Pneumococcal vaccine and using it

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and concerns a pneumococcal vaccine containing saccharide of 4, 6B, 9V, 14, 18C, 19F and 23F serotypes, separately conjugated with CRM197, and at least one Toll-like receptor-9 (TLR-9) agonist as an adjuvant, wherein the above Toll-like receptor-9 agonist represents CpG-oligonucleotide. Also, the group of inventions concerns using the pneumococcal vaccine for preparing the therapeutic agent for preventing or treating diseases caused by S.pneumoniae; a method for individual's immunisation against the diseases caused by S.pneumoniae infection, involving administering the immunoprotective dose of the vaccine into the above individual.

EFFECT: adding B-class CpG to the conjugated pneumococcal vaccine provides the considerable increase of the proportion of highly responsive patients, good tolerance and cell memory induction in relation to pneumococcal polysaccharides.

45 cl, 9 dwg, 4 tbl, 3 ex

 

The SCOPE of the INVENTION

The present invention relates to new pneumococcal vaccines. The invention also relates to the vaccination of subjects, particularly subjects with impaired immunity against pneumococcal infections with the use of these new pneumococcal vaccines.

PRIOR art

Pneumococcal diseases are a major health problem worldwide. Infections caused by pneumococci, are an important cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia and meningitis are the most common manifestations of invasive pneumococcal disease, while the spread of bacteria through the respiratory tract can lead to middle ear infections, sinusitis or recidivisim bronchitis. Compared with invasive non-invasive disease symptoms are usually less severe, but occur much more frequently.

Despite the importance of pneumococcal disease, information about the damage from the disease, particularly in developing countries, is not enough. This is partly due to the inevitable problem of setting an etiologic diagnosis in cases of pneumonia. However, according to available data acute respiratory infections result in the death of approximately 2.6 million children under five every year. Pneum cock is responsible for over 1 million of these deaths, most of which occur in developing countries, where the pneumococcus is probably the most important pathogen in children of early age. In Europe and the United States, pneumococcal pneumonia is the most common community-acquired bacterial pneumonia, which, according to estimates, affects approximately 100 per 100,000 adults per year. The corresponding figures for febrile bacteraemia and meningitis are 15-19 per 100,000 and 1-2 per 100,000, respectively. The risk of one or more than one of these manifestations is much higher in children and the elderly, and people with weakened immunity of any age. Even in economically developed areas of invasive pneumococcal disease causes high mortality rate; adult mortality from pneumococcal pneumonia is an average of 10%-20%, at the same time, it can exceed 50% in high-risk groups. It is recognized that pneumonia is the most frequent cause of death from pneumococcal disease worldwide.

The etiological agent of pneumococcal disease Streptococcus pneumoniae (pneumococcus) is a gram-positive encapsulated cockcom, surrounded by a polysaccharide capsule. The differences of the composition of the capsules allow serologically distinguish approximately 90 capsular types, some of which often include the s with pneumococcal disease, others rarely. Invasive pneumococcal infections include pneumonia, meningitis and febrile bacteremia; frequent non-invasive manifestations are otitis media, sinusitis and bronchitis.

The resistance of pneumococci to the main antimicrobial agents such as penicillins, cephalosporins and macrolides, is a serious and rapidly growing problem throughout the world.

Conditions associated with an increased risk of serious pneumococcal disease include children and old age (children, elderly) and a weakened immune system for any reason, including, without limitation, infection caused by the human immunodeficiency virus (HIV), other chronic viral infections, sickle cell anemia, diabetes, cancer and cancer treatment, Smoking, chronic organ failure, organ transplantation and immunosuppressive therapy.

The recent development of widespread resistance of microorganisms to basic antibiotics and the increasing number of people with weakened immune systems emphasize the urgent need for more effective pneumococcal vaccines.

Some of the disadvantages of modern vaccination include: the need for multiple injections to ensure protective effect; delay production of protective antibodies; the prevalence of people who do not respond to the vaccine (this PR the problem is especially important in individuals with a weakened immune system); the cost of obtaining antigens and vaccine manufacturing, which is a very important constraint in the development of new conjugated pneumococcal vaccines; weak protective properties of antibodies with low affinity; a decrease in antibody titers over time.

The task of the new pneumococcal vaccine according to the invention is at least partially overcome some of these shortcomings, in particular, with the aim of vaccination of subjects with impaired immunity against pneumococcal infections.

SUMMARY of the INVENTION

In the first aspect of the present invention is directed to new pneumococcal vaccines containing one or more than one antigen pneumococcal polysaccharides conjugated to protein carrier, as an antigen and an agonist of Toll-like receptor-9 (TLR-9) as adjuvant.

In another aspect of the present invention is directed to the use of pneumococcal vaccine containing one or more than one antigen pneumococcal polysaccharides conjugated to protein carrier, as antigen and TLR agonist-9 as an adjuvant for vaccination of subjects with weakened immune systems.

In one aspect the invention is directed to any pneumococcal vaccine disclosed here, for use in the vaccination of subjects with a weakened immune system, predpochtitel what about any of the subjects with a weakened immune system, disclosed here.

In another aspect of the present invention is directed to the use of any pneumococcal vaccine, disclosed here for the vaccination of subjects with a weakened immune system, preferably any of the subjects with a weakened immune system disclosed here.

In another aspect of the present invention is directed to any of the vaccine disclosed here, for the prevention or treatment of diseases caused by S. pneumoniae infection, preferably by a subject's immune system is weakened.

In another aspect of the present invention is directed to a method of immunization of a subject, preferably any of the subjects with a weakened immune system disclosed here, against diseases caused by S. pneumoniae infection, including the introduction of a specified subject immunoprotective dose of any vaccine disclosed here.

In another aspect of the present invention is directed to the use of any vaccines disclosed here, for the manufacture of a medicine for the prevention or treatment of diseases caused by S. pneumoniae infection, preferably by a subject's immune system is weakened.

In another aspect of the present invention is directed to any pneumococcal vaccine disclosed here, and at least one agonist of TLR-9, disclosed here.

In another aspect of the present invention is directed to any of pneumococcal vacci is s, disclosed here, and at least one agonist of TLR-9, disclosed here for use in the vaccination of any entity with a weakened immune system disclosed here.

Agonist of Toll-like receptor-9 (TLR agonist-9) according to the invention

In one embodiment of the present invention, the agonist of TLR-9 for use in the present invention is a CpG oligonucleotide. CpG-oligonucleotide when used here refers to immunostimuliruyushhim CpG-oligodeoxynucleotide (one CpG), and, accordingly, these terms are used interchangeably, unless otherwise noted. Immunostimulatory CpG-oligodeoxynucleotide contain one or more than one immunostimulating CpG-motif, representing demetilirovanny dinucleotide cytosine-guanine, possibly surrounded by certain preferred bases. The state of methylation immunostimulating CpG-motif usually refers to casinogame remaining dinucleotide. Immune-stimulating oligonucleotide containing at least one demetilirovanny CpG dinucleotide, is an oligonucleotide that contains a 5'-demetilirovanny cytosine associated phosphate bond with the 3'-guanine, and activates the immune system by binding to Toll-like receptor-9 (TLR-9). In another embodiment of the immune-stimulating oligonucleotide may contain one or more of the eat one methylated CpG dinucleotide, activating the immune system through TLR9, but not as much as if CpG motif(s) was(and is) demetilirovanny(and). Immunostimulatory CpG-oligonucleotides may contain one or more than one palindrome, which, in turn, may include a CpG dinucleotide. CpG oligonucleotides described in several issued patents, published patent applications and other publications, including U.S. patents№6194388, 6207646, 6214806, 6218371, 6239116 and 6339068.

Were identified various classes of immunostimulatory CpG-oligonucleotides. They are called classes a, b, C and P, and they are described in more detail below. The methods of the invention include the use of immunostimulatory CpG-oligonucleotides of these different classes.

Oligonucleotides of any class may be subjected to E-modification, enhancing their activity. E-modification can be a substitution of the 5'-terminal nucleotide halogen; examples of such substitutions include, without limitation, the substitution bromouridine or iodouridine. E-the modification may also include the replacement of the 5'-terminal nucleotide by amiloride.

Immunostimulatory CpG-oligonucleotides "class a" functionally characterized by their ability to induce high levels of secretion of interferon-alpha (IFN-α) plasmacytoid dendritic cells (pDC) and the induction of the activation of natural killer cells (NK-cle is OK) with minimal effects on the activation of b-cells. Structurally, this class of typical stable poly-a sequence at the 5'- and 3'-ends. He also has palindrome sequence with phosphodieterase links that contain CpG dinucleotides, at least 6 nucleotides, for example, but not necessarily, it contains one of the following review of palindromes: GACGTC, AGCGCT or AACGTT described by Yamamoto and colleagues. Yamamoto S et al. J. Immunol 148:4072-6 (1992). The class of immunostimulatory CpG-oligonucleotides and typical sequences of this class have been described in nepredvidatelne application for U.S. patent No. 09/672126 and published PCT application PCT/USOO/26527 (WO 01/22990), filed September 27, 2000

In one embodiment of the CpG-oligonucleotide "class a" according to the invention has the following nucleic acid sequence: 5' GGGGACGACGTCGTGGGGGGG 3' (SEQ ID NO:1).

Some non-limiting examples of oligonucleotides class And include:

5' G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G 3' (SEQ ID NO:2); where * refers to phosphorothioates communication, and _ refers to a phosphodiester bond.

Immunostimulatory CpG-oligonucleotides "class" functionally characterized by their ability to activate b cells and pDC, except for the relatively weak induction of IFN-α and activation of NK-cells. Structurally, the oligonucleotides of this class can usually be fully stabilized phosphorothioate links, but they may also have one or more h is m one fosfodiesterazu communication, preferably between cytosine and guanine CpG motif (CpG motifs), in which case the molecule is called semi-soft ("semi-soft"). In one embodiment, the agonist of TLR-9 for use in the present invention represents a' CpG-oligonucleotide class represented at least by the formula:

5' X1X2CGX3X43', where X1, X2, X3and X4represent nucleotides. In one embodiment X2represents adenine, guanine or thymine. In another embodiment X3represents cytosine, adenine, or thymine.

In another embodiment, the agonist of TLR-9 for use in the present invention is a CpG oligonucleotide class represented at least by the formula:

5' N1X1X2CGX3X4N23', where X1X2, X3and X4are nucleotides and N is any nucleotide and N1and N2are sequences of nucleic acids, each of which consists of approximately 0-25 N. In one embodiment X1X2is a dinucleotide selected from the group consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TPT, and TpG; and X3X4is a dinucleotide selected from the group consisting of TPT, ApT, TpG, ApG, CpG, TPC, APC, CPC, TpA, ApA, and CpA. Preferably, X1X2/sub> is a GpA or GpT, and X3X4represents the TRT. In other embodiments, X1or X2either X1and X2represents a purine, and X3or X4either X3and X4represent a pyrimidine, or X1X2is a GpA, and X3or X4either X3and X4represent pyrimidine. In one preferred embodiment X1X2is a dinucleotide selected from the group consisting of TPA, APA, APC, ApG and GpG. In yet another embodiment X3X4is a dinucleotide selected from the group consisting of TPT, Tra, TpG, Ara, ApG, GpA and CPA. In another embodiment X1X2is a dinucleotide selected from the group consisting of TPT, and TpG, ApT, GpC, CpC, CpT, TpC, GpT and CpG; X3is a nucleotide selected from the group consisting of a and T, and X4represents a nucleotide, but when X1X2represents TpC, GpT or CpG, X3X4is not a TpC, ApT or ARS.

In another preferred embodiment of the CpG oligonucleotide has the sequence 5' TCN1TX1X2CGX3X43'. In some embodiments CpG-oligonucleotides according to the invention contain X1X2selected from the group consisting of GpT, GpG, GpA, APA, and X3X4selected from GRU the dust, consisting of TPT, CpT and TpC.

Sequences of CpG-oligonucleotides class according to the invention are sequences that are widely described above and disclosed in published PCT patent applications PCT/US95/01570 and PCT/US97/19791 and in U.S. patents№6194388, 6207646, 6214806, 6218371, 6239116 and 6339068. Typical sequences include, without limitation, the sequence disclosed in these applications and patents.

In one embodiment of the CpG-oligonucleotide "class" according to the invention has the following nucleotide sequence:

5' TCGTCGTTTTTCGGTGCTTTT 3' (SEQ ID NO:3), or

5' TCGTCGTTTTTCGGTCGTTTT 3' (SEQ ID NO:4), or

5' TCGTCGTTTTGTCGTTTTGTCGTT 3' (SEQ ID NO:5), or

5' TCGTCGTTTCGTCGTTTTGTCGTT 3' (SEQ ID NO:6), or

5' TCGTCGTTTTGTCGTTTTTTTCGA 3' (SEQ ID NO:7).

In any of these sequences, all communication can be phosphorothioate communication. In another embodiment, in any of these sequences, one or more than one link can be fosfomifira, preferably between "C" and "G" CpG motif that makes CpG-oligonucleotide semi-soft. In any of these sequences 5' T can be replaced by amiloride or halogen; examples of the halogen substitutions include, without limitation, the substitution bromouridine or iodouridine.

Some non-limiting examples of oligonucleotides class include:

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3' (SEQ ID NO:8), or

5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3' (SEQ IDNO:9), or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3' (SEQ ID NO:10), or

5' T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3' (SEQ ID NO:11), or

5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3' (SEQ ID NO:12),

where * refers to phosphorothioates connection.

Immunostimulatory CpG-oligonucleotides "class With a" functionally characterized by their ability to activate b-cells and NK-cells and to induce IFN-a. Structurally, the oligonucleotides of this class typically contain a region with one or more than one immunostimulatory CpG motif of class type and the palindrome or region, similar to the palindrome rich in GC, which allows the molecules to form secondary structures (e.g., "stem-loop") or tertiary (e.g., dimer) type. Some of these oligonucleotides have as usual "stimulating" CpG sequence and motif with high GC content or motive, neutralizing b-cells". These oligonucleotides with a combination of motives exert immunostimulatory effects, which occupies an intermediate position between the effects associated with conventional CpG-oligonucleotides class (that is, a strong induction of activated b-cells and activation of dendritic cells (DC), and effects associated with CpG one class (that is, a strong induction of IFN-α and activation of NK cells in a relatively weak induction of activated b cells and DC). Krieg AM et al. (1995) Nature 374:546-9; Ballas ZK et al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992) J Immunol148:4072-6.

