ES2365717T3 - CONJUGATED MENINGOCÓCICA VACCINATION. - Google Patents

Abstract

A composition comprising: (a) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup A and (ii) a diphtheria anatoxin or derivative thereof, which remains immunologically cross-reactive with the diphtheria anatoxin; (b) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup C and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin; (c) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria or derivative anatoxin which remains immunologically cross-reactive with the diphtheria anatoxin; and (d) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup Y and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin, for use in a procedure for immunize a human patient against a disease caused by Neisseria meningitidis, which comprises the step of administering the composition to the human patient, in which the patient has been preimmunized with (a) a diphtheria or derivative anatoxin, which remains immunologically reactive crossed with diphtheria anatoxin and / or (b) a conjugate of (i) a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria or derivative anatoxin thereof, which remains immunologically cross-reactive with diphtheria anatoxin.

Description

Technical field

The present invention relates to vaccines against Neisseria meningitidis. In particular, it refers to vaccines based on conjugated capsular saccharides of multiple meningococcal serogroups.

Prior art

Based on the capsular polysaccharide of the organism, twelve serogroups of N. meningitidis (A, B, C, H, I, K, L, 29E, W135, X, Y and Z) have been identified. Group A is the pathogen most frequently implicated in epidemic diseases in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in more developed countries. Serogroups W135 and Y are responsible for the remaining cases in the United States and in developed countries.

For many years, a tetravalent vaccine of capsular polysaccharides from serogroups A, C, Y and W135 has been known [1,2]. Although it is effective in adolescents and adults, it induces a poor immune response and a short duration of protection and cannot be used in children (for example ref. 3) because polysaccharides are independent antigens of T lymphocytes that induce a weak immune response that cannot be reinforced.In this vaccine, the polysaccharides are not conjugated [4].

Conjugated serogroup C vaccines have been approved for human use and include Menjugate ™ [5], Meningitec ™ and NeisVac-C ™. Mixtures of conjugates of serogroups A + C [6-8] are known and mixtures of conjugates of serogroups A + C + W135 + Y [9-13] have been described.

Although meningococcal conjugates are well known, they have not yet adapted to existing pediatric immunization programs, in which, in developed countries, they typically participate: the hepatitis B vaccine at birth and, after 2 months, all diphtheria / tetanus / pertussis (DTT), H. influenzae type b (Hib), inactivated poliovirus and pneumococcal conjugates at 2 months.

However, when conjugate vaccines are added to existing immunization programs, the problem of vehicle-induced epitopic suppression (or "vehicle suppression," as is generally known) must be addressed, particularly the suppression that arises from vehicle sensitization. "Vehicle suppression" is the phenomenon whereby the preimmunization of an animal with a transport protein prevents it from a late provocation of an immune response against a new antigenic epitope that is present in that vehicle [14].

As described in bibliographic reference 15, when several vaccine antigens contain the same protein component (being used as an immunogen and / or as a transport protein in a conjugate) then there is a possibility of interference between those antigens. In bibliographic reference 15, the immunological response against an antigen, which was conjugated with a tetanus anatoxin vehicle (Ta), was suppressed by preexisting immunity against Ta.

Bibliographic reference 16 describes how a combination of D-T-T vaccines with a Hib conjugate vaccine adversely affected when the vehicle for the Hib conjugate was the same as the tetanus antigen of the D-T-T vaccine. The authors conclude that this “vehicle suppression” phenomenon, which arises from interference by a common protein vehicle, must be taken into account when vaccines that include multiple conjugates are introduced.

Unlike bibliographic references15 and 16, bibliographic reference 17 describes that sensitization with tetanus anatoxin did not have a negative impact on the immune response against a Ta-Hib conjugate subsequently administered, but suppression has been observed in patients with anti-Ta antibodies acquired by the mother. However, in literature reference 18, an effect of "epitopic suppression" for a Ta-based peptide conjugate is described in patients possessing existing anti-Ta antibodies from tetanus vaccination.

In bibliographic reference 19, it was suggested that a conjugate that possessed CRM197 (a non-toxic mutant of diphtheria toxin) as a vehicle could be ineffective in children who had not previously received diphtheria toxin as part of a vaccine (for example as part of a vaccine against DTT or DT). This work was subsequently developed in reference 20, where it was observed that a sensitization effect persisted with vehicle by D-T immunization for subsequent immunization with Hib conjugates.

In the bibliographic reference 21, the authors discovered that preimmunization with a tetanus or diphtheria anatoxin transport protein reduced the increase in anti-Hib antibody levels after subsequent immunization with the Hib capsular saccharide conjugated to these vehicles, similarly affecting with IgG1 and IgG2. The responses against the vehicle parts of the conjugates were also suppressed. Additionally, a more general non-epitopic specific suppression was observed, since it was observed that preimmunization with a conjugate affected immune responses against the vehicle and saccharide parts of a second conjugate which was administered four weeks later.

Reference 22 describes the use of different transport proteins in a single multivalent conjugate pneumococcal vaccine, in which multiple vehicles are used to prevent suppression of the vehicle. The authors predict that, in a multivalent conjugate vaccine, there is a maximum load of a transport protein that can be tolerated without causing negative interference. In bibliographic reference 23, it was described that pneumococcal conjugate vaccines that included mixed transport proteins caused, in parallel to the pneumococcal response, involuntary reinforcement responses against vehicles.

In bibliographic reference 24, an investigation into whether reinforcements could be administered either against diphtheria or tetanus with monovalent meningococcal serogroup C conjugates, it was found that titers against the meningococcal conjugate were reduced when the vehicle was the tetanus anatoxin vehicle and the patient He had received previous immunization with a vaccine containing tetanus.

Finally, bibliographic reference 25 describes that "prior to exposure to the transport protein, the antibody response to polysaccharides administered in protein-saccharide conjugates can be enhanced or suppressed." The conjugates used in bibliographic reference 25 use, as transport protein, tetanus anatoxin or the mutant CRM197.

Therefore, the situation with regard to vehicle sensitization and / or suppression is confusing and remains unclear whether any particular conjugate will experience vehicle suppression or benefit from increased vehicle awareness. Meningococcal conjugate vaccines will not be in a position to integrate or add to existing pediatric immunization programs until this problem is addressed. Additionally, since meningococcal conjugates must be administered as tetravalent mixtures (ie four different conjugates) then the possibility of suppression per vehicle becomes even more risky.

In addition to the sensitization problem with a vehicle that has a negative impact on immune responses against saccharide conjugates, the opposite may also occur, that is, immunization with a conjugate can have a negative impact on immune responses against the vehicle [26].

WO 03/094834 describes immunogenic compositions comprising (a) a capsular saccharide antigen from serogroup C of N. meningitidis and (b) a chitosan adjuvant. The compositions are said to preferably comprise (c) one or more additional antigens and / or (d) one or more additional adjuvants. The compositions are said to be particularly suitable for administration in the mucosa, including intranasal administration. WO 03/094834 also describes immunogenic compositions for mucosal administration comprising capsular saccharides of at least two of serogroups A, C, W135 and Y of N. meningitidis. It is said that, in the compositions of the invention, it is preferred that the capsular saccharides are conjugated to a transport protein (or proteins) and / or that they are oligosaccharides. Conjugated oligosaccharide antigens are said to be particularly preferred.

Disclosure of the invention

Bibliographic reference 27 suggests that, in conjugated meningococcal vaccines, vehicle suppression should be addressed with the use of more than one type of transport protein. In particular, bibliographic reference 27 suggests that H. influenzae D protein should be used as a transport protein for meningococcal conjugates, tetanic anatoxin (Ta) being also a possibility. To avoid epitopic suppression, protein D is also the vehicle of choice in bibliographic reference 28. Similarly, bibliographic reference 29 suggests that Bordetella pertussis fimbriae should be used as a vehicle to prevent suppression by vehicle in multivalent conjugate vaccines. . On the other hand, it has been discovered that diphtheria anatoxin (Ta) and its derivatives can be safely used as a vehicle for meningococcal saccharide conjugates, even when multiple meningococcal conjugates are administered at the same time. None of the monovalent meningococcal serogroup C conjugates studied in bibliographic reference 24 use a Da vehicle.

