FORM 2 THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
PROCESS FOR PRODUCTION OF STRESS PROTEIN AND IMMUNOGENIC COMPOSITION COMPRISING THE SAME
SERUM INSTITUTE OF INDIA PRIVATE LIMITED
An Indian Company
Off 212/2, Soli Poonawalla Road,
Hadapsar, Pune-411028,
Maharashtra, India
THE FOLLOWING SPECIFICATION DESCRIBES THE INVENTION.

FIELD
The present disclosure relates to the field of stress protein manufacturing and use of stress protein in immunogenic compositions.
BACKGROUND
The 23-valent polysaccharide vaccines (Pneumovax 23 (Merck) and Pnu-Imune 23 (Wyeth-Ayerst)), which comprise Capsular Polysaccharides (CPS) as an effective antigen, are commercially available to prevent pneumococcal infections. However, these vaccines were found to be poorly immunogenic when given to infants and young children, due to low antibody production rate and in that they fail to induce immunologic memory. Prevnar-13 conjugate vaccine (Pfizer), which is prepared by conjugating 13 serotypes of Capsular Polysaccharides (CPS) to a carrier protein, has been developed to solve the disadvantages of 23-valent polysaccharide based vaccine as mentioned above. However, this vaccine is very expensive and shows protective effect against only 13, out of 95 or more serotypes of the S. pneumoniae. Therefore, there have been attempts to develop a protein based vaccine possessing high antigenicity and conserved among all serotypes of S. pneumoniae.
The major problem associated with use of protein based vaccines for prevention of pneumococcal infections is low antigenicity or lack of protective effects against all serotypes of the S. pneumoniae. Hence, there remains a strong and urgent need to develop protein based vaccines which have high antigenicity and are conservatively present across all types of the S. pneumoniae.
Stress proteins are a diverse group of proteins that are synthesized at increased levels by cells exposed to a variety of stressful stimuli and which have a protective effect against the stress. These stress proteins have the ability to modulate the cellular immune response. The term stress proteins include the heat shock proteins (HSP, hsp, hsps, HSPs, Hsps), the glucose-regulated proteins (GRPs) ubiquitin and the like. Heat shock proteins are a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. HSPs can be classified into Hsp100, Hsp90, Hsp70, Hsp60, and Small Hsp (20-30kDa) families depending on molecular weight. Hsps are ubiquitously expressed in both prokaryotic and eukaryotic cells subjected to heat stress, where they function as chaperones in the folding and unfolding of polypeptides and are also involved in proteolysis thereby removing

damaged and denatured proteins. Thus, HSPs protect both prokaryotic and eukaryotic cells against adverse effects such as heat stress (elevated temperatures).
There have been various approaches involved in producing vaccine compositions comprising of HSPs for the treatment and prevention of infectious diseases. The use of mammalian HSPs complexes as vaccines against intracellular pathogens has been disclosed in WO 95/24923.WO 97/10000 and WO 97/10001 disclose use of a mixture of HSPs isolated from cancer cells or virally infected cells capable of eliciting protective immune response or cytotoxic T lymphocytes to the cognate tumour or viral antigen. This necessitates the production and purification of HSPs on a large scale. The stimulation of cells by heat stress (elevated temperatures) or other stresses produces a general increase in the level of HSPs.
However, the difficulties associated with large scale production of immunogenic stress protein (HSPs) of interest relative to others, severely limits the use of HSPs as immunogenic/vaccine candidates and remains to be identified. The potential difficulties include high cost of production in case of cells engineered for expression of HSPs. Further, transient expression (expressed for a short time) of HSPs by the cells have been observed limiting its production.
Further previous methods of purifying HSPs include purifying the heat shock proteins without the associated peptides. Other methods that purify HSPs together with their associated peptides are complicated, expensive and are not employable on industrial scale.
Hence, there remains an ever urgent need to develop a process of isolation and purification of immunogenic HSPs complex to be used as immunogenic/vaccine candidates.

OBJECTS
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a process of manufacturing an immunogenic stress protein (such as HSPs) or a stress protein-peptide complex.
Another object of the present disclosure is to provide a process for manufacturing such immunogenic composition comprising stress protein (such as HSPs) or a stress protein-peptide complex against pathogenic compositions.
Yet another object of the present disclosure is to provide an immunogenic composition against pathogenic microorganisms comprising a purified stress protein (such as HSPs) or a stress protein-peptide complex used alone or in combination with other proteins, polysaccharide, monovalent/multivalent polysaccharide-protein conjugate, as part of monovalent/multivalent polysaccharide-HSP protein conjugate, or with whole cell bacteria.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY
The present disclosure provides a process for the production and purification of stress protein (such as HSPs) or a stress protein-peptide complex isolated from a microbial pathogen, previously subjected to a one or more stress inducing stimuli. The stress inducing stimuli is either heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, Hydrostatic pressure, Ethanol shock, chemical stress, UV-stress, cold stress or combination of stress thereof. The process is employable on an industrial scale. The present disclosure further provides an immunogenic composition comprising the stress protein (such as HSPs) or a stress protein-peptide complex used alone or in combination with other proteins, polysaccharide, Monovalent/multivalent polysaccharide-protein conjugate, as part of Monovalent/multivalent polysaccharide-HSP protein conjugate, or with whole cell bacteria.
DESCRIPTION
Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the present disclosure can be considered as an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and processes, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise.

The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure. The present disclosure provides an immunogenic composition and a process for preparing the same.
The present disclosure envisages a process for the production and purification of stress protein (such as HSPs) or a stress protein-peptide complex isolated from a microbial pathogen and immunogenic composition comprising the same used alone or in combination with polysaccharide or polysaccharide-protein conjugate.
The term "stress protein" as used herein, is a protein whose intracellular concentration increases within a cell; when a cell is exposed to stressful stimuli and which have a protective effect against the stress. Stressful stimuli include, but are not limited to, heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, hydrostatic pressure, ethanol shock, chemical stress, UV-stress, cold stress thereof. It is a protein which is capable of binding to other proteins or peptides, and is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH. In the case of diseased cells, stress protein function as chaperonins that guides the viral or tumour-associated peptides to the cell-surface, see Li Z, Srivastava PK. et. al; Behring Inst Mitt. 1994 Jul;(94):37-47 and Przepiorka D, Srivastava PK. Mol Med Today. 1998 Nov; 4(11):478-84. The chaperone function is accomplished through the formation of complexes between stress proteins and the antigenic peptide fragments and between stress proteins and viral or tumour-associated peptide fragments in an ATP-dependent reaction. Stress proteins bind or complex with a wide spectrum of peptide fragments in an ATP dependent manner. The peptides in such complexes appear to be a

random mix of peptide fragments. The complex formation of stress proteins with various peptide fragments has been observed in normal tissues as well; see Przepiorka D, Srivastava PK. Mol Med Today. 1998 Nov; 4(11):478-84.
The term "peptide", as used herein, is understood to mean any amino acid sequence that is present in a cell infected with an intracellular pathogen but which is not present in a similar cell when the cell is not infected with the same pathogen. The definition embraces peptides that not only originate from the intracellular pathogen itself but also peptides which are synthesized by the infected cell in response to infection by the intracellular pathogen. The peptide is an antigenic peptide or an antigenic peptide fragment or a tumour specific antigen.
As used herein, the term "intracellular pathogen" is understood to mean any viable organism, including, but not limited to, viruses, bacteria, fungi, protozoa and intracellular parasites, capable of existing within a mammalian cell and causing a disease in the mammal.
The term "stress protein-peptide complex", as used herein, is any complex containing a stress protein and a peptide that is capable of inducing an immune response in a mammal. The peptides preferably are non-covalently associated with the stress protein.
According to a first embodiment of the present disclosure, a process for the production of stress protein (such as HSPs) or a stress protein-peptide complex may comprise of subjecting the microbial pathogen to a one or more stress inducing stimuli resulting in the expression of stress protein (such as HSPs) or a stress protein-peptide complex by the microbe that may be employed on an industrial scale. As used herein, the term "stress inducing stimuli" means a stimuli which is capable of inducing a stress response within a cell or cells subjected to the stimulus.
Yet preferred aspect of first embodiment, wherein the process for production of stress protein or a stress protein-peptide complex may comprise the steps of:
a) culturing microbial pathogen in an optimized media at optimum pH, osmolarity and temperature,
b) subjecting the said microbial pathogen to a stress inducing stimuli, selected from the group comprising of heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, hydrostatic pressure, pH stress, ethanol shock, chemical stress, UV-stress, cold stress and a combination thereof, and

c) recovering a preparation comprising stress proteins from the stress induced microbial pathogen. According to a first aspect of the first embodiment, the microbial pathogen may be subjected to single stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, pH stress, metabolite restriction, nutrient starvation, media optimization, hydrostatic pressure, ethanol shock, chemical stress, UV-stress, cold stress thereof.
Yet preferred aspect of first embodiment, wherein the microbial pathogen may be subjected to single stress inducing stimuli comprising heat stress.
Yet preferred aspect of first embodiment, wherein the microbial pathogen may be subjected to single stress inducing stimuli comprising osmotic stress.
Yet preferred aspect of first embodiment, wherein the microbial pathogen may be subjected to single stress inducing stimuli comprising pH stress.
Yet alternatively the microbial pathogen may be subjected to multiple stress inducing stimuli which refers to two, three or more stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, hydrostatic pressure, ethanol shock, chemical stress, UV-stress, cold stress thereof.
Yet alternatively the microbial pathogen may be subjected to two or more stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, Hydrostatic pressure, Ethanol shock, chemical stress, UV-stress, cold stress thereof.
Yet alternatively the microbial pathogen may be subjected to two different types of stress inducing stimuli selected from the group comprising of:
a) first stress inducing stimuli is heat stress and second stress inducing stimuli is osmotic stress;
b) first stress inducing stimuli is heat stress and second stress inducing stimuli is pH stress;
c) first stress inducing stimuli is heat stress and second stress inducing stimuli is oxidative stress;

d) first stress inducing stimuli is heat stress and second stress inducing stimuli is metabolite restriction;
e) first stress inducing stimuli is heat stress and second stress inducing stimuli is cold shock;
f) first stress inducing stimuli is heat stress and second stress inducing stimuli is nutrient starvation;
g) first stress inducing stimuli is heat stress and second stress inducing stimuli is heavy metal stress;
h) first stress inducing stimuli is heat stress and second stress inducing stimuli is hydrostatic
pressure; i) first stress inducing stimuli is heat stress and second stress inducing stimuli is UV-stress; j) first stress inducing stimuli is heat stress and second stress inducing stimuli is chemical
stress; and k) first stress inducing stimuli is heat stress and second stress inducing stimuli is ethanol
shock. Yet alternatively the microbial pathogen may be subjected to three or more stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, hydrostatic pressure, ethanol shock, chemical stress, UV-stress, cold stress thereof.
Yet alternatively the microbial pathogen may be subjected to a three different types of stress inducing stimuli selected from the group comprising of:
i) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is pH stress; ii) first stress inducing stimuli is heat stress and second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is media optimization; iii) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is oxidative stress; iv) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is metabolite restriction; v) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is cold shock; vi) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is nutrient starvation;

vii) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is heavy metal stress;
viii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is hydrostatic pressure; ix) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is UV-stress; x) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is chemical stress; xi) first stress inducing stimuli is heat stress, second stress inducing stimuli is osmotic
stress and third stress inducing stimuli is ethanol shock; xii) first stress inducing stimuli is heat stress, second stress inducing stimuli is metabolite
restriction and third stress inducing stimuli is oxidative stress;
xiii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is cold shock;
xiv) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is nutrient starvation; xv) first stress inducing stimuli is heat stress, second stress inducing stimuli is metabolite
restriction and third stress inducing stimuli is heavy metal stress;
xvi) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is hydrostatic pressure;
xvii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is UV-stress;
xviii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is chemical stress;
xix) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is ethanol shock; xx) first stress inducing stimuli is heat stress, second stress inducing stimuli is oxidative
stress and third stress inducing stimuli is pH stress;
xxi) first stress inducing stimuli is heat stress, second stress inducing stimuli is
oxidative stress and third stress inducing stimuli is hydrostatic pressure.
Yet alternatively the microbial pathogen may be subjected to four or more stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic

stress, metabolite restriction, nutrient starvation, media optimization, hydrostatic pressure, ethanol shock, chemical stress, UV-stress, cold stress thereof.
According to a second aspect of the first embodiment, the microbial pathogen may be non-mammalian cells, in particular prokaryotic cells which may be gram positive or gram negative bacteria. Yet alternatively the microbial pathogen may be any pathogen which is capable of inducing an infectious disease. Yet alternatively the microbial pathogen may be virus, protozoa, fungi or a parasitic organism.
Yet alternatively the microbial pathogen may be a virus, selected from the group consisting of, but not limited to: human immunodeficiency virus, hepatitis A virus, hepatitis B, hepatitis C, human papillomavirus, Kaposi's Sarcoma- Associated Herpesvirus, Herpes Simplex virus, Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus, St Louis Encephalitis virus, Rift Valley Fever virus, Influenza viruses, corona virus, rhinovirus, adenovirus, SIV, rotavirus, human papilloma virus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus, varicella virus, huntavirus and cytomegalovirus.
Yet alternatively the microbial pathogen may belong to bacterial species selected from the group consisting of, but not limited to: Escherichia, Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Francisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma.
Yet alternatively the microbial pathogen may be a bacteria selected from the group consisting of, but not limited to Salmonella serovar strains S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. typhimurium and S. Enteritidis, Shigella, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Staphylococcus spp., Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcus spp , Streptococcus pneumoniae (1, 2, 3, 4, 5, 6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8,

