CA2222965A1 - Oral administration of pneumococcal antigens - Google Patents

Oral administration of pneumococcal antigens Download PDF

Info

Publication number
CA2222965A1
CA2222965A1 CA002222965A CA2222965A CA2222965A1 CA 2222965 A1 CA2222965 A1 CA 2222965A1 CA 002222965 A CA002222965 A CA 002222965A CA 2222965 A CA2222965 A CA 2222965A CA 2222965 A1 CA2222965 A1 CA 2222965A1
Authority
CA
Canada
Prior art keywords
pspa
pneumococcal
pneumococci
host
adjuvant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002222965A
Other languages
French (fr)
Inventor
Masafumi Yamamoto
David E. Briles
Larry S. Mcdaniel
Hiroshi Kiyono
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UAB Research Foundation
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/458,399 external-priority patent/US6231870B1/en
Priority claimed from US08/482,981 external-priority patent/US6232116B1/en
Priority claimed from US08/657,751 external-priority patent/US6004802A/en
Application filed by Individual filed Critical Individual
Publication of CA2222965A1 publication Critical patent/CA2222965A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Oral or peroral administration, including intragastrically, of killed whole pneumococci, lysate of pneumococci and isolated and purified PspA, as well as immunogenic fragments thereof, particularly when administered with an adjuvant such as cholera toxin provides protection in a host, animal or human, against pneumococcal infection, including colonization, and systemic infection, such as sepsis. The ability to elicit protection against pneumococcal colonization in a host prevents carriage among immunized individuals, wich can lead to elimination of disease from the population as a whole.

Description

CA 0222296~ 1997-12-01 W O 96/39113 PCT~B96/01051 ORAL ADMINISTRATION OF PNE~MOCOCCAL ANTIGENS
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Serial No. 08/482,981, filed June 7, 1995, which itself is a continuation-in-part of application Serial No. 08/458,399, filed June 2, 1995, the disclosure of each of these applications is incorporated herein by reference.
Reference is made to application Serial No.
08/446,201, filed May 19, 1995 (Attorney Docket No.
454312-2018) as a continuation-in-part of copending United States patent application Serial No. 08/312,949, filed Septem.ber 30, 1994, which itself is a continuation-in-part of copending United States patent application Serial No. 08/246,636 filed May 20, 1994, which itself is a continuation-in-part of copending United States patent application Serial No. 08/048,896 filed April 20, 1993, which itself is a continuation-in-part of copending United States patent application Serial No. 07/835,698 filed February 12, 1992, which itself is a continuation-in-part of United States patent application Serial No.
07/656,773 filed February 15, 1991 (now abandoned). The disclosure of each of such related applications is incorporated herein by reference.
FIELD OF T}IE lNVI~ lON
This invention relates to oral ;mmlln;zation or administration of hosts, animals or hl]m~n~, with pneumococcal antigens to stimulate an immunological response and preferably provide protection against pneumococcal infection, preferably against colonization, and more preferably against systematic and humoral infection; and, to compositions therefor.
Several publications are referenced in this application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned; and each of these publications is hereby incorporated herein by CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 reference.
R~c~ RO ~ OF l~IE lNVI~ ON
Streptococcus pneumoniae causes more fatal infections world-wide than almost any other pathogen (Anonymous, 1991; Fraser, 1982). In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS (Anonymous, 1991). In the U.S.A., most fatal pneumococcal infections occur in individuals over 65 years of age, in whom S. pneumoniae is the most commlln;ty-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less-developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age (Berman et al., 1985; Greenwood et al., 1987; Spika et al., 1989, Bale, 1990). The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospects for control of pneumococcal disease problematical (Munoz et al., 1992; Marton et al., Klugman, 1990).
~ llm~n~ acquire pneumococci through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain for weeks or months. As many as 50~ or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection (Gray et al., 1980; Gray et al., 1981; Hendley et al., 1975). Studies of outbreak strains have suggested that even highly virulent strains can colonize without causing disease (Smillie et al., 1938;
Smillie et al., 1936; Gratten et al., 1980; DeMaria et al., 1984). These expectations have been recently confirmed using molecular probes to fingerprint individual clones (M.J. Crain, personal communication to one of the inventors). In some individuals, however, the carried organism can give rise to symptomatic sinusitis CA 0222296~ 1997-12-01 W O 96/39113 PCTnB96/01051 or middle ear infections. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause meningitis. Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10 of patients die and greater than 50~ of the r~m~;n~er have life-long neurological sequelae (Bohr et al., 1985;
Klein et al., 1981).
In elderly adults, the present 23-valent capsular polysaccharide vaccine is about 60~ effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine (Bolan et al., 1986; Shapiro et al., 1991). The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to elicit adequate responses to most polysaccharides ~Cowan et al., 1978; Gotschlich et al., 1977). Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease. A vaccine that protected against disease but did not reduce pneumococcal carriage rates would not, however, be expected to control the disease in ;mmllnocompromised individuals (Shapiro et al., 1991) and in unimmunized individuals. Such a vaccine would also not be expected to affect the rates of infection in imm.unized children prior to the development of an adequate antibody or immunological response.
A strategy that could control infections in all of these individuals would be any form of immunization that prevented or greatly reduced carriage, and hence transmission of pneumococci. In the case of immunization of young children with Haemophilus influenzae group b CA 0222296~ 1997-12-01 W O 96139113 PCT~B96/01051 polysaccharide-protein conjugates, it has been observed that carriage is reduced from about 4~ to less than 1%, (Barbour et al., 1993), a possible explanation of concomitant herd ;mmlln;ty (Chiu et al., 1994). If a vaccine could prevent colonization by pneumococci, such a vaccine would be expected to prevent virtually all pneumococcal infections in the immunized patients. Since even unimmunized patients must acquire pneumococci from others, a vaccine that reduced carriage should reduce infections in ;mm~lnocompromisedl as well as l~n;mml]n;zed patients. In fact, an aggressive ;mmlln;zation program, coupled with antibiotic treatment of ~emo~.ctrated carriers, might be able to largely eliminate the human reservoir of this organism. It may not be possible, however, to totally eliminate pneumococci since there are a number of reports that they have been found in laboratory rodents (Fallon et al., 1988). Whether these pneumococci are infectious for man, easily transmittable to man, or even pathogens in wild rodents is not known.
S. pneumoniae does not live free in the environment.
Although intramuscular immunization with capsular polysaccharide vaccines has been effective at reducing the incidence of pneumococcal sepsis in the elderly (Shapiro et al., 1991), it has not been reported to affect pneumococcal carriage rates in children up to 54 months of age (Douglas et al., 1986; Douglas et al., 1984). The principal determinant of specific immunity at mucosal surfaces is secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. Mucosal S-IgA response are predominantly generated by the common mucosal immune system (CMIS) (Mestecky, 1987), in which imml~nogens are taken up by specialized lympho-epithelial structures collectively referred to as musoca-associated - 35 lymphoid tissue (MALT). The term common mucosal immune system refers to the fact that ;mml]nization at any mucosal site can elicit an immune response at all other CA 0222296~ 1997-12-01 WO96139113 PCT~B96/01051 mucosal sites (Mestecky, 1987). Thus, ;m~n; zation in the gut can elicit mucosal ;mmlln;ty in the upper airways and visa versa. The best studied MALT structures are the intestinal Peyer's patches (Mestecky, 1987). Further, it is important to note that oral immunization can induce an antigen-specific IgG response in the systemic compartment in addition to mucosal IgA antibodies (McGhee, J.R. and H. Kiyono 1993, "New perspectives in vaccine development:
mucosal ;mmll~;ty to infections", Infectious Aqents and Disease 2:55-73).
Most soluble and non-replicating antigens are poor mucosal immunogens, especially by the peroral route, probably because they are degraded by digestive enzymes and have little or no tropism for the gut associated lymphoid tissue (GALT). A notable exception is cholera toxin (CT). CT is a potent mucosal immunogen (Elson, 1989; Lycke et al., 1986; Wilson et al., 1989) probably because of the GM1 ganglioside-binding property of its binding subunit, CTB, that enables it to be taken up by the M cells of Peyer's patches and passed to the underlying immunocompetent cells. In addition to being a good mucosal immunogen, CT is a powerful adjuvant which greatly enhances the mucosal ;mmllnogenicity of other soluble antigens co-administered with it (Elson, 1989;
Lycke et al., 1986; Wilson et al., 1989). Although it r~m~; n~ somewhat controversial, pure or recombinant CTB
probably does not have these properties when administered intragastrically (i.g.) as an adjuvant. Very small amounts (<l~g) of intact CT, however, can act synergistically with CTB as a powerful oral adjuvant (Wilson et al., 1990). This finding may account for apparent adjuvant activity of many commercial preparations of CTB that usually contain small amounts of contaminating CT.
The mechanisms by which CT and CTB act as adjuvants are not fully understood, but are certainly complex, and appear to depend on several factors CA 0222296~ 1997-12-01 WO96/39113 PCT~B96/0l051 including 1) the toxic activity associated with the ADT-ribosylating property of the Al subunit (Abbas et al., 1991); 2) increased permeability of mucosae (Abbas et al., 1991; Ziegler-Heitbrock et al., 1992); 3) enhanced antigen-presenting cell function (with increased levels of IL-1) (Abbas et al., 1991; Ziegler-HeitbroQk et al., 1992); as well as 4) direct stimulation of T and B cell activities (Elson, 1989; Lycke et al., 1986; Wilson et al., 1989; Wilson et al., 1990). This last point is controversial, however, as the in vitro effects of CT or CTB on T and B cells are generally inhibitory rather than stimulatory (Abbas et al., 1991). Nevertheless, numerous reports attest to the in vivo mucosal immunoenchancing effects of CT and of CTB coupled to antigens (Hakansson et al., 1994; Anderson et al., 1981; Liang et al., 1988;
Dagen et al., 1995; Dillard et al., 1994; Szu et al.
1989). Recent studies have shown that orally-~m; n; stered CT can induce Th2 type responses for antigen-specific serum IgG and mucosal IgA antibodies (Xu-Amano, J., H. Kiyono, R.J. Jackson, H.F. Staats, Fujihashi, P.D. Burrows, C.O. Elson, S. Pillai and J.R.
McGhee. 1993, "Helper T cell subsets for immunoglobulin A responses: Oral ;mmlln; zation with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in ~ucosa associated tissues", J. Exp. Med. 178:1309-1320, Xu-Amano, R.J. Jackson, K. Fujihashi, H. Kiyono, H.F. Staats and J.R. McGhee, 1994, "Helper Thl and Th2 cell responses following mucosal or systemic immunization with cholera toxin", Vaccine 12:903-911). Recently Elson et al. have shown that CT selectively inhibits CD8 cells, and therefore tends to abrogate suppressive effects (Elson et al., 1995).
Since ;~ml~n;ty to carriage would be expected to operate at the mucosal surface, any attempt to identify antigens for vaccines against carriage should include immunizations designed to elicit mucosal immune responses. Accordingly, the oral (including peroral, CA 0222296~ 1997-12-01 WO 96/39ll3 pcrnB96t intragastric) ;mmlln;zation or ~m; n; stration with pneumococcal proteins, as in the present invention has not, it is believed, been heretofore disclosed or suggested or, in addition, the evaluation of adJuvants, as in the present disclo~ure, has not, it is believed, ~ been heretofore taught or suggested.
S~MARY OF T~E INVENTION
In accordance with the present invention, it has now been surprisingly found that oral or peroral ~m; n; stration, preferably into the gut (e.g., stomach, intestines, digestive tract; intragastric), of pneumococcal surface protein A (PspA) or an ;mmllnogenic fragment thereof elicits an immunological response and can even provide protection to a host against pneumococcal infection such as colonization and/or systemic infection.
Accordingly, in one aspect, the present invention provides a method of protecting a host, preferably a hllmAn host, against pneumococci and/or systemic infection by oral or peroral ~m; n; stration to the host, preferably by administration into the gut (stomach, intestines, digestive tract; e.g., intragastrically) of the host, of an effective amount of at least one pneumococcal surface protein A (PspA) and/or an immunogenic fragment thereof containing at least one protection-eliciting epitope.
In another aspect, the present invention provides a method of eliciting an immunological response in a host against pneumococci and/or systemic infection by oral or peroral ~m; n; stration to the host, preferably ~m;ni stration into the gut (stomach, intestines, digestic tract; e.g., intragastrically) of the host, of an effective amount of at least one PspA and/or an immunogenic fragment thereof containing at least one epitope. More preferably, the response is protective and the epitope is protection-eliciting.

CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 The PspA may be in the form of killed whole S.
pneumoniae or a lysate of whole S. pneumoniae.
Alternatively, the PspA may be in the form of purified isolated protein or a fragment thereof (individually and/or collectively, for purposes only of shorthand in this specification, "PspA") may be obtained from bacterial isolates or may be formed recombinantly. The PspA can be from in vivo expression thereof by a suitable vector containing DNA coding for PspA by recombinant techniques. The PspA is preferably in a vaccine or imml~nogenic composition. Such a composition can include a pharmaceutically acceptable adjuvant and/or a pharmaceutically acceptable carrier.
The PspA may be mixed with pharmaceutically acceptable excipients which are compatible with the PspA.
Such excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
In a preferred aspect of the invention, the PspA is a~m; n; stered with non-toxic amounts of cholera toxin as an adjuvant.
The oral ~m; n; stration preferably is effected by delivery into the stomach or gut, i.e., intragastrically to provide protection to the host against infection, preferably colonization, and more preferably against systemic infection. The oral ~m; n; stration also can provide protection to the host against pulmonary infection as well as protection to the host against an infection starting as a pulmonary infection. However, the oral administration can also involve respiratory mucosa, gingival mucosa or alveolar mucosa, and the administration can be perlingual or sublingual or into the mouth or respiratory tract, especially if the composition is administered in a liquid CA 0222296~ 1997-12-01 W096~9113 PCT~B96/01051 form, e.g., as a syrup, elixir etc. However, intragastric ~m; n; stration is preferred.
Thus, compositions of the invention especially for oral administration, are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, or viscous compositions which may be buffered to a select pH. However, since delivery to the digestive tract is preferred, compositions of the invention can be "solid" from the pills, tablets, capsules, caplets, and the like, including "solid"
preparations which are time - release or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach and/or small intestine for delivery into the gut and/or digestive system.
In a particular aspect of the invention, there is provided a method of ;mmlln;zation of a host against Streptococcus pneumoniae which comprises orally or perorally ~m; n; stering to the host an ;mmlln;zing amount of pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a lysate of pneumococci or an isolated PspA or an immunogenic fragment thereof.
The present invention further provides a vaccine composition or immunogenic composition for oral or peroral administration to a host, preferably for ~m;n;stration into the gut (stomach, digestive tract;
e.g., intragastrically) of a host to confer protection or elicit an immunological response, against S. pneumoniae, which comprises:
an effective amount of a pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a pneumococcal lysate, an isolated and purified PspA or an immunogenic fragment thereof containing at least one epitope, preferably protection-eliciting epitope, and optionally an adjuvanting amount of an adjuvant, preferably CT, and optionally CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/01051 a pharmaceutical carrier therefor.
Moreover, the present invention provides a compartmentalized kit comprising PspA or a fragment thereof, and a suitable carrier, diluent or excipient;
and optionally, a pharmaceutically acceptable adjuvant;
and further optionally, instructions for admixture and/or ~m; n; stration.
Other objects and embodiments of the invention are disclosed in or are obvious variants from the following description.
BRIEF DESCRIPTION OF T~E DRAWINGS
In the following description, reference is made to the accompanying Figures, wherein:
Figures 1 and 2 show bar graphs (Reciprocal Log2 ELISA (enzyme linked immunoadsorbant assay) Titers v. Days after ;mmlln;zation) of IgM, IgG and IgA responses of mice orally-immunized by gastric intubation with 7.5 ~g of purified native PspA, with (Fig. 2) and without (Fig. 1) 10~g/mouse CT, 7, 14, 21, 28, 35, 42 and 49 days after immunization (open bars = IgA, darkened bars = IgM, bares with diagonal stripes = IgG) (Log2=4 is just below the limited detection of the assay. This, values of 4 should be considered as indicating that there was no detective antibody);
Figure 3 shows a time course of PspA-specific serum antibody responses in mice subcutaneously immunized with 0.5~g of PspA and CFA (antibody titers were P~mt ned in pooled sera collected from 6 mice by ELISA);
Figure 4 shows the elucidation of PspA-specific antibody forming cells (AFCs) in spleen of mice simultaneously immuntzed with 0.5~g of PspA and CFA (AFCs were assessed on 14 days after 2nd immunization by the ELISPOT assay)i Figure 5 shows the kinetics of antigen-specific serum antibody responses in mice orally immunized with PspA ((a) mice were orally immunized with 7.5~g of PspA
and (b) 7.5~g of PspA together with 10~g of CT. Antibody CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/01051 titers were P~m~ned in pooled sera collected from 6 mice by the ELISA);
Figure 6 shows an analysis of antigen-specific fecal IgA responses in mice orally ;mm~ln;zed with 7.5~g of PspA together with 10~g of CT (antibody titers were ~m; ne~ in pooled fecal extracts collected from 6 mice by the ELISA); and Figure 7 shows the time cource of PspA-specific IgG subclass in mice orally ;~mllnized with 7.5~g of PspA
plus 10~g of CT (antibody titers were Px~m;ned in pooled sera collected from 6 mice by the ELISA).
DET~ TT.lm DESCRIPTION OP INV ~ TION
As discussed above, the principal determinant of specific ;mmlln;ty at mucosal surfaces is secretory IgA
(S-IgA) which is physiologically and functionally separate from the components of the circulatory ;~mune system. S-IgA antibody responses may be induced locally by the application of suitable immunogens to a particular mucosal site. The bulk of mucosal S-IgA responses, however, are the results of ;mmlln;ty generated via the common mucosal ~mmllnP system (CMIS) (Mestecky, 1987), in which immunogens are taken up by specialized lympho-epithelial structures, collectively referred to as mucosa-associated lymphoid tissues (MALT). The best studied immunologic lympho-epithelial structures are the gut-associated lymphoid tissues (GALT), such as intestinal Peyer's patches (Mestecky, 1987). Other structurally and functionally similar lymphoid follicles occur at other mucosal surfaces, including those of the respiratory tract (Croitoru et al., 1994).
Bronchus-associated lymphoid tissue (BALT) was described by Bienenstock (Bienenstock et al., 1973a;
Bienenstock et al. 1973b) in experimental ~nlm~l S, but is ~ apparently not present in the noninfected human bronchial tree (Pabst, 1992). The upper respiratory tract in humans, however, is furnished with Waldeyer's ring of tonsils and adenoids. In rodents, the functional CA 0222296~ 1997-12-01 W096~9113 PCT~B96/0l05 equivalent of these consists of nasal-associated lymphoid tissue (NALT), a bilateral strip of lymphoid tissue with overlying M-like epithelial cells at the base of the nasal passages (Kuper et al., 1992).
According to the present invention, a host can be effectively ;mmlln~zed by oral instillation (e.g., intragastric instillation) of bacterial protein ;mmllnogens~ preferably PspA or a fragment thereof, preferably mixed with an adjuvant, more preferably cholera toxin (CT). Of course, as an adjuvant, the amount of cholera toxin used is non-toxic to the host.
The ability of a vaccine to protect against pneumococcal colonization, as provided herein, means that the active component may protect against disease not only in the immunized host but, by eliminating carriage among immunized individuals, the pathogen and hence any disease it causes may be eliminated from the population as a whole.
Oral or peroral administration (e.g., intragastric administration) can also prevent sepsis resulting from ~m;n; stration of pneumococci, so that the vaccine can protect against both pneumococcal colonization and sepsis (systemic infection).
As mentioned, PspA is the preferred antigen.
WO 92/14488 is incorporated herein by reference. In published International patent application WO 92/14488, there are described the DNA sequences for the pspA gene from S. pneumoniae Rxl, the production of a truncated form of PspA by genetic engineering and the demonstration that such truncated form of PspA confers protection in mice to challenge with live pneumococci.
From sequences of the E~_ gene, it has been shown that PspA proteins are variable in size (roughly 70kDa). The C-terminal 37~ of the molecule is largely composed of the 20-amino acid repeats which form a binding site that permits PspA to attach to the phosphchloine residues of the pneumococcal lipoteichoic CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 acids. The central region of PspA is rich in prolines and is suspected to be the portion of the molecule that passes through the cell wall. The sequence of the N-terminal 80~ of the molecule is largely ~-helical and contains the region of PspA that can elicit antibodies that are protective against sepsis. Although PspA's are almost always at least slightly different from one another, there is enough cross-reactivity between them that antibodies or an ;mmllnological response to one PspA
detect or is effective with respect to PspAs on all pneumococci. Moreover, ;mmlln;zation with one PspA can either protect against death or delay death with virtually all different challenge strains. Accordingly, a mixture of a small number of PspA's could effective lS ;mmlln;ty against most pneumococci.
The immunoprotective truncated PspAs described in WO 92/14488 may be used in the present invention as the PspA fragments described above for oral or peroral ~m; n; stration.
Numerous vector systems for in vitro and in vivo expression of recombinant proteins are known; e.g., bacterial systems such as E. coli; and virus systems such as bacterial viruses, poxvirus (vaccinia, avipox virus, e.g., canarypox virus, fowlpox virus), baculovirus, herpes virus; yeast; and the like; and, these systems may be used for producing recombinant PspA using the coding therefor or of WO 92/14488.
Immunogenicity can be significantly improved if the antigen (PspA) is co-~m; n; stered with an adjuvant, commonly used as .OOl~ to 50~ percent solution in phosphate buffered saline. Adjuvants enhance the immunogenicity of an antigen (PspA) but are not necessarily ;mmllnogenic themselves. Adjuvants may act by retaining the antigen locally near the site of ~m; n; stration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune CA 0222296~ 1997-12-01 W096~9113 PCT~B96/01051 system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune respon~es.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccarides, normally are the components of the killed or attenuated bacteria used as vaccines.
Extrinsic adjuvants are ;mmllnomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and alllm;nllm phosphate (collectively co~mo~ly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to me~mbrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. To efficiently induce humoral imml~ne response (HIR) and cell-mediated immunity (CMI), immunogens are preferably emulsified in adjuvants.
Desirable characteristics of ideal adjuvants include any or all of:
(l) lack of toxicity;
(2) ability to stimulate a long-lasting immune response;
(3) simplicity of manufacture and stability in long-term storage;
(4) ability to elicit both CMI and HIR to antigens ~m; n; stered by various routes;
(5) synergy with other adjuvants;