In addition, bibliographic reference 27 also suggests that meningococcal conjugate vaccines should be administered at the same time as D-T-T-Hib vaccines (for example see example 3), so that there is no prior exposure to the transport protein of meningococcal conjugates. On the other hand, it has now been discovered that meningococcal conjugates can be administered to patients even when they have already received the transport protein, either in the form of a previous immunogen (for example in an immunization against DTT or DT) or as a transport protein previous (for example in a Hib conjugate or pneumococcal conjugate vaccine). The previous study of vehicle-induced epitopic suppression in monovalent serogroup C conjugate vaccines [24] did not consider the effect of any previous conjugate administration.

Also in contrast to bibliographic reference 27, a patient's ability to elicit an immune response against a meningococcal conjugate, even when he has already received a different conjugate, contrasts with bibliographic reference 21.

Thus the invention provides a composition comprising: (a) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup A and (ii) a diphtheria or derivative anatoxin which remains immunologically cross-reactive with the anatoxin diphtheria; (b) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup C and (ii) a diphtheria or derivative anatoxin which remains immunologically cross-reactive with the diphtheria anatoxin; (c) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria or derivative anatoxin that remains immunologically cross reactive with the diphtheria anatoxin and (d) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup Y and (ii) a diphtheria or derivative anatoxin that remains immunologically cross-reactive with diphtheria anatoxin, for use in a procedure to immunize a patient against a disease caused by Neisseria meningitidis, which comprises the step of administering to the patient the composition, in which the patient has been preimmunized with (a) a diphtheria anatoxin or derivative thereof that remains immunologically cross reactive with the diphtheria anatoxin and / or (b ) a conjugate of (i) a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria or derivative anatoxin that remains immunological cross-reactively with diphtheria anatoxin.

The invention also provides the use of: (a) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup A and (ii) a diphtheria or derivative anatoxin which remains immunologically cross reactive with the diphtheria anatoxin; (b) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup C and (ii) a diphtheria or derivative anatoxin which remains immunologically cross reactive with the diphtheria anatoxin; (c) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria or derivative anatoxin which remains immunologically cross reactive with the diphtheria anatoxin and (d) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup Y and (ii) a diphtheria or derivative anatoxin that remains immunologically cross reactive with diphtheria anatoxin, in the preparation of a medicament for immunizing a patient against a disease caused by Neisseria meningitidis in which the patient has been preimmunized with (a) a diphtheria or derivative anatoxin that remains immunologically cross-reactive with the diphtheria anatoxin and / or (b) a conjugate (i) of a capsular saccharide of a an organism other than N. meningitidis and (ii) a diphtheria or derivative anatoxin that remains immunologically cross reactive with diphtheria anatoxin.

The meningococcal disease is preferably meningitis, more preferably bacterial meningitis and more preferably meningococcal meningitis. Therefore the invention can be used to protect against meningococcal infections that cause meningitis.

When the preimmunization antigen is a derivative of a diphtheria anatoxin that remains immunologically cross reactive with Da, then the derivative is preferably CRM197.

The preimmunized patient

The patient to be immunized has been preimmunized with: (a) a diphtheria or derivative anatoxin that remains immunologically cross-reactive with the diphtheria anatoxin and / or (b) a conjugate (i) of a capsular saccharide of a different organism of Neisseria meningitidis and (ii) a diphtheria or derivative anatoxin that remains immunologically cross-reactive with diphtheria anatoxin. Typical preimmunization will have included, a diphtheria anatoxin antigen; a Hib capsular saccharide conjugate using a diphtheria anatoxin carrier or CRM197 and / or a pneumococcal capsular saccharide conjugate using a diphtheria anatoxin carrier or CRM197.

The patient will have received at least one (for example 1, 2, 3 or more) doses of the preimmunization antigen (or antigens) and this dose (or the first multiple dose) will have been administered to the patient at least six (for example 6 , 9, 12, 15, 18, 21, 24, 36, 48, 60, 120, 180, 240, 300 or more) months before immunization with meningococcal conjugates according to the invention. In a preferred group of patients, preimmunization is performed 3 years after birth, for example 2 years after birth, one year after birth, 6 months after birth or even 3 months, 2 months or 1 month after birth. birth.

The patient to be immunized according to the invention will typically be a human being. The human being will generally be at least 1 month old, for example at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least 2 years, at least 5 years, at least 11 years, at At least 17 years, at least 46 years, at least 55 years, etc. A preferred group of patients is at least 6 months old. Another preferred patient group is the 2 to 55 year old group and another preferred patient group is the 11 to 55 year old group. An additional preferred set of patients is less than 11 years, for example, 2 to 11 years. However, in all cases, regardless of age, the patient will have been preimmunized as defined herein.

When the preimmunization antigen is a diphtheria anatoxin then the patient will typically have received the anatoxin as a "D" antigen in a D-T-T or D-T preimmunization. Such immunizations are typically provided to newborn children at 2, 3, and 4 months of age. When the immunization includes a pertussis vaccine, this vaccine may be a cell or whole cell pertussis vaccine (‘Pw’), but preferably it is an acellular pertussis vaccine (‘Pa’). Pa vaccines for preimmunization will generally include one, two or three of the following well-known and well characterized B. pertussis antigens: (1) pertussis anatoxin ('TA'), detoxified by chemical means or by directed mutagenesis, for example the mutant '9K / 129G' [30]; (2) filamentous hemagglutinin (‘FHA’); (3) pertactin (also known as ‘69 kiloDalton outer membrane protein’). Acellular pertussis vaccines may also include agglutinogen 2 and / or agglutinogen 3. The ‘T’ antigen in a D-T-T preimmunization is typically a tetanus anatoxin.

When the preimmunization antigen is a diphtheria anatoxin then the patient may also, or alternatively, have received the anatoxin as a transport protein of a saccharide-protein conjugate. Such conjugates include the ‘PRP-D’ Hib conjugate (see Table 14-7 or bibliographic reference 32], for example the ProHIBIT ™ product.

When the preimmunization antigen is CDM197 then the patient will typically have been preimmunized with a Hib conjugate and / or a multivalent pneumococcal conjugate. Such immunizations are typically provided to newborn children at 2, 3, and 4 months of age. Hib conjugates using a CRM197 vehicle include the ‘HbOC’ conjugates [Table 14-7 of bibliographic reference 32] for example the HibTITER ™ product. Pneumococcal conjugates using a CRM197 vehicle include 7-valent mixtures of PCV7 for example the PrevNar ™ vaccine [31]. The patient may also have preimmunized with a meningococcal (‘MenC’) serogroup C conjugate. MenC conjugates using CRM197 vehicle include Meninvact ™ / Menjugate ™ [5] and Meningitec ™. Preferably, however, the patient has been preimmunized with Hib and / or pneumococcal conjugate, but not with a MenC conjugate. If the patient has been preimmunized with a MenC conjugate then the vaccine administered according to the present invention may or may not include a serogroup C conjugate.

When preimmunization is performed with a conjugated antigen then the patient will have almost inevitably received a small amount of free diphtheria (or derivative) anatoxin as a result of low level contamination of the conjugate (caused, for example, by hydrolysis of the conjugate during storage ) but this small amount will not typically have been adequate to provide a significant immune response.

Diphtheria anatoxin is a well-known and well-characterized protein [see, for example, chapter 13 of bibliographic reference 32] that can be obtained by treating Corynebacterium diphtheriae ribosilant exotoxin with an inactivating chemical, such as formalin or formaldehyde. CRM197 is also well known and well characterized [33-36] and has been widely used as a vehicle in conjugated saccharide vaccines. CRM197 and Da share many vehicle epitopes.

The result of preimmunization is that the patient's immune system has been exposed to preimmunization antigens. For preimmunization with diphtheria anatoxin (Da), this generally means that the patient will have elicited an anti-Da antibody response (typically to provide an anti-Da titration> 0.01 IU / ml) and will possess memory of B and / or lymphocytes. or T specific for Da, that is, preimmunization with Da is typically suitable for eliciting, in the patient, an anamnestic anti-Da immune response. For the preimmunization in which Da (or derivative) is a vehicle for a saccharide within a conjugate then the preimmunization will have elicited an anti-saccharide response and the patient will possess specific memory of B and / or T lymphocytes for the saccharide ie the Preimmunization is typically adequate to elicit an anamnestic anti-saccharide immune response in the patient. The preimmunization was preferably suitable to cause in the patient protective immunity, for example, against diphtheria disease.

