JP7441301B2 - Immunogenic compositions comprising coccal polysaccharide-protein conjugates - Google Patents

Description

The present invention relates to immunogenic compositions comprising different multivalent pneumococcal polysaccharide-protein conjugates, each conjugate comprising a capsule from a different serotype of pneumococcus conjugated to a carrier protein. Contains polysaccharides.

Streptococcus pneumoniae (hereinafter referred to as pneumococcus) is a main causative bacterium of pneumonia. According to the Statistics Agency's 2010 Mortality Rate Trends by Major Cause of Death, the death rate due to pneumonia in 2010 was 14.9 per 100,000 people, which is one of the leading causes of death among teenagers, compared to 2000. It was found that the number increased by 82.9%. Furthermore, according to the WHO in 2012, in 2008, 476,000 HIV-negative children under the age of 5 died from pneumococcal infection, accounting for 5% of all deaths among children under the age of 5. He died from an illness caused by this bacterium.

In 1931, Harold J. White developed the world's first polysaccharide vaccine. However, with the development of penicillin in 1929, microbial infections were treated with antibiotics, but this led to the development of antibiotic-resistant bacterial strains. As a result, existing antibiotic treatments became ineffective, and in 1947 Marie M. A hexavalent polysaccharide vaccine was developed by Dr. Lapi, and in 1977, Dr. A 14-valent polysaccharide vaccine was developed by Robert Austria, and a 17-valent polysaccharide vaccine was developed by Merck, Wyeth, and Pasteur and began to be sold around the world, and was later developed into a 23-valent polysaccharide vaccine. Polyvalent pneumococcal polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients. However, infants and children have poor immune responses to most pneumococcal polysaccharides, and this is due to a T-cell independent immune response phenomenon. This phenomenon occurs in other microbial vaccines as well, and the first polysaccharide-protein conjugate vaccine, encephalmeningitis vaccine (Hib), was developed in 1987 by overcoming this phenomenon. After this, conjugation technology was introduced for microbial vaccines such as meningitides, pneumococcus, and typhoid fever. In the case of pneumococcal conjugate vaccines, before the 7-valent pneumococcal conjugate vaccine was released in the United States in 2000, the developer company Pfizer (Wyeth) began developing a bivalent pneumococcal conjugate vaccine in 1994, and in 2000. The heptavalent form was developed in 2007. The 7-valent pneumococcal conjugate vaccine (Prevenar®) contains capsular polysaccharides from the seven most frequently occurring serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. . Since it was first approved in the United States in 2000, it has proven to be highly immunogenic and effective against invasive disease and otitis media in infants and children. This vaccine is currently approved in approximately 80 countries around the world. Surveillance data accumulated over the years since the introduction of Prevenar has shown, as expected, a clear decline in invasive pneumococcal disease in the United States due to the serotypes included in Prevenar. However, serotype coverage was limited in some regions, and invasive pneumococcal disease due to serotypes not included in Prevenar increased. Since then, Pfizer has selected six main serotypes that cause invasive lung disease after the 7-valent serotype, and released a 13-valent pneumococcal conjugate vaccine (Prevenar 13) in 2010. Since then, it has been reported that serotype replacement, in which serotypes not included in the vaccine become prevalent, has occurred in a manner similar to the case with the existing 7-valent pneumococcal conjugate vaccine.

As pneumococcal infections with serotypes not included in Prevenar 13 increased, research continued to explore additional pneumococcal serotypes. However, by combining conjugates in multivalent injections, competition between different components (immunointerference effects) may occur, which may also adversely affect the immunogenicity of any individual conjugate. Therefore, there has been great difficulty in adding conjugates to immunogenic compositions.

Immune interference effects are a major problem in the development of multivalent vaccines, and have been raised mainly during the development process of pneumococcal vaccines. After PCV7 was first released in 2000, the immune interference effect of pneumococcal vaccines became a major problem when PCV10 was introduced in 2010. PCV11 was scheduled to be released, but Pn6B did not cause an immune reaction due to its immune interference effect, so it was released as PCV10 without 6B. Furthermore, we recently reported the clinical phase 2 results of PCV15, which is currently under development at Merck (HUMAN VACCINES & IMMUNOTHERAPEUTS, 2019, Adose ranging study of 2 different formulations of 15-valen). pneumococcal conjugate vaccine (PCV15) in healthy infants). It has been confirmed that the immunogenicity of the serotype is lower than that of PCV13, which is a positive control group. In the current trend of developing pneumococcal vaccines in multi-valent vaccine form, multivalent vaccine compositions that ensure the immunogenicity of individual serotypes and at the same time do not have immune interference effects are important for vaccines. Essential for development.

Such immune interference phenomena limit the number of conjugates that can be included in a multivalent vaccine. Therefore, although protection against multiple serotypes is of considerable value, it is a fact that it has been very difficult to achieve this while limiting the number of conjugates in the composition.

Therefore, based on the results of a survey of prevalent serotypes in continental Asian countries (Korea, China, Japan, Taiwan, Singapore, Australia, and India), the present inventors identified serotypes 10A, 11A, and 15B as prevalent in Asian countries. , 22F, 23A, and 35B were finally selected and developed in the form of PCV19 in which the six types were added to the existing PCV13, and it was finally confirmed that the combination of the immunogenic compositions did not cause any immune interference effect. .

This will increase the IPD incidence in countries where PCV13 has not been introduced or where NIP has not been introduced, and in countries where serotype substitution has occurred, by final supplementing the serotypes that were not covered by the existing Prevenar 13 products. This means that it is possible to significantly lower the

Therefore, the present inventor confirmed a combination of immunogenic compositions that can cover serotypes that could not be covered by the existing Prevenar 13 products and does not cause immune interference, and completed the present invention. Ta.

Accordingly, the problem sought to be solved by the present invention is a multivalent immunogenic composition comprising polysaccharide-protein conjugates, each conjugate containing Streptococcus pneumoniae of a different serotype conjugated to a carrier protein. An object of the present invention is to provide a multivalent immunogenic composition containing a capsular polysaccharide derived from Streptococcus pneumoniae, wherein the capsular polysaccharide is of a 14- to 19-valent serotype.

Furthermore, another problem to be solved by the present invention is a pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition. It is about providing something.

Furthermore, another problem to be solved by the present invention is to provide a method for preventing or treating a pneumococcal disease, which comprises administering the multivalent immunogenic composition in a prophylactically or therapeutically effective amount. It is to be.

Furthermore, another problem to be solved by the present invention is to provide a use for the prevention or treatment of pneumococcal-related diseases, which comprises administering the multivalent immunogenic composition in a prophylactically or therapeutically effective amount. It is to be.