Immunostimulatory oligonucleotides class With a combination of motives can be fully stabilized frames (for example, when all links are phosphorothioate), chimeric frames (with phosphodieterase connections in the Central region) or semi-soft skeletons (for example, fosfomifira communication within the CpG motif). This class is described in application for U.S. patent US 10/224523, filed August 19, 2002

One incentive domain or motif CpG-oligonucleotide class defined by the formula: 5' X1DCGHX23'. D represents a nucleotide, non-S. is a cytosine. G represents guanine. N is a nucleotide, non-g X1and X2are any nucleotide sequence length 0-10 nucleotides. X1may contain CG, in which case the CG preferably present So In some embodiments DCG is a TCG. X1preferably has a length 0-6 nucleotides. In some embodiments X2contains no poly-G or poly-a motifs. In other embodiments immune-stimulating oligonucleotide has a poly-T sequence at the 5'-end or 3'end. When used here "poly-a or poly-T" will refer to the site of four or more consecutive a or T, respectively, in the example 5" AAAA 3" or 5" TTTT 3'. When used here "poly-To-end" will refer to the site of four or more consecutive G, for example 5" GGGG 3', located at the 5'-end or 3'-end of nucleic acids. When used here "poly-G-oligonucleotide" shall refer to the oligonucleotide having the formula 5' X1X2GGGX3X43', where X1, X2, X3and X4are nucleotides, and preferably at least one of X3and X4is a G. Some preferred variants of the domain, stimulating b cells with this formula include TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT, TCGTCGT.

The second motif CpG-oligonucleotide class called P or N, and it is located just behind the X1closer to the 5'-end or immediately for X2closer to the 3'-end.

N is a sequence, a neutralizing b-cells, beginning with the trinucleotide CGG and having a length of at least 10 nucleotides. Motive, neutralizing B-cell contains at least one CpG sequence, in which CG is present With or after CG is G (Krieg AM et al. (1998) Proc Natl Acad Sd USA 95:12631-12636), or is a CG-containing DNA sequence, in which a pair CG methylated. Neutralizing motifs or sequences have a certain degree of immunostimulatory ability when they are present in the motif which without them is not stimulating, but when they are present in the context of other immunostimulatory motifs, they reduce immune-stimulating potential of other motives.

P is a palindrome with a high content of GC containing a sequence length of at least 10 nucleotides.

When used here "palindrome" and, equivalently, "palindrome sequence" will refer to the inverted repeat, that is, a sequence as ABCDEE'D C B A', where a and a', and b', and so on are bases capable of forming the usual base pairs of Watson-Crick.

When used here "palindrome with a high content of GC" will refer to the palindrome, with the composition of the bases, at least two-thirds presents G and C. In some embodiments domain with a high GC content is preferably closer to the 3'-end from the "domain stimulating b-cells". Thus, in the case of a palindrome with a high GC content length of 10 bases palindrome contains at least 8 G and C. In the case of a palindrome with a high GC content, length 12 bases palindrome also contains at least 8 G and C. In the case of a palindrome with a high GC content, length 14 bases at least ten bases of palindrome are G and C. In some embodiments is a palindrome with a high GC content consists exclusively of G and C.

In some embodiments, the palindrome with a high content of GC is part of the reason, at least 81% of G and C. Thus, in the case of such a palindrome with a high GC content length of 10 bases palindrome consists exclusively of G and C. In the case of such a palindrome with a high GC content, length 12 bases, preferably at least ten bases (83%) palindrome are G and C. In some preferred embodiments of the palindrome with high GC content, length 12 grounds consists exclusively of G and C. In the case of a palindrome with a high GC content, length 14 bases at least twelve foundations (86%) of the palindrome are G and C. In some preferred embodiments of the palindrome with high GC content, length 14 grounds consists exclusively of G and S.-base palindrome with high GC content can be neetilirovannyj, or they can be methylated.

Usually this domain has at least 3 s and G, more preferably 4 C and G, and most preferably 5 or more C and G. In the fact that the number of C and G in this domain was the same, there is no need. Preferably, C and G are arranged so that they are able to form semicomplementary duplex or a palindrome, such as CCGCGCGG. This sequence may contain a or T, but before occhialino semicompetent at least partially preserved, as, for example, in the motives CGACGTTCGTCG or CGGCGCCGTGCCG. When complementarity is not stored, preferably complementary base pairs are TG. In the preferred embodiment are no more than 3 adjacent bases that are not part of the palindrome, preferably not more than 2 and most preferably only 1. In some embodiments, the palindrome with high GC content includes at least one CGG trimer, at least one trimer CCG or at least one tetramer CGCG. In other embodiments, the palindrome with a high content of GC is not CCCCCCGGGGGG or GGGGGGCCCCCC, CCCCCGGGGG or GGGGGCCCCC.

At least one G area with high GC content may be replaced with inosine (I). In some embodiments, R includes more than one I.

In certain embodiments immune-stimulating oligonucleotide has one of the following formulas: 5' NX1DCGHX23', 5' X1DCGHX2N 3', 5' PX1DCGHX23', 5' X1DCGHX2P 3', 5' X1DCGHX2PX33', 5' X1DCGHPX33', 5' DCGHX2PX33', 5' TCGHX2PX3 3', 5' DCGHPX33' or 5' DCGHP 3'.

According to the invention proposed other immunostimulatory oligonucleotides defined by the formula 5' N1PyGN2P 3'. N1represents any sequence of length 1-6 nucleotides. Roux is piramidi is. G represents guanine. N2represents any string of length 0 to 30 nucleotides. P is a palindrome with a high content of GC containing a sequence length of at least 10 nucleotides.

N1and N2can contain more than 50% of the pyrimidine and more preferably more than 50% of T. N1may include CG, in which case the CG preferably present So In some embodiments N1PyG is a TCG, and most preferably TCGN2, where N2 is not G.

NiPyGN2P may include one or more than one insidemy (I) the nucleotide. With a or G in N1can be replaced by inosine, but CpI is more preferable than the IpG. For iesinovich substitutions, such as IpG, optimal activity can be achieved using a "semi-soft" or chimeric frame, where the relationship between IG or CI is fosfomifira. N1may include at least one motive CI, TCI, IG or TIG.

In certain embodiments N1PyGN2is a sequence selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT and TCGTCGT.

In one embodiment of CpG-oligonucleotides "class" according to the invention have the following nucleic acid sequence:

5' TCGCGTCGTTCGGCGCGCGCCG 3' (SEQ ID NO:13), or

5' TCGTCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:14), is whether

5' TCGGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:15), or

5' TCGGACGTTCGGCGCGCCG 3' (SEQ ID NO:16), or

5' TCGCGTCGTTCGGCGCGCCG 3' (SEQ ID NO:17), or

5' TCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:18), or

5' TCGACGTTCGGCGCGCCG 3' (SEQ ID NO:19), or

5' TCGCGTCGTTCGGCGCCG 3' (SEQ ID NO:20), or

5' TCGCGACGTTCGGCGCGCGCCG 3' (SEQ ID NO:21), or

5' TCGTCGTTTTCGGCGCGCGCCG 3' (SEQ ID NO:22), or

5' TCGTCGTTTTCGGCGGCCGCCG 3' (SEQ ID NO:23), or

5' TCGTCGTTTTACGGCGCCGTGCCG 3' (SEQ ID NO:24), or

5' TCGTCGTTTTCGGCGCGCGCCGT 3' (SEQ ID NO:25).

In any of these sequences, all communication can be phosphorothioate communication. In another embodiment, in any of these sequences, one or more than one link can be fosfomifira, preferably between "C" and "G" CpG motif that makes CpG-oligonucleotide semi-soft.

Some non-limiting examples of oligonucleotides class include:

5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:26), or

5' T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:27), or

5' T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:28), or

5' T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3' (SEQ ID NO:29), or

5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3' (SEQ ID NO:30), or

5' T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:31), or

5' T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3' (SEQ ID NO:32), or

5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3' (SEQ ID NO:33), or

5' T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:34), or

5' T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3' (SEQ ID NO:35), or

5' T*C*G*T*C*G*T*T*TT*C*G*G*C*G*G*C*C*G*C*C*G 3' (SEQ ID NO:36), or

5' T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C*G 3' (SEQ ID NO:37), or

5' T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' (SEQ ID NO:38),

where * refers to phosphorate atoi communication and _ refers to a phosphodiester bond.

In any of these sequences 5" T can be replaced by amiloride or halogen; examples of the halogen substitutions include, without limitation, the substitution bromouridine or iodouridine.

Immunostimulatory CpG-oligonucleotides "class P" described in WO 2007/095316 and are characterized by the fact that they contain areas that form the duplex, such as, for example, complete or incomplete palindromes in both 5'- and 3') ends or near both 5'- and 3' - ends, which gives them the ability to form patterns of a higher order, such as concatemer. These oligonucleotides, called oligonucleotides class R, in some cases, have the ability to induce significantly higher levels of secretion of IFN-α than oligonucleotides class C. the Oligonucleotides class R have the ability to spontaneously self-Assembly to form concatemers in vitro and/or in vivo. Without specific theory of mechanism of action of these molecules, one possible hypothesis is that this property gives the oligonucleotides class R greater ability to cross-linking with TLR9 within certain immune cells inducyruya pattern of immune activation, which differs from the previously described classes of CpG-oligonucleotides.

In one embodiment, the agonist of TLR-9 for use in the present invention represents a CpG is oligonucleotid class R, containing 5'-domain that activates TLR, and at least two palindrome area, one of which is a 5'-palindrome region length of at least 6 nucleotides and is linked to the 3'palindrome area length of at least 8 nucleotides, either directly or through a spacer, where the oligonucleotide includes at least one YpR-dinucleotide. In one embodiment the oligonucleotide is not T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO:27). In one embodiment of the CpG-oligonucleotide class P includes at least one demetilirovanny CpG dinucleotide. In another embodiment, the domain that activates TLR is a TCG; TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT or TTTT. In yet another embodiment, the domain that activates TLR, is located in the 5'-palindromes area. In another embodiment, the domain that activates TLR, situated directly at the 5'-palindromes area, closer to the 5'-end. In another embodiment the 5'-palindrome region has a length of at least 8 nucleotides. In another embodiment the 3'-palindrome region has a length of at least 10 nucleotides. In another embodiment the 5'-palindrome region has a length of at least 10 nucleotides. In another embodiment the 3'-palindrome region includes demetilirovanny CpG dinucleotide. In another embodiment the 3'-palindrome region includes two neetilirovannyj CpG-dinucleotide. In another GP is owenii 5'-palindrome region includes demetilirovanny CpG dinucleotide. In another embodiment the 5'-palindrome region includes two neetilirovannyj CpG-dinucleotide. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 25. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 30. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 35. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex of at least 40. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 45. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 50. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 55. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 60. In another embodiment the 5'- and 3'-palindrome region have an index of stability of the duplex is at least 65.

In one embodiment two palindrome region are connected directly. In another embodiment two palindrome region are connected by 3'-3'-link. In another embodiment two palindrome region overlap by one nucleotide. In another embodiment two palindromes overlap on two nucleotides. In another embodiment two palindrome region do not overlap. In another embodiment two palindrome region are connected by a spacer. In one embodiment, the spacer is a nucleic acid having a length of 1-50 nucleotides. In another embodiment, the spacer is a nucleic acid having a length of 1 nucleotide. In another embodiment, the spacer is neucleotides the spacer. In one embodiment neucleotides the spacer is a D-spacer. In another embodiment neucleotides the spacer is a linker. In one embodiment the oligonucleotide has the formula 5' XP1SP2T 3', where X represents a domain that activates TLR, P1is a palindrome, S is a spacer, R2is a palindrome and T represents a 3'-tail length of 0-100 nucleotides. In one embodiment X is a TCG, TTCG or TTTCG. In another embodiment T is the length of 5-50 nucleotides. In yet another embodiment of the T has a length of 5-10 nucleotides. In one embodiment S is a nucleic acid having a length of 1-50 nucleotides. In another embodiment S is a nucleic acid having a length of 1 nucleotide. In another embodiment S is neucleotides the spacer. In one embodiment neucleotides the spacer is a D-spacer. In another embodied the attachment neucleotides the spacer is a linker. In another embodiment the oligonucleotide is not antimuslim the oligonucleotide or a ribozyme. In one embodiment of Pi has a high content of a and T. In another embodiment of the P1includes at least 4 T. In another embodiment R2is a complete palindrome. In another embodiment R2has a high content of G-C. In another embodiment R2represents CGGCGCX1GCGCCG, where X1represents T or missing.

In one embodiment, the oligonucleotide includes at least one phosphorothioate communication. In another embodiment all mezhnukleotidnyh connection of the oligonucleotide are phosphorothioate communication. In another embodiment, the oligonucleotide includes at least one connection, such fosfomifira. In another embodiment of the communication, such fosfomifira is fosfodiesterazu communication. In another embodiment with the oligonucleotide conjugated to a lipophilic group. In one embodiment the lipophilic group is a cholesterol.

In one embodiment, the agonist of TLR-9 for use in the present invention is a CpG oligonucleotide class P with the 5'-domain that activates TLR, and at least two complementarity-containing regions, the 5'- and 3'complementarity-containing region, each of which has a length of at least 8 nucleotides is connected with the other either directly or through a spacer, where the oligonucleotide includes at least one pyrimidine-purine dinucleotide (YpR-dinucleotide) and where at least one of the complementarity-containing regions is not a complete palindrome. In one embodiment, the oligonucleotide includes at least one demetilirovanny CpG dinucleotide. In another embodiment, the domain that activates TLR is a TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT or TTTT. In another embodiment, the domain that activates TLR, is located in the 5'-complementarity-containing region. In another embodiment, the domain that activates TLR, situated directly at the 5'complementarity-containing region, closer to the 5'-end. In another embodiment the 3'complementarity-containing region has a length of at least 10 nucleotides. In another embodiment the 5'complementarity-containing region has a length of at least 10 nucleotides. In one embodiment of the S'-complementarity-containing region includes demetilirovanny CpG dinucleotide. In another embodiment the 3'complementarity-containing region includes two neetilirovannyj CpG-dinucleotide. In another embodiment the 5'complementarity-containing region includes demetilirovanny CpG dinucleotide. In another embodiment the 5'complementarity-containing region includes two neetilirovannyj CpG-dinucleotide. In another embodiment included interest-containing region includes at least one nucleotide analog. In another embodiment of the complementarity-containing region to form an intramolecular duplex. In one embodiment of the intramolecular duplex includes at least one pair of bases, the non-pair basis of Watson-Crick. In another embodiment of the base pair non pair basis of Watson-Crick, is a G-T, G-A, G-G or C-A. In one embodiment of the complementarity-containing region to form intermolecular duplexes. In one embodiment, at least one intermolecular duplex includes at least one pair of bases, the non-pair basis of Watson-Crick. In another embodiment of the base pair non pair basis of Watson-Crick, is a G-T, G-A, G-G or C-A. In yet another embodiment of the complementarity-containing region contain incorrect mating. In yet another embodiment of the complementarity-containing region contains two incorrect mating. In another embodiment of the complementarity-containing region contain intermediate nucleotide. In another embodiment of the complementarity-containing region contains two intermediate nucleotide.

In one embodiment the 5'- and 3'complementarity-containing region have an index of stability of the duplex is at least 25. In another embodiment the 5'- and 3'complementarity-containing region have the indicator is ustoichivosti duplex of at least 30. In another embodiment the 5'- and 3'complementarity-containing region have an index of stability of the duplex is at least 35. In another embodiment of the complementarity-containing region have an index of stability of the duplex of at least 40. In another embodiment of the complementarity-containing region have an index of stability of the duplex is at least 45. In another embodiment of the complementarity-containing region have an index of stability of the duplex is at least 50. In another embodiment of the complementarity-containing region have an index of stability of the duplex is at least 55. In another embodiment of the complementarity-containing region have an index of stability of the duplex is at least 60. In another embodiment of the complementarity-containing region have an index of stability of the duplex is at least 65.