CA 0222296~ 1997-12-01 W 096/39113 PCT~B96/01051 (6) capability of selectively interacting with populations of antigen presenting cells (APC);
(7) ability to specifically elicit appropriate T}I1 or TH2 cell-specific immune responses;
and (8) ability to selectively increase appropriate antibody isotype levels (for example IgA) against antigens.
U.S. Patent No. 4,855,283 granted to Lockhoff et al. on August 8, 1989 which is incorporated herein by reference thereto teaches glycolipid analogs including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immune-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Patent No. 4,855,283) reported that N-glycolipids analogs displaying structural similarities to the naturally occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of eliciting strong ;mml~ne responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain alkylamines and fatty acids that are linked directly with the sugar through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
U.S. Patent No. 4,258,029 granted to Moloney, assigned to Connaught Laboratories Limited and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Octodecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, - enhanced the host immune responses against hepatitis B
virus.
As previously mentioned, compositions of the invention, especially for oral administration, are CA 0222296~ 1997-12-01 WO96~9113 PcTnB96 conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. However, since delivery to the digestive tract is preferred, compositions of the invention can be in the "solid" form of pills, tablets, capsules, caplets and the like, including "solid" preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach and/or small intestine for delivery to the gut and/or digestive system.
Compositions of the invention can contain pharmaceutically acceptable flavors and/or colors for rendering them more appealing. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed.
Viscous compositions have a viscosity preferably of 2500 to 5000 cps, since above that range they become more difficult to ~m;~; ster. However, above that range, the compositions can approach solid or gelatin forms which are then easily administered as a swallowed pill for oral ingestion.
Liquid preparations are normally easier to prepare than gels and other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to ~mi nl ster, especially to ~n;~l S, children, particularly small children, and others who may have difficulty swallowing a pill, tablet, capsule or the like, or in multi-dose situations.
Viscous compositions, on the other hand can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or intestine.

