FIELD OF INVENTION:
A novel immunogenic vaccine composition is provided herein which comprises Streptococcus pneumoniae and one or more additional antigens to simultaneously, concomitantly immunize a subject against more than one pathogen. The present invention thus relates to a novel Streptococcus pneumoniae combination compositions. In particular the present invention relates to novel composition for treating Streptococcus pneumoniae infections and to combination vaccine thereof as a single step immunization in a conjugated and/or unconjugated formulation.
The pneumococcal capsular polysaccharide-protein conjugate referred in this invention comprises a protein carrier conjugated to a capsular polysaccharide, wherein the Streptococcus pneumoniae serotypes are selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9 V, 10A, 10B, IOC, 10F, 11 A, 1 IB, 11C, 1 ID, 1 IF, 12A, 12B, 12F,13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18FJ9A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41F, 42, 43, 44, 45, 46, 47F, 47A, 48; and carrier protein selected from the group of Diphtheria Toxoid, Tetanus toxoid, Cross-reacting material CRM197, Protein D of non-typeable H. influenzae, Exotoxin A of Pseudomonas Aeruginosa (EPA), nontoxic peptide from C. difficile toxin A (CDTA) and Pneumococcal surface protein A (PspA).
The combination described herein also comprises Hepatitis virus antigen. The combination described herein also comprises Salmonella polysaccharide antigen. Furthermore, combination composition of the invention described also provides protection against antigens from one or more additional bacterial antigenic components including tetanus, diphtheria, poliomyelitis, typhoid, O-specific polysaccharide (0:2) of S. Paratyphi A, Haemophilus influenza (Hib), Hepatitis and saccharide antigen from N. meningitidis serogroups in children, adults as well as in diabetic and immunocompromised patients.

A novel immunogenic vaccine composition is provided which comprises (a) Streptococcus pneumoniae serotype(s) components unconjugated or conjugated to carrier proteins (b) hepatitis antigenic components, (c) optionally other antigens, wherein the vaccine components are stable and efficacious and devoid of any antigenic interference.
A novel immunogenic vaccine composition is provided which comprises (a) Streptococcus pneumoniae serotype(s) components unconjugated or conjugated to carrier proteins, (b) Salmonella polysaccharide antigen, (c) optionally other antigens, wherein the vaccine components are stable and efficacious and devoid of any antigenic interference and hindrances amongst each other.
The additional antigens may preferably include but not limited to tetanus, diphtheria, pertussis, poliomyelitis, typhoid, O-specific polysaccharide (0:2) of 5*. Paratyphi A, Haemophilus influenza (Hib) and saccharide antigen from N. meningitidis serogroups. The vaccine composition in the present invention is an ambitious single-step multivalent administration for protection against Streptococcus pneumoniae and other diseases such as hepatitis, tetanus, diphtheria, poliomyelitis, typhoid, paratyphoid, meningococcal disease a and the diseased caused by H. influenzae type b (pneumonia, bacteremia, meningitis, epiglottitis, septic arthritis, cellulitis, otitis media) that children and adults are prone to be exposed to. Vaccine composition is further anticipated to provide protection in immunocompromised and diabetic adults and adolescents. Especially, the vaccine composition provides protection to pregnant women and new born infants and/or provides a family vaccination protection.
Use of such combination vaccines not only helps to decrease the number of injections that is to be administered, but also provide timely optimized vaccination schedule, increases patient compliance and provide family immunization, which concept has not been considered or developed in the art.

Also provided herein are various types of compositions prepared as solutions, suspensions or lyophilized powders for reconstitution, available as a single unit injection/administration containers or as kits, Fixed dose combinations, Prefilled syringe or in single vials with a dual chamber comprising the said immunogenic compositions and its use thereof.
The present invention provides a stable immunogenic vaccine composition for prevention and prophylaxis of infections caused by Streptococcus pneumoniae and hepatitis virus in a single combined vaccine. The present invention provides a stable immunogenic vaccine composition as above which further comprises of antigens selected from tetanus, diphtheria, pertussis, poliomyelitis, typhoid, O-specific polysaccharide (0:2) of S. Paratyphi A, meningococcal disease and diseased caused by H. influenzae type b and others.
The present invention provides a stable immunogenic vaccine composition for prevention and prophylaxis of infections caused by Streptococcus pneumoniae, Hepatitis virus, H. influenzae type b and meningococcal disease in a single combined vaccine. The present invention provides a stable immunogenic vaccine composition as above which further comprises of antigens selected from tetanus, diphtheria, pertussis, hepatitis virus, poliomyelitis and diseases caused by H. influenzae type b and others.
The present invention provides a stable immunogenic vaccine composition for prevention and prophylaxis of infections caused by Streptococcus pneumoniae and Salmonella sp., in a single combined vaccine. The present invention provides a stable immunogenic vaccine composition as above which further comprises of antigens selected from tetanus, diphtheria, pertussis, poliomyelitis, typhoid, O-specific polysaccharide of S. Paratyphi A, meningococcal disease and diseased caused by H. influenzae type b and others.
The process of manufacturing such vaccine compositions of the polyvalent antigens is also disclosed.