In another embodiment two complementarity-containing region are connected directly. In another embodiment two palindrome region are connected by 3'-3'-link. In another embodiment two complementarity-containing region overlap by one nucleotide. In another embodiment two complementarity-containing region overlap by two nucleotides. In another embodiment two complementarity-containing region do not overlap. In another embodiment two komplementaritgtstheorie region are connected by a spacer. In another embodiment, the spacer is a nucleic acid having a length of 1-50 nucleotides. In another embodiment, the spacer is a nucleic acid having a length of 1 nucleotide. In one embodiment, the spacer is neucleotides the spacer. In another embodiment neucleotides the spacer is a D-spacer. In yet another embodiment neucleotides the spacer is a linker.

In one embodiment the oligonucleotide class R has the formula 5' XNSPT 3', where X represents a domain that activates TLR, N represents an incomplete palindrome, P is a palindrome, S is a spacer, and T represents a 3'-tail length of 0-100 nucleotides. In another embodiment X is a TCG, TTCG or TTTCG. In another embodiment T is the length of 5-50 nucleotides. In another embodiment of the T has a length of 5-10 nucleotides. In another embodiment S is a nucleic acid having a length of 1-50 nucleotides. In another embodiment S is a nucleic acid having a length of 1 nucleotide. In another embodiment S is neucleotides the spacer. In another embodiment neucleotides the spacer is a D-spacer. In another embodiment neucleotides the spacer is a linker. In another embodiment the oligonucleotide is not antimuslim oligonucleotid the om or a ribozyme. In another embodiment, N has a high content of a and T. In another embodiment N includes at least 4 T. In another embodiment R is a complete palindrome. In another embodiment, R has a high content of G-C. In another embodiment R represents CGGCGCX1GCGCCG, where X1represents T or missing. In another embodiment, the oligonucleotide includes at least one phosphorothioate communication. In another embodiment all mezhnukleotidnyh connection of the oligonucleotide are phosphorothioate communication. In another embodiment, the oligonucleotide includes at least one connection, such fosfomifira. In another embodiment of the communication, such fosfomifira is fosfodiesterazu communication. In another embodiment with the oligonucleotide conjugated to a lipophilic group. In one embodiment the lipophilic group is a cholesterol.

In one embodiment of the CpG-oligonucleotide "class P" according to the invention has the following nucleic acid sequence: 5' TCGTCGACGATCGGCGCGCGCCG 3' (SEQ ID NO:39).

In these sequences, all communication can be phosphorothioate communication. In another embodiment, one or more than one link can be fosfomifira, preferably between "C" and "G" CpG motif that makes CpG-oligonucleotide semi-soft. In any of these sequences 5' T can be is replaced by amiloride or halogen; examples of the halogen substitutions include, without limitation, the substitution bromouridine or iodouridine.

A non-limiting example of oligonucleotides class P includes:

5' T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ ID NO:40),

where * refers to phosphorothioates communication and _ refers to a phosphodiester bond.

In one embodiment, all mezhnukleotidnyh communication CpG-oligonucleotides disclosed here represent the phosphodiester bond (soft oligonucleotides, as described in PCT application WO 2007/026190). In another embodiment of CpG-oligonucleotides according to the invention has resistance to degradation (e.g., they are stable). "Stabilized oligonucleotide" refers to an oligonucleotide that is relatively resistant to degradation in vivo (e.g., Exo - or endonuclease). Stabilization of nucleic acids can be carried out by modification of the frame. Oligonucleotides with phosphorothioate links provide maximum activity and protect the oligonucleotide from degradation of intracellular Exo - and endonucleases.

Immunostimulatory oligonucleotides may be chimeric frame containing combinations fosfolipidnyh and phosphorothioate links. For the purposes of the present invention chimeric frame refers to a partially stabilized frame, where at least one mezhnukleotidnyh communication is fosfomifira or under the on fosfomifira and where at least one other mezhnukleotidnyh relationship is a stable mezhnukleotidnyh communication, where at least one fosfomifira communication or connection, such fosfomifira, and at least one stable relationship differ from each other. When fosfomifira connection preferably is located in a CpG-motif, such molecules are called "semi-soft", as described in PCT application WO 2007/026190.

Other modified oligonucleotides include combinations fosfolipidnyh, phosphorothioate, methylphosphonate, methylphosphonothioate, phosphorodithioate links and/or p-ethoxy-links.

Since it was reported that boranophosphate links are stable compared to phosphodieterase bonds, for the purposes of the chimeric nature of the frame boranophosphate communication can be classified either as such fosfomifira or stable, depending on the context. For example, in some embodiments the chimeric frame according to the present invention may include at least one fosfodiesterazu (fosfodiesterazu or similar fosfomifira) linkage and at least one boranophosphate (stable) relationship. In other embodiments chimeric frame according to the present invention may include boranophosphate (phosphodieterase or similar fosfomifira) and phosphorothioate (stable) connection. "Stable mezhnukleotidnyh relationship" will mean minule tidow communication, relatively resistant to degradation in viva (for example, Exo - or endonuclease) compared to fosfomifira mezhnukleotidnyh communication. Preferred stabilized mezhnukleotidnyh networks include, without limitation, phosphorothioates, phosphorodithioates, methylphosphonate and methylphosphonothioate. Other stable mezhnukleotidnyh networks include, without limitation, peptide, alkyl, nesvorny (dephospho) and others, as described above.

Modified frames, such as phosphorothioate, can be synthesized by automated methods using either phosphoramidate or H-phosphonate chemistry. Aryl - and alkylphosphonate can be obtained, for example, as described in U.S. patent No. 4469863; and alkylphosphocholine (in which the charged oxygen group is alkylated, as described in U.S. patent No. 5023243 and European patent No. 092574) can be obtained by automated solid-phase synthesis using commercially available reagents. Demonstrated how to implement other modifications and substitutions in the framework of DNA. Uhlmann E et al. (1990) Chem Rev 90:544; J Goodchild (1990) Bioconjugate Chem 1:165. Also known methods for producing chimeric oligonucleotides. For example, such techniques are described in the patents issued by Uhlmann et al.

Modified one with mixed frame can be synthesized as described in PCT-C the turnout WO 2007/026190.

The oligonucleotides according to the invention may also include other modifications. They include non-ionic analogs of DNA, such as alkyl - and arylphosphate (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), fosfomifira and alkylphosphocholine, in which the charged oxygen group is alkylated. It has been shown that nucleic acid-containing diol such as tetraethylene glycol or hexamethyleneimine, on one or both ends, is also substantially resistant to cleavage by nucleases.

The size of the CpG-oligonucleotide (i.e., the number of nucleotide residues in accordance with the length of the oligonucleotide) can also contribute to stimulating the activity of the oligonucleotide. To facilitate penetration into the cells the minimum length of CpG-oligonucleotides according to the invention is preferably 6 nucleotide residues. Oligonucleotides of any size greater than 6 nucleotides (even a length of several thousand bases) is able to induce an immune response in the presence of sufficient immunostimulatory motifs, because of larger Oligonucleotides are susceptible to intracellular degradation. In certain embodiments CpG-oligonucleotide have a length of 6-100 nucleotides, preferably between 8 and 30 nucleotides. In important embodiments of the nucleic acids and oligonucleotides according invented what Yu are no plasmids or expression vectors.

In one embodiment of the CpG-oligonucleotide disclosed here includes the replacement or modification, as, for example, replacement or modification of the bases and/or sugars, as described in 134-147 paragraphs WO 2007/026190.

In one embodiment of the CpG oligonucleotide of the present invention is chemically modified. Examples of chemical modifications are known to the specialist in the art and described, for example, in Uhlmann, E. et al. (1990), Chem. Rev. 90:543, S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke, S. T. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and J. Hunziker et al., (1995), Mod. Synth. Methods 7:331-417. Oligonucleotide according to the invention can have one or more than one modification as compared with the oligonucleotide of the same sequence, composed of natural DNA or RNA, where each modification is located in a certain fosfomifira magnolioideae communication, and/or in particular β-D-ribose unit, and/or position of a particular natural nucleoside base.

In some embodiments of the invention CpG-containing nucleic acid can be simply mixed with immunogenic carriers in accordance with methods known to experts in the art (see, for example, WO 03/024480).

In a specific embodiment of the present invention, any vaccine disclosed here contains from 2 μg to 100 mg CpG oligonucleotide, preferably from 0.1 mg to 50 mg CpG oligonucleotide, predpochtitel is from about 0.2 mg to 10 mg CpG oligonucleotide, preferably from 0.3 mg to 5 mg CpG oligonucleotide, preferably from 0.3 mg to 5 mg CpG oligonucleotide, more preferably from 0.5 to 2 mg CpG oligonucleotide, more preferably from 0.75 to 1.5 mg CpG oligonucleotide. In the preferred embodiment, any vaccine disclosed here contains approximately 1 mg CpG oligonucleotide.

Pneumococcal vaccine

Pneumococcal vaccine according to the present invention will typically contain conjugated antigens capsular saccharides, originating from at least seven serotypes of S. pneumoniae. The number of capsular saccharides S. pneumoniae can vary from 7 different serotypes (or "v", valences) to 23 different serotypes (23v). In one embodiment presents 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 different serotypes. In one embodiment represented by 10 or 11 different serotypes. In one embodiment 7 or 13 different serotypes. Antigens capsular saccharides conjugated to a protein carrier, as described below.

In another embodiment of the invention, the vaccine may contain conjugated S. pneumoniae saccharides and non-conjugated S. pneumoniae saccharides. Preferably, the total number of serotypes of sugars less than or equal to 23. For example, the vaccine may contain 7 serotypes conjugated and 16 unconjugated saccharides. In another embodiment, the vaccine may contain 13 anywhereman the s serotypes and 10 unconjugated saccharides. Similarly, the vaccine may contain 8, 9, 10, 11, 12, 13, 14, 15 or 16 conjugated saccharides and 15, 14, 13, 12, 11, 10, 9, 8 or 7, respectively unconjugated saccharides.

1. In one embodiment, the vaccine according to the invention contains conjugated S. pneumoniae saccharides from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

2. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1 above, conjugated S. pneumoniae saccharides from serotype 1.

3. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1 or 2 above, conjugated S. pneumoniae saccharides from serotype 5.

4. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2 or 3 above, conjugated S. pneumoniae saccharides from serotype 7F.

5. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, or 4, above, conjugated S. pneumoniae saccharides from serotype 3.

6. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4 or 5 above, conjugated S. pneumoniae saccharides from serotype 6A.

7. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5 or 6 above, conjugated S. pneumoniae saccharides from serotype 19A.

8. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6 the sludge is 7, the above conjugated S. pneumoniae saccharides from serotype 22F.

9. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7 or 8 above, conjugated S. pneumoniae saccharides from serotype 15.

10. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8 or 9 above, conjugated S. pneumoniae saccharides from serotype 8.

11. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 above, conjugated S. pneumoniae saccharides from serotype 12F.

12. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 above, conjugated S. pneumoniae saccharides from serotype 2.

13. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11 or 12 above, conjugated S. pneumoniae saccharides from serotype 9N.

14. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12 or 13 above, conjugated S. pneumoniae saccharides from serotype 10A.

15. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 14 above, conjugated S. pneumoniae saccharides from serotype 11A.

16. In another embodiment, the vaccine according to the invention contain what it in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14 or 15 above, conjugated S. pneumoniae saccharides from serotype 11A.

17. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 or 16 above, conjugated S. pneumoniae saccharides from serotype 17F.

18. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16 or 17 above, conjugated S. pneumoniae saccharides from serotype 20.

19. In another embodiment, the vaccine according to the invention contains, in addition to paragraph 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17 or 18 above, conjugated S. pneumoniae saccharides from serotype 33F.

In one embodiment, the vaccine according to the invention contains conjugated S. pneumoniae saccharides from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F.

In one embodiment, the vaccine according to the invention contains conjugated S. pneumoniae saccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

In one embodiment, the vaccine according to the invention contains conjugated S. pneumoniae saccharides from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

In one embodiment, the vaccine according to the invention contains conjugated S. pneumoniae saccharides from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.

In the preferred embodiment the antigen capsular saccharides conjugated to a protein carrier, independently selected from the group, with the standing of tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, fragment TT, PhtD, fused proteins PhtDE (in particular as described in WO 01/98334 and WO 03/54007), neutralized pneumolysin and protein D.

In the preferred embodiment the antigen capsular saccharides conjugated to protein carriers selected from the group consisting of: DT (diphtheria toxoid), TT (tetanus toxoid) or With a fragment TT, CRM197 (a non-toxic, but identical antigenic variant of diphtheria toxin), other point mutants DT, such as CRM176, CRM228, CRM45 (Uchida et al. J. Biol. Chem. 218; 3838-3844, 1973); CRM9, CRM45, CRM102, CRM103 and CRM107, and other mutations described Nicholls and Co. in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletions or substitutions of Glu-148 Asp, Gin, or Ser, and/or 158 Ala to Gly, and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more than one of the Lys residues 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5917017 or US 6455673; or a fragment disclosed in US 5843711, pneumococcal pneumolysin (Kuo et al. (1995) Infect Immun 63; 2706-13), including in some way neutralized pneumolysin, for example dPLY-GMBS (WO 04081515, PCT/ER/010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequence PhtA, PhtB, PhtD or PhtE disclosed in WO 00/37105 or WO 00/39299) and fused proteins Pht proteins, such as fused proteins PhtDE, fused proteins PhtBE, Pht A-E (WO 01/98334, WO 03/54007, WO 2009/000826), OMPC (outer membrane protein meningococcal disease, typically allocated from N. meningitidis CE is gruppy, EP 0372501), PorB (from N. meningitidis), PD (protein D of Haemophilus influenzae, see, for example, EP 0594610), or immunologically functional equivalents, synthetic peptides (EP 0378881, EP 0427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP 0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins containing multiple epitopes recognized by human CD4+T-cells, antigen, originating from different pathogens (Falugi et al. (2001) Eur J Immunol 31; 3816-3824), such as protein N19 (Baraldoi et al. (2004) Infect Immun 72; 4884-7), pneumococcal surface protein PspA (WO 02/091998), proteins associated with the absorption of iron (WO 01/72337), toxin a or b of C. difficile (WO 00/61761).

In one embodiment the antigen capsular saccharides conjugated to DT (diphtheria anatoxin). In another embodiment the antigen capsular saccharides conjugated to TT (tetanus-toxoid).

In another embodiment the antigen capsular saccharides conjugated to a C-fragment TT.

In another embodiment the antigen capsular saccharides conjugated with PD (protein D of Haemophilus influenzae, see, for example, EP 0594610).

