OA12315A - Peptide mimics of conserved gonococcal epitopes and methods and compositions using them. - Google Patents

Abstract

The present invention relates to peptide mimics of a conserved gonoccocal epitode of <i>Neisseria gonorrhoeae</i>, which epitope is not found on human blood group antigens. This invention also relates to methods and compositions using such peptide mimics for the prophylaxis of gonorrheal infections.

Description

_ 4- 012315

PEPTIDE MIMICS OF CONSERVED GONOCOCCAL EPITOPES ANDMETHODS AND COMPOSITIONS USING THEM

TECHNICAL FIELD OF THE INVENTION s The présent invention relates to peptide mimics of conserved epitopes of Neisseria gonorrhoeae,which epitopes are not found on human blood groupantigens. This invention also relates to methods-andcompositions using such peptide mimics for the îo prophylaxis of gonorrheal infections.

BACKGROUND OF THE INVENTION

The sexually transmitted disease, gonorrhea,poses a worldwide risk as one of the most commonlyreported communicable diseases. Gonorrhea is caused by 15 the bacterium Neisseria gonorrhoeae, a gram négativediplococcus. Although the pathogen primarily infectsmucous membranes, it is capable of invading tissues and’evading host defenses.· N. gonorrhoeae is the causâtiveagent of a spectrum of sequelae. These range from 20 asymptomatic mucosal infection to significant diseasesyndromes in both men and women. The more serious ofsuch syndromes include, for example, disseminatedgonococcal infection ("DGI") in men and women, as wellas salpingitis or pelvic inflammatory disease ("PID") 25 in women. Either salpingitis or PID may themselves 2 012315 lead to long-term sequelae, including ectopic pregnancyand infertility. Other important sequelae, sometimesrequiring surgical intervention, include récurrentinfection, chronic pelvic pain, dyspareunia, pelvicadhesions and other inflammatory residua.

It has been estimated that in the UnitedStates, the direct and indirect costs of treating PIDand associated ectopic pregnancy and infertilitytotaled 2.6 billion dollars in 1984 (53). The totaldirect costs were estimated to be 2.18 billion dollarsin 1990, with indirect costs of 1.54 billion dollars.Assuming constant inflation and incidence rates of PID,the total cost of this disease is projected to reach 8billion dollars in the year 2000 (9).

Despite public health efforts to controlgonococcal infections and the availability of effectiveantibiotic thérapies in the United States, there areapproximately 315,000 cases of gonorrhea reportedannually to the Centers for Disease Control ("CDC") (12). A substantial proportion of ail cases ofgonorrhea occur in asymptomatically infected individuals who are the source of most new cases withina community (6). The increasing prevalence ofantibiotic-resistant strains has complicated treatmentof the infection (10, 11, 52). N. gonorrhoeae has multiple virulencefactors. The surface components of this pathogen playan important rôle in attaching to and invading hostcells, while providing potential targets for the hostimmune response. Gonococcal infections elicit localand systemic humoral and cellular immune responses toseveral components which are exhibited as surfaceexposed antigens of the bacterium, particularly pili,porin ("Por") or protein I ("PI"), opacity associated 012315 proteins ("Opas") or protein Ils, Rmp or protein III,and lïpooligosaccharides ("LOSs") (7). Pili, Opa, Porand LOS are ail implicated in attachaient to andinvasion of the host and ail display considérablevariation on their surface exposed régions (26, 45, 46). The intra-and inter-strain variations ofgonococcal surface components hâve led to hypothèsesregarding tissue specificity at different sites and theorganisai's potential for reinfection and continuedvirulence.

In both symptomatic and asyaiptomaticpatients, gonococcal infections hâve been shown tostioiulate increased levels of' anti-gonococcal sérumimoiunoglobulins. The peripheral humoral response ispredominately IgG (mostly subclass IgG3), with lesseramounts of IgM and IgA (13). Quantitatively, theantibody response is primarily directed against thepili, Opa proteins and LOS. Local antibodies areprésent in génital sécrétions, but in reduced amounts(48), and may be directed against different antigenictargets than those in seruoi -(27) . The prédominantclass of antibodies présent in sécrétions is also IgG(mostly IgG3) and not secretory IgA ("slgA") (7).Antibodies against LOS are présent as well, but inlesser amounts than those against pili, Por and Opa.Although patients infected with N. gonorrhoeae may showan antibody response to many- gonococcal antigens, N. gonorrhoeae isolated from patients with disseminatedinfection (DGI) are résistant to the bactericidalaction of normal human sérum ("NHS") and of mostconvalescent sera (38). This serum-resistantphenotype, termed stable sérum résistance ("SR"), mayenable the organisai to évadé local defenses, penetratemucosal barriers and disseminate via the bloodstream. 4 012315

