CA2223041A1 - Immunological combination compositions and methods - Google Patents

Abstract

Immunological compositions and methods for making and using them. The compositions contain at least one antigen and at least one lipoprotein and optionally an adjuvant. The lipoprotein can itself be antigenic or immunogenic. The antigen can be influenza HA and the lipoprotein a recombinantly expressed product having an OspA leader for lipidation and PspA for the protein portion. The antigen can be OspC and the lipoprotein OspA. The components of the composition are co-administered. A potentiated immunological response is obtained by the compositions and methods.

Description

W O 96/~0290 PCTrUS96/08866 IM~n~NOLOGI Q.L co~n3INATIoN COMPOSITIONS ~ND I~ C~S
~K~ TO ~T~ APPLIC~TIONS

This application is a continuation-in-part o~
application Serial No. 08/476,656, ~iled June 7, 1995.
Re~erence, especially with respect to recom.binant Borrelia proteins, is made to each o~
applications Serial Nos. 07/973,338, ~iled October 29, 1992; 08/373,455 (Rule 62 FWC o~ USSN 07/973,338), filed January 17, 1995, 07/888,765, ~iled May 27, 1992;
10 08/211,891, filed October 16, 1992 (national phase of PCT/US92/08697); and 07/779,048, ~iled October 18, 1991.
Re~erence, e~pecially with respect to structural genes of pneumococcal proteins, epitopic regions thereof, and ~m; n;stration o~ pneumococcal proteins, is made to each 15 of applications Serial Nos. 656,773, ~iled February 15, 1991; 835,698, ~iled February 12, 1992; 072,065, filed June 3, 1993; 072,068, ~iled June 3, 1993; 214,222 ~iled March 17, 1994; 214,164, ~iled March 17, 1994; 247,491, ~iled May 23, 1994; 048,896, ~iled April 20, 1993;
20 246,636, ~iled May 20, 1994; 08/458,399 (continuation-in-part o~ application Serial No. 246,636, filed October 7, 1994) ~iled June 2, 1995; 08/446,201 ~iled May 19, 1995;
08/312,949, ~iled September 30, 1994. With respect to Expression o~ Lipoproteins, re~erence is made to 25 application Serial No. 08/475,781, ~iled June 7, 1995.
And, with respect to Compositions and Methods For ~m; n;stering Borrelia Burgdorferi Antigens mucosally, e.g., orally, ~or stimulating an ;mmllnological response, reference is made to Barbour et al., application Serial No. , concurrently ~iled herewith (Attorney Docket No. 454312-2420).
Each o~ the a~orementioned applications is hereby incorporated herein by re~erence. Several documents are cited in the following text, and each is also hereby incorporated herein by re~erence.
FIELD OF '1~; lNVl:~ lON

The present invention relates to compositions ~or eliciting an ;mmllnological response in a host, ~n~m~l or hnm~n, and methods ~or m~k; ng and using the same The invention further relates to such compositions and methods wherein the composition comprises an antigen and a lipoprotein adsorbed to an adjuvant. More preferably, the lipoprotein is also antigenic or ;mml~nogenict and thus the composition can be a combination, multivalent or "cocktail" composition. Accordingly, the invention also relates to co-~m; n; stration of at least one antigen and at least one lipoprotein in a composition which can include additional ingredients, such as an adjuvant.
The lipoprotein can be a naturally occurring lipoprotein or a recom~inant lipoprotein. The recombinant lipoprotein can be from expression by a vector of homologous sequences for the lipidated and protein portions of the lipoprotein, i.e., the sequences for the lipidation and protein can naturally occur together. In such a recombinant lipoprotein, the lipidation thereo~ can be from expression of a first nucleic acid sequence and the protein thereof can be from expression of a second nucleic acid sequence, wherein the first and second nucleic acid sequences, which do not naturally occur together, and such sequences can be expressed as a contiguous lipoprotein. Thus, the invention relates to compositions and methods involving ~m; n; stration of lipoproteins, including recombinant lipoproteins; and the recombinant lipoproteins can be similar to native proteins, or novel hybrid proteins.
The invention further relates to the aforementioned compositions for eliciting an immunological response and methods for making and using the same wherein the lipoprotein is recombinantly expressed lipoprotein from expression of such aforementioned first and second nucleic acid sequences wherein the first nucleic acid sequence encodes a Borrelia lipoprotein leader sequence; preferably such a recombinant lipidated protein expressed using the nucleic acid sequence encoding the OspA leader sequence. In a preferred embodiment the lipoprotein can be OspA; and W O 96/40290 PCTrUS96/08866 thus, the invention also relates to recombinant OspA and uses thereo~ the compositions and methods.
Several publications are re~erenced in this application. Full citation to these re~erences is ~ound at the end o~ the speci~ication immediately preceding the claims or where the publication is mentioned; and each o~
these publications is hereby incorporated herein by re~erence.
R~Rr~o~ND OF THE l~v~NllON
Immunogenicity can be signi~icantly improved i~
an antigen is co-~min; stered with an adjuvant, commonly used as .001~ to 50~ solution in phosphate bu~ered saline (PBS). Adjuvants enhance the immunogenicity o~ an antigen 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 e~ect facilitating a slow, sustained release o~ antigen to cells o~ the immune system. Adjuvants can also attract cells o~ the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used ~or many years to improve the host ;mmllne response to, ~or example, vaccines. Intrinsic adjuvants, such as lipopolysaccarides, normally are the components o~ the killed or attenuated bacteria used as vaccines.
Bxtrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are ~ormulated to ~nh~nce the host ;mmlln~ response. Alllm;nllm hydroxide and alllm;nllm phosphate (collectively commonly re~erred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The e~icacy o~ 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 o~ extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane 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 immune response (HIR) and cell-mediated ;mmlln;ty (CMI), immunogens are preferably emulsified in adjuvants.
Desirable characteristics of ideal adjuvants include any or all of:
(1) lack of toxicity;

(2) ability to stimulate a long-lasting ;mm 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;

(6) capability of selectively interacting with populations of antigen presenting cells (APC);

(7) ability to specifically elicit appropriate TH1 or TH2 cell-specific ;mmlln~ 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 ;mmlln~-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 immune responses in both herpes simplex =
CA 0222304l l997-l2-Ol W 096/40290 PCT~US96/08866 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 re~erence 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.
Bessler et al., "Synthetic lipopeptides as novel adjuvants," in the 44th Forum In Immunology (1992) at page 548 et seq., especially at 548-550, incorporated herein by reference, is directed to employing lipopeptides as adjuvants when given in combination with an antigen. The lipopeptides typically had P3C as the lipidated moiety and up to only 5 amino acids, e.g., P3C-SG, P3C-SK4, P3C-SS, P3C-SSNA, P3C-SSNA. The lipopeptide was coupled with or added to only certain antigens or to non-immunogenic proteins, such as P3C-SSNA supplementing S. typhim7~ri7um vaccine, PC3-SS coupled to VP1(135-154) of foot-and-mouth disease, PC3-SG-OSu coupled to non-immunogenic protein hirudin, P3C-SK coupled to FITC or DNP or P3C-SG coupled to a metabolite from Streptomyces venezuelae. While adjuvant mixing and conjugating procedures of Bessler can be employed in the practice of the present invention, Bessler fails to teach or suggest employing a lipoprotein with at least one antigen in a - composition, especially such compositions wherein the lipoprotein is also antigenic, or the immunological combination compositions and methods of this invention.