CA 0222296~ 1997-12-01 Suitable nontoxic pharmaceutically acceptable carriers, and especially oral carriers, will be apparent to those skilled in the art of pharmaceutical and ~ especially oral or peroral pharmaceutical formations.
For those not skilled in the art, reference is made to the text entitled "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference.
Obviously, the choice of suitable carriers will depend on the exact nature of the particular dosage form, e.g., liquid dosage form [e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, or solid dosage form [e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form].
Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the antigen (PspA). Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and/or flavors may also be present. The compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid.
The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/01051 example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity.
Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
A pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, th;m~rosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02~ to 2~ based on the total weight although there may be appreciable variation depending upon the agent selected.
Those skilled in the art will recognize that the components of the compositions must be selected to be chemically inert with respect to PspA. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure.
The immunologically effective compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally the pH may be from about 3 to 7.5. Compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of W O 96~9113 PCT~B96/01051 the particular patient or ~n;m~l, and the composition form used for ~m;n;stration (e.g., solid vs. liquid).
Dosages for hllmAn~ or other mAmmAls can be determined without undue experimentation by the skilled artisan, from the Examples below (e.g., from the Examples involving mice).
When CT is used as an adjuvant for oral immlln;zations~ specific IgA antibodies are induced in secretions. Strong circulatory ;mmllne responses can also be induced, with IgG and IgA antibodies in the serum, and IgG and IgA antibody-secreting cells in the spleen. The circulatory (or systemic) ;mmllne responses elicited by oral (peroral; intragastric ) A~m; n i stration of PspA along with CT are comparable with, or even stronger than, those induced by the A~m; n; stration of similar immunogens by the intragastric (i.g., peroral) route (Wu et al., 1993;
Russell et al. 1991). Accordingly, it appears that oral (peroral, i.g.) immunization is an effective route for stimulating common mucosal responses as well as circulatory antibody responses and can require less antigen than other ;mmnn;zation routes.
Most soluble or non-replicating antigens are poor immunogens, especially by the peroral route, probably because they are degraded by digestive enzymes and have little or no tropism for the GALT. A notable exception is CT, which is a potent mucosal immunogen (Elson et al., 1984), probably because of the GM1 ganglioside-binding property of this binding subunit, CTB, that enables it to be taken up by the M cells of Peyer's patches and passed to the underlying immunocompetent cells. In addition to being a good - mucosal ;mm~lnogen, CT is a powerful adjuvant (Elson et al., 1989; Lycke et al., 1986; Wilson et al., 1989).
When ~m;n; stered in ~g does, CT greatly enhances immunogenicity of other soluble antigens co-administered with it.

CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/010~1 C~3 is a strong adjuvant when given orally or perorally (e.g., intragastrically) in mice along with antigen, but CTB has no direct adjuvant effect, but can act synergistically with CT (Wilson et al., 1990).
As discussed above, the mechanisms by which CT
and CTB act as adjuvants are not fully understood, but are certainly complex, and appear to depend on several factors, including: 1) the toxic activity associated with the ADP-ribosylating property of the A1 subunit (Lycke et al., 1992; Abbas et al., 1991); 2) increased permeability of mucosae (refs. 46, 47, 48, 49). This last point is controversial, however, as the in vitro effects of CT or CTB on T and B cells are generally inhibitory rather than stimulatory (Woogen et al., 1987; Garrone et al., 1993;
Haack et al., 1993; Quiding et al., 1991; Czerkinsky et al., 1989; Svernerholm et al., 1984). Nevertheless, numerous reports attest to the in vivo mucosal immllnoenhancing effects of CT and of CTB couples to antigens (Lycke et al., 1986; Wilson et al., 1989;
Abraham et al., 1991; Szu et al., 1989; Chen et al., 1g90; Liang et al., 1988; Hakansson et al., 1994;
Anderson et al., 1981; Dagen et al., 1995; Dillard et al., 1994). And, CT can selectively inhibit CD8~ cells, and therefore tend to abrogate suppressive effects (Elson et al., 1995).
Although carriage of pneumococci can be maintained for long periods in the very young and the elderly, it is generally not a permanent condition.
Carriage is much less common in older children and young adults (Gray et al., 1980; Gray et al., 1981; Hendley et al., 1975; Brimblecombe et al., 1958; Masters et al., 1958). One explanation for these findings is that carriage may be interfered with by lmml~n;ty (possibly mucosal immunity) to pneumococci (Gray et al., 1980;
Gwaltney et al., 1975). Most human saliva have IgA
antibodies to type 23 capsular polysaccharide and phosphocholine (an immunodom~ n~nt determinant of CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 pneumococcal cell wall teichoic acids) (Russell et al., 1990). It seems likely, therefore, that hllm~n sera would also contain antibodies to other pneumococcal antigens.
In the cases of group A streptococci, M proteins have been shown to be required for colonization in rats, and antibodies to M proteins can protect against colonization of the throat (Bessen et al., 1988; Hollingshead et al., 1993). In mice, the inventors have shown herein that ;~mlln;ty to PspA can prevent carriage of S. pneumoniae.
Antibodies may be effective against carriage in two ways, namely: 1) they might act at the mucosal surface by opsonizing pneumococci, preventing attachment or surface invasion; 2) they might act via opsonophagocytosis and killing. This latter mechanism could be especially important if carriage is dependent on m;n;m~l invasion of the nasal mucosal surface. The complement fixing antibodies could prevent the invasion and facilitate the killing of any pneumococci that invaded locally. Alternatively, complement fixing antibodies might be able to act and the mucosal surface if inflammation causes a sufficient release of complement, phagocytes, and possibly serum antibody.
One of these mechanisms might play a role in the observation that carriage of H. influenzae can be prevented by an intramuscular vaccine (Barbour et al., 1993). It has recently been reported that significant levels of H. influenzae polysaccharide-specific IgG and IgA are detected in secretions of children following ;mmlln;zation with the group b polysaccharide conjugate vaccine (Chiu et al., 1994; Kauppi et al., 1993).
Existing mouse protection data (Briles et al., 1989; Briles et al., 1981; Lock et al., 1988, Lock et al., 1992) suggests that antibodies that can opsonize pneumococci (e.g. those to the capsule) are generally more protective against sepsis than those that block the activities of toxins (e.g. pneumolysin) or enzymes (e.g.
autolysin or neuraminidase). However, at the mucosal CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/OIOSI

surface, the role played by antibodies that inactivate toxins and enzymes may be greater than that played by opsonic antibodies. The reason to suspect this is that for opsonic antibodies to exert their anti-bacterial effect, complement and phagocytes are required.
Phagocytes are rare on the surface of normal nasopharyngeal tissue, and even if present, the phagocytes do not have the filtering action of the spleen and reticuloendothelial system to increase their chance of interactions with opsonized bacteria. Antibodies that block the virulence enhancing effects of pneumolysin and pneumococcal enzymes should be able to bind their antigens just as effectively whether phagocytes were present or not.
The results provided herein show that oral or peroral (e.g., i.g.) ;mmlln;zation with heat-killed pneumococci, and pneumococcal lysates, and purified PspA
can protect mice against sepsis and therefore carriage too. As noted earlier, the ability of a vaccine to protect against disease not only in the ;mmlln;zed host, but, by eliminating carriage among immunized individuals, the pathogen and hence any disease it causes may be eliminated from the population as a whole.
The vaccine or immunogenic composition which is administered orally, perorally or intragastrically as provided herein may be formulated in any convenient manner and in a dosage formulation consistent with the mode of ~m; n; stration and the elicitation of a protective response (see discussion above and Examples below). The quantity of antigen to be administered depends on the subject to be ;mmlln;zed and the form of the antigen. Precise amounts and form of the antigen to be ~m; n; stered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by those skilled in the art from this disclosure, without undue experimentation, and may be of the order of micrograms to milligrams. Suitable W O 96~9113 PCTnB96/01051 regimes for initial ~m; n; stration and booster doses also are variable, may include an initial ~m;n; stration followed by subsequent ~m; n; strations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, without undue experimentation.
Further, the invention also compr~en~ a kit wherein PspA or a fragment thereof is provided. The kit can include in a container separate from the PspA or fragment thereof, a suitable carrier, diluent or excipient. The kit can also include, in a container separate from the PspA or fragment thereof or from the carrier, diluent or excipient, or already in admixture with either of these components, an adjuvant, such as cholera toxin. Thus, the kit comprises: (i) PspA or fragment thereof, (ii) carrier, excipient or diluent, and optionally, (iii) adjuvant, wherein there are separate containers for (i) and (ii), or for (i), (ii) and (iii), or for (i) and the combination of (ii) and (iii), or for (ii) and the combination of (i) and (iii). Additionally, the kit can include instructions for mixing or combining ingredients and/or a~m; n; stration.
The following Examples are provided for illustration and are not to be considered a limitation of the invention.

