Multivalent glycoconjugate vaccines

Abstract

The present invention refers to new conjugate antigens expressing built-in multiple epitopes and to polyvalent glycoconjugate vaccines and formulations containing the same. In addition, the present invention concerns the use of these vaccines in particular for the protection of the human population, and in particular for the protection of the paediatric population from pulmonary and systemic infections due to S. pneumoniae, N. meningitidis, H. influenzae, K. pneumoniae, M. tuberculosis, S. aereus, or from intestinal infections due to S. typhi, V. cholerae and E. coli. The present invention additionally refers to new polyvalent glycoconjugate vaccines for the protection from C. albicans and E. coli systemic and genitourinary infections or for the protection from M. bovis infections in veterinary medicine.

Claims

1. A vaccine formulation for use in humans or in a veterinary field for protection against pulmonary and systemic infections, said vaccine comprising at least one antigenic multivalent molecular construct for bacterial antigens of Streptococcus pneumoniae or Neisseria meningitidis said vaccine consisting of a basic unit comprising a helper-T dependent carrier protein covalently bound to a minimum of three carbohydrate structures which are capsular poly saccharides of different serological specificity by a linker comprising imine reduced bonds and amide bonds, wherein each carbohydrate structure comprises at least one of the repeating basic epitopes consisting of a minimum of five to twelve monosaccharide residues, where said carrier protein is selected from the group consisting of natural diphtheria mutant 6 protein CRM197, diphtheria toxoid, tetanus toxoid, Protein D from Haemophilus influenzae, Pneumonococcal surface proteins, Pneumonoccal toxin and derivatives thereof including tetanus toxoid derivatized by an adipic acid dihydrazide spacer characterized in that at least one mole or fraction thereof of protein carrier carries at least one mole or fraction thereof of each of the at least three different type-specific carbohydrate antigens in a physiologically acceptable vehicle, optionally together with an adjuvant or pharmaceutically acceptable excipients.

2. A vaccine formulation according to claim 1, wherein the dose of said vaccine ranges between 0.1 to 10 μg.

3. A vaccine formulation according to claim 1, wherein said adjuvant is chosen between a mineral adjuvant selected from aluminium phosphate, aluminium hydroxide; an organic adjuvant selected from squalene-based adjuvants and a biological adjuvant selected from monophosphoryl-lipid A and trehalose dicorynomycolate.

4. A vaccine formulation according to claim 3, wherein the amount of adjuvant ranges between 0.1-1 mg/dose.

5. A vaccine formulation according to claim 1, said formulation being adapted for subcutaneous, intramuscular, intracutaneous or transcutaneous administration.

6. A vaccine formulation according to claim 1 comprising one or more antigenic multivalent molecular constructs of Streptococcus pneumoniae selected among CRM197-3,6A,7F; CRM197-4,5,9V; CRM197-1,6B,14; CRM197-18C,19A,23F; CRM197-6C,19F,22F; CRM197-12F,15B,33F or among CRM197-3,6A,7F; CRM197-5,9V,19F; CRM197-1,14,19A; CRM197-22F,23F,33F; CRM197-4,6B,18C.

7. A vaccine formulation according to claim 2, wherein the dose of each type or group-specific carried carbohydrate antigen is 1.0 μg.

8. A vaccine formulation according to claim 3, wherein the amount of adjuvant is 0.5 mg/dose.

9. A broad-spectrum polyvalent vaccine formulation according to claim 1, comprising bacterial antigens of Streptococcus pneumoniae.

10. A broad-spectrum polyvalent vaccine formulation according to claim 1, comprising bacterial antigens of Neisseria meningitidis.

Description

(1) The present invention will be further illustrated according to preferred embodiments with particular reference to the enclosed figures, wherein:

(2) FIG. 1 shows a comparison between .sup.1H-NMR spectrum of native Ps 3 (grey) and activated Ps 3-DAB 50% Ox (black) of Ps 3 and Ps3-DAB derivative.

(3) FIG. 2 shows .sup.1H-NMR spectra of Ps 3-DAB 50% Ox, with diffusion filters. Spectrum in black 60% GRAD, spectrum in gray 2% GRAD assessing absence of free DAB in the derivative.

(4) FIG. 3 shows Ps 3-DAB derivative, 50% Ox, .sup.1H-NMR spectrum and quantization of DAB activation (2% on molar basis).

(5) FIG. 4 shows a comparison between the .sup.1H-NMR spectrum of native Ps6A (grey) and activated Ps6A-DAB derivative 50% Ox (black).

(6) FIG. 5 shows .sup.1H-NMR spectra of Ps 6A-DAB 50% Ox, with diffusion filters. Spectrum in black 60% GRAD, spectrum in gray 2% GRAD assessing absence of free DAB in the derivative.

(7) FIG. 6 shows Ps 6A-DAB derivative, 50% Ox, .sup.1H-NMR spectrum and quantization of DAB activation (2.3% on molar basis).

(8) FIG. 7 shows a comparison between the .sup.1H-NMR spectrum of native Ps7F (grey) and activated Ps7F-DAB derivative 10% Ox (black). The signal at 1.92 ppm, only present in the native Ps, is due to free acetate ion. OAc signal at 2.24 ppm remains the same after DAB activation.

(9) FIG. 8 shows .sup.1H-NMR spectra of Ps 7F-DAB 10% Ox, with diffusion filters. Spectrum in black 60% GRAD, spectrum in gray 2% GRAD assessing absence of free DAB in the derivative.

(10) FIG. 9: shows Ps 7F-DAB derivative 10% Ox, .sup.1H-NMR spectrum and quantization of DAB activation (1.7% on molar basis). Arrow indicates the reference signal at 5.65 ppm (α-proton of glucose or galactose) for quantization of the DAB activation.

(11) FIG. 10 shows GPC analysis of the antigenic multivalent molecular construct CRM197-3, 6A, 7F.

(12) FIG. 11 shows the GPC analysis of the four components just mixed together (CRM197+Ps 3-DAB+Ps 6A-DAB+Ps 7F-DAB) in order to show absence of any significant amount of complex formation among the single antigens.

(13) FIG. 12 shows SEC-HPLC analysis of the multivalent antigen, following purification on Sepharose 4B-CL, with specific reference to the profile of the carrier protein CRM197.

(14) FIG. 13 shows SEC-HPLC analysis of CRM197 as native protein, when mixed with Ps 3-DAB, Ps 6A-DAB and Ps 7F-DAB.

(15) FIG. 14 shows the SDS-PAGE analysis (9% Glycine buffer) showing the pattern of the purified multivalent antigen CRM197-3, 6A, 7F. Legend of the loaded samples: 1: CRM197 as reference; 2: Polydispersed MW of the purified multivalent conjugate antigen CRM197-3, 6A, 7F; 3: Mixture of CRM197+Ps3-DAB+Ps6A-DAB+Ps7F-DAB as reference.