In the preferred embodiment the antigen capsular saccharides according to the invention conjugated to a protein CRM197. Protein CRM197 is a non-toxic form of diphtheria toxin, but immunologically indistinguishable from diphtheria toxin. CRM197 produced by C. diphtheriae infected netoxygen phage β197 tox-obtained by mutagenesis toxicogenic beta phage corynebacteria using nitrosoguanidine (Uchida, T. et al. 1971, Nature New Biology 233:8-11). Protein CRM197 has the same molecular weight as diphtheria toxin, but differs from it by the substitution of one base (guanine to adenine) in the structural gene. This replacement of one base leads to amino acid substitution (glycine to glutamic acid) in the Mature protein and eliminates toxic properties of diphtheria toxin. Protein CRM197 is a safe and effective media saccharides, dependent on T cells. Additional details of CRM197 and receipt can be detected, for example, in US 5614382.

In one embodiment, if the carrier protein is the same for 2 or more saccharides in the composition, the saccharides can be conjugated to the same molecule of carrier protein (with molecules conjugated to carriers still 2 different saccharide) (see, for example, WO 04/083251).

Alternatively, each saccharide may be separately anywhereman with different molecules of carrier protein (this means that for every molecule of carrier protein konjugierte saccharide only one type). In a specific embodiment of the capsular saccharides called conjugated to protein carrier separately.

In one embodiment the antigen capsular saccharides of the present invention have the origin is s from different serotypes of S. pneumoniae and conjugated with one or more than one protein carrier. In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 different serotypes, where protein D is a protein carrier.

In one embodiment the saccharide from serotype 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F anywhereman with protein D.

In one embodiment the saccharide from serotype 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F anywhereman with CRM197.

In one embodiment of the saccharides from at least serotypes 1 and 3, 1 and 4, 1 and 5, 1 and 6A, 1 6B, 1 and 7, 1 and 9V, 1 and 14, 1 and 22F, 1 and 23F, 3 and 4, 3 and 5, 3 and 6A, 3 and 6, 3 and 7F, 3, and 9V, 3 and 14, 3 and 22F, 3, and 23F, 4, and 5, 4 and 6A, 4 and 6B, 4 and 7F, 4 and 9V, 4 and 14, 4 and 22F, 4, and 23F, 5 and 6A, 5 and 6, 5 and 7F, 5 and 9V, 5 and 14, 5 and 22F, 5, and 23F, 6A, and 6B, 6A and 7F, 9V 6A and 6A and 14, 6A and 22F, 6A and 23F, 6B and 7F, 6V and 9V, 6V and 14, 6B and 22F, 6B and 23F, 7F and 9V, 7F and 14, 7F and 22F, 7F and 23F, 9V and 14, 9V and 22F, 9V and 23F, 14, and 22F, 14 and 23F or 22F and 23F conjugated to CRM197.

In one embodiment of the saccharides from at least serotypes 1, 3, and 4; 1, 3 and 5; 1, 3, and 6A; 1, 3 and 6; 1, 3 and 7F; 1, 3, and 9V; 1, 3 and 14; 3, 4 and 7F; 3, 4 and 5; 3, 4 and 7F; 3, 4 and 9V; 3, 4 and 14; 4, 5 and 7F; 4, 5, 9V; 4, 5, and 14; 5, 7F and 9V; 5, 7F and 14; 7F, 9V and 14; 1, 3, 4 and 5; 3, 4, 5 and 7F; 4, 5, 7F and 9V; 4, 5, 7F and 14; 4, 5, 9V and 14; 4, 7F, 9V and 14; 5, 7F, 9V and 14; or 4, 5, 7, 9V and 14 conjugated to CRM197.

In one embodiment of the saccharides from at least serotypes 1 and 3, 1 and 4, 1 and 5, 1 and 6A, 1 6B, 1 and 7, 1 and 9V, 1 and 14, 1 and 22F, 1 and 23F, 3 and 4, 3 and 5, 3 and 6A, 3 and 6, 3 and 7F, 3, and 9V, 3 and 14, 3 and 22F, 3, and 23F, 4, and 5, 4 and 6A, 4 and 6B, 4 and 7F, 4 and 9V, 4 and 14, 4 and 22F, 4, and 23F, 5 and 6A, 5 and 6, 5 and 7F, 5 and 9V, 5 and 14, 5 and 22F, 5, and 23F, 6A, and 6B, 6A and 7F, 9V 6A and 6A and 14, 6A and 22F, 6A and 23F, 6B and 7F, 6V and 9V, 6V and 14, 6B and 22F, 6B and 23F, 7F and 9V, 7F and 14, 7F and 22F, 7F and 23F, 9V and 14, 9V and 22F, 9V and 23F, 14, and 22F, 14 and 23F or 22F and 23F conjugated to protein D.

In one embodiment of the saccharides from at least serotypes 1, 3, and 4; 1, 3 and 5; 1, 3, and 6A; 1, 3 and 6; 1, 3 and 7F; 1, 3, and 9V; 1, 3 and 14; 3, 4 and 7F; 3, 4 and 5; 3, 4 and 7F; 3, 4 and 9V; 3, 4 and 14; 4, 5 and 7F; 4, 5, 9V; 4, 5, and 14; 5, 7F and 9V; 5, 7F and 14; 7F, 9V and 14; 1, 3, 4 and 5; 3, 4, 5 and 7F; 4, 5, 7F and 9V; 4, 5, 7F and 14; 4, 5, 9V and 14; 4, 7F, 9V and 14; 5, 7F, 9V and 14; or 4, 5, 7F, 9V and 14 conjugated to protein D.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 7 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 7 different serotypes, where protein D is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 10 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 10 different serotypes, where protein D is a protein carrier.

In one embodiment VA is of CIN according to the invention contains conjugates of capsular saccharides from 11 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 11 different serotypes, where protein D is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 13 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 13 different serotypes, where protein D is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 23 different serotypes, where CRM197 is a protein carrier.

In one embodiment, the vaccine according to the invention contains conjugates of capsular saccharides from 23 different serotypes, where protein D is a protein carrier.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F conjugated to protein D.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F conjugated to CRM197.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to protein D.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM197.

In one embodiment of the HAC is in according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to protein D.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to CRM197.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to protein D.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to CRM197.

In one embodiment, the vaccine according to the invention contains a saccharide from serotype 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, conjugated to protein D.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, conjugated to CRM197.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F conjugated to protein D, saccharide from serotype 18C conjugated tetanus toxoid (TT), and saccharide from serotype 19F conjugated with diphtheria toxoid (DT).

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 7F, 9V, 19F and 23F conjugated tetanus toxoid (TT), and saccharide from serotypes 3, 14, 18C and 6B conjugated with diphtheria toxoid (DT).

In one embodiment, the vaccine according to the invention contains a saccharide of serotypes, 4, 5, 6B, 7F, 9V, 14 and 23F individually conjugated to protein D, saccharide from serotype 18C conjugated tetanus toxoid (TT), and saccharide from serotype 19F conjugated with diphtheria toxoid (DT).

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 7F, 9V, 19F and 23F individually conjugated tetanus toxoid (TT), and saccharide from serotypes 3, 14, 18C and 6B conjugated with diphtheria toxoid (DT).

In this description, the term "saccharide" can mean a polysaccharide or oligosaccharide and includes both. Capsular polysaccharides of Streptococcus pneumoniae contain repeating oligosaccharide units that can contain up to 8 sugar residues. Cm. overview of oligosaccharide units for key serotypes of Streptococcus pneumoniae in JONES, Christopher. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. 'an. Acad. Bras. Cienc, June 2005, vol.77, no.2, p.293-324. Table II ISSN 0001-3765.

Antigens capsular saccharides according to the invention receive standard techniques known to experts in this field of technology. Usually conjugates of polysaccharides receive separate ways and include in one pharmaceutical composition. For example, in one embodiment of pneumococcal each polysaccharide serotype grown on soy environment. Then hold up individual polysaccharides by centrifugation, sedimentation, ultrafiltration and number the night chromatography. Purified polysaccharides chemically activate to make the sugars ability to interact with the protein carrier. After activation, each capsular polysaccharide separately kongugiruut with protein carrier to form glycoconjugate. In one embodiment, each capsular polysaccharide kongugiruut with the same protein carrier. In this embodiment the conjugation carried out by reductive amination. Chemical activation of polysaccharides and subsequent conjugation with protein carrier carried out in the usual ways. See, for example, U.S. patent No. 4673574 and 4902506.

After conjugation of capsular polysaccharide to a protein carrier are clean polysaccharide-protein conjugates (enrichment relative to the number polysaccharide-protein conjugate) using a variety of techniques. These techniques include processes of concentration/diafiltration, sedimentation/elution, column chromatography and filtration using porous filters. See, for example, US 2007/0184072 or WO 2008/079653. After purification of the individual glycoconjugates their mix for the manufacture of a vaccine of the present invention. The production of a composition of the immunogenic compositions of the present invention can be carried out using methods recognized in the art. For example, a single pneumococcal conjugates could the t to be mixed with physiologically acceptable excipient for the manufacture of the composition. Examples of such fillers include, without limitation, water, buffered saline, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol) and the solutions of dextrose.

The amount of conjugate in each vaccine dose is chosen as the number of inducing immunoprotective response without significant undesirable side effects in the normal person to be vaccinated. Such amount will vary depending on the specific immunogen and method of its presentation. In one embodiment, each dose contains 0.1 to 1000 μg of each saccharide or sahariana-protein conjugate, preferably 2-100 μg, more preferably 4-40 µg.

In one embodiment, each dose contains 0.1 to 20 μg, 1-10 or 1-5 μg μg of saccharide.

In one embodiment, the vaccine according to the invention contains each capsule of S. pneumoniae saccharide dose of 0.1-20 μg, 0.5 to 10 μg, 0.5 to 5 μg or 1-5 μg of saccharide. In one embodiment of the capsular saccharides may be present in different doses, for example, some capsular saccharides may be present in a dose of approximately or exactly 2 μg, or some capsular saccharides may be present in a dose of approximately or exactly 4 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each is th capsular saccharide S. pneumoniae is present in a dose of 2 µg, with the exception of saccharide from serotype 6, present in a dose of 4 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 4 µg, with the exception of saccharide from serotype 6, present in a dose of 8 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 6 µg, with the exception of saccharide from serotype 6, present in a dose of 12 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 8 μg, except saccharide from serotype 6, present in a dose of 16 ug.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 mcg.

In a specific embodiment of the present invention vaccinederived saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 4 µg, with the exception of saccharide from serotype 6B, present in a dose of 8 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 6 µg, with the exception of saccharide from serotype 6B, present in a dose of 12 mcg.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 8 μg, except saccharide from serotype 6B, present in a dose of 16 ug.

In a specific embodiment of the present invention the vaccine disclosed here contains 5-500 μg, preferably 10-200 μg, even more preferably 20-100 ág of carrier protein CRM197.

In one embodiment of the present invention the vaccine disclosed here contains 20-50 μg, preferably 20-40 μg, even more preferably 25-30 μg, even more preferably about 28 or 29 µg of carrier protein CRM197.

In one embodiment of the present invention the vaccine disclosed here contains 40-100 μg, preferably 40-80 mcg, it is more preferably 50-60 μg, even more preferably about 57 or 58 µg of carrier protein CRM197.

In a specific embodiment of the present invention the vaccine disclosed here, contains a buffer with sodium chloride and/or sodium succinate as the excipients.

In one embodiment of the pneumococcal vaccine for use here is a 7-valent conjugate pneumococcal vaccine (Prevenar) or 13-valent pneumococcal conjugate vaccine disclosed in US 2007/0184072 (Prevenar 13). 7-valent vaccine Prevenar contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197. 13-valent vaccine Prevenar contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individually conjugated to protein D, saccharide from serotype 18C conjugated tetanus toxoid (TT), and saccharide from serotype 19F conjugated with diphtheria toxoid (DT), where each capsule of S. pneumoniae saccharide is present in a dose of 1 μg, except saccharides from serotypes 4, 18C and 19F present in a dose of 3 μg. In a specific embodiment of the present invention this vaccine contains 5-500 μg, preferably 7-100 µg protein D as a protein carrier, 2-200 μg, preferably 4-50 hundred micrograms is brachnogo toxoid (TT) as a protein carrier and 1-100 μg, preferably 2-25 μg of diphtheria toxoid (DT) as a protein carrier. In a specific embodiment of the present invention this vaccine contains 9-16 µg protein D as a protein carrier, 5-10 μg of tetanus toxoid (TT) as a protein carrier and 3-6 µg of diphtheria toxoid (DT) as a protein carrier.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individually conjugated to protein D, saccharide from serotype 18C conjugated tetanus toxoid (TT), and saccharide from serotype 19F conjugated with diphtheria toxoid (DT), where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg except saccharides from serotypes 4, 18C and 19F present in a dose of 6 µg. In a specific embodiment of the present invention this vaccine contains 10-1000 μg, preferably 14-200 µg protein D as a protein carrier, 4-400 mg, preferably 8-100 mcg of tetanus toxoid (TT) as a protein carrier and 2-200 μg, preferably 4-50 µg of diphtheria toxoid (DT) as a protein carrier. In a specific embodiment of the present invention this vaccine contains 18-32 µg protein D as a protein carrier, 10-20 μg of tetanus toxoid (TT) as a protein carrier and 6-12 µg of diphtheria toxoid (DT) as a protein carrier.

In one embodiment, the vaccine according to the invention contains a saccharide from serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F individually conjugated to protein D, saccharide from serotype 18C conjugated tetanus toxoid (TT), and saccharide from serotype 19F conjugated with diphtheria toxoid (DT), where each capsule of S. pneumoniae saccharide is present in a dose of 4 μg, except saccharides from serotypes 4, 18C and 19F, present the respective dose of 12 mcg. In a specific embodiment of the present invention this vaccine contains 20-2000 μg, preferably 28-400 µg protein D as a protein carrier, 8-800 μg, preferably 16-200 mcg of tetanus toxoid (TT) as a protein carrier and 4-400 mg, preferably 8-100 µg of diphtheria toxoid (DT) as a protein carrier. In a specific embodiment of the present invention this vaccine contains 36-64 µg protein D as a protein carrier, 20-40 μg of tetanus toxoid (TT) as a protein carrier and 12-24 µg of diphtheria toxoid (DT) as a protein carrier.

In a specific embodiment of the present invention the vaccine disclosed here, contains a buffer with sodium chloride as excipients.

In one embodiment of the pneumococcal vaccine for use here is a 10-valent pneumococcal conjugate vaccine, commercially available under the trade name Synflorix™.

Extra(s) adjuvant (adjuvants)

In some embodiments pneumococcal vaccine disclosed here, contain at least one, two or three adjuvant, in addition to at least one adjuvant the agonist of TLR-9, opened here. The term "adjuvant" refers to a compound or mixture that enhances the immune response to the antigen. Antigens may act mainly as a delivery system and, mainly as an immunomodulator, or have essential properties of both. Suitable adjuvants include adjuvants suitable for use in mammals, including humans.