EXAMPLE 1 - rR~TERIZATION OF IMM~NE RESPONSES IN
MICE ORALLY IMM~NIZED WITH PsDA

Prior to oral ;mmlln;zation, mice were deprived of food for two hours and then given a solution of sodium bicarbonate to neutralize stomach acid. Thirty minutes later, mice were orally-;mmlln;zed by gastric intubation with 7.5 ~g of purified native PspA with or without CT.
Oral ; mmlln; zations were carried out on day 0, 7, 14, and 35. Serum and fecal extracts were collected prior to immunization on days 7, 14 and 35. Sera and fecal pellets were also collected on days 21, 28, 42 and 49.
Sera and fecal extracts were obtained and assayed as CA 0222296~ 1997-12-01 W O 96~9113 PCTnB96/01051 previously described (Jackson R.J., K. Fujihoski, J. Xu-Amano, H. Kiyono, C.O. Elson and J.R. McGhee. 1993.
"Optimizing oral vaccines: induction of systemic and mucosal B. cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant", Infect. Immun.
61:4272-4279 de Vos et al., 1991). Additionally, the ability of oral immunization with PspA to elicit protective ~mmln;ty was P~m; n~ using the previously described mouse model of pneumococcal sepsis (Briles et al., 1992; Briles et al., 1989).
When mice were orally ;mml~nlzed with PspA
alone, antigen-specific serum IgG responses were detected after the primary ;~lln;zation. The Log2 titers were 5 to 7 (Fig. 1). These responses were not enhanced following the booster oral ;mmlln;zations~ and no IgM
responses were detected during the analysis period (Fig.
1). No IgA antibody to PspA was detected until day 49 when the titer was only Log2-5. The fecal extracts had no detectable levels of antigen-specific responses.
When PspA was given orally with CT, antigen specific IgM, IgG, and IgA responses were induced in the serum. PspA-specific IgM antibodies Log2=9 were detected after the second injection. PspA-specific IgG responses were elevated to Log2=19 after the 4th oral ;mmlln;zation with PspA plus CTi Also, PspA-specific serum IgA
responses of Log2=9 and 13 were detected on day 42 and 49, respectively (Fig. 2). Mucosal IgA responses in fecal extracts (Log2=5) were detected after the fourth immunization.
EXAMPLE 2 - r~rTERIZATION OF PRG~ lON ~TNST

WIT~ P~A
Mice were challenged intravenously with 3.6 x 103 colony forming units (CFU) of S. pneumoniae A66 two weeks following the 4th oral immunization with PspA alone or PspA plus CT. The results (Table 1, below) CA 0222296~ l997-l2-Ol WO96~9113 PCT~B96/0l051 ~emon~trated that oral immlln;zation with purified PspA
(using CT as an adjuvant) can provide systemic protective immllnity to otherwise fatal infections with S.
pneumoniae .

Oral ;mmlln;zation with PspA using cholera toxin as an adjuvant induces protective ;mmlln;ty to systemic challenge with capsular type 3 strain A66 Streptococcus pneumoniae Antigen used for ;mmlln;zation PspA + CT PspA None Log2 Reciprocal titer of IgG anti-PspA 19 6 ~4 Day of Death for individual mice~21,~21,~21,~21 1,1,2,3,4, 1,1,2,2,3 ~21,>21,>21 >21>21,,21 Median Day of Death ~21 3.5 2 Alive:Dead 7:0 3:5 0:5 P value vs. none 0.003 n.s.
P value vs. "PspA" 0.05 n.s.
Note: C57BL/6J Mice were challenged intravenously with 3.5 x 103 A66. P values shown above were calculated by the two tailed Wilcoxon two-sample rank test. Using the Kruskal-Wallis non-parametric analysis of variance the P value is 0.0071.
~XAMPLE 3 - lN~u~lON OF PRG.~llv~ ~MORAL AND ~Y~M
IMhuNl~r IN MICE IM~NIZED WITH P-~pA
For systemic ;mmlln;zation of mice 0.5~g of purified, native PspA with Complete Freund Adjuvant (CFA) was injected subcutaneously on days 0 and 14 (at a ratio of 1:1 CFA to PspA in PBS). For oral ;mmlln; cation, mice were deprived of food for two hours, and then ~m; n; stered by gastric intubation a solution of sodium bicarbonate to neutralize stomach acid prior to the ;mmlln;zation. Thirty minutes later, mice were orally-immlln;zed by gastric intubation with 7.5~g of purified, native PspA, with or without mucosal adjuvant cholera toxin (CT). Oral ;mmllnizations were carried out on day CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 0, 7, 14 and 35. A total of six mice were used in each control and experimental group.
Serum and fecal extracts were prepared as previously descrived (Jackson, R.J. et al, Infect. Immun.
1993) 61: 4272-79). Serum and fecal extracts were collected weekly intervals and assayed for PspA-specific IgM, IgG and IgA antibodies by ELISA. 96 well plates (Nunc Immuno Plate, Roskilde, Denmark) were coated with PspA in PBS for overnight at 4~C. After washing 3 times with PBS, wells were blocked for l hours with PBS
containing lO~ goat serum. Serial two-fold dilutions of samples were tested, starting at a dilution of l:2 (fecal extract) and l:32 (serum). Plates were incubated for 4 hours at room temperature. After incubation, captured antibodies were detected with biotinylated anti-mouse IgM, IgG and IgA (lmg/ml, Southern Biotechnology Associates (SBA), Birmingham, AL) and horseradish peroxidase (HRP)-streptoavidin (0.5mg/ml, Gibco, Gaithersburg, MD). ABTS (2,2 azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), diamonium salt;
Sigma, St. Louis, MO) was used for color development.
Titres were recorded as the last dilution which gave an absorbance (414nm) of O.l units greater than control serum from non immunized mice.
To ~mlne the antigen-specific fecal IgA
responses, the method of lllm;nomPtry was applied.
Briefly, 96 well plates (Dynatech Microlite~, Chantilly, VA) were prepared exactly as described for ELISA.
Following the incubation of the secondary biotinylated antibody, plates were washed with PBS-Tween containing 2mM EGTA, added streptae~uorin and incubated l hour at room temperature. The plates were washed, dried and placed in the luminometer for the color development. The wells containing the aequorin derivatives were developed in a Dynatech ML-3000 luminometer by injection of 50mM
Tris-HCl, pH 7.5, containing 20mM calcium acetate.
Results were expressed as relative light unit (RLU) CA 0222296~ l997-l2-Ol W 096~9113 PCT~B96/01051 following subtraction of blank values. To determine endopoint titer for the ll~m;nometric assay, the straight line generated following background substraction was extrapolated (E titer) to a baseline 1000 relative light units above background.
~ To ~m; ne the number of PspA-specific antibody forming cells (AFCs), ELISPOT assay was performed as previously described (Jackson, R.J. et al., Infect.
Immun. (1993) 61: 4272-79). Single cell suspensions were prepared from spleen by mechanical dissociation. Ninety-six well nitrocellulose plates (Millititer HA; Millipore Corp., Bedford, Mass.) were used for the assay. The plates were coated with 0.5 mg of purified, native PspA.
After incubation overnight at 4~C plates were washed and blocked with 10~ FCS-RPMI 1640. The blocking solution was discarded, and 100 ml of cells in 10~ FCS-RPMI 1640 at various dilutions were added. Cell suspensions were incubated for 4 hours at 37~C in 5~ CO2 atmosphere.
Plates were washed with PBS and PBS-0.05~ Tween-20. The secondary antibody solution consisted of 100 ml of a 1:1,000 dilution of goat anti-mouse IgM, IgG and IgA
conjugated to horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) in PBS-0.05 Tween-20 containing 1~ BSA. Following overnight incubation, plates were washed with PBS, and developed by the addition of 200 ml of 3-amino-9-ethylcarbazole dissolved in 0.lM sodium acetate buffer containing H2O2 per well. Plates were incubated at room temperature for 15-20 min and washed with water, and AFCs were counted with the aid of a stereomicroscope.
Mice which were subcutaneously ;mm~ln;zed with 0.5~g of purified, native PspA with CFA were used in order to establish a base line for systemic ;mm~]n;zation.
PspA-specific IgG responses were detected by 14 days after the primary immunization and levels of antigen-specific titers were increased following secondary immunization. There PspA-specific IgG responses were CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/01051 maintained for greater than 70 days (Fig. 3). In order to confirm the source of these PspA-specific IgG
antibodies, mice were sacrificed 4 days after secondary ;mml~n;zation. Splenic ~o~s~llclear cells were isolated and assayed for the detection of PspA-specific antibody producing cells by the ELISPOT assay. A significant number of antigen-specific IgG producing cells were detected in splenocytes isolated from mice subcutaneously ;mmlln;zed with PspA when compared with control group (nn;mmllnn;zed mice). However, IgM and IgA producing cells were very low or undetectable (Fig. 4). These results support the conclusion that systemic immunization of PspA induces high levels of antigen-specific serum responses.
When mice were orally-;mmlln;zed with purified, native PspA alone, antigen-specific serum IgG responses were elicited after primary immunization, however, the titer of responses was low (1:32-1:128) when compared with the systemic ;mmlln;zation (Figs. 3 and 5A), and 3 2d out of 8 mice were protected from a lethal challenge with 5. pneumoniae. Further, those responses were not enhanced following the booster oral immlln;zation.
Soluble proteins are generally not strong immllnogens when given by the oral route. This result, however, suggests that small amounts of PspA are sufficient to induce serum responses by the oral route which are able to protect against challenge. Very small IgA response (1:32) were detected 3 weeks after 4th oral ;mmlln;zation on day 35.
No antigen-specific IgM responses were detected during the analysis period following ;mmlln;zation (Fig. 5A). In the case of fecal extracts, there was no detectable levels of antigen-specific ;mmllne responses.
Oral ~m; n; stration of mucosal adjuvant CT with soluble protein antigen not only induces antigen-specific serum responses, but it also is an effective immunization regimen for the generation of antigen-specific antibody responses in mucosal sites. Thus, when purified, native CA 0222296~ 1997-12-01 WO96/39113 PcTnB96 PspA was given orally with lO~g CT on day 0, 7, 14 and 35, antigen-specific IgM, IgG and IgA responses were induced in the serum. For example, after secondary ;mmlln;zation, PspA-specific IgM antibodies were induced (1:512) and shifted to secondary type responses where ~ high levels of IgG responses (1:16,384) were elicited on day 21 (Fig. 5B). These PspA-specific IgG responses were elevated up to 1:524,288 after 4th oral immunization with PspA plus CT. Furthermore, PspA-specific IgA serum responses were detected on day 42 and 49 (Fig. SB). When those mice were challenged with a lethal dose of S.
pneumo~iae intravenously, all mice survived challenge (Table 2).
In addition, antigen-specific IgA antibodies were detected in fecal extracts obtained from 2 weeks after the third oral ;mmlln~zation and the responses were increased following the 4th immunization (Fig. 6). When subclass of PspA-specific IgG response was evaluated, main response was noted as IgG1 followed by IgG2b (Fig.
7). Further, IgA anti-PspA appeared in the fecal extracts following the third immunization, and the response was increased after the fourth immunization. In this regard, CT is known to selectively induce antigen-specific Th2 type response which promotes IgG1 in serum as well as mucosal IgA responses when it was given orally together with protein antigen.
Thus, these results showed that oral ;~ml~n;zation with PspA plus adjuvant CT lead to the generation of antigen-specific Th2 type responses accounting for the induction of PspA-specific IgG1 as well as S-IgA responses in systemic and mucosal sites, respectively. Taken together, these results showed that oral immunization of PspA with CT as mucosal adjuvant was capable of inducing PspA-specific serum as well as mucosal antibody responses.

