WO1998033521A1

WO1998033521A1 - Pneumococcal polysaccharide conjugate and pneumococcal polysaccharide cholera toxin b subunit conjugate vaccines

Info

Publication number: WO1998033521A1
Application number: PCT/KR1998/000007
Authority: WO
Inventors: Seo Young Jeong; Ick-Chan Kwon; Yong-Hee Kim; Seung-Yong Seong
Original assignee: Korea Institute Of Science And Technology
Priority date: 1997-01-31
Filing date: 1998-01-17
Publication date: 1998-08-06
Also published as: AU5681298A; KR19980067137A

Abstract

A microencapsulated Streptococcus pneumoniae capsular polysaccharide and Cholera toxin B subunit conjugate and a Streptococcus pneumoniae capsular polysaccharide conjugate vaccine which can effectively protect against pneumococcal respiratory infections, such as meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.

Description

PNEUMOCOCCAL POLYSACCHARIDE CONJUGATE AND PNEUMOCOCCAL POLYSACCHARIDE CHOLERA TOXIN B SUBUN1T

CONJUGATE VACCINES

TECHNICAL FIELD

The present invention relates to a novel microencapsulated conjugate and conjugate vaccines. More particularly, the present invention relates to a microencapsulated pneumococcal capsular polysaccharide conjugate vaccine for oral administration and a pneumococcal capsular polysaccharide conjugate vaccine for intranasal administration, wherein the vaccines evoke mucosal as well as systemic antibody response to antigen conjugates and provide protection against challenges by Streptococcus pneumoniae.

BACKGROUND ART

Acute respiratory infection by S.pneumoniae results in more than one million deaths per year worldwide. It is believed that pneumococcus are the leading cause of pneumonia, meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis and conjunctivitis.

Current vaccines composed of purified pnuemococcal capsular polysaccharide (PS) of the most prevalent 23 serotype provide coverage against more than 90% of invasive pneumococcal isolates. Although pneumococcus vaccines (such as PNEUMOVAX® 23) have been available for more than 10 years, their efficacy is only prevalent among healthy young adults. A major disadvantage with these vaccines are that purified PS of pneumococcus do not reliably induce protective antibody response in children younger than 2 years old or the very old. In addition, the vaccines appear to confer only limited protection on patients with certain underlying diseases such as immunodeficiencies and hematologic malignancies. These limitations are mainly due to the fact that the polysaccharide used are thymus-independent type 2 antigens and consequently induce a relatively weak, short lived response and no immunologic memory.

The S.pneumoniae has been known to colonize in the nasopharynx and to invade into the blood stream after an inflammatory activation of lining cells. A major entry site of pneumococci into the human body is through the mucosal surfaces. Immunization against pneumococci at the mucosal surfaces is most effective because the mucosal immune system is capable of responding to the invading pathogens in the gastrointestinal, respiratory and urogenital tracts by producing pathogen-specific secretory IgA (slgA) and long term serum IgG antibodies. The local slgA has been known to prevent both the colonization at the mucosal tissues and the spread into the systemic circulation more efficiently compared to systemic antibodies. Higher induction of slgA responses could often be achieved through a direct immunization via gut associated lymphoid tissue (GALT), specifically through the Peyer's Patches (PP) of the gastrointestinal (Gl) tract.

Studies have shown promise that mucosal vaccination with PS antigens could be effective against S.pneumoniae. Oral immunization of PS, PS encapsulated with liposomes, and PS co-orally administered with a cholera toxin (CT), and their effects on mice have been studied. Co-oral administration of CT is known in the art to stimulate mucosal IgA responses against itself, serve as an immunity stimulating carrier molecule, and act as an adjuvant for simulating mucosal immune responses against admixed foreign antigens.

Oral administration of PS and PS encapsulated with liposomes induced PS-specific systemic IgM responses, however, an antigen specific

IgG and slgA were not detected, even after a booster immunization was administered. Co-oral administration of PS and CT induced PS specific IgA response. Unfortunately, co-oral administration of CT is not a viable option, because CT is known to cause electrolytes and fluid losses that cause a life-threatening diarrheal disease.

As described above, the induction of slgA and serum IgG could be achieved by the direct immunization of the mucosal surfaces, specifically, through the Peyer's Patches. Therefore, biodegradable and biocompatible microspheres have been used to deliver antigens into the Gl tract. Gelatin, poly^Alactice), poly glycolic acid and albumin, for example, have been used for fabrications of the microspheres. However, the harsh conditions of preparing the microspheres, such as the organic solvent used or the high temperature can denature the antigens. Alginate microspheres (AM) have been preferably used as a carrier of antigens because these microspheres can be prepared in aqueous solutions and at room temperature.

Studies have shown that AM of less than 10 μm in diameter can be uptaken at the small intestine, preferably less than 5 μm in diameter. Microspheres of less than 5 μm in diameter are transported through the efferent lymphatics, while those larger than 5 μm in diameter remain in the Peyer's Patches; therefore, the size of the microspheres are an important consideration, i.e., the smaller in diameter the more effectively can the AM be uptaken in the Peyer's Patches.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide vaccines that can protect against pneumococcal respiratory infections such as meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.

To achieve this object, according to the present invention a microencapsulated Streptococcus pneumoniae capsular polysaccharide and Cholera toxin B subunit conjugate to be administered orally and prepared by: dissolving a S.pneumococcal capsular polysaccharide (PS) serotype in deionized water and activating the PS; coupling the activated PS with a space r m o l ec u le ( S M ) u nde r gen t l e st i rri n g ; add in g 1-ethyl-3(3-dimethylaminoproyl)-carbodiimide (EDC) slowly into a solution containing the PS and a cholera toxin B subunit; stopping the reaction, and thus forming crude conjugates; centrifuging the crude conjugates; dialyzing a supernatant; removing unreacted proteins; adding a microshpere solution dropwise to n-octanol containing an emulsifier; homogenizing the mixture; adding n-octanol containing CaCI₂ into the emulsion while stirring the entire medium slowl with m gnetic stirrer; curing the microspheres; collecting the microspheres o to filters; filteri g a d washi g the microspheres a d the drying in vacuo; and, dissolving aliquot of an alginate in a conjugate aqueous solution, for thereby completing the microencapsulation of the conjugate.