9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11 A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F,
13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C,
19F, 20, 20A, 20B, 21, 22A, 22F, 23A,23B, 23F, 24A, 24B, 24F , 25F, 25A, 27, 28F, 28A,
29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38,
39,40,41F,41A,42,43,44,45,46,47F,47A,48), Group A Streptococcus, Group B
Streptococcus(group Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX.), Neisseria meningitidis, Haemophilus pneumonia, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Enterococcus faecalis, Enterococcus faecium, Enterococcus faecalis Neisseria gonorrhoeae, Bacillus anthracis, Vibrio cholerae, Pasteurella pestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, M. catarrhalis , Klebsiella pneumoniae ,Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Shigella spp., Erlichia spp., Rickettsia spp and N. meningitidis (A, B, C, D, W135, X, Y, Z and 29E), anthrax, Bacillus Calmette–Guérin (BCG).
Yet preferably the microbial pathogen may be Streptococcus pneumoniae serotypes (1, 2, 3, 4, 5,6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A,9L,9F,9N, 9V, 10F, 10B,10C, 10A, 11 A,11F,11B, 11C,11D,11E,12A,12B, 12F,13, 14, 15A,15C ,15B,15F,16A, 16F, 17A,17F, 18, 18C,18F,18A,18B, 19A, 19B, 19C, 19F, 20, 20A,20B,21,22A, 22F, 23A,23B, 23F, 24A, 24B,24F , 25F, 25A,27,28F, 28A, 29, 31,32F, 32A,33A, 33C, 33D, 33E, 33F,33B, 34, 45,38,35A,35B,35C,35F,36,37,38, 39, 40,41F,41A,42,43,44,45,46,47F,47A,48), wherein the Streptococcus pneumoniae has been subjected to a stress inducing stimuli resulting in the expression of stress protein (such as HSPs) or a stress protein-peptide complex by the microbe.
More preferably the microbial pathogen may be Streptococcus pneumoniae serotypes 1, 6B & 19F.
According to a third aspect of the first embodiment, the stress protein (such as HSPs) or a stress protein-peptide complex may be expressed by microbial pathogen in response to a one or more stress inducing stimuli may comprise of stress protein (such as HSPs) of different families of heat shock subtypes. Alternatively there may be a mixture of stress protein (such

as HSPs) derived from different classes based on molecular weight selected from, but not limited to, hsp20-30, HSP40, HSP50, HSP60, HSP70, HSP90 and/or HSP100, or from any other stress protein (such as HSPs) class which is present in microbial pathogen. Preferably the stress protein (such as HSPs) may be selected from, but not limited to, caseinolytic protease ClpP and ClpL, DnaK, Dna J, GroEL, GroES , hspX , acr2, AAA +, clp A / B, HtpG, TRIC, CCT, IbpA, IbpB, calrecticulin, hsp20-30, hsp40, hsp50, hsp60, hsp70, hsp72, hsp90, hsp100, grp94, grp75, BiP/grp78, grp75/mt, gp96. Other stress proteins, includes non native forms, truncated analogs, muteins, fusion proteins as well as other proteins capable of mimicking the peptide binding and immunogenic properties of a stress protein may be used in the preparation of immunogenic composition as disclosed herein. The stress protein-peptide complexes may include, but are not limited to, hsp20-peptide, hsp30-peptide, HSP40-peptide, Hsp60-peptide, Hsp70-peptide hsp72-peptide, Hsp90-peptide and hsp100-peptide complexes or from any other stress protein-peptide complex class.
According to a fourth aspect of the first embodiment, the heat shock protein complex (HspC) enriched preparations (HEPs) may be heat shock proteins or stress protein-peptide complex of a particular molecular weight. In certain embodiments, the heat shock proteins or stress protein-peptide complex have a molecular weight in the range of 10KDa to 1000KDa.
According to a fifth aspect of the first embodiment, where a mixture of stress protein (such as HSPs) or a stress protein-peptide complex is provided, this may comprise heat shock proteins of one particular family, for example, the hsp20-30, hsp40, hsp60, hsp70, hsp72, hsp90 or hsp100 families, or mixture comprises different heat shock proteins derived from different families.
According to a second embodiment of the present disclosure, the heat stress or heat shock may comprise of raising the temperature beyond the normal growth temperature of the microbial pathogen. This temperature increase is achieved by raising the temperature within a fermenter or reactor, of about 3°C to 15°C above the normal growth temperature of the microbial pathogen. The temperature may be raised beyond the normal growth temperature at a rate ranging between or around 0.1°C to 2°C per minute (0.1- 2°C/min). The microbial pathogen may be subjected to a heat stress for a time period ranging from around 10 minutes to 12 hours. Alternatively, the microbial pathogen may be subjected to a two or more heat stress comprising different temperature set-points with gradual increase in temperature and withholding it for a time period ranging from around 10 minutes to 12 hours. For example if

the microbial pathogen are typically grown at 37°C then the heat stress may comprise of increasing the temperature to about 40°C at a rate ranging between or around 0.1°C to 3°C per minute and withholding it for a time period ranging from around 10 minutes to 12 hours, further gradually increasing the temperature to about 42°C at a rate ranging between or around 0.1°C to 2°C per minute and withholding it for a time period ranging from around 10 minutes to 12 hours and lastly increasing the temperature to about 45°C and withholding it for a time period ranging from around 10 minutes to 12 hours.
According to a first aspect of the second embodiment, the microbial pathogen may be subjected to a heat stress or heat shock in the exponential or log phase of growth curve of microbial pathogen.
According to a second aspect of the second embodiment, the microbial pathogen may be subjected to a heat stress or heat shock in the lag phase of growth curve of microbial pathogen.
According to a third embodiment of the present disclosure, the ethanol shock may comprise of exposure of microbial pathogen to ethanol in a concentration range of 0.1% to 20% for a time period ranging from around 10 minutes to 12 hours.
According to a fourth embodiment of the present disclosure, the chemical stress may comprise of exposure of microbial pathogen to DNA damaging agents (0.1% [v/v] ethylmethane sulfonate, 0.01% [v/v] methylmethane sulfonate).
According to a fifth embodiment of the present disclosure, the osmotic stress may comprise of exposure of microbial pathogen to high osmolarity by varying the concentration of salt e.g., Na+, K+, Ca++ or e.g. NaCl (0.1 to 1M gradient range).
Yet another embodiment of the present disclosure, wherein the pH stress may comprise of reducing the pH of the microbial pathogen to a pH below the optimal pH of microbial pathogen by adding an acid e.g. hydrochloric acid to the cells. Optimal pH is the pH at which normal physiological growth or homeostasis of the cells occurs. In certain embodiments, the pH is lowered below 7 going below up to 3. In certain embodiments, the pH is lowered to 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5, 4.5, 4, 3.5 or 3.

According to a sixth embodiment of the present disclosure, the oxidative stress may comprise of exposure of microbial pathogen to hydrogen peroxide (H2O2) or Nitric dioxide (NO2 ) and maintaining dissolved oxygen tension in the range of 1 – 10%.
According to a seventh embodiment of the present disclosure, the media optimization may comprise of optimizing the cell culture media components and pH of the medium to increase cell density, culture viability and HSPs productivity in a timely manner.
According to a one aspect of seventh embodiment, the cell culture media components may comprise of all or in any combination: Hisoya, Soya peptone, Yeast Extract, Dextrose, Potassium dihydrogen phosphate, Sodium chloride, Magnesium sulfate heptahydrate, Calcium chloride dihydrate, L- Lysine, L- Cysteine, Thiamine Hydrochloride, Sodium Bicarbonate.
According to a second aspect of seventh embodiment, the cell culture media components may comprise of:
- Soya peptone in a range of 10-40 gm/L, more preferably 20 gm/L;
- Yeast Extract in a range of 1-10 gm/L, more preferably 5 gm/L;
- Potassium dihydrogen phosphate in a range of 1-10 gm/L, more preferably 5 gm/L;
- Sodium chloride in a range of 1-10 gm/L, more preferably 5 gm/L;
- Dextrose in a range of 1-30 gm/L, more preferably 20 gm/L;
- Magnesium sulfate heptahydrate in a range of 0.1-1 gm/L, more preferably 0.5 gm/L;
- L- Lysine in a range of 0.01 – 1 gm/L, more preferably 0.1 gm/L;
- Sodium Bicarbonate to adjust pH in a range of 6.9 – 7.5.
According to a third aspect of seventh embodiment, the cell culture media components may comprise of:
- Hisoya in a range of 10-40 gm/L, more preferably 23.33 gm/L;
- Yeast Extract in a range of 1-10 gm/L, more preferably 5.83 gm/L;
- Potassium dihydrogen phosphate in a range of 0.1-10 gm/L, more preferably 0.81 gm/L;
- Magnesium sulfate heptahydrate in a range of 0.1-10 gm/L, more preferably 0.58 gm/L;
- Sodium chloride in a range of 1-10 gm/L, more preferably 2.33 gm/L;
- Dextrose in a range of 1-30 gm/L, more preferably 10.76 gm/L;
- Calcium chloride dihydrate in a range of 0.1-1 gm/L, more preferably 0.21 gm/L;
- L- Cysteine in a range of 0.1 – 10 gm/L, more preferably 2.15 gm/L;

- Thiamine Hydrochloride in a range of 0.01 – 10 gm/L, more preferably 0.21 gm/L;
- Sodium Bicarbonate to adjust pH in a range of 6.9 - 7.5.
According to a fourth aspect of seventh embodiment, the cell culture media components may comprise of:
- Hisoya in a range of 10-40 gm/L, more preferably 20 gm/L;
- Yeast Extract in a range of 1-10 gm/L, more preferably 5 gm/L;
- Dextrose in a range of 1-30 gm/L, more preferably 10 gm/L;
- Magnesium sulfate heptahydrate in a range of 0.1-10 gm/L, more preferably 2 gm/L;
- Calcium chloride dihydrate in a range of 0.1-1 gm/L, more preferably 0.2 gm/L;
- Sodium chloride in a range of 0.1-10 gm/L, more preferably 0.5 gm/L;
- Thiamine Hydrochloride in a range of 0.01 – 10 gm/L, more preferably 0.02 gm/L;
- L- Cysteine in a range of 0.01 – 10 gm/L, more preferably 0.02 gm/L;
- Potassium dihydrogen phosphate in a range of 0.1-10 gm/L, more preferably 0.2 gm/L;
- Sodium Bicarbonate to adjust pH in a range of 6.9 - 7.5.
According to a fifth aspect of seventh embodiment, HSPs productivity may be enhanced by use of proteasome inhibitor such as MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal, lactacystin, VSL#3-CM (ELAINE O. PETROF et al. 2004) along with other stress inducing stimuli.
According to an eighth embodiment of the present disclosure, the stress protein (such as HSPs) or a stress protein-peptide complex expressed by microbial pathogen may be further subjected to harvesting and purification.
According to a ninth embodiment of the present disclosure, wherein the step of recovering a preparation comprising stress proteins or a stress protein-peptide complex from the stress induced microbial pathogen may comprise the steps of:
a) harvesting the microbial cellular material by centrifugation, clarification or Tangential Flow filtration (TFF)
b) subjecting the harvested microbial cellular material to lysis comprising sonication or shaking of glass beads to obtain a cell lysate comprising stress proteins,
c) subjecting the cell lysate to centrifugation to obtain a supernatant comprising of stress proteins.