Examples of known suitable adjuvants type "delivery system" that can be used in humans include, without limitation, alum (e.g., aluminum phosphate, aluminum sulfate or a hydrate of aluminum oxide), calcium phosphate, liposomes, emulsions of the type oil-in-water, such as MF59 (4,3%, mass./about., squalene, 0.5 percent, mass./about., Polysorbate 80 (Tween 80), 0,5%, mass./about., sorbitrate (Span 85)), emulsion-type water in oil", such as montanic (Montanide) and microparticles or nanoparticles of poly(D,L-lactide-co-glycolide) (PLG).

Examples of known suitable adjuvant immunomodulatory type, which can be used in humans include, without limitation, saponine extracts from the bark of the tree Aquilla (QS21, Quil A), agonists of Toll-like receptor-4 (TLR4), such as MPL (monophosphorylated A), 3DMPL (3-O-describeany MPL) or GLA-AQ, LT/CT-mutants, cytokines, such as various interleukins (e.g. interleukin-2 (IL-2), interleukin-12 (IL-12)) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and the like.

Examples of known suitable adjuvant immunomodulatory type with properties as delivery systems, and immunolog is the batteries, which can be used in humans include, without limitation, immunostimulating complexes (ISCOM) (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 06/134423 and WO 07/026190) or GLA-EAT, which is a combination of TLR4 agonist and the emulsion of the type oil-in-water".

For veterinary use, including, without limitation, conducting experiments on animals can be used complete adjuvant's adjuvant (CFA), incomplete adjuvant's adjuvant (IFA), emulsion (Emulsigen), N-acetylmuramyl-L-threonyl-O-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-O-isoglutamine (CGP 11637, referred to nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine (CGP 19835A, referred to MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphorylated, dimycolate trehalose and skeletal components of the cell wall (cell wall skeleton, CWS) (MPL+TDM+CWS) in a 2% emulsion of squalene/Tween 80.

Other typical adjuvants to enhance effectiveness of pneumococcal vaccines disclosed here include, without limitation: (1) emulsion compositions of the type oil-in-water (with or without other specific immunostimulating agents such as muramylpeptide (see below) or components of the cell wall of bacteria), such as, for example, (a) SAF, containing 10% squalene, 0.4% of Tween 80, 5% blocked onion skin is nick polymer L121, and thr-MDP, either microfluidized (microfluidized) obtaining submicron emulsion or mixed on the vortex with obtaining emulsion with a large particle size, and (b) adjuvant system RIBI™ (RAS) (Ribi Innmunochem., Hamilton, MT) containing 2% squalene, 0.2% of Tween 80, and one or more than one component of the cell wall of bacteria, such as monophosphorylated A (MPL), dimycolate trehalose (TDM) and skeletal components of cell walls (CWS), preferably MPL+CWS (DETOX™); (2) saponine adjuvants, such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), Abisco® (Isconova, Sweden) or Iscomatrix® (Commonwealth Serum Laboratories, Australia), or can be used obtained from these particles, such as ISCOM (immunostimulating complexes), which can be without any extra detergent, for example, WO 00/07621; (3) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (4) cytokines, such as interleukins (e.g. interleukin-1 (IL-1), IL-2, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), IL-12 (WO 99/44636), and so on), interferons (e.g. gamma interferon), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), and so forth; (5) monophosphorylated A (MPL) or 3-O-describeany MPL (3dMPL) (see, for example, GB-2220221, EP-A-0689454) may essentially without alum when used with pneumococcal saccharides (see, for example, WO 00/56358); (6) combinations of 3dMPL with, for example, QS21 and/or em is lsemi type oil-in-water" (see, for example, EP-A-0835318, EP-A-0735898, EP-A-0761231); (7) simple ether or polyoxyethylene ester of polyoxyethylene (see, for example, WO 99/52549); (8) surface-active substance from a complex ester of polyoxyethylenesorbitan in combination with octoxynol (WO 01/21207) or surfactant from simple or complex polyoxyethyleneglycol ester in combination with at least one additional non-ionic surface-active agent, such as an octoxynol (WO 01/21152); (9) saponin and immune-stimulating oligonucleotide (e.g. a CpG-oligonucleotide) (WO 00/62800); (10) an immunostimulant and a particle of metal salt (see, for example, WO 00/23105); (11) a saponin and an emulsion of the type oil-in-water, for example, WO 99/11241; (12) a saponin (e.g., QS21)+3dMPL+IM2 (possibly+a Sterol), for example, W098/57659; (13) other substances that act as immunostimulating agents, increasing the efficiency of the composition. Muramylpeptide include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine (MTP-PE), and so on.

In the preferred embodiment of the pneumococcal vaccine disclosed here, contain alum, hydrate alumina, aluminum phosphate or aluminum sulfate as an additional adjuvant to at least one adjuvant AG is the NIST TLR-9, opened here.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 μg, and additionally contains 0.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 4 µg, with the exception of saccharide from serotype 6B, present in a dose of 8 mg, and optionally contains 1 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 6 µg, with the exception of saccharide from serotype 6B, present in a dose of 12 μg, and additionally contains 1.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention in which ccina contains saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 8 μg, except saccharide from serotype 6B, present in a dose of 16 μg, and additionally contains 2 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 μg, and additionally contains 0.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 4 µg, with the exception of saccharide from serotype 6B, present in a dose of 8 mg, and optionally contains 1 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, individually con is agiovannii with CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 6 µg, with the exception of saccharide from serotype 6B, present in a dose of 12 μg, and additionally contains 1.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In a specific embodiment of the present invention, the vaccine contains saccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 8 μg, except saccharide from serotype 6B, present in a dose of 16 μg, and additionally contains 1.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

In one embodiment, the pneumococcal vaccine is a 7-valent conjugate pneumococcal vaccine (Prevenar) or 13-valent pneumococcal conjugate vaccine disclosed in US 2007/0184072 (13vPnC).

Subjects with a weakened immune system

In the preferred embodiment of the present invention, the subject to be vaccinated with the vaccine of the present invention, is a subject with a weakened immune system. Preferably, the specified subject with a weakened immune system is a mammal such as a cat, sheep, pig, horse, bull, dog, or man. In the most preferable is lewinii the specified subject is a human.

Individuals with a weakened immune system is generally defined as a person showing a weakened or reduced ability to develop normal humoral or cell-mediated protection against infection by infectious agents.

In one embodiment of the present invention, the subject is immunocompromised subject of vaccination pneumococcal vaccine, suffering from a disease or condition that impairs the operation of the immune system and leading to a humoral response, insufficient to protect against pneumococcal disease or treatment of pneumococcal disease.

In one embodiment the disease is a primary immunodeficiency. Preferably, the specified primary immunodeficiencies selected from the group consisting of: combined T - and b-cell immunodeficiencies, deficits antibodies, clearly defined syndromes, diseases with dysregulation of the immune system, phagocytic disorders, congenital immunodeficiencies, automobiletechnik disorders and deficiency of the complement system.

In one embodiment of the specified combined T - and b-cell immunodeficiencies selected from the group consisting of: deficit γ, JAK3 deficiency, deficiency of α-chain of the receptor for interleukin-7, CD45 deficiency or deficit CD35/CD3E, deficit RAG 1/2, deficit DCLRE1C, deficit adenozindezaminazy (ADA), R is ticularly dysgenesia, syndrome Omenn, deficit DNA ligase IV type, deficiency of CD40 ligand, CD40 deficiency, deficit polynucleotides (PNP) deficiency molecules of the major histocompatibility complex (MHC) class II, CD3γ deficiency, deficiency of CD8, lack of ZAP-70, deficit TAR-1/2 and deficiency of domain type, a "winged helix" ("winged helix").

In one embodiment of this deficit antibody selected from the group consisting of: agammaglobulinemia concatenated with the X-chromosome deficiency of btk, agammaglobulinemia of Bruton, deficits heavy chain µ, deficit I 5, Igα deficiency, deficit BLNK, thymoma with immunodeficiency, variable unclassifiable immunodeficiency (CVID), deficit ICOS, CD19 deficiency, deficit TACI (TNFRSF13B), deficit of BAFF receptor, deficiency of AID, UNG deficiency, deletions heavy chain, deficiency chains Kappa, isolated deficiency of immunoglobulin G subclass (IgG), deficiency of immunoglobulin subclasses of A (IgA) and IgG, selective deficiency of immunoglobulin a deficiency of specific antibodies to specific antigens in normal concentrations of b-cells and immunoglobulins (Ig), the transient hypogammaglobulinemia newborn (THI).

In one embodiment of the specified well-defined syndrome selected from the group consisting of: syndrome Wiskott-Aldrich, ataxia-telangiectasia syndrome, like ataxia, Nijmegenpskov syndrome chromosomal instability, the syndrome is and bloom, syndrome Di George (in connection with defects in the thymus), hypoplasia of cartilage and hair syndrome Since syndrome German-Pudlak type 2 syndrome Hyper-lgE, chronic candidiasis of the skin and mucous membranes.

In one embodiment the disease with dysregulation of the immune system selected from the group consisting of: syndrome Chediak-Higashi syndrome, Griscelli type 2, deficiency perforin, deficit MUNC13D, deficit syntaxin-11, lymphoproliferative syndrome linked to the X chromosome, autoimmune lymphoproliferative syndrome, for example, type 1A (defects CD95), type 1b (defects in the Fas-ligand), type 2A (defects CASP10), type 2b (defects CASP8), APECED (autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy) and IPEX (syndrome polyendocrinopathy with dysregulation of the immune system and disease, coupled with the X-chromosome).

In one embodiment of the specified phagocytic disorder selected from the group consisting of: deficit ELA2 (myelodysplasia), GFI1 deficiency (with T/a-lymphopenia), deficiency of the receptor for granulocyte colony-stimulating factor (G-CSFR) (not modifiable granulocyte colony-stimulating factor (G-CSF)), Kostmann syndrome, hereditary periodic neutropenia, neutropenia/myelodysplasia, coupled with the X-chromosome, leukocyte adhesion deficiency 1, 2 and 3 type defi is it RAC2, deficiency of beta-actin, localized juvenile periodontitis, syndrome Papillon-lefèvre, deficiency of specific granules, syndrome Shwachman-diamond, chronic granulomatous disease (concatenated with the X-chromosome and autosomal forms), deficiency of glucose-6-phosphate dehydrogenase neutrophils, deficiency of IL-12 and β1-chain of the interleukin-23 (IL-23), deficiency of IL-12p40, deficit receptor 1 interferon-γ, deficit receptor 2 interferon-γ and STAT1 deficiency (2 forms).

In one embodiment of the specified congenital immunodeficiency selected from the group consisting of: gipoglikemicheskoy ectodermal dysplasia, NEMO deficiency, IKBA deficiency, deficit, IRAK-4, WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis) and the warty epidermodysplasia.

In one embodiment of the specified autospalatorie disorder selected from the group consisting of: familial Mediterranean fever, periodic syndrome associated with TNF receptor (TRAPS), syndrome of Hyper-IgD (HIDS), diseases associated with CIAS1, syndrome Make wells, family cold autosplitting syndrome, Multisystem inflammatory disease neonatal age, RARA syndrome (sterile pyogenic arthritis, pyoderma gangrenosum, and acne) and Blau syndrome.

In one embodiment of the deficiency of the complement system selected from the group sotoyama is from: deficiency of C1q (lupus syndrome, rheumatoid disease, infection), C1r deficiency (same), deficiency of C4 (same), deficit C2 (lupus syndrome, vasculitis, polymyositis, purulent infection), C3 deficiency (recurrent purulent infection), deficiency of C5 (infection caused by neisseriae, systemic lupus erythematosus (SLE)), deficiency of C6 (same), deficiency of C7 (the same, vasculitis), deficit Sa and C8b (same), deficit C9 (infection caused by neisseriae), deficiency of C1 inhibitor (hereditary angioedema), deficiency of factor I (purulent infection), deficiency of factor H (haemolytic uraemic syndrome, membranosa-proliferative glomerulonephritis), deficiency of factor D (infection caused by neisseriae), deficiency of properdin (infection caused by neisseriae), deficit MBP (purulent infection) and MASP2 deficiency. In one embodiment of the specified autospalatorie disorder selected from the group consisting of deficits C1, C2, C3 and C4.

In one embodiment of the present invention, the subject is immunocompromised subject of vaccination, suffers from a disease that kills the immune system, representing the acquired immunodeficiency syndrome. Acquired immunodeficiency can be caused by several factors, including bacterial or viral infections (such as HIV infection), cancer (such as leukemia or myeloma), other chronic disorders, but also aging, malnutrition or different types of treatment (such as treatment with glucocorticoids, chemotherapeutic drugs).

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, suffers from a disease selected from the group consisting of: HIV infection, acquired immunodeficiency syndrome (AIDS), cancer, chronic disorders of the heart or lungs, congestive heart failure, diabetes, chronic liver disease, alcoholism, cirrhosis, leaks of cerebrospinal fluid, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), dysfunction of the spleen (such as sickle cell disease), insufficiency of the spleen (asplenia), malignant neoplasms of the blood system, leukemia, multiple myeloma, Hodgkin's disease, lymphoma, renal failure, nephrotic syndrome, and asthma.

In a specific embodiment, the subject is immunocompromised subject of vaccination, suffers from a disease selected from the group consisting of: dysfunction of the spleen (such as sickle cell disease), insufficiency of the spleen (asplenia), leukemia, multiple myeloma, Hodgkin's disease and lymphoma.

In the preferred embodiment of the subject's immune system is weakened, to be vaccinated suffers from HIV or Sindh is Ohm acquired immunodeficiency syndrome (AIDS).

In a specific embodiment, the subject is immunocompromised subject of vaccination, is suffering from HIV infection or acquired immunodeficiency syndrome (AIDS) and receives treatment, comprising receiving at least one antiretroviral drug selected from the group consisting of: non-nucleoside reverse transcriptase inhibitors, protease inhibitors and reverse transcriptase inhibitors, non-nucleoside analogue (e.g., ABC). In a particular embodiment said treatment consists of taking at least three drugs belonging to at least two classes of antiretroviral drugs selected from the group consisting of: non-nucleoside reverse transcriptase inhibitors, protease inhibitors and reverse transcriptase inhibitors, non-nucleoside analogue (e.g., ABC). In a particular embodiment said treatment comprises receiving at least two reverse transcriptase inhibitors, non-nucleoside analogues or protease inhibitor or non-nucleoside inhibitor of reverse transcriptase.

In a specific embodiment, the subject is immunocompromised subject of vaccination, is suffering from HIV infection or acquired immunodeficiency syndrome (AIDS) and gets highly efficient is th antiretroviral therapy (WEART). In one embodiment of the specified VAART consists of a regimen of 3 drugs, including non-nucleoside reverse transcriptase inhibitor, a protease inhibitor and/or nucleoside reverse transcriptase inhibitors, non-nucleoside analogue (e.g., abacavir), or regimen 2 medicines, including a combination of non-nucleoside reverse transcriptase inhibitors and protease inhibitors.

In a specific embodiment, the subject is immunocompromised subject of vaccination, is suffering from HIV infection or acquired immunodeficiency syndrome (AIDS), and not receiving highly active antiretroviral therapy (WEART) or not receiving antiretroviral therapy, or the specified subject has never received antiretroviral drugs.