W O 96/40290 PCTrUS96/08866 In this regard, a distinction between a peptide, especially a peptide having up to only about 5 amino acids, and a protein is being made, as is a distinction between an antigenic lipoprotein and a non-antigenic lipopeptide, inter alia. Peptides di~erimmunologically ~rom proteins in that short peptides have the potential for direct presentation by the major histocompatibility complex (MHC), while proteins require processing prior to presentation to T-cells. A peptide ~urther di~ers ~rom a protein in that a protein is large enough that it is capable o~ ~orming ~unctional domains (i.e., having tertiary structure), whereas a peptide cannot.
Nardelli et al. [Vaccine (1994), 12(14):1335-1339] covalently linked a tetravalent multiple antigen peptide containing a gpl20 sequence to a lipid moiety and orally A~m; n; stered the resulting synthetic lipopeptide to mice. It was ~ound that both mucosal IgA response and systemic plasma IgG were stimulated, and cell-mediated ;mm~n; ty, as shown by lymphokine production and generation o~ a speci~ic cytotoxic response, was induced.
Only a short peptide was used, rather than a whole lipoprotein, and there is no teaching or suggestion that the synthetic lipopeptide could be used as an adjuvant ~or other proteins. In ~act, this re~erence actually teaches away ~rom the use o~ lipoproteins, which are more soluble than lipopeptides, as immunogens; see, e.g. p.
1338, last line ('Isoluble proteins are not immunogens by oral routes").
Cro~t et al. [J. Immunol. (1991), 146(5): 793-796] have covalently coupled integral membrane proteins (Imps) isolated ~rom E. coli to various antigens and obtained enhanced ;mml~n~ responses by intramuscular injection into mice and rabbits. However, there are disadvantages to coupling the lipoprotein and the antigen covalently. Important epitopes may be damaged, and the coupling procedure is di~icult to control and o~ten CA 0222304l l997-l2-Ol W 096/40290 PCTrUS96/08866 requires the use of toxic cross-linkers. Thus, it would be advantageous to provide a method for inducing an enhanced immunological response which does not require that the antigen be cross-linked to a protein. Moreover, when the antigen CSP-OVA was merely mixed, rather than covalently linked, with the lipoprotein TraT, only a small increase in antibody response was obtained. Croft et al. therefore concluded that the lipid is not necessary for the adjuvant effect, contrary to the surprising findings of the present inventors.
U.S. Patent No. 4,439,425 relates to lipopeptides having 2 to 10 amino acids and their prophylactic ~m; n; stration by oral or rectal routes.
Bessler et al. ["Synthetic Lipopeptide 15 Conjugates Constitute Efficient Novel Immunogens and Adjuvants in Parenteral and Oral Immunization"
(Abstract), Meeting on Molecular Approaches to the Control oi~ Inf~ectious Diseases, (September 13-17, 1995), Cold Spring, Harbor Laboratory (not prior art in view of 20 June 7, 1995 filing date of USSN 08/476,656)] relates to the oral ~m; n; stration o~ lipopeptides having six amino acids which were covalently coupled to antigens. The lipopeptide-antigen conjugates were found to induce a hapten-specific immune response.
Schlecht et al. [Zbl. Bakt. (1989) 271:493-500]
relates to Salmonella tyrh;m77~ium vaccines supplemented with synthetically prepared derivatives of a bacterial lipoprotein having five amino acids. The vaccines were ~m; n; stered by two intraperitoneal injections and 30 challenged intraperitoneally with graded doses of S.
typhimurium. When the protective capacity of the supplemented vaccines was compared with that of the unsupplemented vaccine, it was found that 90~ of the S.
- typhimurium vaccine could be replaced by the lipopeptide 35 without a recognizable decrease in protective capacity.
Substantial effort has been directed toward the development o~ a vaccine for Lyme disease. Two distinct WO 96/40290 PCT~US96/08866 approaches have been used for vaccine development. One approach is to use a vaccine composed o~ whole inactivated spirochetes, as described by Johnson in US
Patent No. 4,721,617. A whole inactivated vaccine has been shown to protect hamsters ~rom challenge and has been licensed ~or use in dogs.
Due to the concerns about cross-reactive antigens within a whole cell preparation, human vaccine research has ~ocused on the identi~ication and development of non-cross-reactive protective antigens expressed by B. burgdorferi. Several candidate antigens have been identified to date. Much of this ef~ort has focused on the most abundant outer surface protein o~ B.
burgdorferi, namely outer surface protein A (OspA), as described in published PCT patent application WO
92/14488, assigned to the assignee hereof. Several versions o~ this protein have been shown to induce protective ;mmlln;ty in mouse, hamster and dog challenge studies. Clinical trials in hllm~n~ have shown the formulations of OspA to be safe and ;mmllnogeniC in h [Keller et al., JAMA (1994) 271:1764-1768]. Indeed, one formulation containing recombinant lipidated OspA as described in the a~orementioned WO 92/14488, is now undergoing Phase III sa~ety/efficacy trials in hllm~ns While OspA is expressed in the vast majority o~
clinical isolates of B. burgdorferi from North America, a different picture has emerged from ~m;n~tion o~ the clinical Borrelia isolates in Europe. In Europe, Lyme disease is caused by three genospecies o~ Borrelia, namely B. burgdorferi, B. garinii and B. afzelli. In approximately half o~ the European isolates, OspA is not the most abundant outer sur~ace protein. A second outer surface protein C (OspC) is the major surface antigen found on these spirochetes. In fact, a number of European clinical isolates that do not express OspA have been identified. Immunization of gerbils and mice with purified recombinant OspC produces protective ;mmlln;ty to W O 96/40Z90 PCTrUS96/08866 B. burgdorferi strains expressing the homologous OspC
protein [V. Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S. Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspC protein is currently being considered as a possible component of a second generation Lyme vaccine formulation.
Recombinant proteins are promising vaccine or ;mmllnogenic composition candidates, because they can be produced at high yield and purity and manipulated to m~;m;ze desirable activities and m;n;m;ze undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or ;mml~nogenic compositions. Moreover, it would be greatly desired to be able to ~m; ni ster such proteins in combination with other antigens.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviated Pam3Cys. This moiety is found at the amino terminus of the bacterial lipoproteins which are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II.
Synthetic peptides that by themselves are not ;mmllnogenic induce a strong antibody response when covalently coupled to Pam3Cys [Bessler et al. (1992)].
In addition to an antibody response, one often needs to induce a cellular ;mml~n~ response, particularly cytoxic T lymphocytes (CTLs). Pam3Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
The nucleic acid sequence and encoded amino acid sequence for OspA are known for several B.
- burgdorferi clinical isolates and is described, for example, in published PCT application WO 90/04411 (Symbicom AB) for B31 strain of B. burgdorferi and in Johnson et al., Infect. Immun. 60:1845-1853 ~or a comparison o~ the ospA operons o~ three B. burgdorferi isolates o~ di~erent geographic origins, namely B31, ACA1 and Ip90.
As described in WO 90/04411, an analysis of the DNA sequence ~or the B31 strain shows that the OspA is encoded by an open reading frame o~ 819 nucleotides starting at position 151 o~ the DNA sequence and terminating at position 970 o~ the DNA sequence (see Figure 1 therein). The ~irst sixteen amino acid residues o~ OspA constitute a hydrophobic signal sequence o~ OspA.
The primary translation product of the ~ull length B.
burgdorferi gene contains a hydrophobic N-terminal signal sequence which is a substrate ~or the attachment o~ a diacyl glycerol to the sul~hydryl side chain o~ the ad~acent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment o~ a third ~atty acid to the N-terminus occurs. The complete lipid moiety is termed Pam3Cys. It has been shown that lipidation o~ OspA is necessary ~or immunogenicity, since OspA lipoprotein with an N-terminal Pam3Cys moiety stimulated a strong antibody response, while OspA lacking the attached lipid did not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81-90] .
Published international patent application WO
91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence o~ the ospC gene o~ B.
burgdorferi strain Pko and the OspC (termed pC in this re~erence) protein encoded thereby o~ 22 kDa molecular weight. This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used ~or OspA. Based on the ~indings regarding OspA, one might expect that lipidation o~ recombinant OspC would be useful to enhance its ;mmllnogenicity; but, as discussed in above-re~erenced USSN 08/475,781, the therein applicants experienced di~ficulties in obtaining detectable expression o~ recombinant OspC. It would be .

W O 96/40290 PCT~US96/08866 use~ul to enhance the ;mmllnogenicity o~ recombinant OspC.
Moreover, it would be useful to have a multivalent Lyme Disease ~mmllnological composition which contains antigens against both North American and European Borrelia isolates.
Streptoccus pneumoniae causes more ~atal in~ections world-wide than almost any other pathogen. In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS. Most fatal pneumoccal in~ections in the U.S.A. occur in individuals over 65 years o~ age, in whom S. pneumoniae is the most common cause of community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunode~icient patents including those with sickle cell disease. In the less-developed areas o~ the world, pneumococcal in~ection is one o~ the largest causes o~
death among children less than 5 years o~ age. The increase in the ~requency o~ multiple antibiotic resistance among pneumococci and the prohibitive cost o~
drug treatment in poor countries make the present prospect ~or control o~ pneumococcal disease problematical.
The reservoir of pneumococci that in~ect man is maintained primarily via nasopharyngeal human carriage.
~llm~n~ acquire pneumococci ~irst through aerosols or by direct contact. Pneumococci ~irst colonize the upper airways and can remain in nasal mucosa ~or weeks or months. As many as 50~ or more o~ young children and the elderly are colonized. In most cases, this colonization results in no apparent in~ection. In some individuals, however, the organism carried in the nasopharynx can give rise to symptomatic sinusitis of middle ear in~ection.
I~ pneumococci are aspirated into the lung, especially - with ~ood particles or mucus, they can cause pneumonia.
In~ections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially i~ they continue to be shed in large numbers ~rom the original ~ocus o~ in~ection. 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~Pr have life-long neurological sequelae.
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. The 23-valent vaccine is not effective in children less than 2 years of age because o~ their inability to make adequate responses to most polysaccharides. 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.
The S. pneumoniae cell surface protein PspA has been ~mo~Rtrated to be a virulence factor and a protective antigen. In published international patent application W0 92/14488, there are described the DNA
sequences for the pspA yene from 5. 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.
In an effort to develop a vaccine or ;mmllnogenic composition based on PspA, PspA has been recombinantly expressed in E. coli. It has been found that in order to efficiently express PspA, it is useful to truncate the mature PspA molecule of the Rxl strain from its normal length of 589 amino acids to that of 314 amino acids comprising amino acids 1 to 314. This region of the PspA molecule contains most, if not all, of the protective epitopes of PspA. However, immunogenicity and protection studies in mice have demonstrated that the truncated recombinant form of PspA is not immunogenic in CA 0222304l l997-l2-Ol naive mice. Thus, it would be use~ul to improve the immunogenicity o~ recombinant PspA and ~ragments thereo~
Moreover, it would be highly desirable to employ a pneumococcal antigen in a combination or multivalent composition. For instance, in~luenza (Flu) is a problematical in~ection, especially in the elderly and the young, as well as pneumonia; and, yearly Flu shots are common, especially in North America. Thus, it would be desirable to be able to ~m;n;ster Flu and pneumococcal antigens in one preparation.
Helicobacter pylori is the spiral bacterium which selectively colonizes human gastric mucin-secreting cells and is the causative agent in most cases o~
nonerosive gastritis in hllm~n~ Recent research activity indicates that H. pylori, which has a high urease activity, is responsible ~or most peptic ulcers as well as many gastric cancers. Many studies have suggested that urease, a complex o~ the products of the ureA and ureB genes, may be a protective antigen. However, until now it has not been known how to produce a su~icient mucosal immune response to urease without cholera toxin or related adjuvants.
Antigens or immunogenic ~ragments thereof stimulate an immune response when ~m;n;stered to a host Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with adjuvant. Currently, alum is the only adjuvant licensed ~or hllm~n use, although hundreds o~
experimental adjuvants such as cholera toxin B are being tested. However, these adjuvants have de~iciencies. For instance, while cholera toxin is a good adjuvant, it is highly toxic. On the other hand, cholera toxin B, while non-toxic, has no adjuvant activity. It would thus be - desirable to provide immunological compositions capable o~ eliciting a strong response without the need ~or an adjuvant.

