Glycoconjugate vaccines comprising basic units of a molecular construct expressing built-in multiple epitopes for the formulation of a broad-spectrum vaccine against infections due to enteropathogenic bacteria

Abstract

The present invention refers to new glycoconjugate antigens expressing built-in multiple epitopes and to polyvalent glycoconjugate vaccines intended for the protection of mammalians, and particularly for the protection of the human population from enteropathogenic bacteria, such as the Gram-positive anaerobic bacterium Clostridium difficile and the Gram-negative bacteria Salmonella typhi, Escherichia Coli, Vibrio Cholerae, Shigella flexneri, Salmonella typhimurium, Salmonella enteritidis, Salmonella paratyphi A, Shigella sonnei, Shigella dysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter, Pseudomonas aeruginosa and/or from viral gastrointestinal infections due to human noroviruses.

Claims

1. A method for the protection of a subject from systemic and enteric infections due to at least one of the enteropathogenic bacteria selected among Clostridium difficile, Salmonella typhi, Escherichia coli, Vibrio cholerae, Salmonella enteritidis, Shigella flexneri, Shigella sonnei, Salmonella paratyphi A, Salmonella dysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter or a combination thereof, said method comprising administering an effective amount of a vaccine comprising an antigenic multivalent molecular construct consisting of basic units comprising the helper-T dependent carrier detoxified proteins selected between Enterotoxoid A and Cytotoxoid B from Clostridium difficile covalently bound to a minimum of three carbohydrate structures from enteropathogenic bacteria selected between bacterial polysaccharides or detoxified lipopolysaccharides of different serological specificity, wherein each carbohydrate structure comprises at least one of the repeating basic epitopes consisting of a minimum of five to twelve monosaccharide residues, wherein at least one mole of carrier protein is bound to at least one mole of each of the at least three carbohydrate structure or their molar sum to form carried carbohydrate structures of different serological specificity in a physiologically acceptable vehicle, optionally together with a pharmaceutically acceptable adjuvant or excipient.

2. A method for the protection of a subject from systemic and enteric infections due to at least one of the enteropathogenic bacteria according to claim 1 where said subject is a human.

Description

EXAMPLES

Example 1

(1) i) Synthesis of the Tetravalent Conjugate Antigen Comprising Polysaccharides of S. typhi(Vi), E. coli (K1) and V. cholerae (0139) with the carrier protein Enterotoxoid A;

(2) ii) Synthesis of the Tetravalent Conjugate Antigen Comprising Polysaccharides of S. typhi (Vi), E. coli (K1) and V. cholerae (0139) with the Carrier Protein Cytotoxoid B.

(3) Chemical Activation of the Three Ps to the Homologous Ps-DAB (Diamine Butyric Acid Derivative)

(4) This step has been performed according to the process disclosed by the Applicant in the Claim 1 (step A1) of the above mentioned patent EP1501542. Specific controls of such activation as well as the obtained characteristics of the activate Ps structures has been performed using .sup.1H-NMR spectroscopy as reported in the international application No. PCT/EP2014/051670.

(5) .sup.1H-NMR Analysis of Ps-DAB Derivatives

(6) 1. Solution of Ps and Ps-DAB Derivatives for NMR Analysis

(7) 3-4 mg of polysaccharide sample (PS) or PS-DAB is solved in 0.7 ml of D.sub.2O-phosphate buffer and transferred into a 5 mm NMR tube. The concentration of phosphate buffer prepared in D.sub.2O is 100 mM, pH=7. Trimethylsilylpropionic acid sodium salt (TSPA), (CH.sub.3).sub.3Si(CD.sub.2).sub.2COONa is used as an internal reference. The concentration of TSPA is 1 mM.

(8) 2. NMR Equipment

(9) High field NMR spectrometer (600 MHz) is used. A high resolution 5 mm probehead with z-gradient coil capable of producing gradients in the z-direction (parallel to the magnetic field) with a strength of at least 55 G. cm.sup.−1 is employed.

(10) 3. Setup of NMR Experiments

(11) After the introduction of the sample inside the magnet all the routine procedures have been carried out: tuning and matching, shimming, 90 degree pulse calibration. Presaturation can be used to suppress the residual HDO signal. For good presaturation the centre of the spectrum (O1) must be set exactly on the HDO signal (about 4.80 ppm), and good shimming is desirable as well.

(12) After adjustment of parameters for presaturation, the parameters of diffusion gradient experiments are checked. The stimulated echo pulse sequence using bipolar gradients with a longitudinal eddy current delay is used.

(13) 4. Fingerprinting of DAB-Activation

(14) Group —CH.sub.2—NH.sub.2 at 3.08 ppm

(15) Group —CH.sub.2—NH—CH.sub.2— at 3.17 ppm

(16) 5. % of DAB Activation on Ps

(17) % of DAB activation is in the range value of 0.5-5.0% moles DAB/moles BRU (Basic Repeating Unit of the Group-specific Ps) with an optimal molar range 1.5-3.0%.

(18) Derivatization of Ps Vi, Ps 0139, Ps K1 to their Homologous Active Esters as Ps-DAB-MSE Derivatives

(19) This step has been performed according to the process disclosed by the applicant in Claim 8 of the European Patent EP 1501542, herewith included as a reference.