CA 0222296~ 1997-12-01 W O 96~9113 PCTnB96/0105 EXAIIPI.E 4 - OPAL IID~NIZATION WIT~ PspA AND r~C!T.
l~AlN IN ORDER TO ES~RT.T~
PR~ lv~ ~UMORAL IMhu~l~Y ~TNST
PNE~OCOCCAL lN~ lON
The pneumococcal in vivo protection analysis was performed as previously descrived (McDaniel, L.S., et al. Infect. Immun. (1991) 59: 222-228). Briefly, fourteen days after fourth ;mmlln;zation with PspA alone or conjugated to cholera toxin (CT), mice were challenged intravenously with 3. 6x103 CFU of pneumococcal strain A66.
The survival of mice was monitered for 21 days. The mice orally-;mm~]n;zed with PspA plus CT showed antigen-specific serum IgG titer of 19 prior to the challenge (Table 2, below). In the mice orally-immunized with PspA
alone, the levels of antigen-specific IgG response was low (serum titer of 6). When these mice were systemically challenged with a lethal dose of A66 strain, oral ;mmllntzation with PspA and CT protected all the mice from the infection. However, oral ;~mlln;zation with PspA
alone failed to provide complete protection, and three out of eight mice survived challenge (Table 2). These results demonstrated that oral immunization with PspA can provide protective ;mm~ln;ty and mucosal adjuvant CT can further elicit protective humoral immune responses against pnemococcal infection.
Hence, oral imml]n;zation with PspA vaccine can induce a protective, humoral immune response against S.
pneumoniae infection. Further, since it has been shown that PspA elicits cross protection against different capsular serotypes, oral immuization with PspA plus CT
could induce a protective level of systemic responses against different capsular serotypes of pneumococcal infection.

CA 0222296~ 1997-12-01 W O 96~9113 PCTnB96/01051 Table 2. Oral ~mmlln~zation with PspA plus CT induces - protective immunity to systemic challenge with capsular type 3 strain A66 Streptococcus pneuIr?oniae __________________ _____________________ ______________ Serum IgG
Titer Day of Death Median Day P
value Vaccine (1/log2) After Challenge of Death vs.
None*
_________________________________________________________ PspA + CT 19 ~21,>21,~21,~21 ~21,~21,~21 ~21 0.003 PspA 6 1,1,2,3,4,~21 >21,>21 3.5 N.S
None c4 1,1,2,2,3 2 N.A

____ ________________ ________ _ __ ___ _____ __ _______ Note: The mice were challenged intravenously with 3.6x103 CFU of A66. *P values shown above were calculated by the two tailed Wilcoxon two-sample tank test using, N.S: not significant, N.A: not applicable.

These results are analogous to providing humans with solid or solid-with-liquid-center forms which are absorbed primarily in the gut (i.e., swallowed whole for intragastric absorption), and are also indicative of the utility of liquid forms (which may be more advantageous for administration to children, especially children age 2 or less).
In summary of this disclosure, the present invention provides a method of im~lln;zing a host against pneumococci in a host and for protecting against systemic and humoral infection by pneumococci, by oral, particularly intragastric, ~m; n; stration of PspA in various forms.
Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended Wo96/39113 PCT~B96/oloSl claims is not to be limited by particular details set forth above, as many apparent variations thereof are possible without departing from the spirit or scope thereof.

CA 0222296~ 1997-12-01 WO96~9113 PcTnB96 ~ SK~NCES
1. Abbas, A.K., Lichtman, A.H., Pober, J.S., In Cellular and Molecular Immunology, ed. Wonsiewicz M., (W.B. Saunders Co., Philidelphia) (1991).
2. Abraham, E., Robinson, A., Vaccine 9:757-764 (1991).
3. Anastassiou, E.D., Yamada, H., Francis, M.L., Mond, J.J., Tsokos, G.C., J. Immunol. 145:2375-2380 ( 1990 ) .
4. Andersson, B., Eriksson, B., Falsen, E., Fogh, A., Hansen, A., Nylen, 0., Peterson, H., and Svanborg-Eden, C., Infect. Immun. 32:311-317 (1981).
5. Anonymous. Centers for Disease Control HIV/AIDS
Surveillance Report, August :1-18 (1991).
6. Bale, J.R., Rev. Infect. Dis. 12 (Suppl 8):S861-S1083 (1990).
7. Barbour, M.L., Mayon-White, R.T., Crook, D.W., Coles, C., Moxon, E.R., ICAAC Abstracts 33:175 (1993).
8. Berman, S., McIntosh, K., Rev. Infect. Dis. 7:647-691 (1985).
9. Bessen, D., Fischetti, V.A., Infect. Immun. 56:2666-2672 (1988).
10. Bienenstock, J., Johnston, N., Perey, D.Y., Lab.
Invest. 28:686-692 (1973a).
11. Bienenstock, J., Johnston, N. Perey, D.Y., Lab.
Invest. 28:693-698 (1973b).
12. Bohr, V., Rasmussen, N., Hansen, B., Gade, A., Kjersem, H., Johsen, N., Paulson, O., J. Infect.
Dis. 10:143-157 (1985).
13. Bolan, G., Broome, C.V., Facklam, R.R., Plikaytis, B.D., Fraser, W.D., Schlech, W.F.I., Ann. Intern.
Med. 104:1-6 (1986).
14. Briles, D.E., Crain, M.J., Gray, B.M., Forman, C., - Yother, J., Infect. Immun. 60:111-116 (1992).
15. Briles, D.E., Claflin, J.L., Schroer, K., Forman, C., Nature 294:88-90 (1981).
16. Briles, D.E., Forman, C., Horowitz, J.C., Volanakis, J.E., Benjamin, W.H. Jr., McDaniel, L.S., Eldridge, J., Brooks, J., Infect. Immun. 57:1457-1464 (1989).

