VACCINE

专利摘要:
La présente invention se situe dans le domaine des vaccins à base de conjugués de saccharides capsulaires pneumococciques. De façon spécifique, la présente invention concerne des compositions immunogènes et des vaccins comprenant de la pneumolysine detoxifiée adsorbée sur du phosphate d'aluminium et un procédé amélioré pour l'adsorption de la pneumolysine détoxifiée sur du phosphate d'aluminium. Elle concerne en outre l'utilisation des compositions immunogènes et des vaccins dans le traitement ou la prévention d'une infection à Streptococcus pneumoniae.
公开号:BE1024489B1
申请号:E2017/5102
申请日:2017-02-20
公开日:2018-03-12
发明作者:Laurence Danielle Ghenne；Dominique Ingrid Lemoine；Frédéric Mathot；Florence Emilie Jeanne Françoise Wauters
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(30) Priority data:
02/22/2016 GB 1603029.8 (73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA
1330, RIXENSART
Belgium (72) Inventor (s):
GHENNE Laurence Danielle 1330 RIXENSART Belgium
LEMOINE Dominique Ingrid 1330 RIXENSART Belgium
MATHOT Frédéric 1330 RIXENSART Belgium
WAUTERS Florence Emilie Jeanne Françoise
1330 RIXENSART
Belgium (54) VACCINE (57) The present invention is in the field of vaccines based on conjugates of pneumococcal capsular saccharides. Specifically, the present invention relates to immunogenic compositions and vaccines comprising detoxified pneumolysin adsorbed on aluminum phosphate and an improved method for the adsorption of detoxified pneumolysin on aluminum phosphate. It further relates to the use of immunogenic compositions and vaccines in the treatment or prevention of a Streptococcus pneumoniae infection.
Figwe 1: Conipfeti on of adsorption
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Middle Classes & Energy
Publication number: 1024489 Deposit number: BE2017 / 5102
Intellectual Property Office International Classification: A61K 39/00 A61K 39/09 Date of issue: 03/12/2018
The Minister of the Economy,
Having regard to the Paris Convention of March 20, 1883 for the Protection of Industrial Property;
Considering the law of March 28, 1984 on patents for invention, article 22, for patent applications introduced before September 22, 2014;
Considering Title 1 “Patents for invention” of Book XI of the Code of Economic Law, article XI.24, for patent applications introduced from September 22, 2014;
Having regard to the Royal Decree of 2 December 1986 relating to the request, the issue and the maintenance in force of invention patents, article 28;
Considering the patent application received by the Intellectual Property Office on February 20, 2017.
Whereas for patent applications falling within the scope of Title 1, Book XI of the Code of Economic Law (hereinafter CDE), in accordance with article XI. 19, §4, paragraph 2, of the CDE, if the patent application has been the subject of a search report mentioning a lack of unity of invention within the meaning of §ler of article XI.19 cited above and in the event that the applicant does not limit or file a divisional application in accordance with the results of the search report, the granted patent will be limited to the claims for which the search report has been drawn up.
Stopped :
First article. - It is issued to
GLAXOSMITHKLINE BIOLOGICALS SA, Rue de l'Institut 89, 1330 RIXENSART Belgium;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a Belgian invention patent with a duration of 20 years, subject to payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: VACCINE.
INVENTOR (S):
GHENNE Laurence Danielle, c / o GlaxoSmithKline Biologicals s.a., Rue de l'Institut 89, 1330, RIXENSART;
LEMOINE Dominique Ingrid, c / o GlaxoSmithKline Biologicals s.a., Rue de l'Institut 89, 1330, RIXENSART;
MATHOT Frédéric, c / o GlaxoSmithKline Biologicals s.a., Rue de l'Institut 89, 1330, RIXENSART;
WAUTERS Florence Emilie Jeanne Françoise, c / o GlaxoSmithKline Biologicals s.a., Rue de l'Institut 89, 1330, RIXENSART;
PRIORITY (S):
02/22/2016 GB 1603029.8;
DIVISION:
divided from the basic application: filing date of the basic application:
Article 2. - This patent is granted without prior examination of the patentability of the invention, without guarantee of the merit of the invention or of the accuracy of the description thereof and at the risk and peril of the applicant (s) ( s).
Brussels, 12/03/2018, By special delegation:
BE2017 / 5102
VACCINE
Field of 1 ¹ invention
The present invention relates to immunogenic compositions and vaccines comprising detoxified pneumolysin adsorbed on aluminum phosphate and an improved method for the adsorption of detoxified pneumolysin on aluminum phosphate. It further relates to the use of immunogenic compositions and vaccines comprising detoxified pneumolysin adsorbed on aluminum phosphate in the treatment or prevention of infection with Streptococcus pneumoniae.
Context of the invention
Streptococcus pneumoniae (S. pneumoniae) is a Gram positive bacteria responsible for considerable morbidity and mortality (especially in infants and the elderly), causing invasive diseases such as bacteremia and meningitis, pneumonia and other non-invasive diseases, such as acute otitis media. About 800,000 children die each year from pneumococcal disease, especially in emerging countries (O-Brien et al. 2009 Lancet 374: 893-902). The increasing number of strains resistant to antibiotics (Linares et al. 2010 Cin. Microbiol. Infect. 16: 402-410) and the severity of pneumococcal diseases make vaccination the most effective intervention.
BE2017 / 5102
The major clinical syndromes caused by
S. pneumoniae are widely recognized and discussed in conventional medical textbooks (Fedson DS, Muscher DM.
In: Plotkin SA, Orenstein WA, editors. Vaccines.
4th edition. Philadelphia WB Saunders Co, 2004a: 529588). For example, invasive pneumococcal disease (IPD) is defined as any infection in which S. pneumoniae is isolated from blood or another normally sterile site (Musher DM. Streptococcus pneumoniae. In Mandell GL, Bennett JE, Dolin R (editors) Principles and Practice of Infectious diseases (5th ed.) New York, Churchill Livingstone, Chronic bronchopneumopathy
2001, p 2128-2147). Obstructive (COPD) is several conditions recognized as encompassing (obstruction of the respiratory tract, chronic bronchitis, bronchiolitis or disease of the small respiratory tract and emphysema) which often coexist (Wilson ei al., Eur. Respir. J. 2001; 17: 995- 1007). Patients suffer from COPD exacerbations which are often associated with increased shortness of breath, and often have increased coughing which can be productive of mucus or purulent sputum (Wilson, Eur Respir J 2001 17: 995- 1007). COPD is physiologically defined by the presence of an irreversible or partially reversible airway obstruction in patients with chronic bronchitis and / or emphysema (Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society Am J Respir Crit
Care Med. November 1995; 152 (5 Pt 2): S77-121). The
BE2017 / 5102 COPD exacerbations are often caused by a bacterial infection (eg pneumococcal) (Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-theart review. Clin Microbiol Rev. April 2001; 14 (2): 33663).
complement, immunogenic.
Streptococcus pneumoniae, also called pneumococcus, is encapsulated with a chemically bound polysaccharide that confers serotype specificity. There are more than 90 known serotypes of pneumococci, and the capsule is the main determinant of virulence for pneumococci, because the capsule not only protects the internal surface of bacteria against it, but it is itself weakly. -polysaccharide has been considered to be predictive of protection against invasive pneumococcal disease (Jodar et al. Vaccine, (21) 2003, p. 3264-3272). After initial approval of a 7-valent conjugate vaccine containing serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (VCP7), two pneumococcal conjugate vaccines (VCP) designed to expand coverage were authorized protein D of Haemophilus pneumococcus 10-valent (VCP10) contains serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F conjugated to protein D of
Non-typeable H. influenzae, plus serotype 18C conjugated to tetanus toxoid and serotype 19F conjugated to diphtheria toxoid. The 13-valent pneumococcal conjugate vaccine (VCP13) contains the VCP7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F) plus the serotypes
I, 3, 5, 6A, 7F and 19A, combined with the material having
The influenzae conjugate vaccine
BE2017 / 5102 cross-reactivity CRM197. An object of the present invention is to develop improved vaccines against
Streptococcus pneumoniae.
Pneumolysin (ply) is a 53 kDa thiolactivated cytolysin found in all strains of S. pneumoniae, which is released during autolysis and which contributes to the pathogenesis of S. pneumoniae. It is highly conserved with only a few amino acid substitutions appearing between the ply proteins of the different serotypes. Pneumolysin is a multifunctional toxin with distinct cytolytic (hemolytic) and complement activating activities (Rubins et al., Am. Respi. Cit Care Med, 153: 1339-1346 (1996)). The toxin is not secreted by pneumococci, but it is released during lysis of pneumococci under the influence of autolysin. Its effects include, for example, stimulation of the production of inflammatory cytokines by human monocytes, inhibition of eyelash beating on human respiratory epithelia, decrease in bactericidal activity and migration of neutrophils, and in erythrocyte lysis, which involves binding to cholesterol. The expression and cloning of wild type or native type pneumolysin are described in Walker et al. (Infect Immun, 55: 1184-1189 (1987)), Mitchell et al. (Biochim Brophys Acta, 1007: 67-72 (1989) and Mitchell et al (NAR, 18: 4010 (1990)).
The present invention detoxified adsorbed on having properties provides improved aluminum phosphate pneumolysin and method
BE2017 / 5102 improved for the adsorption of detoxified pneumolysin on aluminum phosphate. The present inventors have discovered that by mixing detoxified pneumolysin and aluminum phosphate within a specific pH range, a high rate of adsorption completion (greater than 85%) can be obtained, and it can be produces an adsorbed detoxified pneumolysin having desirable properties, for example in relation to particle size. The particle size of the adsorbed detoxified pneumolysin and the rate of adsorption may affect immunogenicity; therefore, a particle size <10 µm and a high adsorption rate (greater than 85%) is desirable. Furthermore, the present inventors have discovered that it is advantageous to pre-adsorb the detoxified pneumolysin on aluminum phosphate according to the process of the invention before mixing with other antigens. For example, the pre-adsorbed detoxified pneumolysin can be mixed with pre-adsorbed PhtD which has been adsorbed on aluminum phosphate under different conditions.
Brief description of the figures
Figure 1 - Evaluation of the completion of adsorption. Compare the completion of the adsorption of detoxified pneumolysin (dPly) on aluminum phosphate at pH and in different ratios of dPly / Al ³⁺ (from aluminum phosphate) (i) pH 5.5 to 6.1 and 1/1 ratio, (ii) pH 5.5 to 6.1 and 1/2 ratio, (iii) pH 5.5 to 6.1, 1/3 ratio, and (iv) pH 6.5
BE2017 / 5102 and 1/3 ratio. Two different batches of antigen were tested: E-DPLY-P14 and DPLYADA007.
Figure 2 - Antigenicity. Shows the recovery by Elisa for dPly adsorbed on aluminum phosphate at a pH of 5.5 to 6.1, dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/3. The bars correspond to the two different batches of antigen prepared according to the method of Example 1: the bars on the left correspond to dPly E-DPLY-P14 and the bars on the right correspond to dPly DPLYADA007. DPly was stored either for 2 weeks at 4 ° C (T2s4 ° C) or 1 week at 4 ° C followed by 1 week at 37 ° C (Tls4 ° C + ls37 ° C). Note: this figure has been corrected to show that the dPly
DPLYADA007 presented a 110% recovery by Elisa after 1 week to 4 ° C followed by 1 week at 37 ° C (Tls4 ° C + ls37 ° C). Figure 3 - Size of particles. Compared the percentage particles of dPly adsorbed on of
aluminum phosphate less than 10 pm at pH and in different ratios of dPly / Al ³⁺ (from aluminum phosphate): (i) pH 5.5 to 6.1 and ratio of 1/1 and ( ii) pH 5.5 to 6.1, ratio of 1/3. The bars from left to right correspond to T0 (time = zero), T7d4 ° C (7 days at 4 ° C), T7d37 ° C (7 days to 37 ° C), T10d4 + 6d37 ° C (10 days to 4 ° C and 6 days at 37 ° C) and T21d4 ° C (21 days at 4 ° C). Data for the two different batches of antigen are presented: dPly E-DPLY-P14 on the right and dPly DPLYADA007 on the left.
BE2017 / 5102
Description of 1 ¹ invention
The present invention provides an immunogenic composition comprising detoxified pneumolysin having a high rate of adsorption (greater than 85%) on aluminum phosphate. The present invention also provides an improved method for the adsorption of detoxified pneumolysin on aluminum phosphate.
Therefore, in the first aspect of the present invention there is provided an immunogenic composition or a vaccine comprising detoxified pneumolysin adsorbed on aluminum phosphate, in which / which more than 85% (suitably more than 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the detoxified pneumolysin is adsorbed on aluminum phosphate.
In another aspect, the present invention provides a method for the adsorption of detoxified pneumolysin on aluminum phosphate comprising the step of (i) mixing the detoxified pneumolysin and aluminum phosphate at a pH below 6 , 5 (for example, less than 6.4, less than 6.3, less than 6.2, less than 6.1), appropriately less than
pH 6.0, e.g. pH 5.0 at 6.2, pH 5.0 at 6, 1, pH 5.2 at 6.2, pH 5.2 at 6.1, pH 5.4 at 6.2, pH 5.4 at 6, 1, pH 5.5 at 6.1, pH 5.4 at 5.9, pH 5.5 at 5.9, pH 5.4 at 5.7, pH 5.5
at 5.7, or pH 5.4 at 5.6 (for example, pH 5.5). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the pH is pH 5.0 to 6.2, pH 5.0 to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 at 6.2, pH 5.4 at 6.1, pH 5.5 at 6.1, pH 5.4 at 5.9,
BE2017 / 5102 pH 5.5 to 5.9, pH 5.4 to 5.7, pH 5.5 to 5.7, or pH 5.4 to
5.6 does not include extreme values.
In another aspect of the invention there is provided a method for the treatment or prevention of Streptococcus pneumoniae infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition immunogen or vaccine of the invention.
In another aspect of the invention there is provided an immunogenic composition or a vaccine of the invention for use in the treatment or prevention of a disease caused by Streptococcus pneumoniae infection.
The term "fragment" as used in this specification is a smaller part than the whole which is capable of triggering a humoral and / or cellular immune response in a host animal, for example, a human being. Fragments of a protein can be produced using techniques known in the art, for example, by recombination, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be produced by elimination of one or more nucleotides from one end (for a terminal fragment) or from both ends (for an internal fragment) of a nucleic acid which codes for the polypeptide. Generally, the fragments comprise at least 10, 20, 30, 40 or 50 contiguous amino acids of the full length sequence. The fragments can be easily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 amino acids
BE2017 / 5102 from either or both of the C-terminal Net ends.
The term "conservative amino acid substitution" as used in this specification implies the substitution of a native amino acid residue with a non-native residue in such a way that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at this position, and without producing a decrease in immunogenicity. For example, there may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid changes in the sequence of a polypeptide (and corresponding changes in coding nucleotides) can produce polypeptides with functional and chemical characteristics similar to those of a parent polypeptide.
The term "deletion" as used in this specification is the elimination of one or more amino acid residues from the protein sequence. Generally, no more than about 1 to 6 residues (for example, 1 to 4 residues) are deleted at any site within the protein molecule.
The term "insertion" as used in this specification is the addition of one or more non-native amino acid residues to the protein sequence. Generally no more than about 1 to 6 residues
BE2017 / 5102 (for example, 1 to 4 residues) are inserted at any site within the protein molecule.
As used herein, the term "treatment" (including variations thereof, for example, "treat" or "treated") means any one or more of the following: (i) prevention of infection or reinfection, (ii) reducing the severity or eliminating symptoms, (iii) delaying the recurrence of symptoms, and (iii) substantially or completely eliminating the pathogen or disorder in a subject. Therefore, treatment can be done prophylactically (before infection) or therapeutically (after infection).
For the purposes of this invention, "treatment or prevention of exacerbations of COPD" or "reduction of the severity of exacerbations of COPD" refers to a reduction in the incidence or rate of exacerbations of COPD (for example, a reduction in the rate of 0.1, 0.5, 1, 2, 5, 10, 20% or more) or a reduction in the severity of exacerbations of COPD (for example, airway obstruction, chronic bronchitis, bronchiolitis or disease of the small respiratory tract and emphysema), for example within a group of patients immunized with the immunogenic compositions or the vaccines of the invention.
Pneumolysin
By “pneumolysin”, or “ply” or “Ply”, it is meant: native or wild type pneumolysin originating from the pneumococcus or a recombinant pneumolysin having the sequence of the native pneumolysin
BE2017 / 5102 or wild type. Expression and cloning of wild type or native type pneumolysin is known in the art. See, for example, Walker et al. (Infect Immun, 55: 1184-1189 (1987)), Mitchell et al. (Biochim Brophys Acta, 1007: 67-72 (1989) and Mitchell et al. (NAR, 18: 4010 (1990)). WO 2010/071986 describes wild type Ply, for example, SEQ ID NO: 2 to 42 (for example, SEQ ID NO: 34, 35, 36, 37, 41) Furthermore, document EP 1601689B1 describes methods for the purification of bacterial cytolysins such as pneumococcal pneumolysin by chromatography in the presence of detergent and of concentration high in salt. In one embodiment, native or wild type pneumolysin from the pneumococcus or a recombinant pneumolysin having the sequence of native or wild type pneumolysin is used to produce the detoxified pneumolysin. In one aspect, the pneumolysin used for producing the detoxified pneumolysin with the sequence of SEQ ID NO: 1 (SEQ ID NO: 34 from WO 2010/071986) In another aspect, the pneumolysin used to produce the detoxified pneumolysin has the sequence of SEQ ID NO: 2 (SEQ ID NO: 35 from document WO 2 010/071986). In another aspect, the pneumolysin used to produce the detoxified pneumolysin has the sequence of SEQ ID NO: 3 (SEQ ID NO: 36 of WO 2010/071986). In another aspect, the pneumolysin used to produce the detoxified pneumolysin has the sequence of SEQ ID NO: 4 (SEQ ID NO: 37 from WO 2010/071986). In another aspect, the pneumolysin used to produce the
BE2017 / 5102 detoxified pneumolysin has the sequence of SEQ ID NO: 5 (SEQ ID NO: 41 of document WO 2010/071986).
In one embodiment, the pneumolysin used to produce the detoxified pneumolysin comprises fragments and / or variants, having differences in the nucleic acid or amino acid sequences compared to a wild-type sequence (for example, SEQ ID NO: 1 to 5).