To achieve the above object, according to the present inevntion, there is also provided a Streptococcus pneumoniae capsular polysaccharide conjugate to be administered intranasally, which is prepared by: dissolving a serotype S.pneumococcal capsular polysaccharide (PS) in deionized water and activating the PS; coupling the activated PS with a spacer molecule (SM) under gentle stirring; adding 1 -ethyl-3(3-dimethylaminoproyl)-carbodiimide (EDC) slowly into a solution containing the PS and a cholera toxin B subunit; stopping the reaction, and thus forming crude conjugates; centrifuging the crude conjugates, and dialyzing a supernatant; and, removing unreacted proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 (a-c) are scanning electron micrographs of alginate microspheres formed with different alginate concentrations: (a) 1 % (w/v); (b) 3% (w/v); and, (c) 5% (w/v).

Figures 2(a-c) are scanning electron micrographs of alginate microspheres formed with various concentration of CaCI₂ in n-octanol: (a) 2% (w/v); (b) 4% (w/v); and, (c) 8% (w/v).

Figures 3(a-b) are scanning electron micrographs of alginate microspheres formed with various surfactant: (a) Span 80; and, (b) HCO-10.

Figure 4 is a scanning electron micrograph of the alginate microspheres containing PS19-CTB.

Figure 5 is a cumulative release profile of an entrapped PS19-CTB from alginate microspheres at 37° C in phosphate buffered saline (PBS).

Figure 6 is a graph illustrating the antigenicity and binding capacity of PS19-CTB to GM1-gangiioside after microencapsulation.

Figure 7 is a graph illustrating an intestinal anti-PS19 IgA responses and serum IgM responses following peroral immunization with various doses of PS19-CTB entrapped in the alginate microspheres.

Figures 8(a-c) are confocal laser scanned images of alginate microspheres (AM) with FITC-dextran uptaken in the Peyer's Patch. Wherein Fig. 8(a) illustrates the uptake of the microspheres at hour three (3), and Figs. 8(b) and 8(c) at hour five (5), respectively. Figure 8(c) is magnified at x1890.

Figures 9(a-c) are respectively a Sepharcyl S-300 chromatography elution profile of an nonconjugated PS and CTB, a Sepharcyl S-300 chromatography elution profile of a crude conjugate of PS and CTB in the void fraction, and a picture taken by a scanning electron microscope of the microspheres entrapping the PS-CTB conjugates.

Figure 10 shows several graphs illustrating serum anti-PS IgM, IgG, and IgA, and as well as bronchoalveolar IgA and intestinal IgA antibody response of mice two weeks after the 3rd oral vaccination.

Figure 1 1 is a graph showing an anti-CTB antibody response of the mice after an oral immunization with vaccines according to the present invention.

Figure 12 shows several graphs illustrating clearance of pneumococci from the bronchoalveoli (top) and from the blood (bottom) of the mice immunized with vaccines according to the present invention.

BEST MODE(S) FOR CARRING OUT THE INVENTION

The present invention provides compositions which are useful for immunization against pneumococcal respiratory infections, and which are effective against other types of pneumococcal infections such as meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis. It has been found that an oral administration of a microencapsulated pneumococcal capsular polysaccharide (PS) - cholera toxin B subunit (CTB) conjugate vaccine, i.e., AM(PS-CTB), and intranasal administration of a PS-CTB conjugate vaccine, inhibit challenges by live S.pneumoniae. The PS-CTB conjugate vaccines administered intranasally may or may not be administered together with the AM(PS-CTB) conjugate vaccine.

Studies conducted on the composition according to the present invention have demonstrated that oral administration of the AM(PS-CTB) and intranasal administration of the PS-CTB, induced significant increase in levels of serum IgM, serum IgG, serum IgA, bronchoalveolar IgA and intestinal IgA. The AM(PS-CTB) induced bronchoalveolar and intestinal IgA, and prominent serum IgG and serum IgA responses.

Because of the advantageous characteristics of AM such as resistance to the low pH in the stomach and to the proteolytic enzymes of the gut, and the high encapsulation efficiency, alginate microspheres were considered as a proper carrier for the efficient delivery of antigens to the Peyer's Patch (PP) and a concomitant transport through the lymphatics, see FIGS 8(a-c). (The uptake of the microsperes was observed by substituting the PS-CTB conjug te with fluorecein isothioc n te (FIT )-conjug te dextr ne.) As previously described, studies have shown that alginate microspheres of less than 5 μm in diameter can be uptaken at the small intestine; however, the current technology for making the alginate microspheres is still restricted to a size limit of 5 μm in diameter or larger. A method for preparing the alginate microspheres of less than 5 μm in diameter to be used as a carrier for an oral mucosal delivery was developed for the present invention.

Initial studies indicated that serum IgM and intestinal IgA responses were significantly higher in mice immunized with the microencapsulated conjugate [i.e., the AM (PS-CTB)] than in an non-immunized group, and the studies also showed no marked elevation in serum IgG level. Therefore, the ratio of the PS and CTB was changed by inserting a spacer molecule therein between. Preparation of the AM

Various preparation parameters can affect the alginate microshperes' particle size and distribution. The alginate concentration range can vary from 1 % (w/v) to 10% (w/v), more preferably between l %-6% (w/v), but most preferably less than 5% (w/v).