Therefore, the patients to be immunized according to the present invention are different from the patients in general, since they are members of a subset of the general population whose immune system has already created an immune response against preimmunization antigens, so that the Immunization according to the present invention with a meningococcal conjugate that includes a diphtheria (or derivative) anatoxin vehicle causes in the subset a different immune response to that of patients who have not previously created any immunological response against preimmunization antigens. Preferred patients are those who have been preimmunized with Da (or derivative) as the vehicle of a conjugate (particularly of a Hib conjugate). Particularly preferred patients have been preimmunized with Da (or derivative) as the vehicle of a conjugate and also with Da as an unconjugated immunogen.

Just as it has been preimmunized with a diphtheria (or derivative) anatoxin, in conjugate or unconjugated form, the patient may have pre-immunized with other antigens. Such antigens include, but are not limited to: pertussis antigen (or antigens) - see above; tetanus anatoxin - see above; Haemophilus influenzae type B - see above; hepatitis B surface antigen (HBsAg); poliovirus, such as an inactivated poliovirus (IPV) vaccine; Streptococcus pneumoniae - see above; flu virus; BCG; hepatitis A virus antigens; measles virus; mumps virus; Rubella virus; chickenpox virus; etc.

The patient may or may not have preimmunized with one or more meningococcal conjugates. In some preferred embodiments, at the time a patient first receives a meningococcal conjugate, it was already preimmunized with Da (or derivative). In other embodiments, a meningococcal conjugate is administered to a patient who had already been preimmunized with both (i) Da or a derivative and with (ii) a meningococcal conjugate.

The conjugates

The invention immunizes patients with conjugated saccharides. The conjugation is used to enhance the immunogenicity of saccharides, since it transforms them from independent T antigens to dependent T antigens, thereby allowing immunological memory sensitization. Conjugation is particularly useful in pediatric vaccines [eg, bibliographic reference 37] and is well known in the art [review, for example, bibliographic references 38 to 46].

The composition used according to the invention comprises at least two meningococcal conjugates, in which each conjugate comprises a diphtheria anatoxin transport protein (or derivative) and the capsular saccharide. Capsular saccharides are selected from meningococcal serogroups A, C, W135 and Y, so that the compositions include saccharides of 2, 3 or 4 of these four serogroups. Specific compositions comprise saccharides of: serogroups A and C; serogroups A and W135; serogroups A and Y; serogroups C and W135; serogroups C and Y; serogroups W135 and Y; serogroups A and C and W135; serogroups A and C and Y; serogroups A and W135 and Y; serogroups C and W135 and Y; serogroups A and C and W135 and Y. The most preferred compositions are those that include saccharides from the four serogroups.

The capsular saccharides of each of these four serogroups are well characterized. The serogroup A meningococcal capsular saccharide is a homopolymer of (α1�6) -united with N-acetyl-D-mannosamine-1-phosphate, with partial oacetylation at positions C3 and C4. Acetyl groups can be substituted with blocking groups to prevent hydrolysis [47] and said modified saccharides are still saccharides from serogroup A within the meaning of the present invention. The capsular saccharide of serogroup C is a homopolymer of (α2�9) - linked to sialic acid (N-acetyl neuraminic acid, or ‘NeuNAc’). Most strains of serogroup C have O-acetyl groups in the C-7 and / or C-8 residues of sialic acid, but approximately 15% of clinical isolates lack these Oacetyl groups [48,49]. The structure of the saccharide is written �9) -Neu p NAc 7/8 OAc- (α2�. The saccharide of serogroup W135 is a polymer of units of the disaccharide sialic-galactose acid. Like the saccharide of serogroup C, it has Oacetylation variable, but in positions 7 and 9. of sialic acid [50] The structure is written: �4) -DNeup5Ac (7 / 90Ac) -α- (2�6) -D-Gal-α- (1�. The saccharide of serogroup Y is similar to saccharide of serogroup W135 except that the repeat units of the disaccharide include glucose instead of galactose, just like serogroup W135, it has a variable O-acetylation at positions 7 and 9 of sialic acid [ 50] The structure of serogroup Y is written: �4) -D-Neup5Ac (7 / 90Ac) -α- (2�6) -D-Glc-α- (1�.

The saccharides used in accordance with the invention may be O-acetylated as described above (for example with the same O-acetylation model as observed in natural capsular saccharides) or they may be partially or totally de-O-acetylated in one or more positions of the saccharide rings or they may be hyper-oacetylated with respect to the original capsular saccharides.

The saccharides used according to the invention are preferably shorter than the natural capsular saccharides observed in bacteria. Therefore, saccharides are preferably depolymerized, depolymerization occurring after purification but before conjugation. Depolymerization reduces the length of the saccharide chain. A preferred depolymerization process involves the use of hydrogen peroxide [9]. Hydrogen peroxide is added to a saccharide (for example, to provide a final concentration of H2O2 of 1%) and then the mixture is incubated (for example, at approximately 55 ° C) until a desired reduction in chain length is achieved. Another depolymerization process involves acid hydrolysis [10]. Those skilled in the art know other depolymerization processes. The saccharides used to prepare conjugates for use according to the invention can be obtained by any of these depolymerization processes. Depolymerization can be used to provide an optimal chain length for immunogenicity and / or to reduce the chain length for the physical handling of saccharides.

Typical transport proteins for use in conjugates are bacterial toxins or anatoxins such as diphtheria toxin (or its mutant CRM197) and tetanus toxin. Other known transport proteins include the outer membrane protein of N. meningitidis, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4 + T cell epitopes. of various pathogen derived antigens, H. influenzae D protein, PspA pneumococcal surface protein, iron-sensing proteins, C. difficile toxin A or B, etc. However, according to the invention, meningococcal conjugates include a diphtheria anatoxin transport protein (or derivative, such as CRM197). Covalent conjugation is preferred.

In the compositions it is possible to use more than one transport protein. Therefore different transport proteins can be used for different serogroups, for example, saccharides of serogroup A could be conjugated with CRM197 while saccharides of serogroup C could be conjugated with diphtheria anatoxin. It is also possible to use more than one transport protein for a particular saccharide antigen, for example, serogroup A saccharides could be in two groups, some conjugated with CRM197 and others conjugated with diphtheria anatoxin. However, in general, the use of the same transport protein is preferred in the composition for all meningococcal saccharides and more preferably for all saccharides (ie, including any non-meningococcal conjugate that may be present). It is preferred that the compositions of the invention do not include any tetanus anatoxin transport protein. When the composition includes a diphtheria anatoxin transport protein then it is preferred that no CRM197 transport protein be included.

A single transport protein could carry more than one saccharide antigen [51]. For example, a single transport protein could be conjugated with saccharides of serogroups A and C. To achieve this goal, the saccharides can be mixed before the conjugation reaction. However, in general, it is preferred to have different conjugates for each serogroup. The conjugates are preferably mixed to substantially provide a 1: 1: 1: 1 ratio (measured as saccharide mass) for example the mass of each saccharide of the serogroups is within ± 10% relative to each other. A typical amount of meningococcal antigen per serogroup in a composition is between 1 µg and 20 µg for example between 2 and 10 µg per serogroup or about 4 µg. As an alternative for a 1: 1: 1: 1 ratio, a double dose of serogroup A (2: 1: 1: 1) can be used.

Conjugates with a saccharide ratio are preferred: protein (w / w) between 1:15 (for example excess protein) and

15: 1 (ie excess saccharide), preferably between 1:10 and 10: 1, more preferably between 1: 5 and 5: 1. S e prefers excess transport protein. Conjugates with a saccharide: protein ratio of about 1:12 or about 1: 6 or about 1: 3 are preferred, particularly when the carrier is Da a 1: 3 ratio is more preferred.

The conjugates can be used together with a free transport protein [52]. However, when in a composition of the invention there is a transport protein determined in conjugated and free form, the unconjugated form preferably does not exceed 5% of the total amount of the transport protein in the composition as a whole, and more preferably present in less than 2% by weight. Similarly, it is preferred that the unconjugated saccharide does not exceed 15% by weight of the total amount of saccharide.