Various types of vaccines, their form of administration and modes of presentation are described herein and the present invention relates to the production and use of such combination vaccines for prophylaxis against the infections caused by pneumoniae, hepatitis, tetanus, diphtheria, pertussis, poliomyelitis, typhoid, O-specific polysaccharide of S. paratyphi A, meningococcal disease and diseased caused by H. influenzae type b and others.
Most importantly, the invention provides a combination vaccine, wherein any of the each individual antigen is not impaired/compromised by the presence of other components or antigens, thereby conferring an antibody titer which is similar to or preferably better and superior to the criterion for the seroprotection for each antigenic component.
The present invention thus provides an improved vaccine composition for immunizing infants and adults especially protects women against pneumococcal, hepatitis and other infections as described herewith.
BACKGROUND OF THE INVENTION:
Vaccination is the most effective means of prevention and control of infectious diseases; it is an economical and effective public health intervention, which reduces disease and medical costs. It is estimated that immunization can avoid every year from 2 million to 3 million cases of deaths. Increasing types of infections, and increasing number and types of vaccine(s), asks for newer approaches to reduce the number of vaccination and alleviate the suffering of human beings, which lead to development of combined vaccine(s). A combination vaccine is wherein two or more different components/antigens/vaccines have been combined into a single unit. Preparation of such combination vaccines is not just a simple ad-mixture of different antigenic components; it is far more complex, as interactions between the various antigenic components leads to immunogenic competition and interference, which cause reduced and insufficient immune

response, leading to failure of the vaccine when administered to children and adults (or humans).
Combination vaccines are difficult to produce and often suffer from antigenic interferences leading to reduced efficacy and/or unstable compositions. Rappuoli et al (1996) discusses the unpredictable nature of making combination vaccines (Vaccine, 14(7):691-700). Hence it requires time and controlled practices to obtain suitable combination vaccines. Carefully developed combinations containing Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, and optionally Hepatitis B virus and/or Haemophilus influenzae type b have been discussed in PCT application numbers WO 93/24148, WO 99/48525, EP 0594950
The polysaccharide components of vaccine may. be conjugated to carrier protein(s) such as tetanus toxoid, diphtheria toxoid, CRM197, recombinant diphtheria toxin, Haemophilus influenzae protein D preparation and use of which are well known in art {U.S. Patent No. 4,709,017; PCT application number WO 00/48638; WO 93/25210; WO 95/33481). Methods used to conjugate polysaccharide components of antigenic composition to the carrier proteins are also known in art (for example PCT application number WO 95/08348; U.S. Pat. No. 4,372,945, U.S. Patent No. 4,695,624, U.S. Patent No. 4,830,852, U.S. Patent No. 4,882,317, US Patent No. 4,474,757, J. Am. Chem. Soc, 108, 5282 (1986))
US patent number 5,623,057 describes Capsular polysaccharide from Streptococcus pneumoniae are conjugated with immunogenic carrier protein. US patent number 8,398,983 describes an immunogenic composition combining H. influenzae type B and one or more N. meningitis saccharide to provide a combined protection against the multiple antigens.
Vaccine composition comprising meningococcal conjugate antigens and antigens from TRITANRIX-HepB/Hib has been described in Patent Application No.

WO2006/097851. It describes a pentavalent combination containing Diphtheria, Tetanus, whole cell pertussis, hepatitis B surface antigen, heamophilus influenza, combined with at least one meningococcal conjugated antigen of any serogroups selected from A and C. However there is no embodiment discussing the novel composition of the present invention combining Pneumococcal and Hepatitis antigens.
CN patent application number 106110317 describes a combined tetravalent vaccine against diphtheria, tetanus, pertussis and pneumococcal infections. It combines pneumococcus conjugated vaccine with the DPT vaccine including an acellular pertussis component. However none of the aforementioned prior art describes the novel composition of the present invention.
Australian patent application number 2010/235984 describes a vaccine kit* for concomitant administration of two multivalent immunogenic compositions wherein one of the container comprises acellular pertussis component, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, Inactivated polio vaccine and the second container contains Streptococcus pneumonia. This patent includes an acellular pertussis component and Streptococcus pneumoniae antigens is separately and concomitantly administered and not as a combined dose.
However none of the disclosures in the prior art is able to describe the composition of the present invention as described herewith.
Although childhood immunization programs have been very successful, adult immunization is a neglected and under publicized issue all over the world. Adults too need vaccinations to boost efficacy of childhood vaccines, aid immunity for newer comorbidities and afford protection when immunity is suppressed due to acquired illness. Especially most neglected is immunization of women, especially pregnant women. Providing effective and most essential vaccinations to expecting parents, would not only provide protection against the said disease to the mother but also will get effectively transferred to a just born infant providing protection

during the first few weeks of birth, much before the recommended schedule of immunization programme begins.
With additions of booster doses of Tetanus and diphtheria toxoids, by including protection against Salmonella typhi, polio virus, immunizing with present combination of pneumococcal and hepatitis immunogenic compositions the present composition is useful for adult and family immunization programs, which becomes advantageous for neonates and infants who are still to receive the scheduled immunizations and/or have missed the scheduled immunization.
With additions of Haemophilus influenzae and meningococcal serotypes, immunizing with present combination of pneumococcal and hepatitis immunogenic compositions the present composition is useful for adult, immunocompromised or diabetic patients.
Thus it is an object of the present invention to provide an immunogenic vaccine composition which comprises (a) protein/polysaccharide or polysaccharide-protein conjugates of Streptococcus pneumoniae and (b) hepatitis antigens and other antigens included from amongst tetanus, diphtheria, poliomyelitis, typhoid, S. paratyphi A, meningococcal disease and others for use in children and adults, especially for use in pregnant women.
It is also an object of the present invention to provide an immunogenic vaccine composition which comprises (a) protein/polysaccharide or polysaccharide-protein conjugates of Streptococcus pneumoniae and (b) Salomonella Sp. antigens and other antigens included from amongst tetanus, diphtheria, poliomyelitis, typhoid, O-specific polysaccharide of S.paratyphi A, meningococcal disease and others for use in children and adults, especially for use in pregnant women.
Such a combination vaccine is recommended in healthy adults to geriatric population immunization (beyond new born and pediatrics), including especially