Yet preferably sonication may be carried out in PBS buffer of 6.9 - 7.5 pH comprising cell pellet separately for every sample. After the corresponding treatments had been performed, the supernatant of each sample may be filtered through a 0.2 μm filter so as to prevent any contamination when undergoing further analysis.
According to a tenth embodiment of the present disclosure, wherein the step of recovering a preparation comprising stress proteins or a stress protein-peptide complex from the stress induced microbial pathogen may comprise the steps of:
a) harvesting the microbial cellular material,
b) subjecting the harvested microbial cellular material to inactivation to obtain an inactivated whole cell comprising stress proteins.
Yet another aspect of the tenth embodiment, wherein the harvested microbial cellular material may be inactivated using one of the method selected from the group comprising of formaldehyde treatment, heat treatment and treatment with 70 % alcohol.
Yet preferable the harvested microbial cellular material may be inactivated using formaldehyde treatment method comprising treating the harvested microbial cellular material with formaldehyde having concentration in the range of 0.25% to 1.5%.
According to an eleventh embodiment of the present disclosure, wherein the recovered preparation of stress proteins or a stress protein-peptide complex may be subjected to further purification and isolation.
The purification of stress protein (such as HSPs) or a stress protein-peptide complex may be achieved by utilizing purification processes selected from group of but are not limited to, ion exchange chromatography, affinity chromatography, hydroxyapatite chromatography and gelatin chromatography. This purification method maintains the stress protein-peptide association necessary to develop vaccines or immunotherapeutic tools for tumors and for infectious diseases since HSPs have not been shown to be helpful as antigens without the associated peptides.
More preferably the extraction of stress protein (such as HSPs) or a stress protein-peptide complex may be enhanced by use of proteasome inhibitor such as MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal, lactacystin, VSL#3-CM (ELAINE O. PETROF et al. 2004).

According to a twelvth embodiment of the present disclosure, a preferred single or a combination of stress inducing stimuli may be determined by applying the stress inducing stimuli and then quantitating the induced stress proteins (intracellular, cell bound and/or extra-cellular stress proteins). Typically the preferred single or a combination of stress inducing stimuli enhances the quantum of induced stress protein (such as HSPs) or a stress protein-peptide complex produced in a cell (intracellular, cell bound and/or extra-cellular stress proteins). Standard methods which may be used to quantitate the induction of stress proteins include 12% Non-Reducing SDS-PAGE Gel, protein gel analysis, proteomic analysis using mass spectrometry, densitometry (optical density), immunoblotting or ELISA. Quantitative assay to identify effect of Heat Shock Condition on Protein and Polysaccharide Content in PNU Serotype Cells may include Lowry Assay and Anthrone Assay. Characterization and identification of serotypes may be done by NMR spectroscopy. The production of a higher yield of stress protein (such as HSPs) or a stress protein-peptide complex is of significant commercial relevance as large quantities of heat shock protein complexes would be required for large scale production of a prophylactic or therapeutic vaccine.
According to a thirteenth embodiment of the present disclosure, wherein the method may further comprise of mixing the recovered preparation of stress proteins with at least one pharmaceutically acceptable excipient to produce an immunogenic composition.
According to a fourteenth embodiment of the present disclosure, wherein the method may further comprise of combining the recovered preparation of stress proteins with at least one of polysaccharide or polysaccharide-protein conjugate or used as a carrier protein in a polysaccharide-protein conjugate or with whole cell bacteria or fusing with protein to produce an immunogenic composition.
Yet one of the aspect of fourteenth embodiment, wherein the recovered preparation of stress protein is combined with at least one of protein or polysaccharide or polysaccharide-protein conjugate or used as a carrier protein in a polysaccharide-protein conjugate or with whole cell bacteria or fusing with protein derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B,

23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43,44, 45, 46, 47F, 47A, 48 to produce an immunogenic composition.
Yet one of the aspect of fourteenth embodiment, wherein the recovered preparation of stress protein is combined with at least one of protein derived from Streptococcus pneumoniae Example: pneumococcal surface protein comprising of PspA or PspC or pneumolysin (Ply)
Yet one of the preferred aspect of fourteenth embodiment, wherein the recovered preparation of stress protein may be combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A to produce an immunogenic composition.
Yet preferably the immunogenic composition may comprise of:

Table no 1: Description of formulation
Sr. No Name of Formulation Components of formulation
1 Formulation A Inactivated Whole cell bacteria or cell lysate of
Serotype 19F and 6B, 37°C + Pneumo 10 vaccine
2 Formulation B Inactivated Whole cell bacteria or cell lysate of
Serotype 19F and 6B, 45°C + Pneumo 10 vaccine
3 Formulation C Inactivated Whole cell bacteria or cell lysate of
Serotype 19F and 6B, 45°C in buffer with Alum
4 Formulation D Pneumo 10 vaccine
5 Formulation E Inactivated Whole cell bacteria or cell Serotype 19F, 37°C + Pneumo 10 vaccine lysate of
6 Formulation F Inactivated Whole cell bacteria or cell Serotype 19F, 45°C + Pneumo 10 vaccine lysate of
7 Formulation G Inactivated Whole cell bacteria or cell Serotype 19F, 45°C in buffer with Alum lysate of
8 Formulation H Inactivated Whole cell bacteria or cell Serotype 6B, 37°C + Pneumo 10 vaccine lysate of
9 Formulation I Inactivated Whole cell bacteria or cell Serotype 6B, 45°C + Pneumo 10 vaccine lysate of

10 Formulation J Inactivated Whole cell bacteria or cell lysate of Serotype 6B, 45°C in buffer with Alum
11 Formulation K Inactivated Whole cell bacteria or cell lysate of Serotype 1, 37°C + Pneumo 10 vaccine
12 Formulation L Inactivated Whole cell bacteria or cell lysate of Serotype 1, 45°C + Pneumo 10 vaccine
13 Formulation M Inactivated Whole cell bacteria or cell lysate of Serotype 1, 45°C in buffer with Alum
14 Formulation N Inactivated Whole cell bacteria or cell lysate of Serotype 1, 19F and 6B, 37°C + Pneumo 10 vaccine
15 Formulation O Inactivated Whole cell bacteria or cell lysate of Serotype 1, 19F and 6B, 45°C + Pneumo 10 vaccine
16 Formulation P Inactivated Whole cell bacteria or cell lysate of Serotype 1, 19F and 6B, 37°C in buffer with Alum
17 Formulation Q Inactivated Whole cell bacteria or cell lysate of Serotype 1, 19F and 6B, 45°C in buffer with Alum
18 Formulation R Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 37°C + Pneumo 10 vaccine
19 Formulation S Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 45°C + Pneumo 10 vaccine
20 Formulation T Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 37°C in buffer with Alum
21 Formulation U Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 45°C in buffer with Alum
22 Formulation V Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 37°C + Pneumo 10 vaccine
23 Formulation W Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 45°C + Pneumo 10 vaccine
24 Formulation X Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 37°C in buffer with Alum
25 Formulation Y Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 45°C in buffer with Alum

Yet preferably the immunogenic composition may comprise of Inactivated Whole cell bacteria or cell lysate of Serotype 1, 6B and 19F suitably in the range of 101 to 1010cells/ml, added aseptically in the formulation as disclosed above. More preferably 107cells/ml may be added aseptically in the formulation as disclosed above.
Pneumo 10 vaccine added aseptically in the formulation as disclosed above may comprise of SIIPL’s PNEUMOSIL® 10-valent pneumococcal conjugate vaccine comprising Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A.
According to one aspect of the embodiment, the immunogenic composition may additionally comprise of a buffering agent selected from the group consisting of carbonate, phosphate, acetate, HEPES, succinate, histidine, TRIS, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris (hydroxymethyl) aminomethane, or "Tris", formulated to achieve the desired pH. Yet in another example, the buffering agent could be the minimum essential medium with Hanks salts. Other buffers, such as HEPES, piperazine-N, N′-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the present disclosure. The buffer aids in stabilizing the immunogenic composition of the present disclosure. The amount of the buffer may be in the range of 0.1 mM to 100 mM, preferably selected from 5mM, 6mM, 7mM, 22 mM, 23mM, 24mM, 25mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM. The amount of the buffer may be in the range of 0.1 mg - 1.6 mg.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of pharmaceutically acceptable excipient selected from the group consisting of sugars, surfactants, polymers, salts, aminoacids or pH modifiers.
Examples of Surfactants may include ionic and non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethanol, t-Octylphenoxypolyethoxyethanol, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer.
Examples of the polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc.

Examples of the salts may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, etc. Preferably, the salt may be NaCl. Typically the amount of the salt may be in the range of 100 mM to 200 mM.
Examples of the aminoacids as excipient selected from the group of L-Histidine, Lysine, Isoleucine, Methionine, Glycine, Aspartic acid. Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolysed protein or protein such as serum albumin.
Examples of the sugars as excipient selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol, or a combination thereof.
Yet preferably the single dose composition is free of preservative.
Yet preferably the multi-dose immunogenic composition may additionally comprise of preservative selected from the group consisting of 2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters (e.g. methyl-, ethyl¬, propyl- or butyl- paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, or benzyl alcohol or a combination thereof. A vaccine composition may include material for a single immunization, or may include material for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Preferably, the preservative may be 2-phenoxyethanol in the range of 0.1 mg to 50 mg; more preferably 1 – 10 mg.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of auxiliary substances such as wetting or emulsifying agents, diluent pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
Yet preferable aspect of the embodiment, the immunogenic composition may additionally comprise of water for injection as diluent.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of an adjuvant selected from the group of aluminum salt, Alum, aluminum

hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate.
Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of an immunostimulatory component selected from the group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, 3–deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, cholera toxin B subunit, polyphosphazene and derivatives, mycobacterium cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combination with sterols and lipids.
Yet another aspect of the embodiment, the immunogenic composition may be fully liquid. Suitable forms of liquid preparation may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and elixirs that are buffered to a selected pH.
Yet preferably the immunogenic composition may be fully liquid, stable at 2-8°C, 25°C and 40°C for over a period of 2 to 6 months.
Yet preferably the immunogenic composition may be capable of inducing the formation of antibodies against Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43,44, 45, 46, 47F, 47A, 48.

Yet more preferably the immunogenic composition may be capable of inducing the formation of antibodies against Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F, 6A, 2, 3, 15B and 18C.
The use of the expression “one or more” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the composition of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this disclosure.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
Similarly, the components used in purification, e.g., filters, columns, are not intended to be in any way limiting or exclusionary, and can be substituted for other components to achieve the same purpose at the discretion of the practitioner.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustration of the disclosure and not as a limitation.
Strains procurement information: All Strains procured from CDC Atlanta, USA

Table No 2: Serotypes of S. pneumoniae (PNU)
Sr. No Serotype code
1 1
2 19F

3 2
4 4
5 6B
6 3
7 6A
8 23F
9 14
Technical Advantages:
The process for isolation and purification of stress protein (such as HSPs) or a stress protein-peptide complex of the present disclosure described herein above has several technical advantages including, but not limited to, the realization of:
- Simple and cost effective process for isolation and purification of stress protein (HSPs) or a stress protein-peptide complex.
- Improved upstream, downstream process.
- Improved extraction of HSP’s by sonication in presence of PBS, NaCl or proteasome inhibitor such as MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal, lactacystin, VSL#3-CM as compared to homogenizer.
- Use of combination of stress inducing stimuli selected from heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, media optimization, Hydrostatic pressure, Ethanol shock, chemical stress, UV-stress, cold stress results in higher expression of stress proteins (such as HSPs) or

a stress protein-peptide complex that could be used at industrial scale when compared to induced stress proteins produced following exposure to a single type of stress inducing stimulus.
- Enhanced expression of “HSP of interest” relatively to others.
- Enhanced expression of HSP without cell engineering (low cost).
The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “one or more” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.
Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value

assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustration of

Examples:
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1:
1) Identification & Selection of most prevalent serotypes of S. pneumoniae capable of producing stress protein (such as HSPs) in Maximum concentration:

Table No 2: Serotypes of S. pneumoniae (PNU)
Sr. No Serotype code
1 19F
2 2
3 4
4 6B
5 3
6 6A
7 23F
8 14
2) Growth Media for Cell banks of S. pneumoniae serotypes:

Table No 3: Growth Media for cell banking
Sr.No Ingredients gm/L
1 Hisoya 23.33
2 Yeast Extract 5.83
3 Potassium dihydrogen phosphate (KH2PO4) 0.81
4 Magnesium sulfate heptahydrate (MGSO4.7H2O) 0.58
5 Sodium chloride (NaCl) 2.33
6 Dextrose 10.76

7 Calcium chloride dihydrate (CaCl2.2H2O) 0.21
8 L-Cysteine 2.15
9 Thiamine Hydrochloride 0.21
10 Sodium bicarbonate to adjust pH 7.0 ~12

Table No 4: Freezing media for cell banking
Sr.No Ingredients gm/L
1 Hisoya 23.33
2 Yeast Extract 5.83
3 Potassium dihydrogen phosphate (KH2PO4) 0.81
4 Magnesium sulfate heptahydrate (MGSO4.7H2O) 0.58
5 Sodium chloride (NaCl) 2.33
6 Dextrose 10.76
7 Calcium chloride dihydrate (CaCl2.2H2O) 0.21
8 L-Cysteine 2.15
9 Thiamine Hydrochloride 0.21
10 Sodium bicarbonate to adjust pH 7.0 ~12
11 Glycerol 150 ml
After the preparation, media was aseptically filtered through 0.2µ filter before use.