When necessary, any suitable conjugation reaction can be used with any suitable crimp.

The saccharide will typically be activated or functionalized before conjugation. Activation may involve, for example, cyanylation reagents such as CDAP (for example, 1-cyano-4-dimethylamino-pyridinium-tetrafluoroborate [53, 54, etc.])). Other suitable methods use carbodiimides, hydrazines, active esters, norborane, pnitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction of bibliographic reference 44).

Linkages can be made by a linker group using any known method, for example, the procedures described in bibliographic references 55 and 56. One type of linkage involves the reductive amination of the polysaccharide, the coupling of the resulting amino group with one end of a group of adipic acid crimp and then the coupling of a protein to the other end of the adipic acid crimp group [42, 57, 58]. Other linkers include β-propionamido [59], nitrophenyl-ethylamine [60], haloacyl halides [61], glycosidic bonds [62], 6-aminocaproic acid [63], ADH [64], C4 to C12 residues [65] etc. As an alternative to the use of a linker, a direct link can be used. Direct links to the protein may comprise oxidation of the polysaccharide followed by reductive amination with the protein, as described, for example, in bibliographic references 66 and 67.

A process involving the introduction of amino groups into the saccharide is preferred (for example substitution of groups = O-terminals with -NH2) followed by derivatization with an adipic diester (for example N-hydroxysuccinimido diester of adipic acid) and reaction with the protein Of transport.

In a preferred conjugation process, a saccharide reacts with the dihydrazide of adipic acid. For serogroup A, carbodiimide can also be added at this stage. After a reaction period, sodium cyanoborohydride is added. Then, the derivatized saccharide can be prepared, for example, by ultrafiltration. Then the derivatized saccharide is mixed with a transport protein (for example with a diphtheria anatoxin), and carbodiimide is added. After a reaction period, the conjugate can be recovered. Additional details of this conjugation can be found in bibliographic reference 10. The conjugates that can be obtained by this process are preferred conjugates for use in accordance with the present invention, for example, conjugates comprising a diphtheria anatoxin carrier and an adipic acid linker.

Preferably, the conjugates are prepared separately and then mixed. After mixing, the concentration of the mixed conjugates can be adjusted, for example, with sterile phosphate buffered saline, without pyrogen. Before mixing, each conjugate preferably contains no more than 15 µg of vehicle.

The result of administering meningococcal conjugates according to the invention is preferably that, for each serogroup administered, the patient provokes a serum bactericidal antibody (ABS) response, the increase in ABS titration being at least 4 times (compared to the pre-immunized patient before receiving the mixed meningococcal conjugates) and preferably at least 8 times. The ABS test is a conventional correlative concept for protection against meningococci. Additional details of correlative serological concepts for meningococcal vaccines are provided in bibliographic reference 68.

Additional antigenic components of the compositions used in accordance with the invention

In addition to meningococcal conjugates, the compositions used according to the invention may optionally include 1, 2, or 3 of the following additional antigens:

one.

A conjugated capsular saccharide of S. pneumoniae [eg, chapter 23 of bibliographic reference 32; bibliographical references 69-71]. It is preferred to include saccharides of more than one S. pneumoniae serotype. For example, mixtures of polysaccharides of 23 different serotypes are widely used as vaccines conjugated with polysaccharides of between 5 and 11 different serotypes [72]. For example, PrevNar ™ [31] contains seven serotype antigens (4, 6B, 9V, 14, 18C, 19F and 23F) each saccharide being conjugated individually with CRM197 by reductive amination with 2 µg of each saccharide per 0.5 ml dose (4 µg of serotype 6B) and with conjugates adsorbed on an aluminum phosphate adjuvant. When pneumococcal conjugates are included in a composition for use with the present invention, the composition preferably includes at least 6B, 14, 19F and 23F serotypes.

2.

A conjugated capsular saccharide of H. influenzae B [eg chapter 14 of bibliographic reference 32]. The conjugate transport protein can be CRM197, Da, a tetanus anatoxin or an outer membrane complex of N. meningitidis. The saccharide moiety of the conjugate may be a polysaccharide (for example, full length polyribosylribitol phosphate (PRP)), but it is preferred to depolymerize the capsular polysaccharides to form oligosaccharides (for example with a PM of ∼1 to k5 kDa). A preferred Hib conjugate comprises an oligosaccharide covalently linked to CRM197 by an adipic acid linker [73,74]. Administration of the Hib antigen preferably results in an anti-PRP antibody concentration of> 0.15 µg / ml and more preferably> 1 µg / ml. When a composition includes a Hib saccharide antigen, it preferably also does not include any aluminum hydroxide adjuvant. If the composition includes an aluminum phosphate adjuvant then the Hib antigen can be adsorbed [75] or not [27] to the adjuvant. Adsorption prevention can be achieved by selecting the correct pH during the antigen / adjuvant mixture, an adjuvant with an appropriate zero loading point and an appropriate mixing order for the various different antigens in a composition [76].

3.

A serogroup B Neisseria meningitidis protein antigen [for example, bibliographic reference 77].

The composition may comprise one or more of these additional antigens.

Such antigens may or may not be adsorbed to an aluminum salt.

If the meningococcal conjugate is administered in a series of doses then none, any or all doses may include these extra antigens.

Preferably the compositions containing the meningococcal conjugates do not include tetanus anatoxin. Preferably these do not include pertussis antigens. Preferably these do not include hepatitis B virus surface antigens. Preferably these do not include poliovirus. A composition preferably contains no more than 50 µg of diphtheria anatoxin per menincococcal conjugate and more preferably no more than 50 µg of diphtheria anatoxin for all recombinant meningococcal conjugates.

The vaccine composition

The composition used in accordance with the present invention will typically include a pharmaceutically acceptable carrier. Such vehicles include any vehicle that by itself does not induce the production of harmful antibodies to the individual receiving the composition. Suitable carriers are typically slowly metabolized large macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose and lipid aggregates (such as oil drops or liposomes). Such vehicles are well known to a person skilled in the art. Vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like can exist. A typical vehicle is sterile phosphate buffered physiological saline without pyrogen. A detailed analysis of pharmaceutically acceptable vehicles and excipients can be found in bibliographic reference 78.

Compositions used in accordance with the present invention may include an antimicrobial agent, particularly if they are packaged in a multiple dose format.

The compositions used in accordance with the present invention may comprise detergents, for example, a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels for example <0.01%.

Compositions used in accordance with the present invention may include sodium salts (for example sodium chloride and / or sodium phosphate). These can be used for tonicity. A concentration of 10 ± 2 mg / ml of NaCl is typical, for example, approximately 8.8 mg / ml. A concentration of 1.2 mg / ml sodium phosphate is typical.

Compositions used in accordance with the invention will generally include a buffer, for example, a phosphate buffer.

The compositions used according to the invention may comprise a sugar alcohol (for example mannitol) or a disaccharide (for example sucrose or trehalose) for example at about 15-30 mg / ml (for example 25 mg / ml), particularly if they have to be lyophilized or if they include material that has been reconstituted from lyophilized material. However, the preferred compositions are not lyophilized, that is, all meningococcal conjugates are present in aqueous form, from the packaging stage to the administration stage.

Generally the compositions will be administered directly to the patient. Direct administration can be achieved by parenteral injection (for example subcutaneously, intraperitoneally, intravenously, intramuscularly or in the interstitial space of a tissue) or by rectal, oral, vaginal, topical, transdermal, intranasal administration, ocular, otic, pulmonary or other mucosal administration. Intramuscular administration (for example in the thigh or arm) is preferred. The injection can be by means of a needle (for example a hypodermic needle), but as an alternative a needleless injection can also be used. A typical intramuscular dose is 0.5 ml.

Meningococcal conjugates of multiple serogroups are administered in admixture with a single composition. The composition may be administered as a single dose or may be administered more than once in a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. A primary dose schedule may continue with a booster dose schedule of meningococcal conjugates. The appropriate timing between sensitization doses (for example between 4-16 weeks) and between sensitization and reinforcement, can be determined routinely. The conjugates can be conveniently administered at the same time as other vaccines, for example, at the same time as a DTT vaccine or at the same time as a pneumococcal conjugate vaccine or at the same time as a flu vaccine or at the same time as a vaccine against MMR or MMRV. Generally these vaccines will be administered separately but during the same medical consultation.

