MXPA00011664A - Combination meningitidis b/c vaccines - Google Patents

Abstract

The present invention is directed to a combination vaccine for Neisseria meningitidis comprising outer membrane proteins from serogroup B and oligosaccharides from serogroup C, and its use for the prevention or treatment of disease.

Description

COMBINED VACCINES AGAINST MENINGITIDIS B / C FIELD OF THE INVENTION The present invention relates to the combined immunogenic compositions and vaccines against Nei sseri a meningi ti di s B and C and to methods for inducing an immune response when administering them.

BACKGROUND OF THE INVENTION The strains of serogroup B and C of Nei sseri a meni ngi ti di s (Nm) together account for most of the invasive diseases in Europe and the United States. Vaccines against individual serogroups of Nm are currently available. The NmB vaccine from the NIPH (National Institute of Public Health of Norway) is safe, promotes specific immunity in children and adults, and is effective in preventing NmB disease in adolescents. This vaccine has been typically combined with the meningococcal C polysaccharide vaccine and is given with alum. The simple component of the polysaccharide vaccine, however, is not Ref: 125091 _ ^^ g £ effective in infants and young children. The Chiron conjugate NC vaccine (conj.) Is also safe, promotes high serum bactericidal antibody titers in vaccinated infants as young as two and three months of age, and induces immune B cell memory for the unconjugated polysaccharide NmC . Since both serogroups cause disease, a combination vaccine that induces an immune response for both serogroups could be highly advantageous.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention relates to an immunogenic composition or to a vaccine comprising oligosaccharides NmC conjugated to a carrier protein, the outer membrane proteins of NmB, and a carrier. In a preferred embodiment, the carrier protein is CRM? 97, a non-toxic diphtheria toxin, the outer membrane proteins of NmB are presented as proteoliposomal vesicles, and the carrier is aluminum hydroxide or MF59. In still another aspect, the present invention relates to a method for inducing an immune response to NmB and NmC, or vaccination, comprising the administration of an immunologically effective amount of an immunogenic composition comprising NmC oligosaccharides conjugated to a carrier protein, the outer membrane proteins of NmB, and a carrier.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB summarize the IgG antibody titers against NmB and IgG against NmC, respectively, as determined by ELISA. Figures 2A and 2B summarize the titers of the bactericidal antibody in serum for NmB and NmC, respectively. Figure 3 summarizes the comparison of the proportions of antibody to NmB and NmC induced by the vaccine in combination in adjuvant MF59 vs. alum. Figure 4 summarizes the comparison of the proportions of antibody to NmB and NmC induced by the vaccine in combination vs. the respective monovalent vaccine.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES We describe a combination vaccine for NmB and NmC that induces an immune response for both serogroups, which is not significantly different from the immune response induced by each serogroup alone. The immunogenicity of the NmB NIPH vaccine (hereinafter referred to as "NmB" or "MenB" vaccines) and the Chiron NmC conjugate vaccine (hereinafter referred to as "NmC con." Or "MenC conj."), alone, in combination, and in combination with adjuvant MF59 as described herein. The practice of the present invention will employ, unless otherwise indicated, conventional methods of immunology and microbiology. Such techniques are fully explained in the literature. See, for example, Methods In Enzymolgy (S. Colowick and N. Kaplan eds., Academic Press, Inc.) and Handbook of Experimen tal Immunolgy, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications). As used herein, the term "uninogenic in" refers to the material that induces the production of antibodies from administration to a vertebrate, including humans. As used herein, the term "carrier" refers to an acceptable pharmaceutical component different from the immunogenic component of NmB or NmC. The carrier can be organic, inorganic, or both. Suitable carriers well known to those skilled in the art include, without limitation, large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes). ) and inactive viral particles. The carrier can also function as an immunostimulatory agent, for example, adjuvant. Suitable adjuvants are well known to those skilled in the art. As used herein, the term "immunologically effective amount" means the administration of that amount, either in a single dose or as part of a series, which is effective to induce the production of antibody for the treatment or prevention of disease. This amount will vary depending on a variety of factors, including the physical condition of the subject, and can be easily determined by someone skilled in the art. As used herein, the term "vaccine" means an immunogenic composition that is capable of inducing a microbicidal immune response. Preferably, the vaccines of the present invention elicit a bactericidal antibody response. The present invention is directed, in part, to immunogenic compositions that induce an immune response for Meningitidis B and C. In preferred embodiments of the invention, the immunogenic composition comprises the outer membrane protein NmB, and the conjugated NmC oligosaccharide. to a first carrier. The NmB protein preferably comprises partially purified outer membrane proteins from strain 44/76 (B15.P1.7, 16: L3,7,9). The partially purified outer membrane proteins are preferably present as proteoliposomal vesicles as a result of the extraction process using deoxycholate. The dose of NmB is expressed in μg. Preferably, the immune composition of NmB / the components of the vaccine can be obtained from the Norwegian National Health Institute (NIPH). The NmB / alum vaccine comprises 0.05 mg / ml of NmB protein, 3.33 mg / ml of Al (OH) 3 (alum) and 0.10 mg / ml of thiomersalcohol. The Chiron oligosaccharide represents the polysaccharide fragments of NmC preferably from about 12 to about 22 repeat units. Preferably, the NmC oligosaccharide is conjugated to a first carrier. The dose of the NmC conjugate or the polysaccharide thereof is expressed in μg of sialic acid. A NmC vaccine containing the unconjugated polysaccharide (hereinafter referred to as "NmC polysaccharide" or "MenC Ps") may also be used. MenC Ps is a crude isolate comprising polysaccharides preferably from about 60 to about 80 repeating units. In preferred embodiments of the invention, the first carrier is a protein, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymer, lipid aggregate, or inactive viral particle. More preferably, the first carrier is a protein. More preferably, the first carrier is CRM197.