When fragments of pneumolysin are used, these fragments will be at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues in length. In one embodiment of the invention, the immunogenic fragments of pneumolysin comprise at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues of the full length sequence, said a polypeptide capable of triggering an immune response specific for said amino acid sequence. Native pneumolysin is known to consist of four major structural domains (Rossjohn et al. Cell. May 30, 1997; 89 (5): 685-92). These domains can be modified by elimination and / or modification of one or more of these domains. In one embodiment, the or each fragment contains exactly or at least 1, 2 or domains. In another embodiment, the or each fragment contains exactly or at least 2 or 3 domains. In another embodiment, the or each fragment contains at least 3 domains. The or each fragment can be identical to more than 50, 60,
BE2017 / 5102 of identity SEQ ID NO
70, 80, 90 or 100% to a wild type pneumolysin sequence.
According to the present invention, a variant of pneumolysin is a protein in which the native pneumolysin is mutated. The term "mutated" is used here to mean a pneumolysin which has undergone a deletion and / or an addition and / or a substitution of one or more amino acids (for example, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids). An amino acid substitution can be conservative or non-conservative. In one aspect, the amino acid substitution is conservative. Substitutions, deletions, additions and any combination thereof can be combined into a single variant as long as the variant is an immunogenic polypeptide. Variants of pneumolysin generally include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 98, or 99% amino acid sequence identity with a wild type pneumolysin sequence, by example, a wild type pneumolysin sequence disclosed in WO 2010/071986. In one embodiment, the variants of pneumolysin generally include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 96, 97, 98, or 99% of amino acid sequence with 1. In one embodiment, the variants of pneumolysin generally include any pneumolysin or any fragment of pneumolysin which 0, 90, 94, 95, 96, 97, 98, or 99% acid sequence shares at least amino identity with
BE2017 / 5102
SEQ ID NO: 2. In one embodiment, the pneumolysin variants generally include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 96, 97, 98, or 99% identity of amino acid sequence with SEQ ID NO: 3. In one embodiment, the pneumolysin variants generally include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 96, 97, 98 , or 99% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the pneumolysin variants generally include any pneumolysin or any fragment of pneumolysin which shares at least 80, 90, 94, 95, 96, 97, 98, or 99% of amino acid sequence identity with SEQ ID NO: 5. In one embodiment, the present invention comprises fragments and / or variants in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted, or added in any combination not. In another embodiment, the present invention includes fragments and / or variants which include a B cell or T cell epitope. Such epitopes can be predicted using a combination of two-dimensional structure prediction, for example, by using the PSIPRED program (from David Jones, Brunei Bioinformatics Group, Dept. Biological Sciences, Brunel University, Uxbridge UB8 3PH, United Kingdom) and of antigen index calculated on the basis of the method described by Jameson and Wolf (CABIOS 4: 181 -186 [1988]). Variants of pneumolysin are described, for example, in WO 04/43376,
BE2017 / 5102
WO 10/071986, WO 10/109325
46) and WO 10/140119 an embodiment, the
The invention includes one for example, those described
05/108580, WO 05/076696,
WO 05/108580, WO 05/076696,
(SEQ ID NO : 44, 45 and (SEQ ID NO : 50 and 51). In composition immunogenic of variant of pneumolysin, in the documents WO
WO 10/071986.
In one embodiment of the invention, the pneumolysin and its fragments and / or their variants, used to produce detoxified pneumolysin, have an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97 , 98, 99 or 100% identity with the wild type sequence for pneumolysin, for example, SEQ ID NO: 1, 2, 3, 4 or 5. In another embodiment of the invention, pneumolysin and its fragments and / or variants thereof include at least about 15, at least about 20, at least about 40, or at least about 60 contiguous amino acid residues of the wild-type sequence for pneumolysin, for example, SEQ ID NO: 1, 2, 3, 4 or 5.
Because pneumolysin is a toxin, it must be detoxified (i.e. made nontoxic to a mammal, for example, a human, when supplied in an appropriate dosage for protection) before it can be administered in vivo. As used herein, it should be understood that the term "detoxified pneumolysin" or "dPly" refers to detoxified pneumolysin suitable for medical use (i.e. non-toxic when supplied to a mammal, for example example, a human, at an appropriate dosage for protection). Pneumolysin can be detoxified
BE2017 / 5102 chemically and / or genetically. Therefore, the immunogenic compositions of the invention include detoxified pneumolysin (dPly).
The detoxification of pneumolysin can be carried out by chemical means, for example, using a crosslinking agent, such as formaldehyde, glutaraldehyde and a crosslinking reagent containing an N-hydroxysuccinimido ester and / or a maleimide group (for example , GMBS) or a combination thereof, see, for example, EP 1601689 B1, WO 04/081515, WO 2006/032499. Pneumolysin subjected to chemical detoxification can be a native or recombinant protein or a protein that has been genetically modified to reduce its toxicity (see below). The fragments and / or variants of pneumolysin can also be detoxified by chemical means. In one embodiment, the immunogenic compositions of the invention may include pneumolysin which has been chemically detoxified, for example, by treatment with formaldehyde. For example, pneumolysin can be purified and detoxified as described in document WO 2004/081515. Detoxification of pneumolysin using formaldehyde can be accomplished using formaldehyde in the presence of Llysine, for example, by treating purified pneumolysin (ply) with 50 mM L-lysine and 0.1% formaldehyde (w / v) for 21 days at 40 ° C.
Pneumolysin can also be genetically detoxified. Thus, the invention encompasses pneumococcal proteins which may be, for example,
BE2017 / 5102 mutated proteins (as defined here). In one embodiment, the molecule has undergone deletion or substitution from 1 to 15 or any subset thereof, for example, 10 to 15 amino acids. The mutated sequences can eliminate unwanted activities such as membrane permeation, cell lysis, and cytolytic activity against human erythrocytes and other cells, in order to reduce toxicity, while retaining the ability to induce protective antibodies and / or anti-pneumolysin neutralizers following administration to a human. The fusion proteins of pneumolysin or of fragments and / or variants of pneumolysin can also be detoxified by genetic means. For example, as described herein, a mutant pneumolysin protein can be modified to be biologically inactive while retaining its immunogenic epitopes, see, for example, WO 90/06951, Berry et al. (Infect Immun, 67: 981-985 (1999)) and WO 99/03884. Alternatively, a pneumolysin protein can be detoxified by three amino acid substitutions comprising T ₆₅ at C, G293 at C and C428 at A as described in WO 2010/071986. For example, one of SEQ ID NOs: 1 to 5 could be detoxified by three amino acid substitutions comprising Τβ ₅ at C, G293 at C and C428 at A. Another example of a genetically detoxified pneumolysin which can be used in the present invention is SEQ ID NO: 9 of document WO 2011/075823. In another aspect, the modified pneumolysin protein of the invention can be detoxified by amino acid substitutions such as
BE2017 / 5102 as described in Taylor et al. PLOS ONE 8 (4): e61300 (2013), for example A ₃₇₀ in E, W ₄₃₃ in E and / or L ₄₆₀ in E. Thus, in another embodiment, the immunogenic compositions of the invention can comprise pneumolysin which has been genetically detoxified. A combination of techniques can also be used to detoxify pneumolysin. For example, the immunogenic compositions of the invention may include pneumolysin which has been chemically and genetically detoxified.
In one aspect, the detoxified pneumolysin is conjugated to a saccharide, for example, a capsular saccharide of S. pneumoniae. For example, pneumolysin can be conjugated to a capsular saccharide of S. pneumoniae chosen from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,
15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In a particular aspect, pneumolysin can be conjugated to a capsular saccharide of S. pneumoniae of serotype 19A. In another aspect, the pneumococcal protein is unconjugated or present in the immunogenic composition in the form of a free protein.
Aluminum phosphate
Immunogenic compositions comprising an adjuvant of aluminum phosphate phosphate (including anhydrous and hydrated) are often practical reasons "A1PO ₄ ", well hydrated (hydroxyphosphates) can 1'invention aluminum. The forms both called for forms to be distinguished
BE2017 / 5102 of anhydrous ΙΆΙΡΟ4 by the presence of hydroxyl groups (Al (OH) χ (PO4) _y , for example, Al (OH) (PO ₄ )). In one aspect of the invention, aluminum phosphate is aluminum hydroxyphosphate (e.g.
Amorphous aluminum hydroxyphosphate). In another aspect of the invention, aluminum phosphate is aluminum orthophosphate (also known as "aluminum monophosphate"). Aluminum phosphate builders can be purchased from Brenntag, for example, aluminum phosphate gel builder.
Aluminum phosphate can be an insoluble aluminum phosphate precipitate (amorphous, semi-crystalline or crystalline) which can be prepared by mixing soluble aluminum salts and phosphoric acid salts, for example, sodium phosphate or potassium phosphate. In one aspect, the aluminum phosphate is amorphous (for example,
Amorphous hydroxyphosphate). Aluminum hydroxyphosphate is not a stoichiometric compound and its composition in hydroxyl and phosphate groups depends on the reagents and the precipitation conditions. The weight / weight (w / w) phosphate / aluminum (P / Al) ratio of an aluminum hydroxyphosphate adjuvant will generally be between 2/1 and 4/1, suitably between 2.5 / 1 and 3.5 / 1, or between 3/1 and 3.5 / 1. The aluminum content can be determined by atomic absorption spectrophotometry with a nitrous flame, see, for example, May et al. (1984) J. Biol. Stand. 12 (2): 175-83.
In one embodiment, the aluminum phosphate used in the process of the invention
BE2017 / 5102 comprises NaCl, suitably 0.8% to 1.0%, for example, 0.9% (w / w).
In one embodiment, the aluminum phosphate used in the process of the invention has a pH between 4.8 and 6.2. In another embodiment, the aluminum phosphate used in the process of the invention has a pH between 5.5 and 6.1. In another embodiment, the aluminum phosphate used in the process of the invention has a pH between 4.8 and 5.8. In another embodiment, the aluminum phosphate used in the process of the invention has a pH between 5.2 and 5.8.
In one embodiment, the aluminum phosphate used in the process of the invention is subjected to "additional washes" before adsorption of the dPly so that the concentration of phosphate ions is reduced below 10 mM (for example, 3 mM or less, 2.5 mM or less). For example, phosphate ions can be removed either by repeated centrifugation (for example, at least 3 times) and dilution (i.e., removal of the supernatant and resuspension of the pellet in physiological saline), either by diafiltration steps.
In another embodiment, the aluminum phosphate should be sterilized before adsorption of the antigen. In one aspect, the aluminum phosphate is sterilized by autoclaving. In another aspect, the aluminum phosphate is sterilized by irradiation, for example, using ultraviolet (UV) light.
BE2017 / 5102
Completion of the adsorption of a protein antigen (eg, dPly) on aluminum phosphate can be measured by measuring the supernatant (SN) of the samples centrifuged by the Lowry method and comparing the total amount of protein in the sample (measured before adsorption occurs or by desorption of the adsorbed antigen) to the amount that remains in the supernatant after centrifugation, as described in Example 2 here. This methodology is further described in Chapter 4 of Methods in Molecular Medicine, Vol. 42 (edited by D.T. O-Hagan) Vaccine Adjuvants Preparation Methods and Research Protocols. In one aspect, the present invention provides detoxified pneumolysin adsorbed on aluminum phosphate, where more than 85% (suitably more than 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99%) of dPly is adsorbed on aluminum phosphate. In one aspect of the invention, the completion of adsorption is measured on the day of the formulation (T0). In another aspect of the invention, the completion of adsorption is measured after 7 days at + 4 ° C (T7j4 ° C) after formulation. In another aspect of the invention, the completion of the adsorption is measured after 21 days at + 4 ° C (T21j4 ° C) after the formulation. In another aspect of the invention, the completion of adsorption is measured after 7 days under accelerated conditions, for example, 7 days at 37 ° C (7d37 ° C) after formulation. In another aspect of the invention, the completion of the adsorption is measured after 16 days under accelerated conditions,
BE2017 / 5102 for example, 10 days at 4 ° C followed by 6 days at 37 ° C (T10d4 ° C + 6d37 ° C) after formulation.
The size of the particles of a protein antigen (for example, dPly) adsorbed on aluminum phosphate, can be measured by DSL (static light scattering), for example, using a Hydro dispersing unit 2000μ il as it is described in Example 4 here (methods for determining particle size are further described in E. Lindblad, Immunology and Cell Biology (2004) 82: 497505). The intensity of the diffusion is a function of the molecular mass and the concentration. In one aspect, the present invention provides detoxified pneumolysin adsorbed on aluminum phosphate, where more than 80% (suitably more than 81%, 82%,%, 84%, 85%, 86%, 87% , 88%, 89% or 90%) of the particles of the detoxified pneumolysin adsorbed on aluminum phosphate have a size less than 10 μm. In one aspect, the detoxified pneumolysin is unconjugated detoxified pneumolysin. In another aspect, the detoxified pneumolysin is conjugated detoxified pneumolysin.
Method for adsorption
The present invention provides a process for the adsorption of detoxified pneumolysin on aluminum phosphate comprising the step of (i) mixing the detoxified pneumolysin and aluminum phosphate at a pH below 6.5 (e.g. less than 6.4, less than 6.3, less than 6.2, less than 6.1), suitably less than pH 6.0, for example, pH 5.0
BE2017 / 5102
at 6.2, pH 5, 0 at 6, 1, pH 5.2 at 6.2, pH 5.2 to 6.1, pH 5.4 at 6.2, pH 5, 4 at 6, 1, pH 5.5 at 6, 1, pH 5.4 to 5.9, pH 5.5 at 5, 9, pH 5, 4 at 5, 7, pH 5.5 at 5.7, or pH 5.4 to 5.6 (by example - r pH 5 , 5 ). Of way appropriate, the report of
dPly / Al ³⁺ (from aluminum phosphate) in step (i) is between 1 / 1.5 and 1 / 5.5, between 1 / 1.5 and 1/4, between 1/1 , 5 and 1 / 3.5, between 1 / 1.5 and 1 / 2.5, between 1/2 and 1 / 2.5 (for example, 1/2); or between 1 / 2.5 and 1 / 3.5, between 1/3 and 1 / 3.5 (for example, 1/3), or between 1/3 and 1/4 (for example, 1/3, 5) (w / w; weight / weight). In one aspect, dPly is adsorbed on aluminum phosphate in a ratio of 1 µg of dPly to 3 µg of Al ³⁺ (from aluminum phosphate). In one embodiment, the pH is 5.4 to 6.2 (for example, pH 5.5 +/- 0.1) and the ratio of dPly / Al ³ + is between 1 / 2.5 and 1 / 3.5 (e.g. 1/3) (w / w; weight / weight). In another embodiment, the pH is 5.4 to 6.2 (for example, pH 6.1 +/- 0.1) and the ratio of dPly / Al ³⁺ is between 1 / 1.5 and 1 / 3.5 (e.g., 1 / 2.5) (w / w; weight / weight). In another embodiment, the pH is from 5.4 to 6.2 (for example, pH 6.1 +/- 0.1) and the ratio of dPly / Al ³⁺ is between 1/3 and 1 / 4 (for example, 1 / 3.5) (w / w; weight / weight). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the ratio of dPly / Al ³⁺ (originating from aluminum phosphate) in step (i) is between 1 / 1.5 and 1 / 5.5, between 1/1, 5 and 1/4, between 1 / 1.5 and 1 / 3.5, between 1 / 1.5 and 1 / 2.5, between 1/2 and 1 / 2.5; or between 1 / 2.5 and 1 / 3.5, between 1/3 and 1 / 3.5, or between 1/3 and 1/4 (w / w; weight / weight) not including extreme values .
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Suitably, for conjugated detoxified pneumolysin, the ratio of the polysaccharide / Al ³⁺ (from aluminum phosphate) in step (i) is between 1/6 and 1/14, between 1/7 and 1 / 13, between 1 / 7.5 and 1 / 12.5, between 1/8 and 1/12 (for example, 1/10) (w / w; weight / weight). In another aspect, the conjugated dPly is adsorbed on aluminum phosphate in a ratio of 1 µg of polysaccharide to 10 µg of Al ³⁺ (from aluminum phosphate). In one embodiment, the pH is 5.4 to 6.2 (for example, pH 6.1 +/- 0.1) and the ratio of the polysaccharide / Al ³⁺ is between 1 / 7.5 and 1 / 12.5 (for example, 1/10) (w / w; weight / weight).
Suitably, step (i) is carried out at room temperature (18 to 24 ° C). Suitably, step (i) is carried out with stirring at a speed between 60 and 150 rpm, such as 120 to 140 rpm (for example, 130 rpm). Suitably, step (i) is carried out for a period of between 10 minutes and 2 weeks, for example, 10 minutes to 5 hours, 1 to 5 hours, or 2 to 3 hours. Unless otherwise indicated, these ranges include extreme values. In another embodiment, the pH is maintained (and further mixing continues) at that pH (i.e., the pH for adsorption) for a period of between 10 minutes and 2 weeks, by example, 10 minutes to 5 hours, 1 to 5 hours, or 2 to 3 hours not including extreme values. In one embodiment of step (i), the detoxified pneumolysin and the aluminum phosphate (and optionally a buffer) are mixed initially and
BE2017 / 5102 then (for example, after 5 to 15 minutes) the pH is adjusted to a pH of less than 6.5 (for example, less than 6.4, less than 6.3, less than 6.2, less than 6.1), suitably less than pH 6.0, for example, pH 5.0 to 6.2, pH 5.0 to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to 6.1, pH 5.5 to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7, pH 5.5 to 5.7, or pH 5.4 to
5.6 (for example, pH 5.5) (the pH for adsorption), followed by further mixing. The pH can be adjusted using sodium hydroxide (NaOH (aqueous)) and hydrochloric acid (HCl (aqueous)). Suitably, the pH is maintained) and further mixing continues) at that pH (i.e., the pH for adsorption) for a period between
10 minutes and 2 weeks, per example, 10 minutes to 5 hours, 1 at 5 hours, or 2 to 3 hours. Except indication opposite, these ranges include values extremes.
In another embodiment, the pH is maintained (and further mixing continues) at that pH (i.e., the pH for adsorption) for a period of between 10 minutes and 2 weeks, by example, 10 minutes to 5 hours, 1 to 5 hours, or 2 to 3 hours not including extreme values.
In one aspect of the invention, the method of the invention (i.e. adsorption of dPly, step (i)), is carried out in the presence of a buffer, such as a phosphate buffer (for example, NaK2). In one aspect, the concentration of the buffer (e.g. NaK2) is at least 1 mM (e.g. at least 1.5 mM, 2 mM,
2.3 mM, 3 mM, 4 mM) and is suitably at most 10 mM (for example, at most 9 mM, 8 mM, 7 mM, 6 mM,
BE2017 / 5102 mM). In another aspect, the concentration of the buffer (for example, NaK ₂ ) is between 1 mM and 5 mM, or between and 4 mM, or between 1 mM and 3 mM (for example, between mM and 3 mM), for example, between 2 mM and 2.4 mM, for example, 2 mM. The phosphate buffer, NaK ₂ , used in the adsorption of dPly can include NaH ₂ PC> 4 (monobasic sodium phosphate) and K ₂ HPC> 4 (potassium phosphate dibasic). Suitably, the buffer has a pH of 6.5 to 7.5 (for example, pH 7.15). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the buffer concentration (for example, NaK ₂ ) is between 1 mM and 5 mM, or between 1 and 4 mM, or between 1 mM and mM (for example, between 2 mM and 3 mM ) not including extreme values.