CA 0222296~ 1997-12-01 W O 96~9113 PCT~B96/01051 17. Brimblecombe, F.S.W., Cruicshank, R., Masters, P.L., Reid, D.D., Stewart, G.T., British Medical Journal :119-128 (1958).
18. Brom~n~er, A., Holmgren, J., Lycke, N., Journal of Immunology 146:2908-2914 (1991).
19. Chen, K-S., Strober, W., Eur. J. Immunol., 20:433-436 (1990).
20. Chiu, S.S., Greenberg, P.D., Marcy, S.M., Wong, V.K., Chang, S.J., Chiu, C.Y., Ward, J.I., Pediatric Res. Abstracts 35:10A (1994).
21. Converse, G.M. III, Dillon, H.C. Jr., J. Clin.
Micro. 5:293-296 (1977).
22. Cowan, M.J. ~mm~nn, A.J., Wara, D.W., Howie, V.M., Schultz, L., Doyle, N., Kaplan, M., Pediatrics 62:721-727 (1978).
23. Croitoru, K., Bienenstock, J., In Handbook of Mucosal Immunology, ed. Ogra, P.L., Mestecky, J., Lamm, M.E., Strober, W., McGhee, J.R., Bienenstock, J., (Acamdemic Press, Inc., San Diego, CA) pp. 141-149 (1994).
24. Czerkinsky, C., Russell, M.W., Lycke, N., Lindbald, M., Holmgren, J., Infect. Immun. 57:1072-1077 (1989).
25. Czerkinsky, C., Nilsson, L-A., Nygren, H., Ouchterlony, O., Tarkowksi, A., J. Immunol. Methods 65:109-121 (1983).
26. Dagen, R., Melamed, R., Abranson, O., Piglansky, L., Greenberg, D., M~n~elman, P.M., Bohidar, N., Ter-Minassian, D., Cvanovich, N., Lov, D., et al., Pediat. Res. 37:172A (1995).
27. de Vos, T., and Dick, T.A., J. Immunol. Med.
141:285-288 (1991).
28. DeMaria, T.F., McGhee, R.B., Lim, D.J., Arch.
Otolaryngol. 110:279-280 (1984).
29. Dillard, J.P. and Yother, J., Molec. Microbiol.
12:959-972 (1994).
30. Douglas, R.M., Miles, H.B., Paton, J.C., American Journal of Diseases of Children 140:1183-1185 (1986).
31. Douglas, R.M. Miles, H.B., Journal of Infectious Diseases 149:861-869 (1984).

CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 32. Elson, C.O., Curr. Topics Microbiol. Immunol.
146:29-33 (1989).
33. Elson, C.O., Ealding, W., J. Immunol. 132:2736-2741 ~1984).
34. Elson, C.O., Holland, S.P., Dertzbaugh, M.T., Cuff, C.F., A.O., Anderson, J. Immunol. 154:1032-1040 (1995).
35. Fallon, M.T., Reinhard, M.K., Gray, B.M., Davis, T.W., Lindsey, J.R., Laboratory ~n;m~l Science 38:129- ~1988).
36. Francis, M.L., Ryan, J., Jobling, M.G., Holmes, R.K., Moss, J., Mond, J.J., Journal of Immunology 148:1999-2005 (1992).
37. Fraser, D.W., In Bacterial Vaccines, ed. JB Robbins, Hill JC, Sadoff JC, (New York) xix-xxiv (1982).
38. Garrone, P., Banchereau, J., Molecular Immunology 30:627-635 (1993).
39. Gizurarson, S., Tamura, S., Kurata, T., Hasiguchi, K., Ogawa, H., Vaccine 9:825-832 (1991).
40. Gotschlich E.C., Goldschneider, I., Lepow, M.L., Gold, R., In Antibodies in hl~m~n diagnosis and therapy (Raven, New York) pp. 391-402 (1977).
41. Gratten M. Naraqi, S., Hansman, D., Lancet ii:192-195 (1980).
42. Gray, B.M. Converse G.M. III, Dillon, H.C., J.
Infect. Dis. 142:923-933 (1980).
43. Gray, B.M., Converse, G.M. III, Huhta, N., Johnston, R.B. Jr., Pichichero M.E., Schiffman, G., Dillon, H.C. Jr., J. Infect. Dis. 142:312-318 (1981).
44. Greenwood, B.M., Greenwood A.M., Bradley, A.K., Tulloch, S., Hayes, R., Oldfield, F.S.J., Ann. Trop.
Pediatr. 7:91-99 (1987).
45. Gwaltney, J.W., Sande, M.A., Austrian, R., et al., J. Infect. Dis. 132:62 (1975).
46. Haack, B.M., Emmrich, E., Resch, K., Journal of Immunology 150:2599-2606 (1993).
47. Hakansson, A., Kidd, A., Wadell, G., Sabharwal, H., and Svanborg, C., Infect. Immun. 62:2707-2714 (1994).

CA 0222296~ l997-l2-Ol W O 96~9113 PCTAB96/01051 48. Hendley, J.O., Sande, M.A., Stewart, P.M., et al., J. Infect. Dis. 132:55 (1975).
49. Hollingshead, S.K., Simecka, J.W., Michalek, S.W., Infect. Immun. 61:2277-2283 ~1993).
50. Kauppi, M., Eskola, J. Kathty, H.H., ICAAC Abstracts 33: 174 (1993).
0 51. Klein, J.O., Rev. Infect; Dis. 3 :246- (1981).
52. Klugman, K.P., Clin. Microbiol. Rev. (1990).
53. Kuper, C.F., Koornstra, P.J., Hameleers, D.M.H., Biewenga, J., Spit, B.J., Duijvestijn, A.M., van Breda Vriesman, P.J.C., Sminia, T., Immunol. Today 13 :219-224 (1992).
54. Liang, X., Lamm, M.E., Nedrud, J.G., Journal of Immunology 141:1495-1501 (1988).
55. Lock, R.A., Hansman, D., Paton, J.C., Microbial Pathogenesis 12:137-143 (1992).
56. Lock, R.A., Paton, J.C., Hansman, D., Microb.
Pathog. 5 :461-467 (1988).
57. Lycke, N., Holmgren, J., Immunology 59: 301-308 (1986).
58. Lycke, N., Strober, W., J. Immunol. 142:3781-3787 (1989).
59. Lycke, N., Karlsson, U., Sjolander, A., Magnusson, K-E., Scand. Journal of Immunology 33: 691-698 ( 1991) .
60. Lycke, N., Tsuji, T., Holmgren, J., European Journal of Immunology 22:2277-2281 (1992).
61. Marton, A., Gulyas, M., Munoz, R. Tomasz, A., J.
Infect. Dis. 163 :542-548 (1991).
62. Masters, P.L., Brumfitt, W., Mendez, R.L., Likar, M., Brit. Med. J. 1:1200-1205 (1958).
63. Mestecky, J., J. Clin. Immunol. 7:265-276 (1987).
64. Munoz, R., Musser, J.M., Crain, M. Briles, D.E., Marton, A., Parkinson, A.J., Sorensen U., Tomasz, A., Clinic Infect. Dis. 15:112-118 (1992).
65. Munoz, E., Zubiaga, A.M., Merrow, M., Sauter, N.P., Huber, B.T., J. Exp. Med. 172:95-103 (1990).
66. Pabst, R., Immunology Today 13 :119-122 (1992).

CA 0222296~ 1997-12-01 WO96~9113 PCT~B96/01051 67. Quiding, M., Nordstrom, I., Kilander, A., Anderson, G., Hansen, L.A., Holmgren, J., Czerkinsky, C., J.
Clin. Invest. 88:143-148 (1991).
68. Russell, M.W., Prince, S.J., Ligthart, G.J., Mestecky, J., Radl, J., Aging Immunol. Infect. Dis.
2:275-286 (1990).
69. Russell, M.W., Wu, H-Y., Infection and Immunity 59:4061-4070 (1991).
70. Shapiro, E.D., Berg, A.T., Austrian, R., Schroeder, D., Parcells, V., Margolis, A., Adair, R.K., ClemmPn~, J.D., N. Engl. J. Med. 325:1453-1460 (1991).
71. Smillie, W.G., Am. J. Hyg. 24:522-535 (1936).
72. Smillie, W.G., Warnock, G.H., White, H.J., Am. J.
Pub. Hlth. 28:293-302 (1938).
73. Spika, J.S., Munshi, M.H., Wojtyaniak, B., Sack, D.A. Hossain, A., R~hm~n, M., Saha, S.K., Ann. Trop.
Pediatr. 9:33-39 (1989).
74. Svernerholm, A.M., Jertborn, M., Gothefors, L., Karim, A.M.M.M., Sack, D.A., Holmgren, J., J.
Infect. Dis. 149:884-893 (1984).
75. Szu, S.C., Li, X., Schneerson, R., Vickers, J.H., Bryla, D., Robbins, J.B., Infect. Immun. 57:3823-3827 (1989).
76. Wilson, A.D., Clarke, C.J., Stokes, C.R., Scand. J.
Immunol. 31:443-451 (1990).
77. Wilson, A.D., Stokes, C.R., Bourne, F.J., Scand. J.
Immunol. 29:739-745 (1989).
78. Wilson, A.D., Bailey, M., Williams, N.A., Stokes, C.R., European Journal of Immunology 21:2333-2339 ( 1991 ) .
79. Woogen, S.D., Ealding, W., Elson, C.O., Journal of Immunology 139:3764-3770 (1987).
80. Wu, H-Y, Russell, M.W., Infection and Immunity 61:314-322 (1993).
81. Ziegler-Heitbrock, H.W.L., Passlick, B., Kafferiein, E., Coulie, P.G., Izbicki, J.R., Infect. Immun.
60:1692-1694 (1992).