In a specific embodiment, the subject is immunocompromised subject of vaccination, is an HIV-infected patients without viremia. In another embodiment, the subject is immunocompromised subject of vaccination, is an HIV-infected patient with viremia.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, is suffering from tuberculosis or sexually transmitted diseases such as syphilis or hepatitis.

In one embodiment of the crust is asego of the invention the subject with a weakened immune system, to be vaccinated suffers from malnutrition.

In one embodiment of the present invention, the subject is immunocompromised subject of vaccination, suffers from aging. In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, is an adult at the age of 55 years or over, more preferably an adult 65 years of age or older. In one embodiment, the subject is immunocompromised subject of vaccination, is an adult at the age of 70 years or older, at the age of 75 years or older or aged 80 years or older.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, is taking medication or receiving treatment that lowers the body's resistance to infection.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, takes a drug selected from the group consisting of chemotherapeutic drugs (e.g., medicines to treat cancer), disease modifying Antirheumatic drugs, immunosuppressive drugs used after organ transplantation, and Gluck is Rikoito.

In one embodiment of the present invention, the subject is immunocompromised subject of vaccination, taking immunosuppressive drug for oral administration selected from the group consisting of: tacrolimus (prograf), mycophenolate mofetil (CellCept), sirolimus (rapamune), prednisone, cyclosporine (Neoral, Sandimmune, gengraf (Gengraf)) and azathioprine (imuranom). In one embodiment of the subject with a weakened immune system takes at least two or three of these immunosuppressive drugs for oral administration.

In one embodiment of the present invention, the subject is immunocompromised subject of vaccination, taking immunosuppressive drug is selected from the group consisting of: everolimus, mycophenolate acid, corticosteroids (such as prednisone or hydrocortisone), monoclonal antibodies against the α-chain of the receptor for interleukin-2 (IL-2Rα) (such as basiliximab or daclizumab), antithymocyte globulin (ATG) and antilimfocitarnyi globulin (ALG). In one embodiment of the subject with a weakened immune system takes at least two or three of these immunosuppressive drugs.

In a specific embodiment of the present invention the subject is immunocompromised subject to vaccination Tr is splantzia body, or bone marrow transplantation, or cochlear implantation.

In a specific embodiment of the present invention the subject is immunocompromised subject of vaccination, radiation therapy.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, is a smoker.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, suffers from asthma and receives treatment with corticosteroids for oral administration.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, is a native Alaskan or American Indian.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, has a number of cells less than 5×109cells per liter, or less than 4×109cells per liter, or less than 3×109cells per liter, or less than 2×109cells per liter, or less than 1×109cells per liter, or less than 0.5×109cells per liter, or less than 0.3×109cells per liter, or less than 0.1×109cells / litre.

The number of leukocytes. The number of leukocytes (WBC) in blood. The number of WBC is usually defined as part of the clinical blood analysis (CBC). Leukocytes are cells of the blood, b is rushemeza infection and different from red blood cells (carry oxygen), known as erythrocytes. There are different types of leukocytes, including neutrophils (polymorphonuclear leukocytes, PMN), stab cells (slightly immature neutrophils), lymphocytes T-type (T-cells), lymphocytes of b-type (b-cells), monocytes, eosinophils and basophils. The white blood cell count reflects the number of cells of all types. Normal range in the number of cells typically ranges from 4300 to 10800 in a cubic millimeter of blood. It can also be named by the number of cells and is expressed in international units as a 4.3-10,8×109cells / litre.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, suffering from neutropenia. In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, has the number of neutrophils less than 2×109cells per liter, or less than 1×109cells per liter, or less than 0.5×109cells per liter, or less than 0.1×109cells per liter, or less than 0.05×109cells / litre.

Reduced the number of leukocytes or neutropenia is a condition characterized by abnormally reduced levels of neutrophils in the circulating blood. Neutrophils are a certain type of white blood cells to help prevent and fight infections. Often the only cause of neutropenia in patients with cancer are the side effects of chemotherapy. Induced by chemotherapy neutropenia increases the risk of infection in the patient and violates the treatment of cancer.

The smaller the number of neutrophils in the blood and the longer patients don't have enough neutrophils, the higher the susceptibility of a patient to the development of bacterial or fungal infection. Neutrophils are a key component of the mechanisms of antibacterial protection. When the decrease in the number of neutrophils to 1.0, with 0.5 and 0.1×109/l frequency of life-threatening infections sharply increases from 10% to 19% and 28%, respectively.

In a specific embodiment of the present invention, the subject is immunocompromised subject of vaccination, has a CD4+ count less than 500 cells/mm3or a CD4+ count less than 300 cells/mm3or a CD4+ count of less than 200 cells/mm3the number of CD4+ cells less than 100/mm3the number of CD4+ cells less than 75/mm3or a CD4+ count of less than 50 cells/mm3.

The results of the study CD4 cells usually reported as number of cells / mm3. A normal CD4 cell count is between 500 and 1600, and the number of CD8 cells ranges from 375 to 1100. The number of CD4 cells is greatly reduced in people with HIV infection.

In one embodiment of the invention, any entity with a weakened immune system, unveiled here is a human male or human as the ski sex.

Treatment

In some cases it may be required only one dose of vaccine according to the invention, but in some circumstances, such as conditions are more pronounced immunodeficiency, there may be second, third or fourth dose.

In one embodiment, the first dose is administered at 0 day and one or more than one booster dose administered at intervals ranging from about 2 to about 24 weeks, preferably with an interval between doses 4-8 weeks.

In one embodiment, the first dose is administered at 0 day and a booster dose is administered over approximately 3 months.

As shown in the "Examples" section, some of the shortcomings of modern vaccination can be overcome with the use of a vaccine according to the invention. In particular, the vaccine according to the invention can reduce the number of vaccinations required to achieve seroprotection, accelerate seroconversion, possibly allowing vaccination after infection, reduce the proportion of subjects who do not respond to vaccination, to reduce the required amount of antigen to enhance the avidity and protective activity of antibodies and/or to obtain more stable antibody levels.

These advantages are of particular interest in the treatment of subjects with weakened immune systems.

EXAMPLES

Example 1: the Immune response to pneumococcal vaccine with an agonist of Toll-podobno the receptor 9 adjuvant in HIV-infected adults

A study was conducted phase II 96 HIV-infected patients.

Tasks

Main task:

- to compare the proportion of patients with a strong response to the vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 µg/ml (according to the quantitative measurement of IgG), in the group of CpG 7909 and the control group.

Additional tasks:

- conduct qualitative (functional) comparison of the humoral response to the introduction of pneumococcal vaccine with CpG 7909 or without CpG 7909;

- to evaluate the safety and tolerability of CpG 7909 adjuvant for pneumococcal vaccines;

to analyse changes in the state carriage of pneumococci after the introduction of pneumococcal vaccine.

The main estimated parameters

Efficiency

Primary: a quantitative measure of specific protivokashlevyh antibodies (7 serotypes).

Secondary: functional activity of specific protivokashlevyh antibodies (against pneumococcal serotypes 6B, 14, 19F and 23F); the number and severity of undesirable and serious adverse events; microbiological changes colonization of the pharynx by pneumococci; initial CD4 cell count and record sCD163.

Safety/tolerability

Undesirable phenomenon is the s (AE); serious adverse events (SAE); laboratory tests (Hematology, clinical chemistry, i.e. viral load (HIV RNA and CD4 cell count); physical examination.

Study design: placebo-controlled, randomized, double-blind study. Total sample size: 96 participants (48 per group).

Investigational medicinal product and composition: CpG 7909, a synthetic agonist of Toll-like receptor 9) in phosphate buffered saline solution (PBS buffer). CpG 7909 is a CpG one class with the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:5), and it is synthesized with a fully phosphorothioate frame.

Dose of the investigational medicinal product: 1 mg CpG 7909 (100 µl) was mixed with each pneumococcal vaccine.

Controls: 100 µl of neutral PBS buffer (identical in color and viscosity of the investigational medicinal product) with each pneumococcal vaccine.

Route of administration: intramuscular injection. Masking: double-blind study. Inclusion; randomization.

Conducted the randomization of eligible patients in a 1:1 ratio to receive pneumococcal vaccine with CpG 7909 or without CpG 7909.

Immunization

Vaccines are kept in their original packaging as described by the manufacturer and mixed with adjuvant (CpG 7909 or placebo) immediately before immunization. And is anizatio carried out by introducing into the upper part of the left or right deltoid muscle on the choice of the subject.

The duration of the study for each participant: 10 months from first vaccination to the last observation.

The termination of the participation of subjects in the study

For analysis "entity that has ceased to participate in the study," is any entity, not arrived at the final visit, provided by the Protocol.

Entity named entity that has ceased to participate in the study if no procedures were conducted according to the study, there was follow-up, and this subject has not been received subsequent information, starting from the date of termination of participation in the study and/or last contact.

Subjects who have discontinued participation in the study were not replaced. Stop getting investigational product subject

Entity that has ceased to receive the investigational product is any entity that has not received the full treatment, that is, if the date of termination of the receipt of the investigated product was not entered any subsequent scheduled dose. Stop getting the investigational product by the subject is not necessarily the termination of participation in the study because they can be carried out other procedures according to the study or follow-up (safety or immunogenicity), if they are scheduled in the Protocol.

Data, Podles is appropriate for inclusion in the individual registration card:

- date of birth, sex, race, height, weight, number in the study;

- adverse events reported by the subject, including the start and duration (time to resolution);

- positive findings upon physical examination;

- history of the disease;

- other vaccines received outside the study during the study period;

- any changes to the regular treatment throughout the study;

- pre-condition or signs and/or symptoms present in the subject prior to the study/first vaccination;

all laboratory data obtained during the study.

Inclusion criteria participants:

1) written informed consent and statement of authority provided in accordance with local regulatory and ethical practice using forms information sheet participant and informed consent approved by the responsible ethics Committee;

2) participants are male or female, aged at least 18 years of age;

3) HIV-seropositive individuals.

Exclusion criteria participants:

1) pregnancy defined by a positive result of the test for the beta-subunit of human chorionic gonadotropin (beta-HCG) in urine (for female subjects);

2) failure of the participant application is to be reliable methods of contraception for further research; reliable methods of birth control include: pharmacological contraception, including contraception oral, parenteral and transdermal delivery; condoms with spermicide; diaphragm with spermicide; surgical sterilization; vaginal ring, intrauterine device; abstinence; and the state after the menopause (for female subjects);

3) breastfeeding during the study (for female subjects);

4) last result of determining the number of CD4 cells less than 200×106cells/µl;

5) viral load (HIV RNA) more than 50 copies/ml when conducting VAART (defined as the introduction of at least three antiretroviral drugs, including either a protease inhibitor or non-nucleoside reverse transcriptase inhibitors (NNRTIS), i.e. Combivir 300/150 mg x 2 + stocrin 600 mg×1 for at least 6 months);

6) prior to inclusion in this study;

7) any medical, psychiatric, social or professional status or other responsibility that, in the opinion of the chief investigator (PI), will prevent the evaluation of research problems (such as serious alcohol abuse, severe drug addiction, dementia);

8) failure to comply with the treatment regimen according to the Protocol;

9) introduction pnev cockboy vaccine for 5 or less years before inclusion;

10) planned participation in other studies of vaccines during the study.

Procedures

The study included patients that agreed to participate in the study meet the criteria of inclusion/exclusion. Before immunization was obtained blood samples for measurement of initial parameters. When randomization of participants was distributed at a ratio of 1:1 in the group with one of the two study regimens:

experimental group: two doses of 7-valent pneumococcal conjugate vaccine (Prevenar®, Wyeth)+1 mg CpG 7909 (0 day), two doses of 7-valent pneumococcal conjugate vaccine (Prevenar®, Wyeth)+1 mg CpG 7909 (90 day) and a single dose of 23-valent polysaccharide vaccine (Pneumo Novum®, Sanofi Pasteur MSD)+1 mg CpG 7909 (270 days);

- control group: two doses of 7-valent pneumococcal conjugate vaccine (Prevenar®, Wyeth)+100 μl placebo (0 day), two doses of 7-valent pneumococcal conjugate vaccine (Prevenar®, Wyeth)+100 μl placebo (90 day) and a single dose of 23-valent polysaccharide vaccine (Pneumo Novum®, Sanofi Pasteur MSD)+100 μl placebo (270 days).

Received the blood samples and subsequent medical follow-up included physical examination and medical history, registration AE (adverse events)/AE (serious adverse events), the collection of data on vaccination outside of the study and any other information that may is be important for inclusion in the CRF. The final visit was carried out on 300 days.

The subject arrived at the final visit or available for the final contact, under this Protocol, considered as completed the study.

Injections of vaccines and investigational medicinal product/placebo

All subjects injections were performed at 0, 90 and 270 days. All immunization was performed by injection in the deltoid muscle of the right or left shoulder (on the participant's choice).

- At 0 and 90 day participants spent one intramuscular injection of double doses of Prevenar 1.0 ml+0.1 ml of an investigational medicinal product (CpG 7909)/placebo. In both cases, the amount entered in the shoulder, is 1.1 ml.

- 270 day the participants of the studies were single intramuscular injection of 0.5 ml Pneumo Novum+0.1 ml of an investigational medicinal product (CpG 7909)/placebo. In both cases, the amount entered in the shoulder, is 1.1 ml.

Researchers and participants did not know which experimental or control, the injection was performed. The volume and appearance of each injection of the product were identical.

Main parameter of efficiency and the analysis of the humoral response

The study was designed to detect differences between experimental and control groups in the number of patients with a strong response to the pneumococcal vaccine, certain to improve the peer IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 µg/ml (according to the quantitative measurement of IgG). The study was not designed to detect differences in the incidence of pneumonia or confirmed invasive/non-invasive pneumococcal disease. This required a significant number of participants and longer follow-up period. The most widely used assessment of immune response to pneumococcal vaccine is a quantitative determination of the serotype-specific protivokashlevyh antibodies. Recent evidence shows that the specificity of the method can be enhanced by the inclusion of stage absorption 22F with removal through this cross-reacting antibodies with low avidity. Quantitative determination of serotype-specific IgG was held at Statens Serum Institut (SSI), Copenhagen, Denmark using enzyme-linked immunosorbent assay (ELISA), including the stage of absorption 22F. In the SSI was not aware of the allocation to treatment groups.

Additional performance parameters and analysis of the humoral response

Quantitative determination of the serotype-specific protivokashlevyh antibodies does not give any information about the functional properties of antibodies. They can be estimated flow-cytometrical analysis opsonophagocytic activity, allowing to obtain indirect information about the ability of antibodies to opsonizing penetrated the body of pneumococci to promote their destruction.