To solve the above problems, the present invention provides a multivalent immunogenic composition comprising polysaccharide-protein conjugates, each conjugate containing Streptococcus pneumoniae of a different serotype conjugated to a carrier protein. a) from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F; and b) a capsule of at least one serotype selected from the group consisting of serotypes 10A, 11A, 15B, 22F, 23A and 35B. Multivalent immunogenic compositions comprising membrane polysaccharides are provided.

In the multivalent immunogenic composition according to the invention, a) the capsular polysaccharide consists of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. There may be 13 serotypes, but the present invention is not limited thereto.

In the multivalent immunogenic composition according to the present invention, the carrier protein is diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, inactivated toxoid derived from Escherichia coli, inactivated toxoid derived from Pseudomonas aeruginosa. and bacterial outer membrane protein (OMP), but is not limited thereto.

In the multivalent immunogenic composition according to the present invention, the diphtheria toxoid may be any one selected from the group consisting of CRM ₁₉₇ , CRM ₁₇₃ , CRM ₂₂₈ and CRM ₄₅ , but is not limited thereto. It is not something that will be done. According to one embodiment of the invention, said carrier protein may be CRM ₁₉₇ .

In the multivalent immunogenic composition according to the present invention, the method for conjugating the capsular polysaccharide and the carrier protein includes a CDAP conjugation method, a reductive amination method, and a Thiol-Malemide method. It may be any one selected from the group, but is not limited thereto.

The multivalent immunogenic composition according to the invention may further contain an adjuvant, for example, an aluminum salt as an adjuvant. The aluminum salt may be selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide, and preferably may be aluminum phosphate.

To solve another object of the present invention, the present invention provides a pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of a multivalent immunogenic composition. A composition is provided.

In one embodiment, the pharmaceutical composition comprises 2 μg of each saccharide with the exception of 4 μg of 6B; about 34 μg of CRM ₁₉₇ carrier protein; 0.125 mg elemental aluminum (0.5 mg aluminum phosphate) adjuvant; The immunogenic composition may be formulated to contain sodium chloride and a sodium succinate buffer, but is not limited thereto.

To solve another object of the present invention, the present invention provides for the prevention or treatment of pneumococcal-related diseases, comprising administering the multivalent immunogenic composition as described above in a prophylactically or therapeutically effective amount. provide a method.

Furthermore, to solve another object of the present invention, the present invention provides for the prevention of pneumococcal-related diseases, comprising the step of administering said multivalent immunogenic composition in a prophylactically or therapeutically effective amount. or provide therapeutic uses.

The multivalent immunogenic composition according to the present invention can induce immune responses against more various serotypes than the existing Prevenar 13. In particular, while the existing Prevenar 13 is designed and produced mainly for serotypes that frequently occur in Europe and North America, the immunogenic composition of the present invention is designed to target not only Europe and North America but also all of Asia. It is an immunogenic composition with high coverage. Therefore, the multivalent immunogenic composition according to the present invention can be usefully used to prevent diseases caused by pneumococci in infants, children, and adults.

FIG. 7 shows IgG ELISA results for 13-valent pneumococcal vaccine serotype. It is a figure showing OPA results for 13-valent pneumococcal vaccine serotypes.

The present invention will be explained in more detail below.

However, this description is provided for the understanding of the present invention, and the scope of the present invention is not limited in any way by what is described in the detailed description of the present invention.

Interregional deviations in serotype distribution have raised limitations in the regional coverage of Prevenar. Therefore, there is no reason to exclude any serotype from existing pneumococcal conjugate vaccines, but rather there is a need to add serotypes to further expand the range of applicability.

The serotype of Prevenar 13 was developed as a serotype prevalent in Europe and the United States, and since the introduction of Prevenar 13, a serotype replacement phenomenon has occurred in each country. This is reported to show differences between Asia and Europe/USA. In the present invention, we selected serotypes that have been prevalent in Asia and Europe/USA since the introduction of Prevenar 13. The criteria for the incidence rate of pneumococcus in each country are based on 1) children under 5 years of age, 2) the results of the incidence rate since the introduction of Prevenar 13 in each country, and 3) prevalent/non-prevalent serotypes, and the prevalence in each country. / Non-endemic serotypes were selected, and common prevalent serotypes in Asia/Europe/USA were selected.

Serotype Replacement is known to be influenced by age, country, time of vaccine introduction, and whether or not the National Immunization Program (NIP) has been introduced. Therefore, in order to overcome this phenomenon, pneumococcal vaccine development companies are developing new vaccines with a strategy of adding representative serotypes that cause IPD (invasive pulmonary disease), and Merck's PCV15 is similar to PCV13. Pfizer's PCV20 is being developed by adding 8, 10A, 11A, 12F, 15B, 22F, and 33F to PCV13. When checking the serotype selection criteria of Merck and Pfizer, they selected prevalent serotypes that cause IPD in the United States and Europe, and in particular, in the case of Merck, they selected 22F and 33F, which are US/European cross-serotypes. This is a characteristic.

In the case of Japan, which is evaluated as a vaccine-developed country, PCV7 and PCV13 were registered in Japan's NIP immediately after they were released, and after registration, the number of IPD patients with the serotypes included in PCV13 rapidly decreased. confirmed. This phenomenon also occurs in the United States/Europe. However, although the serotypes included in PCV13 have similar IPD incidence trends, the serotypes prevalent in Non-PCV13 (non-vaccine-containing serotypes) differ in the degree of prevalence by country/continent. I can do it. (Source 1, Vaccines 34 (2016) 67-76, Source 2, Vaccines 4 (2016), 2000-2014: A Pooled Data Analysis, Source 3. Plos one (2017) A systematic review and meta -analysis)

In Japan, PCV7 was introduced in 2010 and PCV13 was introduced in 2013, and it is known that there are large differences in the prevalent serotypes before and after the introduction of the PCV13 vaccine. During the period from 2011 to 2013, when PCV13 was introduced, the frequency of PCV13-containing serum was high, but from 2014 to 2016, two to three years after the introduction of the vaccine, serotype replacement ) phenomenon has occurred. Among the serotypes included in PCV13, the serotypes that have been causing disease until recently have occurred in the order of 3>6A, 6B, 19F, and 23F, but the number of cases has been reduced to 1/10 of the number before the introduction of the vaccine. be. Since the introduction of the vaccine, non-PCV13 serotypes have been prevalent, occurring in the order of 24F>15A>12F>15B>22F.

For example, in the case of the United States, unlike the case in Japan, 22F and 33F have been prevalent since the introduction of PCV13, but 33F has not become widely prevalent in the Asian continent, including Japan. (Source 1. Clin Microbiol Infect 2016;22:60.e9-60.e29, Source 2. WHO 2011, Global review of the distribution of pneumococcal disease by age and region, Source 3. CDC (US) homepage)

In Europe, the timing of vaccine introduction and NIP introduction are different, but if we refer to the cases of the UK/Germany/France, the serotypes that will be prevalent after the introduction of PCV13 are 8>22F>33F>24>9N. Yes, showing differences from the Asian continent. (Expert review of vaccines.2019).