Claims (21)

WHAT IS CLAIMED IS:
1. A method of immunization of a host against pneumococcal infection, which comprises orally administering to the host an immunizing amount of pneumococcal surface protein A (PspA) or at least one fragment of such PspA containing at least one protection-eliciting epitope.
2. The method of claim 1 wherein said PspA is in the form of killed whole pneumococci or of a lysate of pneumococci.
3. The method of claim 1 wherein said PspA is in the form of an isolated and purified PspA.
4. The method of claim 1 wherein said PspA is administered in conjunction with an adjuvant.
5. The method of claim 4 wherein the adjuvant is a cholera toxin.
6. The method of claim 1 wherein said mucosal administration is effected intragastrically to provide protection to the host against both pneumococcal colonization and systemic infection.
7. The method of claim 1 wherein said mucosal administration is effected intragastrically to provide protection to the host against pulmonary infection.
8. The method of claim 1 wherein said oral administration is effected intragastrically to provide protection to the host against an infection starting as a pulmonary infection.
9. A method of immunization of a host against Streptococcus pneumoniae, which comprises intragastrically administering to the host an immunizing amount of pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a lysate of pneumococci or an isolated and purified PspA, or an immunogenic fragment thereof.
10. The method of claim 9 for the prevention of pulmonary infection and pneumococcal disease.
11. The method claim 9 for the prevention of pulmonary infection and pneumococcal disease originating from pulmonary infection.
12. The method of claim 9 wherein said PspA is administered intragastrically in conjunction with an adjuvanting amount of a cholera toxin.
13. The method of claim 9 for the prevention of carriage of pneumococci and prevention of pneumococcal disease.
14. A vaccine composition for intragastric administration to a host to confer protection against S.
pneumoniae which comprises:
an effective amount of pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a pneumococcal lysate, an isolated and purified PspA or an immunogenic fragment thereof containing at least one protection-eliciting epitope, and optionally an adjuvanting amount of cholera toxin, and optionally a pharmaceutical carrier therefor.
15. A vaccine or immunogenic composition for oral administration to a host to confer protection or elicit an immunological response against S. pneumoniae which comprises:
an effective amount of a pneumococcal surface protein A (PspA) in the form of a killed whole pneumococci, a pneumococcal lysate, an isolated and purified PspA or an immunogenic fragment thereof containing at least one protection-eliciting epitope or immunological response-eliciting epitope.
16. The composition of claim 15 wherein the oral administration is intragastric.
17. The composition of claim 15 further comprising:
a pharmaceutically acceptable adjuvant, or a pharmaceutically acceptable carrier, or both a pharmaceutically acceptable carrier and a pharmaceutically acceptable adjuvant.
18. The composition of claim 17 wherein the adjuvant is a cholera toxin.
19. A method for eliciting an immunological response in a host against S. pneumoniae which comprises orally administering to the host an effective amount of pneumococcal surface protein A (PspA) or at least one fragment of such PspA containing at least one immunologically active epitope.
20. The method of claim 19 wherein the orally administering is performed by intragastrically administering and, the PspA is in the form of killed i whole pneumococci, a lysate of pneumococci, an isolated and purified PspA, or an immunogenic fragment thereof containing at least one immunologically active or protection-eliciting epitope.
21. A kit for a composition 17 comprising PspA
and the carrier or diluent separately packaged, and optionally, instructions for admixture or administration.
CA002222965A 1995-06-02 1996-05-31 Oral administration of pneumococcal antigens Abandoned CA2222965A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US08/458,399 1995-06-02
US08/458,399 US6231870B1 (en) 1995-06-02 1995-06-02 Oral administration of pneumoccal antigens
US08/482,981 1995-06-07
US08/482,981 US6232116B1 (en) 1991-02-15 1995-06-07 Oral administration of pneumococcal antigens
US08/657,751 US6004802A (en) 1995-06-02 1996-05-30 Oral administration of pneumococcal antigens
US08/657,751 1996-05-30

Publications (1)

Publication Number Publication Date
CA2222965A1 true CA2222965A1 (en) 1996-12-12

Family

ID=27412732

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002222965A Abandoned CA2222965A1 (en) 1995-06-02 1996-05-31 Oral administration of pneumococcal antigens

Country Status (6)

Country Link
EP (1) EP0871479A2 (en)
AU (1) AU709368B2 (en)
CA (1) CA2222965A1 (en)
FI (1) FI974376A (en)
NO (1) NO975522L (en)
WO (1) WO1996039113A2 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19703437A1 (en) * 1997-01-30 1998-08-06 Luitpold Pharma Gmbh Mixtures of outer membranes and / or cell walls of bacteria for oral immunization against mucosal infections
KR19980067137A (en) * 1997-01-31 1998-10-15 박원훈 Vaccine against pneumococcal pneumoniae using alginate ultrafine granulocytes
DE19708537A1 (en) * 1997-03-03 1998-09-10 Biotechnolog Forschung Gmbh New surface protein (SpsA protein) from Streptococcus pneumoniae etc.
WO2008109958A1 (en) * 2007-03-15 2008-09-18 Hunter Immunology Limited Methods for evaluation of oral vaccines

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6027734A (en) * 1991-02-15 2000-02-22 Uab Research Foundation Mucosal administration of pneumococcal antigens
DK0571538T5 (en) * 1991-02-15 2002-03-04 Uab Research Foundation The structural gene for pneumococcal protein
US5980909A (en) * 1991-02-15 1999-11-09 Uab Research Foundation Epitopic regions of pneumococcal surface protein A

Also Published As

Publication number Publication date
WO1996039113A2 (en) 1996-12-12
AU709368B2 (en) 1999-08-26
NO975522L (en) 1998-02-02
WO1996039113A3 (en) 1997-06-05
FI974376A0 (en) 1997-12-01
EP0871479A4 (en) 1998-11-11
FI974376A (en) 1998-01-26
AU6998596A (en) 1996-12-24
NO975522D0 (en) 1997-12-01
EP0871479A2 (en) 1998-10-21

Similar Documents

Publication Publication Date Title
Frasch Vaccines for prevention of meningococcal disease
Alexander et al. Immunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae
US6984385B2 (en) Immunological combination compositions and methods
LEVINE et al. Typhoid vaccines come of age
US20090098157A1 (en) Helicobacter Pylori Vaccination
AU2018201768A1 (en) Protein antigens that provide protection against pneumococcal colonization and/or disease
US20050260225A1 (en) Intranasal recombinant Salmonella vaccine encoding heterologous polysaccharide antigens
JP2012501959A (en) Composition comprising Yersinia pestis antigen
KR20120093141A (en) Vaccines and compositions against streptococcus pneumoniae
US20180271969A1 (en) Streptococcus suis polysaccharide-protein conjugate composition
EP0682950B1 (en) Mucosal administration of pneumococcal antigens
US6232116B1 (en) Oral administration of pneumococcal antigens
US9310381B2 (en) Engineered type IV pilin of Clostridium difficile
US6004802A (en) Oral administration of pneumococcal antigens
US6231870B1 (en) Oral administration of pneumoccal antigens
AU709368B2 (en) Oral administration of pneumococcal antigens
US5429818A (en) Non-capsulated mutants of Actinobacillus pleuropneumoniae useful as vaccines
US20140161836A1 (en) Carious tooth vaccine and preparation method
Kaplonek et al. A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae
AU734488B2 (en) Mucosal administration of pneumococcal antigens
Hara et al. Outer membrane proteins as potential candidate vaccine targets
Henriksen et al. Vaccination with Protein‐Conjugated and Native Type 3 Capsular Polysaccharide in an Ethanol‐Fed Rat Model of Pneumococcal Pneumonia
RU2005779C1 (en) Strain of bacterium leptospira interrogans of serogroup icterohaemorrhagiae, serovara copenhagent used for preparing of vaccine against leptospirosis

Legal Events

Date Code Title Description
EEER Examination request
FZDE Discontinued