patients at risk of disease conditions as diabetics, respiratory diseases, transplant patients, Chronic kidney diseases (CKD) patients, HIV positive AIDS patients, tuberculosis patients, patients on immunosuppressive therapy, hepatitis, cancer and other life threatening conditions, including patients with risk of respiratory infection in immune-compromised patients, in Chronic Obstructive Pulmonary disease (COPD) patients, in dialysis patients, pulmonary infection in HIV patients, infections in alcoholics, infection after solid organ transplantation, community acquired infections such as common cold, bacterial lower respiratory infections and such others. Prophylactic combination vaccine is advised in conditions as such above.
Such population is on various medicines including but not limited to steroids, immune- suppressants which further lower immune system, exposed to additional risks due to pollution in developing countries, comorbid conditions and consequently response of vaccines to be given may get impaired / compromised in such patients. Hence there is a need of development of vaccine with combination of appropriate antigens addressing various diseases. Such patients cannot wait for vaccination time schedule to fall in place for the respective vaccines to get the immunization done. Hospitalizations and surgical interventions lead to additional risks of acquiring infection. By way of combining two or more antigens, the aforesaid problems associated with multiple injections are overcome or at least mitigated and a stable, highly immunogenic combined formulation is provided, wherein the composition of the invention are particularly suitable for administration to adults.
In the current invention, the advantage is that no substantial decrease in the immunogenicity of the Hepatitis antigens and/or tetanus, diphtheria, poliomyelitis, typhoid, paratyphoid, meningococcal disease would occur in the said combination vaccine formulation, in-spite of inclusion of various numbers of serotypes of pneumococcal antigens.

Accordingly, in a further object of the invention, there is provided a stable and effective combined vaccine composition directed to the prevention of two or more diseases, comprising pneumococcal, HBsAg antigens and such others as described herewith.
ANTIGENS
Pneumococcal (PCV) antigens:
The pathogenic bacteria classified as Streptococcus pneumoniae is a gram positive bacteria (Pneumococci, Pn) have been subdivided into more than 90 antigenic serotypes, based on the capsular polysaccharide (Pn-Ps) of the organism. In order to prevent pneumococcal disease, multivalent pneumococcal polysaccharide vaccines has been in development since 1977, and over a period of time three PCV vaccines have been approved and available globally. These capsular polysaccharide vaccine(s) has been proven to be useful in the prevention of pneumococcal disease in infants, young children, elderly adults and high-risk patients.
Capsular polysaccharide is linked in determining the pneumococcal virulence which also protects inner surface of the bacteria by preventing phagocytosis by host immune cells. However this capsule is poorly immunogenic. With more than 90 serotypes identified, an unconjugated polysaccharide vaccine composition selecting 23 more prevalent serotypes was introduced, as Pnemovax or Pneumovax®23. Distribution of these serotypes is determined by various factors as age, disease syndrome, and its geographic distribution along with time. It was reported that the serotypes from 6 toll accounted for >70% of all invasive pneumococcal disease occurring in children worldwide (Johnson HL et al. Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five: the pneumococcal global serotype project. PLoS Medicine, 2010, 7: pii: el000348).

An unconjugated pneumococcal vaccine which was first introduced in market was a 23 polysaccharides vaccine, marketed under the trade name Pneumovax®23 by Merck. Pneumovax®23 comprises of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, II A, 12F, 14, 15B, I7F, 18C, 19F, 19A, 20, 22F, 23F and 33F.
In 2000, Pfizer's first 7-valent pneumococcal conjugate vaccine (7vPnC, Prevnar ) was approved which contains seven most prevalent serotypes 4, 6B, 9V, 14, 18C, 19F and 23F. In order to increase coverage Pfizer later introduced a 13-valent pneumococcal conjugate vaccine (Prevnar 13®) by adding six additional serotypes 1, 3, 5,7F, 6A, 19A. Glaxo-Smith Kline commercialized a
(H)
pneumococcal vaccine, Synflorix which is a 10-valent conjugate vaccine with serotypes 1,4, 5, 6B,7F,9V, 14, 18C, 19F and 23F (lOvPnCD).
As the pneumococcal polysaccharide vaccine exhibited poor immunogenicity in children below 2 years of age, increased efforts were directed towards conjugated vaccines which are based upon chemical coupling of S. pneumoniae polysaccharide to an immunogenic protein carrier. Such a polysaccharide-protein conjugate increased antibody response as well as induced immune memory. The major advantage of conjugated vaccine in children is that, conjugated vaccines are able to elicit T- cell dependent response and which institutes memory B cell and thus long term protection in the recipient.
In order to confer wider and broader protection, there are many newer conjugated vaccines under development to provide appropriate protection against 5*. pneumoniae serotypes which includes serotypes additional to the above in an attempt to cover all the 23 serotypes known through Pneumovax®23.
Streptococcus pneumonia infection is globally identified as major cause of morbidity and mortality through 2 years to 65 years of ages, but also in co-morbid populations like HIV and hematological malignancy groups (Daniel Curcio et al., International Journal of Infectious Diseases Volume 37, August 2015, Pages 30-35; K.L. O'Brien et al., Lancet, 374 (2009), pp. 893-902; J.J. Drijkoningen, Clin