Preferably, 10 μg of oligosaccharide is used per dose at 12.5-33 μg of CRM197 (for example, to maintain an oligo / protein ratio of from about 0.3 to about 0.8). More preferably, it can be used in the preferred embodiments of the invention, the immunogenic composition comprises a second carrier, preferably aluminum hydroxide (alum) or MF59. Alum can be obtained from Superfos, Bedbaek, Denmark, and is a 3% solution. When present, about 1 mg to about 1.67 mg of alum is used per dose. MF59 is a microfluidized emulsion of squalene in water that has been shown to be safe and that increases antibody responses in serum to a variety of investigational vaccines. MF59 comprises about 5% squalene, 0.5% Tween 80 and about 0.5% Span 85. Adjuvant MF59 is described in PCT publication No. WO 90/14837, incorporated by reference herein in its entirety. MF59 can be prepared according to the procedures described for example in Ott et al., Vaccine desi gn: The Subunit and Adj uvant Approach, 1995, M.F. Powell and M.J. Newman, Eds., Plemam Press, New York, p. 277-296; Singh et al., Vacine, 1998, 16, 1822-1827; Ott et al., Vacci ne 1995, 13, 1557-1562; and Valensi et al., J. Immunol, 1994, 153, 4029-39, the descriptions of which are incorporated by reference herein in their entirety. The immunogenic composition of the invention will employ an immunologically effective amount of the antigens. That is, an amount of antigen will be included, which in combination with the adjuvant, will cause the subject to produce a specific and sufficient immune response, preferably a T or B lymphocyte response, to impart protection to the subject from the subsequent exposure to Nei sseri a. A simple dose designation providing a specific guidance for each and every antigen that can be employed in this invention can not be assigned. The effective amount of the antigen will be a function of its inherent activity and purity, and is empirically determined by those of ordinary skill in the art by means of routine experimentation.

The immunogenic compositions according to the present invention comprise an immunostimulatory amount of Nei sseri a antigen. An immunostimulatory amount is that amount which is sufficient to induce a measurable humoral or cellular immune response. For example, the immunogenic compositions of the present invention comprise about 1 nanogram to about 1000 micrograms of antigen or about 10 nanograms to about 800 micrograms of antigen. In some preferred embodiments, the immunogenic compositions contain about 0.1 to about 500 micrograms of antigen. In some preferred embodiments, the immunogenic compositions contain about 1 to about 350 micrograms of antigen. In some preferred embodiments, the immunogenic compositions contain from about 25 to about 250 micrograms of antigen. In some preferred embodiments, the immunogenic compositions contain about 100 mocrograms of antigen. One skilled in the art can easily formulate an immunogenic composition comprising any desired amount of antigen, which can be empirically determined by those of ordinary skill in the art by means of routine experimentation. Immunogenic compositions can be conveniently administered in unit dose form and can be prepared by any of the methods well known in the pharmaceutical art, for example, in Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980), description of which is incorporated by reference herein in its entirety. The present invention is also directed to vaccines comprising any of the immunogenic compositions described above. The present invention is also directed to methods for inducing an immunological response to NmB and NmC comprising administering an immunologically effective amount of an immunogenic composition described above., a human. The administration can be by any mode known to those skilled in the art, including by the oral, parenteral, pulmonary, transdermal, rectal, intraperitoneal, intramuscular, or subcutaneous routes.