(16) FIG. 15 shows immunoblot analysis (Western-blot) of the multivalent antigen as qualitatively revealed by type-specific serum polyclonal antibody. Legend of the loaded samples: 1: Multivalent conjugate CRM197-3, 6A, 7F (from line 2 of FIG. 14, above); 2: Mixture of CRM197+Ps3-DAB+Ps6A-DAB+Ps7F-DAB as reference.

(17) FIG. 16 shows the comparison between the % inhibition (expressed as MIC.sub.50) for CRM197 native and in its conjugated form as CRM197-Ps, 3, 6A, 7F.

(18) FIG. 17 shows the sigmoidal curve (log scale) referred to the graph of FIG. 16.

(19) FIG. 18 shows the comparison between the % inhibition (expressed as MIC) of Ps 3-DAB and CRM-3, 6A, 7F vs. native Ps3 showing Type 1 Antigenicity or Antigenic Identity of Ps3 following either DAB activation or conjugation.

(20) FIG. 19 shows the sigmoidal curve (log scale) referred to the graph of FIG. 18.

(21) FIG. 20 shows the comparison between the % inhibition (expressed as MIC.sub.50) of Ps 6A-DAB and CRM-3, 6A, 7F vs. native Ps 6A showing Type 1 Antigenicity or Antigenic Identity of Ps6A following either DAB activation or conjugation

(22) FIG. 21 shows the sigmoidal curve (log scale) referred to the graph of FIG. 20.

(23) FIG. 22 shows the comparison between the % inhibition (expressed as MIC.sub.50 of Ps 7F-DAB and CRM-3, 6A, 7F vs. native Ps 7F showing Type 1 Antigenicity or Antigenic Identity of Ps7F following either DAB activation or conjugation.

(24) FIG. 23 shows the sigmoidal curve (log scale) referred to the graph of FIG. 22.

(25) In the following experimental section the invention will be disclosed in more detail according to preferred embodiments, that should be considered not to be limitative for the scope of protection but merely for illustrative purpose.

EXAMPLES

Example 1: Synthesis of the Tetravalent Conjugate Antigen Comprising Polysaccharides 3, 6A, 7F of S. pneumoniae and the Carrier Protein CRM197

Chemical Activation of Ps 3, 6A, 7F to the Homologous Ps-DAB (Diamine Butyric Acid Derivative)

(26) 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, herewith included as a reference.

(27) Specific controls of such activation as well as the obtained characteristics of the activate Ps structures is here below reported using .sup.1H-NMR spectroscopy.

.SUP.1.H-NMR Analysis of Psi-DAB, Ps6A-DAB, Ps7F-DAB

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

(29) 2. NMR Equipment 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.Math.cm.sup.−1 is employed.

(30) 3. Setup of NMR Experiments 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. 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.

(31) 4. Fingerprinting of DAB-Activation Group —CH.sub.2—NH.sub.2 at 3.08 ppm Group —CH.sub.2—NH—CH.sub.2— at 3.17 ppm

(32) 5. % of DAB Activation on Ps 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%.

Derivatization of Ps3-DAB, Ps6A-DAB, Ps7F-DAB to Their Homologous Active Esters as Ps-DAB-MSE Derivatives

(33) 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.

Simultaneous Coupling of the Three Activated (Poly-Functional) Ps to the (Poly-Functional) Carrier Protein CRM197

(34) 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 herewith included as a reference.

(35) 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).

(36) 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).

(37) 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% (or 8 out of 40) of the amino groups available in the structure of CRM197 (as an example) in order for the protein to optimally conserve its antigenic repertoire.

(38) Based on experimental data, the coupling reaction is consistent with the following stoichiometry:
CRM197+4 Ps-DAB-derivatives.fwdarw.CRM197-(Ps).sub.3+Ps-DAB-derivatives

(39) Where the entity Ps-DAB-derivatives refer to the total of equal parts of each of the three type-specific carbohydrate 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-derivatives, as ruled by the equilibrium constant:

(40) $\begin{array}{c}{K}_{\mathrm{eq}}=\frac{\left[\mathrm{CRM}197-{\left(\mathrm{DAB}-\mathrm{Pa}\right)}_3\right]\left[\mathrm{Ps}-\mathrm{DAB}-\mathrm{derivatives}\right]}{{\left[\mathrm{CRM}197\right]\left[\mathrm{Pa}-\mathrm{DAB}-\mathrm{derivatives}\right]}^a}\\ =\frac{\left[\mathrm{CRM}197-{\left(\mathrm{DAB}-\mathrm{Pa}\right)}_3\right]}{{\left[\mathrm{CRM}197\right]\left[\mathrm{Pa}-\mathrm{DAB}-\mathrm{derivatives}\right]}^b}\end{array}$

(41) The chemical equation makes evidence for the complete glycosylation of the CRM197 carrier protein. The equation also shows that the conjugation reaction depends from the concentrations of both reagents, the nucleophile (CRM197 through the epsilon-NH.sub.2 groups of its Lys residues) and the electrophile (the carbonyl moiety of the ester groups of Ps-DAB-derivative) therefore being defined as S.sub.N2 reaction.

(42) The above considerations are consistent with the experimental observation that the highest yield in the glycosylation reaction obtained with CRM197 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.

(43) 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.). However, in the present case using CRM197 as carrier protein, polar protic solvents and polar aprotic solvents work very well when experimentally compared vis-à-vis.

(44) The temperature of the 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.

(45) 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 hydrogen uncoupling) or the use of heterologous and chemically complementary linkers that may serve to activate the Ps and the protein.

(46) 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 activated at their end-reducing group for then being coupled to the carrier protein, as the applicant did show earlier in another model of conjugate antigen in the above mentioned paper Porro M. et al. in Molecular Immunology, 23: 385-391, 1986, herewith enclosed as a reference.

(47) 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: Physical-Chemical Analysis of the Antigenic Multivalent Molecular Construct CRM197-3, 6A, 7F

(48) The GPC (Gel Permeation Chromatography) analysis has been employed to perform the physical analysis of the antigenic multivalent molecular construct of Example 1. FIG. 10 shows GPC analysis of the multivalent antigen as it is obtained from the conjugation reaction, before purification. The chromatogram comes from Sepharose 4B-CL equilibrated in 0.14M NaCl buffered at pH=7.50. Purification of the multivalent antigen is simply obtained by collecting and pooling the eluted fractions from Kd=0.00 to Kd=0.30.