In one aspect of the invention, the process of the invention (i.e., adsorption of dPly, step (i)) is carried out in the presence of a sodium salt, for example, NaCl. In one aspect, the concentration of the sodium salt is between 20 and 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60 mM (for example, 55 mM). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the concentration of the sodium salt is between 20 and 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60 mM not including the extreme values.
In one aspect of the invention, the method further comprises step (ii) of adjusting the pH of the composition to a pH between 6 and 7 (for example, pH 6, 0 to 6.5, pH 6 , 0 to 6.3, pH 6.1). Step (ii) is carried out in an appropriate manner following step (i).
BE2017 / 5102
Suitably, step (i) is carried out at room temperature (18 to 24 ° C). Suitably, step (i) is carried out with stirring at a speed between 60 and 150 rpm (for example, 130 rpm).
The pH can be adjusted using NaOH and HCl.
Following steps (i) and (ii), the adsorbed dPly is maintained at a pH between 6 and 7 (for example, pH 6, 0 to 6.5, pH 6, 0 to 6.3, pH 6 , 1) for at least 7 days, suitably at a temperature between 2 and 8 ° C (maturation step). Therefore, the immunogenic compositions of the invention may have a pH between 6 and 7 (for example, pH 6.0 to 6.5, pH 6.0 to 6.3, pH 6.1).
In one embodiment, the adsorption of dPly on aluminum phosphate (with or without sodium salt or potassium salt) is carried out in the absence of other additives, for example, in the absence of histidine.
The present invention also provides a method of preparing an immunogenic composition of the invention comprising the method of the invention for the adsorption of detoxified pneumolysin on aluminum phosphate as described herein.
Immunogenic compositions
In one embodiment, the present invention provides an immunogenic composition comprising detoxified pneumolysin adsorbed on aluminum phosphate prepared by the method of the invention.
In one aspect, the present invention provides an immunogenic composition comprising detoxified pneumolysin adsorbed on aluminum phosphate, in
BE2017 / 5102 which more than 85% (suitably more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the detoxified pneumolysin is adsorbed on aluminum phosphate. In another aspect, the present invention provides an immunogenic composition in which more than 80% (suitably more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 % or 90%) of the particles of the detoxified pneumolysin adsorbed on aluminum phosphate have a size less than 10 μm. In one embodiment, the pH of the immunogenic composition is between pH 6 and pH 7 (for example, pH 6, 0 to 6.5, pH 6, 0 to 6.2, pH 6.1). The immunogenic compositions can be buffered at this pH, for example, using a phosphate buffer. In one aspect, the detoxified pneumolysin in the immunogenic composition is unconjugated detoxified pneumolysin. In another aspect, the detoxified pneumolysin in the immunogenic composition is conjugated detoxified pneumolysin.
The immunogenic composition of the invention (i.e., comprising adsorbed dPly), may also include a buffer, such as a phosphate buffer (eg, NaK2). In one aspect, the concentration of the buffer (e.g. NaK2) is at least 1 mM (e.g. at least 1.5 mM, 2 mM, 2.3 mM, 3 mM, 4 mM) and is so suitable for more than 10 mM (for example, more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM). In another aspect, the concentration of the buffer (for example, NaK2) is between 1 mM and 5 mM, or between 1 mM and 3 mM (for example, between 2 mM and 3 mM), for example, between 2 mM and
2.4 mM. The phosphate buffer, NaK2, used in
BE2017 / 5102
DPly adsorption can include NaELPCp (monobasic sodium phosphate) and K2HPO4 (dibasic potassium phosphate). Other buffers which may be used include histidine, sodium phosphate, potassium phosphate, carbonate, NaHCCb buffers. Other buffers that may be used also include maleate, succinate, tartrate and Tris-maleate buffers.
In one aspect of the invention, the immunogenic composition of the invention (i.e., comprising adsorbed dPly) may also include a sodium salt, for example, NaCl. In one aspect, the concentration of the sodium salt is between 20 and 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60 mM (for example, 55 mM). In another aspect, the concentration of the sodium salt is between 100 and 200 mM, 120 to 180 mM, 140 to 160 mM (for example, 150 mM). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the concentration of the sodium salt is between 20 and 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60 mM not including the extreme values. In another aspect, the immunogenic composition comprises less than 2 mg of Al ³⁺ / ml, suitably between 100 and 2000 pg of Al ³ + / ml, 500 to 2000 pg of Al ³ + / ml, 800 at 2000 pg of Al ³ + / ml, 800 to 1500 pg of Al ³ + / ml, 800 to 1200 pg of Al ³ + / ml, 1000 to 2000 pg of Al ³⁺ / ml, 1500 to 2000 pg of Al ³ + / ml or 1700 to 2000 pg of Al ³⁺ / ml (aluminum, Al ³⁺ ) in the form of aluminum phosphate. Unless otherwise indicated, these ranges include extreme values. In another embodiment, the immunogenic composition comprises
BE2017 / 5102 between 100 and 2000 pg of Al ³⁺ / ml, 500 to 2000 pg of Al ³⁺ / ml, 800 to 2000 pg of Al ³ + / ml, 800 to 1500 pg of Al ³ + / ml, 800 to 1200 pg of Al ³⁺ / ml, 1000 to 2000 pg of Al ³ + / ml, 1500 to 2000 pg of Al ³⁺ / ml or 1700 to 2000 pg of Al ³⁺ / ml ( aluminum, Al ³⁺ ) in the form of aluminum phosphate, not including extreme values.
In another aspect, the immunogenic composition includes water for injection (PPI).
The immunogenic compositions of the invention can be lyophilized or in aqueous form, that is to say, solutions or suspensions. The immunogenic compositions of the invention can be lyophilized in the presence of a stabilizing excipient such as sucrose or trehalose. The immunogenic compositions can be presented in vials, or they can be presented in ready-filled syringes.
The present invention also provides a method of preparing an immunogenic composition comprising detoxified pneumolysin, comprising the method of the invention.
Additional antigens
The immunogenic compositions of the present invention may include additional antigens capable of triggering an immune response against a human or animal pathogen. These additional antigens include, for example, additional antigens of S. pneumoniae, for example, protein antigens of S. pneumoniae. Such proteins can be used as carrier proteins, or they can be present in the form
BE2017 / 5102 of a free protein (unconjugated), or they can be present both as a support protein and as a free protein. When the additional antigen is a pneumococcal protein, the protein can be conjugated, for example, to a saccharide. In one embodiment, the immunogenic composition of the invention further comprises one or more unconjugated proteins of S. pneumoniae, for example, protein D of the polyhistidine pneumococcal triad (PhtD). In another embodiment, the immunogenic composition of the invention further comprises one or more conjugated proteins of S. pneumoniae, for example, protein D of the polyhistidine pneumococcal triad (PhtD) conjugate.
Additional Streptococcus pneumoniae antigens are either exposed on the surface, at least during part of the pneumococcal cell cycle, or are proteins that are secreted or released by the pneumococcus. In one embodiment, the antigens of S. pneumoniae are chosen from the following categories, such as proteins comprising a type II signal sequence motif of LXXC (where X represents any amino acid, for example, the family of the polyhistidine triad (PhtX)), choline binding proteins (eg, CbpX (family of choline binding proteins), PcpA (pneumococcal choline binding protein A)), proteins with a sequence motif type I signal (for example, SplOl), and proteins comprising an LPXTG motif (where X represents any amino acid, for example, Spl28, Spl30). The
BE2017 / 5102
LytA (NLytC), the preferred examples within these categories (or motifs) are the following proteins, or their immunologically functional equivalents. Thus, the immunogenic composition of the invention may comprise one or more proteins of S. pneumoniae chosen from the family of the polyhistidine triad (PhtX), the family of choline binding proteins (CbpX), the truncated forms of CbpX, the family of pneumococcal autolysins (LytX) (for example, acetylmuramoyl-l-alanine amidase), LytB, truncated forms of LytX, chimeric proteins truncated form of CbpX-truncated form of LytX, PspA (pneumococcal surface protein A), PsaA (pneumococcal surface adhesion protein A), Spl28, SplOl, Spl30, Spl25 and Spl33. In another embodiment, the immunogenic composition of the invention comprises 2 or more proteins chosen from the group consisting of the polyhistidine triad family (PhtX), the family of choline binding proteins (CbpX), the forms truncated CbpX, the LytX family, truncated forms of LytX, chimeric proteins (or fusions) truncated form of CbpX-truncated form of LytX, PspA (pneumococcal surface protein A), PsaA (surface adhesion protein pneumococcal A), and Spl28. In another embodiment, the immunogenic composition comprises 2 or more proteins chosen from the group consisting of the polyhistidine triad family (PhtX), the family of choline binding proteins (CbpX), the truncated forms of CbpX, the LytX family, the truncated forms of LytX, the
BE2017 / 5102 chimeric proteins (or fusions) truncated form of
CbpX-truncated form of LytX, and Spl28.
The Pht family (polyhistidine triad) includes the proteins PhtA, PhtB, PhtD, and PhtE. The family is characterized by a lipidation sequence, two domains separated by a proline-rich region and several histidine triads, possibly involved in binding to metals or nucleosides or enzymatic activity, (3 to 5) coiled regions, a conserved N-terminal end and a heterogeneous Cterminal end. It is present in all the strains of pneumococci tested. Homologous proteins have also been found in other streptococci and Neisseria. In one embodiment of the invention, the immunogenic composition comprises PhtD. However, it is understood that the terms Pht A, B, D, and E refer to proteins having sequences disclosed in the citations below as well as their variants which have a sequence homology which is at least 90% identical to the proteins described below, for example, amino acids 21 to 838 of SEQ ID NO: 4 from document WO 00/37105. In one embodiment, it is at least 95% identical and in another embodiment, it is 97% identical to the proteins described below, for example, amino acids 21 to 838 of SEQ ID NO: 4 of document WO 00/37105.
With regard to PhtX proteins, PhtA is disclosed in WO 98/18930, and it is also called Sp36. As noted here, this is a protein from the polyhistidine triad family
BE2017 / 5102 and it includes the type II signal motif of LXXC. PhtD is disclosed in WO 00/37105, and it is also called SpO36D. As noted here, it is also a protein of the polyhistidine triad family and has the type II signal motif LXXC. PhtB is disclosed in WO 00/37105, and it is also called SpO36B. Another member of the PhtB family is the C3 degradation polypeptide, such as disclosed in document WO 00/17370. This protein is also of the polyhistidine triad family and it has the type II signal motif LXXC. A preferred immunologically functional equivalent is the protein Sp42 disclosed in WO 98/18930. A trongue form of PhtB (a "trongue form" being part of a protein with an N-terminal and / or C-terminal deletion) (approximately 79 kD) is disclosed in WO 99/15675, laguelle is also considered as a member of the PhtX family. PhtE is disclosed in WO 00/30299 and is called BVH-3. When reference is made here to a long-lived Pht protein, it is meant that immunogenic fragments or fusions thereof of the Pht protein can be used.
In one embodiment, the S. pneumoniae antigen chosen from one or more members of the polyhistidine triad family is PhtD. The term "PhtD" as used herein includes the full length protein with the fixed signal sequence or the mature full length protein with the signal peptide (for example, 20 amino acids at the N-terminus)
BE2017 / 5102 eliminated, and its immunogenic fragments, variants and / or fusion proteins, for example, SEQ ID NO: 4 of document WO 00/37105. In one aspect, PhtD is the full-length protein with the signal sequence fixed, for example, SEQ ID NO: 4 from WO 00/37105. In another aspect, PhtD is a sequence comprising the full-length mature protein with the signal peptide (e.g., 20 amino acids at the N-terminus) removed, e.g., amino acids 21-838 of SEQ ID NO : 4 of document WO 00/37105. Suitably, the PhtD sequence includes an N-terminal methionine. The present invention also includes PhtD polypeptides which are immunogenic fragments of PhtD, variants of PhtD and / or fusion proteins of PhtD. For example, as described in documents WO 00/37105, WO 00/39299, US 6,699,703 and WO 09/12588.
When immunogenic fragments of PhtD proteins are used (separately or as part of a fusion protein), these immunogenic fragments will be at least about 15, or at least about 20, at least about 40, or d '' at least about 60 contiguous amino acid residues, for example, from an amino acid sequence of PhtD in document WO 00/37105 or WO 00/39299, as SEQ ID NO: 4 of document WO 00 / 37105. In one embodiment of the invention, the immunogenic fragments of the protein PhtD comprise at least approximately 15, at least approximately 20, at least approximately 40, or at least approximately 60 contiguous amino acid residues of the sequence represented by SEQ ID NO: 4 of WO 00/37105, where said
BE2017 / 5102 polypeptide is capable of triggering an immune response specific for said amino acid sequence. In one embodiment, the immunogenic composition of the invention comprises an immunogenic fragment of PhtD, for example described in documents WO 09/12601, WO 01/98334 and WO 09/12588. When immunogenic fragments of the PhtD proteins are used (separately or as part of a fusion protein), each immunogenic fragment optionally contains one or more histidine triad motifs of these polypeptides. A histidine triad motif is the part of the polypeptide which has the sequence HxxHxH where H represents histidine and x represents an amino acid other than histidine. In one embodiment of the present invention, the or each immunogenic fragment contains exactly or at least 2, 3, 4 or 5 histidine triad motifs (optionally, with the native sequence of PhtD between the 2 or more triads, or a intra-triad sequence) where the immunogenic fragment is more than 50, 60, 70, 80, 90 or 100% identical to a native pneumococcal intra-triad PhtD sequence (for example, the intra-triad sequence represented by SEQ ID NO: 4 of document WO 00/37105). The immunogenic fragments of the PhtD proteins optionally contain one or more supercoiled regions of these polypeptides. An overwound region is a region predicted by the "Coils" algorithm of Lupus, A et al. (1991) Science 252; 1162-1164. In one embodiment of the present invention, each immunogenic fragment contains exactly or at least 2, 3 or 4 supercoiled regions. In one embodiment of
BE2017 / 5102 the present invention, the or each immunogenic fragment contains exactly or at least 2, 3 or 4 supercoiled regions where the immunogenic fragment is identical to more than 50, 60, 70, 80, 90, 95, 96 or 100% at a native pneumococcal PhtD sequence (for example, the sequence represented by SEQ ID NO: 4 from document WO 00/37105). In another embodiment of the present invention, the immunogenic fragment comprises one or more histidine triad motifs as well as at least 1, 2, 3 or 4 supercoiled regions.
In the case where the PhtD polypeptide is a variant, the variation is generally in a part of it other than the residues of the histidine triad and the coiled region, although variations in one or more of these regions can be realized. According to the present invention, a variant is a protein in which the native pneumolysin is mutated. An amino acid substitution can be conservative or non-conservative. In one aspect, the amino acid substitution is conservative. Substitutions, deletions, insertions or any combination thereof can be combined into a single variant as long as the variant is an immunogenic polypeptide. Variants generally include polypeptides that share at least 80, 90, 94, 95, 98, or 99% amino acid sequence identity with a wild type sequence. PhtD variants generally include any immunogenic fragment or any variation of PhtD which shares at least 80, 90, 95, 96, 98, or 99% amino acid sequence identity with a wild type PhtD sequence, for example,
BE2017 / 5102
SEQ ID NO: 4 from document WO 00/37105. In one embodiment, the present invention comprises immunogenic fragments and / or variants in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted, or added in a any combination. In another embodiment, the present invention includes immunogenic fragments and / or variants which include a B cell or T cell epitope. These epitopes can be predicted using a combination of two-dimensional structure prediction, for example, by using the PSIPRED program (from David Jones, Brunei Bioinformatics Group, Dept. Biological Sciences, Brunel University, Uxbridge UB8 3PH, United Kingdom) and of antigen index calculated on the basis of the process described by Jameson and Wolf (CABIOS 4: 181-186 [1988]).
In one embodiment of the invention, the PhtD and its immunogenic fragments, variants and / or fusion proteins thereof comprise an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, or 100% identity with the sequence of amino acids 21 to 838 of SEQ ID NO: 4 from document WO 00/37105. In another embodiment of the invention, the PhtD and its immunogenic fragments, variants and / or fusion proteins thereof have an amino acid sequence sharing at least 80, 85, 90, 95, 96, 97 , 98, or 100% identity with the sequence of amino acids 21 to 838 of SEQ ID NO: 4 from document WO 00/37105. Suitably, the PhtD and its immunogenic fragments, variants and / or fusion proteins thereof
BE2017 / 5102 comprise an amino acid sequence comprising an N-terminal methionine. In another embodiment of the invention, the PhtD and its immunogenic fragments, variants and / or fusion proteins thereof comprise at least about 15, at least about 20, at least about 40, or at least about 60 or at least at least about 100, or at least about 200, or at least about 400 or at least about 800 contiguous amino acid residues of the sequence represented by SEQ ID NO: 4 from WO 00/37105.
In one aspect, PhtD is conjugated to a saccharide, for example, a capsular saccharide from S. pneumoniae. For example, PhtD can be conjugated to a capsular saccharide of S. pneumoniae chosen from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In particular, PhtD can be conjugated to a capsular saccharide of S. pneumoniae of serotype 22F. In another aspect, PhtD is unconjugated or present in the immunogenic composition in the form of a free protein. In one aspect of the invention, more than 80% (for example, more than 82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of PhtD is adsorbed on aluminum phosphate. In another aspect of the invention, more than 80% (e.g., more than 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89% or 90%) of particles of the PhtD (by example, the PhtD no conj ugue ) adsorbed sure of phosphate aluminum have a size less than 10 pm.
Regarding the family of choline binding proteins (CbpX), members of this family have been