In order to confirm the prevalent serotypes on the Asian continent, we are investigating cases in South Korea, Japan, Taiwan, China, Australia, Singapore, and India, and the criteria for investigation are: 5 years old or younger, 65 years or older, and after the introduction of PCV13. The IPD prevalent serotypes were confirmed. As a result, in the case of Japan, PCV13 was introduced in 2013, and the prevalent serotypes of non-PCV13 were confirmed to be 24F>15A>12F>15B>22F, and in the case of South Korea, 10A>15A, 23A>15B, It was confirmed that 35B is prevalent, in Australia, 23B>22F>35B>33F is prevalent, and in Taiwan, 23A>15B>15A>22F, 11A are prevalent. In the case of Singapore, there are few applicable cases, but 15B and 15A are reported to be prevalent among Non-PCV13, and in the case of China/India, NIP was introduced late in both countries (after 2017), and since NIP was introduced. It was not possible to calculate the prevalent serotypes due to the lack of epidemiological survey results, but if we refer to the prevalent serotypes in the preceding countries, there is a high possibility that serotype substitution will occur in both China and India. It is expected that the European/US prevalent serotypes and other Asian prevalent serotypes are likely to become prevalent.

As a result of analyzing the prevalent serotypes in Asian countries, the serotypes that overlap in three or more countries in Asia are 15B, 15A, 22F, and 23A, and 15B and 15A are cross-reactive ( There is a report that there is a cross-reactivity), so we selected only the 15B serotype and identified a total of six serotypes (10A, 11A, 15B, 22F, 23A, 35B) were selected.

In South Korea, Prevenar 13 was introduced in 2010, and similar to Japan, serotype replacement occurred rapidly. An increasing trend of non-prevenar 13 has been occurring since 2016-2017. Among the 13 serotypes of Prevenar, the serotypes that have been causing disease until recently are 19F and 6A, and the number and frequency of these serotypes have decreased significantly compared to before the introduction of the vaccine. The serotypes that have become prevalent since the introduction of the non-Prevenar 13 vaccine have occurred in the order of 10A>15A, 23A>15B, and 35B.

In Australia, Prevenar 13 was introduced in 2011. A serotype substitution has occurred since 2014, with an increasing trend of non-Prevenar 13 serotype. Among the non-prevenar 13, the serotypes that have been prevalent since the introduction of the vaccine have occurred in the order of 23B>22F>35B>33F.

In Taiwan, Prevenar 13 was introduced in 2010. A serotype substitution phenomenon has been occurring since 2016. Among non-PV13, the serotypes that have become prevalent since the introduction of the vaccine have occurred in the order of 23A>15B>15A>22F, and 11A.

In Singapore, Prevenar 13 was introduced in 2011. As in other Asian countries, serotype substitution has occurred since the introduction of the vaccine. The prevalent serotypes of non-prevenar 13 occur in the order of 15B>15A.

In India, Prevenar 13 was introduced in 2017. In the case of India, since only one to two years have passed since the introduction of Prevenar 13, serotype replacement has not completely progressed, and serotype replacement will increase as the vaccine penetration rate increases. I predict something. According to currently reported data, the prevalent serotypes of non-prevenar 13 occur in the order of 23A>22F>15B>6D.

In the case of China, Prevenar 7 was approved for sale in 2008 and Prevenar 13 was approved for sale in 2016, but as they are not included in the national free immunization program, vaccination rates and penetration rates are reported to be significantly low. . However, as China's economic growth rate increases, health expectations rise, and the vaccination rate increases, we expect that the serotype substitution phenomenon will occur over time, just like in other countries.

After enumerating the frequency of occurrence of non-Prevenar 13 serotypes since the introduction of Prevenar 13 in six Asian countries (South Korea, Japan, Taiwan, Singapore, India, and Australia), it was confirmed that 15B was the most prevalent in the six Asian countries. This was followed by 22F, 23A, and 35B in that order. In the United States and Europe, the most common serotypes are 22F and 33F, and the next most common serotypes are 10A and 11A. Therefore, we added six serotypes prevalent in Asia (10A, 11A, 15B, 22F, 23A, 35B) and four serotypes prevalent in Europe and the United States (10A, 11A, 22F, 35B) to finalize the serotypes. The following configuration was selected.

Therefore, based on the survey results of prevalent serotypes in continental Asian countries (Korea, China, Japan, Taiwan, Singapore, Australia, and India), the present inventor identified the serotypes prevalent in Asian countries as 10A, 11A, 15B, and 22F. , 23A, and 35B were finally selected, and these six types were added to the existing PCV13 to finally develop the PCV19 form.

Since vaccines are a national industry, whether or not the vaccine is included in the national free vaccine program is important for vaccination rates. This shows that the types of vaccines included in the National Immunization Program (NIP) and the targets to be vaccinated vary greatly depending on each country's economic level and the position of health authorities. Among these, the pneumococcal conjugate vaccine is one of the more expensive vaccines, and only a few countries around the world have included it in their national free vaccine programs. In the case of Japan, it operates a national free vaccination program comparable to that of developed countries, and has shown a pneumococcal vaccination rate similar to that of the United States/Europe, resulting in serotype replacement occurring at a similar time to Europe and the United States. Occurred. Differences in serotype substitution among Asian countries can also be said to be due to racial differences. However, a common denominator of serotype substitution could be found, which shows differences with the non-prevenar 13 prevalent serotypes in Europe and the United States. Of the 22F and 33F serotypes that are commonly prevalent in Europe and the United States, the 33F serotype occurs only in Australia, the only Asian country where serotype substitution has occurred, and in South Korea, Japan, and Taiwan. , it has not occurred in Singapore or India. Unlike Europe and the United States, serotypes 15A, 15B, 22F, and 23A are prevalent in Asian countries, showing differences from the serotypes prevalent in Europe and the United States.

The 19-valent pneumococcal conjugate composition containing six novel serotypes (10A, 11A, 15B, 22F, 23A and 35B) of the present invention provides functional immunity in humans against diseases induced by pneumococcal infection. induce a reaction. More specifically, in one embodiment, the human subject is an elderly subject and the disease is pneumonia or invasive pneumococcal disease. More specifically, in one embodiment, the elderly subject is at least 50 years old. More specifically, in one embodiment, the elderly subject is at least 55 years old. More specifically, in one embodiment, the elderly subject is at least 60 years old.

More specifically, in one embodiment, the human subject is an infant and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM).

More specifically, in one embodiment, the infant is between 0 and 2 years old, or between 2 and 15 months old.