(49) The technique of SEC-MALLS helps to define the dispersity (Ð) of the molecular system obtained, calculated using the equation Ð=M.sub.m/M.sub.n, where M.sub.m is the mass-average molar mass and M.sub.n is the number-average molar mass and also allows to determine some intrinsic properties of the above molecular system since the intensity of the light scattering angles carries information about the molar mass, while the angular dependence carries information about the size of the macromolecule. In fact, if a given macromolecule of mass M is made up of elements m.sub.i, then the basic light scattering equation shows that:

(50) $<r_g^2>=\underset{i}{.Math.}{r}_i^2{m}_i/\underset{i}{.Math.}{m}_i=\frac{1}{M}\underset{i}{.Math.}{r}_i^2{m}_i$

(51) where r.sub.i is the distance of element m.sub.i from the center of mass of the molecule of total mass M. According to this equation, the relationship between mass, size, and the quantities measured is defined.

(52) The following Table 1 shows the characterization of the dispersed molecular mass of the above purified multivalent antigen (fractions in the range Kd=0.00−0.30) analyzed by SEC-MALLS.

(53) TABLE-US-00001 TABLE 1 Upper Mass Average Mass Lower Mass (g/mol) (g/mol) (g/mol) 5.92 × 10.sup.6 9.67 × 10.sup.5 2.69 × 10.sup.5 (66.4%) (26.6%) (7.0%)

(54) The experimental data collected by SEC-MALLS show that the dispersed mass of the antigenic multivalent molecular construct encompasses the basic unit [CRM197-3, 6A, 7F].sub.n-1 for about 7% of the mass dispersion, and polymers of it with composition [CRM197-3, 6A, 7F].sub.n-3.6, for about 27% of the mass dispersion, and up-to [CRM197-3, 6A, 7F].sub.n-22 for the rest of the mass dispersion which represents the main form (66%) of the molecular construct in terms of product of reaction. 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 shown by H-NMR spectroscopy) and the polyfunctionality of the carrier protein (40 reactive amino groups/mole, available as 39 Lysine residues+1 amino terminal aa within the structure encompassing the whole 535 aa of the sequence).

(55) FIG. 11 shows the GPC analysis, as an example, on the same gel Sepharose 4B-CL of the four components just mixed together (CRM197+Ps 3-DAB+Ps 6A-DAB+Ps 7F-DAB) in order to show absence of any significant amount of complex formation among the single antigens. Chemical methods for titration of the three Ps structures involved the analysis of uronic acid (type 3), phosphorous (type 6A) and hexosamines (type 7F) according to the requirements of the WHO guidelines.

(56) The following Table 2 shows the characterization of the dispersed molecular masses of the three mixed type-specific Ps-DAB derivatives as analyzed by SEC-MALLS, for reference.

(57) TABLE-US-00002 TABLE 2 Upper Mass Average Mass Lower Mass (g/mol) (g/mol) (g/mol) 2.75 × 10.sup.5 7.27 × 10.sup.4 1.81 × 10.sup.4 (17.0%) (70.5%) (12.5%) Considering the different BRU of the three Ps structures, the mean number ± SD of BRU/Ps is 112 ± 62

(58) FIG. 12 shows SEC-HPLC analysis of the multivalent antigen, following purification on Sepharose 4B-CL, with specific reference to the profile of the carrier protein CRM197.

(59) The following experimental conditions were used in the SEC-HPLC analysis:

(60) Column: Phenomenex, Biosep-SEC-S3000, 300×7.80 mm (Vo 6.92 min.; Vt 12.5 min.)

(61) MW Sizing range: 700 K-5 K

(62) Eluent: NaCl 0.14M+NaH.sub.2PO.sub.4 0.05M pH 6.80

(63) Flow: 1 ml/min

(64) Detector: 280 nm (detection of the protein CRM197)

(65) FIG. 13 shows SEC-HPLC analysis of CRM197 as native protein, when mixed with Ps 3-DAB, Ps 6A-DAB and Ps 7F-DAB. Experimental conditions are the same as above reported for the analysis carried out in FIG. 12.

(66) In light of the above the conjugate under analysis is a polydispersed, monomeric to polymeric, molecular entity which contains the basic unit of the molecular construct reported in the chemical equation [CRM197-(Ps).sub.3], with a calculated average MW of ca. 2.7×10.sup.5 when considering the average MW (estimated by SEC-MALLS) of the poly-functional DAB-activated Ps structures (ca. 0.7×10.sup.5) and that of CRM197 (5.85×10.sup.4) accounting for 535 aa); accordingly, the several cross-linked units of such basic structure is reaching a MW of ca. 6 millions as evaluated by SEC-MALLS. The w/w ratio between the carrier protein and each of the three type-specific Ps is ca. 1.0; this w/w ratio yields an average molar ratio (R) protein/type-specific Ps of 1.0, corresponding to an average ratio of one mole of protein/mole of type-specific Ps, as well suggested by the chemical equation.

(67) 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 3: Immunochemical Analysis of the Antigenic Multivalent Molecular Construct CRM197-3, 6A, 7F

(68) The immunochemical analysis of the antigenic multivalent molecular construct CRM197-3, 6A, 7F was carried out by SDS-PAGE using the analytical conditions according to Laemmli U. K., Nature 227, 680-685 (1970), herewith enclosed as reference.

(69) FIG. 14 shows the SDS-PAGE analysis (9% Glycine buffer) showing the pattern of the purified multivalent antigen.

(70) FIG. 15 shows immunoblot analysis (Western-blot) of the multivalent antigen as qualitatively revealed by type-specific serum polyclonal antibody. The analytical conditions employed were according to Towbin H. et al., PNAS 76: 4350-4354 (1979). Silver staining according to Porro M. et al., Anal. Biochem. 118: 301-306 (1981). The serum polyclonal, Ps type-specific, antibodies are described in the below section dedicated to the inhibition-ELISA method.

Qualitative and Quantitative Determination of Each Antigenic Ps Component on the Basis of Inhibition-ELISA Using Polyclonal (or Monoclonal) Antibodies

(71) As well known since the birth of Immunochemistry, branch of the wider field of Immunology in the Thirties' of the past Century, capsular Ps antigens are composed of Basic Repeating Units (BRU) which may be constituted by homologous monosaccharides (e.g.: meningococcal Ps) as well as by more complex hetero-polysaccharide sequences involving bi/tri/tetra/penta/esa/epta-saccharide residues (e.g.: pneumococcal Ps). An average sequence of 5 (preferably 8) to 12 monosaccharide residues form the basic structural epitope of (Ps) carbohydrate antigens, which confer the due immunological specificity to each (Ps) structure. This size, typical of a single epitope within the human ABO system or of a sequence of epitope-repeating structures within complex bacterial capsular Ps, is coherent with the size of the binding site of an antibody (Kabat E. A., “The nature of an antigenic determinant” J. Immunol. 97: 1-11, 1966) and, on these basis, it was possible to describe the reactivity of a Ps structure toward a specific polyclonal population of antibodies, by inhibiting the binding reaction of the system Ps-Ab using different MW of the Ps polydispersed system, in order to document the relation existing between antigenicity of a Ps structure containing repeating BRU (thus forming repeated epitopes of identical antigenicity) and the specificity for it of the homologous polyclonal antiserum (Porro M. et al, Mol. Immunol. 22: 907-919, 1985); by comparison of the MIC50 of the various MW of the polydispersion of a given Ps, it was then possible to define the relative specificity of a polyclonal (or monoclonal) population of antibodies for such MW and finally calculating the relative concentration of the different Ps structures for a quantitative determination of it.