(20) Simultaneous Coupling of the Three Activated (Poly-Functional) Ps to the (Poly-Functional) Carrier Protein Enterotoxoid A or Cytotoxoid B

(21) The chemical synthesis of the conjugate, also known as coupling reaction, has been performed according to the process disclosed by the applicant in Claim 8 of the European Patent EP1501542. The procedure, however, can be here considered as innovative because the three coupling reactions are simultaneously run, rather than proceeding in one coupling reaction at the time (or step-by-step process).

(22) This procedure may be preferred to the step-by-step coupling of each Ps-activated antigen for the simple reason of shorting the reaction time, therefore improving the efficiency of the reaction, provided that the three activated-Ps are in the condition to comparatively compete at the equilibrium for the coupling reaction (this feature include comparable average MW, comparable range of Ps-DAB activation and comparable stoichiometric ratios among the reacting groups of the protein and those of the activated Ps).

(23) The appropriate stoichiometry of reaction keeps in consideration the total amount of succinimidyl esters relative to the three Ps antigens activated and the amino groups of the carrier protein available. Stoichiometry is preferentially set as to consider the reactivity of no more than 20-25% of the amino groups available in the structure of Enterotoxoid A or Cytotoxoid B (as an example) in order for the protein to optimally conserve its antigenic repertoire.

(24) The coupling reaction of Enterotoxoid A or Cytotoxoid B (briefly indicated as Toxoid) with Ps-DAB derivatives (Ps-DAB) is consistent with the following stoichiometry:
Toxoid+4 Ps-DAB(MSE).fwdarw.Toxoid-(Ps).sub.3+Ps-DAB(MSE)

(25) Where the entity Ps-DAB(MSE) derivatives refer to the total of equal parts of each of the three type-specific Ps structures in reaction yielding a conjugate averaging 1 mole of protein for the total of 3 moles of type-specific Ps carried, plus the due excess of Ps-DAB(MSE) derivatives, as ruled by the equilibrium constant:

(26) $\mathrm{Keq}=\frac{\left[\mathrm{Toxoid}\text{-}{\left(\mathrm{Ps}\right)}_3\right]\left[\mathrm{Ps}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}{{\left[\mathrm{Toxoid}\right]\left[\mathrm{Ps}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}^4}=\frac{\left[\mathrm{Toxoid}\text{-}{\left(\mathrm{Ps}\right)}_3\right]}{{\left[\mathrm{Toxoid}\right]\left[\mathrm{Ps}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}^3}$

(27) The equation refers to the concentration of the total active esters (MSE) deriving from the sum of equal parts of the DAB-activated Ps antigens, which are in turn comparable to the amount of the DAB linker quantitated by .sup.1H-NMR spectrometry which is present in each activated Ps antigen (conversion rate of Ps-DAB to Ps-DAB(MSE) ≥98% on molar basis).

(28) The chemical equation makes evidence for the complete glycosylation of the Toxoid carrier protein. The equation also shows that the conjugation reaction depends from the concentrations of both reagents, the nucleophile (Toxoid through the epsilon-NH.sub.2 groups of its Lys residues) and the electrophile (the carbonyl moiety of the ester groups of Ps derivatives) therefore being defined as S.sub.N2 reaction.

(29) The above considerations are consistent with the experimental observation that the highest yield in the glycosylation reaction obtained with Toxoid as carrier protein has been 100% of the carrier protein and about 80% (w/w) of the Ps-DAB-derivatives present in reaction, with the remaining part of them being a low amount of uncoupled Ps-derivatives necessary for pushing to the right side the equilibrium.

(30) In this type of reactions, the solvent affects the rate of reaction because solvents may or may not surround the nucleophile, thus hindering or not hindering its approach to the carbon atom. Polar aprotic solvents, are generally better solvents for this reaction than polar protic solvents because polar protic solvents will be solvated by the solvent's hydrogen bonding for the nucleophile and thus hindering it from attacking the carbon with the leaving group. A polar aprotic solvent with low dielectric constant or a hindered dipole end, will favor S.sub.N2 manner of nucleophylic substitution reaction (preferred examples are: DMSO, DMF, tetrahydrofuran etc.).

(31) The temperature of reaction, which affects K.sub.eq, is the lowest compatible with the use of the solvent chosen, when considering that the reaction is a spontaneous one (therefore being exothermic) and therefore is generally set between a temperature of 4° and 20° C.

(32) In addition to the conjugation chemistry above detailed, other chemistries can be used to achieve the synthesis of the multivalent conjugate antigen; among these, the direct coupling of the protein (via reductive amination) to the oxidized Ps (via O-de-hydrogen uncoupling) or the use of heterologous and chemically complementary linkers that may serve to activate the Ps and the protein.

(33) Also, in addition to the strategy of using chemistries leading to obtain multivalent cross-linked protein-Ps conjugates via the poly-functionality of the protein and that of the Ps components, one may consider the synthesis of the presently disclosed antigenic multivalent molecular construct as based on oligosaccharides derived from capsular Ps or from Lipid A-deprived oligosaccharides of LPS, activated at their end-reducing group for then being coupled to the carrier protein, as the applicant showed in another model of conjugate antigen in the above mentioned paper Porro M. et al. in Molecular Immunology, 23: 385-391, 1986.

(34) Finally, the disclosed molecular construct might be thought to be prepared by enzymatic glycosylation in bacterial or yeast cells or other engineered living cells, using “ad hoc” DNA-recombinant techniques.