The choice of a proper organic solvent is an important factor in determining the particle size and distribution. Certain solvents can denature the encapsulated antigens, and the particle size decreases as the polarity of the organic solvent increases. In the present invention, n-octanol is used as the organic solvent in which CaCI₂ is dissolved and slowly diffused into an aqueous phase containing the alginate for a diffusion controlled interfacial gelation. The stirring rate is important to form microemulsion droplets with the desired diameter. The stirring rate range is between 5,000 rpm and 10,000 rpm, preferably at a stirring rate of 8,000 rpm for one (1 ) hour. In addition, further continuous stirring at a low speed is required until the gelation is completed to ensure that the hardened microspheres are isolated as discrete particles.

The particle size is also dependent upon the concentration of CaCI₂ in the n-octanol and the rate of the addition into the medium. The smallest microspheres(<5 μm) are formed when 1 % (w/v) of CaCI₂ in n-octanol is used. The size of the microspheres could be further reduced by spraying CaCI₂ solution onto the emulsion rather than by adding dropwise using a syringe.

Preparation of the PS-Spacer conjugate

Five to fifty milligrams, more preferably 5 to 25 milligrams, but most preferably 10 mg of pneumococcal capsular polysaccharide (PS) serotype selected from 1 , 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10F, 11A, 11 B, 1 1 C, 1 1 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43, 44, 45, 46, 47A, 47F, and 48 (Danish designation) is dissolved in 3 to 10 ml of deionized water and activated with 5 to 15 mg of cyanogen bromide at a pH of 10.5 for 6 to 15 minutes, preferably 10 minutes, in an ice cold water bath. The activated PS is coupled to a spacer molecule (SM), such as 6-aminocaproic acid (AC), or molecules of C4 to C18, such as but not limited to glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, β-alanine, poly-L-lysine, alanine, sistine, homocysteine etc., at a ratio between 1 :10 to 1 :1000, preferably at 1 :750 (mol/mol) under gentle stirring at room temperature for more than 4 hours but less than 15 hours at a pH of 7.2. The reaction product is dialyzed against three changes of deionized water.

Preparation of the PS-CTB conjugate

The conjugation between the PS-SM and CTB is performed using the EDC method as described in, Peeter, C.C. et al. A comparative study of the immunogenicity of pneumococcal type 4 polysaccharide and oligosaccharide tetanus toxoid coniuαates in adult mice. J. Immunol. 146, 4308-4314 (1991 ); Beuvery, E.C., van Rossum, F. & Nagel, J. Comparison of the induction of immunoglobulin M and G antibodies in mice with purified pneumococcal type 3 and meninαococcal group C polysaccharide and their protein conjugates. Infect. Immun. 37, 15-22 (1982), both the disclosures of which are incorporated herein by reference. The PS-CTB is conjugated by slowly adding 5-50 mg, more preferably 5-35 mg, but most preferably 20 mg, of the PC-CTB to a final concentration of 0.1 M 1 -ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) into 0.5 to 10 ml, more preferably 0.5 to 3 ml, but most preferably 1 ml solution containing equal amounts (5-50 mg, more preferably 5-20 mg, most preferably 10 mg) of the PS-SM and cholera toxin B subunit (CTB), and incubated. The pH is maintained at 4.7 with the addition of 0.1 M HCI acid. The reaction is stopped by the addition of 4.8 mg of ethanolamine, or any base that can increase the pH, such as but not limited to NaOH, KOH, etc., after 4 hours, for thus forming crude conjugates. The crude conjugates are centrifuged, and the supernatant is removed and dialyzed against a phosphate buffer saline (PBS) solution. Unreacted proteins are removed by using a Sephacryl S-300 column chromatography in the PBS. Fractions that contain both the protein and PS are pooled and concentrated by using an Am icon Centriprep^®.

Preparation of the alginate microspheres containing PS-CTB

A l t h o u g h a n a l g i n ate of a n i o n i c copo lym e rs of 1 ,4-linked- β-D-mannuronic acid and α-L-guluronic acid is used as the microspheres, a person skilled in the art will appreciate that various kinds of polymers could be substituted as the alginate microspheres, such as but not limited to ethylene-vinyl acetate, polyiminocarbonate, poly (DL-lactide-co-glycolides), a polysaccharide such as dextran, puliuran, chitosan, cellulose, modified cellulose, a natural polyamide such as collagen, gelatin, albumin, synthetic polymers such as polylactide, polyglycolide and copolymers of polylactides and glycolides, polyorthoesters, polyanhidride, polyvinylpyrrolidone, polyphosphagene. As described above, the important factor is preparing microspheres with a diameter of less than 5 μm.

The alginate microspheres are prepared by a modified water in oil

(w/o) emulsion technique. Five to 10 milliliters of an alginate solution of 1 .0 - 5.0% (w/v) is added dropwise to 30 ml of n-octanol containing 5% (w/v) hydrogenated caster oil 10 (HCO-10), hydrogenated caster oil 60 (HCO-60), or Span-80^® which is used as an emulsifier, respectively. Aliquot of the alginate 1 -5% (w/v) is dissolved in 5 ml of the conjugate solution, thereby completing the microencapsulation of the conjugate.

The w/o microemulsions are prepared by homogenizing the mixtures for variable times and at various rpms with a homogenizer. Then, n-octanol containing CaCI₂ 1.0 - 8.0 (w/v) is added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres are cured after mixing with addition of 5.0 ml of 1-8% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres are collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm, or any hyrophobic filter membrane. Finally, the microspheres on the filters are washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours. See FIGS. 5 and 9(a-c), and wherein the anthrone reaction (solid line) and BCA protein assay (dotted line) were used to monitor the PS and protein contents before the conjugation. The arrow denotes the void volume. The GM1 -ELISA was used after the conjugation with anti-PS antibody (solid line) or with anti-CTB antibody (dotted line) as the primary antibody.