Upon subculture, many strains of gonococcibecome phenotypically sensitive to killing by NHS orsérum sensitive (38). These organisms are termed sérumsensitive ("SS") or unstably serum-resistant. Suchorganisms are frequently isolated from women withsevere manifestations of local inflammation orclinically évident PID. Acute salpingitis, thepathologie counterpart of PID (caused by SS gonococci),rarely progresses to bacteremic illness or DGI. Thissuggests that the intense local inflammatory response,generated by SS gonococci, may serve to contain theinfection and prevent bacteremia, although at the costof damaging the local tissues. SS gonococci generatesignificantly greater amounts of the complément derivedchemotactic peptide, C5a, than do SR gonococci (16) .This may be responsible for the polymorphonuclearleukocyte ("PMN") mediated inflammatory response thatis produced by SS gonococci.

The development of antibiotic-resistantstrains of N. gonorrhoeae, has rendered control of thisinfection increasingïy difficult. The potential toundertreat gonococcal infection has accelerated theneed for an anti-gonococcal vaccine. The prévention ofgonococcal infection, particularly the severe complications of PID, has been the goal of many investigators. Ongo.ing attempts to develop an effective anti-gonococcal vaccine, however, hâve beenplagued with several difficulties.

Attempts to use individual surface componentsof the pathogen as targets for conventional vaccineshâve been unsuccessful because of their antigenicvariability. Pilus vaccines hâve been protective onlyagainst infection with the homologous strain (used tomake the pilus vaccine) and Por vaccination has been 5 012315 unsuccessful even in human experimental challenge. Inaddition, N. gonorrhoeae express marked phenotypicheterogeneity, typically shifting from one antigenicform to another at a frequency of >1 in 103 organisais(4 9, 50) making the surface of this organism a movingtarget for most vaccine strategies. Although thevaccine candidates hâve provoked antibody responses,the antibodies and immune responses produced hâve notbeen broadly protective. LOS is an important virulence' déterminant ofN. gonorrhoeae. Considérable evidence supports therôle of LOS as a major target of bactericidal antibodydirected to the surface of N. gonorrhoeae (2, 16, 18, 37, 47) . Antibodies to LOS hâve several importantfunctions: bactericidal activity, complémentactivation through the classical or alternativecomplément pathways (2), and opsonic activity (16).Additionally, LOS has been shown to be the mosteffective gonococcal antigen to induce a functionalantibody response to homologous and heterologousgonococci (51).

The monoclonal antibody ("mAb") 2C7 (30),detects » LOS derived oligosaccharide ("OS") epitopethat appears to be widely conserved and expressedamongst clinical isolâtes of gonococci. Typically,saccharides are T-cell independent antigens. Whenadministered alone as immunogens,- they generally elicitonly a primary antibody response. In addition,oligosaccharides are small (<10 saccharide units) (19) ,and would likely require additional biochemicalderivatization to render them immunogenic. The use ofsuch oligosaccharides as vaccine candidates, therefore,is limited in several respects. 6 0123 1 β

Internai image déterminants hâve beenproposed for use in vaccines (36) . By means of mAbtechnology, a protective antibody (Abl) to an epitopeof interest on the pathogen can be produced. Theparticular antibody (Abl) can be purified andsubsequently used as an immunogen to elicit an anti-idiotypic antibody (Ab2) which may be an internai imageof the original epitope on the pathogen.

As predicted by the Jerne "network" theory(23), immunization with an anti-idiotypic antibody(Ab2) that is directed against antigen combining sitesof primary antibody (Abl), may elicit a humoral immuneresponse spécifie for the nominal antigen. Theresuiting anti-anti-idiotypic antibody (or Ab3) shouldreact with the original primary antigen. If theprimary antigen is an oligosaccharide (and thereforeexpected to give a T-cell independent immune response),then immunization with Ab2 (the protein équivalent) mayelicit a T-cell dépendent response.