Example 2

(35) iii) Synthesis of the Tetravalent Conjugate Antigen Comprising LPS (Lipopolysaccharides) of S. enteritidis, S. paratyphi A and S. dysenteriae with the Carrier Protein Cytotoxoid B;

(36) iv) Synthesis of the Tetravalent Conjugate Antigen Comprising LPS (Lipopolysaccharides) of S. enteritidis, S. paratyphi A and S. dysenteriae with the Carrier Protein Enterotoxoid A.

(37) Chemical Activation of the Three LPS to the Homologous LPS-DAB Derivatives (Diamine Butyric Acid Derivative)

(38) The three LPS, were chemically derivatized in their O-antigen carbohydrate moiety to the corresponding -DAB derivatives (see the below scheme showing the DAB-activated area within the O-antigen carbohydrate moiety which is the most hydrophilic part of the LPS molecule). The “core” structure is difficult to be activated because is very close to the hydrophobic area (Lipid A), which is a quite kriptic structure responsible for the micelle-like structure of LPS, which is also responsible for the biological toxicity of LPS (e.g. local and systemic inflammation, TNF- and IL6-mediated, followed by pyrogenicity) as well as for the optimal expression of antigenicity and immunogenicity. In a preferred embodiment of the present Application, the biological toxicity of LPS is then selectively blocked through the high affinity binding with SAEP, which preserves such optimal features of LPS linked to its supramolecular, micelle-like, structure.

(39) The step of DAB-activation has been performed according to the process disclosed by the Applicant in the Claim 1 (step A1) of the above mentioned patent EP1501542. Specific controls of such activation as well as the obtained characteristics of the activate Ps structures has been performed using .sup.1H-NMR spectroscopy as reported in the international patent application No. PCT/EP2014/051670.

(40) .sup.1H-NMR analysis on the -DAB derivatives were conducted as above reported for Example 1.

(41) The schemem set forth in the Figure, represents the general LPS structure of Enterobacteriaceae with the located sites of DAB-activation (necessary for conjugation to the carrier protein) and the necessary biological detoxification, preferentially performed by SAEP (Synthetic Anti Endotoxin Peptide), which allows to achieve detoxification while LPS retaining its supramolecular, micelle-like, antigenic structure).

(42) Derivatization of LPS of S. enteritidis, S. paratyphi a and S. dysenteriae to their Homologous Active Esters as LPS-DAB-MSE Derivatives

(43) This step has been performed according to the process disclosed by the applicant in Claim 8 of the European Patent EP 1501542.

(44) Simultaneous Coupling of the Three Activated (Poly-Functional) LPS to the (Poly-Functional) Carrier Protein Enterotoxoid A

(45) The chemical equation reported above in Example 1, also applies to the conjugates of Toxoids and LPS-derivatives:
Toxoid+4 LPS-DAB(MSE).fwdarw.Toxoid-(LPS).sub.3+LPS-DAB(MSE)

(46) So that:

(47) $\mathrm{Keq}=\frac{\left[\mathrm{Toxoid}\text{-}{\left(\mathrm{LPS}\right)}_3\right]\left[\mathrm{LPS}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}{{\left[\mathrm{Toxoid}\right]\left[\mathrm{LPS}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}^4}=\frac{\left[\mathrm{Toxoid}\text{-}{\left(\mathrm{LPS}\right)}_3\right]}{{\left[\mathrm{Toxoid}\right]\left[\mathrm{LPS}\text{-}\mathrm{DAB}\left(\mathrm{MSE}\right)\right]}^3}$

(48) The equation refers to the concentration of the total active esters (MSE) deriving from the sum of equal parts of the DAB-activated LPS antigens, which are in turn comparable to the amount of the DAB linker quantitated by .sup.1H-NMR spectrometry which is present in each activated LPS antigen (conversion rate of Ps-DAB to Ps-DAB(MSE) ≥98% on molar basis).

(49) The chemical synthesis of the conjugate, also known as coupling reaction, has been performed according to the process disclosed by the applicant in Claim 8 of the European Patent EP1501542. The procedure, however, can be here considered as innovative because the three coupling reactions are simultaneously run, rather than proceeding in one coupling reaction at the time (or step-by-step process). This procedure may be preferred to the step-by-step coupling of each Ps-activated antigen for the simple reason of shorting the reaction time, therefore improving the efficiency of the reaction, provided that the three activated-Ps are in the condition to comparatively compete at the equilibrium for the coupling reaction (this feature include comparable average MW, comparable range of LPS-DAB activation and comparable stoichiometric ratios among the reacting groups of the protein and those of the activated LPS). The molecular constructs obtained in this way, however, result to be toxic because the Lipid A moiety of LPS is actively present in the molecular structure. In order to pursue and achieve the safe use of the Toxoid-LPS conjugate entity, the LPS structure must therefore undergo detoxification alternatively through cleaving out the Lipid A moiety, or by saturation of the Lipid A-binding site through a specific strategy that use the Synthetic Anti-Endotoxin Peptides (SAEP). The latter is the preferred embodiment in the context of the present invention (see next example 3).

Example 3: Preparation of Enterotoxoid A-Endotoxoid Conjugates and Cytotoxoid B-Endotoxoid Conjugates from their Homologous Enterotoxoid A-LPS (Endotoxin)/Cytotoxoid B-LPS (Endotoxin) Conjugates

(50) Endotoxoids are non-toxic antigens able to induce specific immunological activity against their homologous LPS which are the native main toxic antigens exposed on the surface of the Gram (−) bacteria.