(72) By having a reliable immunochemical method for mapping and titering the Ps structures present in such a molecular construct, there are practical advantages of determining the qualitative and quantitative characteristics of such model of conjugates, over the chemical methods, especially in cases of Ps with very close structural features for their sequences, like in the case of type 6A and 6B or type 19A and 19F or in any other case where structural similarities among Ps antigens are present as in the case of type-specific Ps belonging to a given reference group (e.g.: Group 6 includes the type-specific Ps 6A, 6B, 6C, 6D; Group 19 includes the type-specific Ps 19A, 19B, 19C, 19F; Group 23 includes the type-specific Ps 23A, 23B, 23F). In fact, the exquisite specificity of an antibody can easily discriminate between such structural similarities without ambiguity and in short time, unlike chemical methods.

(73) The recent development of monoclonal antibodies to the Ps antigens of S. pneumoniae (Pride M. W. et al., Clin. And Vaccine Immunol. 19(8): 1131-1141, 2012) would further increase the potential of this powerful method of analysis.

(74) The comparison between chemical titration and immunochemical titration of carbohydrate antigens for testing their quantitative equivalence, is 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 accuracy and precision of the immunochemical method with respect to the chemical one in the analytical control of such a kind of molecular construct.

Inhibition-ELISA Protocol

(75) The following ELISA protocol was applied in order to determining the value of MIC.sub.50 of each of the three Ps-DAB derivatives and the protein CRM197 or the multivalent conjugate CRM197-3, 6A, 7F as inhibitors of the homologous reference reaction type-specific Polysaccharide-Antibody (Ps-Ab) or Protein-Antibody (Prot-Ab).

(76) Reference type-specific Ps-derivative (Ps-DAB) and the multivalent conjugate CRM197-3, 6A, 7F were prepared according to the mentioned process reported by Porro M. in claim 8 of the Patent EP1501542.

(77) Chemical methods for titration of the three Ps structures involves analysis of Uronic acid (type 3), Phosphorous (type 6A) and Hexosamines (type 7F) according to the requirements of the WHO guidelines. The inhibition reaction is based on the principle for a given carbohydrate structure, of a given molecular mass, of inhibiting the homologous reference reaction system according to the immunochemical equation:

(78) ##STR00004##

(79) So that the difference in reactivity between the reference reaction and the inhibited-one is representative for the different or identical specificity of the antibody population for the inhibitor. By using carbohydrate structures of different molecular mass, one can describe the sigmoidal curve typical of that specific reaction and calculate the MIC.sub.50 of the inhibitor for then comparing it with the one of the carbohydrate structure of reference and establishing the parameter of Antigenicity of the inhibitor (on qualitative basis) and Specificity of the antibody (on quantitative basis). All these concepts and the relative practical use are reported in the following publications, herewith incorporated as references: Berzofsky J. A. and Schechter A. N. Mol. Immunol., 18: 751-763 (1981); Porro M. et al. Mol. Immunol., 22: 907-919 (1985).

Method of Analysis (Illustrative)

(80) Stock Solutions: Ps-DAB or CRM197 at 1 mg/ml in PBS pH 7.2-7.4 PBS 1× (1 L) 8.0 g NaCl 0.31 g KH.sub.2PO.sub.4 2.06 g Na.sub.2HPO.sub.4.7H.sub.2O 0.16 g KCl Do not adjust pH TBS-Brij 0.1% (v/v) TBS 10× (11) 80 g NaCl 1.6 g KCl 0.94 g Tris 14.56 g Tris-HCl 33 ml Brij-35 (30% v/v) Stable at r.t. for 12 months Dilute 50 ml buffer to 500 ml MilliQuf water PBS-Tween20 0.05% (v/v) Goat Anti-Mouse IgG or IgM peroxidase labeled Phosphate-Citrate Buffer 0.05M pH 5.0 H.sub.2O.sub.2 30% (v/v) O-Phenilenediamine 1 mg/ml in Phosphate-Citrate Buffer 0.05M pH 5.0 H.sub.2SO.sub.4 3M

Procedure

(81) 1. Coating Plates (GREINER 65001 polystyrene plate SIGMA cod. M4436) Ps at 20 μg/ml PBS pH 7.4 37° C. 2 h+o.n. 4° C. CRM197 at 10 μg/ml PBS pH 7.4 o.n. 4° C. Coat 100 μl/well

(82) 2. Washing 5× with TBS-Brij 0.1% (v/v) (1 wash 20 sec.)

(83) 3. Pneumococcal reference polyclonal antisera from Statens Serum Institute, Copenhagen, DK (www.ssi.dk) in PBS-Tween 0.05% (v/v) 2 h 37° C. Final dilutions (as examples):

(84) a. Rabbit antiserum to group 3 1:100,000 v/v (Positive˜1.0 OD490.sub.nm)

(85) b. Rabbit antiserum to group 6A1:25,000 v/v (Positive˜1.0 OD490.sub.nm)

(86) c. Rabbit antiserum to group 7F 1:800,000 v/v (Positive˜1.0 OD490.sub.nm)

(87) 4. Unknown samples: the unknown samples are interpolated versus the reference sigmoidal regression curve obtained by the reference reaction.

(88) 5. Murineserum anti-CRM197, final dilution, 1:100,000 v/v (Positive˜1.0 OD490.sub.nm).

(89) TABLE-US-00003 TABLE 3 Inhibitor stock solution Anti-Ps Inhibitor 1 10 100 1 10 100 200 400 1 2 stock final/well ng/ml ng/ml ng/ml μg/ml μg/ml μg/ml μg/ml μg/ml mg/ml mg/ml solution  0.5 ng/ml 50 μl 50 μl    5 ng/ml 50 μl 50 μl   50 ng/ml 50 μl 50 μl  0.5 μg/ml 50 μl 50 μl    5 μg/ml 50 μl 50 μl   50 μg/ml 50 μl 50 μl  100 μg/ml 50 μl 50 μl  200 μg/ml 50 μl 50 μl  500 μg/ml 50 μl 50 μl   1 mg/ml 50 μl 50 μl

(90) 6. Incubation×Inhibition time: 15 min

(91) 7. Washing 5× with TBS-Brij 0.1% (v/v) (1.sup.st wash 20 sec.)