3) Establishment of Cell banks of S. pneumoniae serotypes
One vial of above serotype research cell bank was taken out from -70°C deep freezer and thawed the vial content in the bio safety cabinet. Aseptically withdrawn loop full of culture from the vial and done streaking on the Chocolate Agar plate (CAP)/Blood Agar plate (BAP) and incubated in incubator at 37ºC for 48 hr. After 48 hrs, the incubated plate was observed for the bacterial colonies and picked up 3 isolated colonies and inoculated into 25 mL growth medium in 125 mL flask. This inoculated medium was then incubated at 37°C at 135 rotation per minute (RPM) in a shaker. OD was measured hourly after initial inoculation, once OD reached about 0.3 and above. At this time point the cell were harvested, the grown culture was then centrifuged at 4500 RPM at 8°C for 10 minute. The cell pellets are then resuspended into the 40 mL freezing media (15% glycerol) for storing the cells for further experiments. The 2 mL cell suspension was then distributed into 20 vials (numbered sequentially), each vial culture with 2.0 OD/mL. These vials were labelled with serotype code and 01 to 20. Initially these vials were kept at -20ºC for 30 minutes and then shifted to -70ºC deep freezer for long term storage.
4) Optimization of fermentation process for Maximizing the harvest OD
Selection and optimization of media is an important activity as it is one of the key factors for getting the better cell growth. Various experiments were conducted for selecting suitable media. Based on the available literature, media were supplemented with different nutrients to enhance the bacterial growth and the pH of the medium was adjusted to 6.9 to 7.1. During the medial preparation, a usual issue like precipitation of the media was occurred (usually due to trace elements). Those were resolved by changing the order of addition of media ingredients, slight warming of the medium and pH adjustment.
After dissolving all the media components, pH of the medium was adjusted and finally volume makes up as per the requirement and sterile filtered through 0.2µm before use at the time of study.
4.1 Flask study Experiment for serotype 19F
One vial of above serotype working cell bank was taken out from -70°C deep freezer and thawed the vial content in the bio safety cabinet. Aseptically withdrawn loop full of culture

from the vial and done streaking on the Chocolate Agar plate (CAP)/Blood Agar plate (BAP) and incubated in incubator at 37ºC for 48 hr.
Following media was used in the experiment. After dissolving all the media components, pH of the medium was adjusted to 6.9-7.1 and finally volume makes up as per the requirement and sterile filtered through 0.2µm before use at the time of study.
Cultivation media- :
Following three different media tried to obtain the better bacterial growth.

Table No 5: Media 1, 2, 3
Sr. No. Media pH =7.0
Media 1 Ingredients gm/L
1 Soya peptone 20
2 Yeast Extract 5
3 Potassium hydrophosphate monobasic 5
4 Sodium chloride 5
5 Dextrose 20
6 Magnesium sulfate 0.5
7 L- Lysine 0.1
8 Sodium Bicarbonate to adjust pH 7.0 ~20

Media 2 Ingredients gm/L
1 Hisoya 23.33
2 Yeast Extract 5.83
3 KH2PO4 0.81
4 MGSO4.7H2O 0.58
5 NaCl 2.33
6 Dextrose 10.76
7 CaCl2.2H2O 0.21
8 L-Cysteine 2.15
9 Thaimine HCL 0.21
10 Sodium bicarbonate to adjust pH 7.0 ~12

Media 3 Ingredients gm/L
1 Hisoya 20
2 Yeast Extract 5
3 Dextrose 10
4 MgSO4.7H2O 2
5 CaCl2.2H2O 0.2

6 NaCl 0.5
7 Thaimine HCL 0.02
8 L-Cysteine 0.02
9 KH2PO4 0.2
10 Sodium bicarbonate to adjust pH 7.0 ~2
After preparing the media in water the pH was adjusted to 6.9-7.1 and made up the volume. It was then sterile filtered through 0.2µm membrane prior to use in the experiment.
One vial of working cell bank of above serotype was taken out from -70°C deep freezer and thawed the vial content in the bio safety cabinet. Aseptically withdrawn loop full of culture from the vial and done streaking on the Chocolate Agar plate (CAP)/Blood Agar plate (BAP) and incubated in incubator at 37ºC for 48 hr. After 48 hrs, the incubated plate was observed for the bacterial colonies and picked up 3 isolated colonies and inoculated into 25 mL growth media-1, media-2 and media-3 in 125 mL flask. This inoculated media flasks was then incubated at 37°C at 135 rotation per minute (RPM) in a shaker.
The optical density (OD) was recorded periodically at 590nm and OD expansion was done by the addition of the fresh media as given in below, the OD measurement continued till the decline phase.

Table N o 6: Bacterial growth and expansion study shown in table Media-1 Media-2
Sr. no.
Media-3
Culture
Age
(hrs) OD @590 nm Culture Age (hrs) OD @590 nm Culture Age (hrs) OD @590 nm
1 0 0 0 - 0 -
2 4 0.174 4 0.274 4 0.204
3 5 0.04 5 0.05 5 0.04
4 6 0.06 6 0.143 6 0.09
5 7 0.08 7 0.25 7 0.182
6 8 0.08 8 0.29 8 0.21
7 9 0.06 9 0.30 9 0.23
8 10 0.05 10 0.32 10 0.265
9 12 0.05 12 0.35 12 0.291

Interpretation:
Based on the flask experimental findings it is observed that the media-1 and media-3 did not support the bacterial cell growth.
Media -2 shown better growth as compared to other two media and indicate that media-2 can be further used for the experimentation. The maximum OD recorded in media-2 was 0.35, further experiments needs to be conducted to optimize the media-2 so that the OD can be increased to about 0.5 -1.0.
All the flasks and other accessories were soaked with 0.5M NaOH and then discarded by autoclaving.
Refer Figure 1: Bacterial Growth curve of Serotype PNU 19F
4.2 Flask study Experiment for serotype 2
Observation and Interpretation:
Like serotype 19 F, growth & expansion studies were carried out using Media 1, Media2 and media-3 as mentioned in the previous section for serotypes such as Serotype 2
Based on the flask experimental findings it is observed that the media-1 and media-3 did not support the bacterial cell growth.
Media -2 shown better growth as compared to other two media and indicate that media-2 can be further used for the experimentation. The maximum OD recorded in media-2 was 0.35, further experiments needs to be conducted to optimize the media-2 so that the OD can be increased to about 0.5 -1.0.
All the flasks and other accessories were soaked with 0.5M NaOH and then discarded by autoclaving.
Refer Figure 2: Bacterial growth curve of PNU Serotype 2
4.3 Flask study Experiment for serotype 4
Observation and Interpretation:
Like serotype 19 F, growth & expansion studies were carried out using Media 1, Media2 and media-3 as mentioned in the previous section for serotypes such as Serotype 4

Based on the flask experimental findings it is observed that the media-1 and media-3 did not support the bacterial cell growth.
Media -2 shown better growth as compared to other two media and indicate that media-2 can be further used for the experimentation. The maximum OD recorded in media-2 was 0.35, further experiments needs to be conducted to optimize the media-2 so that the OD can be increased to about 0.5 -1.0.
All the flasks and other accessories were soaked with 0.5M NaOH and then discarded by autoclaving.
Refer Figure 3: Bacterial growth curve Serotype 4
4.4 Flask study Experiment for serotype 6B
Observation and Interpretation:
Like serotype 19 F, growth & expansion studies were carried out using Media 1, Media2 and media-3 as mentioned in the previous section for serotypes such as Serotype 6B
Based on the flask experimental findings it is observed that the media-1 and media-3 did not support the bacterial cell growth.
Media -2 shown better growth as compared to other two media and indicate that media-2 can be further used for the experimentation. The maximum OD recorded in media-2 was 0.40, further experiments needs to be conducted to optimize the media-2 so that the OD can be increased to about 0.5 -1.0.
All the flasks and other accessories were soaked with 0.5M NaOH and then discarded by autoclaving.
Refer Figure 4: Bacterial growth curve Serotype 6B
4.5 Flask study Experiment for serotype 3
Observation and Interpretation:
Like serotype 19 F, growth & expansion studies were carried out using Media 1, Media2 and media-3 as mentioned in the previous section for serotypes such as Serotype 3

Based on the flask experimental findings it is observed that the media-1 and media-3 did not support the bacterial cell growth.
Media -2 shown better growth as compared to other two media and indicate that media-2 can be further used for the experimentation. The maximum OD recorded in media-2 was 0.45, further experiments needs to be conducted to optimize the media-2 so that the OD can be increased to about 0.5 -1.0.
All the flasks and other accessories were soaked with 0.5M NaOH and then discarded by autoclaving.
Refer Figure 5: Bacterial growth curve Serotype 3

Example 2
Enrichment of Stress proteins - Heat shock proteins (HSPs) methodology:
Details of the experiments carried out using heat shock on the various serotype are described below.

Table No. 7: S. pneumoniae serotype
Sr. No Serotype code
1 19F
2 2
3 4
4 6B
5 3
6 6A
7 23F
8 14
Fermentation process carried out using Media-2 after having been optimized for growth for these serotypes.
Media Preparation:

Table No. 8: Media 2
Media Ingredients Seed Media (1X) g/L Fermentation
Media (2X)
g/L Feed Media (3X) g/L
1 HY-SOYA 20 40 100
2 Yeast Extract 5 10 25
3 KH2PO4 0.7 1.4 -
4 MgSO4.7H2O 0.5 1.0 2.5
5 NaCl 5.0 10 -
6 Dextrose 10 20 200
7 CaCl2.2H2O 0.02 0.04 -
8 L-Cysteine 0.2 0.4 -
9 Thiamine HCL 0.02 0.04 -

( Base prepared for pH adjustment was 0.2 μ filtered solution of 2 N NaOH and 20% sodium carbonate)
After preparing the media in water for injection, the pH was then adjusted in the range of 7.0-7.1 with Sodium Carbonate and the volume was then made up to the required amount. The seed media and feed media prepared were 500 mL each and the fermentation media prepared was 1 L. The media and base was then filtered through a 0.2 μm membrane in a bio safety cabinet to ensure sterility and prevent any contamination. After which, it was placed on an incubator shaker at 135 rpm at 37°C overnight to check for any contamination. The pH probe and DO probe were washed and cleaned and kept for sensing.
The next day the fermenter and all required parts were washed and cleaned with tap water twice and then water for injection. The fermenter was then assembled and autoclaved at 121°C at 15 psi for 30 minutes. Around 750 mL of water for injection was added for autoclaving. It was allowed to cool for 3 hours and then placed onto the hot plate and connected to the monitor. The pH probe and DO probe are calibrated prior to autoclaving and then connected to the ports.
The sterile fermenter can now be used to to carry out the fermentation process. 350 mL of water for injection was removed by the sampling bottle and then 400 mL of fermentation media was passed through sterile tubes into the fermenter to make up the final volume of the fermenter to 1 L.
A vial of working cell bank of above serotype was taken out from -70°C deep freezer and thawed the vial content in the bio safety cabinet. The contents of the vial were then inoculated into 100 mL seed media and kept at 37°C, 145 rpm at 22:35 and kept for incubation.
The OD of the seed media obtained after incubation. The seed media was then passed into the fermenter and the batch fermentation process had begun.
The batch was inoculated with the ~100 ml seed in 800 ml of fermentation media. The base and feed was allowed to pass at pH stat mode from 2nd hour.

similar OD values & average of OD values was summarized in the table no. 9 after 5 hrs of incubation.

Table No. 9: Growth and Expansion of expansion study shown in table Culture: Bacterial growth and
Hour OD Temperature (°C) Agitation pH Air Feed (%) Base/ Feed
0 - 37.0 50 7.0 0.05 - -
2 0.343 37.0 50 7.0 0.05 5
3 1.21 37.0 50 7.0 0.05 5
4 3.94 36.9 50 6.99 0.04 5 2.418
5 6.44 37.0 50 7.0 0.04 5
Procedure for Enrichment of HSP by Heat Shock
Procedure for enrichment of HSP in tested serotypes was optimized. Prior to the Heat shock treatment around 50 mL of sample was removed and centrifuged at 8,000 rpm for 20 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
The heat shock was performed at 5th Hour with the shift of temperature to 42°C. After 15 minutes 50 mL of sample was removed and centrifuged at 8,000 rpm for 20 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively. Second heat shock treatment was performed by setting temperature at 45°C
30 minutes after reaching 45°C, 100 ml of sample was again removed having OD 3.51 and centrifuged at 8,000 rpm for 20 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
The total harvest volume obtained at 8 hour age was = 600 mL. It was centrifuged at 8000 rpm for 30 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
Sonication was carried out in 40 ml PBS buffer of 7.4 pH along with = 2 g of cell pellet separately for every sample.