Confirmation of the conjugation and intact immunoαenicitv of the microencapsulated PS-CTB

The conjug tion of the PS nd the TB w s confirmed Seph cc I S-300 elution profiles. The completion of the conjugation was also confirmed by using an enzyme-linked immunosorbent assay with a GM1 -ganglioside (GMIg)-coated plate (GM1 -ELISA). As shown in FIG. 6, the released conjugate was neutralized (rectangle) or was not neutralized (circle) before applying into wells, respectively. The conjugate bound to the wells coated with GM1 g was detected by anti-PS antibody (filled symbol) and anti-CTB antibody (open symbol). The nonspecific binding of the conjugate was evaluated by using wells coated with bovine serum albumin as a control (triangle).

A solution containing the PS-CTB was applied to wells coated with GM1g and bound conjugates were detected with anti-CTB antibodies or with anti-PS antibodies. In one set of experiments, the conjugates were preincubated with a soluble GM1 g (3 μM) before adding to the GM1 g coated wells. To exclude the possibility of nonspecific binding of the conjugates to the wells, the binding of the conjugates to bovine serum albumin (BSA) was set as a control. The binding capacity was determined after two-fold dilutions of the conjugates. The optical density obtained from 2.4 mg/ml of the conjugates was regarded arbitrarily as representing 100% binding. The released conjugate showed binding affinities to GM1g and to anti-PS19 antibodies as well as to anti-CTB antibodies. The conjugates did not bind to GM1g if it was preincubated with the soluble GM1 g. The GM1 g neutralized the binding capacity of the conjugates completely. There was no specific binding of the conjugate to the BSA. This suggests that the binding activity of the conjugates toward GM1g and the immunoreactivity to the anti-PS19 antibodies or to the anti-CTB antibodies were maintained after the encapsulation.

Oral vaccination of Mice

Groups of 10 female Balb/c mice (Korea Advanced Institute of Science and Technology, Daeduk, Korea), 6-8 weeks old, were respectively orally administered three times at an interval of two weeks with mock microspheres, naked PS-CTB (i.e., without AM), AM(PS), AM(PS-CTB), and AM(PS-BSA). Five μg of cholera toxin (CT) was co-administered with antigens to four groups of the mice. The mice were kept under standardized conditions at ad libitum with pelletted food throughout the experiment. All the mice received 20 μg equivalent doses of the PS diluted in 500 μl of 0.1 M sodium bicarbonate buffer (pH 8.1) at a single time for peroral immunization. The doses were in the range of 20 - 500 μg/mouse for dose titration. The mice were orally administered with microspheres via a blunt-tipped feeding needle inserted into the stomach. Figure 11.

Intranasal immunization of mice

Two groups of the mice were vaccinated with three intranasal administrations of 20 μg PS (in 20 μl) in either encapsulated AM(PS-CTB) or naked form (PS-CTB). A group of mice immunized intranasally with mock microspheres served as a control.

Efficacy of the oral vaccination

The effects of the microencapsulation on the immunogenicity of the orally administered PS revealed that the oral immunization of the mice with naked conjugates (PS-CTB) induced significant increases in levels of serum IgM, serum IgA and intestinal IgA, however, no significant serum IgG and Bronchoalvelor IgA were detected. As shown in FIG. 10 the mean concentration (ng/ml) and standard error of mean (SEM) are represented by column and error bars, respectively. The encapsulated conjugates AM(PS-CTB) induced bronchoalveolar and intestinal IgA as well as serum antibody responses significantly higher than those vaccinated with PS-CTB (p<0.05), indicating that the microencapsulation of the PS-CTB enhances not only systemic IgG responses responsible for the long term immunity but also mucosal IgA responses in remote effector sites where the bacteria colonize, see FIGS. 7 and 10. The carrier effect on the immunogenicity of the orally administered PS was compared with the immune responses of the mice vaccinated with the AM(PS), AM(PS-CTB) and AM(PS-BSA), as shown in FIG. 10. The physical mixtures of the CTB and PS when microencapsulated and .used in immunization induced serum and mucosal immune responses not statistically different from those induced by the the AM(PS) (data not shown). The AM (PS-CTB) induced prominent serum IgG, serum IgA and intestinal IgA responses among these groups (p<0.05). Each bronchoalveolar IgA responses induced by the AM(PS), AM(PS-CTB), and AM(PS-BSA) was significantly higher than of the PS-CTB (FIG. 10). The serum IgG levels (15.4 ± 2.3 ng/ml) of the mice immunized with the AM(PS-CTB) remained significantly higher than those (4.0 ± 1.2 ng/ml) of the mock immunized mice at 14 weeks after the first immunization.

Efficacy of the intranasal vaccination

Systemic antibody responses of the mice were examined after the intranasal immunization. Immunization with the PS-CTB yielded significantly higher serum IgM responses (2,902 + 331 ng/ml) than that with the mock microspheres (460 + 82 ng/ml). However, the level of the serum IgM antibody induced by the PS-CTB was not significantly different from that of the AM(PS-CTB) (2,071 ± 294 ng/ml). The serum IgA (240 + 41 ng/ml) and serum IgG (31 ± 15 ng/ml) antibody responses of the mice immunized with the PS-CTB were significantly higher than those with mock microspheres (IgA: 25 ± 16 ng/ml and IgG 4 ± 1 ng/ml) and than those with the AM(PS-CTB) (IgA: 96 + 6 ng/ml and IgG 15 + 4 ng/ml). The higher immunogencity of the PS-CTB over the AM(PS-CTB) is ascribed to the fact that the unencapsulated vaccine antigen degrades less in the nasal cavity than in the intestine and also to the fact the smaller sized PS-CTB are uptaken preferentially to the microencapsulated ones. The effect of Cholera Toxin (CT)