Bacterial infections can affect various areas of the body and therefore the compositions can be prepared in various ways. For example, the compositions can be prepared as injectables, either as suspensions or as liquid solutions. Prior to injection, solid forms suitable for solution or suspension in liquid vehicles (for example a lyophilized composition) can also be prepared. The composition can be prepared for topical administration, for example, as an ointment, cream or powder. The composition can be prepared for oral administration, for example, as a tablet or capsule or as a syrup (optionally flavored). The composition can be prepared for pulmonary administration, for example, as an inhaler, using a fine powder or a spray. The composition can be prepared as a suppository or pessary. The composition can be prepared for nasal, otic or ocular administration for example as sprays, drops, gel or powder [eg, bibliographic references 79 and 80]. However, in general, meningococcal conjugates are formulated for intramuscular injection.

The compositions used according to the invention may or may not include a vaccine adjuvant. Adjuvants that can be used in the compositions of the invention include, but are not limited to:

A. Compositions containing minerals

Suitable compositions containing minerals for use as adjuvants in the invention include mineral salts, such as aluminum salts and calcium salts. The invention includes mineral salts such as hydroxides (for example oxyhydroxides), phosphates (for example hydroxyphosphates, orthophosphates), sulfates, etc. [for example see chapters 8 and 9 of bibliographic reference 81] or mixtures of different mineral compounds, the compounds adopting any suitable form (for example gel, crystalline, amorphous, etc.) and adsorption being preferred. Mineral-containing compositions can also be formulated as a metal salt particle [82].

Particularly preferred are aluminum phosphates and a typical adjuvant is amorphous aluminum hydroxyphosphate with a PO4 / Al molar ratio between 0.84 and 0.92, including approximately 0.6 ml Al3 + / ml. An adsorption with a low dose of aluminum phosphate can be used, for example between 50 and 100 µg Al3 + per dose conjugate. When a composition includes conjugates of multiple bacterial species not all conjugates need to be adsorbed

B. Oilseed emulsions

Oleaginous emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of bibliographic reference 81; see also bibliographic reference 83] (5% squalene, 0.5% Tween 80 and 0.5% Span 85, formulated in submicron particles using a microfluidizer). Freund's complete adjuvant (ACF) and incomplete Freund's adjuvant (AIF) can also be used.

C. Formulations with saponin [Chapter 22 of the bibliographic reference 81]

Saponin formulations can also be used as adjuvants in the present invention. Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides found in wood, leaves, stems, roots and even flowers of a wide variety of plant species. The wood saponin from the Quillaia saponaria Molina tree has been widely studied as adjuvants. Saponin can also be obtained from the market from Smilax ornata (sarsaprilla), Gypsophilla paniculata (bridal veil) and Saponaria officianalis (soap dish). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations such as ISCOMs. QS21 is marketed as Stimulon ™.

Compositions with saponin have been purified using HPLC and RP-HPLC. Using these techniques, specific purified fractions have been identified including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, the saponin is QS21. In ref. 84 describes a production procedure of QS21. Saponin formulations may also comprise a sterol, such as cholesterol [85].

The combinations of saponins and cholesterols that can be used to form single particles are called immunostimulatory complexes (ISCOM) [Chapter 23 of the bibliographic reference 81]. ISCOMs also typically include a phospholipid, such as phosphatidyl ethylamine or phosphatidyl choline. Any known saponin can be used in ISCOMs. Preferably, ISCOMs include one or more of QuilA, QHA and QHC. ISCOMs are further described in bibliographic references 85-87. Optionally, ISCOMs may lack additional detergents [88].

A review of the development of adjuvants based on saponin can be found in bibliographic references 89 and 90.

D. Virosomes and virus-like particles

Virosomes and virus-like particles (VLP) can also be used as adjuvants in the invention. These structures generally contain one or more virus proteins optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any natural viral genome. Viral proteins can be produced recombinantly or isolated from whole viruses. These viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as Ha or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot and Mouth Disease virus, Retrovirus, Norwalk virus, human papillomavirus, HIV, RNA phage, Qβ phage (such as cover proteins), GA phage, fr phage, phage AP205 and Ty (such as the P1 protein of the Ty retrotransposon). VLPs are further analyzed in bibliographic references 91-96. Virosomes are further analyzed, for example, in bibliographic reference 97.

E. Bacterial or microbial derivatives

Adjuvants suitable for use in the invention include bacterial or microbial derivatives, such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides and ribosilating ADP toxins and their detoxified derivatives.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and MPL 3-O-deacylated (3dMPL). The 3dMPL is a mixture of monophosphoryl lipid A 3-des-O-acylated with 4, 5 or 6 acylated chains. In bibliographic reference 98 a preferred form of "small particle" of monophosphoryl lipid A 3-de-O-acylated is described. Said "small particles" of 3dMPL are small enough to be sterilized by filtration through a 0.22 µm membrane [98]. Other non-toxic LPS derivatives include monophosphoryl lipid A mimetics, such as aminoalkyl glucosaminide phosphate derivatives for example RC-529 [99,100].

Lipid A derivatives include Escherichia coli lipid A derivatives such as OM-174. OM-174 is described, for example, in bibliographic references 101 and 102.

Immunostimulatory oligonucleotides suitable for use as adjuvants in the present invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated ctiosine linked by a phosphate bond to a guanosine). It has also been shown that double-stranded RNAs and oligonucleotides containing palindromic or poly (dG) sequences are also immunostimulants.

CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double stranded or single stranded. Bibliographic references 103, 104 and 105 describe possible analog substitutions, for example, the substitution of guanine for 2'-deoxy-7-desazaguanosine. The adjuvant effect of CpG oligonucleotides is further analyzed in bibliographic references 106-111.

The CpG sequence can be directed to TLR9, such as the GTCGTT or TTCGTT motif [112]. The CpG sequence may be specific to induce a Th1 immune response, such as CpG-A ODN or it may be more specific to induce a C lymphocyte response, such as CpG-B ODN. CpG-A and CpG-B ODN are analyzed in bibliographic references 113-115. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed such that the 5 'end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can at its 3' ends to form "immunomers." See, for example, bibliographic references 112 and 116-118.

ADP bacterial ribosilant toxins and their detoxified derivatives can be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (thermolabile enterotoxin "TL" from E. coli), cholera ("CT") or whooping cough ("PT"). The use of detoxified ribosilant ADP toxins as mucosal adjuvants is described in bibliographic reference 119 and as parenteral adjuvants in bibliographic reference 120. The toxin or anatoxin is preferably in the form of a holotoxin comprising both subunits A and B. Preferably, the subunit A contains a detoxifying mutation, preferably subunit B is not mutated. Preferably, the adjuvant is a detoxified TL mutant such as LT-K63, LT-R72 and LT-G192. The use of ribosilating ADP toxins and their detoxifying derivatives, particularly LT-K63 and LT-R72, as adjuvants can be found in references 121-128. The numerical reference for amino acid substitutions is preferably based on the alignments of subunits A and B of the ribosilating ADP toxins set forth in bibliographic reference 129.

F. Human immunomodulators

Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleukins (for example IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [130 ], etc.) [131], interferons (for example interferon-γ), macrophage colony stimulating factor and tumor necrosis factor.

G. Bioadhesives and mucoadhesives

In the invention, bioadhesive and mucoadhesive adjuvants can also be used. Suitable bioadhesives include esterified hyaluronic acid microspheres [132] or mucoadhesives such as crosslinked derivatives of poly (acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethyl cellulose. Chitosan and its derivatives can also be used as adjuvants in the invention [133].

H Microparticles

In the invention microparticles can also be used as adjuvants. Microparticles (i.e. a particle with a diameter of ∼100 nm to ∼150 µm, more preferably ∼200 nm to ∼30 µm and more preferably with a diameter of ∼500 nm to ∼10 µm) formed with materials that are biodegradable are preferred and non-toxic (for example a poly (α-hydroxy acid), a poly (hydroxybutyric acid), a polyortoester, a polyanhydride, a polycaprolactone, etc.) with poly (lactide-co-glycolic acid), optionally treated to have a charged surface negatively (for example with SDS) or a positively charged surface (for example with a cationic detergent such as CTAB).