Analysis of Samples: The supernatant and cell pellets were stored at 8°C and -20°C respectively.
The supernatants were filtered with 0.2μ filter and analyzed further.
Further analysis was performed on a 12% Non-Reducing SDS-PAGE Gel to ascertain the enrichment of HSPs.
This procedure was applied for enrichment of HSPs from serotypes such as Serotype 4, 3, 2, 19F, 14, 6B and 1.
Protein marker used was ‘Bio-Rad precision plus protein standards ( dual color )’ containing ten recombinant protein bands of 10 kD, 15 kD, 20 kD, 25 kD, 37 kD, 50 kD, 75 kD, 100 kD, 150 kD and 250 kD.
Refer:
Figure No. 6: Analysis of HSP enrichment in serotype 6A and 23F Figure No. 7: Analysis of HSP enrichment in serotype 4 and 3
Figure No. 8: Analysis of HSP enrichment in serotype 2 and 19F
Figure No. 9: Analysis of HSP enrichment in serotype 14 and 6B
Interpretation:
The heat shock methodology seems to be working better, however as soon as temperature was raised upto 45°C, decrease in OD was observed and the cells could not sustain longer time at this temperature and also the intensity of the HSP’s was not significantly higher as compared to normal temperature (37°C). Out of 08 serotype tested, preliminary data shows that Serotype 19F and 6B showed increase in band intensity between 37kD to 75 kD. To overcome this limitation, new procedure was developed and adopted as given below for Serotype 19F and 6B.

Example 3: Preparation of HSP enriched working seed (WS) of S. pneumoniae
In the present study, fermentation was carried out using serotypes 19F, 6B and additionally Serotype 1 using standardized procedure as mentioned in the previous section after attaining the desired growth cells were subjected to heat shock i.e. temperature was increased to 45 Deg C for about 1 hour. Then cells were collected and were frozen. Enrichment of HSPs was reassured using SDS-PAGE analysis. Cells were stored at -70°C and labeled as HSP enriched cells. Aseptically withdrawn loop full of culture from the flask and done streaking on the Chocolate Agar plate (CAP)/Blood Agar plate (BAP) and incubated in incubator at 37ºC for 48 hr. After 48 hrs, the incubated plate was observed for the bacterial colonies and picked 2 isolated colonies and inoculated into 25 mL growth medium in 125 mL flask. This inoculated medium was then incubated at 37°C at 135 rotation per minute (RPM) in a shaker.
New working seed was prepared for serotype 19F, 6B and 01 as previously prepared for 08 serotypes and characterized. In addition to the routine characterization, serotypes were also identified by NMR spectroscopy and are comparable with reported NMR spectra.
Refer:
Figure No. 10: NMR spectra for serotype 1
Figure No. 11: NMR spectra for serotype 6B
Figure No. 12: NMR spectra for serotype 19F

Results: Serotype 19F:

Table No.10: Growth and Expansion of Culture: Bacterial growth and expansion
Culture Age (Hr.) Temperature(0C) OD at 590 nm Remark
0 37 - -
1 37 1.59 -
2 37 3.25 -
3 37 7.02 -
4 37=> 45 10.32 Temp set to 450C
5 45 13.75 -
6 45 13.5 -
7 45 13 -
8 45 10.37 -
9 45 9.75 -
10 45 8.5 -
11 45 7.2 Terminated
Sonication was carried out in 35 ml PBS buffer of 7.4 pH along with 2.3 g of cell pellet separately for every sample. After the corresponding treatments had been performed, the supernatant of each sample was then filtered through a 0.2 μm filter so as to prevent any contamination when undergoing further analysis.
Conclusion:
After heat shock, increase in band intensity at about 60 and 70 KD observed.
Refer
Figure No. 13: Bacterial Growth Curve Serotype 19F Figure No.14: Analysis of HSP enrichment in serotype 19F

Serotype 6B:

Table No.11: Growth and Expansion of Culture: Bacterial growth and expansion study shown in table
Culture Age (Hr.) Temperature(0C) OD at 590 nm Remark
0 37 - -
2 37 1.5 -
3 37 4.2 -
4 37=> 45 6.62 Temp set to 450C
5 45 6.2 -
6 45 5.1 -
7 45 2.3 Terminated
Sonication was carried out in 35 ml PBS buffer of 7.4 pH along with 1.0 g of cell pellet separately for every sample. After the corresponding treatments had been performed, the supernatant of each sample was then filtered through a 0.2 μm filter so as to prevent any contamination when undergoing further analysis.
Conclusion:
After heat shock, increase in band intensity at about 37, 60 and 70 KD observed.
Refer
Figure No. 15: Bacterial Growth Curve Serotype 6B Figure No.16: Analysis of HSP enrichment in serotype 6B

Serotype 1:

Table No.12 Growth and Expansion of Culture: Bacterial growth and expansion study shown in table
Culture Age (Hr.) Temperature(0C) OD at 590 nm Remark
0 37 - -
3 37 1.7 -
4 37 3.6 -
5 37 => 42 5.1 Temp set to 420C
6 42=> 45 6.4 Temp set to 450C
7 45 6
8 45 3 Terminated
Sonication was carried out in 40 ml PBS buffer of 7.4 pH along with 1.0 g of cell pellet separately for every sample. After the corresponding treatments had been performed, the supernatant of each sample was then filtered through a 0.2 μm filter so as to prevent any contamination when undergoing further analysis.
Conclusion:
After heat shock, increase in band intensity at about 25kD, 37 kD, 50 kD, 60 kD and 70 kD observed.
Figure No. 17: Bacterial Growth Curve Serotype 1 Figure No.18: Analysis of HSP enrichment in serotype 1

Example 4: Quantitative assay to identify effect of Heat Shock Condition on Protein and Polysaccharide Content in S. pneumoniae (PNU) Serotype Cells

Table 13: Effect of Heat Shock Condition on Protein and Polysaccharide Content in PNU Serotype Cell Lysates after Exposure to Heat Shock Conditions
Sr. No. Parameter Method Cell Lysate of PNU 1+6B+19F (Combined) % Increase Fold Increase

Normal Condition (37°C) Heat Shock Condition (45°C)

1 Protein Content Lowry Assay 31.3 37.9 121 1.2
2 Polysaccharide Content Anthrone Assay 17.8 23.4 131 1.3

Sr. No. Parameter Method Cell Lysate of Serotype PNU 1 % Increase Fold Increase

Normal Condition (37°C) Heat Shock Condition (45°C)

1 Protein Content Lowry Assay 9.7 11.1 114 1.1
2 Polysaccharide Content Anthrone Assay 6.3 2.7 43 0.4

Sr. No. Parameter Method Cell Lysate of Serotype PNU 6B % Increase Fold Increase

Normal Condition (37°C) Heat Shock Condition (45°C)

1 Protein Content Lowry Assay 6.5 12.6 194 1.9
2 Polysaccharide Content Anthrone Assay 6.4 16.2 253 2.5

Sr. No. Parameter Method Cell Lysate of Serotype PNU 19F % Increase Fold Increase

Normal Condition (37°C) Heat Shock Condition (45°C)

1 Protein Content Lowry Assay 15.0 14.3 95 1.0
2 Polysaccharide Content Anthrone Assay 5.0 4.7 94 0.9
Observations:
1. The cell lysates of three of the PNU serotypes indicated response to heat shock condition (45°C) versus 37°C.
2. The combined cell lysate of three PNU serotypes of 1, 6B and 19F showed an overall 1.3 fold increase in total protein content in response to heat shock condition (45°C) versus 37°C.

Conclusions:
1. The heat shock condition of "45°C" is able to trigger increase in overall protein content by
PNU serotypes indicating that HSPs might be effectively induced by this heat shock
procedure.
2. The increase in heat shock induced protein content by PNU serotypes was seen in
following order: PNU 6B > PNU 1 > PNU 19F.
3. The cocktail of combination of three PNU serotypes of "1 + 6B + 19F", exposed to 45°C,
exhibited global increase in total protein and polysaccharide content. This suggests that this
cocktail may be added as an adjuvant to the current vaccine formulation as a synergistic
approach.

5) Example 5: Effect of other stress inducing stimuli on production of stress proteins in microbial pathogen (S. pneumoniae serotype 1)
A) Effect of pH stress on production of stress proteins in S. pneumoniae serotype 1

Refer Figure no 25 for Bacterial Growth Curve Analysis for Serotype 1 (pH stress) B) Effect of osmotic stress on production of stress proteins in S. pneumoniae serotype 1

Table 14: Bacterial growth and expansion study on effect of pH stress on production of stress proteins in S. pneumoniae serotype 1
Culture Age (Hr.) Temperature(0C) OD at 590 nm Sample
storage
volume
mL) pH Remarks
0 37 0.35 7.16 -
3 37 2.12 7.16 -
4 37 4.26 7.13 -
5 37 6.1 50 mL x 4 no 6.7 pH
maintained
at 6.5
6 37 5.6 50 mL x 4 no 6.5 -
7 37 3.8 50 mL x 4 no 6.5 -
Terminated -

Table 15: Bacterial growth and expansion study on effect of osmotic stress on production of stress proteins in S. pneumoniae serotype 1
Culture Age (Hr.) Temperature(0C) OD at 590 nm Sample
storage
volume
mL) OSMO (mOsm/L)
0 37 0.82 - 290
3 37 2.21 - 289
4 37 4.36 - 287
5 37 5.63 50 mL x 4 no 320
6 37 6.9 50 mL x 4 no 380
7 37 3.8 50 mL x 4 no 428
Terminated
Refer Figure no 26 for Bacterial Growth Curve Analysis for Serotype 1 (osmotic stress)

C) Effect of Heat and osmotic stress on production of stress proteins in S. pneumoniae serotype 1

Table 16: Bacterial growth and expansion study on effect of Heat and Osmotic stress on production of stress proteins in S. pneumoniae serotype 1
Culture Age (Hr.) Temperature(0C) Osmo (mOsm/L) OD at 590 nm Sample
storage
volume
mL) Remark
0 37 290 0.33 - -
2 37 286 0.68 - -
3 37 282 2 - -
4 37 280 3.9 - -
5 37 => 45 300 5.8 50 mL x 3 no Temp set to 450C,
and
osmotic
stress
increased
6 45 322 7.6 50 mL x 3 no -
7 45 353 5.8 50 mL x 3 no -
8 45 427 1.9 50 mL x 5 no -
Terminated
Refer Figure No. 27: Bacterial Growth Curve Analysis for Serotype 1 (Heat and Osmo Stress)
D) Effect of Heat and pH stress on production of stress proteins in S. pneumoniae serotype 1

Table 17: Bacterial growth and expansion study on effect of Heat and pH stress on production of stress proteins in S. pneumoniae serotype 1
Culture Age (Hr.) Temperature(0C) pH OD at 590 nm Sample
storage
volume
mL) Remark
0 37 7.12 0.4 - -
2 37 7.11 0.71 - -
3 37 7.13 2.2 - -
4 37 7.15 4.2 - -

5 37 => 45 6.68 6.5 50 mL x 3 no Temp set to
450C and pH
maintained
at 6.7
6 45 6.69 7.8 50 mL x 3 no -
7 45 6.5 7.4 50 mL x 3 no -
8 45 6.5 3.5 50 mL x 5 no -
Terminated
Refer Figure No. 28: Bacterial Growth Curve Analysis for Serotype 1 (Heat and pH Stress)
E) Effect of Heat, osmotic and pH stress on production of stress proteins in S. pneumoniae serotype 1

Table 18: Bacterial growth and expansion study on effect of Heat, osmotic and pH stress on production of stress proteins in S. pneumoniae serotype 1
Culture Age (Hr.) Temperature(0C) Osmo (mOsm/L) pH OD
at 590 nm Sample
storage
volume
mL) Remark
0 37 291 7.13 0.33 - -
2 37 290 7.12 0.76 - -
3 37 288 7.11 2.3 - -
4 37 287 7.13 4.3 - -
5 37 => 45 322 6.68 6.9 50 mL x 3 no Temp set
to 450C
and pH
maintained
at 6.5,
osmotic
increased
6 45 353 6.5 7.8 50 mL x 3 no -
6.5 45 402 6.5 7 - -
7 45 421 6.5 6.1 50 mL x 3 no -
8 45 458 6.5 3 50 mL x 5 no -
Terminated
Refer Figure No. 28: Bacterial Growth Curve Analysis for Serotype 1 (Heat osmotic and pH Stress)

Procedure for enrichment of HSP in tested serotypes was optimized. Prior to the Heat shock, osmotic shock or/and pH stress treatment around 50 mL of sample was removed and centrifuged at 8,000 rpm for 20 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
The Heat shock, osmotic shock or/and pH stress treatment was performed at 5th Hour. Every 30 minutes 50 mL of sample was removed and centrifuged at 8,000 rpm for 20 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
The total harvest volume obtained at 8 hour age was = 600 mL. It was centrifuged at 8000 rpm for 30 minutes. The pellet and supernatant were then separated and kept at 8°C and -20°C respectively.
Sonication was carried out in 40 ml PBS buffer (pH 7.4) along with ^2 g of cell pellet separately for every sample.
Analysis of Samples: The supernatant and cell pellets were stored at 8°C and -20°C
respectively.
The supernatants were filtered with 0.2y filter and analyzed further.
Further analysis was performed on a 12% Non-Reducing SDS-PAGE Gel to ascertain the enrichment of HSPs.
This procedure was applied for enrichment of HSPs from serotypes such as S. pneumoniae serotype 1.
Protein marker used was ‘Bio-Rad precision plus protein standards ( dual color )’ containing ten recombinant protein bands of 10 kD, 15 kD, 20 kD, 25 kD, 37 kD, 50 kD, 75 kD, 100 kD, 150 kD and 250 kD.