Microbiol Infect, 20 (2014), pp. 45-51). Significant increase in pneumococcal septicaemia cases during 2007-2017 with 18.4%, when compared to 9.4% of 1993-2008 (K. Thomas et al., J Clin Epidemiol, 66 (2013), pp. 36-43)
A study published on epidemiology of invasive pneumococcal disease (IPD), prevalent serotypes, and pattern of antimicrobial resistance in Indian adults reported that Pneumonia and meningitis were the most common clinical syndromes associated with IPD, accounting for 39% (146/374) and 24.3% (91/374) of IPD cases. 18.4% (69/374) of IPD patients had clinical features suggestive of pneumococcal septicaemia with an unknown infective focus (R. Jayaraman et al., Journal of Microbiology, Immunology and Infection (2008), pp. 1-7).
Hepatitis (Hep) antigens:
Hepatitis terminology is used to indicate the inflammation to the liver. There are viruses identified which are causative agents of hepatitis liver infections. Such strains of Hepatitis virus include A, B, C, D, and E hepatitis viruses. It is said to have caused around 34,000 deaths in 2005 (Thomas H.C., Lok A.S.F., Locarnini S.A., Zuckerman J.A. Viral Hepatitis, Fourth Edition. John Wiley & Sons, Ltd.; Oxford, UK: 2013). The severity of the disease is dependent on age of the infected patient. While the acute infection in the children below 5 years might go asymptomatic in most of the cases, few (1%) also leads to acute liver failures and might require liver transplants. Vaccines as Havrix , Vaqta , Avaxim , and Epaxal are inactivated hepatitis A virus vaccines.
Hepatitis B (Hep B) is a disease caused by hepatitis B virus (HBV) which infects the liver of hominoid, including humans, and causes an inflammation called hepatitis. It ranges in severity from a mild illness, lasting a few weeks (acute), to a serious long-term (chronic) illness that can lead to liver disease or liver cancer. The vaccine against the disease contains one of the viral envelope proteins, hepatitis B surface antigen (HBsAg). The FDA approved Hepatitis B vaccines as Recombivax HB® and Comvax® by Merck, Engerix-B® and Pediarix® by Glaxo SmithKline Biologicals.

Hepatitis B virus (HBV) vaccine is available as a single antigen recombinant vaccine or combination with Hepatitis A vaccine. It is to be administered as a 3-dose series of Hep B to those persons not previously vaccinated.
As on current date, there is no disclosure of a stable and immunogenic
combination vaccine comprising antigens affording protection against
Streptococcus pneumoniae and Hepatitis antigens and other antigens including
tetanus, diphtheria, poliomyelitis, typhoid, meningococcal disease and such other
antigens.
In the present disclosure, the term Hepatitis antigens include inactivated hepatitis
A virus and recombinant hepatitis B surface antigen protein.
Tetanus and Diphtheria antigens:
Tetanus or lockjaw is caused by soil bacterium Clostridium tetania causes muscle spam beginning from jaws to rest of the body. Diphtheria infection is caused by gram positive rod shaped bacteria Cornybacterium diphtheriae. The disease causing toxins by these bacteria are converted to toxoids and utilized in multiple combination vaccines. Multiple combinations for these infections are available such as DTaP for Diphtheria, Tetanus, acellular pertussis, DTwP for Diphtheria, pertussis, and whole pertussis. DT and Td contain only diphtheria and tetanus vaccines.
Tdap is an inactive tetanus, diphtheria, acellular pertussis vaccine available as Adacel® and Boostrix®; for adults between 18 and 64 years, who have completed their primary vaccination schedule, a booster dose of Td vaccine is indicated once every 10 years till the age of 65. Tdap vaccine can substitute any one of the Td doses. It is also present in tetravalent combination with Hepatitis B or Heamophilus influenza.
Poliomyelitis Antigens

Polio or poliomyelitis is another infectious disease, notoriously affecting children for years not. It is caused by three serotypes of viruses belonging to family enteroviruses. The three strains are termed as poliovirus type 1 (PV1), poliovirus type 2 (PV2), poliovirus type 3 (PV3) vary from each other in terms of Capsid protein. All the three strains of polio virus are virulent. It varies from minor illness to affecting Central Nervous system. It may also be paralytic.
• A live attenuated (weakened) oral polio vaccine (OPV) developed by Dr. Albert Sabin in 1961. OPV, comprising the Sabin strains, is given orally.
• An inactivated (killed) polio vaccine (IPV) developed in 1955 by Dr. JonasSalk.
IPV, comprising the Salk strains, is given as an injection. The first polio vaccine was an inactivated polio vaccine developed by Jonas Salk. After which came, oral polio vaccine developed by Albert Sabin. Oral Polio Vaccine or OPV is live attenuated sabin strain vaccine orally administered. Trivalent OPV is active against all 3 serotypes while bivalent is active against Type 1 and type 3. Inactivated polio vaccine based on Salk strain, commonly referred to as IPV, is formulation with all 3 types of polio virus which are grown in monkey kidney tissue cultures termed as Vero cell lines and then inactivated by formalin. Mahoney type 1, MEF Type 2 and the Saukett type 3 are the Salk strains that have been used in the vaccine against the poliomyelitis disease. The Sabin strains include the Sabin 1 and Sabin 2 strains.
Typhoid antigens:
Salmonella enterica subsp. enterica, serovar Typhi is a gram negative bacteria and causative agent of systemic infection or typhoid fever. It affects the reticuloendothelial system, intestinal lymphoid tissue and gall bladder. Typhoid vaccine includes an injectable typhoid conjugate vaccine with Vi polysaccharide antigen linked to Tetanus toxoid. It may be given to children from 2 weeks to adults up to 45 years. Then there is also unconjugated polysaccharide typhoid vaccine with purified Vi antigen recommended for ages 2 years and above. There is also an oral attenuated Ty21a vaccine in a capsule formulation for 6 years and