In other embodiments, the human subject is 6 weeks to 17 years old and the disease is pneumonia, invasive pneumococcal disease (IPD), or acute otitis media (AOM). In certain embodiments, the human subject is between 6 weeks old and 5 years old. In another embodiment, the human subject is between 5 weeks old and 17 years old.

The present invention provides a capsular polysaccharide in which Prevenar 13 contains all the serotypes included in the vaccine and further contains one or more serotypes selected from the group consisting of 10A, 11A, 15B, 22F, 23A and 35B. - Provide multivalent immunogenic compositions comprising protein conjugates. That is, the present invention provides multivalent immunogenic compositions comprising 14 or more different polysaccharide-protein conjugates together with a physiologically acceptable vehicle, each conjugate being conjugated to a carrier protein. Contains capsular polysaccharides from different serotypes of pneumococci.

Capsular polysaccharides can be manufactured by standard techniques known to those skilled in the art. The capsular polysaccharide can be reduced in size to reduce viscosity or to increase the solubility of the activated capsular polysaccharide. In an embodiment of the invention, the capsular polysaccharide is a polysaccharide of 13 pneumococcus serotypes consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. and a capsular polysaccharide of one or more serotypes selected from the group consisting of serotypes 10A, 11A, 15B, 22F, 23A, and 35B.

The pneumococcal conjugate is manufactured by a separate process and formulated into a single dosage form. For example, each pneumococcal polysaccharide serotype is grown in a soybean-based medium and the individual polysaccharides are then purified by centrifugation, precipitation, and ultrafiltration.

The carrier protein is preferably a protein that is non-toxic, non-reactogenic, and can be obtained in sufficient quantity and purity. The carrier protein must be suitable for standard conjugation methods. In the multivalent immunogenic composition according to the invention, the carrier protein may be CRM ₁₉₇ . CRM ₁₉₇ is a nontoxic variant of diphtheria toxin isolated from a culture of Corynebacterium diphtheria strain C7 (β197) grown in casamino acid and yeast extract-based medium. CRM ₁₉₇ is purified by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. CRM ₁₉₇ can be produced recombinantly according to US Pat. No. 5,614,382.

Other diphtheria toxoids can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid, exotoxin A from E. coli and Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane conjugate c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or group B C5a peptidase from Streptococcus or Haemophilus influenzae protein D can also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins. Variants of diphtheria toxin such as CRM ₁₇₃ , CRM ₂₂₈ , CRM ₄₅ can also be used as carrier proteins.

Existing known conjugation methods can be used to conjugate the carrier protein and polysaccharide. As an example, but not limited to, a reductive amination method, a CDAP conjugation method or a Thiol-Malemide method can be used. Purified polysaccharides can be chemically activated to produce saccharides that can react with carrier proteins. Once activated, each capsular polysaccharide is conjugated one by one to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. Chemical activation of the polysaccharide and subsequent conjugation to the carrier protein can be performed by known methods (eg, US Pat. Nos. 4,673,574, 4,902,506).

The resulting polysaccharide-protein conjugate can be purified (ie, enriched for the amount of polysaccharide-protein conjugate) by various methods. Examples of these methods include concentration/diafiltration steps, column chromatography, and multilayer filtration. The purified polysaccharide-protein conjugates can be mixed together and formulated into an immunogenic composition of the invention, which can be used as a vaccine. Formulation of the immunogenic compositions of the invention can be carried out using art-recognized methods. For example, 14 to 19 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to produce a composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In one embodiment, the immunogenic compositions of the invention include one or more adjuvants. An "adjuvant" as defined herein is a substance used to increase the immunogenicity of the immunogenic compositions of the invention. Accordingly, adjuvants are sometimes provided to boost the immune response and are well known to those skilled in the art. Adjuvants suitable for increasing the effectiveness of the composition include, but are not limited to:

In certain embodiments, aluminum salts are used as adjuvants. The aluminum salt adjuvant may be an Alum-precipitated Vaccine or an Alum-adsorbed Vaccine. Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), and the like. Mixing aluminum chloride and sodium phosphate in a 1:1 ratio precipitates aluminum hydroxyphosphate sulfate. The precipitate is made to have a size of 2 to 8 μm using a High Shear Mixer, and then dialyzed against physiological saline and sterilized. In one embodiment, commercially available Al(OH) ₃ (eg, Alhydrogel or Superfos) is used to adsorb proteins. 50-200 g of protein can be adsorbed per mg of aluminum hydroxide, the ratio depending on the pI of the protein and the pH of the solvent. Low pI proteins bind more strongly than high pI proteins. Aluminum salts form antigen depots that gradually release antigen over a period of 2-3 weeks, non-specifically activating macrophages, complement, and innate immune mechanisms.

The present invention provides a pharmaceutical composition (e.g., a vaccine formulation) for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of the multivalent immunogenic composition. .

The vaccine formulation according to the invention can be used to protect or treat persons susceptible to pneumococcus infection by administering it by systemic or mucosal routes. An "effective amount," as defined herein, refers to a dose necessary to induce antibodies to a degree that can significantly reduce the probability of infection with pneumococci or the severity of infection. Administration may include injection through intramuscular, intraperitoneal, intradermal or subcutaneous routes; or mucosal administration to the oral/gastrointestinal tract, respiratory tract or urogenital tract. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media, as it can more effectively prevent nasopharyngeal carriage of pneumococci and attenuate the infection in its early stages. .

The amount of the conjugate in each vaccine dose is selected to induce an immunoprotective response without serious side effects. Such amounts may vary depending on the pneumococcal serotype. Generally, each dose may contain, but is not limited to, 0.1-100 μg, preferably 0.1-10 μg, more preferably 1-5 μg of polysaccharide. Optimal amounts of components for a particular vaccine can be ascertained by standard studies including observation of appropriate immune responses in subjects. For example, animal test results can be extrapolated to determine vaccination doses for humans. Doses can also be determined empirically.

In one embodiment of the invention, the vaccine composition of the invention comprises serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM ₁₉₇ , respectively. and capsular polysaccharides of one or more serotypes selected from the group consisting of serotypes 10A, 11A, 15B, 22F, 23A and 35B. It is in a sterile liquid dosage form. In each 0.5 mL dose, 2 μg of each saccharide except 4 μg of 6B; approximately 34 μg of CRM ₁₉₇ carrier protein; 0.125 mg elemental aluminum (0.5 mg aluminum phosphate) adjuvant; sodium chloride as excipient. and sodium succinate buffer. The liquid can be filled into single dose syringes without preservatives. Upon shaking, the vaccine becomes a homogeneous white suspension ready for intramuscular administration.

Compositions of the invention can be formulated in single-dose vials, multi-dose vials, or prefilled syringes.