CA 0222304l l997-l2-Ol In certain instances when multiple antigens (two or more) are ~m;n; stered in the same preparation or se~uentially, a phenomenon called efficacy interference occurs. Simply, due to the interaction of one or more antigens in the preparation with the host immunological system, the second or other antigens in the preparation fail to elicit a sufficient response, i.e., the efficacy of the latter antigen(s) is inter~ered with by the former antigen(s). It would thus be desirable to provide multivalent ;mmnnological compositions which do not give rise to this efficacy interference phenomenon; for instance, without wishing to necessarily be bound by any one particular theory, because the second antigen is a lipoprotein and as such is having an adjuvanting effect on the first antigen and, when in a combination composition with an adjuvant, a synergistic potentiating ef~ect is obtained (whereby the first antigen is not inter~ering with the second antigen and vice versa) .
More generally it would be desirable to enhance the ~mmllnngenicity of antigens by methods other than the use o~ an adjuvant, and to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater ;mml~nologica response.
Above-referenced USSN 08/446,201 discloses that mucosal ~min; stration of killed whole pneumococci, lysate of pneumococci or isolated and purified PspA, as well as immunogenic frayments thereof, particularly when ~m; n; stered with an adjuvant, provides protection in ~n;m~l S against pneumococcal colonization and systemic infection. It has now been surprisingly found that mucosal ~m;n; stration of other antigens, such as urease, along with a lipoprotein, elicits systemic and local responses in ~n;m~l S without the use of an adjuvant.
It is believed that heretofore the art has not taught or suggested: immunological compositions comprising at least one antigen and a lipoprotein, and, CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 optionally, an adjuvant, more pre~erably an antigen, an antigenic lipoprotein and, optionally, an adjuvant, and methods ~or ~m;n; stering the same as a multivalent composition, or ~or ~m;n; stering those components simultaneously or sequentially, especially such compositions and methods having enhanced immunogenicity.
OBJECTS AND SI~I~RY OF T}IE lNV~;NllON
It is an object o~ the invention to provide immunological compositions and methods ~or making and using the same.
It is a ~urther object o~ the invention to provide ;mmllnological compositions having enhanced immunogenicity; or, compositions the ~m;n; stration o~
which potentiates the immunological response.
It is another object o~ the invention to provide methods for inducing an immunological response, pre~erably a potentiated response, involving ~m;n; stration to a suitable host such ;mml]nological compositions.
It is yet an additional object o~ the invention to provide an ;mml~nological composition comprising at least one antigen and at least one lipoprotein and, optionally, an adjuvant, pre~erably such compositions wherein the lipoprotein is antigenic.
It is still a ~urther object o~ the invention to provide a method for inducing or potentiating an ;mmllnological response comprising ~m;n; stering to a host, ~n;m~l or hllm~n, at least one antigen and at least one lipoprotein, and optionally, an adjuvant; and more pre~erably such methods wherein the lipoprotein is antigenic.
It has surprisingly been ~ound that administration to a host o~ at least one lipoprotein with - at least one antigen provides an immunological response 35 by the host. The immunological response is generally better than that obtained by ~m;n; stration o~ the antigen alone.

CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 Moreover, it has also surprisingly been found that ~m;n;stration to a host of at least one antigen, at least one lipoprotein and, optionally an adjuvant by either co-~m;n;stration or by sequential ~m;n;stration (over a suitable time period such that each of the antigen, adjuvant and lipoprotein are present within the host at the same time) obtains an ;mml~nQlogical response to the antigen by the host. This ;mmllnological response is generally better than that obtained by administration of the antigen alone or by ~m;n;stration of the antigen and adjuvant. Lipidated proteins appear to stimulate the ;mmllne response, in the m~nn~r of the adjuvant cholera toxin B.
Furthermore, it has additionally been surprisingly found that in these administrations the lipoprotein itsel~ can be ;mmllnQgenic or antigenic, e.g., be an antigen, and that not only is the ;mmllnQlogical response to the antigen by the host obtained; but also, an ;mml~nological response to the antigenic lipoprotein is obtained. The ;mmllnological response to the antigenic lipoprotein can be as good as, or better than, that obtained by ~m; n;stration of the lipoprotein alone or with an adjuvant; and, the immunological response to the antigen can be better than that obtained by administering the antigen alone or the antigen and adjuvant.
The term lipoprotein as used herein is meant to exclude prior art lipopeptides; ergo, a lipoprotein can have more than 2 to 10 amino acids, or more than 18 to 20 amino acids, or greater than 24 amino acids, or 30 or more amino acids. Lipoproteins are larger molecules which reduce the amount of antigen and/or ~m; n;strations of the antigen, despite a prejudice in the art against lipoproteins, e.g., Vaccine (1994) 12(14):1335, 1338 last line, column 1, to first line, column 2 ("soluble proteins ... not ;mmllnQgenic [by oral routes]"). Prior lipopeptides, due to their small size, can have at most one epitope whereas lipoproteins that can be used in the CA 0222304l l997-l2-Ol present invention can have more than one epitope, e.g.
one B and one T, or can even be antigenic in their own right. Lipopeptides, in addition to being shorter and having less molecular weight than lipoproteins, and being di~icult to synthesize because usually are made by Merrifield or other synthesis methods, di~er ~rom lipoproteins in that lipoproteins are larger, generally not made by Merri~ield synthesis methods, and can be ~rom isolation ~rom natural sources or ~rom recombinant techniques. That is, lipopeptides o~ the prior art were synthetically made, which limits their size to no more than about thirty amino acids. Lipoproteins are larger and o~ greater molecular weight than lipopeptides, and, unlike lipopeptides, are generally not made by Merrifield synthesis methods. Lipoproteins can be isolated ~rom natural sources or produced by recombinant techniques.
Further, lipoproteins are more soluble than lipopeptides.
Additionally, peptides do not have quaternary or tertiary structure whereas proteins can have quaternary and/or tertiary structure. Based upon their ability to ~orm tertiary structure, proteins have the ability to ~orm ~unctional domains which peptides cannot. Thus, there are several di~ferences between prior "lipopeptides" and "lipoproteins" as used in this invention.
The lipoproteins ~ormulations of the invention can be ~m; n; stered nasally and this is advantageous.
According to the present invention, it also has been ~ound that a lipoprotein administered with an antigen according to the present invention is 500 times more potent then administration of a lipopeptide and an antigen.
Accordingly, the present invention provides an immunological composition comprising at least one antigen - and at least one lipoprotein. The composition can ~urther optionally, but not necessarily, comprise an adjuvant. Pre~erably the lipoprotein is an antigen. The ;mmllnological composition can be a vaccine.