above. Typherix™ is a Vi polysaccharide typhoid vaccine. Combination vaccines prepared with the Vi polysaccharide typhoid vaccine have often been found being unstable as a result of the presence of the phenol. Typhim Vi® for adults and children is a single injection of 0.5 mL. A reimmunizing dose is 0.5 mL Re-immunization consisting of a single dose for US travelers every two years under conditions of repeated or continued exposure to the S. typhi organism is recommended at this time. Another vaccine Vivotif is given in adults and children greater than 6 years of age.
Confounding conditions where Pneumococcal infections accompanied with typhoid fevers have often been reported. Typhoid fever is a concerning systemic communicable infection, the causative agent as mentioned above, has more than 2600 Salmonella enterica serotypes. The serotypes are distinguished on the basis of presence of Lipopolysaccharide O antigen and flagellar H antigen. Salmonella enterica serovar Typhi and S. Paratyphi are grouped as Typhoidal Salmonella, manifesting in only humans and causing what is commonly termed as Typhoid fever and paratyphi fevers. The other group consists of Non-typhoidal Salmonella (NTS), including S. Enteritidis, S. Typhimurium, responsible for causing gastroenteritis, which manifest in humans as well as in animals.
Haemophilus influenza e (Hib) antigen:
This is an antigen that can be used for vaccine purposes in man in order to protect infections caused by Haemophilus influenzae type b. This antigen is formed by a capsular polysaccharide of the bacteria, polyribosylribitol phosphate (or PRP), which is made T-dependent due to coupling to a carrier protein, tetanus toxoid. To solve the problem of stability of the PRP-T, it was used lyophilized in the prior art. This solution allows antigen to retain its immunogenic character over time.
Neisseria meningitides antigens:
The vaccines of the invention may further comprise a capsular saccharide of a bacterium selected from the group consisting of N. meningitidis type A (MenA, optionally conjugated to a carrier protein), N. meningitidis type C (MenC,

optionally conjugated to a carrier protein), N. meningitidis type W- 135 (MenW,
optionally conjugated to a carrier protein), and N. meningitidis type Y (MenY,
optionally conjugated to a carrier protein).
Paratyphoid Antigens:
Salmonella enterica serovars Typhi and Paratyphi (S. Paratyphi) A and B cause enteric fever in humans. Of the paratyphoid group, S. Paratyphi A is the most common serovar. In 2000, there were an estimated 5.4 million cases of S. Paratyphi A worldwide. More recently paratyphoid fever has accounted for an increasing fraction of all cases of enteric fever. Although vaccines for typhoid fever have been developed and in use for decades, vaccines for paratyphoid fever have not yet been licensed. Several S. Paratyphi A vaccines, however, are in development and based on either whole cell live-attenuated strains or repeating units of the lipopolysaccharide O-antigen (0:2) conjugated to different protein carriers.
While vaccination seems to be a strategically relevant route, but due to long-term and persistent nature of these infections, constant manifestations and spontaneous outbreaks are often reported. For which there is still need for coherent, concentrated efforts, as outlined in the present invention platform. Additionally, there is an implicit necessity to target multiple age and condition cohorts at the time of vaccine introduction in order to accelerate the impact of vaccine use. This strategy may also increase indirect herd protection of unvaccinated individuals.
It is therefore an object of the present invention to strengthen the routine immunization strategy and effective coverage of affected population.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the manufacture of the most ambitious polyvalent immunogenic combination vaccine to date, the administration of which can prevent or treat infection by Streptococcus pneumoniae and Hepatitis virus and

other bacteria and virus including tetanus, diphtheria, pertussis, haemophilus, poliomyelitis, typhoid and others, wherein the components of the vaccine do not significantly interfere with the immunological performance of any one component of the vaccine.
Accordingly, in a one aspect of the invention there is provided a polyvalent immunogenic combination vaccine for conferring protection in a host against disease caused by Streptococcus pneumoniae and Hepatitis virus.
In a second aspect of the invention there is provided advantageous kits comprising two or more multi-valent immunogenic compositions, said kits being capable of conferring protection in a host against disease caused by Streptococcus pneumoniae and hepatitis.
Accordingly, in a one aspect of the invention there is provided a polyvalent immunogenic combination vaccine for conferring protection in a host against disease caused by Streptococcus pneumoniae and Salmonella typhi.
In a second aspect of the invention there is provided advantageous kits comprising two or more multi-valent immunogenic compositions, said kits being capable of conferring protection in a host against disease caused by Streptococcus pneumoniae and Salmonella typhi.
A stable and effective combination vaccine offers patients the advantage of receiving a reduced number of injections, which leads to the clinical advantage of increased compliance. Two pathogens of particular concern which has been combined in the present invention are Streptococcus pneumoniae antigenic component (Pneumococci, Pn) and hepatitis virus antigenic component (especially 'HepA or HepB1, viral hepatitis). The pathogens of particular concern which has been combined in the present invention are Streptococcus pneumoniae antigenic component (Pneumococci, Pn) and Salmonella typhi. Other relevant and important

pathogens which can be further added in the combination include tetanus, diphtheria, pertussis, poliomyelitis, meningococcal, Haemophilus influenzae, S. Paratyphi A and others. The earlier research has been focused on the development of the vaccine which gives individual effect directed towards different diseases and infections. Such a combination as described herein has not been mentioned or disclosed in the art.
It is an object of the present invention to provide an improved, better and superior
combination vaccine for protection against Streptococcus pneumoniae and
hepatitis virus.
It is an object of the present invention to provide an improved, better and superior
combination vaccine for protection against Streptococcus pneumoniae and
Salmonella sp. virus.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae, hepatitis
virus and tetanus.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae,
Salmonella typhi and tetanus.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae, hepatitis
virus, tetanus and diphtheria.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae,
Salmonella typhi, tetanus and diphtheria.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae, hepatitis
virus, tetanus, diphtheria and poliomyelitis.
It is an object of the present invention to provide an improved and stable
combination vaccine for protection against Streptococcus pneumoniae, hepatitis