Interpretation:
The combined Heat shock osmotic shock and pH stress treatment is able to trigger increase in overall protein content of PNU serotype 1 indicating that HSPs might be effectively induced by this procedure.
After heat shock, increase in band intensity at about 25kD, 37 kD, 50 kD, 60 kD and 70 kD observed.

Example 5: Characterization of novel HSPs found in S. pneumoniae (PNU) protein lysates
The novel HSPs probably induced in response to the HS treatments were further investigated by Proteomic Analysis using Mass Spectrometry (via MS based Protein/Peptide Identification Using a LC-MS set-up).
The following three S. Pneumoniae strains were investigated for effect of variable temperature conditions on expression of immunogenic proteins. Each of the strains exhibited induction of various proteins in response to the elevated temperature conditions. The following table summarizes the heat shock treatment.
The spectrum of proteins induced as Heat Shock Proteins (HSPs) from three distinct S. Pneumoniae serotype, namely S. Pneumoniae serotype 1 (PNU01), S. Pneumoniae serotype 6B (PNU6B) and S. Pneumoniae serotype 19F (PNU19F), were further studied for proteomic analysis. The objective of this approach was to identify potential HS protein/s candidate responsible for direct or indirect role in inducing/improving immunogenicity of the vaccine prepared from HSPs from these distinct strains.
The protein lysate samples from the PNU01, PNU16B and PNU19F strains, post-exposure to various temperature conditions, were subjected to following steps to identify expression of novel HSPs.
Step 1: SDS-PAGE analysis to optimize the condition for visualization of protein-band of interest extraction
SDS PAGE Analysis of Whole Cell Extracts from PNU01, 6B and 19F at Varying Temperature Conditions: S. Pneumoniae protein antigens identified from the protein extracts from three different strains; 01, 6B, and 19F. The whole cell proteins collected from S.

Pneumoniae strains heat-shocked at temperature >37°C such as 42°C/45°C (at varying times) were immune-blotted by SDS-PAGE. The whole cell extracts were loaded at two different protein concentrations. The position of possible HSPs is indicated by the arrows at the left of the target protein band. The proteins induced in response to the heat conditions were identified and are indicated by marked gel-bands on the gel image (indicated in Figure No. 19).
Step 2: Selective extraction of specific protein bands
Initially, proteins were separated following the routine standard operating protocol that allows separation of wide-range of molecular weight of protein (250 kDa to 10 kDa). SDS-PAGE separated protein profile of different samples is shown in Figure 19. The HSP proteins of interest, in the range of 100 kDa to 25 kDa, were further separated with increased run time. The marked gel protein bands were carefully excised; proteins in the bands were digested in sample buffer and further subjected to identification using mass-spectrometry based method.
Step 3: Subjecting the extracted proteins to Tandem mass spectrometry
Proteomic Analysis using Mass Spectrometry (via MS based Protein/Peptide Identification Using a LC-MS set-up):
a) The digested peptides were separated on a reverse phase liquid chromatographic column through a linear gradient of 0.1%FA and acetonitrile developed over a period of 110 minutes.
b) Data were collected in data-dependent mode and acquired MS/MS data were searched against Streptococcus pneumoniae proteome database using Morpheus software. Peptide Identification was performed with the following criteria: (a) Trypsin digested peptides with 2 missed cleavages allowed, (b) peptide tolerance < 10 ppm, (c) > 1 unique peptide, (d) FDR < 1%, and (e) Fixed Modification -carbamidomethylation of Cysteine and Variable Modification – Oxidation of Methionine
c) The protein concentration of the whole cell extract samples from S. Pneumoniae strains of PNU19F, PNU6B and PNU01 were found to be 4.7µg/µl, 3.0 µg/µl, 3.5 µg/µl respectively.
Step 4: Data analysis using Uniprot software for identification of specific HSP proteins.

Altogether, from all three strains, 9 separate gel bands (group of proteins) were subjected to MS analysis. A total number of 244 protein candidates were screened for MS analysis. The following table indicates the total number of proteins per S. Pneumoniae strain processed for MS browser. The tandem mass tag (TMT) labelling was used to assess relative protein quantities.
The following table indicates total number of band observed in three types of PNU cell lysates.

Table No.20: Table indicates total number of band observed in three types of PNU cell lysates.
Sr. No. Strain MW (kDa) Total Bands
1 19F 70 0
2 19F 60 3
3 6B 70 24
4 6B 55-60 47
5 6B 37 48
6 1B 70 22
7 1B 60 24
8 1B 50 29
9 1B 37 47
Total Proteins 244
The following table indicates various important protein candidates identified by MS in three different S. Pneumoniae strains in response to variable heat conditions.
Strain PNU 01:

Table No.21: Table indicates various important protein candidates identified by MS in S. Pneumoniae strain PNU 01
No. Strain MW (kDa) Protein Name Significance

1. PNU01 37 HRCA_STRZJ Heat-inducible transcription repressor Not yet reported for novel induction. Negative regulator of class I heat shock genes (grpE-dnaK-dnaJ and groELS operons). Prevents heat-shock induction of these operons.
2. PNU01 70 Tyrosine-protein kinase This protein is involved in the pathway capsule polysaccharide biosynthesis, which is part of Capsule biogenesis.
3 PNU01 50 Chaperone protein DnaK It binds to different regions of CD40 on macrophages and dendritic cells to trigger memory. It can induce the upregulation of cytokines, MHC class II co-stimulatory molecules, and promoting DC migration to the T-cell area of the lymph node (Colaco, 2013).
Observations:
The proteomic analysis of cell lysate from serotype 1 of S. Pneumoniae showed presence of three proteins corresponding to 37, 50 and 70kDa.
The proteomic analysis of cell lysate was analyzed using proteome database and mass spectrometric analysis.
Conclusions:
1. The heat shock treatment of 45°C was found to induce heat-shock proteins such as chaperones in serotype 1 of S. Pneumoniae.
2. The HSPs observed in serotype 1 of S. Pneumoniae might be directly responsible for immunogenicity observed in rabbit model and synergistic potential in mice model.
3. The further correlation of above HSPs to biological activity warrants further investigation.

Strain PNU 6B:

Table No.22: Table indicates various important protein candidates identified by MS in
S. Pneumoniae strain PNU 6B
No. Strain MW (kDa) Protein Name Significance
1. PNU6B 70 Translation initiation factor Leads to increased production of proteins
2. PNU6B 55 Putative
competence-damage inducible protein Susceptibility tolerance of Pneumo bacteria [(UniProtKB - C1CGM5 (CINA_STRZJ)]
3. PNU6B 50 Chaperone protein DnaK It binds to different regions of CD40 on macrophages and dendritic cells to trigger memory. It can induce the upregulation of cytokines, MHC class II co-stimulatory molecules, and promoting DC migration to the T-cell area of the lymph node (Colaco, 2013).
4. PNU6B 37 Manganese ABC transporter substrate-binding lipoprotein This protein is responsible for immunostimulatory effect (Chan, 2019)
Observations:
The proteomic analysis of cell lysate from serotype 6B of S. Pneumoniae showed presence of three proteins corresponding to 37, 50 and 70kDa.
The proteomic analysis of cell lysate was analyzed using proteome database and mass spectrometric analysis.
Conclusions:
1. The heat shock treatment of 45°Cwas found to induce heat-shock proteins such as chaperones in serotype 6B of S. Pneumoniae.

2. The HSPs observed in serotype 6B of S. Pneumoniae might be directly responsible for immunogenicity observed in rabbit model and synergistic potential in mice model.
3. The further correlation of above HSPs to biological activity warrants further investigation.
Strain PNU 19F:

Table No.23: Table indicates various important protein candidates identified by MS in S.
Pneumoniae strain PNU 19F
No. Strain MW (kDa) Protein Name Significance
1. PNU19F 60 Protein translocase subunit SecA Encoded in RD10, a pathogenicity island with an atypical GC content that is associated with invasive pneumococcal disease. Pathogenicity islands account for greater than half the genomic diversity observed between isolates (PubMed:11463916, PubMed:16861665). The main function of this island seems to be correct synthesis and export of pneumococcal serine-rich repeat protein PsrP (PubMed:18507531, PubMed:20714350).
2. PNU19F 60 GroL (chaperonin) This protein has been known to be responsible for immunogenicity against (Khan, 2009) lethal injection in mice.
3 PNU19F 37 HRCA_STRZ J Heat-inducible transcription repressor Not yet reported for novel induction. Negative regulator of class I heat shock genes (grpE-dnaK-dnaJ and groELS operons). Prevents heat-shock induction of these operons.
Observations:
The proteomic analysis of cell lysate from serotype 19F of S. Pneumoniae showed presence of three proteins corresponding to 37 and 60kDa.
The proteomic analysis of cell lysate was analyzed using proteome database and mass spectrometric analysis.

Conclusions:
1. The heat shock treatment of 45°C was found to induce heat-shock proteins such as
chaperones in serotype 19F of S. Pneumoniae.
2. The HSPs observed in serotype 19F of S. Pneumoniae might be directly responsible for immunogenicity observed in rabbit model and synergistic potential in mice model.
3. The further correlation of above HSPs to biological activity warrants further investigation.
4. The proteomic analysis provides important information about the pro-immunogenic
contents in the cell lysates of PNU serotypes. The various HSPs revealed by MS analysis
strongly suggest that the HS treatment might be able to directly or indirectly aid in inducing
the immunogenicity in the preclinical and clinical settings. Further studies would be
warranted to isolate, purify and test the specific HSPs, observed in cell lysates of PNU
serotypes, for studying the underlying mechanism for pro-immunogenic properties.

Example 5: Inactivation of whole cells
Rationale:
In the previous section, cells were enriched with HSPs using heat shock method. It is essential to remove pathogenicity of the cells, exposed to heat shock conditions, without altering the immunogenicity potential. This is important because the whole cells as well as cell lysates would be used as antigen in the pr-clinical immunogenicity studies. The inactivation was performed using different methods such as formaldehyde treatment, heat treatment and treatment with 70 % alcohol.
Conclusion:
Process of inactivation by formaldehyde treatment will be adopted for further experimentation.

Example 6: Designing of Final Formulation & stability studies
Rationale:
The final formulation is considered as the composition of the final dose used for injection in clinical setting. This section describes components of different formulations standardized for further pre-clinical or clinical use. The stability studies were essential to establish shelf life of vaccine and to assign expiry date to vaccine. For this purpose, formulation/s is kept at varying storage temperatures for different time intervals. After each time interval, testing of samples was carried out using tests assessing quality of the vaccine or final formulation.