Qualitative analysis was carried out using a flow-cytometrical analysis opsonophagocytic activity, allowing to measure functional (opsonophagocytic activity (OPA) serotype-specific antibodies. Briefly, 10 μl of the test serum is purchasing eight twofold dilutions in ORA-buffer. To each well add aliquots of 20 μl of the suspension of many bacteria or multiple granules containing 1 x 105pneumococcal each target serotype or granules with conjugated pneumococcal polysaccharides, and the plate is incubated for 1 hour at 37°C with horizontal shaking (200 rpm). Then to each well add 20 ál of sterile serum from rabbits at the age of 3-4 weeks (Pel-Freez, Brown Deer, Wis.), except the control wells with cells HL60, add 20 ál of OPA-buffer. After incubation at 37°C for 20 minutes with shaking (200 rpm on an orbital shaker) to each well add 30 ál of washed polymorphonuclear leukocytes (PMN) HL60 (2,5×104ml) to obtain the ratio effector:target 1:4 (for each type of target). The final volume in the well is 80 μl, the final dilution in the first hole of a series of dilutions are 1:8. The tablet is then incubated for 60 minutes with shaking at 37°C. To each well add 80 ál of PR-b is Fehr to provide sufficient volume for flow-cytometrical analysis and the contents of the wells are transferred into microtiter tubes (Bio-Rad, Hercules, Calif.). On each tablet, you can analyze up to 12 serum samples, including sample quality control. Flow-cytometrics analysis was performed using Flow Applications, Inc., III, USA51.

Carriage of pneumococci

The introduction of the pneumococcal vaccine may affect the carriage of pneumococci in the throat. Colonization of the pharynx by pneumococci may also affect the immune response to the introduction of pneumococcal vaccines. For this reason, it is important to establish the status of the carriage before and after the introduction of pneumococcal vaccines. Estimated colonization of the oropharynx on the back of the throat using a swab for culture BBL (Becton Dickson Microbiology Systems, Cockeysville, MD, USA) through the oral cavity. The samples are labelled according to individual rooms (ID), frozen at -20°C for several hours and then sent to Statens Serum Institut, where they spent the isolation, cultivation and serotyping. This was carried out on 0 day and again at the follow-up to 270 days.

Adverse events (AE)

AE is any unwanted medical manifestation of the subject participating in a clinical study, which coincides with the application of the drug, regardless of, consider whether this is a manifestation associated with the drug.

Thus, As the can be any unwanted or unintended sign (including abnormal laboratory findings), symptom or disease (new or aggravated) that coincides with the use of drugs.

In this study, AE classified in accordance with the General criteria of toxicity (Common Toxicity Criteria version 2.0.

Definition of serious adverse events (SAE)

An adverse event occurring during the clinical trials, is any undesirable event associated with the use of party drugs. The phenomenon seriously and will be reported to regulatory agencies, if the outcome for a participant is:

1) death;

2) the threat of life;

3) hospitalization (initial or prolonged);

4) disability;

5) the need for intervention for the prevention of persistent deterioration or damage;

6) congenital disorder/abnormality (for pregnant women).

The definition of a suspected unexpected serious adverse reaction (SUSAR)

Suspected unexpected serious adverse reaction Suspected Unexpected Serious Adverse Reaction SUSAR) occurring during the study and which must be reported:

The phenomenon should be an SAE.

- Required a certain degree of probability that the event is an undesirable reaction to the administered drug.

Unwanted response should be unexpected, t which is not provided in the investigator's brochure (Investigator''s Brochure) (unlicensed medicines).

Evaluation data: evaluation criteria task

All endpoints were compared between the group receiving the experimental vaccine (CpG 7909), and the group receiving the control vaccine (placebo).

In pozyskiwanie (substudy) were compared endpoints in two (non-randomized) treatment groups (with VAART or without VAART).

Primary endpoints

Six months after the second vaccination with Prevenar.

- Patients with a strong response to the pneumococcal vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 µg/ml (according to the quantitative measurement of IgG).

Secondary endpoint

Immunogenicity

Three months after the first vaccination with Prevenar.

- Patients with a strong response to the pneumococcal vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 µg/ml (according to the quantitative measurement of IgG).

- Opsonophagocytic activity against serotypes 6B, 14, 19F and 23F, presented in the caption.

- The serotype-specific humoral response, defined by increased levels of IgG in 2 times and levels of IgG at least 1 µg/ml.

- The serotype-specific the th humoral response, a certain change levels of IgG.

Six months after the second vaccination with Prevenar.

- Opsonophagocytic activity against serotypes 6B, 14, 19F and 23F, presented in the caption.

- The serotype-specific humoral response, defined by increased levels of IgG in 2 times and levels of IgG at least 1 µg/ml.

- The serotype-specific humoral response, defined by the change in the levels of IgG.

One month after vaccination Pneumo Novum.

- Patients with a strong response to the pneumococcal vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 µg/ml (according to the quantitative measurement of IgG).

- Opsonophagocytic activity against serotypes 6B, 14, 19F and 23F, presented in the caption.

- The serotype-specific humoral response, defined by increased levels of IgG in 2 times and levels of IgG at least 1 µg/ml.

- The serotype-specific humoral response, defined by the change in the levels of IgG.

- Geometric average concentration of antibodies in a standard ELISA analysis for serotypes 1, 4, 7F, 9V, 14, 18C and 19F.

Colonization of the pharynx

Six months after the second vaccination with Prevenar.

The number of individuals with colonization by pneumococci.

Prognostic facto the s humoral response

Source

- Risk factors in relation to the response to the vaccine six months after the second vaccination with Prevenar.

Secondary endpoint

Reactogenicity and safety in all subjects.

The analyzed population

The population for safety assessment: all patients who received at least one vaccination.

- Frequency requested and General symptoms during the period of 4 days (from 0 days to 3 days) after each vaccination.

- Frequency of unsolicited symptoms within 1 month after each vaccination.

- Change the number of CD4 cells and viral load during the study.

Safety was evaluated by physical examination, analysis of adverse events (according to the Common toxicity criteria version 2.0), laboratory studies and monitoring indicators of HIV infection (HIV RNA and CD4 cells).

Statistical analyses

Baseline

Differences between study groups on the 0 day will be judged by the criterion of the sum of ranks Mann-Whitney (continuous variables) and Chi-square (dichotomous and categorical variables).

Primary endpoint

The prevalence of patients with a strong response six months after the second vaccination with Prevenar when comparing two groups with different schemes is mi vaccination (with or without CpG 7909) were assessed by Chi-square. Plan on using a regression model Poisson correction for age, baseline CD4 cell count and WEART (WEART or without VAART) in the beginning of the study.

Secondary endpoint

Comparison of endpoints between the study groups was performed by Chi-square. Plan on using Poisson regression (dichotomous endpoints) or linear regression (continuous endpoints) with a correction for possible relevant factors that may affect the study results.

Risk factors about getting a strong response to the vaccine (classified as a strong response to the vaccine) six months after the second vaccination with Prevenar will be evaluated by multivariate Poisson regression.

Safety data

Safety data were recorded and compared by Chi-square.

The size of the evaluated sample

The population started to receive treatment (ITT population): all randomized participants.

Calculate the sample size for the primary endpoint (the prevalence of patients with a strong response six months after the second vaccination with Prevenar when comparing two groups with different vaccination schedules). Were selected following error probability of type I and type II:

- the probability of error of 1 type (a)=0,05 (two-sided);

probably the th type II error (β)=0,20 (the exponent (power)=1-β=0,80).

- Primary endpoint: proportion of patients with a strong response to the vaccine (defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes of at least 1 μg/ml).

N represents the minimum number of participants in each group.

ControlCpG0,500,550,600,650,70
0,203929231815
0,255841312419
0,309361423124
0,3517096624331
0,40 388173976242

Assuming a rate of 30% in control group vaccine and 60 per cent in the group of experimental vaccines, to identify differences in performance when estimating the regression Poisson required sample size is 42 patient. Estimated percentage of drop-out is 10%. Therefore, research was needed in total 94 of the subject.

According to the method recommended by the regulatory authorities, the calculated bilateral 95% confidence interval (CI) of the differences of the immune response.

Example 2: Immunogenicity and safety of pneumococcal vaccine with TLR9 agonist adjuvant in HIV-infected adults. The results of a randomized, double-blind, placebo-controlled study

Clinical study described in Example 1. The study was a placebo-controlled randomized phase II trials in HIV-infected patients who were vaccinated with two doses of PCV (pneumococcal conjugate vaccine) (Prevnar)±1 mg CpG 7909 at 0 and 3 months and one dose of PPV (pneumococcal polysaccharide vaccine)±1 mg CpG 7909 in 9 months. Immunogenicity and safety were evaluated at 0, 3, 4, 9 and 10 months. The primary end-that is what was the proportion of patients with a strong response to the vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes included in PCV, 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes included in PCV, at least 1 µg/ml (quantitative determination of IgG by ELISA, Statens Serum Institute, Copenhagen, Denmark) at 9 months.

Results. As shown in Table 1, the study included 96 participants. In each group there were 48 participants, 38 of whom were receiving antiretroviral therapy (APT).

Table 1
Baseline characteristics at time of entry
The placebo groupGroup CPG
N4848
Floor
Men38 (79,2)43 (89,6)
Women10 (20,8)5 (10,4)
Race
Caucasian43 (89,6)47 (97,9)
Neuropenia5 (10,4)1 (2,1)
Mean age, years (IQR)48,9 (42,0-59,0)48,9 (43,0-58,8)
The average number of CD4+cells, ×106per ml (IQR)617 (500-848)673 (393-817)
VAART
38 (79,2)38 (79,2)
No10 (20,8)10 (20,8)
The mean log HIV RNA, IQR
1,601,60
Without VAART4,47 (of 3.73-4,86)4,25 (3,70-4,59)

Prior immunization with PPV-23 *1 (2,1)2 (4,2)
Smoking at the present time17(35,4)and 18(37.5)
* less than 5 years before inclusion; IQR - interquartile span

As shown in Table 3 and Fig.1, the proportion of patients with a strong response to the vaccine after immunization with PCV in the group CpG was significantly higher than in the adjuvant group-placebo (48,8% compared to 25.0%, p=0,018). Reinforced responses were also observed in 3 (51.1% vs. 39.6 per cent, p=0.26), 4 (77.3 per cent against 56,3%, p=0.033) and 10 (87,8% compared to 51.1%, p<0,001) months.

Table 3
The proportion of patients with a strong response to the vaccine at each time point
n (%)The placebo groupGroup CpGHR prior to the first vaccination with PCVYes00
no00
HR 3 months after the first vaccination with PCVYes19 (39,6)24 (51,1)0,26
no29 (60,4)23 (48,9)
HR 1 month after the second vaccination with PCVYes27 (56,3)34 (77,3)0,03
no21 (43,7)10 (22,7)
HR 6 months after the second vaccination with PCVYes12 (25,0)21 (48,8)0,02
no 36 (75,0)22 (51,2)
HR at 1 month after vaccination with PPV-23Yes24 (51,1)36 (87,8)0,001
no23 (48,9)5 (12,2)

HR-patients with a strong response to the pneumococcal vaccine, defined by increased levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes included in Prevnar, 2 times and levels of IgG antibodies to pneumococcus at least 5 of the 7 serotypes included in Prevnar, at least 1 µg/ml (quantitative measurement of IgG); PCV - conjugated pneumococcal vaccine; PPV-23 - 23-valent pneumococcal polysaccharide vaccine

In Fig.2 and 3 shows the relative difference of lgG response to two serotypes included in PCV (9v and 14), CPG groups and placebo.

In Fig.4 and 5 show the relative lgG-response to two serotypes not included in PCV (1 and 7f), CPG groups and placebo (as would be expected, increased IgG in connection with immunization with PCV were not observed).

After immunization with PPV both groups (+/-CpG) show significant responses. However, CpG did not enhance the humoral response to serotypes not included in PCV(1 and 7f), after immunization with PPV.

As shown in Table 4 (pages 67-71), data on average geometric concentrations (GMC) of IgG antibodies revealed increased GMC-relations from baseline to 3, 4, 9, and 10 months for almost all serotypes included in PCV-7, for the experimental group compared with the control group. As expected, the GMC antibody to 3 serotypes not included in PCV (1, 7F and 19A), after immunization with PCV-7 was not changed. After PPV-23 in both groups was increased GMC antibodies to serotypes not included in PCV-7, 2-5 times (lowest to serotype 19A), but significant differences GMC-relations between groups was not.

Participants were immunized with two doses of PCV-7 (Prevnar®, Wyeth)±1 mg CPG 7909 at 0 and 3 months and one dose of PPV-23 (Pneumo Novum®, Sanofi Pasteur MSD)±1 mg CPG 7909 at 9 months, and all included in the PPV-23; ORA - opsonophagocytic activity; PS - serotype pneumococci; GM-ratio - the ratio of average geometric; PCV-7 - 7-valent conjugated pneumococcal vaccine; PPV-23 - 23-valent pneumococcal polysaccharide vaccine.

As shown in Table 2, the lungs systemic and local reactions at the injection PCV were more frequent in the group CpG (100% against 81.3%, p=0.002). Moderate-severe flu-like symptoms were observed in the group CpG after PPV.

None of the group was not niela the nutrient effects on CD4+cells (see Fig.6) or organ functions.

Table 2
Unwanted effects associated with injections
The first introductionThe second introductionPPV-23
PCVpPCVpP
PCVPCV+CPGPCVPCV+CPGPPV-23PPV-23+CPG
n (%)n=48n=47n=48n=44n=47n=41
At least one adverse event44(93,6)0,00230 (62,5)40 (90,9)0,00128 (59,6)41 (100)<0,001
Pain at the injection site32 (66,7)43 (91,5)0,00330 (62,5)37 (84,1)0,0227 (57,5)36 (87,8)0,002
Erythema at the injection site3 (6,3)10 (21,3)0,045 (10,4)11 (25,0)0,077 (14,9)25 (61,0)<0,001
Burning at the injection site6 (12,5)18 (38,3)0,0047 (14,6)16 cases (36.4)0,029 (19,2)27 (65,9)<0,001
the UD at the injection site 0 (0)1 (2,1)0,500(0)1 (2,3)0,480 (0)3 (7,3)0,10
Flu-like symptoms*3 (6,3)17 (36,2)0,0013 (6,3)17 (38,6)<0,0012 (4,3)37 (90,2)<0,001
Headache2 (4,2)1 (2,1)1,000(0)0(0)1,000,05 (5,7)0,02
Nausea2 (4,2)1 (2,1)1,000(0)1 (2,3)0,481 (2,1)0(0)1,00
*Flu-like but the ohms included fever, arthralgia, chills and fatigue

The conclusions. In the population, which we know that it responds poorly to immunization, the addition of CPG 7909 to pneumococcal conjugate vaccine significantly increased the proportion of patients with a strong response to the vaccine.

CPG 7909 and conjugated pneumococcal vaccine (Prevnar) were safe, and during the study did not observe adverse effects on the function of organs or the progression of HIV infection. Patients received a combination of CPG 7909 and pneumococcal conjugate vaccine (Prevnar), and adverse events were local light reactions and flu-like symptoms. In this study, CPG 7909, apparently, did not enhance the response to serotypes not included in Prevnar, after the introduction of pneumococcal polysaccharide vaccine.