The invention is further illustrated by way of the following examples, which are intended to elucidate the invention. It is understood that the following examples illustrate the invention and are not to be considered as limiting the invention in any way. Those skilled in the art will recognize the modifications that are within the spirit and scope of the invention. All references cited herein are incorporated herein by reference, in their entirety.

EXAMPLES Example 1: ELISA results Groups of guinea pigs (n = 15 animals) were assigned to receive one of the following vaccines described in Table 1: Table 1 Group Component e s Q u sity s Group 1 NmC conj. / alum 10 μg / 1 mg Group 2 NmB / alum 25 μg / 1 mg Group 3 Polysaccharide 10 μg / 25 μg / 1 mg NmC / NmB / alum Group 4 NmC conj. / NmB / alum 10 μg / 25 μg / 1 mg Group 5 NmC conj./NmB/MF59 10 μg / 25 μg / 0.5 ml. Group 6 (n = 5) comprised control animals that received alum alone. Eighty guinea pigs were randomized into the groups described above and received one of six vaccine combinations. For the data presented in Table 2, each animal received two injections, IM, separated by 28 days. The serum samples were obtained before each injection, and 18 days after the second injection. For the data presented in Figures 1A and IB, each animal received two immunizations separated by six weeks. Each dose consisted of two intramuscular injections of 0.25 ml. Serum samples were obtained immediately before each injection, and 14 or 18 days after the second injection. Serum samples were evaluated for the IgG anticapsular antibody concentrations for NmC (Table 2 and Figure 1A) and for the concentrations of Igm anti-outer membrane vesicle antibody for NmB, by ELISA (Figure IB). The ELISA data were generated in a representative assay of sera from individual animals (Table 2) and also expressed as averages from a plurality of assays (Figures 1A and IB). The summarized ELISA data described in Table 2 are, therefore, expressed as geometric means. For ELISA, the MCPS-ADH conjugate (NmC polysaccharide-adipic acid dihydrazide) 0 the components of the outer membrane vesicle (OMV) were coated on polystyrene microtiter plates overnight at 4 ° C, 1 μg / ml, 100 μl / well. On each coated plate, 100 μg / well of each reference standard (for example, the combined serum of the guinea pigs), a positive control, a negative control, and serum samples diluted serially in half in a buffer containing 75 μM ammonium thiocyanate, and incubated for two hours at room temperature. Anti-guinea pig IgG antibody conjugated to peroxidase was added to the wells (100 μl / well). After 2 hours, the 3, 3 ', 5, 5' -tetramethylbenzidine (TMB) colorimetric substrate (100 μl / well) was added, and the color was developed for 15 minutes. Antibody levels for MCPS and for OMV present in controls and samples were obtained from a standard form using the reference standard having an assigned value of 100 ELISA units / ml. The results are shown in Table 2 and Figures 1A and IB. The results are summarized in Table 2 and Figures 1A and IB and reveal that the vaccine in combination was immunogenic, as measured by the IgG antibody titers against NmC and NmC, respectively. Figure 1A shows that the specific response of anti-eningococcus B antibody was induced by vaccine combinations comprising NmB. Figure IB shows that a specific response of anti-meningococcus C antibody was induced by vaccine combinations comprising NmC. In particular, the antibody response induced by the combination of NmC conjugate and NmB in the presence of adjuvant MF59 (Group 5) was significantly greater than the antibody response induced either by the NmC conjugate alone (Group 1) or the combination of the conjugate NmC, and NmB in the presence of alum (Group 4). When adjuvant MF59 was present, the antibody titer for the vaccine in combination was increased approximately six-fold.