Table 24: Description of formulation
Sr. No Name of Formulation Components of formulation
1 Formulation A Inactivated Whole cell bacteria or cell lysate of
Serotype 1, 19F and 6B, 37°C + Pneumo 10 vaccine
2 Formulation B Inactivated Whole cell bacteria or cell lysate of
Serotype 1, 19F and 6B, 45°C + Pneumo 10 vaccine
3 Formulation C Inactivated Whole cell bacteria or cell lysate of
Serotype 1, 19F and 6B, 45°C in buffer with Alum
4 Formulation D Pneumo 10 vaccine
5 Formulation E Inactivated Whole cell bacteria or cell Serotype 19F, 37°C + Pneumo 10 vaccine lysate of
6 Formulation F Inactivated Whole cell bacteria or cell Serotype 19F, 45°C + Pneumo 10 vaccine lysate of
7 Formulation G Inactivated Whole cell bacteria or cell Serotype 19F, 45°C in buffer with Alum lysate of
8 Formulation H Inactivated Whole cell bacteria or cell Serotype 6B, 37°C + Pneumo 10 vaccine lysate of
9 Formulation I Inactivated Whole cell bacteria or cell Serotype 6B, 45°C + Pneumo 10 vaccine lysate of
10 Formulation J Inactivated Whole cell bacteria or cell Serotype 6B, 45°C in buffer with Alum lysate of
11 Formulation K Inactivated Whole cell bacteria or cell lysate of

Serotype 1, 37°C + Pneumo 10 vaccine
12 Formulation L Inactivated Whole cell bacteria or cell lysate of Serotype 1, 45°C + Pneumo 10 vaccine
13 Formulation M Inactivated Whole cell bacteria or cell lysate of Serotype 1, 45°C in buffer with Alum
14 Formulation N Inactivated Whole cell bacteria or cell lysate of Serotype 19F and 6B, 37°C + Pneumo 10 vaccine
15 Formulation O Inactivated Whole cell bacteria or cell lysate of Serotype19F and 6B, 45°C + Pneumo 10 vaccine
16 Formulation P Inactivated Whole cell bacteria or cell lysate of Serotype 19F and 6B, 37°C in buffer with Alum
17 Formulation Q Inactivated Whole cell bacteria or cell lysate of Serotype 1, 19F and 6B, 45°C in buffer with Alum
18 Formulation R Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 37°C + Pneumo 10 vaccine
19 Formulation S Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 45°C + Pneumo 10 vaccine
20 Formulation T Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 37°C in buffer with Alum
21 Formulation U Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 6B, 45°C in buffer with Alum
22 Formulation V Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 37°C + Pneumo 10 vaccine
23 Formulation W Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 45°C + Pneumo 10 vaccine
24 Formulation X Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 37°C in buffer with Alum
25 Formulation Y Inactivated Whole cell bacteria or cell lysate of Serotype 1 and 19F, 45°C in buffer with Alum

Preparation of formulation
107cells/ml, added aseptically in the formulation as disclosed above and mixing @ 120 RPM for 1 hour at room temperature.
Pneumo 10 vaccine:
Pneumo 10 vaccine is the SIIPL’s PNEUMOSIL® is a 10-valent pneumococcal conjugate vaccine comprising Streptococcus pneumoniae serotypes (1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A), which covers over 70% of invasive pneumococcal-disease causing serotypes. The product covers over 71% IPD causing serotypes, and targeting the Indian Universal Immunization Program and Asian, African and other low and middle-income countries under the GAVI Advanced Market Commitment (AMC). SIIPL’s Pneumosil has received World Health Organization (WHO) prequalification on 18 December, 2019.
Stability studies of Formulation

Table No. 25: Formulation A: 3 Months @ 25°C +2°C/ 65%+5% RH
Parameter Appearance pH Osmolality (mOsmol/Kg) Al content Thiomersal content
Initial Whitish liquid turbid 5.72 360 0.135 0.0050
1 Month Whitish liquid turbid 5.78 371 0.132 --
2 Month Whitish liquid turbid 5.68 355 0.140 --
3 Month Whitish liquid turbid 5.73 368 0.138 0.0053

Table No. 26: Formulation B: 3 Months @ 25°C +2°C/ 65%+5% RH
Parameter Appearance pH Osmolality (mOsmol/Kg) Al content Thiomersal content
Initial Whitish turbid liquid 5.79 371 0.130 0.0051

1 Month Whitish liquid turbid 5.63 382 0.138 --
2 Month Whitish liquid turbid 5.72 369 0.143 --
3 Month Whitish liquid turbid 5.76 374 0.133 0.0049

Table No. 27: Formulation C: 3 Months @ 25°C +2°C/ 65%+5% RH
Parameter Appearance pH Osmolality (mOsmol/Kg) Al content Thiomersal content
Initial Whitish liquid turbid 5.83 368 0.142 0.0049
1 Month Whitish liquid turbid 5.87 372 0.139 --
2 Month Whitish liquid turbid 5.79 388 0.147 --
3 Month Whitish liquid turbid 5.86 373 0.142 0.0052
Conclusion:
Present study indicated that formulation is stable till 3 months when stored at 25°C +2°C/ 65%+5% RH.

Example 7: Assessment of Immunogenicity Potential of Streptococcus Pneumoniae vaccine (proteins and whole cells) in Animal Model (BalbC mice)
Determination of Immunogenicity of Heat Shock Proteins in mouse model (in form of whole cells and cell lysates)
The whole cells and cell lysates obtained from three separate strains of S. Pneumoniae (1, 19F, 6B) exposed to heat shock conditions were investigated for potential to trigger immunogenicity in mouse model (BalbC mice). Additionally, the ability of whole cells and cell lysates in boosting the immunogenicity of PNEUMOSIL, an existing pneumococcal vaccine (10 valent), was assessed in BalbC mouse model.

Table No. 28: Table illustrates the various treatment groups involved Mice Immunogenicity Study
Sr. No. Group Treatment Group Temperature Condition Sample Type Animals / Group Dose per animal# Injection Volume Day of Injection Blood
(serum)
collection
day
1 G1 Vehicle (PBS) alone - PBS 8 NA 100µL 1, 14, 21 21, 42
2 G2 Buffer with alum alone - Alum Buffer 8 NA 100µL

3 G3 PNEUMOSIL - Commercial Vaccine 8 NA 100µL

4 G4 PNU 1 37°C Whole Cells 8 107 cells 100µL

5 G5 PNU 1 45°C
8 107 cells 100µL

6 G6 PNU 6B 37°C
8 107 cells 100µL

7 G7 PNU 6B 45°C
8 107 cells 100µL

8 G8 PNU 19F 37°C
8 107 cells 100µL

9 G9 PNU 19F 45°C
8 107 cells 100µL

10 G10 PNU 1, 19F, 6B 45°C
8 107 cells 100µL

11 G11 PNEUMOSIL + 1, 19F, 6B 37°C
8 107 cells 100µL

12 G12 PNEUMOSIL + 1, 19F, 6B 45°C
8 107 cells 100µL

13 G13 PNU 1 37°C Cell Lysates 8 25|ag 100µL

14 G14 PNU 1 45°C
8 25|amg 100µL

15 G15 PNU 6B 37°C
8 25|amg 100µL

16 G16 PNU 6B 45°C
8 25|amg 100µL

17 G17 PNU 19F 37°C
8 25|amg 100µL

18 G18 PNU 19F 45°C
8 25|amg 100µL

19 G19 PNU 1, 19F, 6B 45°C
8 25|amg 100µL

20 G20 PNEUMOSIL + 1, 19F, 6B 37°C
8 25|amg 100µL

21 G21 PNEUMOSIL + 1, 19F, 6B 45°C
8 25|amg 100µL

# Dose of "Whole Cells" is 10^7 cells for respective serotype cells and 25|ig for respective serotype.

Table No. 29: Summary of Immunogenicity Potential of S. Pneumoniae PNU Serotypes 1, 6B and 19F HSPs in Mouse Model
Sr. No. Sample Type PNU Serotype Temperature Condition Sera Collection Time Point Immunogenicity Fold
Increase in
IgG over
Buffer Fold Increase
in IgG over
37°C
1 Buffer Alone - - 21 0.003 - -
2 Pneumosil Commercial Vaccine -
1.127 376 -
3 Whole Cells Vaccine + (1+6B+19F) 37°C
1.151 384 -
4 Whole Cells
45°C
1.783 594 2
5 Cell Lysates Vaccine + (1+6B+19F) 37°C
0.696 232 -
6 Cell Lysates
45°C
2.083 694 3
7 Buffer Alone - - 42 0.001 - -
8 Pneumosil Commercial Vaccine -
1.832 1832 -
9 Whole Cells Vaccine + (1+6B+19F) 37°C
2.109 2109 -
10 Whole Cells
45°C
2.48 2480 1
11 Cell Lysates Vaccine + (1+6B+19F) 37°C
1.445 1445 -
12 Cell Lysates
45°C
2.425 2425 2
Observations:
The immunogenicity data is assessed in terms of IgG antibody levels observed in response to the PNU whole cells and cell lysates in mouse model.
The IgG induction is measured as Geometric Mean (geometric mean) of colorimetric response (OD) observed in IgG ELISA.
At 21 Day time point, the GM observed in response to Pneumosil + Whole Cells and Cell Lysate of 1+6B+19F Serotype exposed to (45°C) is considered as immunogenic if the fold rise in OD is higher than 4 fold over OD observed in response to buffer condition. (Refer Figure No. 20)
At 42 Day time point, the GM observed in response to Pneumosil + Whole Cells and Cell Lysate of 1+6B+19F Serotype exposed to (45°C) is considered as immunogenic if the fold rise in OD is higher than 4 fold over OD observed in response to buffer condition. (Refer Figure No. 21)

At 21 and 42 Day time point, the Whole Cells and Cell Lysate of 1+6B+19F Serotype exposed to (45°C) display the additive or synergistic potential with Pneumosil when added to Pneumosil towards the overall immunogenicity.
Conclusions:
1. The heat shock proteins produced in response to 45°C treatment was found to induce immunogenic response observed via a serological assay in mice.
2. The HSPs in whole cells as well as protein lysates in S. Pneumoniae serotypes possess immunogenic potential.
3. The induction of immunogenicity response (GM and >4 fold rise) was observed in
following order: Pneumosil+ Cell Lysate 1+6B+19F > Pneumosil+ Whole Cells 1+6B+19F.
4. As compared to Pneumosil alone, Pneumosil when combined with the cocktail of HSPs of
PNU serotypes, 1+6B+19F, exhibited synergistic immunogenicity potential. This indicates
that S. Pneumoniae HSPs might provide an additive value to the current composition of
Pneumosil.

Example 8: Effect of PNU Serotype 1, 6B and 19F inactivated whole cells and cell lysate in inducing immunogenicity against PNU serotypes not covered by PNEUMOSIL (Pneumo 10 Vaccine)
To understand the breadth of the immunogenicity induction, shown predominantly by heat shock proteins in whole cells and cell lysates, we further investigated the effectiveness of HSPs in PNU whole cells and cell lysates for IgG induction against non-vaccine PNU serotypes such as Serotype 2, 3, 15B and 18C.
The mice sera, at Day 21 time point, which exhibited higher immunogenicity induction than Pneumosil (Figure No. 22) was investigated for efficacy against the serotypes which are not present in Pneumosil vaccine (non-vaccine serotypes) such as Type 2, 3, 15B and 18C available at the time of study. Pneumosil has not previously shown to induce antibodies against these Type 2, 3, 15B and 18C PNU serotypes. The mice sera obtained from various treatments was studied for immunogenicity (IgG) induction above four PNU serotypes, namely Type 2, 3, 15B and 18C.
Refer Figure No. 22: Induction of Immunogenicity by PNU Heat Shock Proteins against novel PNU Serotypes in Mice
Interpretation:
The effect of inducing the IgG’s against “Type 2, 3, 15B and 18C PNU Serotype” was evaluated using and indirect ELISA method. Figure 22 shows that, as compared to PNEUMOSIL alone, the combination of PNEUMOSIL and mixture of cell lysates of PNU 1, 6B and 19F, exhibited higher induction of immunogenicity than Pneumosil alone (rightmost bar in Figure No. 22). A response was observed for IgG induction against the non-vaccine serotypes Type 2, 3, 15B and 18C PNU. This suggested that the HSPs might possess cross-reactivity with other PNU serotypes and have a capability to broaden the activity of currently licensed vaccine, Pneumosil.