(92) 8. Goat Anti-Rabbit or anti Mouse IgG peroxidase labelled in PBS-Tween 0.05% (v/v) 2 h 37° C.

(93) 9. Washing 5× with TBS-Brij 0.1% (v/v) wash 20 sec.)

(94) 10. O-Phenilenediamine 1 mg/ml in Phosphate-Citrate Buffer 0.05M pH 5.0, H.sub.2O.sub.2; 0.03% (v/v)

(95) 11. After 5′ Stop the reaction with H.sub.2SO.sub.4 3M 50 μl/well

(96) 12. Read at OD 490 nm

(97) 13. Interpolate unknown values vs the reference sigmoidal line regression obtained by the reference reaction:

Calculation of % Inhibition

(98) $\frac{\mathrm{Inhibited}\mathrm{OD}\mathrm{Value}-\mathrm{Blank}\mathrm{OD}\mathrm{value}}{\mathrm{Positive}\mathrm{OD}\mathrm{Value}-\mathrm{Blank}\mathrm{OD}\mathrm{value}}$

(99) Thus, % Inhibited=100−(%)

Calculation of MIC.SUB.50

(100) This inhibitory concentration is determined at 50% of either the regression function or the related sigmoidal curve. Method's SD is within 20% of the mean value.

Results

(101) The results of the MIC50 for CRM197 native and in its conjugated form as CRM197-Ps, 3, 6A, 7F show that the conjugation reaction did not affect the antigenic features of CRM197 (Type 1 Antigenic Identity), as may be inferred from the analysis of the graphs set forth in FIG. 16-17. FIG. 17 illustrates the non-linear regression function of the sigmoidal curve.

(102) The results of the MIC50 for each of the three conjugated Ps-DAB derivatives are illustrated in the graphs set forth in FIGS. 18-23. FIGS. 19, 21 and 23 illustrate the non-linear regression function of the sigmoidal curve.

Example 4: Determination of the Concentration for the Carbohydrate Antigen in Either Activated or Multivalent Conjugated Form: Comparison of Chemical Titration vs. Immunochemical Titration

(103) Immunochemical titers are obtained according to the method reported above in Example 3 dedicated to the Inhibition-ELISA method; chemical titers are obtained according to the methods above reported in Example 2; 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. The reported values are the mean of several independent assays. Results on determination of quantitative equivalence of the two methods are summarized in the following Table 4.

(104) TABLE-US-00004 TABLE 4 Chemical Immunochemical determination determination* (μg/ml) (μ/ml) Ps1 1.0  0.9 (−10.0%) 2.0  2.3 (+13.1%) 4.0 3.7 (−7.5%) *Lowest amount Ps1 detected: 0.02 ug Ps3 0.80 0.91 (+13.4%) 1.60 1.77 (+10.6%) 3.20 3.31 (+3.4%)  6.40 6.71 (+4.8%)  *Lowest amount Ps3 detected: 0.01 ug Ps4 2.0 2.25 (+11.2%) 4.0 3.80 (−5.0%)  8.0 7.40 (−7.5%)  *Lowest amount Ps4 detected: 0.01 ug Ps5 3.1  3.3 (+6.1%) 6.25 5.7 (−8.8%) 12.5 10.8 (−13.6%) *Lowest amount Ps5 detected: 0.015 ug Ps6A 0.63 0.60 (−4.8%)  1.72 1.92 (+11.6%) 3.43 3.63 (+5.8%)  6.87 7.31 (+6.4%)  *Lowest amount Ps6A detected: 0.01 ug Ps6B 2.0  2.4 (+16.7%) 4.0 4.3 (+7.0%) 8.0  9.2 (+13.0%) *Lowest amount Ps6B detected: 0.10 ug Ps7F  1.34 1.43 (+6.7%)   2.68 3.00 (+11.9%)  5.37 5.47 (+1.9%)  10.75 11.07 (+3.0%)   *Lowest amount Ps7F detected: 0.01 ug Ps9V  3.8  4.2 (+9.6%)   7.5  6.4 (−15.0%) 15.0 12.2 (−18.7%) *Lowest amount Ps9V detected: 0.10 ug Ps14  3.4  3.8 (+10.6%)  6.8  6.5 (−5.0%)  13.5 16.2 (+16.5%) *Lowest amount Ps14 detected: 0.10 ug Ps18C  2.5  2.8 (+10.8%)  5.0 4.7 (−6.0%) 10.0  8.9 (−11.0%) *Lowest amount Ps18C detected: 0.02 ug Ps19A  3.8 4.1 (+8.7%)  7.5  6.5 (−13.4%) 15.0 13.3 (−11.4%) *Lowest amount Ps19A detected: 0.02 ug Ps19F  3.8  3.5 (−7.9.%)  7.5 8.3 (+9.4%) 15.0 17.0 (+11.8%) *Lowest amount Ps19F detected: 0.02 ug Ps23F  3.8  4.3 (+11.7%)  7.5  6.6 (−12.0%) 15.0 13.3 (−11.4%) *Lowest amount Ps23F detected: 0.02 ug CRM.sub.197  1.3 1.2 (−7.7%)  2.5  2.7 (+11.7%)  5.0 5.3 (+5.7%) 10.0 9.6 (−4.0%) *Lowest amount CRM.sub.197 detected: 0.10 ug *Lowest amount immunochemically detectable for the type-specific Ps in the assay conditions.

(105) Note: Physical-chemical determination of the protein CRM197 was performed by Folin reagent and/or amino acid analysis using hydrophobic reverse-phase HPLC to separate fluorescein-labeled amino acids following acid hydrolysis (Pico-Tag method by Millipore). SD for the physical-chemical determinations is within 10% of the mean values; SD for the immunochemical determinations is within 20% of the mean values, that is within the estimated SD of the day-by-day variation of the ELISA method and in agreement with the guidelines of the European Pharmacopoeia 5th Edition (2008) for the Pneumococcal Polysaccharide Conjugate Vaccine.

(106) 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 several (4 or 5 or 6 or more) molecular constructs in order to get the complete characterization of an exemplificative 12-valent or 15-valent or 18-valent vaccine.

Example 5: Immunological Analysis in a Murine Model of the Antigenic Multivalent Molecular Construct, as an Example

Vaccine Formulation

(107) Mean ratio Protein/each of the type-specific Ps: 1.1±0.1 (w/w).

Dose of the Molecular Construct CRM197-3, 6A, 7F

(108) The injected dose is 0.01 μg and 0.1 μg of each type-specific conjugated Ps, with and without AlPO.sub.4 as adjuvant at the fixed dose of 0.5 mg/dose (equivalent to ca. 0.120 mg of Alum). Adsorption of the multivalent molecular construct to the mineral adjuvant occurred at ≥80%, on weight basis, as estimated by ELISA.