BE2017 / 5102 originally identified as pneumococcal proteins which could be purified by affinity chromatography for choline. All choline binding proteins are non-covalently linked to phosphorylcholine radicals from cell wall teichoic acid and membrane-associated lipoteichoic acid. Structurally, there are several regions in common across the entire family, although the exact nature of proteins (amino acid sequence, length, etc.) may vary In general, choline binding proteins include a region N-terminal (N), conserved repeat regions, a proline-rich region (P) and a conserved choline binding region (C), consisting of several repeats, which makes up approximately half of the protein. As used in this application, the term “family of choline binding proteins (CbpX)” is chosen from the group consisting of choline binding proteins as identified in document WO 97/41151, the protein A, CbpA (also called PbcA (C3 A binding protein), SpsA (Streptococcus pneumoniae secretory IgA binding protein), PspC (pneumococcal surface protein C), binding protein choline D (CbpD), and the choline G binding protein
(CbpG). The CbpA is disclosed in the document WO 97/41151. The CbpD and CbpG are disclosed in the document WO 00/29434. The PspC is disclosed in the document WO 97/09994. The PbcA is disclosed in the
document WO 98/21337. SpsA is a binding protein
BE2017 / 5102 of choline disclosed in document WO 98/39450. In one embodiment, the choline binding protein is CbpA. Another choline binding protein is the pneumococcal A choline binding protein (PcpA) (Sanchez-Beato et al. FEMS Microbiology Letters 164 (1998) 207-214).
Another preferred embodiment consists of truncated forms of CbpX where "CbpX" represents CbpA, CbpD or CbpG and the "truncated forms of CbpX" refer to CbpX proteins which lack 50% or more of the binding region choline (C). Another preferred embodiment consists of truncated forms of PcpA where the "truncated forms of PcpA" refer to PcpA in which 50% or more of the choline binding region (C) is missing. In one embodiment, the truncated forms of CbpX or the truncated forms of PcpA lack the binding region of the whole choline. In another embodiment, the truncated forms of CbpX or the truncated forms of PcpA lack (i) the choline binding region and also (ii) part of the Nterminal half of the protein, while retaining however at least one repeat region. In another embodiment, the truncated shape has at least 2 repeating regions. Examples of these preferred embodiments are illustrated in WO 99/51266 or WO 99/51188, however, other choline binding proteins lacking a similar choline binding region are also contemplated within the scope of this invention.
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The LytX family consists of membrane-associated proteins associated with cell lysis. The N-terminal domain includes one or more choline binding domains, however, the LytX family does not have all of the characteristics found in the CbpA family noted here and so for the present invention, the LytX family is considered to be distinct from the CbpX family. Unlike the CbpX family, the C-terminal domain contains the catalytic domain of the LytX family of proteins. The family includes LytA, LytB and LytC. With respect to the LytX family, LytA is disclosed in Ronda et al., Eur J Biochem, 164: 621-624 (1987). LytB is disclosed in WO 98/18930, and it is also called Sp46. LytC is also disclosed in WO 98/18930, and it is also called Sp91. A favorite member of this family is LytC.
Another preferred embodiment consists of truncated forms of LytX where "LytX" represents LytA, LytB or LytC and "truncated forms of LytX" refer to LytX proteins which lack 50% or more of the binding region choline. Suitably, such proteins lack the binding region of the entire choline. Yet another preferred embodiment of this invention consists of chimeric proteins (or fusions) truncated form of CbpX-truncated form of LytX. In one embodiment, the chimeric protein truncated form of CbpX-truncated form of LytX comprises the repeat regions of CbpX and the C-terminal part (Cterm, i.e., which lacks the binding domains of
BE2017 / 5102 choline) from LytX (for example, LytCCterm or Sp91Cterm)
In a other fashion of achievement, CbpX is chosen in the group made up of CbpA, PbcA, SpsA and PspC. In a other fashion of achievement, it's about the CbpA. In a fashion of achievement, the LytX is the LytC
(also called Sp91). Another embodiment of the present invention is a truncated form of PspA (pneumococcal surface protein A) or PsaA (pneumococcal surface adhesion protein A) which lacks the binding domain of choline ( C) and expressed as a fusion protein with LytX. In one embodiment, the LytX is the LytC.
PsaA (pneumococcal surface adhesion protein A) and its variants by transmembrane deletion have been described by Berry and Paton, Infect Immun December 1996; 64 (12): 5255-62. PspA (pneumococcal surface protein A) and its transmembrane deletion variants have been disclosed, for example, in US 5,804,193, WO 92/14488, and WO 99/53940.
Spl28 and Spl30 are disclosed in document WO 00/76540. Spl25 is an example of a pneumococcal surface protein with the motif anchored to the cell wall of LPXTG (i.e., leucineproline-X-threonine-glycine where X represents any amino acid). It has been found that any protein within this class of pneumococcal surface proteins is useful in the context of this invention, and therefore is considered another protein of the invention. The Spl25 itself is
BE2017 / 5102 disclosed in WO 98/18930, and is also known as ZmpB - a zinc metalloproteinase. The SplOl is disclosed in document WO 98/06734 (where it has the reference # y85993). It is characterized by a type I signal sequence. The Spl33 is disclosed in document WO 98/06734 (where it has the reference # y85992). It is also characterized by a type I signal sequence.
Antigens of S. pneumoniae can also be beneficially combined. By combined, it is meant that the immunogenic composition comprises all the proteins within the combination, either in the form of carrier proteins or in the form of free proteins or a mixture of the two. For example, in a combination of two proteins as presented below, the two proteins can be used as carrier proteins, or the two proteins can be present as free proteins, or both can be present as that support proteins and in the form of free proteins, or one may be present as a support protein and in the form of free protein while the other is present only as a support protein or only in the form of free protein, or one may be present as a carrier protein and the other as a free protein. When a combination of three similar possibilities proteins exist, preferred include, but are not limited to, PhtD + repeat regions of CbpX, PhtD + dPly, PhtD + Spl28,
PhtD + PsaA, PhtD + PspA, PhtA + repeat regions of is given, combinations
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CbpX, PhtA + CbpX repeat regions - Sp91Cterm chimeric or fusion proteins, PhtA + dPly, PhtA + Spl28, PhtA + PsaA, PhtA + PspA, CbpX + LytC repeat regions, CbpX + PspA repeat regions, CbpX + PsaA, CbpX + Spl28 repeat regions, CbpX + LytC repeat regions, CbpX + PspA repeat regions, CbpX + PsaA repeat regions, CbpX + Spl28 repeat regions, CbpX + PhtD repeat regions, CbpX repeat regions + PhtA. In one embodiment, the repeat regions of CbpX are from CbpA. In another embodiment, they come from CbpA. Other combinations include combinations of 3 proteins such as PhtD + repeat regions of CbpX + dPly, and PhtA + repeat regions of CbpX + PhtD. In one embodiment, the immunogenic composition includes detoxified pneumolysin and PhtD as carrier proteins. In another embodiment, the immunogenic composition comprises detoxified pneumolysin and PhtD in the form of free proteins.
The immunogenic compositions of the invention can also comprise capsular saccharides of S. pneumoniae (suitably conjugated to a support protein), for example as described in document WO 2007 / 071707A2. The bacterial capsular saccharide from Streptococcus pneumoniae can be chosen from the capsular saccharides from Streptococcus pneumoniae of serotype 1, 2, 3, 4, 5, 6A,
6B, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F,
11A, 11B, 11C, HD, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B,
15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A,
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19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 26, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45, 46, 47A, 47F or 48. Saccharides (for example, polysaccharides (PS)) can be derived from pneumococcal serotypes such as serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14 , 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In one embodiment, at least four serotypes are included in the composition, for example, 6B, 14, 19F and 23F (suitably conjugated to a carrier protein). In another embodiment, at least 7 serotypes are included in the composition, for example, 4, 6B, 9V, 14, 18C, 19F and 23F (suitably conjugated to a carrier protein). In another embodiment, the immunogenic composition comprises 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 19 or more, or 20 polysaccharides capsular from different serotypes of S. pneumoniae (appropriately conjugated to a carrier protein). In one embodiment, the immunogenic composition comprises 10 to 23 capsular polysaccharides from different serotypes of S. pneumoniae (suitably conjugated to a carrier protein). In one embodiment, the vaccine can be a 11valent vaccine. For example, an 11-valent vaccine may include polysaccharides from serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F and 23F. In one embodiment, the vaccine can be a 12-valent vaccine or a 13-valent vaccine. A pediatric vaccine (infants)
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12- or 13-valent can also include the 11-valent formulation supplemented with serotypes
19A, or 22F, or 15 (for example, PS1-PD, PS4-PD, PS5-PD,
PS6A-CRM197, PS6B-PD, PS7F-PD, 9V-PD, 14-PD, 18C-TT,
19A-CRM197, 19F-DT, 23F-PD), while a 13valent vaccine for the elderly may include the 11-valent formulation supplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15, or 8 and 19A, or 8 and 22F, or 12F and 15, or 12F and 19A, or 12F and 22F, or 15 and 19A, or 15 and 22F. In one embodiment, the vaccine can be a 14-valent or 15-valent vaccine. A 14- or 15-valent pediatric vaccine may include the 11-valent formulation described above supplemented with serotypes 3, 19A and 22F; serotypes 8, 19A and 22F; serotypes 12F, 19A and 22F; serotypes
15, 19A and 22F; serotypes 3, 8, 19A and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15, 19A and 22F. In one embodiment, the vaccine can be a 16-valent vaccine. A 16-valent vaccine may include the 11-valent formulation described above supplemented with serotypes 3, 15B, 19A, 22F and 23F. A vaccine
16-valent may include the 11-valent formulation described above supplemented with serotypes 3, 15B, 19A, 22F and 33F. In one embodiment, the vaccine can be a 19-valent vaccine. A 19-valent vaccine may include the 11-valent formulation described above supplemented with serotypes 8, 10A, 11A, 12F, 15B,
19A, 22F and 23F. A 19-valent vaccine may include the 11-valent formulation described above supplemented with serotypes 8, 10A, 11A, 12F, 15B, 19A, 22F and 33F. In one embodiment, the vaccine can be a
BE2017 / 5102 20-valent vaccine. A 20-valent vaccine may include the 11-valent formulation described above supplemented with serotypes 3, 8, 10A, 11A, 12F, 15B, 19A, 22F and 23F. A 20-valent vaccine may include the 11-valent formulation described above supplemented with serotypes 3, 8, 10A, 11A, 12F, 15B, 19A, 22F and 33F. In one embodiment, the vaccine can be a 21-valent vaccine. In one embodiment, the vaccine can be a 22-valent vaccine. In one embodiment, the vaccine can be a 23-valent vaccine.
Suitably, each of the saccharides is conjugated to a carrier protein. Examples of carrier proteins which can be used in the present invention are TT, DT, CRM197, PhtD, detoxified pneumolysin and protein D. In another embodiment, each capsular saccharide of Streptococcus pneumoniae is conjugated to a selected carrier protein independently in the group consisting of TT, DT, CRM197, PhtD and protein D. In another embodiment, each capsular saccharide of Streptococcus pneumoniae is conjugated to a support protein independently selected from the group consisting of TT, DT, CRM197 and protein D. In one embodiment, the immunogenic composition of the invention comprises two or more different support proteins. In one embodiment, the immunogenic composition of the invention comprises 2, 3, 4, 5 or 6 different support proteins.
In one embodiment, the carrier protein is protein D of Haemophilus influenzae (PD), for example, the sequence of protein D of Figure 9
BE2017 / 5102 (Figures 9a and 9b together, 364 amino acids) of document EP 0594610 (SEQ ID NO: 6). The inclusion of this protein in the immunogenic composition can provide a level of protection against otitis media associated with Haemophilus influenzae (Pyrmula et. Al. Lancet 367; 740-748 (2006)). Protein D can be used in the form of a full-length protein or in the form of a fragment (for example, protein D can be as described in WO 0056360). For example, a protein D sequence can comprise (or be made up) the protein D fragment described in document EP 0594610 which begins with the sequence SSHSSNMANT (SerSerHisSerSerAsnMet AlaAsnThr) (SEQ ID NO: 8), and which is missing 19 Nterminal amino acids of FIG. 9 of document EP 0594610, optionally with the tripeptide MDP of NSI fused at the N-terminal end of said protein D fragment (348 amino acids) (SEQ ID NO: 7) appearance, protein D or a fragment of protein D is non-lipidized. Protein D may be present in the immunogenic composition in the form of a free protein or as a support protein. In one aspect, protein D is present in the immunogenic composition as a free protein. In another aspect, protein D is present both as a carrier protein and as a free protein. In another aspect, protein D is present as a support protein for one or more of the polysaccharides. In another aspect, 2 to 9 of the capsular polysaccharides chosen from different serotypes are conjugated to protein D. In another aspect, the of the
In one
BE2017 / 5102 protein D is present as a support protein for the majority of polysaccharides, for example, 6, 7,
8, 9 or more of the polysaccharides can be conjugated to protein D.
In one embodiment, the carrier protein is CRM197. CRM197 is a non-toxic form of the diphtheria toxin but from an immunological point of view it cannot be distinguished from the diphtheria toxin (DT). Genetically detoxified analogues of diphtheria toxin include CRM197 and other mutants described in US 4,709,017, US 5,843,711, US 5,601,827, and US 5,917,017. CRM197 is produced by C. diphtheriae infected with non-toxigenic phage ß197tox- created by mutagenesis with nitrosoguanidine of the toxigenic corynephage b (Uchida et al. Nature New Biology (1971) 233; 8-11). The protein CRM197 has the same molecular mass as the diphtheria toxin but it differs from it by the change of a single base in the structural gene. This leads to an amino acid change from glycine to glutamine at position 52 which makes fragment A incapable of binding NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983 p560-564).
In one embodiment, the carrier protein is tetanus toxoid (TT). Tetanus toxoid is a single peptide of approximately 150 kDa, which is made up of 1315 amino acid residues. Tetanus toxin can be cleaved by papain to produce two fragments; one of them, fragment C, is approximately 50 kDa. The C fragment of TT is
BE2017 / 5102 described in Neubauer et al. Biochim. Brophys. Acta 1981,
27, 141-148.
The conjugates can be prepared by direct reductive amination methods as described in US 200710184072 (Hausdorff) US 4,365,170 (Jennings) and US 4,673,574 (Anderson). Other methods are described in documents EP-0161-188, EP-208375 and EP-0-477508. The conjugation process may alternatively be based on the activation of the saccharide with 1-cyano-4dimethylamino-pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Such conjugates are described in the published PCT application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094. See also Chu C. et al Infect. Immunity, 1983 245 256. The activated saccharide can thus be coupled directly or via a spacer group (linker) to an amino group on the support protein. For example, the spacer can be a cystamine or a cysteamine to give a thiolated polysaccharide which can be coupled to the support via a thioether bond obtained after reaction with a support protein activated by maleimide (for example, using GMBS (N-hydroxysuccinimide ester of 4-maleimidobutyric acid)) or a haloacetylated support protein (for example using SIAB ((4-iodoacetyl) aminobenzoate or SIA (iodoacetate or SBAP (bromoacetamide) propionate)). In one embodiment, the cyanate ester (optionally produced by CDAP chemistry) is coupled with hexane (succinimidyl-3 a mode of succinimidyl), succinimidyl),
BE2017 / 5102 diamine or DHA (adipic acid dihydrazide) and the amino derivative saccharide is conjugated to the support protein using carbodiimide chemistry (e.g. l-ethyl-3- (3-dimethylaminopropyl ) carbodiimide (EDAC or EDC)) via a carboxyl group on the support protein. Such conjugates are described in the published PCT application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094.
In one aspect of the invention, the dPly is preadorbed individually on aluminum phosphate in accordance with the present invention, before being mixed with other antigens (for example, before mixing with the PhtD protein of Streptococcus pneumoniae) . Thus, in one aspect of the invention, the method of the invention further comprises the step (iii) of mixing the detoxified pneumolysin adsorbed with one or more antigens other than the detoxified pneumolysin (for example, PhtD). In one embodiment, step (iii) is carried out in an appropriate manner following step (i). In another embodiment, step (iii) is carried out in an appropriate manner following step (ii). The PhtD protein can be prepared and purified as described in document WO 2007/071710 (see example lb).
In one embodiment, step (iii) comprises mixing the detoxified pneumolysin adsorbed with pre-adsorbed PhtD (i.e., PhtD which has previously been adsorbed on aluminum phosphate ). The PhtD pre-adsorbed on aluminum phosphate may have been prepared by a process using
BE2017 / 5102 adsorption conditions different from those used for the adsorption of detoxified pneumolysin. Thus, in one aspect, the pre-adsorbed PhtD is pre-adsorbed on aluminum phosphate using different adsorption conditions (for example, a different pH and / or a different protein / Al ³⁺ ratio (from aluminum phosphate)) of the adsorption conditions used for the adsorption of the detoxified pneumolysin on aluminum phosphate. For example, in one aspect, the pre-adsorbed PhtD is prepared by mixing the PhtD with aluminum phosphate at pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5, 2 or pH 4.9 to 5.1 (for example, pH 5.0) and / or using a PhtD / Al ³⁺ ratio (coming from aluminum phosphate) between 1/1 and 1/3, suitably between 1/1 and 1 / 2.5, or between 1 / 1.5 and 1 / 2.5, or between 1/2 and 1 / 2.5 (e.g. 1/2) (p / p; weight / weight). In another aspect, PhtD is pre-adsorbed on aluminum phosphate at pH 4.9 to 5.1 and / or in a ratio of 1 pg of PhtD to 2 pg of Al ³⁺ (from phosphate d 'aluminum). In one aspect, PhtD is unconjugated PhtD. In another aspect, PhtD is conjugated PhtD. Unless otherwise indicated, these ranges include extreme values. In another embodiment, the pre-adsorbed PhtD is prepared by mixing the PhtD with aluminum phosphate at pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5, 2 or pH 4.9 to 5.1 and / or using a PhtD / Al ³⁺ ratio (from aluminum phosphate) between 1/1 and 1/3, suitably between 1/1 and 1 / 2.5, or between 1 / 1.5 and 1 / 2.5, or between 1/2 and 1 / 2.5 (w / w; weight / weight) not including extreme values.