Example 9: Determination of Immunogenicity of Heat Shock Proteins in rabbit model (in form of whole cells and cell lysates)
The whole cells and cell lysates obtained from three separate serotypes (1B, 19F, 16B) of S. Pneumoniae exposed to heat shock conditions (3h; 45°C) were investigated for potential to trigger immunogenicity in rabbit model. Additionally, the ability of whole cells and cell lysates in boosting the immunogenicity of PNEUMOSIL, an existing pneumococcal vaccine, was also assessed in rabbit model.
Results:

Sr. No.
1 2 3 4 5 6 7 8 9 10 Sample Type PNU Serotype Temperature Condition Sera Collection Time Point Immunogenicity Fold Increase in IgG over Buffer

Buffer Alone - - 21 0.05 -

Whole Cells 6B+19F 45°C
0.19 4.0

Cell Lysates 6B+19F 37°C
0.09 2

Cell Lysates 6B+19F 45°C
0.17 4

Cell Lysates 1+6B+19F 45°C
0.28 6

Buffer Alone - - 42 0.06 -

Whole Cells 6B+19F 45°C
0.11 2

Cell Lysates 6B+19F 37°C
0.06 1

Cell Lysates 6B+19F 45°C
0.21 4

Cell Lysates 1+6B+19F 45°C
0.36 7
Observations:
The immunogenicity data is assessed in terms of IgG antibody levels observed in response to the PNU whole cells and cell lysate in NZW rabbit model.
The IgG induction is measured as Geometric Mean (geometric mean) of colorimetric response (OD) observed in IgG ELISA.
At 21 Day time point, the GM observed in response to Whole Cells and Cell Lysate of 1+ 6B+19F Serotype exposed to (45°C) is considered as immunogenic if the fold rise in OD is higher than 4 fold over OD observed in response to buffer condition. (Refer Figure 23)
At 42 Day time point, the GM observed in response to Cell Lysate of 1+6B+19F Serotype exposed to (45°C) is considered as immunogenic if the fold rise in OD is higher than 4 fold

over OD observed in response to buffer condition. (Refer Figure 24)
Conclusions:
1. The heat shock proteins produced in response to 45°C treatment were found to induce immunogenic response observed via a serological assay in rabbits.
2. The HSPs in whole cells as well as protein lysate in S. Pneumoniae serotypes possess immunogenic potential.
3. The cell lysate of PNU serotype 6B+19F, exposed to 45°C, exhibited 7-fold higher immunogenicity response than the cell lysate of PNU serotype 6B+19F, exposed to 37°C. This clearly indicates that the HSPs have immunogenicity potential in rabbit model.
3. The induction of immunogenicity response (GM and >4 fold rise over buffer) was observed in following order: Cell Lysate 6B+19F > Whole Cells of 6B+19F.

We Claim,
1. A method for production of stress protein, wherein the said method comprising the steps
of:
d) culturing microbial pathogen in an optimized media at optimum pH, osmolarity and temperature,
e) subjecting the said microbial pathogen to a stress inducing stimuli, selected from the group comprising of heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, hydrostatic pressure, pH stress, ethanol shock, chemical stress, UV-stress, cold stress and a combination thereof, and
f) recovering a preparation comprising stress proteins from the stress induced microbial pathogen.

2. The method as claimed in claim 1, wherein the stress proteins is selected from the group comprising of stress protein-peptide complex, Heat shock proteins (HSPs) of one particular family or mixture comprising different heat shock proteins derived from different families of heat shock subtypes or derived from different classes based on molecular weight or any other stress protein including non native forms, truncated analogs, muteins, fusion proteins as well as other proteins capable of mimicking the peptide binding and immunogenic properties of a stress protein which is present in microbial pathogen.
3. The method as claimed in claim 2, wherein the Heat shock proteins is selected from the group comprising of ClpP, ClpL, DnaK, Dna J, GroEL, GroES , hspX , acr2, AAA +, clp A / B, HtpG, TRIC, CCT, IbpA, IbpB, calrecticulin, hsp20-30, hsp37, hsp40, hsp50, hsp60, hsp70, hsp72, hsp90, hsp100, grp94, grp75, BiP/grp78, grp75/mt, gp96 or heat shock proteins have a molecular weight in the range of 10KDa to 1000kDa more particularly 37 kDa, 50kDa, 60 kDa or 70 kDa.
4. The method as claimed in claim 2, wherein the stress protein-peptide complexes is selected from the group comprising of hsp20-peptide, hsp30-peptide, hsp37-peptide, HSP40-peptide, HSP50-peptide, Hsp60-peptide, Hsp70-peptide hsp72-peptide, Hsp90-peptide and hsp100-peptide complexes or from any other stress protein-peptide complex class.

5. The method as claimed in claim 1, wherein the microbial pathogen is subjected to a single stress inducing stimuli selected from the group comprising of heat stress, cold shock, oxidative stress, heavy metal stress, osmotic stress, metabolite restriction, nutrient starvation, pH stress, hydrostatic pressure, ethanol shock, chemical stress, UV-stress and cold stress.
6. The method as claimed in claim 1, wherein the microbial pathogen is subjected to a two different types of stress inducing stimuli selected from the group comprising of:
l) first stress inducing stimuli is heat stress and second stress inducing stimuli is osmotic
stress; m) first stress inducing stimuli is heat stress and second stress inducing stimuli is pH
stress; n) first stress inducing stimuli is heat stress and second stress inducing stimuli is
oxidative stress; o) first stress inducing stimuli is heat stress and second stress inducing stimuli is
metabolite restriction; p) first stress inducing stimuli is heat stress and second stress inducing stimuli is cold
shock; q) first stress inducing stimuli is heat stress and second stress inducing stimuli is nutrient
starvation; r) first stress inducing stimuli is heat stress and second stress inducing stimuli is heavy
metal stress; s) first stress inducing stimuli is heat stress and second stress inducing stimuli is
hydrostatic pressure; t) first stress inducing stimuli is heat stress and second stress inducing stimuli is UV-stress; u) first stress inducing stimuli is heat stress and second stress inducing stimuli is
chemical stress; and v) first stress inducing stimuli is heat stress and second stress inducing stimuli is ethanol
shock.
7. The method as claimed in claim 1, wherein the microbial pathogen is subjected to a three
different types of stress inducing stimuli selected from the group comprising of:
xxii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is pH stress;

xxiii) first stress inducing stimuli is heat stress and second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is media optimization;
xxiv) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is oxidative stress;
xxv) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is metabolite restriction;
xxvi) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is cold shock;
xxvii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is nutrient starvation;
xxviii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is heavy metal stress;
xxix) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is hydrostatic pressure;
xxx) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is UV-stress;
xxxi) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is chemical stress;
xxxii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
osmotic stress and third stress inducing stimuli is ethanol shock;
xxxiii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is oxidative stress;
xxxiv) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is cold shock;
xxxv) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is nutrient starvation;
xxxvi) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is heavy metal stress;
xxxvii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is hydrostatic pressure;
xxxviii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is UV-stress;

xxxix) first stress inducing stimuli is heat stress, second stress inducing stimuli is
metabolite restriction and third stress inducing stimuli is chemical stress; xl) first stress inducing stimuli is heat stress, second stress inducing stimuli is metabolite
restriction and third stress inducing stimuli is ethanol shock; xli) first stress inducing stimuli is heat stress, second stress inducing stimuli is oxidative
stress and third stress inducing stimuli is pH stress;
xlii) first stress inducing stimuli is heat stress, second stress inducing stimuli is
oxidative stress and third stress inducing stimuli is hydrostatic pressure.
8. The method as claimed in any one of the claims 1, 5, 6 and 7, wherein the heat stress comprises raising the temperature of about 3°C to 15°C above the normal growth temperature of the microbial pathogen at a rate ranging between 0.1°C to 3°C per minute for a time period ranging between 10 minutes to 12 hours.
9. The method as claimed in claim 1, 5, and 8, wherein the microbial pathogen is subjected to a two or more heat stress comprising different temperature set-points.
10. The method as claimed in any one of the claims 1, 5, 6 and 7, wherein the osmotic stress comprises subjecting the microbial pathogen to high osmolarity by raising the concentration of salts selected from the group of Na+, K+, Ca++ in the range of about 0.1 to 1M.
11. The method as claimed in any one of the claims 1, 5, 6 and 7, wherein the pH stress comprises reducing the pH of the microbial pathogen to a pH below the optimal pH of microbial pathogen by adding an acid.
12. The method as claimed in any one of the preceding claims, wherein said microbial pathogen is selected from the group comprising of viruses, bacteria, fungi, protozoa and intracellular parasites causing a disease in the mammal.
13. The method as claimed in claim 12, wherein said microbial pathogen is virus selected from the group comprising of human immunodeficiency virus, hepatitis A virus, hepatitis B, hepatitis C, human papillomavirus, Kaposi's Sarcoma- Associated Herpesvirus, Herpes Simplex virus, Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus, St Louis Encephalitis virus, Rift Valley Fever virus, Influenza viruses, corona virus, rhinovirus, adenovirus, SIV, rotavirus, human papilloma virus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus, varicella virus, huntavirus and cytomegalovirus.

14. The method as claimed in claim 12, wherein said microbial pathogen is bacteria selected from the group comprising of Salmonella serovar strains S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. typhimurium and S. Enteritidis, Shigella, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Staphylococcus spp., Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcus spp , Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus(group Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX.), Neisseria meningitidis, Haemophilus pneumonia, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Enterococcus faecalis, Enterococcus faecium, Enterococcus faecalis Neisseria gonorrhoeae, Bacillus anthracis, Vibrio cholerae, Pasteurella pestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, M. catarrhalis , Klebsiella pneumoniae ,Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Shigella spp., Erlichia spp., Rickettsia spp and N. meningitidis (A, B, C, D, W135, X, Y, Z and 29E), anthrax, Bacillus Calmette–Guérin (BCG).
15. The method as claimed in claims 12 and 14, wherein said microbial pathogen is bacteria selected from the group comprising of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43,44, 45, 46, 47F, 47A, 48.
16. The method as claimed in claims 12, 14 and 15, wherein said microbial pathogen is bacteria selected from the group comprising of Streptococcus pneumoniae serotypes 1, 6B and 19F.

17. The method as claimed in any one of the preceding claims, wherein the step of recovering
a preparation comprising stress proteins from the stress induced microbial pathogen
comprises the steps of:
d) harvesting the microbial cellular material by centrifugation, clarification or Tangential Flow filtration (TFF)
e) subjecting the harvested microbial cellular material to lysis comprising sonication or shaking of glass beads to obtain a cell lysate comprising stress protein complexes,
f) subjecting the cell lysate to centrifugation to obtain a supernatant comprising of stress proteins.
18. The method as claimed in any one of the preceding claims, wherein the step of recovering
a preparation comprising stress proteins from the stress induced microbial pathogen
comprises the steps of:
a) harvesting the microbial cellular material,
b) subjecting the harvested microbial cellular material to inactivation to obtain an inactivated whole cell comprising stress proteins.

19. The method as claimed in claim 18, wherein the harvested microbial cellular material are inactivated using one of the method selected from the group comprising of formaldehyde treatment, heat treatment and treatment with 70 % alcohol.
20. The method as claimed in any one of the preceding claims, wherein the recovered preparation of stress proteins is subjected to further purification and isolation.
21. The method as claimed in any one of the preceding claims, wherein the method further comprising mixing the recovered preparation of stress proteins with at least one pharmaceutically acceptable excipient to produce an immunogenic composition.
22. The method as claimed in any one of the preceding claims, wherein the method further comprising mixing the recovered preparation of stress proteins with an adjuvant selected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate or a mixture thereof to produce an immunogenic composition.
23. The method as claimed in any one of the preceding claims, wherein the method further comprising combining the recovered preparation of stress proteins with at least one of protein, polysaccharide or polysaccharide-protein conjugate or used as a carrier protein in a polysaccharide-protein conjugate or with whole cell bacteria or fusing with protein to produce an immunogenic composition.

24. The method as claimed in claim 23, wherein the recovered preparation of stress protein is combined with at least one of protein, polysaccharide or polysaccharide-protein conjugate or used as a carrier protein in a polysaccharide-protein conjugate or with whole cell bacteria or fusing with protein derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5,6, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F,16A, 16F, 17A, 17F, 18, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48 to produce an immunogenic composition.
25. The method as claimed in claim 23, wherein the recovered preparation of stress protein is combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A to produce an immunogenic composition.
26. An immunogenic composition selected from the group comprising of:
a) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 19F
b) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 6B
c) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1
d) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1 and 6B
e) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 19F and 6B
f) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1 and 19F
g) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1, 6B and 19F
h) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1, 6B and 19F; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A

i) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1 and 19F; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A
j) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1 and 6B; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A
k) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 19F and 6B; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A
l) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 19F; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A
m) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 1; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A
n) Inactivated Whole cell bacteria or cell lysate of Streptococcus pneumoniae Serotype 6B; combined with polysaccharide-protein conjugate derived from Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F and 6A.
27. The immunogenic composition as claimed in claim 26, wherein the composition comprises of a pharmaceutically acceptable buffer selected from the group of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer saline, borate, Histidine buffer, Succinate buffer, HEPES, TRIS or Citrate-phosphate.
28. The immunogenic composition as claimed in claim 26, wherein the composition additionally comprises an adjuvant selected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate or a mixture thereof.
29. The immunogenic composition as claimed in claim 26, wherein the said immunogenic composition is capable of inducing the formation of antibodies against Streptococcus pneumoniae serotypes 1, 5, 6B, 9V, 14, 19A, 19F, 23F, 7F, 6A, 2, 3, 15B and 18C.