(109) According to the stoichiometry of the multivalent conjugate, the total dose of CRM197 is ca. 0.01 μg in the case of the lowest dose of each type-specific conjugated Ps and ca. 0.1 μg in the case of the highest dose of each type-specific conjugated Ps.

(110) It is remarked that the dose injected of 0.01 μg Type-specific Ps is the lowest-one, immunogenic in mice, which is acknowledged by US-FDA and EMEA for the currently licensed pneumococcal conjugate vaccines, which use Aluminum Phosphate as adjuvant.

Animals

(111) Each group of animals containing 10 female Balb/c mice (alternatively CD1) and 6 female New Zealand white rabbits.

Route

(112) i.p. (mice) and s.c. (rabbits)

Immunization Schedule

(113) 0, 2, 4 weeks; bleeding at week 0, 2, 4, 6 (mice).

(114) 0, 4 weeks; bleeding at week 0, 4, 6 (rabbits).

(115) 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.

ELISA Titers

(116) Titers expressed as end-point reaction showing O.D.≥2.0 relative to the control reactions for each type-specific Ps and CRM197 or DT (Diphtheria Toxoid), the antigen immunogenically identical and in statistical correlation with CRM197 (Porro M. et al. J. Infect. Dis., 142 (5), 716-724, 1980). Sera pool dilutions are performed serially, in twofold fashion, starting from dilution 1/200.

MOPA (Functionality Assay)

(117) For testing Opsonic activity of the murine and rabbit polyclonal antibodies raised following immunization with the multivalent molecular construct, the MOPA-4 test (4-fold Multiplexed Opsono Phagocytic killing assay) was run, as recommended by WHO guidelines, using HL60 cells. Titers expressed as geometric mean of the end-point dilution showing ≥50% killing activity for each sera pool at each dose, as referred to a standard curve built in parallel for calculating the titer values of the various samples by linear interpolation.

Immunological Results

(118) Dose of 0.01 μg Ps/type-specific conjugated Ps. Geometric Mean Titers of IgG or IgM to type-specific Ps or to CRM197 in murine sera pool as determined by ELISA. SD is within ±25% of the reported Geometric Mean. MOPA titers are reported in parenthesis as calculated by linear interpolation in the assay procedure. Unless otherwise indicated, the statistical significance among sera titers (determined by t-test) was <0.01. Results are summarized in the following Table 5.

(119) TABLE-US-00005 TABLE 5 Without Adjuvant With Adjuvant Ag W0 W2 W4 W6 W0 W2 W4 W6 3 <200 <200 200   800 <200 200 800  3,200 <200 <200 <200   <200 (12) (124) 6A <200 <200 200   800 <200 200 400  3,200 <200 <200 <200   <200 (6) (135) 7F <200 <200 200   800 <200 200 1,600  6,400 <200 <200 200    400 (26) (248) CRM197 <200 <200 800 3,200 <200 1,600 12,800 25,600 <200 200 800    800

(120) Dose of 0.10 μg/type-specific conjugated Ps. Geometric Mean Titers of IgG or IgM to type-specific Ps or to DT in murine sera pool as determined by ELISA. SD is within ±25% of the reported Geometric Mean. MOPA titers are reported in parenthesis as calculated by linear interpolation in the assay procedure. Results are summarized in the following Table 6.

(121) TABLE-US-00006 TABLE 6 Type Without Adjuvant With Adjuvant Ps W0 W2 W4 W6 W0 W2 W4 W6 3 <200 200   800  6,400 <200 800 6,400 25,600 <200 200 400 800 (16) (254) (1,824) 6A <200 200   800  3,200 <200 800 3,200 12,500 <200 200 200 800 (22) (120) (1,150) 7F <200 200 1,600  3,200 <200 1,600 6,400 25,600 <200 200 400 800 (48) (168) (1,580) CRM197 <200 800 3,200 12,800 <200 6,400 25,600 102,400 <200 200 800 1,600

(122) The above Tables 5 and 6 show the anamnestic induction of biologically functional IgG isotype antibodies for each of the four components of the multivalent molecular construct.

(123) 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 effect of the mineral adjuvant is particularly evident at such low doses of the multivalent antigen, another feature of helper T-dependent antigens like proteins which do generate a stronger immune response taking further advantage from the antigen slow-release over time in the host's body.

(124) Furthermore, the effect of glycosylation on the carrier protein CRM197, as generally known for glycoproteins, can be beneficial for the improved resistance of this protein to proteolytic enzymes, since CRM197 is a fragile protein when exposed to the serine proteases widely present in mammalians (Porro M. et al., J. Infect. Dis., 142 (5), 716-724, 1980).

(125) The booster effect obtained against CRM197 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 diphtheria toxin, a well documented property of CRM197 in animal models (see the above bibliographic reference), in which case the multivalent antigen might be also used for the immunization of infants and young children in replacement of the diphtheria toxoid vaccine (present in the DTP vaccine) so that the antigenic burden of the paediatric vaccines in use could be further reduced. Finally, according to the immunological features of helper T-dependent antigens, IgM isotype antibody were neither significantly induced nor boosted by the carrier protein or the carried Ps of the multivalent molecular construct.

(126) Rabbit sera were specifically used to assess the four-fold increase of IgG isotype antibody ELISA titers to type-specific Ps, with the parallel increase of OPA titers, following the first booster dose of the molecular construct. The following results were collected, expressed as fold-increase of the sera GMT obtained with respect to the titers detected following the immunological priming dose and reported in the following Table 7.

(127) TABLE-US-00007 TABLE 7 IgG Ab to Ps OPA to Ps Type Ps (fold increase) (fold increase) 3 12 40 6A 18 48 7F 28 52

Example 6: Vaccine Formulation of a Quadrivalent Meningococcal Conjugate Vaccine (QMCV) and of an up-to 25-Valent Pneumococcal Polyvalent Conjugate Vaccine (PPCV)

(128) The composition/formulation of QMCV may be limited to one single molecular construct where one mole (or fractions of it) of carrier protein carries at least one mole (or fractions of it) of each of the four different carbohydrate structures. The related pondered amount of the multivalent antigen depends upon the selected MW of the activated carbohydrate structures which may vary from LMW haptens constituted by a few (8-12) monosaccharide residues or BRU (Basic Repeating Units) encompassing the respective basic epitopes [Porro M. et al. Molecular Immunology, 22: 907-919 (1985); Porro M. et al. Molecular Immunology, 23: 385-391 (1986)] and up-to HMW carbohydrate structures composed of 200 BRU or more for containing the repeated structure of the basic epitope.

(129) In such a case the amount of carrier protein per human dose, can be reduced to at least 25% of the amount present in a formulation which uses the association of single, group-specific, conjugates.