Cholera toxins can modulate systemic and mucosal immune responses against the PS. Therefore, the CT was co-administered with the vaccines, as described above. See also FIG. 10. The PS-specific serum and mucosal antibody responses of the mice were significantly reduced in level when the CT was co-administered orally with the AM(PS-CTB) (p<0.05). Co-administration of the CT with the AM(PS-BSA), however, significantly enhanced the systemic antibody responses of the mice (p<0.05) without significant change in the mucosal IgA responses. The systemic anti-CTB antibody responses of the mice with the PS-CTB or with the AM(PS-CTB) were evaluated after the co-administration of the CT (Fig. 1 1 ). The serum anti-CTB IgG and IgA as well as mucosal anti-CTB IgA were observed to be increased when the CT was co-administered. This suggested that the down-regulation of the immune responses of the mice to the PS on the co-administration with the CT results from the effect of the administered of CT rather than from experimental artifacts.

Challenges with live ^.pneumoniae

Vaccination with oral administration of the AM(PS-CTB) or intranasal vaccination with the PS-CTB showed successful protection against challenges from live S.pneumoniae. A control group of mice receiving mock AMs were highly susceptible to infection with S.pneumoniae, while immunized mice were protected. Viable pneumococcus recovered from the lungs and blood of all groups of immunized mice showed significant reduction (>95%) in numbers as compared with the control (p<0.05). When delivered orally, the PS-CTB conferred limited protection on mice when compared with the AM(PS-CTB). Seventy five percent of the colonizing bacteria were inhibited by the oral immunization with the PS-CTB. On the contrary, more than 90% of the bacterial colonization in the lung tissue was inhibited by the oral immunization with the AM(PS-CTB). The bacterial colonization at the lung mucosa was most profoundly inhibited by the intranasal immunization with the PS-CTB. The mice vaccinated orally with the AM(PS-CTB) were significantly better protected against pneumococcal bacteremia than those intranasally immunized with the PS-CTB or with the AM(PS-CTB) (p<0.05).

ELISA

Blood was collected from a puncture of the retroorbital plexuses. The serum was harvested following coagulation at 4°C for 18 hours and centrifuged. Anti-PS19 antibodies were measured by ELISA. The quantity of the antibodies are expressed as the geometric mean of the optical density. Before assaying serum for the anti-PS19 antibodies, anti-cell wall polysaccharide (CPS) activity was neutralized by incubating the serum with a CPS solution (20 μg/ml in PBS, Statens Seruminstitut, Copenhagen, Denmark) for 2 hours at 37° C in an ELISA plate which was blocked with 5% skim milk (Difco, Detroit, Mich.). To calibrate the specific IgA, IgG or IgM levels, a purified immunoglobulin with known concentrations of isotopes served as a standard. The absorbance values of the standard immunoglobulin were determined by using a sandwich ELSIA.

Polystyrene microplates (Nunc Products, Roskilde, Denmark) were coated with PS19F by incubating 100 μl of the appropriate antigen solution (10 μg ml in PBA) per well for 2 hours at 37°C, and then over night at 4°C. Plates coated with the PS19F were washed six times with PBS containing 0.05% Tween 20 (PBST). The same washing was carried out after each subsequent antibody reaction. After washing, plates were coated with 5% skim milk for 1 hour at 37° C. Diluted specimens were added to paired wells and incubated for 2 hours at 37° C. After washing with PBST, 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin

(Cappel, Durham, NC) was incubated in each well for 2 hours at 37° C. After washing with PBST, 200 μl of substrate solution (0.04 mg of o-phenylenediamine dihydrochloride Sigma Chemical Co., St. Louis, MO, in substrate buffer from PIERCE Chemical Co.) was incubated in each well for 1 hour. The enzyme reaction was stopped by adding 50 μl of 2.0 N NH₂S0₄. The optical density (OD) of the wells were measured at 492nm. Serum specimens were diluted 10 times. Bronchoalveolar lavage fluid and gut samples were diluted 2 times. The net OD of each samples were determined by subtracting the OD from the OD sample which showed the lowest value among the control mice.

The sera, intestinal and bronchoalveolar lavage were pooled from each group of mice and analyzed for the mice immunized with naked PS-CTB and AM(PS-CTB). Five μg of the CT was co-administered with antigens in two groups of mice. Anti-CTB antibodies were measured by ELISA. Cut-off values for the antibody titer were determined by analyzing the sample from the mice immunized with mock microspheres (0.004 for serum IgG, 0.135 for serum IgA, 0.0145 for lung IgA, and 0.045 for intestinal IgA). All antibody titers of the samples derived from mock-immunized mice were less than 1:16 by these cut-off values.

Protection Assay

Protection of mice against intranasal challenge by live pneumococcus was assessed by recovering viable organism from lungs and blood of mice immunized with viable vaccines. Two weeks after the last immunization, the mice were anesthetized and challenged intranasally with 1 x 10⁶ colony forming units of S.pneumoniae 19F in 20 μl of medium. The CFU of pneumococci in the bronchoalveolar lavage fluid and in the blood were counted 18 hours after the challenge. As shown in figure 12, the gray and white bars represent the CFU of pneumococci in specimens obtained from the mice after intranasal or the oral vaccination, respectively. The extent of inhibition of bacteremia or of bacterial colonization in the broπchoalveoli are denoted by %inhibition representing 100 x (1-CFUtest/CFUcontrol). The filled and open circles depict ^inhibition of bacterial infection after the intranasal or oral vaccination, respectively.