I. Liposomes (Chapters 13 and 14 of bibliographic reference 81)

Examples of suitable formulations with liposomes for use as adjuvants are described in bibliographic references 134-136.

J. Formulations of polyoxyethylene ether and polyoxyethylene ester

Suitable adjuvants for use in the invention include polyoxyethylene ether and polyoxyethylene ester [137]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol

[138]

as well as polyoxyethylene alkyl ether or ester surfactants in combination with at least one other nonionic surfactant such as octoxynol [139]. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether-23-lauryl ether ether.

L.

Muramyl Peptides

Examples of suitable muramyl peptides for use as adjuvants in the invention include Nacethylmuramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normulamil-L-alanyl-D-isoglutamine (nor-MDP) and Nacetilmuramil-L-alanyl-D-isoglutamyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) ethylamine MTP-PE).

K Polyphosphazene (PCPP)

PCPP formulations are described, for example, in bibliographic references 140 and 141

M. Imidazoquinolone compounds.

Examples of imidazoquinolone compounds suitable for use as adjuvants in the invention include Imiquamod and its counterparts (eg "Resiquimod 3M"), and are further described in bibliographic references 142 and 143.

N. Thiosemicarbazone Compounds

Examples of thiosemicarbazone compounds are described in literature reference 144, as well as methods for formulating, preparing and selecting compounds suitable for use as adjuvants in the invention. Thiosemicarbazones are particularly effective in stimulating human peripheral blood mononuclear cells for the production of cytokines such as TNF-α.

O. Tryptantrine compounds.

Examples of tryptantrin compounds as well as methods for preparing, formulating and selecting compounds suitable for use as adjuvants in the invention are described in literature reference 145. Triptrantrin compounds are particularly effective in stimulating human peripheral blood mononuclear cells for the production of cytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or more of the adjuvants identified above. For example, the following adjuvant compositions can be used in the invention: (1) a saponin and an oil-in-water emulsion [146]; (2) a saponin (for example QS21) + a non-toxic LPS derivative (for example 3dMPL) [147]; a saponin (for example QS21) + a non-toxic LPS derivative (for example 3dMPL) + a cholesterol; (4) a saponin (for example QS21) + 3dMPL + IL-12 (optionally + a sterol) [148],

(5) combinations of 3dMPL, for example, with QS21 and / or oil-in-water emulsions [149]; (6) SAF, which contains 10% squalene, 0.4% Tween 80 ™, 5% L121 pluronic block polymer and thr-MDP, microfluidified in a submicron emulsion or subjected to vortexing to generate an emulsion of greater particle size (7) the Ribi ™ adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80 and one or more components of the bacterial cell wall of the group consisting of monophosphoryl lipid A (MPL ), trehalose dimicolate (TDM) and cell wall skeleton (EPC), preferably MPL + EPC (Detox ™); (8) one or more mineral salts (such as an aluminum salt) + a non-toxic LPS derivative (such as 3dMPL); and (9) one or more mineral salts (such as an aluminum salt) + an immunostimulatory oligonucleotide (such as a nucleotide sequence that includes a CpG motif).

Chapter 7 of bibliographic reference 81 describes other substances that act as immunostimulatory agents.

The use of an aluminum hydroxide or aluminum phosphate adjuvant is particularly preferred and the conjugates are generally adsorbed to these salts [eg, examples 7 and 8 and bibliographic reference 9; Example J of bibliographic reference 10]. It is also possible to mix with aluminum salts without adsorption [27, 76]. Another preferred adjuvant is calcium phosphate. The conjugates can be mixed with the adjuvants (and optionally adsorbed thereto) separately and then the conjugates can be mixed together or the conjugates can be mixed together and then mixed with the adjuvant.

The pH of the compositions used according to the invention is preferably between 6 and 8, preferably about 7. Using a buffer the pH can be kept stable. When a composition comprises an aluminum hydroxide salt, it is preferred to use a histidine buffer [150]. The composition may be sterile and / or pyrogen free. The compositions may be isotonic with respect to humans.

The compositions may include a preservative (eg, thiomersal, 2-phenoxyethanol) or may not include preservatives. Preferred compositions of the invention do not include any mercuric material, for example they do not have thiomersal.

To avoid interference between antigens, particularly conjugated antigens, it is possible to include a polyanionic polymer, such as poly (L-glutamic acid) [151].

The compositions may be presented in vials or they may be presented in syringes already filled. Syringes can be supplied with or without needles. A syringe will include a single dose of the composition, while a vial may include a single dose or multiple doses. Injectable compositions will normally be liquid solutions or suspensions. Alternatively, they can be presented in solid form (for example lyophilized) for solution or suspension in liquid vehicles before injection.

The compositions can be packaged in single-dose form or in multi-dose form. For multi-dose forms, the vials are preferred over the already filled syringes. Effective dosage volumes can be routinely established, but a typical dose of the composition for humans for injection has a volume of 0.5 ml.

When a composition is to be prepared improvisedly before use (for example when a component is presented in lyophilized form) and is present as a kit, the kit may comprise two vials or may comprise an already filled syringe and a vial using the contents of the syringe to reactivate the contents of the vials before injection. For compositions that include a capsular saccharide of serogroup A the saccharide of serogroup A may be lyophilized, while saccharide (or saccharides) of another serogroup (or serogroups) may be present in liquid form.

The compositions will comprise an immunologically effective amount of the meningococcal conjugates, as well as any other component, as necessary. An "immunologically effective amount" means that the administration of that amount to an individual, either in a single dose or as part of a series, causes a protective anti-meningococcal immune response in patients. This amount varies depending on the health and physical condition of the individual to be treated, the age, the taxonomic group of the individual to be treated (for example, non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the evaluation of the doctor treating the medical situation and other relevant factors. It is expected that the amount will be in a relatively wide range that can be determined by routine tests and a typical amount of each meningococcal antigen per dose is between 1 µg and 20 µg per serogroup (measured in saccharide terms) for example between 2 and 10 µg by serogroup. A dose of approximately 4 µg per serogroup is preferred (ie a total of 16 µg in a tetravalent mixture).

Preferably the total amount of transport protein in a composition is not more than 100 µg / dose for example is <90 µg / dose, <80 µg / dose, <70 µg / dose, <60 µg / dose, ≤50 µg / dose, etc. The total amount of transport protein in a composition will generally be at least 10 µg / dose.

Generalities

The term "comprises" includes "including" as well as "consisting of", for example a composition that "comprises" X may consist exclusively of X or may include something additional for example X + Y.

The term "approximately" with respect to a numerical value X means, for example, x ± 10%.

The word "substantially" does not exclude "completely", for example, a composition that "substantially lacks" Y may completely lack Y. When necessary, the word "substantially" may be omitted from the definition of the invention.

MODES FOR CARRYING OUT THE INVENTION

Absence of suppression by vehicle using tetravalent conjugate mixture A / C / W135 / Y

Mixtures of meningococcal conjugates for serogroups A + C, C + W + Y or A + C + W + Y can be prepared as described in references 9 and 10. If desired, these can be mixed with hydroxide adjuvants of aluminum or aluminum phosphate, as also described in bibliographic references 9 and 10. These vaccines have CRM197 or diphtheria anatoxin (Da) as transport protein, covalently bound to saccharides.

Patients who received pediatric DTT vaccination (DT-Pa or DT-Pw) were selected including those who received vaccines containing DTT and other antigens (for example tetravalent DTT-Hib, tetravalent DTT-HBsAg, pentavalent DTT-Hib-HBsAg, hexavalent DTT-Hib-HBsAg-IPV, etc.) to receive the conjugate mixture that a Da vehicle had. This tetravalent mixture of conjugates is immunogenic in humans [9, 11, 13].

Patients who received pediatric vaccination against Hib were selected (either as monovalent Hib, with or without DT-Pa or DT-Pw or as part of a combination vaccine such as tetravalent DTT-Hib, pentavalent DT-THib-HBsAg, hexavalent DTT- Hib-HBsAg-IPV) to receive the conjugate mixture that a CRM197 vehicle had.