virus, tetanus, diphtheria, poliomyelitis, Haemophilus influenzae and disease caused by meningococcal bacteria typhoid and paratyphoid.
It is an object of the present invention to provide an improved and stable combination vaccine for protection against streptococcus pneumoniae, Salmonella typhi, tetanus, diphtheria, and poliomyelitis.
It is an object of the present invention to provide an improved and stable combination vaccine for protection against Streptococcus pneumoniae, Salmonella typhi, tetanus, diphtheria, poliomyelitis, Haemophilus influenza, disease caused by meningococcal bacteria, paratyphoid and Hepatitis.
It is a specific object of the present invention to provide an improved, better and superior combination vaccine comprising antigens for a polyvalent pneumococcal-hepatitis and which further comprises TT, diphtheria, IPV, typhoid, paratyphoid antigens, Haemophilus influenza (Hib), disease caused by meningococcal bacteria and others.
It is a specific object of the present invention to provide an improved, better and superior combination vaccine comprising antigens for a polyvalent pneumococcal- Salmonella typhi cell surface Vi polysaccharide and which further comprises TT, diphtheria, IPV, Hepatitis, Haemophilus influenza (Hib), disease caused by meningococcal bacteria, paratyphoid antigens and others.
Thus, as per the first one aspect, in an embodiment of the present invention is provided a combination vaccine comprising one or more pneumococcal capsular saccharide, hepatitis antigen, adjuvant and/or a carrier. The pneumococcal polysaccharide is such that it is preferably conjugated to a carrier protein. As per the one aspect, in an embodiment of the present invention is provided a combination vaccine comprising one or more pneumococcal capsular saccharide, Salmonella typhi cell surface Vi polysaccharide, adjuvant and/or a carrier. The

pneumococcal polysaccharide is such that it is preferably conjugated to a carrier protein.
Along with the antigenic components the vaccine may comprise a number of supplementary non-antigenic components that are pharmaceutical^ acceptable excipients. These include but are not restricted to pH modifiers, buffers, adjuvants, preservative, surfactants, carrier and tonicity modifying agents. It is also an object of the present invention, to include indirect protective effects, whether herd or community immunity strategies and ease of reiteration of vaccination for a detail oriented scheduled as well as for outbreak controls.
Along with the antigenic components the vaccine may comprise a number of non-antigenic components that are pharmaceutically acceptable excipients. These include but are not restricted to pH modifiers, buffers, adjuvants, preservative, surfactants, carrier and tonicity modifying agents.
Adjuvants
The pneumococcal adjuvant may or may not be adsorbed on an adjuvant. These adjuvants serve multiple purposes as bringing the antigen in contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration); decrease the toxicity of certain antigens; and also provides solubility to some vaccines components. There are many adjuvants in the art, aluminum salts, calcium salts. Iron salts, zinc salts, acylated sugars, derivatised polysaccharides and so. The antigen in an immunogenic composition may be adsorbed on the salt (EPO 576478BI, EPO 689 454BI and EPO 633 784BI). Such examples are also presented in US7357936B1, US20090214592A1 , US6635261B2, US6306404B1, ES2228497T3, KR101321732B1, US20110180430A1, CA2628206A1, ES2377884T3 , EP2368572A3, WO 199400015 3A1, WO2004110480A2, Ewasyshyn, et al., Vaccine, 10, Issue 6, pp. 412-420 (1992). Ewasyshyn, et ai, Vaccine , 10, Issue 6, pp. 412 420 (1992). Other examples also include adjuvants that promote a Thl

responses. For example 3-de-O-acylated monophosphoryl lipid A with aluminium phosphate, monophosphoryl lipid A and/or in combination with saponin derivative (WO 94/00153; WO 96/33739), QS2I, 3D-MPL and tocopherol (WO 95/17210), oil in water emulsion and tocopherol (WO 95/17210), CpG containing oligonucleotides (WO 96/02555).
Preservatives
Preservatives avoid the potentially life threatening contamination with harmful microbes, that may be introduced in a vaccine incorporated during the event of accidental contamination. Examples of such preservatives include but are not limited to Benzethonium chloride (Phemerol), thiomersal, Phenol and 2-phenoxyethanol (2-POE).
Thimerosal is a mercury-containing organic compound (an organomercurial) that has been used in many vaccines as a preservative. There are reports pertaining to certain allergic reactions to thiomersal primarily in the form of delayed-type local hypersensitivity reactions, including redness and swelling at the injection site. There are also conflicting reports on linking autism to mercury. 2-phenoxyethanol (2-POE) is also known as i-hydroxy-2-phenoxyethane', '2-hydroxyethyl phenyl ether', 'ethyleneglycol phenyl ether', etc. The safety profile of 2-phenoxyethanol is better than that of mercurial preservatives (e.g. thiomersal). Thus, there is a need of avoiding thiomersal and using 2- phenoxyethanol in the vaccines.
pH Modifiers and/or Buffers
Various pH modifiers known to person skilled in the art may be used to adjust the pH of the vaccine composition as desired, such as sodium hydroxide or hydrochloric acid. Various buffers such as sodium phosphate, potassium phosphate and citrate buffers may be used in the formulation of the vaccine. Suitable diluents are used as PBS or saline. Surfactants like non-ionic surfactants selected from a group comprising polysorbate, Cremophore, poloxamer, etc. may

also be added. Trace constituents includes polysorbate-80, gelatin and remnants from chemical toxoidation (e.g., if PT is chemically toxoided) such as glutaraldehyde, formaldehyde.
In an embodiment the present invention provides a combination vaccine which contains one or more pneumococcal capsular saccharide serotypes selected from: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 9A, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F, 39 and/or 45. Isolates of these serotypes are available in ATCC.
Furthermore, the novel combination of the present invention includes one or more of pneumococcal capsular saccharide serotypes from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, IOC, 10F, 11 A, 11B, 11C, 11D, 1 IF, 12A, 12B, 12F,13, 14, I5A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18FJ9A, 19B, 19C, 19F,20 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F,29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41F, 42, 43, 44, 45, 46, 47F, 47A, 48.
These serotypes are obtained at the end of the growth cycle the cells from lysates in a broth which is then sent for downstream harvesting (PCT application number WO 2006/110381, U.S. Patent App. Pub. Nos. 2006/0228380, 2008/0102498 and 2008/0286838. The routine process for growing these capsular polysaccharides includes growing each S. pneumoniae serotypes in a medium (e.g., in a soy-based medium), the polysaccharides are then prepared from the bacteria culture. This can also be found in aforementioned these prior arts. Such methods of culturing pneumococci are known in art (Chase, M. W., Methods of Immunology and Immunochemistry 1, 52 (1967)).
Preparation of serotype saccharides can be directly from bacteria using isolation procedures known to one of ordinary skill in the art and as also disclosed in U.S.