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In one embodiment, the pre-adsorption of PhtD is carried out by a process in which the PhtD and the aluminum phosphate (and optionally a buffer) are mixed initially and then (for example, after 5 to 15 minutes, the pH is adjusted to pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5.2 or pH 4.9 to 5.1 (for example, pH 5.0) (the pH for adsorption), followed by further mixing. The pH can be adjusted using sodium hydroxide (NaOH (aqueous)) and hydrochloric acid (HCl (aqueous)). , the pH is maintained (and the additional mixing continues) at that pH (i.e., the pH for adsorption) for a period between
10 minutes and 2 weeks, per example, 10 minutes to 5 hours, 1 at 5 hours, or 2 to 3 hours. Except indication opposite, these ranges include values extremes.
In another embodiment, the pH is maintained (and further mixing continues) at that pH (i.e., the pH for adsorption) for a period of between 10 minutes and 2 weeks, by example, 10 minutes to 5 hours, 1 to 5 hours, or 2 to 3 hours not including extreme values.
In one aspect of the invention, the pre-adsorption of PhtD (i.e., mixing of PhtD with aluminum phosphate) is carried out in the presence of a buffer, such as a buffer phosphate (for example, NaK ₃ ). In one aspect, the concentration of the buffer (e.g. NaK2) is at least 1 mM (e.g. at least 1.5 mM, 2 mM, 2.3 mM, 3 mM, 4 mM) and is so suitable for more than 20 mM (for example, more than 19 mM, 18 mM, 17 mM, 16 mM, 15 mM). In another aspect, the
BE2017 / 5102 buffer concentration (for example, NaK2) is between 1 mM and 25 mM, or between 5 mM and 15 mM (for example, between 8 mM and 12 mM), for example, 10 mM. The phosphate buffer, NaK2, used in the adsorption of PhtD may include NaPhPCh.lPhO (monobasic sodium phosphate) and K2HPO4 (dibasic potassium phosphate) or K2HPO4.3H2O. Suitably, the buffer has a pH of 6.5 to 7.5 (for example, pH 7.15). Unless otherwise indicated, these ranges include extreme values. In another embodiment, the buffer concentration (for example, NaK2) is between 1 mM and 25 mM, or between 5 mM and 15 mM (for example, between 8 mM and 12 mM) not including the values extremes.
In one aspect of the invention, after preadsorption of PhtD on aluminum phosphate, the pH of the pre-adsorbed PhtD is adjusted to a pH between 6 and 7 (for example, pH 5.9 to 6, 5, pH 5.9 to 6.3, pH 6.0) before mixing the pre-adsorbed PhtD and the pre-adsorbed dPly.
In another aspect of the invention, a mixture of pre-adsorbed dPly and PhtD can be prepared, according to steps (i) to (iii) described above, before mixing with other antigens, for example, capsular saccharides of S. pneumoniae (suitably conjugated to a carrier protein) as described herein.
Dosage
The total content of protein antigens in the immunogenic composition or vaccine of the invention will generally be in the range of 1 to 100 µg, or 5
BE2017 / 5102 different, at 80 pg, for example, in the range of 50 to 70 pg. In one aspect, the immunogenic composition or vaccine of the invention comprises 1 pg to 50 pg (for example, 26 pg to 45 pg, 26 pg to 40 pg, 28 pg to 35 pg or around 30 pg) of detoxified pneumolysin (e.g. dPly), per human dose. In another aspect, the immunogenic composition or vaccine of the invention comprises 1 pg to 50 pg (for example, 26 pg to 45 pg, 26 pg to 40 pg, 28 pg to 35 pg or around 30 pg) of each protein of S. pneumoniae, per human dose. For example, the immunogenic composition or the vaccine of the invention can comprise 1 pg to 50 pg (for example, 26 pg to 45 pg, 26 pg to 40 pg, 28 pg to 35 pg or around 30 pg) of PhtD, per human dose.
In one embodiment, the immunogenic composition or the vaccine of the invention may comprise capsular saccharides of S. pneumoniae, each of which may be at a dose of between 0.1 and 20 μg; 0.5 to 10 µg; 0.5 to 5 pg or 1 to 3 pg of saccharide. In one embodiment, the capsular polysaccharides may be present for example, some at capsular polysaccharide dosages may be present at a dose around or exactly 1 pg or certain capsular polysaccharides may be present at a dose around or exactly 3 pg. "Around" or "approximately" are defined as being more or less 10% of the figure given for the purposes of the invention.
By the term "human dose" is meant a dose which is in a volume suitable for human use. Generally, this is between 0.25 and
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1.5 ml, although for administration to the skin a smaller volume between 0.05 ml and 0.2 ml can be used. In one embodiment, a human dose is 0.5 ml. In another embodiment, a human dose is greater than 0.5 ml, for example, 0.6, 0.7, 0.8, 0.9 or 1 ml. In another embodiment, a human dose is between 1 ml and 1.5 ml. In another embodiment, in particular when the immunogenic composition is intended for the pediatric population, a human dose can be less than 0.5 ml as between 0.25 and 0.5 ml.
Method of administration
The vaccine preparations containing the immunogenic compositions of the present invention can be used to protect or treat a mammal, for example, a human being, susceptible to infection, by means of the administration of said vaccine via a systemic route or mucosal. These administrations can include an injection by the intramuscular (IM), intraperitoneal (IP), intradermal (ID) or subcutaneous (SC) routes; or by mucosal administration to the oral / food, respiratory, urogenital systems. Although the vaccine of the invention can be administered as a single dose, its components can also be co-administered together at the same time or at different times (for example, conjugates of pneumococcal saccharides can be administered separately, at the same time or 1 to 2 weeks after administration of any bacterial protein component of the vaccine
BE2017 / 5102 for optimal coordination of immune responses to each other). For a co-administration, the possible adjuvant may be present in any one or all of the different administrations. In addition to a single route of administration, 2 different routes of administration can be used. For example, polysaccharide conjugates can be administered by IM (or ID) and bacterial proteins can be administered by IN (or ID). In addition, the vaccines of the invention can be administered by IM for sensitization doses and by IN for booster doses.
Following an initial vaccination, subjects may receive one or more adequately spaced booster immunizations.
Vaccine
The present invention further provides a vaccine containing the immunogenic compositions of the invention and a pharmaceutically acceptable excipient or carrier.
The pharmaceutically acceptable excipients and carriers are well known and can be chosen by a person skilled in the art. For example, the pharmaceutically acceptable excipient or carrier may comprise a buffer, such as Tris (trimethamine), phosphate (for example, sodium phosphate), acetate, borate (for example, sodium borate), citrate, glycine, histidine and succinate (e.g. sodium succinate), suitably sodium chloride, histidine, sodium phosphate or sodium succinate. The pharmaceutically acceptable excipient can comprise a salt,
BE2017 / 5102 for example, sodium chloride, potassium chloride or magnesium chloride. Optionally, the pharmaceutically acceptable excipient contains at least one component which stabilizes the solubility and / or the stability. Examples of solubilizers / stabilizers include detergents, for example, laurel sarcosine and / or polysorbate (for example, Tween ™ 80). Examples of stabilizers also include Poloxamer (for example, Poloxamer 124, Poloxamer 188, Poloxamer 237, Poloxamer 338 and Poloxamer 407). The pharmaceutically acceptable excipient may comprise a nonionic surfactant, for example, fatty acid esters of polyoxyethylene sorbitan, Polysorbate-80 (Tween ™ 80), Polysorbate-60 (Tween ™ 60), Polysorbate-40 (Tween ™ 40 ) and Polysorbate20 (Tween ™ 20), or polyoxyethylenated alkyl ethers (suitably, polysorbate-80). Alternative solubilizing / stabilizing agents include arginine, and glass forming polyols (such as sucrose, trehalose and the like). The pharmaceutically acceptable excipient can be a preservative, for example, phenol, 2-phenoxy-ethanol, or thiomersal. Other pharmaceutically acceptable excipients include sugars (for example, lactose, sucrose), and proteins (for example, gelatin and albumin). Pharmaceutically acceptable carriers include water, saline solutions, aqueous dextrose and glycerol solutions. Many pharmaceutically acceptable excipients and carriers are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, by E. W.
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Martin, Mack Publishing Co., Easton, PA, 5th Edition (975).
According to another aspect of the invention, there is provided a process for manufacturing the immunogenic composition or the vaccine of the invention comprising the step of mixing detoxified pneumolysin adsorbed on aluminum phosphate according to the invention with an excipient or a pharmaceutically acceptable carrier.
The vaccines of the present invention can be stored in solution or freeze-dried. In one embodiment, the solution is lyophilized in the presence of a sugar such as sucrose or lactose. It is also further preferable that they are lyophilized and reconstituted extemporaneously before use. Lyophilization can produce a more stable composition (vaccine) and can possibly lead to higher antibody titers in the presence of 3D-MPL and in the absence of aluminum-based adjuvant.
The vaccine or immunogenic composition of the invention may also include an antimicrobial, generally when packaged in multiple dose format. For example, the immunogenic composition or the vaccine of the invention can comprise 2phenoxyethanol.
The vaccine or immunogenic composition of the invention may also include a detergent, for example, polysorbate, such as Tween ™ 80. Detergents are generally present at low levels, for example, <0.01%, but higher levels have been suggested for stabilization of antigen formulations, for example, up to 10%.
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In one aspect of the invention, there is provided a vaccine kit, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilized form, and further comprising a vial containing an adjuvant as described herein. It is envisaged in this aspect of the invention that the adjuvant is used to reconstitute the lyophilized immunogenic composition.
Although the vaccines of the present invention can be administered by any route, administration of the vaccines described in the skin (ID) forms an embodiment of the present invention. Human skin has an outer “horny” cuticle, called the stratum corneum, that covers the epidermis. Under this epidermis, there is a layer called the dermis, which in turn covers the subcutaneous tissue. Researchers have shown that injecting a vaccine into the skin, especially the dermis, stimulates an immune response, which may also be associated with a number of additional benefits. Intradermal vaccination with the vaccines described herein forms a preferred feature of the present invention.
The traditional technique of intradermal injection, the "Mantoux procedure", includes the steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (gauge 26 to 31) facing up, the needle is inserted at an angle between 10 and 15 °. Once the needle bevel is inserted, the needle cylinder is lowered and advanced further while providing slight pressure to lift it
BE2017 / 5102 under the skin. The liquid is then injected very slowly, forming a vesicle or bump on the surface of the skin, followed by a slow withdrawal of the needle.
More recently, devices which are specifically designed to deliver liquid agents into or through the skin have been described, for example, the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described , for example, in
documents WO 01/13977 ; US 5 480 381, US 5 599 302, US 5 334 144, US 5 993 412, US 5,649 912, US 5 569 189, US 5 704 911, US 5 383 851, US 5,893 397, US 5 466 220, US 5 339 163, US 5 312 335, US 5,503 627, US 5 064 413, US 5 520 639, US 4 596 556, US 4,790 824, US 4 941 880, US 4 940 460, WO 97/37705 and WC > 97/13537. The variants
of methods for intradermal administration of vaccine preparations may include traditional syringes and needles, or devices designed for ballistic administration of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037 ); or applied to the surface of the skin (transdermal or transcutaneous administration, WO 98/20734; WO 98/28037).
When the vaccines of the present invention are to be administered to the skin, or more specifically to the dermis, the vaccine is in a small volume of liquid, particularly a volume between about 0.05 ml and 0.2 ml.
The content of the immunogenic composition in the skin or of the intradermal vaccines of the present invention
BE2017 / 5102 may be similar to the traditional doses found in intramuscular vaccines (see above). However, a characteristic of skin or intradermal vaccines is that the formulations can be "low dose". Therefore, protein antigens in "low dose" vaccines are suitably present in as little as 0.1-10 pg, or 0.1-5 pg per dose, and polysaccharide antigens (suitably conjugated) may be present in the range of 0.01 to 1 pg, and suitably between 0.01 and 0.5 pg of saccharide per dose.
As used herein, the term "intradermal administration" means administration of the vaccine or immunogenic composition to the region of the dermis in the skin. However, the vaccine or the immunogenic composition will not necessarily be located exclusively in the dermis. The dermis is the layer in the skin located between about 1.0 and about 2.0 mm from the surface of human skin, but there is a certain amount of variation between individuals and in different parts of the body. In general, you can expect to reach the dermis by going 1.5mm below the surface of the skin. The dermis is located between the stratum corneum and the epidermis on the surface and the subcutaneous layer below. Depending on the mode of administration, the vaccine or the immunogenic composition may ultimately be located only or mainly within the dermis, or it may finally be distributed within the epidermis and the dermis.
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The present invention further provides an improved vaccine for the prevention or amelioration of otitis media caused by Haemophilus influenzae by the addition of Haemophilus influenzae proteins, for example, protein D in conjugated form or in the form of free protein. (unconjugated). One or more protein antigens of Moraxella catarrhalis can also be included in the vaccine or the immunogenic composition of the invention in free or conjugated form. Thus, the present invention is an improved method for triggering an immune response against otitis media in infants.
Examples of preferred protein antigens of Moraxella catarrhalis which can be included in a vaccine or a combined immunogenic composition of the invention (especially for the prevention of otitis media) are: the outer membrane protein 106 ( OMP106) [WO 97/41731 (Antex) and WO 96/34960 (PMC)]; the outer membrane protein 21 (OMP21) or its fragments (WO 0018910); lactoferrin A binding protein (LbpA) and / or lactoferrin B binding protein (LbpB) [WO 98/55606 (PMC)]; transferin A binding protein (TbpA) and / or transferin B binding protein (TbpB) [WO 97/13785 and WO 97/32980 (PMC)]; the CopB protein of Moraxella catarrhalis [Helminen ME, et al. (1993) Infect. Immun. 61: 2003-2010]; ubiquitous surface protein A1 (UspAl) and / or ubiquitous surface protein A2 (UspA2) [WO 93/03761 (University of Texas)]; CD outer membrane protein (OmpCD); HasR (PCT / EP99 / 03824); PilQ (PCT / EP99 / 03823); protein
BE2017 / 5102 of the outer membrane 85 (OMP85) (PCT / EP00 / 01468);
lipo06 (GB 9917977.2); lipolO (GB 9918208.1); lipoll (GB 9918302.2); lipol8 (GB 9918038.2); the outer membrane protein P6 (P6) (PCT / EP99 / 03038);
surface antigen D15 (D15) (PCT / EP99 / 03822); the outer membrane protein Al (OmpAl) (PCT / EP99 / 06781); Hly3 (PCT / EP99 / 03257); and outer membrane protein E (OmpE). Examples of proteins or non-typable Haemophilus influenzae fragments thereof which can be included in a combined vaccine (especially for the prevention of otitis media) include: the fimbrin protein [(US 5,766,608 - Ohio State Research Foundation)] and fusions comprising peptides derived therefrom [for example, fusions of the LB1 peptide (f); US 5,843,464 (OSU) or WO 99/64067]; outer membrane protein 26 (OMP26) [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of New York)]; TbpA and / or TbpB; adhesin from H. influenzae (Hia); Haemophilus surface fibrils (Hsf); the Hin47 protein of Haemophilus influenzae; the Haemophilus influenzae Hit protein; the Hmwl protein of Haemophilus influenzae; the Hmw2 protein of Haemophilus influenzae; the Hmw3 protein of Haemophilus influenzae; the Hmw4 protein of Haemophilus influenzae; Haemophilus influenzae autotransportant adhesin (Hap); D15 (WO 94/12641); P2;
and P5 (WO 94/26304).
Processing procedures and use
The present invention provides a method for treating or preventing infection with
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Streptococcus pneumoniae in a subject in need thereof comprising administration to said subject of a therapeutically effective amount of an immunogenic composition or the vaccine of the invention. The present invention also provides a method of immunizing a human host against Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of the immunogenic composition or the vaccine of the invention. The present invention also provides a method of inducing an immune response against Streptococcus pneumoniae (e.g., Streptococcus pneumoniae pneumolysin) in a subject, the method comprising administering a therapeutically effective amount of the immunogenic composition or the vaccine of the invention.
In one embodiment, the present invention is an improved method for triggering an immune response in infants (defined as 0-2 years of age in the context of the present invention) by administering a therapeutically effective amount of an immunogenic composition or a vaccine of the invention (a pediatric vaccine). In one embodiment, the vaccine is a pediatric vaccine. In one embodiment, the immune response is protective (i.e., it prevents or reduces the infection caused by S. pneumoniae).
In one embodiment, the present invention is an improved method for triggering an immune response in the elderly population (in the context of the present invention, a patient is
BE2017 / 5102 considered as an elderly person if he is 50 years of age or over, generally over 55 years and more generally over 60 years) by the administration of a therapeutically effective amount of the immunogenic composition or vaccine of the invention.
In one embodiment, the present invention provides a method of protecting a subject against a disease caused by infection with Streptococcus pneumoniae, or a method of preventing infection with Streptococcus pneumoniae, or a method of reducing severity or delayed onset of at least one symptom associated with an infection caused by Streptococcus pneumoniae, the methods comprising administering to an subject an immunogenic amount of an immunogenic composition or a vaccine of the invention .
In one embodiment, the present invention provides immunogenic compositions and vaccines of the invention for use in the prevention or treatment of a disease caused by S. pneumoniae infection. In one embodiment, the present invention provides the use of an immunogenic composition or a vaccine of the invention in the manufacture of a medicament for the prevention (or treatment) of a disease caused by a S. pneumoniae infection.
The disease caused by Streptococcus pneumoniae infection can be chosen from pneumonia, invasive pneumococcal disease (MPI), exacerbations of chronic obstructive pulmonary disease (eBPCO), otitis media, meningitis,