CA 0222304l l997-l2-Ol The present invention ~urther comprises a method ~or inducing an ;mml]nological response in a host comprising ~m;n;stering the aforementioned ;mml7nslogical composition. The method can be ~or inducing a protective response, e.g., when the immunological composition is a vaccine.
The present invention ~urther comprises a method for inducing an ;mml1nslogical response comprising sequentially administering a ~irst composition comprising an antigen, and a second composition comprising a lipoprotein. Optionally either the ~irst or second composition, or both the ~irst and second compositions can ~urther comprise an adjuvant. Pre~erably the lipsprotein is an antigen. The sequential ~m;n;stration should be undertaken over a suitable period of time whereby each o~ the antigen, lipoprotein and optional adjuvant is present at the same time in the host; and, such a time period can be determined by the skilled artisan, ~rom this disclosure, without undue experimentation and by methods within the ambit o~ the skilled artisan, such as host sera titrations involving analysis thereo~ ~or the presence o~ antigen or antibody by, ~or instance, ELISA analysis. The administration may be mucosal, e.g., intragastric or intranasal.
The present invention particularly involves methods ~or inducing an ;mml1nological response in a host comprising the steps o~ mucosally ~m;n;stering to the host at least one antigen, and mucosally ~m;n;stering to the host at least one lipoprotein. The ~m;n;stration can be simultaneous or sequential. The antigen may be a bacterial protein or ~ragment thereo~, e.g. urease.
The "antigen" in the inventive compositions and methods can be any antigen to which one wishes to elicit an ;mm11nnlogical response in a host, ~n; m~l or human.
For instance, without wishing to necessarily limit the invention, the antigen can be: a Borrel ia antigen, e.g., OspA, OspC, OspB, OspD; a pneumococcal antigen, e.g., W O 96/40290 PCT~US96/08866 PspA; an influenza (Flu) antigen such as HA; a pertussis or whooping cough antigen such as the pertussis 69KD
polypeptide; a hepatitis antigen, e.g., hepatitis B
antigen such as hepatitis B surface antigen; a Helicobacter pylori antigen such as urease; a rabies virus antigen, e.g., rabies G antigen; a flavivirus antigen, e.g., a Japanese encephalitis virus, Dengue virus or yellow fever virus antigen; a chicken pox virus antigen; a diphtheria antigen; a C. tetani antigen, e.g., tetanus toxoid; a mumps virus antigen; a measles virus antigen; a malaria antigen; a herpes virus antigen, such as an alphaherpesvirus, betaherpesvirus or g~mm~h~rpesvirus antigen, e.g., a herpes virus glycoprotein, for instance an equine herpesvirus antigen, e.g., gpl3, gpl4, gD, gp63, or gE, a pseudorabies virus antigen, e.g., gp50, gpII, gpIII, gpI, a herpes simplex virus antigen, e.g., gC, gD, a bovine herpes virus antigen, e.g., gI, a feline herpes virus antigen, e.g., gB, an Epstein-Barr virus antigen, e.g., gp220, gp340, or gH, or a hllm~n cytomegalovirus antigen, e.g., gB; a human ;mmllnQde~iciency virus antigen, e.g., gpl60 or gpl20; a simian immunode~iciency virus antigen; a bovine viral diarrhea virus antigeni an equine influenza virus antigen; a feline leukemia virus antigen; a canine distemper virus antigen, e.g., HA or F glycoproteins; a c~n; ne adenovirus antigen, e.g., canine adenovirus type 2 antigen; a c~n; n~ coronavirus antigen; a canine parainfluenza antigen; a canine parvovirus antigen; a Hantaan virus antigen; an avian influenza virus antigen e.g., a nucleoprotein antigen; a Newcastle Disease virus antigen, e.g., F, HN; an antigen o~ rous associated virus, e.g., an RAV-l envelope antigen; an infectious bronchitis virus antigen, e.g., a matrix antigen or a - preplomer antigeni an infectious bursal disease virus antigen; a cholera antigen; a tumor associated antigen; a feline ;mmllnodeficiency virus antigen; a foot-and-mouth disease virus antigen; a Marek's Disease Virus antigen; a Staphylococci antigen; a Streptococci antigen; a Haemophilus influenza antigen, e.g., group b polysaccharide-protein conjugates; a papilloma virus; a poliovirus antigen; a rubella virus antigen; a poxvirus, such as smallpox antigen, e.g., vaccinia; a typhus virus antigen; a typhoid virus antigen; a tuberculosis virus antigen; an HTLV antigen; or, other bacteria, virus or pathogen antigen, such as a bacterial or viral sur~ace antigen or coat protein.
The antigen can be a known antigen; can be isolated from the bacteria, virus or pathogen; or, can be a recombinant antigen ~rom expression o~ suitable nucleic acid coding there~or by a suitable vector, and isolation and/or purification o~ the recombinant antigen. The selection of the antigen is, o~ course, dependent upon the immunological response desired and the host.
The lipoprotein can be any lipoprotein which is compatible physiologically with the host. Most preferably it is a bacterial lipoprotein or a lipoprotein having a bacterial lipid moiety.
The lipoprotein is pre~erably itsel~ also an antigen. Thus, the lipoprotein is pre~erably an outer membrane component o~ a pathogen, e.g., virus or bacteria, more pre~erably a lipoprotein which has an extrinsic or peripheral protein such that the lipoprotein is extracted with mild conditions or detergent without substantial denaturation or loss o~ lipid moiety (so as to retain epitopes). However, any antigenic lipoprotein can be employed in the practice of the invention. And, the lipoprotein can be isolated ~rom a suitable physiological source, or ~rom an organism, e.g., bacteria; or can be recombinantly produced. Thus, the lipidated Borrelia antigens, e.g., recombinant OspA, and, the lipidated OspA and Borrelia ~ractions containing lipidated proteins (isolated by mild conditions) disclosed in the applications re~erenced in the Reference to Related Applications, and in W0 90/04411 (incorporated CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 herein by reference) can be used as the lipoprotein in the practice o~ the invention. Of course, the "antigen"
and the "lipoprotein" in the invention are separate, different ingredients (such that, for instance, when the "lipoprotein" is OspA, it is not also the "antigen").
In application Serial No. 08/475,781 filed June 7, 1995 and incorporated herein by reference, recombinant lipoproteins, especially antigenic recombinant lipoproteins, for instance, those from expression of the leader sequence of OspA for the lipidation thereof, are disclosed; and, those recombinant lipoproteins may be employed in the practice of the invention. As to expression of recombinant proteins, it is expected that the skilled artisan is familiar with the various vector systems available for such expression, e.g., bacteria such as E. col i and bacterial viruses, and the like.
The adjuvant can be any vehicle which would typically enhance the antigenicity of the antigen, e.g., a suspension or gel of minerals (for instance, alum, alllm;nllm hydroxide or phosphate) on which the antigen is adsorbed; or a water-in-oil emulsion in which antigen solution is emulsified in mineral oil (e.g., Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (e.g., Freund's complete adjuvant);
or cholera toxin (sometimes with cholera toxin B, which may enhance the effect); or, any of the other adjuvants known in the art, or discussed in the Background of the Invention. The antigen and/or the lipoprotein can be absorbed onto or coupled with the adjuvant.
Presently preferred embodiments of the invention involve: alum as the adjuvant if an adjuvant is present; OspA, or a recombinant OspA leader/PspA, a recombinant OspA leader/OspC, a recombinant OspA
- leader/UreA of H. pyl ori, or, a recombinant OspA
leader/UreB of H. Pyl ori as the lipoprotein (OspA
leader/PspA is a recombinant lipoprotein having a lipidated moiety from expression of the OspA leader nucleic acid sequence and a protein moiety ~rom expression o~ a pspA nucleic acid sequence; OspA
leader/OspC is analogous to OspA leader/PspA, except that the protein moiety is ~rom expression o~ an ospC nucleic acid sequence and OspA leader/ureA and OspA leader/ureB
are also analogous to OspA leader/PspA, except that the protein moiety is ~rom expression o~ a ureA or ureB
nucleic acid sequence); and OspC or another Borrelia antigen, or an in~luenza antigen, e.g., HA (such as from in~luenza A, e.g., Texas strain), or urease as the antigen. Particular e~odiments can include compositions: (i) comprising alum [adjuvant], OspA
[lipoprotein] and another Borrelia antigen such as OspC
[antigen]; (ii) comprising alum [adjuvant], OspA
[antigen], and OspA leader/OspC [lipoprotein]; (iii) comprising alum [adjuvant], OspA leader/PspA
[lipoprotein] and in~luenza antigen, e.g., in~luenza A HA
[antigen] (iv) OspA [lipoprotein] and an H. pylori antigen, e.g., urease [antigen].
Other objects and embodiments o~ the invention are disclosed in or are obvious variants ~rom the ~ollowing description.
BRIEF DESCRIPTION OF THE DRAWINGS
In the ~ollowing detailed description, re~erence is made to the accompanying drawings, wherein:
Figure 1 is a graphical representation o~ the immune response o~ mice ;mmlln;zed with OspC formulations with or without puri~ied lipidated OspA and with or without alum as an adjuvant as measured in an anti-OspC
ELISA at day 63 a~ter ;mmlln;zation; and Figure 2 is a graphical representation o~ the ;mmlln~ response o~ mice ;mmlln;zed with OspC ~ormulations with or without puri~ied lipidated OspA and with or without alum as an adjuvant as measured in an anti-OspC
ELISA at day 91 after ;mmnn;zation~
Figure 3 is a graphical representation o~ the ;mmlln~ response o~ mice ;mmlln;zing twice, intranasally, CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 with either lipidated or non-lipidated OspA as measured in an anti-OspA ELISA at day 9 after the second ;mmlln;zation.
Figure 4 is a graphical representation of the immune response of mice immunized twice, both intranasally and intragastrically, with either jack bean urease alone or both urease and OspA, as measured in an anti-urease ELISA at day 9 after the second ;mmlln;zation~
Figure 5 iS a graphical representation of the immune response of mice ;mmlln;zed twice, intranasally, with jack bean urease, either above or with OspA or cholera toxin, as measured in an anti-urease ELISA at day 9 after the second ;mmlln;zation.
DET~TT-T~'n DESCRIPTION OF TEIE lNV~N~ lON
As discussed above, the invention involves ;mmllnological compositions and methods for making and using (e.g., ~m;n;stering) them which, in a broad sense, include ;mmllnological compositions comprising an antigen and a lipoprotein and optionally including an adjuvant;
20 and the methods broadly include ~m;n;stering such compositions to a suitable host such that there is co-~m;n;stration of the antigen and lipoprotein and optional adjuvant, or sequentially ~m;n;stering the components thereof.
It has now surprisingly been found that mucosal administration of an antigen, e.g., a bacterial protein or fragment thereof, and a lipoprotein produces both local and serum ;mmllne responses. The principal determ;n~nt of specific immunity at mucosal surfaces is 30 secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. S-IgA antibody responses may be induced locally by the application of suitable - ;mmllnogens to a particular mucosal site. The bulk of 35 mucosal S-IgA responses, however, are the results of ;mmlln;ty generated via the common mucosal immune system (CMIS) [Mestecky, J. J. Clin Immunol. (1987) 7:265-276], in which immunogens are taken up by specialized lympho-epithelial structures, collectively re~erred to as mucosa-associated lymphoid tissue (MALT). The best studied ;mmllnologic ly~pho-epithelial structures are the gut-associated lymphoid tissues (GALT), such as intestinal Peyer's patches. It is now clear, however, that other structurally and ~unctionally similar lymphoid ~ollicles occur at other mucosal sur~aces, including those o~ the respiratory tract [Croituru, K., et al., in "Handbook o~ Mucosal Immunology" (Bienenstock, J., ed.) San Diego, CA:Academic Press, Inc. (1994), 141-149].
In the experimental results set ~orth in the Examples below, it is shown that mice can be ef~ectively ; mmlln; zed by intranasal (i.n.) or intragastric (i.g.) installation of bacterial protein ;mmllnogens in conjunction with a lipoprotein such as OspA. Speci~ic IgA- and IgG- secreting cells are induced in the salivary glands and stomachs (stomachs not shown) and specific IgA
antibodies are induced in saliva (not shown). Strong circulatory ;mmlln~ responses are also induced with IgG
and IgA antibodies in the serum. Accordingly, it appears that mucosal ;mmlln;zation with antigens along with lipoproteins is an e~ective route ~or stimulating common mucosal responses as well as circulatory antibody responses. Such ;mmlln;zation may be both therapeutic and prophylactic.
The determination o~ the amount o~ antigen, lipoprotein and optional adjuvant in the inventive compositions and the preparation o~ those compositions can be in accordance with standard techniques well known to those skilled in the pharmaceutical or veterinary arts. In particular, the amount o~ antigen, lipoprotein and adjuvant in the inventive compositions and the dosages ~m; n; stered are determined by techniques well known to those skilled in the medical or veterinary arts taking into consideration such ~actors as the particular antigen, the lipoprotein, the adjuvant, the age, sex, CA 0222304l l997-l2-Ol weight, species and condition of the particular patient, and the route of ~m; n; stration. For instance, dosages of particular antigens listed above for suitable hosts in which an ;mml~nological response is desired, are known to those skilled in the art, as is the amount of adjuvant typically ~m; n; stered therewith. Thus, the skilled artisan can readily determine the amount of antigen and optional adjuvant in compositions and to be administered in methods of the invention. Typically, an adjuvant is commonly used as 0.001 to 50 wt~ solution in phosphate buffered saline, and the antigen is present on the order of micrograms to milligrams, such as about 0.0001 to about 5 wt~, preferably about 0.0001 to about 1 wt~, most preferably about 0.0001 to about 0. 05 wt~ (see, e.g., the Examples below).
The skilled artisan can refer to a known dosage for the particular antigen for a particular host to determine the amount of lipoprotein in compositions and ~m; n; stered in methods of the present invention, (if the lipoprotein is antigenic) such as the known dosages for OspA from the documents cited herein, or can scale the dosage for a particular host from the documents cited herein and the Examples below (e.g., with respect to OspA
leader/PspA, OspA leader/OspC, OspA leader/ureA, and OspA
leader/ureB. Typically, however, the antigenic and/or recombinant lipoprotein is present in an amount on the order of micrograms to milligrams, or, about 0.001 to about 20 wt~, preferably about 0.01 to about 10 wt~, and most preferably about 0. 05 to about 5 wt~ (see, e.g., Examples below).
Of course, for any composition to be ~m;n; stered to an ~nimAl or human, including the components thereof, and for any particular method of - ~m;n;stration, it is preferred to determine therefor:
toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable ~n i m~ l model e.g., rodent such as mouse; and, the dosage of the composition(s), CA 0222304l l997-l2-Ol WO 96/40290 PCT~US96/08866 26 concentration of components therein and timing o~
administering the composition(s), which elicit a suitable immunological response, such as by titrations o~ sera and analysis thereo~ ~or antibodies or antigens, e.g., by 5 ELISA. Such determinations do not rec~uire undue experimentation from the knowledge o~ the skilled artisan, this disclosure and the documents cited herein.
And, as discussed above, the time ~rom ~or secluential ~m; n; strations can be ascertained without undue experimentation.
Examples o~ compositions o~ the invention include lic~uid preparations ~or ori~ice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or 15 respiratory mucosa) etc., ~lm; n; stration such as suspensions, syrups or elixirs; and, preparations ~or parenteral, subcutaneous, intradermal, intramuscular or intravenous ~m;n;stration (e.g., injectable ~m;n;stration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting or 25 emulsi~ying agents, pH lbuEi~ering agents, gelling or viscosity enhancing additives, preservatives, ~lavoring agents, colors, and the like, depending upon the route o~
administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by re~erence, may be consulted to prepare suitable preparations, without undue experimentation.
Compositions of the invention, are conveniently provided as licluid preparations, e.g., isotonic aclueous 35 solutions, suspensions, emulsions or viscous compositions which may be bu~ered to a selected pH. I~ digestive tract absorption is pre~erred, compositions o~ the CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/Q8866 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 for delivery to the gut.
If nasal or respiratory (mucosal) ~m; n;stration is desired, compositions may be prepared as inhalables, sprays and the like and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or, a dose having a particular particle size.
Compositions within the scope of this invention can contain a humectant to inhibit drying of the mucous membrane and to prevent irritation. Any o~ a variety of pharmaceutically acceptable humectants can be employed including, for example sorbitol, propylene glycol or glycerol. As with the thickeners, the concentration will vary with the selected agent, although the presence or absence of these agents, or their concentration, is not an essential ~eature o~ this invention.
~nh~nced absorption across the mucosal and especially nasal membrane can be accomplished employing a pharmaceutically acceptable surfactant. Typically use~ul sur~actants for compositions include polyoxyethylene derivatives of ~atty acid partial esters of sorbitol anhydrides such as Tween 80, Polyoxynol 40 Stearate, Polyoxyethylene 50 Stearate and Octoxynol. The usual concentration is form 1~ to 10~ based on the total weight.
. 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, ~or example, Parabens, th;m~rosal~ chlorobutanol, or benzalkonium CA 0222304l l997-l2-Ol W 096/40290 PCT~US96/08866 chloride may also be employed. A suitable concentration of the preservative will be from O. 02~ to 2~ based on the total weight although there may be appreciable variation depending upon the agent selected.
Compositions o~ the invention can contain pharmaceutically acceptable flavors and/or colors ~or rendering them more appealing, especially if they are A~m; n; stered orally. The viscous compositions may be in the ~orm of gels, lotions, ointments, creams and the like and will typically contain a su~icient amount o~ a thickening agent so that the viscosity is ~rom about 2500 to 6500 CpS, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity pre~erably of 2500 to 5000 cps, since above that range they become more di~ficult to ~lm;n; 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, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to A~m; n; ster, especially by injection or orally, to An;mAls, children, particularly small children, and others who may have di~iculty 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 o~ the stomach or nasal mucosa.
Obviously, the choice of suitable carriers and other additives will depend on the exact route of -administration and the nature o~ 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 ~orm [e.g., whether the composition is to be ~ormulated CA 0222304l l997-l2-Ol W O 96/40290 PCT~US~G/'033G6 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, lipoprotein and optional adjuvant. 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 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, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration o~ the preservative will be ~rom 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 the antigen, lipoprotein and optional adjuvant. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by re~erence to standard texts or by simple experiments (not involving undue experimentation), ~rom this disclosure and the documents cited herein.
The ;mmllnologically e~ective compositions o~
this invention are prepared by mixing the ingredients ~ollowing 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 ~inal concentration and viscosity by the addition o~ water or thickening agent and possibly a bu~er to control pH or an additional solute to control tonicity. Generally the pH may be ~rom about 3 to 7.5. Compositions can be ~3~m; n; stered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such ~actors as the age, sex, weight, and condition o~
the particular patient or ~n;m~l, and the composition ~orm used ~or administration (e.g., solid vs liquid) Dosages ~or hllm~ns or other m~mm~l S can be determined without undue experimentation by the skilled artisan, ~rom this disclosure, the documents cited herein, the Examples below (e.g., ~rom the Examples involving mice), and the knowledge o~ antigens and lipoproteins and adjuvants herein mentioned.
The present invention also includes a method ~or inducing an immunological response in a host wherein the antigen and lipoprotein are ~m; n; stered at one CA 0222304l 19s7-l2-ol W 096/40290 pcTrus96/o8866 mucous membrane and a response is detectable at another mucous membrane, e.g. nasal ~m;n; stration and vaginal response. This aspect of the invention is particularly useful for the treatment or prevention of sexually transmitted diseases.
Suitable regimes for initial ~m; n; stration and booster doses or for sequential ~m; n; strations also are variable, may include an initial ~m; n; stration followed by subsequent ~m; n; strationsi but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, the Examples below, and the knowledge of antigens, lipoproteins and adjuvants herein mentioned without undue experimentation.
The following Examples are provided for 15 illustration and are not to be considered a limitation of the invention.