A group of control patients was selected to receive one of the two conjugate mixtures. Control patients had not previously received either diphtheria anatoxin or CRM197, either as individual antigens or as transport proteins in conjugates.

The ability of tetravalent conjugates to elicit an immune response in patients was evaluated. Suppression by vehicle is indicated if the test groups show significantly lower anti-meningococcal immune responses than those of control patients and in particular if the conjugates do not cause a useful ABS response in patients.

In the V52P2 clinical trial, conducted in Finland and Germany with 620 subjects aged 12-16 months, five formulations were tested. Vaccines used CRM197 vehicle and an aluminum phosphate adjuvant [10]. The doses of each saccharide serogroup, expressed as µg saccharide mass per 0.5 ml dose, were as follows:

Group

Ore MenC MenW135 Meny

one

10 10 10 10

2

0 10 10 10

3

10 5 5 5

4

5 5 5 5

5

2.5 2.5 2.5 2.5

5

10

fifteen

twenty

25

30

35

The subjects received an injection at time zero, and then 25% of the subjects received a second dose of the vaccine 4 weeks later.

Patient sera were collected 1 month after the administration of the vaccine and tested in an ABS test against N. meningitidis for each serogroup using human complement. The increase in the titration of ABS with respect to serum in zero time was evaluated, being the criteria ≥1: 4 and ≥1: 8, the anti-capsule titres (MGT) were also measured for each serogroup. The following Table 1 shows the results.

Therefore trivalent and tetravalent vaccines were immunogenic in young children. Conjugates are immunogenic at doses of saccharides as low as 2.5 µg per conjugate. The immune response is reinforcible, with large increases in ABS titration after the second dose. In this trial no evidence of suppression by vehicle was observed.

Lack of suppression of anti-Da responses

A study conducted in Belgium in 1999 [26] showed in newborns modified immunity against tetanus, which persisted in early childhood, after receiving a primary treatment of Hib vaccine conjugated with tetanus anatoxin. Therefore, the conjugate transport protein had a negative immune impact. On the other hand, the opposite effect was observed in a study of pneumococcal conjugate vaccines [152]. The possibility that a meningococcal conjugate could interfere with immunity against diphtheria [153] was studied using a serogroup C meningococcal saccharide conjugate with a CRM197 vehicle (Menjugate ™).

The children were divided into five groups, which were immunized during the first year of life as follows: (1) four doses of Menjugate ™; (2) three doses of Menjugate ™ plus one dose of a bivalent unconjugated A / C mixture; (3) three doses of HBsAg vaccine after one dose of Menjugate ™; (4) three doses of HBsAg vaccine then one dose of a bivalent unconjugated A / C mixture; (5) controls that did not receive meningococcal vaccines. All children also received three doses of diphtheria vaccine during the first four months of life, but did not receive any preschool diphtheria booster.

Patients received a single dose of Mejugate ™ at 4 years of age and a blood sample was taken before and after (∼30 days) to this Mejugate ™ injection. The diphtheria antibodies were measured by ELISA and the geometric mean concentrations were evaluated. The percentage of subjects with an antibody level ≥ 0.1 IU / ml was also evaluated. The results were the following:

Group

Prevaccination Post vaccination

GMC

% ≥0.1  GMC % ≥0.1

one

0.35 94% 9.00 100%

2

0.18 87% 8.55 100%

3

0.20 81% 2.82 100%

4

0.10 51% 4.55 99%

Baseline anti-Da degrees were higher in patients who had previously received Menjugate ™ compared to those who had not received it (compare, for example, groups 1 and 4). A simple regression analysis revealed significant linear relationships between the number of previous Menjugate ™ doses (4, 3, 1 and 0 for groups 1, 2, 3 and 4, respectively) and (a) anti-Da titers before vaccination and (b) anti-Da degrees after vaccination.

5

10

fifteen

twenty

25

30

35

Therefore, there is persistence of enhanced immunity against diphtheria at 4 years of age in children who received four doses of Mejugate ™ in childhood. In addition, there is a trend towards increased anti-Da antibody responses after a booster dose of Menjugate ™ in patients who had received at least three doses of Menjugate ™ as children. No evidence of immunological interference was found between meningococcal and diphtheria conjugates.

The bivalent A / C conjugate mixture shows no interference with DTT

In Nigeria [154] a mixture of capsular saccharides from serogroups A and C [8] was administered to newborns (5 to 11 weeks of age) who had not previously received the DTT vaccine. The children received unconjugated saccharides (50 µg of each serogroup) or saccharides conjugated with Da-no adjuvant (4 µg of each), with six different programs:

(one)

Four conjugate doses: 6 weeks, 10 weeks, 14 weeks, 9 months.

(2)

Three conjugate doses: 6 weeks, 10 weeks, 14 weeks.

(3)

Two conjugate doses: 14 weeks, 9 months.

(4)

One conjugate dose: 14 weeks.

(5)

One dose of conjugate: 9 months.

(6)

One unconjugated dose: 9 months.

The children received oral vaccines against DTT and against polio at 6, 10 and 14 weeks, with a booster at 9 months and meningococcal vaccines were administered at the same time as these existing vaccines. To assess the anamnestic responses, children were also given an unconjugated vaccine at 24 months.

Serum bactericidal antibody responses were measured at 18 weeks (ie 4 weeks after the third vaccination against DTT / polio) at 10 months (ie 1 month after booster against DTT / polio at 9 months) and 1 week after administration of unconjugated material at 24 months. The percentages of patients who showed an ≥ 128 fold increase in ABS titers were as follows:

%

18 weeks 10 months 24¼

Program

Ore MenC Ore MenC Ore MenC

(one)

56 84 89 73 100 95

(2)

56 86 6 9 96 82

(3)

68 64 85 85 100 95

(4)

61 57 4 8 100 93

(5)

3 7 62 26 100 99

(6)

2 2 5 eleven 97 52

There was no difference between the six groups in terms of antibody responses against diphtheria anatoxin [8].

Therefore there is no evidence of interference resulting from the use of diphtheria anatoxin as both a protective antigen and a carrier for the conjugates. For example, in group 5 patients had received diphtheria anatoxin in DTT vaccines at 6, 10 and 14 weeks before receiving the first dose of meningococcal conjugate, but showed an anti-meningococcal ABS response of> 99% at 24 months

Non-negative impact on diphtheria responses

As mentioned above, patients receiving meningococcal conjugate vaccines at the same time as DTT vaccines showed no reduction in immune responses against diphtheria anatoxin. In another study, meningococcal and DTT conjugates had been administered at different times. A 3-dose DTT program had been followed at 2, 3, and 4 months of age with a single dose of a bivalent A / C vaccine with Da conjugated saccharides or unconjugated saccharides. The immune responses against diphtheria anatoxin were measured by ELISA and the MG (geometric means) of the titers were as follows:

Antigens

Pre-immunization Post-immunization

Conjugates

0.05 21.2

Unconjugated

0.06 0.06

Unconjugated saccharides produced no anti-Da response (as might be expected), however conjugated saccharides resulted in a strong anti-Da response. The administration of these conjugates may therefore provide immunity against diphtheria in untreated patients or may take the place of a booster against Da.

It will be understood that the above invention is described by way of example only and that modifications can be made while remaining within the scope of the invention defined in the appended claims.