Example 3: Adjuvant TLR9 agonist induces cell anamnestic response in response to pneumococcal conjugate vaccine in HIV-infected adults

The inventors investigated the effect of CPG 7909 to the induction of cellular anamnestic response to pneumococcal conjugate vaccine.

Methods. At 0 and 4 months of the study was received and stored (frozen) mononuclear cells of peripheral blood (RVMS) from 40 HIV-infected individuals included in a double-blind, placebo-controlled study phase Ib/IIa from When the EPA 1 (20 subjects in each group).

Frozen RWMS was unfrozen, tested for viability and transferred into 96-well plates to tissue cultures with a flat bottom. Cells were incubated over night at 37°C and stimulated the next day, purified pneumococcal polysaccharides (serotypes (ST) 6B and 14). After 48 hours of incubation were collected supernatant and measured concentrations of cytokines using Luminex. The relative response was calculated as the ratio of the concentrations of cytokines before and after immunization, whereas preexisting immunity to Streptococcus pneumoniae, as well as eliminating the error due to innate recognition.

Results. As shown in Fig.7, 8 and 9, one month after the second introduction of the pneumococcal conjugate vaccine relative cytokine response in the group of CPG 7909 was significantly higher than in the group with adjuvant placebo for IFN-gamma (ST6B): 1,22 against of 0.82, p=0.004; (ST14): 1,21 against 0,89, p=0.04; TNF-alpha (ST6B): 1,49 against of 0.82, p=0.03; (ST14): 1,76 against of 0.85, p=0.01); IL-6 (ST6B): 2,11 against 0,83, p=0,0084; (ST14): 1,64 against 0,81, p=0,0357), IFN-alpha (ST6B): 1,55 against 0,84, p=0.0014; (ST14): 1,43 against of 0.90, p=0,0466). Cytokine responses in the group of CPG 7909, a significantly elevated compared with the control group, was also observed for IL-1B, IL-2R, inflammatory protein macrophage-1-alpha (MIP-1-alpha), an inflammatory protein macrophage-beta (MIP-β), macrophage hamot xeonsaga protein-1 (MCP-1) and interferon-γ inducible protein-10 (IP-10).

Conclusion. The results obtained by the inventors show that in HIV-infected people adjuvant TLR9 agonist, administered with pneumococcal conjugate vaccine induced cellular anamnestic response to pneumococcal polysaccharides, which was not observed after administration of the vaccine itself.

1. Pneumococcal vaccine containing saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, and at least one agonist of Toll-like receptor-9 (TLR-9) as an adjuvant, where the specified agonist of Toll-like receptor-9 is a CpG oligonucleotide has the nucleotide sequence selected from the group consisting of:
5' TCGTCGTTTTTCGGTGCTTTT 3' (SEQ ID NO: 3),
5' TCGTCGTTTTTCGGTCGTTTT 3' (SEQ ID NO: 4),
5' TCGTCGTTTTGTCGTTTTGTCGTT 3' (SEQ ID NO: 5),
5' TCGTCGTTTCGTCGTTTTGTCGTT 3' (SEQ ID NO: 6) and
5' TCGTCGTTTTGTCGTTTTTTTCGA 3' (SEQ ID NO: 7).

2. Pneumococcal vaccine under item 1, where the specified CpG-oligonucleotide selected from the group consisting of:
5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3' (SEQ ID NO: 8), or
5 ' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3' (SEQ ID NO: 9), or
5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3' (SEQ ID NO: 10), or
5' T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T 3' (SEQ ID NO: 11), or
5' T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G*A 3' (SEQ ID NO: 12),
where * refers to phosphorothioates connection.

3. Pneumococcal vaccine for p. 1, containing from 0.2 mg to 10 mg CpG oligonucleotide.

4. Pneumococcal vaccine at p., containing approximately 1 mg CpG oligonucleotide.

5. Pneumococcal vaccine for p. 1, additionally containing saccharide from serotypes 1, 3, 5, 6A, 7F and 19A, individually conjugated to CRM197.

6. Pneumococcal vaccine for p. 1, additionally containing saccharide from serotypes 1, 5 and 7F, individually conjugated to CRM197.

7. Pneumococcal vaccine for p. 1, additionally containing saccharide from serotypes 1, 3, 5 and 7F, individually conjugated CRM197.

8. Pneumococcal vaccine under item 1, where the amount of conjugate in each vaccine dose is 0.1-1000 μg of each sahariana-protein conjugate.

9. Pneumococcal vaccine under item 8, where the amount of conjugate in each vaccine dose is 2-100 μg.

10. Pneumococcal vaccine under item 8, where the amount of conjugate in each vaccine dose is 4-40 µg.

11. Pneumococcal vaccine for p. 1, where each dose contains 0.1 to 20 μg, 1-10 or 1-5 μg μg of saccharide.

12. Pneumococcal vaccine under item 1 that contains each capsule of S. pneumoniae saccharide dose of 0.1-20 μg, 0.5 to 10 μg, 0.5 to 5 μg or 1-5 μg of saccharide.

13. Pneumococcal vaccine for p. 1 containing saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 mcg.

14. Pneumococcal vaccine is .1, optionally containing saccharide from serotypes 1, 3, 5, 6A, 7F and 19A, individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 mcg.

15. Pneumococcal vaccine for p. 1 containing buffer with sodium chloride and/or sodium succinate as the excipients.

16. Pneumococcal vaccine for p. 1, containing at least one, two or three adjuvant in addition to at least one adjuvant the agonist of TLR-9.

17. Pneumococcal vaccine for p. 1 containing an additional adjuvant.

18. Pneumococcal vaccine for p. 1, containing alum, hydrate alumina, aluminum phosphate or aluminum sulfate as an additional adjuvant to at least one adjuvant the agonist of TLR-9.

19. Pneumococcal vaccine for p. 1 containing saccharide from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F individually conjugated to CRM197, where each capsule of S. pneumoniae saccharide is present in a dose of 2 µg, with the exception of saccharide from serotype 6B, present in a dose of 4 μg, optionally containing 0.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

20. Pneumococcal vaccine for p. 1, additionally containing saccharide from serotypes 1, 3, 5, 6A, 7F and 19A, individually conjugated to CRM197, where ka is every capsule of S. pneumoniae saccharide is present in a dose of 2 mg, with the exception of saccharide from serotype 6B, present in a dose of 4 μg, optionally containing 0.5 mg of aluminum phosphate and possibly buffer with sodium chloride and sodium succinate as the excipients.

21. Pneumococcal vaccine according to any one of paragraphs.1-20 for the prevention or treatment of diseases caused by S. pneumoniae infection.

22. Pneumococcal vaccine according to any one of paragraphs.1-20 for the prevention or treatment of diseases caused by S. pneumoniae infection, the subject is immunocompromised.

23. Pneumococcal vaccine for p. 22, where the specified entity with a weakened immune system represents a person.

24. Pneumococcal vaccine for p. 22, where the specified entity with a weakened immune system suffers from a disease that kills the immune system.

25. Pneumococcal vaccine for p. 24, where the disease is a primary immunodeficiency.

26. Pneumococcal vaccine for p. 24, where the disease is an acquired immunodeficiency.

27. Pneumococcal vaccine for p. 22, where the subject is immunocompromised subject of vaccination, is an adult at the age of 55 years or over, more preferably an adult 65 years of age or older.

28. Pneumococcal vaccine for p. 22, where the subject is immunocompromised subject of vaccination, the stand is made by an adult aged 70 years or older, at the age of 75 years or older or aged 80 years or older.

29. The use of pneumococcal vaccine according to any one of paragraphs.1-20 for the manufacture of a medicine for the prevention or treatment of diseases caused by S. pneumoniae infection.

30. The use of pneumococcal vaccine according to any one of paragraphs.1-20 for the manufacture of a medicine for the prevention or treatment of diseases caused by S. pneumoniae infection, the subject is immunocompromised.

31. The use of pneumococcal vaccine according to any one of paragraphs.1-20 for vaccination of a subject with a weakened immune system.

32. Application under item 30 or 31, where the specified entity with a weakened immune system represents a person.

33. Application under item 30 or 31, where the specified entity with a weakened immune system suffers from a disease that kills the immune system.

34. Application under item 33, where the disease is a primary immunodeficiency.

35. Application under item 33, where the disease is an acquired immunodeficiency.

36. Application under item 30 or 31, where the subject is immunocompromised subject of vaccination, is an adult at the age of 55 years or over, more preferably an adult 65 years of age or older.

37. Application under item 30 or 31, where the subject is immunocompromised, the lying vaccination, is an adult at the age of 70 years or older, at the age of 75 years or older or aged 80 years or older.

38. The method of immunization of a subject against disease caused by S. pneumoniae infection, including the introduction of a specified subject immunoprotective doses of the vaccine according to any one of paragraphs.1-20.

39. The method of immunization of a subject with a weakened immune system against diseases caused by S. pneumoniae infection, including the introduction of a specified subject immunoprotective doses of the vaccine according to any one of paragraphs.1-20.

40. The method according to p. 39, where the specified entity with a weakened immune system represents a person.

41. The method according to p. 39 or 40, where the specified entity with a weakened immune system suffers from a disease that kills the immune system.

42. The method according to p. 41, where the disease is a primary immunodeficiency.

43. The method according to p. 41, where the disease is an acquired immunodeficiency.

44. The method according to p. 39 or 40, where the subject is immunocompromised subject of vaccination, is an adult at the age of 55 years or over, more preferably an adult 65 years of age or older.

45. The method according to p. 39 or 40, where the subject is immunocompromised subject of vaccination, is an adult at the age of 70 years or older is e, at the age of 75 years or older or aged 80 years or older.



 

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2 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: pharmaceutical composition possessing antiviral and antibacterial activity and containing L-N-2,6-diamino-hexanoyl-D-glucosamine, L-y-glutamyl-cysteinyl-glycyl-proline, lysine glycyrisinate, desoxyribonuclease, palmitoyl hydroxypropyl trimmonium amylopectin/glycerin cross polymer, hydroxypropyl-beta-cyclodextrin, D,L-pyrrolidone carboxylate N-cocoyl ethylarginate, escin, an emulsifying agent, a preserving agent, a pH control agent, demineralised water in certain amounts. The pharmaceutical composition possessing antiviral and antibacterial activity and containing L-N-2,6-diamino-hexanoyl-ascorbyl phosphate, N-pyrrolidine-carbamoyl-D-glucosamine, lysine ursolate, ribonuclease, a modified acrylic thickening agent, hydroxypropyl-beta-cyclodextrin, D,L-pyrrolidone carboxylate N-cocoyl ethylarginate, glycyrrhizic acid, an emulsifying agent, a preserving agent, a pH control agent, demineralised water in certain amounts. The pharmaceutical composition possessing antiviral and antibacterial activity and containing L-N-2,6-diamino-hexanoyl-hinokitiol, N-pyrrolidine-carbamoyl-D-glucosamine, lysine betulinate, collagenase, palmitoyl hydroxypropyl trimmonium amylopectin/glycerin cross polymer, hydroxypropyl-beta-cyclodextrin, chlorhexidine bigluconate, D-panthenol, an emulsifying agent, a preserving agent, a pH control agent, demineralised water in certain amounts. The pharmaceutical composition possessing antiviral and antibacterial activity and containing fusidoyl lysine, lysyl-valyl-proline pomolate, proline escinate, lysozyme, hydroxypropyl trimmunium maltodextrin, cross polymer, hydroxypropyl-beta-cyclodextrin, D,L-pyrrolidone carboxylate N-cocoyl ethylarginate, cedar extract, an emulsifying agent, a preserving agent, a pH control agent, demineralised water in certain amounts.

EFFECT: compositions possess expressed antiviral and antibacterial activity and are stable.

4 cl, 2 tbl, 11 ex

Antimicrobial gels // 2535013

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, particularly to aspects covering antimicrobial compositions, and described antimicrobial compositions, antimicrobial silicone gel based on the above antimicrobial composition, a wound dressing and methods for preparing them. Among other things, the antimicrobial compositions contain at least one alkenyl- and/or alkynyl-substituted polysiloxane, at least one polysiloxane containing silicone-linked hydrogen atoms, and at least one hydroxylation catalyst, at least one hydrophilic ingredient, at least one silver salt.

EFFECT: invention can be used for preparing a drug preparation to be used in treating burns, scars, bacterial infections, viral infections and/or mycotic infections.

19 cl, 5 dwg, 8 ex, 6 tbl

FIELD: veterinary science.

SUBSTANCE: method involves intramuscular injections of a drug preparation. The drug preparation is presented by a mixture of a medical solution of formaldehyde and normal saline at the weight portion ratio (2-6):(994-998). First, the preparation is injected into ill, latently ill and healthy animals starting from 3-month-old calves in two doses of 5-6 ml every 7-8 days during the period from September to November. Thereafter, the injections are continued for the ill animals only, but for no more than 5 weeks, and 5-7 days after the last injection the remaining ill animals are detected and isolated.

EFFECT: method enables recovering the farm effectively from necrobacteriosis.

1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to veterinary science and aims at treating subclinical mastitis in cows. A preparation and a method for using it are declared. The preparation contains silver nanoparticles stabilised by submicrone titanium dioxide particles, poly-N-vinylpyrrolidone-2, and water in the following proportions, wt %: silver nanoparticles having a size of 200-300 nm 2.0×10-3, titanium dioxide particles having a size of 2-4 mcm 60.0×10-3, poly-N-vinylpyrrolidone-2 10.0, water - the rest up to 100.0. The method involves intracisternal introduction of the declared drug preparation in a dose of 10.0 ml twice a day for 2-4 days.

EFFECT: using the method enables providing the higher clinical effectiveness and reduces the length of recovery.

2 cl, 4 dwg, 7 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared group of inventions refers to veterinary science and is applicable for treating the animals suffering from bacteriosis and yeast mycosis. A declared preparation contains oxytetracycline hydrochloride, sulphadimine, ampicillin sodium, nistatine, a solvent and the active substance conduit dimethyl sulphoxide, the quick-relief anaesthetic lidocaine in the following proportions, wt %: ampicillin sodium 4.0-8.0, oxytetracycline hydrochloride 2.0-4.0, nistatine 1.0-2.0, sulphadimine 2.0-4.0, novocaine 0.25-0.5, lidocaine 0.25-0.5, dimethyl sulphoxide 10.0-20.0, 1,2-propylene glycol - the rest. A method of treating the animals consists in administering the declared preparation in a dose of 0.1-0.2 cm3 per 1 kg of body weight.

EFFECT: using the declared group of inventions is high-efficiency for treating the animals suffering from bacteriosis and yeast mycosis and enables improving livestock farms with an unfavourable incidence of bacteriosis and yeast mycosis.

5 cl, 9 ex

FIELD: medicine.

SUBSTANCE: composition containing an activated-potentiated form of antibodies to the brain-specific S-100 protein, and an activated-potentiated form of antibodies to endothelial NO-synthase as an additional exalting component is administered.

EFFECT: effective treatment of organic diseases of the nervous system by the synergetic neurotropic action of the components.

18 cl, 5 ex, 5 tbl

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