Sample 1

Ten milligrams of pneumococcal capsular polysaccharide (PS19) (American Type Culture Collection, Rockville, MD) was dissolved in 3 ml of deionized water and activated with 10 mg of cyanogen bromide at a pH of 10.5 for 10 minutes in an ice cold water bath. The activated PS 19 was coupled to 6-aminocaproic acid (AC)(Sigma Chemical Co., St. Louis, MO) at a ratio of 1 :1 (w/w), i.e., 1 :750 mol/mol, under gentle stirring at room temperature for 12 hours at a pH of 7.2. The reaction product was dialyzed against three changes of deionized water.

The PS19-AM was conjugated by slowly adding 20 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma Chemical Co., St. Louis, MO) into 1 ml of a solution containing 10 mg of the PS19 and 10 mg of a cholera toxin B subunit (CTB) (List Biologicals Inc., Campbell, CA), and incubated for 4 hours. The pH was maintained at 4.7 by the addition of 0.1 M HCI. The reaction was stopped by the addition of 4.8 mg of ethanolamine after 4 hours, for thus forming crude conjugates. The crude conjugates were centrifuged (30 min; 50,000 x g), and the supernatant was removed and dialyzed against phosphate buffer saline [PBS, 0.20g/L KCI, 0.20g/L KH₂P0_4> 8.0g/L NaCI, 2.92 g/L Na₂HP0₄(12H₂0)]. Unreacted proteins were removed by using a Sephacryl S-300 column (Pharmacia Biotech, Uppsala, Sweden) chromatography in PBS. Fractions that contained both the protein and PS was pooled and concentrated by using an Am icon Centriprep^®. Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 1.0% (w/v) was added dropwise to 30 ml of n-octanol (Junsei Chemical Co., Tokyo, Japan) containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) (Nikko Chemical Co. Ltd., Tokyo, Japan) as an emulsifier as shown in FIG. 1. The mixture was homogenized (Ultr-turrax® disperser, IKA Werke, Staufen, Germany). Then, n-octanol containing CaCI₂ 1% (w/v) was added into the emulsion by an air sprayer (Fuso Seiki Co. Ltd., Tokyo, Japan) while stirring the entire medium slowly with a magnetic stirrer. The microspheres were cured after mixing by the addition of 5.0 ml of 8% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol (Oriental Chemical Industry, Seoul Korea). The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm (Alltech Associates, Inc., IL). Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Aliquot of alginate 1 % (w/v) was dissolved in 5 ml of the conjugate solution (prepared as described above), for thereby completing the microencapsulation of the conjugate.

Sample 2

The PS19-spacer conjugation and PS19-CTB conjugation were prepared as in sample 1.

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 3.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier, see FIG. 1. The mixture was o homogenized. Then, n-octanol containing CaCI₂ 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Aliquot of alginate 3% (w/v) was dissolved in 5 ml of the conjugate solution, for thereby completing the microencapsulation of the conjugate. See FIG. 4, 5% (w/v) HCO-60 and 1% (w/v) CaCI₂ in n-octanol as well as

5% (w/v) of an alginate solution in distilled water was used in the microspheres formation.

Sample 3

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier, see FIG. 1. The mixture was homogenized. Then, n-octanol containing CaCI₂ 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours. Aliquot of alginate 5% (w/v) was dissolved in 5 ml of conjugate solution, for thereby completing the microencapsulation of the conjugate.

Sample 4

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI₂ 2% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2. The microspheres were cured after mixing by the addition of 5.0 ml of 2% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Aliquot of alginate 5% (w/v) was dissolved in 5 ml of conjugate solution, for thereby completing the microencapsulation of the conjugate.

Sample 5

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI₂ 4% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2. The microspheres were cured after mixing by the addition of 5.0 ml of 4% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Sample 6

The PS19-spacer conjugation and PS19-CTB conjugation were prepared in sample 1.

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 60 (HCO-60) 5% (w/v) as an emulsifier. The mixture was homogenized. Then, n-octanol containing CaCI₂ 8% (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer, see FIG. 2. The microspheres were cured after mixing by the addition of 5.0 ml of 8% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Aliquot of alginate 5% (w/v) was dissolved in 5 ml of conjugate solution, for thereby completing the microencapsulation of the conjugate. Sample 7

The PS19-spacer conjugation and PS19-CTB conjugation prepared as in sample 1.

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing SPAN-80® 5% (w/v) (Nikko Chemical Co. Ltd., Tokyo, Japan) as an emulsifier, see FIG. 3. The mixture was homogenized. Then, n-octanol containing CaCI₂ 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres were cured after mixing by the addition of 5.0 ml of 1 % (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 mi of isopropyl alcohol and dried in vacuo for 18 hours.

Sample 8

The PS19-spacer conjugation and PS19-CTB conjugation prepared as in sample 1.

Alginate microspheres were prepared by a modified water in oil (w/o) emulsion technique. Five milliliters of alginate solution 5.0% (w/v) was added dropwise to 30 ml of n-octanol containing hydrogenated caster oil 10 (HCO-10) 5% (w/v) (Nikko Chemical Co. Ltd., Tokyo, Japan) as an emulsifier, see FIG. 3. The mixture was homogenized. Then, n-octanol containing CaCI₂ 1 % (w/v) was added into the emulsion by an air sprayer while stirring the entire medium slowly with a magnetic stirrer. The microspheres were cured after mixing by the addition of 5.0 ml of 1% (w/v) CaCI₂ followed by slow addition of 6 ml of isopropyl alcohol. The microspheres were collected onto polyvinylidene difluroide (PVDF) membrane filters with a pore size of 0.44 μm. Finally, the microspheres on the filters were washed with 20 ml of isopropyl alcohol and dried in vacuo for 18 hours.