Patent App. Pub. Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340, and 2008/0102498 and WO 2008/118752). In addition, they can be produced using synthetic protocols. The pneumococcal polysaccharide is bound covalently to a carrier protein selected from tetanus toxoid (TT), diphtheria toxoid (DT), cross-reactive mutant diphtheria toxin material 197 (CRM197), pneumococcal surface protein A (rPspA), group B meningococcal outer membrane protein (MenB-OMP), Haemophilus influenzae protein D (HiD), Pseudomonas exotoxin A (PEA), E. coli heat-sensitive toxin, pneumolysin, pertussis toxin, Pseudomonas aeruginosa pili, fimbriae or P-3-2 Neisseria gonorrhoeae HBsAg. The polysaccharides are typically purified through centrifugation, precipitation, ultra-filtration, and/or column chromatography (see for example WO 2006/110352 and WO 2008/118752).
The preparation of Hepatitis antigen is documented in prior publications as EPA0226846, EP 299108, Harford et al in Develop. Biol. Standard 54, page 125 (1983), Gregg et al in Biotechnology, 5, page 479 (1987). The term "hepatitis antigen" refers to any antigenic material derived from hepatitis antigen to induce an immunogenic reaction in the host administered with the present novel composition.
In the following preferred embodiment, immunologically effective compositions include one or more hepatitis antigenic material.
In a preferred embodiment the present invention provides a method for preparing the above-described combination vaccine.
Typically, the vaccines are administered by injection, either intravenously or intraperitoneally.
In preferred embodiments, formulations and combinations possible, but are not limited to, include -

Formulation Type: Fully liquid vaccine of PCV and HepB in a single vial;
Formulation Type: Kit: Lyophilized PCV and Liquid HepB (Two vials in a kit);
Formulation Type: Kit: Lyophilized HepB and Liquid PCV (Two vials in a kit);
Formulation Type: Fully Lyophilized HepB and PCV in a single vial;
Formulation Type: Fully liquid vaccine of PCV, HepB and Typhoid Vi
Polysaccharide in a single vial;
Formulation Type: Fully liquid vaccine of PCV and Typhoid Vi Polysaccharide in
a single vial;
Formulation Type: Fully liquid vaccine of PCV, HepB, Tetanus toxoid, Diphtheria toxoid, Typhoid antigenic component(s); Poliomyelitis antigenic component(s);
Formulation Type: Kit lyophilized PCV, Typhoid Vi Polysaccharide;
Formulation Type: Fully liquid vaccine of PCV, Typhoid Vi Polysaccharide, N.
meningitis antigens, Hib antigen;
Formulation Type: Fully liquid vaccine of PCV, Typhoid Vi Polysaccharide,
HepB, N. meningitis, Hib antigen, Hib, N. meningitis antigens, Tetanus toxoid,
Diphtheria toxoid;
Formulation Type: Fully liquid vaccine of PCV, Paratyphoid Polysaccharide
conjugates and inactivated Hepatitis A in a single vial
Formulation Type: Fully Lyophilized PCV, Paratyphoid Polysaccharide conjugate
and inactivated Hepatitis A in a single vial
Vaccines of the invention can be prepared in liquid form (preferably where all antigens are in aqueous solution or suspension) during manufacture and are presented in one pack, vial or syringe or any suitable mode of delivery, as a single unit combination vaccine.
In another embodiment, the vaccines can be prepared extemporaneously at the time of use by mixing together two components (a) a first component comprising pneumococcal (PCV) antigens and (b) a second component comprising hepatitis antigens (HBsAg) or Hep A. The person administering an extemporaneous

combination vaccine mixes the different vaccine components either in the same syringe (e.g. the by-pass syringe) or in the same vial, just before injection.
In another embodiment, the vaccines can be prepared extemporaneously at the time of use by mixing together two components (a) a first component comprising pneumococcal (PCV) antigens and (b) a second component comprising Typhoid Vi Polysaccharide from salmonella typhi. The person administering an extemporaneous combination vaccine mixes the different vaccine components either in the same syringe (e.g. the by-pass syringe) or in the same vial, just before injection.
The two components are preferably in separate containers (e.g vials and/or syringe) and the invention provides a kit comprising components (a) and (b) described above. The contents of the first container are preferably lyophilized such that vaccine of the invention can be prepared by reconstituting the lyophilized component with the aqueous component.
In an embodiment the two components to be mixed can be either as a lyophilized form or a liquid form, provided as two vials in a kit form.
Thus in contrast to an association of different vaccines, in certain embodiments, a combination vaccine is a single product, preferably prepared as a single unit dosage form by mixing the components into a single unit vaccine. The two components in the single unit container can be either in fully lyophilized form or fully liquid form.

We claim:
1. An immunogenic vaccine composition comprising:
(a) Streptococcus pneumoniae polysaccharides from different S.
pneumoniae serotypes
(b) Salmonella typhi Vi polysaccharide antigen and/or
(c) Hepatitis antigen
(d) One or more antigens selected from tetanus, diphtheria, pertussis,
inactivated poliomyelitis, O-specific polysaccharide (0:2) of S. Paratyphi
A, Haemophilus influenza (Hib) and saccharide antigen from N.
meningitidis serogroups; Wherein the vaccine components are stable and
do not interfere with each other.
2. An immunogenic vaccine composition according to the claim 1; comprises up to 30 capsular saccharides from different S. pneumoniae serotypes.
3. An immunogenic vaccine composition according to the claim 1; wherein up to 30 capsular saccharides selected from S. pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 8, 9B, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 24B, 33F, 34, 35F, 45.
4. An immunogenic vaccine composition according to the claim 1; wherein capsular saccharides from different S. pneumoniae serotypes are conjugated or non-conjugated.
5. The immunogenic composition of claims 1; wherein the Streptococcus pneumoniae polysaccharides are conjugated to a carrier protein selected from the group comprising diphtheria toxoid, tetanus toxoid, recombinant diphtheria toxin, OMPC from N. meningitidis, pneumolysin from S. pneumoniae and protein D from H. influenzae.