EXAMPLES

20 Construction of a pET9a Expression Vector Cont:~;n;n~ a ~ybrid ospA/ pspA Gene Specifically designed oligonucleotide primers were used in a PCR reaction to amplify the portion o~ the 25 pspA gene of interest (in this case from amino acid 1 to 314) f~rom the S. pneumoniae strain RX1.
The 5'-end primer had the nucleotide sequence:
5'-GGG ACA GCA TGC GAA GAA TCT CCC GTA GCC AGT- 3~ ( PspN1) (SEQ ID NO: 1).
The 3'-end primer had the nucleotide sequence:
5'-GAT GGA TCC TTT TGG TGC AGG AGC TGG TTT-3' (PspC370) (SEQ ID NO: 2).
The PCR reaction was as Eollows: 94~C for 30 seconds to denature DNA; 42~C for one minute for 35 annealing DNA; and 72~C for one minute for extension of DNA. This was carried out for 25 cycles, followed by a 5 minute extension at 72~C. This procedure introduced a stop codon at amino acid 315. The PCR product was W O 96/40290 PCT~US96/08866 puri~ied using the Gene Clean II method (BiolO1), and digested with SphI and BamHI.
The plasmid pLF100 was prepared as follows.
Plasmid pBluescript KS+ (Stratagene) was digested with XbaI and BamHI and ligated with a 900 bp XbaI-BamHI DNA fragment containing the complete coding region of B. burgdor~eri strain ACA1 ospA gene, to form a lipoprotein fusion vector pLF100. This procedure is shown schematically in Figure 1 of application Serial No. 08/475,781, filed June 7, 1995 and incorporated herein by reference.
The vector pLF100 has been deposited with the American Type Culture Collection at Rockville, Maryland on February 2, 1995 under Accession No. 69750. This deposit was made under the terms of the Budapest Treaty.
pLF100 was digested with SphI and BamHI and the amplified pspA gene was ligated to this plasmid to form the plasmid pLF321, which contained the hybrid ospA-pspA
gene. The hybrid gene was excised ~rom pLF321 by digestion with NdeI and BamHI and cloned into the NdeI
and BamHI sites of the plasmid vector pET9a to place the ospA-pspA hybrid gene under the control of a T7 promoter.
The resulting plasmid is called pPA321-L. This process is shown schematically in Figure 9 o~ application Serial 25 No. 08/475,781 filed June 7, 1995 and incorporated herein by re~erence.