It has been demonstrated that an anti-idiotope of mAb 2C7 elicits anti-LOS antibodies in miceand rabbits that together with complément are bactericidal for gonococci, and that sérum from animaisimmunized with this anti-idiotypic antibody alsosupports opsonophagocytosis by human PMNs (20).

It has also been shown that syntheticpeptides which mimic a nominal antigen through bindingto a spécifie antibody directed to the nominal antigencan also elicit an immune response against the nominalantigen (29, 24, 54).

The need exists for an agent useful for theprévention of gonorrhea targeted to the prévention ofgonococcal salpingitis, an infection that may beassociated with debilitating and chronic pelvic pain, 7 072315 infertility and ectopic pregnancy (42). Another important objective is to prevent transmission of the organism from an infected but asymptomatic host to an otherwise immune sexual consort. This is important 5 because a substantial fraction of ail cases of gonorrhea in both men and women are asymptomatic, andasymptomatically infected, sexually active persons areprobably the major source of most new infections.Accordingly, a gonococcal vaccine that only atténuâtes ' îo the severity of symptomatic gonorrhea could resuit in ahigher ratio of asymptomatic/symptomatic cases and as aresuit, such a vaccine might promote the spread ofgonorrhea, unless it also prevents transmission (41).

SUMMARY OF THE INVENTION 15 The présent invention generally solves the problems referred to above by providing peptide mimicsof widely conserved oligosaccharide epitopes ofN. gonorrhoeae which are not présent in human bloodgroup antigens. Also provided are methods for 20 producing the peptide mimics according to this.invention.

The peptide mimics according to thisinvention are useful in methods and compositions forthe prophylaxis of N. gonorrhoeae infections. 25 BRIEF DESCRIPTION OF THE DRAWINGS '

Figure 1 shows a Western blot analysis of thebinding of mAb 2C7 to E. coli clones. The seven uniqueE. coli clones (PEP1-PEP7) [SEQ ID NOS: 1-7] were grownin IMC media containing 100 gg/ml ampicillin, and then 30 induced to express fusion proteins. Bacterial lysâtesfrom each of the clones were prepared and loaded onto14% SDS-PAGE gels. After electrophoresis, the proteins 8 012315 were transferred to Immobilon PVDF transfer membranesusing a Biorad electrophoretic transfer apparatus(Biorad, Hercules CA) . The membranes were probed withmAb 2C7 (A) or anti-thioredoxin antibody (B). Anégative clone that did not bind mAb 2C7 was used as acontrol [SEQ ID NO:9].

Figure 2 shows the peptide mimic sequencesderived from the seven E.coli clones that bind to mAb2C7.

Figure 3 shows FACS analysis of mAb 2C7binding to E.coli clones expressing peptide mimicfusions. E. coli clones were grown in IMC mediacontaining 100 /zg/ml ampicillin, and then induced toexpress fusion proteins. The bacterial cells werefixed with 1% paraformaldéhyde before staining with mAb2C7, followed by FITC-conjugated anti-mouse IgG. Anégative clone that did not bind mAb 2C7 was used as acontrol [SEQ ID NO: 9] . The number below the E. coliclones represents the médian fluorescent intensity inthe populations that bind to mAb 2C7 compared to thecontrol; the number in parenthesis shows the percentageof the cells in the population (total population = 100%).

Figure 4 shows inhibition of mAb 2C7 bindingto LOS by E. coli clones expressing peptide fusions. E. coli clones were grown in IMC media containing 100μg/ml ampicillin, and then induced to express fusionproteins. E. coli cells were incubated with mAb 2C7for 30 min. before loading onto LOS coated plates. Anégative clone that did not bind mAb 2C7 was used as acontrol [SEQ ID NO:9]. The data represent means fromat least 2 experiments (duplicate wells). PEP1 clonesshowed the maximum inhibition of mAb 2C7 binding to LOS(66%) [SEQ IDNO:1], PEP7, PEP3, PEP4, PEP2, PEP6, and 9 0Î2315 PEP5 showed respective decreases in inhibition ofbinding [SEQ ID NOS:7, 3, 4, 2, 6 and 5, respectively].

Figure 5 shows inhibition of mAb 2C7 bindingto LOS by a peptide comprising the consensus sequence 5 (DE_GLF) [SEQ ID NO:8]. The data represent means +SEfrom 3 experiments (duplicate wells). Peptide PEP1inhibited the binding of mAb 2C7 to LOS in a doseresponsive manner.