BE2017 / 5102 bacteremia, pneumonia and / or conjunctivitis. When the human host is an infant (defined as being 0 to 2 years old in the context of the present invention), the disease can be chosen from otitis media, meningitis, bacteremia, pneumonia and / or conjunctivitis. In one aspect, when the human host is an infant (defined as 0-2 years of age in the context of the present invention), the disease is selected from otitis media and / or pneumonia. When the human host is an elderly person (that is, aged 50 years or more, generally over 55 years and more generally over 60 years), the disease can be chosen from pneumonia, invasive pneumococcal disease (IPD), and / or exacerbations of chronic obstructive pulmonary disease (eBPCO). In one aspect, when the human host is an elderly person, the disease is invasive pneumococcal disease (IBD). In another aspect, when the human host is an elderly person, the disease consists of exacerbations of chronic obstructive pulmonary disease (eBPCO).
The embodiments herein relating to the "vaccine compositions" of the invention are also applicable to the embodiments relating to the "immunogenic compositions" of the invention, and vice versa.
All references and patent applications cited within this patent specification are incorporated herein by reference.
In order that this invention may be better understood, the following examples are presented. These examples are
BE2017 / 5102 for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.
The embodiments of the invention are further described in the following numbered paragraphs:
1. An immunogenic composition comprising adsorbed on phosphate (eg, plus 96%, 97%, 98% detoxified aluminum pneumolysin, in which more than 85 to 90%, 91%, 92%, 93%, 94 %, 95 or 99%) of the detoxified pneumolysin is adsorbed on aluminum phosphate
2. An immunogenic composition according to paragraph 1, in which more than 95% of the detoxified pneumolysin is adsorbed on aluminum phosphate.
3. An immunogenic composition according to paragraph 1 or paragraph 2, in which the immunogenic composition has a pH between 6 and 7 (for example, pH 6.0 to 6.5, pH 6.0 to 6.2, pH 6.1).
4. An immunogenic composition according to any one of paragraphs 1 to 3, in which more than 80% (for example, more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89% or 90%) of the detoxified pneumolysin adsorbed on aluminum phosphate has a particle size of less than 10 µm.
5. An immunogenic composition according to any one of paragraphs 1 to 4, wherein more than 85% of the detoxified pneumolysin adsorbed on aluminum phosphate has a particle size of less than 10 µm.
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6. An immunogenic composition according to any one of paragraphs 1 to 5, wherein the detoxified pneumolysin has been chemically detoxified.
7. An immunogenic composition according to any one of paragraphs 1 to 6, wherein the detoxified pneumolysin has been genetically detoxified.
8. An immunogenic composition according to any one of paragraphs 1 to 7, wherein the detoxified pneumolysin is unconjugated.
9. An immunogenic composition according to any one of paragraphs 1 to 8, wherein the detoxified pneumolysin is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
10. An immunogenic composition according to any one of paragraphs 1 to 9, further comprising PhtD adsorbed on aluminum phosphate.
11. An immunogenic composition according to paragraph 10, in which the PhtD is unconjugated.
12. An immunogenic composition according to paragraph 10, in which the PhtD is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
13. An immunogenic composition according to any one of paragraphs 1 to 12 further comprising 10 or more capsular polysaccharides of S. pneumoniae from different serotypes of S. pneumoniae conjugated to one or more support proteins.
14. A process for the adsorption of detoxified pneumolysin on aluminum phosphate comprising the step of (i) mixing the detoxified pneumolysin and aluminum phosphate at a pH below 6.5 (by
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example, less than pH 6.0, pH 5.0 at 6.2, pH 5.0 at 6, 1, pH 5.2 at 6.2, pH 5.2 at 6.1, pH 5.4 at 6.2, pH 5.4 at 6, 1, pH 5.5 at 6.1, pH 5.4 at 5.9, pH 5.5 at 5, 9, pH 5.4 at 5.7, pH 5.5 at 5.7, pH 5.4 at 5.6 or pH 5, 5)
15. The process according to paragraph 14 in which the detoxified pneumolysin and the aluminum phosphate are in a ratio of dPly / Al ³⁺ (originating from the aluminum phosphate) in step (i) situated between 1/1, 5 and 1/4 (for example, 1 / 1.5 to 1 / 3.5, 1 / 1.5 to 1 / 2.5, 1/2 to 1 / 2.5, 1 / 2.5 to 1 / 3.5, 1/3 to 1 / 3.5, 1/2 or 1/3) (w / w;
any of step (i) is weight / weight).
16. The method according to one of paragraphs 14 or 15 in which carried out in the presence of a phosphate buffer, optionally comprising NaEhPCq and K2HPO4, and optionally at a concentration between 1 mM and 5 mM (for example, between 1 mM and 3 mM, between 2 mM and 2.4 mM, or mM).
17. The method according to any one of paragraphs 14 to 16 followed by step (ii) of adjusting the pH of the composition to a pH between 6 and 7 (for example, pH 6.0 to 6.5 , pH 6.0 to 6.3, or pH 6.1).
18. The method according to any one of paragraphs 14 to 17 followed by step (iii) of mixing the detoxified pneumolysin adsorbed with one or more antigens other than the detoxified pneumolysin (for example, PhtD).
19. The method according to paragraph 18 in which step (iii) comprises mixing the detoxified pneumolysin adsorbed with pre-adsorbed PhtD.
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20. The process according to paragraph 19 in which the pre-adsorbed PhtD is prepared by mixing PhtD with aluminum phosphate at a pH of 4.5 to 5.5 (for example, pH 4.5 to 5, 4, pH 4.7 to 5.2, pH 4.9 to 5.1, or pH 5.0) and / or using a PhtD / Al ³⁺ ratio (from aluminum phosphate) between 1 / 1 and 1/3 (for example, 1/1 to 1 / 2.5, 1 / 1.5 to 1 / 2.5, 1/2 to 1 / 2.5, or 1/2) (p / p; weight / weight).
21. The method according to paragraph 20 in which the mixing of PhtD with aluminum phosphate is carried out in the presence of a phosphate buffer, optionally comprising NaPhPCy. IH2O, K2HPO4 and / or K2HPO4.3H2O, and optionally at a concentration between 5 mM and 15 mM (for example, between 8 mM and 12 mM, or 10 mM).
22. The process according to Mon any of paragraphs 19 to 21 in which PhtD is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae. 23. The process according to Mon any of paragraphs 19 to 21 in which PhtD is no conj ugue. 24. The process according to Mon any of paragraphs 14 to 23 in which pneumolysin detoxified is unconjugated. 25. The process according to Mon any of paragraphs 14 to 23 in which pneumolysin detoxified < is conjugate has a saccharide, for example : a
capsular saccharide of S. pneumoniae.
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26. A process for the preparation of an immunogenic composition comprising detoxified pneumolysin, comprising the process according to paragraphs 14 to 25.
27. An immunogenic composition according to any one of paragraphs 1 to 13 prepared by the method according to paragraphs 14 to 26.
28. A vaccine comprising the immunogenic composition according to any one of paragraphs 1 to 13 or 27 and a pharmaceutically acceptable excipient or carrier.
29. A method for the treatment or prevention of a Streptococcus pneumoniae infection in a subject in need thereof (for example, a human) comprising administering to the said subject a therapeutically effective amount of an immunogenic composition according to the invention. any of paragraphs 1 to 13 or 27 or of the vaccine according to paragraph 28.
30. A method of immunizing a human host against a Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of the immunogenic composition according to any one of paragraphs 1 to 13 or 27 or of the vaccine according to paragraph 28.
31. A method of inducing an immune response against Streptococcus pneumoniae in a subject (eg, a human), the method comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition according to any one paragraphs 1 to 13 or 27 or the vaccine according to paragraph 28.
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32. The immunogenic composition according to paragraphs 1 to 13 or 27 or the vaccine according to paragraph 28 for use in the treatment or prevention of a disease caused by an infection with Streptococcus pneumoniae.
33. Use of the immunogenic composition according to paragraphs 1 to 13 or 27 or of the vaccine according to paragraph 28 in the manufacture of a medicament intended for the treatment or prevention of a disease caused by an infection with Streptococcus pneumoniae.
Other embodiments of the invention are also described in the following numbered paragraphs:
the. An immunogenic composition comprising adsorbed on phosphate% (for example, plus%, 96%, 97%, 98% detoxified aluminum pneumolysin, in which more than 85 of 90%, 91%, 92%, 93%, 94%, 95 or 99%) of the detoxified pneumolysin is adsorbed on aluminum phosphate.
2a. An immunogenic composition according to paragraph la, in which the pH of the composition is between 6 and 7 (for example, pH 6.0 to 6.5, pH 6.0 to 6.2, pH 6.1).
3a. An immunogenic composition according to paragraph la or paragraph 2a, in which more than 80% (for example, more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%) of the particles of the detoxified pneumolysin adsorbed on aluminum phosphate have a size less than 10 μm.
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4a. An immunogenic composition according to any of paragraphs 1a to 3a, wherein the pneumolysin has been chemically detoxified.
5a. An immunogenic composition according to any one of paragraphs 1a to 4a in which the pneumolysin has been genetically detoxified.
6a. A process for the adsorption of detoxified pneumolysin on aluminum phosphate comprising the step of (i) mixing the detoxified pneumolysin and
aluminum phosphate at a pH inferior at 6, 5 (by example, below pH 6 , 0, pH 5.0 at 6.2, pH 5.0 at 6, 1, pH 5.2 to 6.2, pH 5.2 to 6, 1, pH 5.4 at 6.2, pH 5.4 at 6, 1, pH 5.5 to 6.1, pH 5.4 to 5, 9, pH 5.5 at 5.9, pH 5.4 at 5.7,
pH 5.5 to 5.7, pH 5.4 to 5.6 or pH 5.5).
7a. The method according to paragraph 6a in which the ratio of dPly / Al ³⁺ (from aluminum phosphate) in step (i) is between 1 / 1.5 and 1/4 (for example, 1/1 , 5 to 1 / 3.5, 1 / 1.5 to 1 / 2.5, 1/2 to 1 / 2.5, 1 / 2.5 to 1 / 3.5, 1/3 to 1/3 , 5, 1/2 or 1/3) (w / w; weight / weight).
8a. The method according to paragraph 6a or 7a in which step (i) is carried out in the presence of a phosphate buffer, optionally comprising NaPLPCy and K2HPO4, and optionally at a concentration between 1 mM and 5 mM (for example, between 1 mM and 3 mM, between 2 mM and 2.4 mM, or 2 mM).
9a. The method according to any one of paragraphs 6a to 8a followed by step (ii) of adjusting the pH of the composition to a pH between 6 and 7 (for example, pH 6.0 to 6.5, pH 6.0 to 6.3, or pH 6.1).
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10a. The method according to any one of paragraphs 6a to 9a followed by step (iii) of mixing the detoxified pneumolysin adsorbed with one or more antigens other than detoxified pneumolysin (for example, PhtD).
lia. The method according to paragraph 10a in which step (iii) comprises mixing the adsorbed detoxified pneumolysin with pre-adsorbed PhtD.
12a. The method according to paragraph 11a in which the pre-adsorbed PhtD is prepared by mixing PhtD with aluminum phosphate at a pH of 4.5 to 5.5 (for example, pH 4.5 to 5.4, pH 4.7 to 5.2, pH 4.9 to 5.1, or pH 5.0) and / or using a PhtD / Al ³⁺ ratio (from aluminum phosphate) between 1/1 and 1/3 (for example, 1/1 to 1 / 2.5, 1 / 1.5 to 1 / 2.5, 1/2 to 1 / 2.5, or 1/2) (w / w; weight / weight).
13a. The method according to paragraph 12a in which the mixing of PhtD with aluminum phosphate is carried out in the presence of a phosphate buffer, optionally comprising NaPhPCy.IH2O, K2HPO4 and / or K2HPO4.3H2O, and optionally at a concentration between 5 mM and 15 mM (for example, between 8 mM and 12 mM, or 10 mM).
14a. A method of preparing an immunogenic composition comprising detoxified pneumolysin, comprising the method of paragraphs 6a to 13a.
15a. An immunogenic composition according to any one of paragraphs 1a to 5a prepared by the method according to paragraphs 6a to 14a.
16a. A vaccine comprising the immunogenic composition according to any one of paragraphs la to 5a or 15a and
BE2017 / 5102 a pharmaceutically acceptable excipient or carrier.
17a. A method for the treatment or prevention of a Streptococcus pneumoniae infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any of paragraphs la to 5a or 15a or the vaccine according to paragraph 16a.
18a. A method of immunizing a human host against a Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of the immunogenic composition according to any one of paragraphs la to 5a or 15a or of the vaccine according to paragraph 16a.
19a. A method of inducing an immune response against Streptococcus pneumoniae in a subject, the method comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition according to any of paragraphs 1a to 5a or 15a or the vaccine according to paragraph 16a.
20a. The immunogenic composition according to paragraphs la to 5a or 15a or the vaccine according to paragraph 16a for use in the treatment or prevention of a disease caused by an infection with Streptococcus pneumoniae.
21a. Use of the immunogenic composition according to paragraphs la to 5a or 15a or of the vaccine according to paragraph 16a in the manufacture of a medicament
BE2017 / 5102 intended for the treatment or prevention of a disease caused by an infection with Streptococcus pneumoniae.
Examples
Example 1a - Adsorption of detoxified pneumolysin on aluminum phosphate
Detoxification of pneumolysin using formaldehyde: a stock of purified pneumolysin at a concentration of approximately 0.4 mg / ml in 25 mM phosphate buffer pH 7.0 was treated with 50 mM L-lysine and 0, 1% formaldehyde (w / v) for 21 days at 40 ° C.
Aluminum phosphate (AlPCy), 1890 pg of Al ^{3 + /} ml together with PO ₄ (Na / K ₂ ) at 2 mM pH 7.15 and dPly (detoxified pneumolysin) at 630 pg of dPly / ml (ratio of 1 μg of dPly / 3 μg of Al ³⁺ ) were mixed with magnetic stirring (130 rpm) for 5 to 15 minutes at room temperature (18 to 24 ° C). The pH was adjusted to pH 5.5 +/- 0.1 with 0.05 M or 0.5 M NaOH / 0.03 M or 0.3 M HCl with magnetic stirring (130 rpm) for 5 to 15 minutes at room temperature (18 to 24 ° C). The pH was maintained at pH 5.5 + / 0.1 for 120 to 150 minutes at room temperature (18 to 24 ° C) with magnetic stirring (130 rpm). The pH was then adjusted to pH 6.1 +/- 0.1 with 0.05 M or 0.5 M NaOH / 0.03 M or 0.3 M HCl with magnetic stirring (130 rpm ) for 5 to 15 minutes at temperature (18 to 24 ° C). The maturation was carried out for at least 7 days at a temperature of 2 to 8 ° C (maturation step) without any agitation.
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Process :
AIPO4 (Al ³⁺ ) ^ 1890 pg Al ³ + / ml +
PO ₄ (Na / K ₂ ) 2 mM pH 7.15 +
dPly - ^ 630 pg dPly / ml (ratio of 1 pg dPly / 3 pg Al ³⁺ )
j. (magnetic stirring - time (min): 5 to 15 temperature (° C): room temperature from 18 to 24 ° C) adjust the pH to 5.5 +/- 0.1 with 0.05 M or 0 NaOH .5 M / 0.03 M or 0.3 M HCl
j. (magnetic stirring - time (min): 5 to 15 temperature (° C): room temperature from 18 to 24 ° C) check the pH and adjust if necessary j. (magnetic stirring - time (min): 120 to 150 temperature (° C): room temperature from 18 to 24 ° C) adjust the pH to 6.1 +/- 0.1 with 0.05 M or 0 NaOH .5 M / 0.03 M or 0.3 M HCl
j. (magnetic stirring - time (min): 5 to 15 temperature (° C): room temperature from 18 to 24 ° C) check the pH and adjust if necessary i
maturation: time (min): for at least 7 days - temperature (° C): +2 to +8 - agitation: no i
sampling
Note: the bulk of the vaccine is kept under slight agitation throughout the formulation process, the ambient temperature is 18 to 24 ° C.
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Table 1
Components Concentration Other Last name Component Antigen dPly 630 pg / ml Al ³⁺ from ΙΆΙΡΟ4 1890 pg / ml NaCl 55 mM PO ₄ Na / K ₂ NaH ₂ PO ₄ K ₂ HPO ₄ 2.4 mM pH 6.1 (+/- 0.1)
Specifications: aluminum 0.50%, phosphate 1.59%, NaCl 0.9%.
Example lb - Adsorption of PhtD on aluminum phosphate ¹
The PhtD was taken from a storage at -70 ° C and thawed in a thermostatically controlled bath at 25 ° C. Aluminum phosphate (AIPO4), 4000 pg of Al ^{3 + /} ml together with PO4 (Na / K2) at 10 mM pH 7.15 and PhtD at 2000 pg of PhtD / ml (PhtD / ratio Al ³⁺ of 1/2) were mixed with mechanical stirring (130 rpm) for 5 to 15 minutes at room temperature (18 to 24 ° C). The pH was adjusted to pH 5.0 +/- 0.1 with 0.03 M or 0.3 M HCl, this followed by magnetic stirring (130 rpm) for 120 to 150 minutes at temperature ambient (18 to 24 ° C). The pH was then adjusted to pH 6.0 +/- 0.1 with 0.05 M or 0.5 M NaOH. Sampling was carried out followed by maturation for at least 7 days at a temperature from 2 to 8 ° C (maturation step) without any agitation.
Process :
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A1PC> 4 (Al ³⁺ ) not pH adjusted - ^ 4000 pg of Al ^{3 + /} ml +
PO ₄ (Na / K ₂ ) 10 mM pH 7.15 +
PhtD - ^ 2000 pg PhtD / ml (1/2 PhtD / Al ³⁺ ratio)
j. (magnetic stirring - time (min): 5 to 15 temperature (° C): room temperature) adjust and check the pH 5.0 +/- 0.1 with 0.03 M or 0.3 M HCl
j. (magnetic stirring - time (min): 120 to 150 temperature (° C): room temperature) adjust and check the pH 6.0 + / - 0.1 with 0.05 M or 0.5 M NaOH;
sampling;
maturation: time (min): for at least 7 days - temperature (° C): +2 to +8 - agitation: none
The frozen bulk of PhtD (storage temperature: -70 ° C) is thawed in a thermostatically controlled bath at 25 ° C.
Note: the bulk of the vaccine is kept under stirring throughout the formulation process, the ambient temperature is 18 to 24 ° C.
Table 2
Components Concentration Other Last name Component Antigen PhtD 2000 pg / ml Al ³⁺ AIPO4 4000 pg Al ³ + / ml
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NaCl Residual (+ / -120 mM) PO ₄ Na / K ₂ NaH ₂ PO ₄ .2H ₂ O K ₂ HPO4 or K ₂ HPO ₄ .3H ₂ O Residual (+ / -4.56 mM) pH 6.0 (+/- 0.1)
The DPly / PhtD-AlPCh vaccine can be formulated by mixing sterile NaCl solutions and water for injection (to reach a final concentration of 150 mM NaCl), before adding the required amount of sterile AlPO4 which is added to obtain a final concentration of 0.5 mg of Al ³⁺ per unit dose (0.5 ml) of the final vaccine. The mixture is stirred, the pH is adjusted to 6.1 ± 0.1 and the adsorbed dPly and PhtD are added. After the addition of the monovalent bulk of the antigens, the mixture is slightly stirred at room temperature and if necessary, the pH is adjusted to 6.1 ± 0.1 before the storage of the final bulk in glass containers at a temperature of 2 at 8 ° C.
Example le - Formulation of dPly and PhtD adsorbed on aluminum phosphate
The antigen was pre-adsorbed separately on ΙΆΙΡΟ4 and then pooled to obtain the final vaccine according to the following process.
Process :
PPI water (water for injection) +
1500 mM NaCl +
ad 150 mM
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A1PC> 4 ad 1000 pg Al ³⁺ / ml
j. (agitation magnetic - time (min) : 5 at 15 - temperature (° C) : temperature ambient) adjust and check pH 6, 1 +/- 0.1 with of NaOH 5 to 0.05 M or 0.5 Μ / 0.03 M HCl or 0.3 M j. (agitation magnetic - time (min) : 5 at 15 - temperature (° C) : temperature ambient)
PhtD pre-adsorbed (on AIPO4) - ^ 60 pg of PhtD / ml dPly pre-adsorbed (on AIPO4) - ^ 60 pg of dPly / ml
j. (magnetic stirring - time (min): 15 to 20 temperature (° C): room temperature) adjust and check the pH 6.1 + / - 0.1 with 0.05 M NaOH or 0.5 Μ / HCl at 0.03 M or 0.3 M ï
sampling ï
storage at temperature (° C): +2 to +8