(130) The composition of PPCV depends from its polyvalent formulation. For instance, for a 15-valent vaccine containing selected 15 serotypes or for a 18-valent vaccine containing selected 18 serotypes, as above considered, only five to six entities of the multivalent antigenic molecular construct will be necessary, since in each of them, one mole (or fractions of it) of carrier protein will carry an average of one mole (or fractions of it) of each of three different type-specific carbohydrate structures. The related pondered amount of the multivalent antigen depends upon the selected MW of the activated carbohydrate structures which may vary from LMW haptens constituted by a few BRU (Basic Repeating Units) encompassing the respective epitopes (Arndt B. and Porro M. in: Immunobiology of Proteins and Peptides, Edited by M. Z. Atassi, Plenum Press, New York and London, pg. 129-148, 1991) and up-to HMW carbohydrate structures composed of 200 BRU or more for containing the repeating structure of the basic epitope. In any case the amount of carrier protein per human dose, can be reduced to at least 30% of the amount present in a formulation which uses the association of single, type-specific, conjugates.

(131) New emerging serotypes of S. pneumoniae according to the public available data on epidemiology and antibiotic resistance, are type 6C, 6D (Satzke C. et al., J. Clin. Microbiol., 48(11): 4298, 2010; Yao K H et al., Diag. Microbiol. Infect. Dis., 70(3):291-8, 2011); serogroups 11 (type 11A, 11B, 11C, 11F) (Richter S. et al., Clin. Infect. Dis., 48:23-33, 2009); Calix J. J. et al. J. Bacteriol. 193:5271-5278, 2011); serogroup 15 (type 15B and type 15C); type 23A, serogroups 33 (33F) and 35 (type 35B) (Swanson D., IDSA meeting, Boston, 2011); such antigen Ps might be likely included in a further up-dated broad-spectrum vaccine formulation prepared according to the molecular construct disclosed in the present Application.

(132) While the presently licensed 13-valent vaccine covers about 61% of IPD in children younger than 5 years, an up-dated formulation containing the Ps from the newly emerging types of S. pneumoniae, might well elevate the bar on coverage to 75-80%; in fact, it has been estimated that a formulation containing the 23-valent types of Ps today present in the polysaccharide-based vaccine, accounts for 88% of the bacteremic pneumococcal diseases which then cross-react with types of Ps causing an additional 8% of disease due to S. pneumoniae (source US-Center for Disease Control: www.cdc.gov.). Such kind of up-dated, very broad, formulations 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 an increased number of Ps antigens. For instance, when considering the dose of 2 μg of CRM197 (similar to Prevnar composition)/molecular construct, six molecular constructs carrying 18 Ps antigens would contain a total amount of 12 μg of protein, that is ca. 40% of that present in the 13-valent Prevnar vaccine, composed of single-conjugates of each type-specific Ps antigen.

(133) As specifically referred to an exemplified formulation of PPCV containing a 15-valent formulation which includes nowadays the most prevalent, epidemiologically significant, type-specific capsular polysaccharides of S. pneumoniae, the following molecular constructs have been synthesized and analyzed as an extended exemplification of the preferred embodiments, according to the methods reported above in Example 1, 2 and 3 for the molecular construct CRM197-3, 6A, 7F. The total amount of carrier protein exemplified in this exemplified 15-valent vaccine prepared and formulated according to the procedures reported in this application and defined by the stoichiometry of the resulting five 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 CRM197 carrier protein (MW=58.5 K) and ca. 1 ug of each of the three selected DAB-activated, type-specific, polysaccharide antigens (average MW=70.0 K based on two different criteria of analysis, that is estimating sizing by molecular filtration on calibrated filter membranes and estimating sizing by SEC-MALLS, in all cases using reference carbohydrate molecules like Dextrans of various MW).

(134) TABLE-US-00008 TABLE 8 Molecular Average (w/w) ratio Average molar construct CRM197/Ps ratio CRM197/Ps* CRM197-3,6A,7F CRM197/Ps3 = 1.20 1.44 CRM197/Ps6A = 0.98 1.17 CRM197/Ps7F = 1.09 1.30 CRM197-5,9V,19F CRM197/Ps5 = 1.03 1.23 CRM197/Ps9V = 0.93 1.11 CRM197/Ps19F = 1.05 1.26 CRM197-1,14,19A CRM197/Ps1 = 1.19 1.42 CRM197/Ps14 = 0.97 1.16 CRM197/Ps19A = 0.92 1.10 CRM197-22F,23F,33F CRM197/Ps22F = 1.00 1.20 CRM197/Ps23F = 1.14 1.37 CRM197/Ps33F = 1.11 1.32 CRM197-4,6B,18C CRM197/Ps4 = 1.18 1.41 CRM197/Ps6B = 1.19 1.42 CRM197/Ps18C = 1.08 1.20

(135) In the exemplified molecular constructs, the Mean of the (w/w) Protein/type-specific Ps ratio is: 1.07±0.097 (9.1%) corresponding to the Mean of the (mol/mol) ratio: 1.27±0.12.

(136) In the case when the carrier protein selected is CRM197 and the average MW of each Ps antigen is twice of the above reported value, or 140 K, the molar ratio protein to each Ps increases to an average of 2.5; in contrast, when the average MW of each Ps antigen is half of the above reported value, or 35 K, the molar ratio protein to each Ps decreases to an average of 0.64.

(137) 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. Accordingly, depending from the average MW of each type-specific Ps antigen and that of the protein carrier, 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).

(138) According to the above considerations, another way to change the stoichiometry, and therefore the molar ratio among the components (the carrier protein and each of the carried carbohydrate antigens) of the molecular construct, without changing the average MW of the Ps antigens selected, is the one which refers to the following exemplified molecular model. This model was synthesized by virtue of a modified stoichiometry in the reagents of the chemical reaction above reported, in favor of the protein component which was present in reaction at the reversed (w/w) ratio of the reaction reported in the above chemical equation, with each of the Ps-activated antigens, in order to make evidence of the flexibility of such chemical reaction which may also lead to a product showing the molar ratio between the carrier protein and the carried Ps antigens in favor of the former component. When referring to the vaccine dose related to the stoichiometry of this exemplified molecular construct, it still contains ca. 1.0 ug of CRM197 carrier protein (MW=58.5 K) but only ca. 0.3 ug of each of the three selected DAB-activated, type-specific, polysaccharide antigens (average MW=70.0 K).