6. The immunogenic composition as claimed in claims 1 and 2, wherein the amount of polysaccharide from each of the serotypes is lug to 25p.g.
7. The immunogenic vaccine composition of claims I and 2 is either a fully liquid composition in a single vial or a kit comprising (a) a first component in a lyophilized form (b) a second component in a liquid form, in separate containers.
8. An immunogenic vaccine composition according to the claim 1; comprises Following compositions
Table 1:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 |ig
to 30 valent
Purified Hepatitis B surface antigen protein (HBsAg) 10-40 ng
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s. to 0.5 ml
Table 2:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 (ig
to 30 valent
Purified Hepatitis B surface antigen protein (HBsAg) 10-40 \ig
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Lactose 25 mg
Sucrose 20 mg
Mannitol 20 mg
Buffer Saline q.s. to 0.5 ml

Table 3:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 |ag
to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi NLT 5 (ig
conjugate
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s. to 0.5 ml
Table 4:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 \xg
to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi NLT 5 \ig
conjugate
Purified Hepatitis B surface antigen protein (HBsAg) NLT 10 (ig
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s. to 0.5 ml
Table 5:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 jag
to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi NLT 5 \xg
conjugate
Purified Hepatitis B surface antigen protein (HBsAg) NLT 10 |ig
Purified Diphtheria Toxoid 3-5 Lf
Purified Tetanus Toxoid NLT 5 Lf

Inactivated Polio Virus (Salk strain)
Type 1 2.5-40DU
Type 2 0.8-8 DU
Type 3 3.2-32DU
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s.to0.5 ml
Table 6:
Each dose of 0.5 ml contains
Streptococcus pneumoniae polysaccharide conjugates up I to 5 (ig
to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi NLT 5 ng
conjugate
Lactose 25 mg
Sucrose 35 mg
Mannitol 20 mg
Buffer Saline q.s. to 0.5 ml
Table 7:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up . 1 to 5 |ig
to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi NLT 5 jag
conjugate
N. meningitidis serogroup A, C, Y, and W conjugate NLT 5 (ig
Haemophilus influenza type b Conjugate NLT 10 ^g
Aluminum content (As Aluminum phosphate) NMT 1.25 mg

Buffer Saline

q.s. to 0.5 ml

Table 8:
Each dose of 0.5 ml contains
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 jag to 30 valent
Purified Vi Capsular Polysaccharide of S. typhi conjugate NLT 5 (ig

Purified Hepatitis B surface antigen protein (HBsAg) Haemophilus influenza type b Conjugate N. meningitidis serogroup A, C, Y, and W conjugate Aluminum content (As Aluminum phosphate)
Buffer Saline

10-40ng NLT 10 ^ig
NLT 5 ^g NMT 1.25 mg q.s. to 0.5 ml

Table 9:
Each dose of 0.5 ml contains:
10-40 ug NLT 5 ^g 7-lO^g 1.25 mg q.s. to 0.5 ml
Streptococcus pneumoniae polysaccharide up to 30 valent 25 (ig (non-conjugated)
Purified Hepatitis B surface antigen protein (HBsAg) N. meningitidis serogroup A, C, Y, and W conjugate Haemophilus influenza type b Conjugate Aluminum content (As Aluminum phosphate)
Buffer Saline

Table 10:
Each dose of 0.5 ml contains
Purified Polysaccharide of S. Pneumoniae serotypes up to 25 ug each

30 valent (non-conjugated)

Purified Hepatitis B surface antigen protein (HBsAg) Purified Diphtheria Toxoid
Purified Tetanus Toxoid
Inactivated Polio Virus (Salk strain)
Type 1
Type 2
Type 3 Typhoid Polysaccharide Conjugate Haemophilus influenza type b Conjugate Aluminum content (As Aluminum phosphate)
Buffer Saline

NLT 10 ng
3 -5 Lf NLT 5 Lf
4 - 40 DU
0.8 - 8 DU
3.2-32DU
25 Mg
10 \xg
NMT 1.25 mg
q.s. to 0.5 ml

Table 11:
Each dose of 0.5 ml contains
Purified Polysaccharide of S. Pneumoniae serotypes up to 25 jig each 30 valent (non-conjugated)
Purified Hepatitis B surface antigen protein (HBsAg) NLT 10 jig

Purified Diphtheria Toxoid Purified Tetanus Toxoid Inactivated Polio Virus (Sabin strain)
Type 1
Type 2
Type 3 Typhoid Polysaccharide conjugate

3-5 Lf NLT 5 Lf
NLT 2.5 DU NLT 4 DU NLT 5 DU
25 ^g

N. meningitidis serogroup A, C, Y, and W conjugate NLT 5 p.g
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s. to 0.5 ml
Table 12:
Each dose of 0.5 ml contains
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 |ig
to 30 valent (Each)
Purified Paratyphoid Polysaccharide conjugate 2.5-5 ^g
Hepatitis A inactivated 100-500 U
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Buffer Saline q.s. to 0.5 ml
Table 13:
Each dose of 0.5 ml contains:
Streptococcus pneumoniae polysaccharide conjugates up 1 to 5 |ig
to 30 valent (Each)
Purified Paratyphoid Polysaccharide conjugate 2.5 -5 |ig

Hepatitis A inactivated 100-500 U
Aluminum content (As Aluminum phosphate) NMT 1.25 mg
Lactose 25 mg
Sucrose 20 mg
Mannitol 20 mg
Buffer Saline q.s. to 0.5 ml