(51) A comprehensive, publically available, textbook which is exhaustive on the many scientific aspects of Endotoxin antigens originating from Gram-negative bacteria is “Endotoxins” by Kevin L. Williams, Editor, Informa Health Care USA Inc., publisher, New York (2007).

(52) An Endotoxoid is a molecular entity composed of an equimolar complex of SAEP, Synthetic Anti Endotoxin Peptides, with the Lipid A moiety of LPS (Endotoxin):
Toxoid-(LPS).sub.3+3 SAEP.fwdarw.Toxoid-(Endotoxoid).sub.3

(53) An Endotoxoid, originating from a given species-specific (immunotype), Endotoxin (Lipopolysaccharide), is prepared according to the scientific concept reported by Rustici et al. (Science 259: 361-365, 1993) and in the previously disclosed molecular details reported in the U.S. Pat. No. 6,951,652 and in the U.S. Pat. No. 7,507,718.

(54) The immunological activity of an Endotoxoid involves polyclonal antibodies of the IgG (mainly) and IgM isotypes having biological activity (bactericidal effect) via the mechanism known in immunology as Opsonophagocytosis (OP, or antibody-mediated engulfing of bacteria in macrophages and PMC) and Direct Bactericidal (DB, antibody-mediated lysis of the bacterial cell wall), both mechanisms being mediated by activation of the complement pathway.

(55) Endotoxoids are helper-T dependent antigens in animal models but not yet experienced in human infants, where the immune system is not fully developed until an age over 2 years. For this reason, the conjugation to helper-T dependent carrier proteins like the two above reported protein Toxoids of C. difficile has been considered in the present Application for preparing the desired vaccine product.

(56) Accordingly, the conjugates of Enterotoxoid A or Cytotoxoid B with selected species-specific LPS (Endotoxin) have been reacted with SAEP2 (Rustici et al., Science 259: 361-365, 1993) in the conditions generally reported in the U.S. Pat. No. 7,507,718 (see pages 33-34 and Claim 17), in order to achieve the detoxification of the Toxoid-conjugated LPS so that the relative homologous Toxoid-conjugated Endotoxoids are formed.

(57) The following Toxoid-conjugated Endotoxoids have been prepared: Enterotoxoid A covalently conjugated to Endotoxoids of S. paratyphi A, S. dysenteriae, S. enteritidis; Cytotoxoid B covalently conjugated to Endotoxoids of S. paratyphi A, S. dysenteriae, S. enteritidis;

(58) CRM197 covalently conjugated to Endotoxoids of S. paratyphi A, S. dysenteriae, S. enteritidis as a well established helper-T dependent carrier protein useful in controlling the immunization experiments in animal models.

Example 4: Combination of the Tetravalent Conjugate Antigen Comprising Polysaccharides of S. typhi (Vi), E. coli (K1) and V. cholerae (0139) Conjugated to the Carrier Protein Enterotoxoid A, with the Tetravalent Conjugate Antigen Comprising LPS/Endotoxoids of S. enteritidis, S. paratyphi A and S. dysenteriae Conjugated to the Carrier Protein Cytotoxoid B

(59) The combination is prepared by associating the two kind of molecular models at the dose as appropriate for immunogenic studies in animal models below reported in the Example 8.

Example 5: Physical-Chemical Analysis of the Antigenic Multivalent Molecular Construct Comprising the Polysaccharides of S. typhi (Vi), E. coli (K1) and V. cholerae (0139) Conjugated to the Carrier Protein Enterotoxoid A or Cytotoxoid B

(60) The GPC analysis (Gel Permeation Chromatography) on Sepharose 4B-CL has been used to perform the physical analysis of the antigenic multivalent molecular construct of Example 1. Purification of the High Molecular Weight (HMW)-multivalent antigen is simply obtained by collecting and pooling the eluted fractions from Kd=0.00 to Kd=0.30.

(61) Polymers of the basic unit of the molecular construct are obtained as cross-linked molecular entities because of the polyfunctionality of the Ps antigens (about 2% of DAB activation, on molar basis, as evidenced by .sup.1H-NMR spectroscopy) and the polyfunctionality of the carrier protein (ca. 104 reactive amino groups/mole Toxoid A, as determined by TNBS reaction, remaining from the native 223 Lys residues of the Toxin A +1 amino terminal AA, within the structure encompassing the whole 2,710 AA of the sequence; ca. 85 reactive amino groups/mole Toxoid B, as determined by TNBS reaction, remaining from the native 156 Lys residues of the Toxin B+1 amino terminal AA, within the structure encompassing the whole 2,366 AA of the sequence).

(62) In light of the above, the conjugate under analysis appears as a polydispersed, monomeric to polymeric, molecular entity which contains the basic unit of the molecular construct reported in the chemical equation, with a HMW which derives from the basic polymerized unit encompassing the Enterotoxoid A (MW=3.08×10.sup.5) or Cytotoxoid B (MW=2.70×10.sup.5) and an average of MW=10.sup.5 for each of the three Ps/LPS antigens (or a total of ca. 3.0×10.sup.5) resulting in a comprehensive average MW of 6.10×10.sup.5 per basic unit; accordingly, the several cross-linked units of such basic structure is reaching several millions and are mainly eluted at the Vo of the Sepharose 4B-CL column.