Construction of a pET9a Expression Vector Cont~;n;n~ the pspA Gene Specifically designed oligonucleotide primers were used in a PCR reaction to ampli~y the portion of the pspA gene o~ interest (in this case from amino acid 1 to 314) from the S. pneumoniae strain RX1.
The 5'-end primer had the nucleotide sequence:
5'-GCT CCT GCA TAT GGA AGA ATC TCC CGT AGC C-3' (PspNL-2) (SEQ ID NO: 3) The 3'-end prlmer had the nucleotide sequence:

5'-GAT GGA TCC TTT TGG TGC AGG AGC TGG TTT-3' (PspC370) (SEQ ID NO: 4).
The PCR reaction was as follows: 94~C for 30 seconds to denature DNA; and 72~C for one minute for annealing and extension of DNA. This was carried out for 25 cycles, which was followed by a 5 minute extension at 72~C. This procedure introduced a stop codon at amino acid 315. The PCR product was purified using the Gene Clean II method (Bio 101), and digested with NdeI and BamHI. The digested PCR product was cloned into the NdeI
and BamHI sites of the plasmid vector pET9a to place the pspA gene under the control of a T7 promoter. The resulting plasmid is called pPA321-NL. This process is shown schematically in Figure 10 of application Serial No. 08/475,781, filed June 7, 1995 and incorporated herein by reference.

Expression and Purification of Lipidated PspA
Plasmid pPA321-L was used to transform E. coli strain BL21(DE3)pLyS. The transformed B. col i was inoculated into LB media containing 30~g/ml kanamycin sulfate and 25 ~g/ml chloramphenicol. The culture was grown overnight in a flask shaker at 37~C.
The following morning 50ml of overnight culture was transferred to lL LB media containing 30~g/ml kanamycin sulfate and the culture was grown in a flask shaker at 37~C to a level of OD 600nm of 0.6-1.0, in approximately 3-5 hours. To the culture medium was added IPTG to a final concentration of 0.5mM and the culture was grown for an additional two hours at 30~C. The cultures were harvested by centrifugation at 4~C at ~ --10,000xG and the cell pellet collected. Lipidated PspA
was recovered from the cell pellet.
- The cell pellet was resuspended in PBS at 30g wet cell paste per liter PBS. The cell suspension was frozen and stored at -20~C. The cells were thawed to room temperature to effect lysis. DNaseI was added to CA 0222304l l997-l2-Ol the thawed material at a ~inal concentration o~ l~g/ml and the mixture incubated ~or 30 minutes at room temperature, which resulted in a decrease in viscosity of the material.
The material was then chilled in an ice bath to below 10~C and TritonT~ X-114 was added as a 10~ stock solution to a ~inal concentration o~ 0.3 to 1~. The mixture was kept on ice for 20 minutes. The chilled mixture was then heated to 37~C and held at that temperature ~or 10 minutes. This caused the solution to become very cloudy as phase separation occurred The mixture was then centri~uged at about 20~C for 10 minutes at 12,000xG, which caused a separation o~ the mixture into a lower detergent phase, an upper clear aqueous phase and a pellet. The lipidated PspA partitioned into the detergent phase. The detergent phase was separated ~rom the other two phases, diluted 1:10 with a bu~er comprising 50mM Tris, 2mM EDTA, lOmM NaCl pH 7.5, and was stored at -20~C.
A Q-Sepharose column was prepared in a volume o~ 1 ml per 5 ml diluted detergent phase. The column was washed with 2 column volumes o~ a bu~er comprising 50mM
Tris, 2mM EDTA, 0.3~ Triton~ X-100, lM NaCl pH 4.0, and then equilibrated with 5 to 10 column volumes 5OmM Tris, 25 2mM EDTA, 0.3~ Triton~ X-100, lOmM NaCl pH 4Ø The pH
o~ the diluted detergent phase material was adjusted to 4.0, at which time a precipitation occurred. This material was passed through a 0.2~M cellulose acetate ~iltering unit to remove the precipitated material. The ~iltered diluted detergent phase was applied to the Q-Sepharose column and the ~low through (containing PA321-L) was collected. The column was washed with 1-2 column volumes o~ 50mM Tris, 2mM EDTA, 0.3~ Triton~ X-100, lOmM
NaCl pH 4.0, and the flow through was pooled with the previous ~low through ~raction. The pH o~ the ~low through pool was adjusted to 7.5. The bound material, contaminating E. coli proteins, was eluted ~rom the Q-CA 0222304l l997-l2-Ol W 096/40290 pcTrus96/o8866 Sepharose with 2 column volumes of~ 50mM Tris, 2mM EDTA, 0.3~ TritonT~ X-100, lM NaCl pH 4Ø A schematic o~ the puri~ication process described in this Example is shown in Figure 11 of application Serial No. 08/475,781, ~iled June 7, 1995 and incorporated herein by re~erence.

Expression and Purification o~ Non-lipidated PspA
Plasmid pPA321-NL was used to trans~orm E. col i strain BL21(DE3)pLyS. The trans~ormed E. col i was inoculated into LB media containing 30~g/ml kanamycin sul~ate and 25~g/ml chloramphenicol. The culture was grown overnight in a ~lask shaker at 3 7~C.
The ~ollowing morning 50ml o~ overnight culture was transferred to lL LB media containing 30,ug/ml kanamycin sul:Eate and the culture was grown in a ~lask shaker at 37~C to a level of OD 600nm o~ 0.6-1.0, in approximately 3-5 hours. To the culture medium was added IPTG to a ~inal concentration o:E 0 .5mM and the culture was grown ~or an additional two hours at 30~C. The cultures were harvested by centri~ugation at 4~C at 10,000xG and the cell pellet collected. Non-lipidated PspA was recovered ~rom the cell pellet.
The cell pellet was resuspended in PBS at 30g wet cell paste per liter PBS. The cell suspension was ~rozen and stored at -20~C. The cells were thawed to room temperature to ef~ect lysis. DNaseI was added to the thawed material at a ~inal concentration o~ l~g/ml and the mixture incubated for 30 minutes at room temperature, which resulted in a decrease in viscosity o~
30 the material. The mixture was centrii~uged at 4~C at 10,000xG, and the cell supernatant saved, which contained non-lipidated PspA. The pellet was washed with PBS, centri~uged at 4~C at 10,000xG and the cell supernatant - pooled with the previous cell supernatant.
A MonoQ column (Pharmacia) was prepared in a volume o~ 1 ml per 2 ml cell supernatant. The column was washed with 2 column volumes of a bu~er comprising 50mM

WO 96/40290 PCT~US96/08866 Tris, 2mM EDTA, lM NaCl pH 7.5, and then equilibrated with 5 to 10 column volumes o~ a bu~er comprising 50mM
Tris, 2mM EDTA, lOmM NaCl pH 7.5. The cell supernatant pool was applied to the Q-Sepharose column and the ~low through was collected. The column was washed with 2-5 column volumes o~ 50-m-M Tris, 2mM EDTA, 10 mM NaCl pH 7.5, and the ~low through pooled with the previous ~low through.
The elution o~ bound proteins began with the ~irst step o~ a 5-10 column volume wash with 50mM Tris, 2mM EDTA, lOOmM NaCl pH 7.5. The second elution step was a 5-10 column volume wash with 50mM Tris, 2mM EDTA, 200mM
NaCl pH 7.5. The non-lipidated PspA was contained in this ~raction. The r~m~;n;ng bound contaminating proteins were removed with 50mM Tris and 2-m-M EDTA pH 7.5 with 30 OmM-lM NaCl.
A schematic of the puri~ication process described in this Example is shown in Figure 12 o~
application Serial No. 08/475,781, ~iled June 7, 1995 and incorporated herein by re~erence.

T ogenicity o~ Recombinant Lipidated PspA
Puri~ied recombinant lipidated PspA, prepared as described in Example 3, was tested ~or immunogenicity in mice and compared to that ~rom non-lipidated PspA
prepared as described in Example 4. For this study, CBA/N mice were immunized subcutaneously in the back o~

W O 96/40290 PCTrUS96/08866 the neck with 0.5 ml o~ the ~ollowing ~ormulations at the indicated PspA antigen concentrations.
Fo l~tion PspA Antigen Concentration Native PspA molecule o~ the RX1 200 ng/ml strain (Native RX1) Non-Lipidated Recombinant PspA 200 and 1000 (pPA-321-NL) Alone in PBS* ng/ml Non-Lipidated Recombinant PspA 200 and 1000 (pPA-321-NL) Adsorbed to Alum ng/ml Lipidated Recombinant PspA (pPA- 200 and 1000 321-L) Alone in PBS ng/ml Lipidated Recom~inant PspA 200 and 1000 (pPA0321-NL) Adsorbed to Alum* ng/ml Alum* 0 ng/ml 15 PBS 0 ng/ml *Alum was Hydrogel at a concentration o~ 200 ~g/ml Four mice were ;mmlln;zed on days 0 and 21 ~or each dosage o~ the ~ormulations. The mice were then bled on day 35 and subsequently challenged with S. pneumoniae o~ A66 strain. The days o~ survival a~ter challenge ~or the mice were recorded and surviving mice were bled on days 36, 37, 42 and 46. From these subsequent bleeds the blood was assayed ~or the number o~ colony ~orming units (CFU) o~ S. pneumoniae/ml. The sera taken on day 35 were assayed by ELISA ~or antibodies against PspA using ELISA.
The days to death ~or the challenged mice are shown in the ~ollowing table.