Formulations of the invention may include a surfactant or a mixture of surfactants such as Tween 80 or Span 85.

In one embodiment of the present invention, the present invention provides a method for preventing or treating a pneumococcal disease, comprising administering the multivalent immunogenic composition in a prophylactically or therapeutically effective amount.

In one embodiment of the present invention, the present invention provides a use for preventing or treating pneumococcal-related diseases, comprising administering the multivalent immunogenic composition in a prophylactically or therapeutically effective amount.

Inclusion of Prevenar 13 serotype In Northern California, Prevenar 13 serotype accounted for more than 90% of all cases of invasive pneumococcal disease in infants and children. Furthermore, in Western Europe, Prevenar serotype 13 accounted for more than 70% of all cases of invasive pneumococcal disease in infants and children. Since the United States and Europe are the largest vaccine sales markets, there is no reason to exclude any Prevenar 13 serotypes from next-generation pneumococcal conjugate vaccines, but rather it is preferable to add additional serotypes to further expand the scope of application.

Addition of serotype hexavalent Prevenar 13 serotype was developed as a serotype prevalent in Europe and the United States, and since the introduction of Prevenar 13, a serotype substitution phenomenon has occurred in each country, and this is the case in Asia and the United States. It is reported that there are differences between Europe and the United States. In the present invention, we selected serotypes that have been prevalent in Asia and Europe/USA since the introduction of Prevenar 13. The criteria for the incidence rate of pneumococcus in each country are based on 1) children under 5 years of age, 2) the results of the incidence rate since the introduction of Prevenar 13 in each country, and 3) prevalent/non-prevalent serotypes, and the prevalence in each country. / Non-endemic serotypes were selected, and common prevalent serotypes in Asia/Europe/USA were selected.

Hereinafter, the present invention will be explained in more detail through Examples. However, the following examples are for illustration of the present invention, and the present invention should not be construed as being limited by these examples.

Example 1. Production and purification of pneumococcus capsular polysaccharide Cultivation of pneumococci and purification of capsular polysaccharide were performed by methods known to those skilled in the art. Each serotype of pneumococcus can be obtained from a depository organization (CDC, Center for Disease Control and Prevention). Pneumococci were identified by capsular and nonmotile, Gram-positive, lancet-shaped diplococci by alpha hemolysis on blood agar. Serotypes were confirmed based on Quellung Test using specific antisera.

Example 1-1. Production of cell bank 19 different serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B) Streptococcus pneumoniae was obtained from the US CDC (Center for Disease Control and Prevention, US), which is a contract institution.

Pneumococcus strains were plated on blood agar plates and single colonies were isolated. After selecting a single colony with good growth from among 10 or more single colonies, it is inoculated and cultured in a liquid medium that does not contain animal-derived components, and then it is placed in a research cell bank containing synthetic glycerol. , RCB) were manufactured.

One vial from a research cell bank that was confirmed to express a polysaccharide with a unique serotype was taken out, the cells were grown in a liquid medium that did not contain animal-derived components, and then synthetic glycerol was added. A master cell bank was produced, one vial of the master cell bank was taken out, cells were grown in a liquid medium containing no animal-derived components, and synthetic glycerol was added to produce a production cell bank. The produced cell bank was stored at -70°C or lower for use in the next step.

Example 1-2. Fermentation and Separation of Polysaccharides One vial from the production cell bank was thawed and inoculated into a liquid medium containing no animal-derived components to begin seed fermentation. Seed culture was carried out at 37±2° C. without stirring until a certain bacterial cell concentration (Optical Density, OD600) was reached and the end point of the intermediate-exponential growth phase was reached. Main fermentation was started by inoculating the culture solution obtained in the seed culture into a fermenter containing a liquid medium containing no animal-derived components.

Next, main culture was performed at 37±2° C. while adjusting the pH of the medium with a potassium hydroxide solution. The optimal cell density and the glucose concentration contained in the medium were measured every 2 hours. Fermentation was terminated when glucose in the medium was depleted.

After fermentation was complete, 12% Sodium Deoxycholate was added to the culture over 1 hour to a final concentration of 0.12% to lyse the cells and release polysaccharides bound to the cells. .

Example 1-3. Purification of Capsular Polysaccharide Phosphoric acid was added to the sodium deoxycholate-treated sample, and the supernatant was collected through centrifugation. The collected supernatant liquid was passed through a depth filter, and then concentrated and buffer exchanged with a phosphate buffer solution. After buffer exchange, the sample was passed through an active carbon filter, and impurities were removed using the following two methods.

In the case of 17 serotypes 1, 3, 4, 5, 6A, 6B, 9V, 10A, 11A, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B, the ionic bond with CTAB (Cetyltrimethylammonium Bromide) is As possible, a CTAB step was performed. CTAB treatment, centrifugation, sodium chloride (NaCl) and sodium iodide (NaI) treatment, and centrifugation steps were performed.

Two serotypes, 7F and 14, which do not react with CTAB were reacted by adding an aluminum phosphate gel (Algel) solution, and then the supernatant obtained through centrifugation was used.

After completing the process of removing the two types of impurities, the sample was subjected to a depth filter and an ultrafiltration (UF/DF) process, the amount of ethanol and sodium chloride was adjusted, and the sample was stored in bulk powder form.

Example 1-4. Dissolution and Hydrolysis of Capsular Polysaccharide The bulk capsular polysaccharide derived from each serotype was dissolved in water for injection so that the final concentration range was as shown below, and filtered through a 0.45 μm filter.

Specifically, serotypes 1, 3, and 4 are dissolved in a range of 0.8 to 2.0 mg/ml, and serotypes 5, 6B, 9V, 18C, and 19F are dissolved in a serum concentration of 4 to 8 mg/ml. Types 6A and 19A were dissolved at 8 to 12 mg/ml, and serotypes 7F, 10A, 11A, and 23F were dissolved at 2 to 4 mg/ml and filtered. In the case of serotypes 15B, 22F, and 35B, they were dissolved in a range of 2 to 5 mg/ml and filtered.

For each serotype, solutions were subjected to constant temperature treatment within the following pH and temperature ranges. Specifically, for serotypes 1, 3, 5, 6B, 7F, 10A, 11A, 14 and 23F, 70-80°C overnight; for serotypes 6A and 19F, 70-80°C for 1-4 hours. For serotypes 9V and 18C, a constant temperature treatment process was performed at pH 2.0 and 65-80°C for 1-3 hours using a phosphoric acid solution. For serotypes 22F, 23A and 35B, 75-85°C overnight; for serotypes 4, 15B and 19A, no hydrolysis was performed. The hydrolysis was then stopped by cooling to 21-24° C. and adding sodium hydroxide at a target pH of 6.0±1.0.