(63) The w/w ratio between the carrier protein and each of the three type-specific Ps is ca. 3.6 (Table 1, below); this w/w ratio yields an average molar ratio (R) protein/type-specific Ps of ca. 1.0, corresponding to an average ratio of one mole of protein/mole of type-specific Ps, as well suggested by the chemical equation. Accordingly, the experimentally obtained, cross-linked, molecular entity responds to a molecular model constituted by several polymeric units of the basic unit just consisting of one mole of carrier protein carrying a total of three moles of type-specific Ps (one mole for each type-specific Ps).

Example 6: Immunochemical Analysis of the Antigenic Multivalent Molecular Construct Enterotoxoid A-PsVi, PsK1, Ps0139 or Cytotoxoid B-PsVi, PsK1, Ps0139

(64) The GPC purified molecular construct was analyzed by inhibition-ELISA for determining the serological specificity of the four serum different polyclonal antibodies (PAbs) and for determining the qualitative and quantitative presence of each antigen of the construct, as disclosed in the international patent application PCT/EP2014/051670.

(65) The comparison between chemical titration and immunochemical titration of carbohydrate antigens for testing their quantitative equivalence, was performed by the use of inhibition-ELISA, through the experimentally determined parameter MIC.sub.50 (Minimal Inhibitory Concentration of the selected carbohydrate antigen working as inhibitor of the homologous reference Ps-Ab reaction) in order to evaluate and correlate accuracy and precision of the immunochemical method with respect to the chemical one in the analytical control of such a kind of molecular construct.

Example 7: Determination of the Concentration for the Carbohydrate Antigen in Either Activated or Multivalent Conjugated Form: Comparison of Chemical Titration Vs. Immunochemical Titration

(66) Immunochemical titers are obtained according to the method reported above relative to the Inhibition-ELISA as compared to chemical titers obtained according to the methods reported in the specific sections of the international patent application PCT/EP2014/051670; immunochemical titers of unknown samples of each of the three carbohydrate-specific antigens, either in activated or conjugated form, were determined by interpolation on the linear part of a reference standard curve built by inhibition-ELISA using known, chemically titred, carbohydrate antigen amount.

(67) The same methodology described for the qualitative and quantitative immunochemical analysis of each molecular construct above reported, is then used for characterization of the final formulation of the polyvalent vaccine containing the association of the two molecular constructs, each constituted by a triad of Ps/LPS (Endotoxoid) conjugates of the two Toxoids of C. difficile used as carrier proteins, in order to get the complete characterization of an exemplificative 4-valent or 8-valent vaccine.

Example 8: Vaccine Formulation as Related to the Stoichiometry of the Multi-Valent Molecular Constructs

(68) Such kind of broad-spectrum formulations for an Enteric Vaccine can be safely prepared by the use of molecular constructs of the present invention, which allows a reduced use of protein carrier for carrying such a number of conjugated Ps and LPS (Endotoxoids) antigens. As specifically referred to an exemplified formulation of an Enteric Vaccine containing an 8-valent formulation which includes the most prevalent, epidemiologically significant, specific Ps and LPS/(Endotoxoids), the following molecular constructs (Table 1) have been synthesized and analyzed as an extended exemplification of the preferred embodiments, according to the methods reported above in the various Examples detailing the molecular constructs based on Enterotoxoid A and Cytotoxoid B carrying Ps/LPS (Endotoxoids), as well as the combination of the two.

(69) The total amount of the two carrier protein Toxoids exemplified in this 8-valent Enteric Vaccine prepared and formulated according to the procedures reported in this application and defined by the stoichiometry of the resulting molecular constructs, each one expressing built-in multiple epitopes, is coherent with the following molar composition relatively to the dose of each molecular construct containing ca. 1 ug of each of the two carrier protein Toxoids (MW=308K and 270K, respectively) and ca. 0.3 μg of each of the three selected DAB-activated, type-specific, Ps/LPS (Endotoxoid) antigens (average MW=100K based on two different criteria of analysis, that is estimating the average sizing by molecular filtration on calibrated filter membranes and estimating sizing by GPC, in all cases using reference carbohydrate molecules like Dextrans of various MW).

(70) TABLE-US-00001 TABLE 1 Molecular Average weight ratio Average molar ratio Construct Toxoid/Ps Toxoid/Ps EnteroTox A for: Ps.sub.E. coli 3.30 1.08 Ps.sub.S. typhi 3.80 1.24 Ps.sub.V. cholerae 4.05 1.33 CytoTox B for: EndoTox.sub.S. enteritidis 3.65 1.36 EndoTox.sub.S. paratyphi A 3.01 1.12 EndoTox.sub.S. dysenteriae 3.90 1.45

(71) In the exemplified molecular constructs, the mean of the (w/w) ratio Protein to Ps/LPS is: 3.61±0.39 (10.8%) corresponding to the mean of the (mol/mol) ratio: 1.26±0.14 (11.1%).

(72) The concept of calculating and comparing the features of conjugate antigens on molar basis is fundamental because the immune system processes antigens on molar basis, as Nature does in each chemical or biochemical reaction of transforming matter, therefore referring to the antigen's MW.

(73) Accordingly, depending from the average MW of each type-specific Ps/LPS antigen and that of the protein carrier Toxoids, the molar ratios of conjugate antigens are subject to change by the selection of their antigen components. It is mostly preferred that molar ratios between carrier protein and each type-specific Ps antigen be equal to or higher than 1.0 for a likely optimal expression of helper T-dependency. In addition to this molar parameter, it is also important considering the average amount of covalent bonds interposed between the protein and each type-specific carbohydrate antigen, which parallels the activation rate of the type-specific polysaccharide, since this hybrid molecular region is the one experimentally suggested as responsible for the acquired helper T-dependent properties of a conjugate molecule (Arndt and Porro, 1991).