CA 0222304l l997-l2-Ol Survival in I-vuno a~d Non-I une CBA/N hice T' 7~; ~n B~ica y Group Antigendo3eAlumDayn to P value ti~e Alive: P value in ~g Deathto death~ Doad Survival~
#lApPA-321-L 1.0 - 4x~14 0.01 4:0 0.01 #lB PpA-321-L 0.2 - 4x~14 0.01 4:0O.ol #2A pPA-321-L 1.0 + 4x~14 0.01 4:00.01 #2B pPA-321-D 0.2 + 4x~14 o.Ol 4:00.01 #3ApPA-321-NL 1.0 - 1,1,2,2 n.3. 0:4n.3.
#3BpPA-321-NL 0.2 - 1,1,2,215 n.3. 1:3n.s.
1 0#4ApPA-321-NL 1.0 + 4x~14 o.Ol 4:00.01 #4BpPA-321-ND 0.2 ~ 4x~14 0.01 4:00.01 #5 FL-Rxl 0.2 - 4x~14 0.01 4:00.01 #6 none o.o + 1,1,3,6 n.3. 0:4 n.3 #7 none 0.0 - 1,1,1,215 n.3. 1:3n.s.
pooled o.o 5x1,3,6,2 -- 1:7 none 15 Note: * indicates versus pooled controls; time to death, by one tailed two sample rank test; survival, by one tailed Fisher Exact test. Calculations have been done using "one tail" since we have never observed anti-PspA
;mmnn;ty to consistently cause susceptibility The number o~ CFU in the blood o~ the mice are shown in the table below.

W O 96/40290 PCTrUS96/08866 ~a c c c c r~ ~ c c c '~ ~' ~ r ~
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~ol z W O 96/40290 PCTrUS96/08866 These results indicate that the recombinant protein was not protective when injected alone. The recombinant antigen adjuvanted with alum and/or PAM3cys lipidation was ;mmnnogenic and protective. The native ~ull length PspA
antigen did not need an adjuvant to be protective. The CFU results indicate that mice protected by ;mml~n;zation cleared the challenging S. pneumoniae ~rom the blood in two days.
ELISA analysis o~ sera taken on day 35 indicated that there was a good correlation between protection o~ the mice from 5. pneumoniae challenge and the induction o~ measurable antibody responses. No detectable antibody responses were observed in the sera o~ mice ;mmnn;zed with the non-lipidated antigen (pPA-321-NL) in saline or to the negative controls that did not contain PspA antigen, (as shown in the table below).
Good antibody responses were detected to the Native RX1 PspA antigen and to the recombinant PspA when it was lipidated with PAM3cys and/or adsorbed to alum.

CA 0222304l l997-l2-Ol W O 96/40290 4~ PCTAUS96/08866 o _ ~ _ ~ _ _ _ _ _ _ _ .~ O0~ t~ t~ m ~ N m O ~ N O
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WO 96/40290 PCT~US96/08866 To determine whether protection was at least in part mediated by the anti-PspA antibody responses, a passive experiment was run. BALB/c mice were ;mml7n;zed with 0.5 ~g of recombinant lipidated PspA alone or absorbed to alum, or with recombinant non-lipidated PspA
adsorbed to alum on days 0 and 21; and were bled on day 35. The anti-sera were diluted 1:3 or 1:15 in saline and 0.1 ml of the dilution was injected i.p. into two mice for each dilution. A 1/3 dilution of normal BALB/c mouse serum was used as a negative control. Subsequently one hour after passive ;mmlln;zation, the ~n;m~ls were challenged i.v. with the WU2 strain of S. pneumoniae (15,000 CFU). Mice passively ;mmlln;zed with anti-PspA
sera were protected as compared to those mice that received dilutions of normal mouse sera as shown in the following table.
Passive Protection of BALB/c to WU2 Immunizing Formulation PspA DoseDilution Days to Death PspA AntigenAlum(~g/animal)o~ Serum Post Challenge pPA-321-L No 0.5 3 4, ~7 2, 4 pPA-321-L Yes 0.5 3 ~7, ~7 4, ,7 pPA-321-NL Yes 0.5 3 2, 4 ~7, ~7 None No 0 3 2,2 CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 Combination PspA/Flu Vaccine Purified recombinant lipidated PspA, prepared as described in Example 3, and non-lipidated PspA
prepared as described in Example 4 were combined with split flu antigen from the A/Texas strain.
These combinations and the flu antigen alone were formulated either in saline or adsorbed to alum in saline. The alum when added was kept constant at 100 ~g/injection and the PspA was kept constant at 0. 5 ~g/injection. The flu antigen was diluted to concentrations of 0. 5, 0.1, 0. 02 and 0. 004 ~g/injection.
Four BALB/c mice for each of the formulations were ;mmlln;zed on days 0 and 21, and were then bled on day 35.
The sera from the ;mmlln;zed mice were then assayed for their ability to inhibit the agglutination of chicken red blood cells by A/Texas HA antigen. The resulting hemagglutination inhibition (HAI) titers are shown in the following table.

W O 96/40290 PCT~US96/08866 44 ~AI Titers from Combination-~ of Recombinant Ps~A and Flu Flu An~gen PspAAr~genAlum Flu HA Dose GMT oF STD of GMr A~ L ~ 9/i~ ) HAI rlter o~ HAI rlter A/Texas - + 0.5 28.1 3 A/Texas - + 0.1 21.8 6.6 A/Texas - + 0.02 22.8 5.2 A/Texas - + 0.004 16.1 3.8 A/Texas - _ 0.5 12.4 5.3 A/Texas - _ 0.1 23.8 3.3 A/Texas - _ 0.02 19.2 2.8 1 0 A/Texas - _ 0.004 11.9 3 7 A/TexaspPA-321-L + 0.5 794.8 2.6 A/TexaspPA-321-L + 0.1 452.5 2.7 A/TexaspPA-321-L + 0.02 54.2 6.9 A/TexaspPA-321-L + 0.004 36.7 4.9 A/TexaspPA-321-L - 0.5 51.9 4 A/TexaspPA-321-L - 0.1 27.1 5.1 A/TexaspPA-321-L - 0.02 19.2 3.3 A/TexaspPA-321-L - 0.004 15.4 3.4 A/TexaspPA-321-NL + 0.5 174.5 2.7 2 0 A/TexaspPA-321-NL + 0.1 59.1 3.4 A/TexaspPA-321-NL + 0.02 19.2 5.1 A/TexaspPA-321-NL + 0.004 14.8 3.1 A/TexaspPA-321-NL - 0.5 35.1 2.7 A/TexaspPA-321-NL - 0.1 23.8 3 A~'Texas pPA-321-NL - 0.02 14.8 2.9 A/TexaspPA-321-NL - 0.004 10.2 2.6 None None - 0 7.1 1.9 Expression and Purification of Non-lipidated OspC.
E. coli JM 109 transformants containing plasmid vector containing chromosomal gene fragment encoding non-lipidated OspC were prepared and grown as described in W0 3 5 9 1 / 0 9 870. The cultures were harvested, the culture medium centrifuged at 10,000 XG for 10 minutes at 4~C, the supernatant discarded and the pellet collected.
The cell pellet was first resuspended in lysis buffer A, namely 50nM Tris-HCI pH 8.0, 2mM EDTA, 0.lmM
DTT, 5~ glycerol and 0.4mg/ml lysozyme, and the suspension stirred for 20 minutes at room temperature.
TRITON~ X-100 then was added to the cell suspension to a CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 concentration of 1 wt~, DNase I was added to a concentration o~ l~g/ml, and the suspension stirred at room temperature for a further 20 minutes to e~f~ect cell lysis. Sodium chloride next was added to the cell suspension to a concentration of lM and the suspension again stirred at 4 C ~or a further 20 minutes. The suspension then was centri~uged at 20,000 x G for 30 minutes, the resultant supernatant separated from the pellet and the pellet was discarded.
The separated supernatant was dialyzed against a buf~er comprising 50 mM Tris pH 8, 2 mM EDTA. The supernatant next was loaded onto a DEAE-Sepharose CL-6B
column and the non-lipidated OspC was collected in the column ~low-through. The flow-through was dialyzed against a 0.1 M phosphate bu~er, pH 6Ø
The dialyzed flow-through next was bound to a S-Sepharose fast flow column equilibrated with O.lM
phosphate buffer, pH 6Ø Purified non-lipidated OspC
then was eluted from the S-Sepharose column usin~ the dialysis buffer with 0.15 M NaCl added.
The aqueous solution o~ highly purified non-lipidated OspC was analyzed by Coomassie stained gels.
The purity o~ the product was estimated to be greater than 80~.

Potentiation of Response to Non-lipidated OspC with Lipidated OspA
Purified recombinant non-lipidated OspC, prepared as described in Example 7, was tested ~or immunogenicity in mice in combination with or without purified lipidated OspA (prepared as described in WO
92/14488). Formulations were ~3~m;n;stered with or without alum as an adjuvant. The antigen dose tested in this experiment was 1 ~g per dose. For this study, 4 to 8 week old ~emale C3H/He mice were ;mmllnized on day 0 and boosted on days 21 and 42.

WO 96/40290 PCT~US96/08866 Three representative ~n;m~l S were exsanguinated on days 21, 42, 63 and 91. ELISA testing was performed on these sera using purified non-lipidated OspC as the coating antigen.
The only detectable OspC ELISA responses generated in this study were with the formulation of OspC
on alum. However, when lipidated OspA was included on the alum the OspC ELISA response was 20-fold higher on day 63 (as shown in Figure 1) and 5-fold higher on day 91 (as shown in Figure 2). When lipidated OspA was included in the formulation without alum there was no apparent ef~ect on the immune response.