Sample 9

Ten milligrams of pneumococcal capsular polysaccharide (PS19) (American Type Culture Collection, Rockville, MD) was dissolved in 3 ml of deionized water and activated with 10 mg of cyanogen bromide at a pH 10.5 for 10 minutes in an ice cold water bath. The activated PS19 was coupled to 6-aminocaproic acid (AC)(Sigma Chemical Co., St. Louis, MO) at a ratio 1 :1 (w/w), i.e., 1 :750 mol/mol, under gentle stirring at room temperature for 12 hours at a pH of 7.2. The reaction product was dialyzed against three changes of deionized water.

The PS19-CTB was conjugated by slowly adding 20 mg 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma Chemical Co., St. Louis, MO) into 1 ml of solution containing 10 mg of the PS19 and 10 mg of a cholera toxin B subunit (CTB) (List Biologicals Inc., Campbell, CA), and incubated for 4 hours. The pH was maintained at 4.7 by the an addition of 0.1 M HCI. The reaction was stopped by the addition of 4.8 mg of ethanolamine after 4 hours, for thus forming crude conjugates. The crude conjugate was centrifuged (30 min; 50,000 x g), and the supernatant was removed and dialyzed against phosphate buffer saline [PBS, 0.20g/L KCI, 0.20g/L KH₂P0₄, 8.0g/L NaCI, 2.92 g/L Na₂HP0₄(12H₂0)]. Unreacted proteins were removed by using a Sephacryl S-300 column (Pharmacia Biotech, Uppsala, Sweden) chromatography in PBS. Fractions that contained both the protein and PS was pooled and concentrated by using an Am icon Cent prep®. Thus, completing the conjugation of the PS19 and CTB.

Statistics

Unpaired, two-tailed Student's t-tests were used to compare the mean values of antibody levels and CFUs between the groups of mice. The values were considered statistically significant when p<0.05.

Although the preferred embodiment of the present invention has been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as recited in the accompanying claims.

INDUSTRIAL APP ICABILTY

The microencapsulated AM(PS-CTB) and PS-CTB conjugates prepared as described herein can be used as vaccines against

S.pneumoniae for the general public, but specifically, forchildren and infants, th e e l de rly , an d th ose w it h i m m u n o def i c ie n c i es o r immunodeficiency-associated diseases, such as AIDS or the like.

The compositions of the invention, i.e., the vaccines, can be administered to prevent respiratory infections. In accordance with one embodiment of the invention the vaccine can be administered orally, and in accordance with another embodiment the vaccine can be administered intranasally.

Claims

CLAIMSWhat is claimed is:

1. A microencapsulated Streptococcus pneumoniae capsular polysaccharide and cholera toxin B subunit conjugate composition comprising: a S.pneumoniae capsular polysaccharide (PS) linked to a spacer molecule, and the PS being covalently linked to a cholera toxin B subunit (CTB), and wherein the PS-CTB conjugate is microencapsulated in a microsphere.

2. The microencapsulated conjugate of claim 1 , wherein the pneumococcal capsular polysaccharide is a S.pneumoniae serotype selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10F, 11A, 11 B, 11 C, 11 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

3. The microencapsulated conjugate of claim 1 , wherein the microsphere is an alginate selected from the group consisting of anionic copolymers of 1 ,4-linked-╬▓-D-mannuronic acid and ╬▒-L-guluronic acid, ethylene-vinyl acetate, polyiminocarbonate, poly (DL-lactide-co-glycolides), dextran, pulluran, chitosen, cellulose, modified cellulose, collagen, gelatin, albumin, polylactide, polyglycolide, copolymers of polylactides, glycolides, polyorthoesters, polyanhydride, polyvinylpyrrolidone, and polyphosphagene.

4. The microencapsulated conjugate of claim 3, wherein a size of the alginate microspheres is between 0.1 to 5 ╬╝m.

5. The microencapsulated conjugate of claim 1 , wherein the spacer molecule is selected from the group consisting of a 6-aminocaproic acid, glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, ╬▓-alanine, poly-L-lysine, alanine, Sistine, and homocysteine.

6. The microencapsulated conjugate of claim 1 , wherein a ratio between the spacer molecule and the PS is between 1 :0.1 to 1 :10 (w/w), namely,

7500:1 to 75:1 (mol/mol).

7. The microencapsulated conjugate of claim 1 , wherein a ratio of the PS and CTB is between 10:1 to 1 :20 (mol/mol).

8. A Streptococcus pneumoniae capsular polysaccharide and cholera toxin B subunit conjugate composition comprising: a S.pneumoniae capsular polysaccharide (PS) linked to a spacer molecule, the PS being covalently linked to a cholera toxin B subunit.

9. The conjugate of claim 8, wherein the pneumococcal capsular polysaccharide is a S.pneumoniae serotype selected from group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10F, 11 A, 11 B, 11C, 11 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

10. The conjugate of claim 8, wherein the spacer molecule is selected from the group consisting of 6-aminocaproic acid, glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, ╬▓-alanine, poly-L-lysine, alanine, Sistine, and homocysteine.

11. The conjugate of claim 8, wherein a ratio between the spacer molecule and the PS is between 1 :0.1 to 1 :10 (w/w), namely, 7500: 1 to 75: 1 (mol/mol).