(74) It is however possible to synthesize the molecular constructs according to different stoichiometries of synthesis, as detailed in the international patent application PCT/EP2014/051670, by addressing the amount of reagents participating to the chemical equilibrium reported in the above chemical equation, which may lead to a molecular construct of different stoichiometry, where the amount of helper T-dependent carrier protein in the molecular construct can be optimally selected according to the optimal expression of immunogenicity of such molecular construct in the various age groups of the human population. In both, above exemplified, 4-valent to 8-valent formulations, containing one to two molecular constructs each carrying three type-specific Ps/LPS, the total amount of each carrier protein Toxoid is ca. 1 μg, while the conjugated type-specific Ps/LPS (Endotoxoid) are in the amount of ca. 0.3 μg, respectively.

(75) Accordingly, it is the purpose of the above reported embodiments to provide evidence of the fact that the disclosed multivalent antigenic molecular construct with built-in epitopes can be synthesized in a broad range of stoichiometric parameters in order to then properly define, in mammalian hosts and particularly in humans, the optimal dose of the construct even when considering the different age-groups (from infants to elders) to be immunized by such a broad-spectrum vaccine formulation.

(76) Table 2 below, shows different molecular models obtained for the above concept, by making use of the same chemical reaction of synthesis, although using different “ad hoc” chosen stoichiometries for the reagents participating to the equilibrium.

(77) Here below, are reported some considerations on the two Toxoids used in the present application, Enterotoxoid A and Cytotoxoid B, since they are (or may be) chemically-treated derivatives of the homologous Toxins. This historic procedure, used for historic vaccines like Tetanus Toxoid and Diphtheria Toxoid, is necessary for having the Toxins purposely detoxified for a safe human use as immunogens. In the present Application, we have considered the average MW of the purified Toxoids as being comparable to that of the Toxins from which they derive.

(78) However, among other features, the marked difference between Toxoids and Toxins resides in the amount of residual primary amino groups from the Lysine residues which remain in the Toxoid structures after the chemical detoxification. An average of 47% to 54% reactive amino groups are about to be detected in the Toxoids with respect to those originally present in the structure of the homologous Toxins, which work as nucleophylic groups in the coupling reaction with the activated Ps/LPS antigens. When comparing the structure of the two Toxoids to that of a consolidated, historic, carrier protein like CRM197, in terms of capability to compete in the coupling reaction as nucleophylic reagent, one may determine that Toxoid A has ca. 104 amino groups/mole (MW=3.08×10.sup.5 for 2,710 AA) while Toxoid B has ca. 85 amino groups/mole (MW=2.7×10.sup.5 for 2,366 AA), so that the molar density of them (which we define as “molar nucleophile activity”) is 3.84% in Enterotoxoid A and 3.60% in Cytotoxoid B, two parameters that are significantly lower than that calculated for CRM197 (7.47%) which does have a higher capability to serve as nucleophylic reagent in a given coupling reaction (as detailed in the international patent application No. PCT/EP2014/051670). However, given the significant difference in the MW of the two protein Toxoids (basically a factor=5.3 and 4.7 in their favor with respect to CRM197) the molar ratios of the protein carrier, for each of the carried carbohydrate antigens selected in the molecular constructs, may result advantageous for the Toxoids when one is willing to limit the amount of carrier protein/dose in a polyvalent formulation. In fact, at comparable weight doses of the two carrier protein Toxoids, they result to be about 5.0 times lower than CRM197 on molar basis. Accordingly, attention must be paid to the fact that the carrier MW is an important parameter affecting the physical-chemical features of the conjugates and may limit the possibility to obtain a molar ratio Toxoid/specific Ps/LPS with a value ≥1.0 for the optimal induction of T-helper dependency in the host's immune system. Table 2 lists all the molecular models synthesized for the work detailed in the present Application, representative of the various stoichiometries used for the purpose, which are dependent from: i) the MW of the carrier protein used; ii) the molar nucleophile activity of such carrier proteins (expressing the amount of —NH.sub.2 groups/mole of protein); iii) the average MW of the activated Ps/LPS antigens and, iv) the respective activation rate of the Ps/LPS antigens (DAB-MSE groups for then reacting with the —NH.sub.2 groups of the protein). The exemplified molecular models make evidence for the flexibility of the chemistry adopted and the fact that the carrier protein may be present in the conjugate entity in a broad variety of ponderal and molar ratios, above 1.0 and below 1.0. In particular, the molar ratio Protein/Ps ranged from at least 0.3 to 1.0 when considering each type-specific or group-specific Ps present in the glycoconjugate, and from at least 0.3 to 1.0 when considering the total of the three Ps, each Ps contributing for about one third to the total amount finally present in the glycoconjugate.

