50 - 150 pg per dose; j. a human papillomavirus antigen, preferably in a quantity of10 - 60 pg per dose; k. a hepatitis A antigen, preferably in a quantity of 1 - 100 pg per dose; l. an ebola antigen, preferably in a quantity of at least 72xl06plaque forming units; m. a dengue antigen, preferably in a quantity of 2.0 - 9.0 logio of the 50% cell culture infectious dose; n. an anthrax antigen; o. a cholera antigen; p. a rabies antigen; q. a rubella antigen.

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