12. The conjugate of claim 8, wherein a ratio of the PS and CTB is between 10:1 to 1 :20 (mol/mol).

13. A process for preparing a microencapsulated Streptococcus pneumoniae capsular polysaccharide and Cholera toxin B subunit conjugate, comprising: a) dissolving a pneumococcal capsular polysaccharide (PS) in deionized water and activating the PS with cyanogen bromide in an ice cold water bath; coupling the activated PS with a spacer molecule (SM) with gentle stirring; b) adding 1-ethyl-3(3-dimethylaminoproyl)-carbodiimide (EDC) slowly into a solution containing PS-SM conjugates and a cholera toxin B subunit (CTB); maintaining the pH at 4.7 with an addition of 0.1 M HCI; stopping the reaction by the addition of ethanolamine, for thus forming crude conjugates; centrifuging the thusly formed crude conjugates, and dialyzing supernatant against phosphate buffer saline (PBS); removing unreacted proteins with a column chromatography in PBS; c) dissolving aliquot of an alginate in a conjugate aqueous solution; d) adding a mixed solution of the alginate and conjugate dropwise to n-octanol containing an emulsifier; homogenizing the mixture; adding n-octanol containing CaCI₂ into the emulsion with an air sprayer while stirring the entire medium slowly with a magnetic stirrer; curing microspheres; adding additional CaCI₂ followed by a slow addition of isopropyl alcohol into the mixture; collecting the microspheres onto filters; filtering and washing the microspheres with isopropyl alcohol, and then drying in vacuo; and, thereby completing the microencapsulation of the conjugate.

14. The process of claim 13, wherein the pneumococcal capsular polysaccharide is a S.pneumoniae serotype and selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10F, 11A, 11 B, 11 C, 1 1 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21 , 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

15. The process claim of 13, wherein the spacer molecule of step a) is selected from the group consisting of a 6-aminocapric acid, glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, ╬▓-alanine, poly-L-lysine, alanine, Sistine, and homocysteine.

16. The process of claim 13, wherein a ratio between the SM and the PS in step a) is 1 :0.1 to 1 :10 (w/w), namely, 7500:1 to 75:1 (mol/mol).

17. The process of claim 13, wherein a ratio between the PS and CTB in step b) is 10:1 to 1 :20 (mol/mol).

18. The process of claim 13, wherein the microsphere solution of step c) is a solution of an alginate selected from the group consisting of an anionic copolymers of 1 ,4-linked-╬▓-D-mannuronic acid and ╬▒-L-guluronic acid, ethyiene-vinyl acetate, polyiminocarbonate, poly (DL-lactide-co-glycolides), dextran, pulluran, chitosen, cellulose, modified cellulose, collagen, gelatin, albumin, polylactide, polyglycolide, copolymers of polylactides, glycolides, polyorthoesters, polyanhyd╧Çde, polyvinylpyrrolidone, and polyphosphagene.

19. The process of claim 13, wherein the alginate microsphere solution of step c) is between 1-5% (w/v).

20. The process of claim 13, wherein the emulsifier of step d) is selected from the group consisting of HCO-10, HCO-60 and Span-80┬«.

21. The process of claim 13, wherein in step d) the n-octanol containing CaCI₂ is between 1-8% (w/v).

22. The process of claim 13, wherein a size of the alginate microspheres produced is between 100nm to 5 ╬╝m.

23. The process of claim 13, wherein the filter of step d) is a polyvinylidene difluroide (PVDF) membrane filter with a pore size between

0.10 to 0.44 ╬╝m.

24. A process for preparing a Streptococcus pneumoniae capsular polysaccharide and Cholera toxin B subunit conjugate, comprising: a) dissolving a pneumococcal capsular polysaccharide (PS) in deionized water and activating the PS with cyanogen bromide in an ice cold water bath; coupling the activated PS with a spacer molecule (SM) under gentle stirring; b) adding 1-ethyl-3(3-dimethylaminoproyl)-carbodiimide (EDC) slowly into a solution containing PS-SM conjugates and a cholera toxin B subunit (CTB); maintaining the pH at 4.7 with an addition of 0.1 M HCI; stopping the reaction by an addition of ethanolamine, for thus forming crude conjugates; centrifuging the crude conjugates, and dialyzing supernatant against phosphate buffer saline (PBS); removing unreacted proteins with a column chromatography in PBS; and, c) dissolving aliquot of an alginate in a conjugate aqueous solution.

25. The process of claim 24, wherein the pneumococcal capsular polysaccharide is a S.pneumoniae serotype and selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10F, 11A, 11 B, 11C, 11 F, 12A, 12F, 13, 14, 15, 15A, 15B, 15C, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28F, 29, 31 , 32A, 32F, 33, 33A, 33B, 33C, 33F, 34, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41 A, 41 F, 42, 43, 44, 45, 46, 47A, 47F, and 48.

26. The process of claim 24, wherein the spacer molecule of step a) is selected from the group consisting of a 6-aminocapric acid, glycine, 4-aminobutanoic acid, aminoadipic acid, aminobenzoic acid, diaminodipropylamine (DADPA), succinic acid, 1 ,3-diamino-2-propanol, 1 ,6-diaminohexane (DAH), ethylenediamine (EDA), poly-L-aspartic acid, poly-L-glutamic acid, ╬▓-alanine, poly-L-lysine, alanine, Sistine, and homocysteine.

27. The process of claim 24, wherein a ratio between the SM and the PS in step a) is 1:0.1 to 1 :10 (w/w), namely, 7500:1 to 75:1 (mol/mol).

28. The process of claim 24, wherein a ratio between the PS and CTB in step b) is 10:1 to 1:20 (mol/mol).

29. A method for vaccinating against respiratory infection, comprising: administering a microencapsulated Streptococcus pneumoniae (PS) capsular polysaccharide and Cholera toxin B subunit (CTB) conjugate [AM(PS-CTB)]to inhibit said infections.

30. The method of cl im 29, wherein the infection is pneumoni , meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.

31. The method of claim 29, wherein the AM(PS-CTB) is administered orally.

32. A method for vaccinating against respiratory infection, comprising: administering a Streptococcus pneumoniae capsular polysaccharide (PS) and Cholera toxin B subunit (CTB) conjugate (PS-CTB) to inhibit said infection.

33. The method of claim 32, wherein the infection is pneumonia, meningitis, otitis media, bacteremia and acute exacerbations of chronic bronchitis, sinusitis, arthritis or conjunctivitis.

34. The method of claim 32, wherein the PS-CTB is administered intranasally.