Example 2. Conjugation and purification of pneumococcal capsular polysaccharide and CRM ₁₉₇ protein carrier Example 2-1. Preparation of CRM ₁₉₇ Protein Vehicle CRM ₁₉₇ protein vehicle was purchased and prepared from Wyeth, Sanford, NC, USA.

Example 2-2. Conjugation of Capsular Polysaccharide and CRM ₁₉₇ A 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to all serotypes. Appropriate CDAP (1-Cyano-4-dimethylaminopyridinium Tetrafluoroborate) for each serum was dissolved at a rate of 1 g of CDAP per 100 ml of 50/50 acetonitrile/water for injection (v/v) solution. Specifically, for serotypes 6A and 14, CDAP is 1w/w% to polysaccharide, for serotype 4, 2w/w%, and for serotypes 1, 3, 6B, 7F15B and 19A, 3w/w%. was dissolved in a weight ratio of 4 w/w % for other serotypes and added to each polysaccharide solution. Then, after 1-3 minutes, sodium hydroxide solution was added to raise the pH to 9.4-9.7, followed by stirring for 3-7 minutes so that the hydroxyl groups of the polysaccharide could be sufficiently activated by CDAP. Polysaccharide contrast CRM ₁₉₇ 0.5-1.0 w/w% was added to the polysaccharide solution of each serotype, a conjugation reaction was performed for 1 to 4 hours, and the reaction conversion rate was measured using HPLC-SEC. Additional CDAP was added as needed.

Example 2-3. Termination of Conjugation Reaction The reaction was terminated by adding 3 to 6 molar equivalents of glycine solution to 1 molar equivalent of CDAP added for all serotypes and adjusting the pH to 9.0. The conjugation solution was stirred at 21-24°C for 1 hour and then stored overnight at a low temperature of 2-8°C.

Example 2-4. Ultrafiltration The diluted conjugation mixture was concentrated and diafiltered onto an ultrafiltration filter using a minimum of 20 volumes of buffer. Here, the buffer solution was maintained at a pH range of 5.5 to 6.5 and contained 0.9% sodium chloride. Ultrafiltration filter molecular weight cutoffs were performed using 300 kDa for all serotypes, and the permeate was discarded.

Example 2-5. Sterilization filtration The residual solution after diafiltration was diluted with a buffer solution to a polysaccharide content concentration of less than 0.4 g/L, and filtered through a 0.22 μm filter. Controls (sugar content, residual DMAP) were performed on the filtered product during the manufacturing process. Controls were performed on the filtered retentate during the manufacturing process to determine whether additional concentration, diafiltration and/or dilution was required.

Example 3. Formulation of 1.1 Trivalent Pneumococcal Vaccine and Immunogenicity Studies Example 3-1. Formulation of 1.1 Trivalent Pneumococcal Vaccine The final volume of the final bulk concentrate obtained from Example 2 was , calculated based on batch volume and bulk sugar concentration. 0.85% Sodium Chloride (Saline), Polysorbate 80 and Succinate Buffer were added to the pre-labeled dosage form container followed by the bulk concentrate. The formulation was then thoroughly mixed and sterile filtered using a 0.2 μm membrane. The formulated bulk was gently mixed during the bulk aluminum phosphate addition process and after the final addition was completed, and the pH was checked and adjusted as necessary. Additionally, the formulated bulk product was final stored at 2°C to 8°C. The table below shows the polyvalent pneumococcal conjugate vaccine dosage forms produced.

(*Synflorix is a GSK product and falls under the 11-valent category)

The resulting vaccine composition contained, in a total of 0.5 mL, 2 μg of each saccharide, with the exception of 4 μg of 6B; approximately 35 μg of CRM ₁₉₇ carrier protein; 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate). Adjuvant; approximately 4.25 mg sodium chloride; approximately 295 μg succinate buffer; and approximately 100 μg polysorbate 80.

Example 3-2. Measurement of serotype-specific IgG concentration ELISA In order to confirm whether each of the 13-valent pneumococcal vaccine compositions prepared in the above examples had the ability to induce an immune response in mice, the following experiment was conducted. . The immunogenicity was confirmed by measuring serum IgG concentration through antigen-specific ELISA.

The planned human clinical dose of each formulated polyvalent pneumococcal vaccine composition or control group Prevenar 13 (4.4 μg/mL for each polysaccharide, exception: 100 μg/mL for 6B 8.8 μg/mL) %, the content of some serotypes was adjusted as necessary), and mice (C57BL/6) were immunized intramuscularly at 0, 2 and 4 weeks, and after the last inoculation. One week later, each serum was collected. The serotype-specific IgG concentration was measured for the collected serum using ELISA.

This will be explained in detail as follows. Each serum was analyzed using a dilution factor that reduced the absorbance value to less than 0.175. A 96-well immunoplate was coated with serotype capsular polysaccharide at a concentration of 5 μg/ml and then left at 4°C. To block nonspecific binding, serum diluted with 5 μg/ml cell wall capsular polysaccharides (CWPS) and 22F was added to the coated plate, and 2 hours later, After treatment with anti-mouse IgG, which is a secondary antibody to which HRP was attached, the plate was left at room temperature for 1 hour. After 1 hour, the substrate that reacts with HRP (3,3'5,5'-Tetramethylbenzidine; TMB) was treated for 10 minutes, the reaction was stopped using 2N H ₂ SO ₄ , and the absorbance was measured at 450 nm after 10 minutes. was measured.

(*+: 1 to 3 times more effective than the control group)

Prior to producing a multivalent combination vaccine, the present inventors compared the efficacy of an existing commercially available 13-valent conjugate with Prevenar 13. As a result, we identified serotypes that correlated with the concentration-dependent IgG titer of the 13-valent immunogenic composition and serotypes that did not. Furthermore, in comparison with Prevenar 13, it was confirmed that it had similar IgG titers regardless of the concentration. (Table 2 and Figure 1)

Example 3-3. Antibody function opsonization test (MOPA assay)
Antibody function was assessed by testing serum with the MOPA assay. The analysis method was performed using a known method (multiplexed opsonophagocytic killing assay (UAB-MOPA) test method for measuring antibody function against pneumococci, 2013), and most of the experimental methods were as follows.

The process in which phagocytes engulf pathogens using complement and antibodies is called opsonization, and Single is an experimental method that uses the opsonization process to confirm the ability of serotype-specific antibodies produced by vaccination. /Multiplex opsonophagocytic assay. The phagocytic process was induced by culturing one or more serotypes of pneumococci with different antibiotic resistances together with serum, complement, and phagocytes, and the antibiotics to which each serotype was resistant were added. Spread it on an agar medium and measure the number of colonies based on the serum dilution factor. The 50% killing rate of the bacterial strain was measured from the number of colonies in which the strain survived 100% (0% killing) in the control group, and the opsonization index was determined through the dilution ratio that indicates the 50% removal rate of each sample. ;OI) were compared.