Note: the bulk of the vaccine is kept shaking throughout the formulation process. Note: 20 ad means "up to".
Table 3
Components Concentration Other Last name Component Antigen PhtD 60 pg / ml of 30 pg of dPly each each protein protein by human dose both 300 pg 150 pg of Al ³⁺protein Al ³ + / ml per dose are pre- human
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adsorbedon AIPO4 Al ³ + AIPO4 1000 pg / ml Total: 500 pg Al ³ + per human dose NaCl 150 mM 4.38 mg perhuman dose PO ₄ Na / K ₂ NaH ₂ PO ₄ K ₂ HPO ₄ Residual Water forpreparationinjectable ad 500 μΐ pH 6.1 (+/- 0.1)
(phosphate formulation
Example 2 - Completion of adsorption
Method - Completion of adsorption was measured by measuring the supernatant (SN) of the samples centrifuged by the Lowry method. To estimate the percentage of each antigen adsorbed on the aluminum adjuvant), samples of the (with dPly) of 250 μΐ were centrifuged for approximately 10 minutes at 6000 rpm to separate the non-adsorbed protein (pellet ) of the adsorbed protein (supernatant). 210 μΐ of the supernatant (SN1) were collected for the determination of the completion of the adsorption. The protein concentration in the supernatant was determined by the Lowry method (see details of the method below).
The percentage of adsorption was determined by comparing the amount of detoxified pneumolysin protein in the
BE2017 / 5102 supernatant after centrifugation compared to a control (non-adsorbed detoxified pneumolysin). The percentage of adsorption was calculated as follows:% A = 100 - ([PrSN] x 100 / [PfCtr]) where, [PrSN] represents the concentration of protein in the supernatant and [PfCtr] represents the concentration in the control corresponding non-adjuvanted.
The completion of the adsorption was measured on the day of the formulation (T0), after 21 days at + 4 ° C (T21d4 ° C) and under accelerated conditions (T10d4 ° C + 6d37 ° C). Among the drawings, Figure 1 illustrates the data at T0.
The adsorption of detoxified pneumolysin on aluminum phosphate under different conditions has been studied. The results are presented in FIG. 1. FIG. 1 compares the completion of the adsorption of detoxified pneumolysin (dPly) on aluminum phosphate at different pH and in different ratios of dPly / Al ³⁺ (originating from phosphate aluminum): (i) pH 5.5 to 6.1 and ratio of 1/1, (ii) pH 5.5 to 6.1 and ratio of 1/2, (iii) pH 5.5 to 6.1 , ratio of 1/3, and (iv) pH 6.5 and ratio of 1/3 to T0 (the day of the formulation). Two different lots of the antigen were tested: E-DPLY-P14 and DPLYADA007.
Conclusion - Using the method of Example 1 (pH 5.5 to 6.1, dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/3), the completion was improved by ~ 20 % compared to other methods (pH 5.5 to 6.1 and dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/1; pH 5.5 to 6.1 and dPly / ratio Al ³⁺ (from aluminum phosphate) 1/2;
BE2017 / 5102 pH 6.5 and dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/3) without addition of additional alum
Completion of the adsorption of 96% was observed with the method of Example 1.
After 1 week of storage at 37 ° C, dPly remained adsorbed at more than 95%.
In comparison, a 78 to 83% adsorption completion was obtained from the pH 6.5 adsorption according to the following method.
Process :
AIPO4 (Al ³ +) pre-adjusted to pH 6.5 +/- 0.1 +
PO ₄ (Na / K ₂ ) 2 mM pH 7.15 +
dPly (ratio of 1 pg of dPly / 3 pg of Al ³⁺ ) j. (agitation magnetic - time (min) : 5 at 15 - temperature (° C) : temperature ambient) check or adjust pH 6.5 +/- 0.1 with of NaOH at 0.05 M or 0.5 Μ / 0.03 M HCl or 0.3 M j. (agitation magnetic - time (min) : 5 at 15 - temperature (° C) : temperature ambient) check or adjust pH 6.5 +/- 0.1 with of NaOH at 0.05 M or 0.5 Μ / 0.03 M HCl or 0.3 M j. (agitation magnetic - ' time (min): 120 at 150 - temperature (° C) : temperature ambient)
ï maturation: time (min): for at least days - temperature (° C): +2 to +8 - agitation: none;
sampling
BE2017 / 5102
Note: the bulk of the vaccine is kept under agitation (130 rpm) throughout the formulation process.
BE2017 / 5102
Table 4
Components Concentration Other Last name Component Antigen dPly 1 pg of dPly / 3 pg of Al ³ + Al ³⁺ AlPO ₄ 1 pg of dPly / 3 pg of Al ³ + NaCl Residual PO ₄ Na / K ₂ NaH ₂ PO ₄ K ₂ HPO ₄ ad 1000 pg / mfrom dPly pH 6.5 (+/- 0.1)
Lowry process:
1. To a 200 μί sample, add 200 μί of 10% SDS.
2. Add 1 ml of mixture A and stir. Let sit for 10 minutes.
3. Add 100 μί of reagent B and shake. Let sit for 30 minutes.
4. Place in a cuvette and take the reading at 750 nm.
Mixture A = 50 ml of 2% NaCCb / 0.1 N NaOH + 500 μί of 2% potassium tartrate + 500 μί of CuSO ₄ .5H ₂ O
Reagent B = foline diluted 2x in H ₂ O.
Example 3 - Antigenicity
BE2017 / 5102
The antigenic activity of the adsorbed pneumolysin prepared according to Example 1 (M-dPLY-PO3 and E-DPLY-P01) was determined according to the following method.
Method - The antigenic activity was determined based on the ratio between the protein content by an ELISA test and the protein content by the Lowry method. The ELISA test was used to measure antigenicity after desorption as described below.
Sensitization of the wells - Purified polyclonal guinea pig anti-dPly sera (1807 pg / ml) at 5 pg / ml were diluted 1/400 in PBS and 100 μΐ were added to each well of a microtiter plate. The plate was then incubated for 2 h at 37 ° C.
Four washes were then carried out using 0.9% NaCl + 0.05% Tween ™.
Reference samples:
• M-dPLY-P03 (534 pg of dPly / ml) diluted + / 0.7 pg of dPLY / ml in PBS Tween ™ 20 0 at 0.0 5% (dilution 1/800).
• E-DPLY-P01 (944 pg of dPly / ml) diluted + / 0.5 pg of PS / ml in PBS Tween ™ 20 0 at 0.05% (dilution 1/1800).
The two above reference samples were diluted in 0.05% PBS Tween ™ 20 to reach a concentration of +/- 0.5 μg of dPly / ml and 100 μl were added in the first and the second. well. 100 µl of buffer was added to the other wells. A 1/2 dilution was carried out until the second well ll ^th wells. The plate was kept for 30 minutes at 25 ° C +/- 2 ° C with shaking.
BE2017 / 5102
Four washes were then carried out using 0.9% NaCl + 0.05% Tween ™.
Detection - Detection was carried out using anti-dPly polyclonal rabbit sera diluted to 1/1000 + 1% negative guinea pig serum. The plate was kept for 30 minutes at 25 ° C +/- 2 ° C with stirring.
Four washes were then carried out using 0.9% NaCl + 0.05% Tween ™.
Conjugation - Conjugation was carried out by adding fragment F (ab ') 2 anti-rabbit Ig linked to horseradish peroxidase (donkey (Amersham. NA 9340V) diluted to 1/1000 + 1% guinea pig serum plate negative (negative control) The plate was held for 30 minutes at 25 ° C +/- 2 ° C with shaking.
Four washes were then carried out using 0.9% NaCl + 0.05% Tween ™.
Substrate - OPD (o-phenylenediamine (dihydrochloride)) (Sigma P8787) was used as the chromogenic substrate. A 4 mg tablet was dissolved in 9 ml of H ₂ O, and 1 ml of 1 M citrate buffer pH 4.2, and 5 μΐ of H ₂ O ₂ were added. 100 μΐ of the substrate solution were added to each well of the microtiter plate. The plate was kept for 15 minutes at room temperature in the absence of light.
The reaction was stopped using 50 μΐ of H ₂ SO ₄ 1 N. The spectrophotometer was read at 490 nm and 620 nm.
Calculation - Use of the 4-parameter process using SoftMaxPro software. Only the
BE2017 / 5102
values included Between 25 and 85 o0 of curves of reference and of samples have summer taken in consideration (asymptote superior and lower than the
curve).
The results are shown in Figure 2. Figure 2 shows the Elisa recovery for dPly adsorbed on aluminum phosphate at a pH of 5.5 to 6.1, a ratio of dPly / Al ³⁺ (from phosphate of aluminum) of 1/3. The bars on the left correspond to two different batches of the antigen: dPly E-DPLY-P14 and on the right correspond to dPly DPLYADA007.
Conclusion - Using the method of Example 1 (pH 5.5 to 6.1, dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/3), the Elisa recovery was located at within the acceptable rate.
Example 4 - Particle size
Process - The particle size was measured by DSL (static light scattering) using a Hydro 2000μΡ dispersing unit (Malvern Instruments) at a temperature of 20 to 25 ° C for 20 s using a circulation pump speed of 1500 rpm. 5 µm polymer latex microspheres were used as the size standard. Five measurements were used to calculate the mean size distributions for each sample.
The results are presented in FIG. 3. FIG. 3 compares the percentage of particles of dPly adsorbed on aluminum phosphate less than 10 μm at different pH and in different ratios of dPly / Al ³⁺ (originating from phosphate d 'aluminum):
BE2017 / 5102
(i) pH 5.5 at 6, 1 and 1/1 ratio and (ü) pH 5.5 to 6.1, report of 1/3 • The bars of left to the right match at T0 (time = zero), T7d4 ° C (7 days to 4 ° C), T7d37 ° C (7 day at 37 ° C), T10j4 + 6d37 ° C (10 days to 4 ° C and 6 days at 37 ° C) and T21d4 ° C (21 days at 4 ° C).Conclusion - - Using the process of Example 1
(pH 5.5 to 6.1, dPly / Al ³⁺ ratio (from aluminum phosphate) of 1/3), on average> 95% of the particles of detoxified pneumolysin adsorbed on aluminum phosphate were < 10 pm, within the acceptable rate.
Example 5 Adsorption of Pneumolysin and PhtD Conjugated on Aluminum Phosphate
The preparation of the monovalent bulks of the adsorbed conjugates consisted in the separate adsorption of each of the monovalent bulks of the sterile purified conjugates on AlPCy in a ratio of 1.0 μg of conjugate to 10 μg of Al ³⁺ (as it is presented in Table 3) according to the method presented below. The monovalent bulk of the purified conjugates were mixed with ΙΆΙΡΟ4 (previously adjusted to the specific adsorption pH of the serotype), and with the 150 M sodium chloride solution. The mixture was stirred for 15 to 45 minutes at room temperature (AT ).
The mixture was then adjusted to a pH in the range of 5.2 to 6.1 (see Table 3) and slightly stirred for 2 hours at room temperature for adsorption to occur.
BE2017 / 5102
A final adjustment of the pH to 6.1 ± 0.1 took place before the storage of the monovalent bulk of the conjugates adsorbed at a temperature of 2 to 8 ° C. The maturation of the monovalent bulks of the adsorbed conjugates lasted at least 7 days at a temperature of 2 to 8 ° C.
Process :
AIPO4 (pH pre-adjusted as described in Table 5) +
diluent NaCl 150 mM i
stirring until homogenized at RT (room temperature) i
addition of bulk of conjugate i
agitation until homogenized at TA i
pH adjustment (serotype specific pH adjustment, Table 5) i
shaking 2 h ± 15 min at RT i
pH adjustment to 6.1 ± 0.1 i
minimum maturation 7 days at a temperature of 2 to 8 ° C i
storage at 2 to 8 ° C Table 5
BE2017 / 5102
Conjugate ofserotype PH adjustment PS / A1 ^{3+ report} PS19A-dPly 6.1 1/10 PS22F-PhtD 6.1 1/10
Identity of S. pneumoniae polysaccharides by an ELISA test
The samples were centrifuged and the supernatants were collected and stored at 2-8 ° C before use. Analysis was performed on the supernatant. For the monovalent bulk of 19A-dPly, the microtiter plates were sensitized with polyclonal anti-Ply guinea pig antibodies and incubated for 2 hours at 37 ° C. and for the monovalent bulk of 22F-PhtD, the microtiter plates were sensitized with anti-PhtD guinea pig polyclonal antibodies and incubated for 2 hours at 37 ° C. The plates were washed with an NaCl solution containing 0.05% of polysorbate 20 (Na Tween 20) after each incubation step. After washing the plate, serial dilutions of the standard material, the internal control of the supernatant samples, were prepared in phosphate buffer solution supplemented with 0.05% polysorbate 20 (PBS Tween 20). These serial dilutions were tested in duplicate. The plates were incubated overnight at 2-8 ° C. After washing with the Na Tween 20 solution, the microtiter plates were incubated. For 19A-dPly, the incubation was carried out with anti-PS rabbit polyclonal antibodies, supplemented, if necessary, with guinea pig serum
BE2017 / 5102 negative for 30 minutes at 25 ° C. For 22F-PhtD, the incubation was carried out with anti-PS rabbit polyclonal antibodies, supplemented, if necessary, with negative guinea-pig serum for 30 minutes at 25 ° C. Following washing, the captured 19A-dPly antigen was incubated with peroxidase-conjugated goat anti-rabbit IgG, supplemented, if necessary, with negative guinea-pig serum for 30 minutes at 25 ° C. The captured 22F-PhtD was incubated with anti-rabbit goat IgG conjugated to peroxidase, supplemented, if necessary, with negative guinea-pig serum for 30 minutes at 25 ° C. After washing, the enzyme substrate, ortho-phenylenediamine supplemented with 30% H 2 O 2, is added. After incubation in the dark for 15 minutes at room temperature, the reaction was stopped with 1.0 N H2SO4. The absorbance was measured by spectrophotometry at 490 nm and 620 nm. The identity was positive when the absorbances were higher than those of the background.
Total polysaccharide content by resorcinol Dilutions of the standard material, a batch of purified polysaccharide (PS) of the serotype concerned, in 150 mM NaCl were used to establish the calibration curve. The test samples of monovalent bulk adsorbed were diluted in 150 mM NaCl in order to have a content lying within the range of the calibration curve. Resorcinol reagent and sulfuric acid were added to each of the samples. After homogenization, the samples were incubated for 30 min at 100 ° C. For the
BE2017 / 5102 serotypes 4 and 5, the samples are incubated for minutes at 100 ° C. The temperature of the samples was then lowered to room temperature for 30 min.
The yellow-orange color was measured by spectrophotometry at 430 nm.
The PS content was calculated from the PS calibration curve.
Completion of adsorption on the adjuvant (% of unbound PS)
Adsorption completion is determined on monovalent bulk adsorbed by an ELISA test (Enzyme Linked Immunosorbent Assay). The test was an anti-support / anti-PS ELISA test and the same as that used for identity analysis. After centrifugation of the monovalent bulk adsorbed, the non-adsorbed conjugates present in the supernatant were measured by an appropriate ELISA test (anti-dPly / anti-PS, anti-PhtD / anti-PS). The completion of the adsorption was expressed in% (quantity of the conjugate measured in the supernatant over the total PS content of the monovalent bulk adsorbed measured by the resorcinol test).
BE2017 / 5102
Table 6
Tests for bulk adsorbed PS19A-dPly
Tests D19AFAA001 Identity of the polysaccharide conjugate ofS. pneumoniae 19A-dPly by ELISA Positive Content of type 19A polysaccharide ofS. pneumoniae free by ELISA <1.0% Completion of adsorption onadjuvant (% of polysaccharide conjugateunbound S. pneumoniae 19A-dPly) <1.0%
Table 7
Tests for bulk adsorbed PS22F-PhtD
Tests D22FHAA001 Identity of the polysaccharide conjugate ofS. pneumoniae 22F-PhtD by ELISA Positive 22F polysaccharide content ofS. pneumoniae free by ELISA <1.0% Completion of adsorption onadjuvant (% of polysaccharide conjugateunbound S. pneumoniae 22F-PhtD) <1.0%
BE2017 / 5102
SEQ ID NO: 1
GenBank EF413952
MANKAX ^ O-lLAbWiMKKELiHGGESIENREiKEGNGLRBEFWIERKKRBLSTMï'SDfB
VTATNBSRLYPGAU.YYDEKLERNPTLLAVBRAPGTYBIDIPGLABSDSFLGYFDPBRB
SVRGAVWLLAKyWIGEWQVYWPARMQYEKITAHSMEQt ^ KFÖSDFEKTGNSLDID
FFWHSGEKGiGIVNFKOlWTVSVDAVKNPGBVFGDT'YPYEDLKGRGlSAERR.WiBSV
ÄYGRGWLKtETTSKSDBYEAAfEÄEiKGVKVAPGTEWKGILDNTEVKÄY'lLGGDRSSGA
RVVTGFVGMVEDLjGEGxARFTADHPGLRIGYTTSFLRDRVYATFGRSTDYYETRVTAYPN
GDLLLDRSGAYYAGYYFPYHELSYDRQGKfcVLTPkAWDRNGQDtTAHETTBsPLKGRVR
RLGYKIRECTGLAWEWWRTVYERIGLRlVRKRTISIWGT'ilYRGYEBRYEHD
SEQ ID NO: 2
GenBank EF4I39S3
MANKAVNL> nLAMNyDKKKLt'THaGEGI £ NRfMEGNŒ.PDEEWIERKKRGLSWFSŒS
Y4ATRGGRL.YRGAG.VYDETG..ERRRTG.AY0RAPF1TYBIDI.PGLABS0SFLGYEDPBGB
SVRGAVGDtLAKWRGOYGGVGWPARFIGYERGAHSMBGiRVEFGSDFEETGHSLOlO
FRFYBGGERGKJFFRFPGÎYYTYGVDAYKRPGGYFGDTVTVFGÎRGRGISAERPLVYIBGV
AYGRGYYLKLETTBKSGEYEAAFEAGKGYKYAPGTEWRGEDNYEYRAVEGGÖRSGGA
RWlGRYOGVFDLIGEGBRF'rAEPIRGLRIBY'nSFLRBGVVATFGRSl'OYVElRYIAYRR
GDLl.LDRSGAWAGYYlTYVRELSYDHQGFFVLJPKAWDRRGGDFTARFTTSäPL.RGRVR
RLBVKIREGTGÎAWEWWRP2YERTDLPLVRKRTk5FY3GTTLYRGVEDKVERD
SEQ ID NO: 3
GARBî EF413954
F1ARRAvWDFIlAPP1YDKRKLFTRGGESiERl7E1REGRG (.PDEFV53ERRRRBLBTRTSDIG
VTATNDSRLYPGM.LWDEÎlLEHNPTLLÂVORARMTVSIOLPGtASSOSFLGVEDPSHS
SVEGAVRDLLÂKWKODYGOVRRVPARMÛYEKGAHSGEGLKVKFGSDFEKÏGGStGD
FRSYHBGERG1OfYRFRGiYYTVSVöAVRRPGÖYFGDTVTVEGLRGRGiSAER.Pt5 ⁱ YIBSV
AYGRGVYEELETÏBRSÔEYFAAEEAUFGVKVAPGTEWKGF.DRÏEVRAVILGGDRBGGA
RWTGODMVEDLiGEGBRFTAORRGLPiSYTTSFLPGRYYATFGRSTOYYETKVTAYPR
GBLLtEYIGGAYVAGYYiTWDELSYGRGGKEYEYPRAWDRRGGrOAHFTTSFR.KGRVB
NtGYRIRBGTGLAWEWWRWYEKTDLPtYRRRTIRWGTTLYPGVEôRVERO
SEQ ID NO: 4 GenBank EF413955
FIARKAYNOFÏLAMRYÜRKRLLTHGGEeeNRFlREGRGLPDEFVYiERRRRSLSTRTSGiS
VTATGÔSRLYPGALlÀR ^ DETEtERGPÏtLAÀ'GRAPPGYSIDLPGLASSBSFLGVEDPSRB
SVRGAYRDLtARWRQDYGQIiRRYPARRGYFRRARSBIEGLKYEFGBDFEKTGRSFRIR
FRGYHSGERGIGIvRFKGiYYTYGvGAYRRRGOYFGGIVTVEGLRGRGIGAERPLWISGY
AYGRGVYLKLFTTBRROBYEAAFBAtiRGYKYAPGTElYRatGNTBYKAVEGGORSSGÄ
PVvTGKWMVEDtfâEGSRFTAÔRPGLRSYÎTSFLRDRYYATFGHSTGYVETRYTAYRN
GDtDLDE3GAYVAGYY1TWDELSYDRQGR £ VLTPKAWDRHGÖDLIAHFTTSäFLRGRVR
RLBVRiREGTÔtÂWEWWRTYYEKÏDLPLVRKRGAïWGTîLYRGYEDKVEHü
SEQ ID NO: 5
BE2017 / 5102
GenBank ER 13959 (ply-2) ^ AHKAVNDFILMWfMKKLLIHCX ^ . SIE "FIKEGN <>. PDEFW'r: RKKRSLSll <SDkS
VWWSREYPGALLVVDeTLtENHPTELAVDRAPMIYEiiOLPGLASSDSFLOVEaPSNS svRGAynDLtAKWHQDYGQyanvFARGGYEKnAnsGFGLFARFGSDFEKTGNSLDiD
FNSVHSGEKQIŒVNFKOiy’mwnÂVKNPGDVFQDTVTVESLKGRGkSÂERPLWfSSV
AyGPGVYiKŒTÏSKSŒVEAAFGAUAGYYAGPGTEWQGCAŒVKAVkGGDPSSGA
RVVTGKVDMVEOtlŒGSRfTAGHPGLRSVTTSELRDNVVÂTFQHSTDYVETKWAYRM
GnLtLLY-GGAYVAGYYPWN £ LSYFyiGŒEVLl’AK.AWDRGGGPL'FAFfPrïGPLKGNVR
NLSYKIREGTGLAWE'AAYRTVYeKTnLR..YnKRT AFA / GTTLYPQvEOA'YEND
SEQ ID NO: 6: kteLy8L “} LysThrtsuAi <.ei S“ <“uLeaAIa ^ a <” yv “ILsuAis <5ify
CYsSarSerHGSnrSerAsnkfetAGAFnTnfGInkfeGyeSerAspLysIG
ItelleAlsHisArgGlyAlaSerGlyTyftwProGfoHteThrtwGIuS ^ FtysAia
LnuAGPheAbQnGGGAAepTyrLnuGluGh'sAApteuAGGeGhrlysAspGIy ^ • gL © “VaiyaOfeHteÄspHisRh © L ^ uÄ ^ GIyi“ uThrÄspVMAIaLysLysPh &
Pn3HGArgHAApiLy'SAspGlyAA3TyrTyrValkAAspPhnThrLetk.y'SGk.kG
GlnSefteuGtaMetTb'GluAsnPheGlyOirLysÄ ^ pC ^ yLysGlaAlöOnysITyr
PnYAsAAR ^ PlYsPföLauTrpLysSerHIsPhaÄfglGHGTG'PhaGluAspGkilte
GkiPhalGGInGM.enGkfLysSerTkrGlyLysLy3yesGlyknTyrProGkkte
LyvAGPfoTrpPnaHfeHGGkiAsnGlyLyAAspkeAbAGGGThi'LnnLysyel
L en L ysL y vTyrGiy Ty r.AepL y <.ysThfAspPI eGGOyrL mOnThrPheAepPhe ·
AsnGh.CenLysAigkeLyFlYiiGksteutAnRöGlnMGGGPfeWpteuLyFteu
VGGGtaukeAGTyii'hAAspG'ptysGYinY'GInGbLyFAspRötyFGGTyi
TipVckAsnTyiAsnTyï.AspTîpFtetlAieLyYR'GAâyAtePGtAGGfaVaGnAys
TyäGGÄ3pGfyY <3äGlyPfnGBTipTy 'MÄeuVAlAsnLysGkäGkfSeilysPRi
AspAsikGVsG'yG'hrPfnLeisYAGyFGkätAuAGGinTyrAsä ^ YGGkiVaäHG
PmTyrThfValÄr ^ LysAs ^ AlaLwR ^ GI "Ph8Ph @ ThFAspWIAsfiGfe> fclTyF
AspAGLetGewkAnLysGFYGkAGThfGGVdlPneThrAnpPnePreAspTnrGly ⁵
VGGlöPhetnuLysGGlteLyA
3EQ ID NO7FGtGppRGsGsinAS ^ GiSfAss'iYfeGisAssAhrGsnPkfSLysetefAsptysSfe
OalkïAteHiSiAnjGiyAkfSerGlyTyrtauProGâuHIsîhrtauGluSeïLysÂÎÎf teuAiaPneAlaGinGlnAbAspïyrLeuGluCilnAspLeuAîaMeiïhrLysAspGiy
ArgLeaVary'a ^ eHiGAspHGPheLeuAspGlyLeaThFAepYYilAlaLysLysPhe
FFoHBArgHisArgLysA ^ pGlyArgTyFTyrVàOb ^ PhiëThfLmiEysGfelle
GlnSen_eui3kiMeiThfGSuAsnPheGluThrLysAspGlyLysGkiÂiaGlnValTyr
Pf'oAi-fWgPhePFoLeuTFpLysSAiHlsPhe ^ 'glleHlsThfPheGiuAspGIul ^
GluPhelieGInGiyteuGluLysSe & ThrGlytysiysVsIGlyläeTyrPFoGiulte
100
BE2017 / 5102
LysAJaPfoTrpPheHisHisGinAsnGiyLysAspHeAfeAiaGiuThfteuLysVal
Leul..ysLysTy5GiyTyiAspi..ysLysThrAspMetValTyrteuGhiThrPheAspPh ^
AsaGiuLaaLysAigllatysThrGiaLeuLqaProGiaMelGiyMetAspLeuLysUîa
VoiGsnLêuheÂioiyr'rhrAspIï |: îLy Giu'ïhrGlnGKCys.AspProLysGiyiÿr
TrpValAsnTyrAsaTyrAspTrphtelPheLysProGlyAiök ^ tAiaGiuValV'aiLys
TyrAiàAsp (3iyVoOyPfoGlyT PTyr ^ 1etLeuValAsntysGluGlüSefLysPrô
ÄspA & nh £ A / aiTyfThrPmLbuVaU.ysGiuLeuAiaGinTyrAsnVaiGkiVaiHis
ProTyrThrVâiArgtysAspAïqlêiiProGkîPhePhêThrAspVolAsnGInhlêlTyr
ÂspAh <.euLeaAsnLysSeBGiyAkïihiGiy'VafPaeïhfAspPhePsûAspThK’My
ValGluPheLe ^ LyaGfyifeLyts
SEQ ID NO
SqrSbfHGSerSerAsnMetAisAsnThr
101
BE2017 / 5102