(79) TABLE-US-00002 TABLE 2 Molecular Average weight ratio Average molar ratio Construct Toxoid/Ps Toxoid/Ps EnteroTox A for: Ps.sub.E. coli 3.30 1.08 Ps.sub.S. typhi 3.80 1.24 Ps.sub.V. cholerae 4.05 1.33 Ps.sub.E. coli 1.05 0.34 Ps.sub.S. typhi 1.15 0.37 Ps.sub.V. cholerae 1.03 0.33 EndoTox.sub.S. enteritidis 3.35 1.09 EndoTox.sub.S. paratyphi A 3.00 0.97 EndoTox.sub.S. dysenteriae 3.20 1.04 EndoTox.sub.S. enteritidis 1.13 0.37 EndoTox.sub.S. paratyphi A 1.20 0.39 EndoTox.sub.S. dysenteriae 1.05 0.34 CytoTox B for: EndoTox.sub.S. enteritidis 3.65 1.36 EndoTox.sub.S. paratyphi A 3.01 1.12 EndoTox.sub.S. dysenteriae 3.90 1.45 EndoTox.sub.S. enteritidis 1.23 0.46 EndoTox.sub.S. paratyphi A 1.02 0.38 EndoTox.sub.S. dysenteriae 1.15 0.43 Ps.sub.E. coli 3.60 1.33 Ps.sub.S. typhi 3.45 1.28 Ps.sub.V. cholerae 3.85 1.43 Ps.sub.E. coli 1.25 0.46 Ps.sub.S. typhi 1.10 0.40 Ps.sub.V. cholerae 1.43 0.53

Example 9: Immunological Analysis in Animal Models of the Antigenic Multivalent Molecular Constructs of Enterotoxoid a and Cytotoxoid B (Originating from the Homologous Toxins of C. difficile) Carrying Polysaccharides (S. typhi, V. cholerae, E. coli) or LPS/Endotoxoids (S. paratyphi A, S. dysenteriae, S. enteritidis)

(80) The two kind of conjugates using the two protein Toxoids from C. difficile, have been experienced in a murine animal model for active immunization experiments. As helper-T dependent control immunogen, the homologous conjugates of CRM197 were used in parallel experiments.

(81) Vaccine Formulation for Ps-Conjugates

(82) Enterotoxoid A and Cytotoxoid B conjugates of PsVi, Ps0139 and PsK1 were combined. Stoichiometric features of the conjugates showed a mean ratio Protein/each of the type-specific Ps of 3.61±0.39 (w/w) as shown in Table 1, above.

(83) Vaccine Formulation for LPS/Endotoxoids-Conjugates

(84) Enterotoxoid A and Cytotoxoid B conjugates of LPS S. enteritidis, S. dysenteriae and S. paratyphi A were combined. Stoichiometric features of the conjugates showed a mean ratio Protein/each of the type-specific LPS/Endotoxoid of 3.61±0.39 (w/w) as shown in Table 1, above.

(85) Combined Broad-Spectrum Enteric Vaccine Formulation for Ps-Conjugates and LPS (Endotoxoids)-Conjugates Using the Carrier Proteins Enterotoxoid A and Cytotoxoid B

(86) Enterotoxoid A conjugates of PsVi, Ps0139 and PsK1 and Cytotoxoid B conjugates of LPS (Endotoxoids)S. enteritidis, S. dysenteriae and S. paratyphi A were combined for the purpose.

(87) Dose and Formulations of the Exemplary Vaccines

(88) According to the stoichiometry of the molecular constructs reported above in Table 1, the injected dose is ca. 1.0 μg for each Ps/LPS (Endotoxoid) conjugated present in each molecular construct and for each Toxoid (ca. 3.0 μg) contained in the Vaccine Formulation; the dose becomes ca. 6.0 μg of total protein amount when the Vaccine Formulation contains the combined Toxoids for the same or different triads of carried Ps/LPS (Endotoxoid) antigens (Broad-spectrum Vaccine); AlPO.sub.4 is used as adjuvant at the fixed dose of 0.5 mg/dose (equivalent to ca. 0.120 mg of Alum). Adsorption of each multivalent molecular construct to the mineral adjuvant occurred at ≥80%, on weight basis, as estimated by inhibition-ELISA.

(89) Animals

(90) Each group of animals selected for each of the below reported immunization experiments, contained 10 female Balb/c mice.

(91) Route

(92) i. p.

(93) Immunization Schedule

(94) 0, 2, 4 weeks; bleeding at week 0, 2, 4, 6.

(95) Control immunization with plain Ps antigens were omitted on the basis of the historical knowledge that highly purified Ps antigens are not significantly immunogenic in mammalians and do not “boost” IgG isotype antibodies following repeated injections of it.

(96) ELISA Titers

(97) Titers expressed as end-point reaction showing O.D. ≥2.0 relative to the control reactions for each type-specific Ps/LPS (Endotoxoid) and the two protein Toxoids. Sera pool dilutions are performed serially, in twofold fashion, starting from dilution 1/200.

(98) Immunological Results

(99) Geometric Mean Titers of IgG to specific Ps/LPS (Endotoxoid) or to each of the two Toxoids, in murine sera pool, as determined by ELISA. SD is within ±25% of the reported Geometric Mean. Unless otherwise indicated, the statistical significance among sera titers (determined by t-test) was <0.01. Results are summarized in the following Table 3 and 4.

(100) In Vitro Neutralization of the Homologous Toxins

(101) Performed as reported by Porro et al. (1980) for Diphtheria Toxin and as Pavliakova et al. (2000) for C. difficile Toxins.

(102) Table 3 illustrates the immunoresponse of mice to the molecular model involving Enterotoxoid A and Cytotoxoid B as carrier protein for Ps antigens of E. coli, V. cholerae, S. typhi.