(*+: Confirmed an effect 1 to 3 times greater than the control group / ++: Confirmed an effect 3 to 5 times greater than the control group / +++: Confirmed an effect 5 to 7 times greater than the control group / +++++: Confirmed an effect 5 to 7 times greater than the control group ~7 times more effective) confirmed)

As a result, in all serotypes, antibody titers and OPA titers higher than or similar to those of Prevenar 13 were confirmed, and no large immune interference effect was confirmed with the 13-valent immunogenic composition. Although some groups showed low titers depending on the administered concentration and serotype, this phenomenon was confirmed to be a well-known phenomenon based on experiments with heptavalent immunogenic compositions and some papers. . Previous ELISA titer assays have confirmed similar levels of titers to Prevenar 13 titers, but for OPA titers, some serotypes have significantly higher serotype and similar serotypes than Prevenar 13. It was confirmed that the types were distributed differently. Furthermore, it was confirmed that the reliability/discrimination power of the OPA results was higher than that of the ELISA results (Table 3 and FIG. 2).

Example 4. Study on formulation and immunogenicity of polyvalent pneumococcal vaccine Example 4-1. Formulation of Multivalent Pneumococcal Vaccine The final volume of the final Bulk concentrate obtained from Example 2 was calculated based on the Batch volume and bulk saccharide concentration. 0.85% Sodium Chloride (Saline), Polysorbate 80 and Succinate Buffer were added to the pre-labeled dosage form container followed by the bulk concentrate. The formulation was then thoroughly mixed and sterile filtered using a 0.2 μm membrane. During the bulk aluminum phosphate addition process and after the final addition, the formulated bulk was gently mixed and the pH was checked and adjusted as necessary. The formulated bulk product was stored at 2-8°C. The table below shows four types of multivalent pneumococcal conjugate vaccine formulations.

The resulting vaccine composition contained, in a total of 0.5 mL, 2 μg of each saccharide, except 4 μg of 6B; approximately 35 μg of CRM ₁₉₇ carrier protein; 0.125 mg elemental aluminum (0.5 mg aluminum phosphate). Adjuvant; approximately 4.25 mg sodium chloride; approximately 295 μg succinate buffer; and approximately 100 μg polysorbate 80.

Example 4-2. Measurement of Serotype-Specific IgG Concentration ELISA In order to confirm whether each of the polyvalent pneumococcal vaccine compositions produced in the above examples had the ability to induce an immune response in rabbits, an experiment was conducted as follows. . The immunogenicity was confirmed by measuring serum IgG concentration through antigen-specific ELISA.

The planned human clinical dose of each formulated polyvalent pneumococcal vaccine composition or control group Prevenar 13 (4.4 μg/ml of each polysaccharide, exception: 100 μg/ml for 6B 8.8 μg/ml) %, the content of some serotypes was adjusted as necessary) and was intramuscularly inoculated into New Zealand White rabbits at 0, 2 and 4 weeks, and 2 days after inoculation. Serum of each was collected at weekly intervals. The serotype-specific IgG concentration was measured for the collected serum using a multiplex bead assay. This will be explained in detail as follows.

A solution in which 13 or 17 types of polysaccharide antigens were conjugated to magnetic beads was placed in a 96-well plate and allowed to adhere thereto. The serum of each individual was adsorbed by reacting with 1 mg/mL of CWPS multi solution (CWPS multi (registered trademark), Statens Serum Institute) for 30 minutes at room temperature to minimize non-specific antigen-antibody reactions. After that, the antibody was diluted at an appropriate dilution ratio using an antibody dilution buffer containing Tween 20. The plate to which the 13, 17, 18, or 19 types of polysaccharide antigen-coupled magnetic beads were attached was washed twice with washing buffer, and 50 μl of pre-adsorbed and diluted serum was added to the plate, followed by reaction at room temperature for 30 minutes. .

The reacted plate was washed three times in the same manner, R-PE goat anti-rabbit IgG (1:500) was added to each well, and the plate was incubated at room temperature for 30 minutes. Ta. The plate was washed three times as described above, 80 μl of buffer was added to each well, and then fluorescence was measured using a multiplex reader. Blood samples collected after immunization with Prevenar 13 (registered trademark) were also analyzed as a control group for objective immunogenicity evaluation.

(*+: Confirmed an effect 1 to 3 times greater than the control group / ++: Confirmed an effect 3 to 5 times greater than the control group / +++: Confirmed an effect 5 to 7 times greater than the control group)

As a result, it was confirmed that PCV17-CRM ₁₉₇ exhibited an excellent level of serotype-specific IgG concentration against all 17-valent serotypes. In the case of PCV17-CRM ₁₉₇ , serotypes in common with Prevenar 13 show similar or higher serotype-specific IgG concentrations than Prevenar 13, and newly added serotypes 10A, 11A, 15B and Each of the 22Fs exhibited excellent levels of serotype-specific IgG concentrations.

23A and 35B are serotypes that are not included in PPV23, the pneumococcal polysaccharide vaccine (PPV), and are serotypes that have not been used in pneumonia vaccines to date. It is one of the serotypes that arrived after the replacement.

It was confirmed that PCV18-CRM ₁₉₇ and PCV19-CRM ₁₉₇ containing 23A and 35B exhibited an excellent level of serotype-specific IgG concentration against all serotypes. Serotypes in common with Prevenar 13 have similar or higher serotype-specific IgG concentrations than Prevenar 13, and each of the newly added serotypes 10A, 11A, 15B, 22F, 23A and 35B It was confirmed that both showed excellent levels of serotype-specific IgG concentration. (Table 5)

Example 4-3. Antibody function opsonization test (MOPA assay)
Antibody function was assessed by testing the serum with the MOPA (Multiplex Opsonophagocytosis Assay) assay. Streptococcus pneumoniae MOPA strains stored at below -70°C were diluted to the corresponding final dilution so that the concentration of each strain was approximately 50,000 CFU/mL. Equivalent amounts of serum were sampled from each subject, pooled by group, and serially diluted 2-fold so that 20 μl of serum remained in the U-bottom plate. After diluting the sample, 10 μl of the strain prepared for each serotype was mixed with the diluted sample, and the mixture was allowed to react at room temperature for 30 minutes to ensure good mixing of S. pneumoniae and antibodies. A mixture of predifferentiated HL-60 cells and complement was added and reacted in a CO ₂ incubator (37° C.) for 45 minutes. Phagocytosis was interrupted by lowering the temperature, and 10 μl of the reaction solution was spotted on a pre-dried agar plate for 30-60 minutes, and then allowed to absorb onto the plate for 20 minutes until dry. A 25 mg/mL TTC stock solution was added to the prepared Overlay Agar and to this was added an antibody compatible with the corresponding strain. After the mixture was thoroughly mixed, approximately 25 mL of the mixture was added to the plate and allowed to cure for approximately 30 minutes. Colonies were counted after fully hardened plates were incubated in a CO ₂ incubator (37° C.) for 12-18 hours. MOPA titer was expressed as the dilution at which 50% killing was observed. As a comparative example, a commercially available 13-valent vaccine (Prevenar 13) was applied in the same process.