权利要求:
Claims (30)
[1]
1/2 to 1 / 2.5, or 1/2) (w / w; weight / weight).
1 / 1.5 to 1 / 2.5, 1/2 to 1 / 2.5, 1 / 2.5 to 1 / 3.5, 1/3 to 1 / 3.5, 1/2 or 1 / 3) (w / w; weight / weight).
1. Immunogenic composition having a pH between 6 and 7 (for example a pH 6.0 to 6.5, a pH 6.0 to 6.2, or a pH 6.1) and comprising detoxified pneumolysin adsorbed on aluminum phosphate, in laguelle more than 85% (for example, more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of the detoxified pneumolysin is adsorbed on aluminum phosphate and more than 80% (e.g. more than 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89% or 90%) of the detoxified pneumolysin adsorbed on aluminum phosphate has a particle size of less than 10 µm.
[2]
2. Immunogenic composition according to claim 1 in laguelle more than 95% of the detoxified pneumolysin is adsorbed on aluminum phosphate.
[3]
3. Immunogenic composition according to claim 1 or 2, in laguelle the detoxified pneumolysin was chemically detoxified.
[4]
4. Immunogenic composition according to claim 1 or 2, in laguelle the detoxified pneumolysin was genetically detoxified.
[5]
5. An immunogenic composition according to claim 1 or claim 2, in which the detoxified pneumolysin is unconjugated.
[6]
6. Immunogenic composition according to one guelcongue of claims 1 to 5 in laguelle the detoxified pneumolysin is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
i.
[7]
Immunogenic composition according to one of the claims 1 to 6, further comprising PhtD adsorbed on aluminum phosphate.
[8]
8. Immunogenic composition according to claim i, in laguelle the PhtD is unconjugated.
102
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[9]
9. Immunogenic composition according to claim 7, in laguelle the PhtD is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
[10]
10. Immunogenic composition according to one guelcongue of claims 1 to 9 further comprising 10 or more capsular polysaccharides of S. pneumoniae of different serotypes of S. pneumoniae conjugated to one or more support proteins.
[11]
11. A method for the adsorption of detoxified pneumolysin on aluminum phosphate comprising the step of (i) mixing the detoxified pneumolysin and aluminum phosphate having a particle size of less than 10 μm, at a pH of less than 6 , 5 (for example, below pH 6.0, pH 5.0 to 6.2, pH 5.0 to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to 6.1, pH 5.5 to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5 , 7, pH 5.5 to 5.7, pH 5.4 to 5.6 or pH 5.5).
[12]
12 in leguel step (i) is carried out in the presence of a phosphate buffer, optionally comprising NaPhPCy and K2HPO4, and optionally at a concentration between 1 mM and 5 mM (for example, between 1 mM and 3 mM, between 2 mM and 2.4 mM, or 2 mM).
12. The method of claim 11 in leguel detoxified pneumolysin and aluminum phosphate are in a ratio of dPly / Al ³⁺ (from aluminum phosphate) in step (i) between 1 / 1.5 to 1/4 (for example, 1 / 1.5 to 1 / 3.5,
[13]
13 followed by step (ii) of adjusting the pH of the composition to a pH between 6 and 7 (for example, pH 6, 0 to 6.5, pH 6, 0 to 6.3, or pH 6 , 1).
13. Method according to one guelcongue of claims 11 or
[14]
14 followed by step (iii) of mixing the detoxified pneumolysin adsorbed with one or more antigens other than the detoxified pneumolysin (for example, PhtD).
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14. Method according to one guelcongue of claims 11 to
[15]
15. Method according to one guelcongue of claims 11 to
[16]
16. The method of claim 15 wherein step (iii) comprises mixing the adsorbed detoxified pneumolysin with pre-adsorbed PhtD.
[17]
17. The method of claim 16 wherein the pre-adsorbed PhtD is prepared by mixing PhtD with aluminum phosphate at a pH of 4.5 to 5.5 (for example, pH 4.5 to 5.4 , pH 4.7 to 5.2, pH 4.9 to 5.1, or pH 5.0) and / or using a PhtD / Al ³⁺ ratio (from aluminum phosphate) between 1 / 1 and 1/3 (for example, 1/1 to 1 / 2.5, 1 / 1.5 to 1 / 2.5,
[18]
18. The method of claim 17 wherein the mixing of PhtD with aluminum phosphate is carried out in the presence of a phosphate buffer, optionally comprising NaPhPCy · IH2O, K2HPO4 and / or K2HPO4.3H2O, and optionally a concentration between 5 mM and 15 mM (for example, between 8 mM and 12 mM, or 10 mM).
[19]
19. Method according to any one of claims 16 to 18 in which the PhtD is unconjugated.
[20]
20. A method according to any one of claims 16 to 18 wherein the PhtD is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
[21]
21. A method according to any one of claims 11 to 20 wherein the detoxified pneumolysin is unconjugated.
[22]
22. A method according to any one of claims 11 to 20 wherein the detoxified pneumolysin is conjugated to a saccharide, for example a capsular saccharide of S. pneumoniae.
[23]
23. A method of preparing an immunogenic composition comprising detoxified pneumolysin, comprising the method according to claims 11 to 20.
[24]
24. Immunogenic composition according to any one of claims 1 to 10 prepared by the method according to claims 11 to 23.
104
BE2017 / 5102
[25]
25. A vaccine comprising the immunogenic composition according to any one of claims 1 to 10 or 24 and a pharmaceutically acceptable excipient or carrier.
[26]
26. A method for the treatment or prevention of a Streptococcus pneumoniae infection in a subject in need thereof (for example, a human being) comprising administering to said subject a therapeutically effective amount of an immunogenic composition according to the invention. any of claims 1 to 10 or 24 or the vaccine of claim 25.
[27]
27. A method of immunizing a human host against a Streptococcus pneumoniae infection comprising administering to the host an immunoprotective dose of the immunogenic composition according to any one of claims 1 to 10 or 24 or of the vaccine according to claim 25.
[28]
28. A method of inducing an immune response against Streptococcus pneumoniae in a subject (eg, a human), the method comprising administering an effective amount of any vaccine according to the therapeutically or prophylactically immunogenic composition according to one of claims 1 to 10 or 24 or claim 25.
[29]
29. An immunogenic composition according to claims 1 to 10 or 24 or a vaccine according to claim 25 for use in the treatment or prevention of a disease caused by infection with Streptococcus pneumoniae.
[30]
30. Use of the immunogenic composition according to claims 1 to 10 or 24 or of the vaccine according to claim 25 in the manufacture of a medicament intended for the treatment or prevention of a disease caused by an infection with Streptococcus pneumoniae.
105
BE2017 / 5102

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同族专利:

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公开号 | 公开日

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BE1024489A1|2018-03-07|

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US11266731B2|2022-03-08|

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US20210187090A1|2021-06-24|

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WO2017144394A1|2017-08-31|

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GB201603029D0|2016-04-06|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2004081515A2|2003-03-13|2004-09-23|Glaxosmithkline Biologicals S.A.|Purification process for bacterial cytolysin|

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WO2006032499A1|2004-09-22|2006-03-30|Glaxosmithkline Biologicals S.A.|Purification process for bacterial cytolysin|

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US20140105927A1|2012-10-17|2014-04-17|Glaxosmithkline Biologicals S.A.|Immunogenic composition|

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法律状态:
2018-03-21| FG| Patent granted|Effective date: 20180312 |

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2019-11-20| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190228 |

优先权:

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申请号 | 申请日 | 专利标题

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GBGB1603029.8A|GB201603029D0|2016-02-22|2016-02-22|Vaccine|

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GB1603029.8|2016-02-22|

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