(139) TABLE-US-00009 TABLE 9 Molecular Average (w/w) ratio Average molar construct CRM197/Ps ratio CRM197/Ps CRM197-3,6A,7F CRM197/Ps 3 = 2.85 3.41 CRM197/Ps 6A = 3.15 3.77 CRM197/Ps 7F = 2.70 3.22 CRM197-5,9V,19F CRM197/Ps 5 = 3.20 3.82 CRM197/Ps 9V = 2.90 3.47 CRM197/Ps 19F = 3.47 4.15 CRM197-1,14,19A CRM197/Ps 1 = 2.87 3.43 CRM197/Ps 14 = 3.15 3.77 CRM197/Ps 19A = 3.45 4.13 CRM197-22F,23F,33F CRM197/Ps 22F = 3.25 3.89 CRM197/Ps 23F = 2.80 3.35 CRM197/Ps 33F = 3.05 3.64 CRM197-4,6B,18C CRM197/Ps 4 = 2.90 3.47 CRM197/Ps 6B = 3.41 4.07 CRM197/Ps 18C = 3.10 3.71

(140) In the exemplified molecular constructs, the Mean of the (w/w) Protein/type-specific Ps ratio is: 3.08±0.24 (7.8%) corresponding to the Mean of the (mol/mol) ratio: 3.69±0.29.

(141) The above examples make evidence that different stoichiometries of synthesis, as addressed by the amount of reagents participating to the chemical equilibrium reported in the above chemical equation, 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, 15-valent formulations, containing five molecular constructs each carrying three type-specific Ps, the total amount of carrier protein CRM197 is ca. 5 μg, while the conjugated type-specific Ps are in the amount of ca. 1.0 and ca. 0.3 μg, respectively. Thus, at the dose of CRM197 equivalent to the one present in the Prevnar vaccine for each type-specific Ps conjugated, ca. 2 μg/dose, the total amount of CRM197 here exemplified in the 15-valent formulations would be ca. 10 μg or about 33% of the total amount present in the dose of the 13-valent Prevnar vaccine. Even in the hypothesis of a 23-valent formulation of a conjugate vaccine that would use the molecular model reported here, at comparable amount of protein/dose, the total amount of carrier protein would be significantly lower (ca. 50%) of the amount present in the today's reported 13-valent or 15-valent vaccines formulated by association of separate, single type-specific, conjugate antigens.

(142) 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, the optimal dose of the construct even when considering the different age-groups to be immunized by a broad-spectrum vaccine formulation. It may be here important to recall that past clinical studies had demonstrated that, in adults and toddlers, the immune system could not discriminate, in terms of immunogenicity, among different sizes of the conjugated Ps to the protein carrier CRM197 as well as among multi-point (cross-linked) or mono-point (not cross-linked) models of conjugates (Eby R. et al., in: Modern Approaches to New Vaccines, CSH Ed., 119-123, 1994) even though such studies are not publically available for infants in the range 2-24 months of age.

Example 7: Multivalent Molecular Construct with Built-in Epitopes Based on the Carrier Protein Tetanus Toxoid

(143) In addition to the carrier protein CRM197, other well established helper T-dependent carrier proteins may be used in a polyvalent formulation which considers the molecular construct disclosed in this application. As an example, the Applicant has here considered Tetanus Toxoid (TT) as carrier protein, an universal immunogen safely used in paediatric immunization since many decades ago. In contrast to the carrier protein CRM197, TT has never been formulated in a 13 or 15-valent conjugate vaccine, so that the safety of such a potential high-dose protein vaccine in humans remains to be eventually established. Accordingly, the use of the disclosed multi-valent molecular construct for a protein like TT represents a rational approach for limiting the amount of carrier protein in a 13 or 15-valent (or more) possible formulation based on such helper-T dependent carrier protein.

(144) TT is a derivative of the homologous toxin, chemically treated for having the toxin purposely detoxified for a human use of the immunogen. The MW of the purified toxoid is quite comparable to that of the toxin, that is 1.51×10.sup.5, encompassing 1,375 amino acids. However, among other features, the marked difference between toxoid and toxin resides in the amount of residual primary amino groups from the Lysine residues which remain in the toxoid structure after the chemical detoxification. An average of 50 reactive amino groups are about to be detected in the toxoid or about 50% of those originally present in the structure of the toxin, which work as nucleophylic groups in the coupling reaction with the activated bacterial Ps. When comparing the structure of TT to that of CRM197 in terms of capability to compete in the coupling reaction as nucleophylic reagent, one may determine that TT has ca. 50 amino groups/mole (MW=1.51×10.sup.5 for 1,375 aa) while CRM197 has 40 amino groups/mole (MW=58.5×10.sup.4 for 535 aa), so that the molar density of them (which we define as “molar nucleophile activity”) is 3.6% in TT and 7.5% in CRM197, showing a much higher capability of the latter protein to serve as nucleophylic reagent in a given coupling reaction. However, given the significant difference in the MW of the two proteins (basically a factor=2.6 in favor of TT) the molar ratios of the protein carrier, for each of the carried carbohydrate antigens selected in the molecular constructs, may result advantageous for TT when one is willing to limit the amount of carrier protein/dose in a polyvalent formulation. In fact, at comparable weight dose of the two carrier proteins, TT results to be 2.6 times lower than CRM197 on molar basis. In contrast, attention must be paid to the fact that its MW may limit the possibility to obtain a molar ratio TT/type-specific Ps with a value 1.0 for the optimal induction of T-helper dependency in the host's immune system.

(145) Here below, the Applicant reports on the physical-chemical features of such molecular construct using TT as carrier protein, synthesized according to the method above used for the CRM197-based molecular construct, with a stoichiometry in the reagents which allows the complete glycosylation of the carrier protein. Such a molecular construct can be considered as the basic component for a polyvalent formulation based on the TT carrier protein:

(146) TABLE-US-00010 TABLE 10 Molecular Average (w/w) ratio Average molar construct TT/Ps ratio TT/Ps TT-6A,9V,23F TT/Ps 6A = 2.08 0.96 TT/Ps 9V = 1.90 0.90 TT/Ps 23F = 2.15 1.00

(147) In the case of multivalent conjugates of N. meningitidis Ps and H. influenzae Ps, as additional examples, here below is a comparison between the carrier proteins CRM197 and TT highlighting the relevance of the carrier protein in the different constructs (synthesized according to different stoichiometries as allowed by the general chemical equation above reported), as related to their MW in the definition of the molar ratio (protein/Ps), when considering for the protein and the Ps the MW values above reported in Tables 8-9:

(148) TABLE-US-00011 TABLE 11 Molecular Average (w/w) ratio Average molar ratio construct TT/Ps or CRM/Ps TT/Ps or CRM/Ps TT-A,C,Hib TT/PsA = 1.79 0.83 TT/PsC = 2.05 0.95 TT/PsHib = 1.91 0.89 CRM197-A,C,Hib CRM197/PsA = 2.18 2.60 CRM197/PsC = 1.87 2.24 CRM197/PsHib = 1.95 2.33 CRM197-A,C,W135,Y CRM197/PsA = 0.78 0.93 CRM197/PsC = 0.97 1.16 CRM197/PsW135 = 0.75 0.90 CRM197/PsY = 0.88 1.05

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