(*+: Confirmed an effect 1 to 3 times greater than the control group / ++: Confirmed an effect 3 to 5 times greater than the control group / +++: Confirmed an effect 5 to 7 times greater than the control group)

As a result, it was confirmed that all serotypes exhibited excellent levels of functional immunogenicity in PCV17-CRM ₁₉₇ . In the case of PCV17-CRM ₁₉₇ , serotypes in common with Prevenar 13 have shown similar or better functional immunogenicity than Prevenar 13, and the newly added serotypes 10A, 11A, 15B and 22F each exhibited high levels of functional immunogenicity.

In the case of 23A and 35B, it is known that antibiotic-resistant strains do not exist because they are serotypes that are not included in PPV23, a pneumococcal polysaccharide vaccine. The resistant strains used in the analysis were created based on the UAB antibiotic-resistant strain manufacturing method (MOPA Protocol), and after creation, the characteristics of each 23A and 35B serotype were confirmed by strain characterization analysis and identification.

Through the above experiment, it was confirmed that PCV18-CRM ₁₉₇ and PCV19-CRM ₁₉₇ containing 23A and 35B produced excellent levels of serotype functional antibodies against all serotypes. Serotypes in common with Prevenar 13 have shown similar or higher functional antibodies to Prevenar 13, and the newly added serotypes 10A, 11A, 15B, 22F, 23A and 35B each have superior levels of functional antibodies. demonstrated functional antibodies.

Furthermore, the results show that the PCV17, 18, and 19 compositions do not show reduced immunogenicity of the serotypes against the positive control PCV13, but similar or higher levels of titer against PCV13. As a result, it was finally confirmed that the PCV19 composition had no immune interference effect (Table 6).

Example 5. Study on formulation and immunogenicity of polyvalent pneumococcal vaccine Example 5-1. Formulation of polyvalent pneumococcal vaccine With reference to the above examples, the following immunogenic compositions were prepared, and the formulations of the polyvalent pneumococcal conjugate vaccine of each composition are shown in the table below.

Example 5-2. Measurement of serotype-specific IgG concentration by ELISA As in the previous example, ELISA was performed using serum obtained from mice, and the results are as follows.

(*+: 1 to 3 times more effective than the control group / ++: 3 to 5 times more effective than the control group)

The results confirmed that PCV19-CRM ₁₉₇ Full dose produced excellent levels of serotype-specific IgG concentrations for all 19 serotypes. In the case of PCV19-CRM ₁₉₇ , serotypes in common with Prevenar 13 show similar or higher serotype-specific IgG concentrations than Prevenar 13, and newly added serotypes 10A, 11A, 15B, 22F. Each of these showed excellent levels of serotype-specific IgG concentrations.

Serotypes common to Prevenar 13 have similar or higher serotype-specific IgG concentrations than Prevenar 13, and the newly added serotypes 10A, 11A, 15B, 22F and 23A, 35B, respectively. It was confirmed that an excellent level of serotype-specific IgG concentration was exhibited (Table 8).

Example 5-3. Antibody function opsonization test (MOPA assay)
Referring to the above example, OPA analysis was performed using serum obtained from mice, and the results are as follows.

(*+: Confirmed an effect 1 to 3 times greater than the control group / ++: Confirmed an effect 3 to 5 times greater than the control group / +++: Confirmed an effect 5 to 7 times greater than the control group)

As a result, all serotypes showed similar or higher levels of functional antibody titers than Prevenar 13 in all groups at Full dose, 1/4 dose, and 1/16 dose of PCV19-CRM ₁₉₇ . It was confirmed.

Confirmed that full dose, 1/4 dose, and 1/16 dose groups of PCV19-CRM ₁₉₇ containing 23A and 35B induce excellent levels of serotype functional antibodies against almost all serotypes. did. Serotypes common to Prevenar 13 have shown similar or higher functional antibodies than Prevenar 13, and the newly added serotypes 10A, 11A, 15B, 22F and 23A, 35B each have superior levels. showed functional antibodies.

Furthermore, the above results show that the PCV19 composition does not show reduced immunogenicity of the serotype against the positive control group PCV13, and it is a final confirmation that the PCV19 composition has a similar or higher level of titer against PCV13. Therefore, it was confirmed again that the PCV19 composition had no immune interference effect (Table 9).

Claims (11)

A multivalent immunogenic composition comprising polysaccharide-protein conjugates, each conjugate comprising a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein. polysaccharide),
The serotypes include 19 serotypes consisting of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F and 35B. including a type, and
characterized in that the capsular polysaccharides derived from Streptococcus pneumoniae of serotypes 4, 15B and 19A are not hydrolyzed ;
Multivalent immunogenic compositions. The carrier protein is selected from the group consisting of diphtheria toxoid, tetanus toxoid, pertussis toxoid, cholera toxoid, an inactivated toxin from Escherichia coli, an inactivated toxin from Pseudomonas aeruginosa, and a bacterial outer membrane protein (OMP). The multivalent immunogenic composition according to claim 1, characterized in that it is any one of: The immunogenic composition according to claim 2, wherein the diphtheria toxoid is one selected from the group consisting of _CRM197 , _CRM173 , _CRM228 , and _CRM45 . The conjugation method for the capsular polysaccharide and the carrier protein is at least one selected from the group consisting of a CDAP conjugation method, a reductive amination method, and a thiol-maleimide method. The immunogenic composition according to claim 1. 2. The multivalent immunogenic composition of claim 1, further comprising an adjuvant. Multivalent immunogenic composition according to claim 5, characterized in that the adjuvant is an aluminum salt. The multivalent immunogenic composition according to claim 6, wherein the aluminum salt is one selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. 2. The multivalent immunogenic composition of claim 1, wherein the multivalent immunogenic composition comprises a dose of polysaccharide from 0.1 to 100 μg. A pharmaceutical composition for inducing an immune response against a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising an immunologically effective amount of a multivalent immunogenic composition according to any one of claims 1 to 8. . A prophylactic or therapeutic agent for pneumococcal diseases, comprising the multivalent immunogenic composition according to any one of claims 1 to 8. Use of the multivalent immunogenic composition according to any one of claims 1 to 8 in the manufacture of a prophylactic or therapeutic agent for pneumococcal diseases.