(103) TABLE-US-00003 TABLE 3 Enterotoxoid A Cytotoxoid B Ps W0 W2 W4 W6 W0 W2 W4 W6 Vi <200 200 2,600 15,800 <200 200 2,200 18,900 K1 <200 200 3,200 12,400 <200 200 2,400 20,000 0139 <200 200 1,800 11,600 <200 200 1,200 14,800 Tox <200 2,800 25,800 84,400 <200 3,200 32,600 95,400

(104) Table 4 shows the immunoresponse of mice to the molecular model involving Enterotoxoid A and Cytotoxoid B as carrier for LPS/Endotoxoids antigens of S. enteritidis, S. paratyphi A, S. dysenteriae.

(105) TABLE-US-00004 TABLE 4 Enterotoxoid A Cytotoxoid B LPS(Endotoxoid) W0 W2 W4 W6 W0 W2 W4 W6 S. enteritidis <200 400 3,600 12,800 <200 200 1,800 10,400 S. paratyphi A <200 200 2,400 14,800 <200 400 3,600 16,400 S. dysenteriae <200 200 2,200 16,400 <200 400 2,800 14,200 Toxoid <200 3,400 28,200 66,400 <200 2,400 24,800 84,200

(106) The results depicted in the above Tables 3 and 4 show the anamnestic induction of biologically functional IgG isotype antibodies for each of the four components of the two multivalent molecular constructs (Toxoid-Ps and Toxoid-Endotoxoid multivalent conjugates).

(107) Particularly, any boosting activity on the immune system observed for the carrier protein is in parallel observed for each of the carried Ps antigens, typical and well known behavior of helper T-dependent antigens. The booster effect obtained against the two Toxoids and the biological activity of the induced anti-Toxoid antibodies also strongly supports the fact that the multivalent molecular construct has the potential to work as antigen in humans for the prevention of toxicity due to the homologous Toxins. The following results were collected, expressed as fold-increase in respect to pre-immunization titers, of the sera GMT obtained following the second booster dose and reported in the following Table 5 as anti-toxic titers.

(108) TABLE-US-00005 TABLE 5 Abs to homologous Toxin (fold increase for toxin Toxoid neutralization, in vitro) Enterotoxoid A 456 Cytotoxoid B 562 CRM197 824

(109) The above detailed results, although just focusing on some specific examples, support the preparation and use of a broad-spectrum enteric vaccine for inducing immunity in a mammalian host against the carrier proteins Enterotoxoid A and Cytotoxoid B of C. difficile as well as against the carried Ps of E. coli, V. cholerae, S. typhi and the carried Endotoxoids of S. paratyphi A, S. dysenteriae, S. enteritidis. Based on the above, the capsular Ps of C. difficile may be also considered as Ps antigens carried by the two Toxoids of the homologous pathogen, according to the detailed molecular construct.

(110) The formulation of a broad-spectrum vaccine as the one above reported in Examples 8 and 9, has objective advantages on a vaccine formulation which considers the simple and eventual association of each of the six different Ps/LPS (Endotoxoid) conjugates of each of the two Toxoid proteins:

(111) A) by using the molecular model with built-in multiple-epitopes one may actually reduce the amount of carrier protein present in the broad-spectrum formulation (e.g.: the use of just two triads of conjugates does reduce the amount of protein carrier to ⅓ or 33% of the amount of carrier protein present in the associated formulation of the six conjugates);

(112) B) the number of injections would be reduced to a total of 3 injections with an obvious saving of materials and resources in addition to the lower stress of the mammalian host involved (a minimum of 3 injections, one priming dose and two booster doses, for each of the six individual type-specific vaccines, would result in a total of 18 injections).

BIBLIOGRAPHY

(113) Arndt and Porro, Immunobiology of Proteins and Peptides, Edited by M. Z. Atassi, Plenum Press, New York and London, pages 129-148, 1991. Chan M. eta al. Scientific reports (www.nature.com), DOI 10.1038/srep11507 of Jun. 17, 2015. Dagan R. et al. Vaccine, 28:5513-5523 (2010) Donald R. et al. Microbiology, 159: 1254-1266, 2013. Endotoxins. Kevin L. Williams, Editor, Informa Health Care USA Inc., publisher, New York, 2007. European patent EP 1,501,542. U.S. Pat. No. 6,951,652. U.S. Pat. No. 7,507,718. Jones M. K. et al., Science, 346: 755-759, 2014. Lee L. H. and Blake M. S., Clinical and Vaccine Immunol., pg. 551-556 (2012) Libby J. M. et al., Infect. Immun. 36: 822-829, 1982. Lindesmith L C et al. (2015), PLoS Med 12 (3): e1001807. doi:10.1371/journal.pmed.1001807 Liverly D. M. et al., Infect. Immun. 47:349-352, 1985. Lowy I. et al., New England J. Med. 362: 197-205, 2010. Pavliakova D. et al., Infect. Immun., 68:2161-2166, 2000. International patent application WO2004/052394 A1. International patent application No. PCT/EP2014/051670. Porro M. et al. Molecular Immunology, 23: 385-391, 1986. Porro M. et al. J. Infect. Dis., 142:716-724,1980 Romano M. et al., Toxins, 6:1385-1396, 2014. Rupnick M. et al. Nat Rev Microbiol. 7(7):526-536, 2009. Rustici A. et al., Science 259: 361-365, 1993. Salcedo J. et al., Gut, 41:366-370-1997. Simor A. E. et al. Infection Control and Hospital Epidemiology, Vol. 23, No. 11,696-703, 2002. Sougioultzis S. et al., Gastroenterology, 128: 764 770, 2005.