Table 2: IgG Antibody Responses to MenC (GMT) Example 2: Bactericidal Titers Serum samples were tested for bactericidal titers mediated by complement for MenC strain 60E and MemB strain 44/76. Bactericidal titers were evaluated on combined sera from each group. The bactericidal data were generated using the human complement. Assay components (eg, buffer, antibody, complement, and bacteria) were added to sterile, 96-well tissue culture plates with lids (Nunc # 167008). The plates were maintained at room temperature during the test. To each well, 50 μl of Gey buffer (Gibco) containing 1% BSA Grade RIA (Sigma), 25 μl of diluted test antibody, 25 μl of bacteria diluted to 1: 8000 in Gey buffer / 1 were added sequentially. % of BSA. Control wells include 1) Gey buffer / 1% BSA and bacteria alone (to determine if the organisms are viable in the diluent alone); 2) a time control 0 containing 75 μg of buffer, 25 μl of human complement inactivated by heat (56 ° C, 30 minutes), and 25 μl of bacteria; and 3) a toxicity control that tests the complement at 20% and 40% with buffer and bacteria, to verify that the complement source is non-toxic for the test strain. All antibody samples (at the highest concentration tested) were also tested with the heat-inactivated complement to show that a decrease in colony forming units (cfu) in the presence of the antibody is complement dependent. After all the reagents were added, 22 μl was taken from each control well and plated on Mueller-Hinton agar plates by allowing the sample to run from the top to the bottom of the plate to determine the cfu in the well at 0 minutes. The microtiter plates were then covered and sealed with parafilm paper, and gently rotated for 1 hour at 37 ° C in a 4% C02 incubator. The plates were then removed, and a 22 μl sample from each well was seeded on Mueller-Hinton agar. Culture plates were incubated for approximately 18 hours at 37 ° C with 4% C02. Colonies were counted, and% survival was determined for each test well:% survival = ([cfu from the sample well at 60 minutes] / [cfu in the control well with heat-inactivated complement at time 0 minutes] ) x 100. The reported bactericidal titers are those that resulted in 50% survival. The results from a simple experiment are presented in Table 3. The results are also presented in Figures 2A and 2B, with Figure 2B representing the average titres from a plurality of experiments. As revealed by the results summarized in Table 3, the vaccine in combination promoted high titers of bactericidal antibody in serum for NmB and Nmc. The bactericidal antibody titer against NmC was slightly higher for the vaccine in combination using MF59 as the carrier, but there was essentially no effect on the bactericidal titer of NmB using MF59. Interestingly, bactericidal titers were obtained two to five times higher for NmB with the vaccine in combination than with the vaccine for NmB alone. Figure 2A demonstrates that antibodies directed to meningococcus B by vaccine combinations comprising NmB were bactericidal. Figure 2B demonstrates that the antibodies directed to meningococcus C induced by the vaccine combinations comprising the NmC conjugate were also bactericidal.

Table 3 Table 3 (continued) Example 3: Comparison of alum adjuvants and MF59 The sera from the animals described above in Figures 1A and IB were compared and the antibody responses MenC and MenB generated by NmB / NmC conj. either in alum adjuvant or MF59 were detected as described above in Examples 1 and 2. The results, shown in Figure 3, demonstrate that the antibody response to meningococcus C was approximately 6 times higher than the vaccines comprising the adjuvant MF59.

Example 4: Comparison of Antibody Responses Generated by the Combination Vaccine for Monovalent Vaccines The sera from the animals described above in Figures 1A and IB were compared and the MenC and MenB antibody responses generated by NmB / NmC conj. were compared with the antibody responses generated either by the NmB vaccine alone or the NmC conj. only in alum, as described above in Examples 1 and 2. The results, shown in Figure 4, demonstrate that there was no significant difference in antibody responses to components of the Nmb / NmC conj. in comparison to the responses induced by the respective monovalent vaccines (either NmB or NmC conj.).

It is stated that in relation to this date, the best method known by the. Applicant for carrying out the said invention is the one that is clear from the present description of the invention.

Claims (16)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An immunogenic composition, characterized in that it comprises the oligosaccharide NmC conjugated to a first carrier and the outer membrane protein of NmB.

2. The immunogenic composition according to claim 1, characterized in that the first carrier is selected from the group consisting of protein, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymer, lipid aggregate, and inactivated viral particles.

3. The immunogenic composition according to claim 2, characterized in that the first carrier is a protein.

4. The immunogenic composition according to claim 3, characterized in that the first carrier is CRM197.

5. The immunogenic composition according to claim 1, characterized in that the outer membrane protein of NmB is presented as proteoliposomal vesicles.

6. The immunogenic composition according to claim 1, characterized in that the composition comprises a second carrier.

7. The immunogenic composition according to claim 6, characterized in that the second carrier is aluminum hydroxide or MF59.

8. A method for inducing an immunological response for Nmb and NmC, characterized in that the method comprises administering an immunologically effective amount of an immunogenic composition according to claim 1.

9. The method according to claim 8, characterized in that the first carrier is selected from the group consisting of protein, polyscaride, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymer, lipid aggregate, and inactivated viral particles.

10. The method according to claim 9, characterized in that the first carrier is a protein.

11. The method according to claim 10, characterized in that the first carrier is CRM197.

12. The method according to claim 8, characterized in that the outer membrane protein of NmB is presented as proteoliposomal vesicles.

13. The method according to claim 8, characterized in that the composition comprises a second carrier.

14. The method according to claim 13, characterized in that the second carrier is aluminum hydroxide or MF59.

15. A vaccine, characterized in that it comprises an immunogenic composition according to any of claims 1-7.

16. A method for vaccinating an individual, characterized in that it comprises administering to said individual an immunogenic composition according to any of claims 1-7.