TABLE 1 - Results of the V59P2 test

Group

TO C W135 Y

MGT (1 month after 1 dose)

one

3.9 6.4 7.1 8.9

2

2

6.1 8.3 8.5

3

5.7 5.2 6.9 12

4

3.8 4,5 7.0 9.6

5

3.9 5.3 7.0 12

MGT (1 month after 2 doses)

Group

TO C W135 Y

one

27 89 22 37

2

2

80 twenty 57

3

29 76 28 58

4

14 47 twenty 35

5

17 71 2. 3 52

% of patients with ABS image1 1: 4 (1 month after 1 dose)

one

33 56 57 58

2

0 57 60 61

3

55 49 53 70

4

37 42 54 64

5

40 51 57 67

% of patients with ABS image2 1: 4 (1 month after 2 doses)

one

100 100 96 96

2

0 100 73 92

3

91 96 95 95

4

84 96 88 96

5

80 100 80 92

% of patients with ABS image2 1: 8 (1 month after 1 dose)

one

25 44 46 48

2

0 40 fifty 49

3

39 3. 4 Four. Five 64

4

2. 3 30 44 51

5

26 35 40 60

% of patients with ABS image2 1: 8 (1 month after 2 doses)

one

92 100 85 93

2

0 100 64 92

3

87 96 95 82

4

60 92 77 92

5

72 92 72 88

5

10

fifteen

twenty

25

30

35

40

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Claims (23)

1. A composition comprising: (a) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup A and (ii) a diphtheria or derivative anatoxin thereof, which remains immunologically cross-reactive with the diphtheria anatoxin; (b) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup C and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin; (c) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria or derivative anatoxin which remains immunologically cross-reactive with the diphtheria anatoxin; and (d) a conjugate (i) of the N Meningitidis capsular saccharide of serogroup Y and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin, for use in a procedure for immunize a human patient against a disease caused by Neisseria meningitidis, which comprises the step of administering the composition to the human patient, in which the patient has been preimmunized with (a) a diphtheria or derivative anatoxin, which remains immunologically reactive crossed with diphtheria anatoxin and / or (b) a conjugate of (i) a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria or derivative anatoxin thereof, which remains immunologically cross-reactive with diphtheria anatoxin. 2. A composition according to claim 1 for the use of claim 1, wherein the composition comprises: (a) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup A and (ii) a diphtheria anatoxin ; (b) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup C and (ii) a diphtheria anatoxin; (c) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria anatoxin; and (d) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup Y and (ii) a diphtheria anatoxin, and in which the patient has been preimmunized with (a) a diphtheria anatoxin and / or (b) a conjugate of (i) a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria anatoxin or CRM197.

3.

A composition according to claim 1 for the use of claim 1, wherein the composition comprises: (a) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup A and (ii) CRM197; (b) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup C and (ii) CRM197; (c) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup W135 and (ii) CRM197; and (d) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup Y and (ii) of CRM197, and in which the patient has been preimmunized with a conjugate of (i) a capsular saccharide of an organism other than N. meningitidis and (ii) CRM197 or a diphtheria anatoxin.

Four.

The use of (a) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup A and (ii) of a diphtheria anatoxin or a derivative thereof, which remains immunologically cross-reactive with the diphtheria anatoxin; (b) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup C and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin; (c) of a conjugate (i) of the N. meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria or derivative anatoxin that remains immunologically cross reactive with the diphtheria anatoxin and (d) a conjugate (i ) of the capsular saccharide of N. meningitidis of serogroup Y and (ii) a diphtheria anatoxin or a derivative thereof that remains immunologically cross reactive with diphtheria anatoxin, in the manufacture of a medicament for immunizing a human patient against a disease caused by

N. meningitidis, in which the patient has been preimmunized with (a) a diphtheria or derivative anatoxin that remains immunologically cross reactive with diphtheria anatoxin and / or (b) a conjugate (i) of a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria or derivative anatoxin, which remains immunologically cross-reactive with the diphtheria anatoxin.

5.

The use according to claim 4, wherein the use is: (a) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup A and (ii) a diphtheria anatoxin; (b) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup C and (ii) a diphtheria anatoxin; (c) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup W135 and (ii) a diphtheria anatoxin; and (d) a conjugate (i) of the N. meningitidis capsular saccharide of serogroup Y and (ii) a diphtheria anatoxin, and in which the patient has been preimmunized with (a) a diphtheria anatoxin and / or (b) a conjugate (i) of a capsular saccharide from an organism other than N. meningitidis and (ii) a diphtheria anatoxin or CRM197.

6.

The use according to claim 4, wherein the use is: (a) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup A and (ii) CRM197; (b) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup C and (ii) CRM197; (c) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup W135 and (ii) CRM197; and (d) a conjugate (i) of the capsular saccharide of N. meningitidis of serogroup Y and (ii) CRM197, and in which the patient has been preimmunized with a conjugate (i) of a capsular saccharide of an organism other than N Meningitidis and (ii) CRM197 or a diphtheria anatoxin.

7.

The composition of any one of claims 1 to 3 for the use of this claim, or the use of any one of claims 4 to 6, wherein the conjugates are mixed to give a ratio of 1: 1: 1: 1 or

2: 1: 1: 1 (measured as saccharide mass).

8.

The composition of any one of claims 1 to 3 and 7 for the use of this claim, or the use of any one of claims 4 to 7, wherein each meningococcal antigen per dose is comprised between 2 and 10 µg per serogroup ( measured in terms of saccharide).

9.

The composition of any one of claims 1 to 3 and 7 to 8, for the use of this claim or the use of any one of claims 4 to 8, wherein the patient has been preimmunized with a vaccine comprising an anatoxin diphtheria

10.

The composition of any one of claims 1 to 3 and 7 to 9 for the use of this claim, or the use of any one of claims 4 to 9, wherein the patient has been preimmunized with a vaccine comprising a conjugate of Hib.

eleven.

The composition of any one of claims 1 to 3 and 7 to 10 for the use of this claim or the use of any one of claims 4 to 10, wherein the patient has been preimmunized with a vaccine comprising at least one pneumococcal conjugate.

12.

The composition of any one of claims 1 to 3 and 7 to 11 for the use of this claim, or the use of any one of claims 4 to 11, wherein the patient has been preimmunized at least six months before the procedure. or use.

13.

The composition of claim 12 for the use of claim 12, or the use of claim 12, wherein the patient has been preimmunized at least 8 years before the procedure or use.

14.

The composition of any one of claims 1 to 3 and 7 to 13 for the use of this claim or the use of any one of claims 4 to 13, wherein preimmunization occurs during the year following the birth of the patient.

fifteen.

The composition of any one of claims 1 to 3 and 7 to 14 for the use of this claim, or the use of any one of claims 4 to 14, wherein the saccharides in the meningococcal conjugates (a) to

(d) they have been depolymerized so that they are shorter than the natural capsular saccharides observed in a meningococcus.

16.

The composition of any one of claims 1 to 3 and 7 to 15 for the use of this claim, or the use of any one of claims 4 to 15, wherein the meningococcal conjugates comprise a diphtheria anatoxin carrier and a linker. of adipic acid.

17.

The composition of claim 16 for the use of claim 16, or the use of claim 16, comprising no more than 60 µg of diphtheria anatoxin carrier.

18.

The composition of any one of claims 1 to 3 and 7 to 17 for the use of this claim, or the use of any one of claims 4 to 17, wherein the composition or medicament further comprises a conjugated capsular saccharide of Streptococcus pneumoniae.

19.

Composition of any one of claims 1 to 3 and 7 to 18 for the use of this claim, or the use of any one of claims 4 to 18, wherein the composition or medicament further comprises a conjugated capsular saccharide of Haemophilus influenzae type B.

twenty.

The composition of any one of claims 1 to 3 and 7 to 19 for the use of this claim, or the use of any one of claims 4 to 19, wherein the composition or medicament further comprises a serogroup protein antigen B of Neisseria meningitidis.

twenty-one.

The composition of any one of claims 1 to 3 and 7 to 20 for the use of this claim, or the use of any one of claims 4 to 20, wherein the composition or medicament comprises an aluminum hydroxide adjuvant and / or an aluminum phosphate adjuvant.

22

The composition of any one of claims 1 to 3 and 7 to 21 for the use of this claim, or the use of any one of claims 4 to 21, wherein the disease caused by Neisseria meningitidis meningococcal meningitis.

2. 3.

The composition of any one of claims 1 to 3 for the use of this claim, or the use of any one of claims 4 to 6, wherein:

-the patient has been preimmunized, at least five years before the procedure or use, with a vaccine that comprises a diphtheria anatoxin; - Meningococcal conjugates comprise a diphtheria anatoxin vehicle and optionally, a adipic acid crimper; -Meningococcal conjugates are present at a rate of 8 µg / ml (measured as meningococcal saccharide) by serogroup; -The saccharide weight ratio: vehicle for each conjugate is 1: 3; -the composition or medicament includes 96 µg / ml of diphtheria anatoxin; -the composition or medicament includes 1.2 mg / ml sodium phosphate; and -the composition or medicament includes 8.8 mg / ml of sodium chloride.