Salivary Gland ELISPOT Analysis of Response to Urease with OspA
Mice (CH3/HeN; 4-5/group) were ;mmlln;zed by mucosal routes with the antigens indicated in the table below, on days 0 and 28. Proteins were diluted in PBS to a final volume of 25 ~l for intranasal and 0.5 ml for intragastric. The mice were sacrificed for ELISPOTS at 15-17 days after the second immllnization~
The ELISPOT protocol was derived from the one described by Mega et al., J. Immunol. (1992), 148:2030-2039. The salivary glands were taken just after sacrifice of the mouse, and placed immediately in a large volume of RPMI 1640 medium (Gibco). The organs were cut in small pieces (lxl mm) using an automated tissue chopper (Mc Illwain tissue chopper, The Mickle Laboratory Engineering, Gilford, U.K.), and then digested in 2 ml of RPMI 1640 medium containing 5~ FCS and lmg/ml of collagenase type IV (Sigma) for 30 minutes at 37~C with gentle agitation. The digested cells and fragments were passed through a 70 ~M filter (Falcon), and the digestion was repeated three more times. The digested cells were pooled and washed twice in a large volume of medium. The pooled cells were then lysed using Gey's solution for 4 minutes on ice. After two more washes, the cells were CA 0222304l l997-l2-Ol W O 96/40290 PCT~US96/08866 resuspended in 2ml of medium (+5~ FCS), counted and aliquoted in 96 well nitrocellulose plates (MILLIPORE).
The plates had been coated overnight with 20~g/ml of jackbean urease (Boehringer M~nnh~im) or 10~g/ml OspA
(Connaught) in PBS at 4~C, and then saturated with complete medium for 1 hour at 37~C. Two five-fold dilutions of the cells were loaded in the wells (100 ~l/well) in quadruplicate for each dilution and each isotype. After 4-16 hours at 37~ under 5~ C02, the cells 10 were lysed 2x5 minutes in PBS/Tween 20 (0.005~) and biotinylated anti-isotypes antibodies (Amersham) added for two hours at room temperature (dilution 1/1000).
After 3 washes with PBS Tween, biotinylated streptavidin peroxydase complex (Amersham) was added for 1 hour 15 (dilution 1/500), and then spots revealed with 3,9-aminoethylcarbazole (SIGMA). Once the plates dried, the spots were numerated under a dissecting microscope (magnification 16 or 40X). The values represent the means for 4 wells averaged for each group of i:~n;m~1s As shown in the table below, it was found that lipidated OspA lipoprotein ;~lmin; stered by mucosal routes without any added adjuvant induced very strong local IgG
and IgA responses, while non-lipidated OspA did not induce any detectable responses. It was also found that OspA had a powerful adjuvant effect on the local response to urease.

Anti Ure Anti Osp Spots/106 Spots/106 Cells Cells ~q Jackbean ~re ~ Route IaA IaG IaA IaG
OspA

- lL i.n. n.d n.d 583 345 - lNL i.n. n.d. n.d 4 2 CA 0222304l l997-l2-Ol - i.n. 11 0 n.d. n.d lL i.n. 18918 742 257 lOL i.n. 19139 1237 174 20+CT 10 ,ug - i.n. 47842 n.d. n.d.
- i.n., O 1 25 0 i.g.
lOL i.n.,32231 1919 177 i.g.
i.n. = intranasal i.n., i.g. = intranasal & intragastric (the indicated dose was given by each route) L = lipidated OspA; NL = non-lipidated OspA
CT = cholera toxin (10 ~Lg CTB + 10 ng CTX/mouse (PMSV)) n.d. = not determined W O 96/40290 PCT~US96/08866 ELISA Assay to Measure Serum Antibodies Against OspA and Urease Mice (CH3/HeN; 4-5/group) were immlln;zed by mucosal routes with the antigens indicated in the table in Example 9 on days 0 and 28. Proteins were diluted in PBS to a final volume of 25 ~1 for intranasal and 0.5 ml for intragastric. Blood was taken 9 days after the second ;mmlln;zation.
For the ELISA assay, flat-bottomed 96 well microliter plates (Dynatech) were coated with 100 ~l/well of 1 ~g/ml OspA (Connaught) or 2 ~g/ml jackbean urease (Boehringer ~nnh~im), diluted in 0.1 M sodium carbonate buffer, pH 9.6. Plates were coated overnight at room temperature.
The following day, plates were washed 4x with PBS/0.05~ Tween 20 and blocked with PBS with 1~ BSA for 30 min. at room temperature. After another wash, each well received 100 ~l of PBS with 0.05~ Tween 20 and 0.1 BSA (PBS/T/B). Sera were pooled within each group of mice and serially diluted, and plates were incubated for 3 hr. at room temperature. After washing, 2~ antibody biotinylated goat anti-mouse IgG or IgA (Amersham) diluted 1:5,000 in PBS/T/B was added, and plates were incubated for two hours at room temperature. Plates were washed again and incubated with streptavidin horseradish peroxidase (Amersham) diluted 1:2,000 in PBS/T/B for 1.5 hr. at room temperature. After a final wash the substrate, OPD (Sigma), was added and plates were incubated 10-20 min. Finally, the reaction was stopped with 50 ~l 2 N H2SO4 and plates were read at 490/650 nm - with a Molecular Devices plate reader. The results shown in Figures 3, 4 and 5 demonstrate that lipidated OspA, but not non-lipidated OspA, ~m; n;stered mucosally induces a very strong serum IgG response. Additionally, W O 96/40Z90 PCT~US96/08866 lipidated OspA had a strong adjuvant e~ect on the serum IgG response to urease.
Having thus described in detail certain pre~erred embodiments of the present invention, it is to be understood that the invention def~ined by the appended claims is not to be limited by particular details set ~orth in the above description, as many apparent variations thereo~ are possible without departing ~rom the spirit or scope thereo~.

Claims (43)

WHAT IS CLAIMED IS:

1. A method for inducing an immunological response in a host comprising the steps of:
administering to the host at least one antigen;
and administering to the host a lipoprotein.

2. The method of claim 1 wherein the antigen and the lipoprotein are administered simultaneously.

3. The method of claim 1 wherein the antigen exhibits epitopes of a bacterial protein.

4. The method of claim 3 wherein the antigen is urease.

5. The method of claim 3 wherein the antigen is a Borrelia antigen other than OspA.

6. The method of claim 5 wherein the antigen is OspC.

7. The method of claim 1 wherein the lipoprotein is naturally lipidated.

8. The method of claim 1 wherein the lipoprotein is not naturally lipidated.

9. The method of claim 1 wherein the lipoprotein is an expression product of a hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by the first nucleic acid sequence.

10. The method of claim 9 wherein, in the hybrid nucleic acid molecule, the signal sequence is the signal sequence of an OspA protein of a Borrelia species, and the sequences are contiguous.

11. The method of claim 10 wherein, in the hybrid nucleic acid molecule, the first nucleic acid sequence and the second nucleic acid sequence are coupled in a translational open reading frame relationship.

12. The method of claim 11 wherein, in the hybrid nucleic acid molecule, the mature protein is an OspC protein of a Borrelia species, or a fragment thereof.

13. The method of claim 12 wherein, in the hybrid nucleic acid molecule, the mature protein is an OspC protein from a strain of Borrelia burgdorferi.

14. The method of claim 13 wherein the strain of Borrelia burgdorferi is selected from the B31, ACA1 and Ip90 families of strains.

15. The method of claim 11 wherein, in the hybrid nucleic acid molecule, the mature protein is PspA
or a fragment thereof.

16. The method of claim 15 wherein the antigen is an influenza antigen.

17. The method of claim 16 wherein the antigen is an HA antigen.

18. The method of claim 1 wherein the lipoprotein is antigenic.

19. The method of claim 18 wherein the lipoprotein is OspA.

20. The method of claim 1 wherein the antigen and lipoprotein are administered mucosally.

21. The method of claim 20 wherein the antigen and lipoprotein are administered intranasally.

22. The method of claim 20 wherein the antigen and lipoprotein are administered intragastrically.

23. The method of claim 20 wherein the antigen and lipoprotein are administered both intranasally and intragastrically.

24. The method of claim 1 wherein the immunological response is therapeutic.

25. The method of claim 1 wherein the immunological response is prophylactic.

26. A vaccine or immunogenic composition comprising:
an effective amount of at least one antigen;
and an effective amount of at least one lipoprotein.

27. The composition of claim 26 wherein the antigen exhibits epitopes of a bacterial protein.

28 The composition of claim 27 wherein the antigen is urease.

29. The composition of claim 28 wherein the antigen is a Borrelia antigen other than OspA.

30. The composition of claim 29 wherein the antigen is OspC.

31. The composition of claim 26 wherein the lipoprotein is naturally lipidated.

32. The composition of claim 26 wherein the lipoprotein is not naturally lipidated.

33. The composition of claim 26 wherein the lipoprotein is an expression product of a hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by the first nucleic acid sequence.

34. The composition of claim 33 wherein, in the hybrid nucleic acid molecule, the signal sequence is the signal sequence of an OspA protein of a Borrelia species, and the sequences are contiguous.

35. The composition of claim 34 wherein, in the hybrid nucleic acid molecule, the first nucleic acid sequence and the second nucleic acid sequence are coupled in a translational open reading frame relationship.

36. The composition of claim 35 wherein, an in the hybrid nucleic acid molecule the mature protein is an OspC protein of a Borrelia species, or a fragment thereof.

37. The composition of claim 37 wherein, the hybrid nucleic acid molecule, the mature protein is an OspC protein from a strain of Borrelia burgdorferi.

38. The composition of claim 37 wherein the strain of Borrelia burgdorferi is selected from the B31, ACA1 and Ip90 families of strains.

39. The composition of claim 35 wherein, in the hybrid nucleic acid molecule, the mature protein is PspA or a fragment thereof.

40. The composition of claim 39 wherein the antigen is an influenza antigen.

41. The composition of claim 40 wherein the antigen is an HA antigen.

42. The composition of claim 26 wherein the lipoprotein is antigenic.

43. The composition of claim 42 wherein the lipoprotein is an OspA protein of a Borrelia species.