Figure 6 shows binding of mAb 2C7 to the10 multiple antigen peptide ("MAP") MAPI.

Figure 7 shows inhibition of mAb 2C7 bindingto LOS by multiple antigen peptides.

Figure 8 shows octa-MAPl-induced IgG anti-LOSantibody responses in mice. (A) Eight mice received a 15 dose of 50 μ<3 of Octa-MAPl emulsified in Freund's adjuvant on day 0 and again on day 21. (B) Four mice were immunized with purified LOS as a positive control.

Mice were immunized with either Freund's adjuvant (C)or an unrelated octa-MAP control peptide (D) as 20 négative Controls.

Figure 9 shows IgG anti-LOS antibody responses in ail immunized mice. IgG anti-LOS antibodyresponses (mean + SE) are shown for ail mice (includinganimais that exhibited no response). 25 Figure 10 shows IgG anti-LOS antibody •responses in responder mice only. Antibody responsewas defined as IgG anti-LOS (mean ± SE) greater than0.4 pg/ml (4 fold above baseline IgG anti-LOS levels).

Mice were immunized with Octa-MAPl, LOS, Freund's 30 adjuvant alone or unrelated octa-MAP control peptide.Elicited IgG anti-LOS antibody levels were plotted as afunction of concentration over time.

Figure 11 shows IgM anti-LOS antibodyresponses in responder mice only. Mice were immunized 10 012315 with Octa-MAPl, LOS, Freund's adjuvant alone orunrelated octa-MAP control peptide. Elicited IgG anti-LOS antibody levels were plotted as a function ofconcentration over time.

Figure 12 shows survival of gonorrhoeaestrain 15253 and its lgtG mutant (2C7 epitope négative)exposed to mouse immune sérum (67% [100 μΐ of sérum in150 μΐ total reaction volume) plus added human complément from normal human donor sérum [giving afinal human complément concentration of 17% byvolume] ) . A bactericidal assay was performed using (A)mAb 2C7 mice against strain 15253 (positive control)and strain 15253 lgtG (négative control) (4) . 25 pg/ml of mAb 2C7 (100 μΐ in 150 μΐ of total volume ofreaction mixture) mediated 100% killing of strain15253, and no killing of strain 15253 lgtG. (B) Normalmouse sérum (pool of 2 0 mouse sera, mean concentrationof IgG anti-LOS antibody, 0.1 μg/ml) failed to killeither strain. (C) Sérum taken from a single mouseimmunized with Octa-MAPl (containing 5.05 pg/ml of IgGanti-LOS antibody, pooled from bleeds taken betweenweeks 7-11) showed 92% killing (8% survival) of strain15253, whereas strain 15253 lgtG survived fully. (D) Sérum taken from a single mouse immunized with LOS-(containing 21.98 μg/ml of IgG anti-LOS antibodies,poolèd from bleeds taken between weeks 7-11) showed nokilling of strain 15253 (179% survival) and strain15253 lgtG (133% survival) . Single mice immunized withnégative control antigens (E) Freund's adjuvant aloneor (F) unrelated octa-MAP control peptide did not killeither strain. Figure 12 Controls included theComplément source without antibody (137.9% ± 1.0% 11 ο 12315 survival (no killing) for strain 15253, and 132,5% ± 14.3% survival (no killing) for the IgtG mutant of 15253).

Figure 13 shows a plot of IgG anti-LOS5 antibody concentration versus killing of N. gonorrhoeae

strain 15253. IgG anti-LOS antibody levels from eachof three mice immunized with Octa-MAPl are plottedversus percent bacterial killing. Mouse seracontaining 1.38, 2.50 and 5.05 μg/ml of anti-LOS îo antibodies showed 31, 74 and 92 % killing respectivelyof strain 15253. Killing by mAb 2C7 is shown at 5separate LOS antibody concentrations as a positivecontrol.

DETAILED DESCRIPTION OF THE INVENTION 15 Définitions

As used herein, an "antibody" is an intactimmunoglobulin molécule comprising two each ofimmunoglobulin light and heavy chains. Accordingly,antibodies include intact immunoglobulins of types IgA, 20 IgG, IgE, IgD, IgM (as well as subtypes thereof ) , wherein the light chains of the immunoglobulin may beof types kappa or lambda.

As used herein, "monoclonal antibodies" aremonospecific antibodies produced initially by a single 25 clone of antibody forming cells.

As used herein, "immunoprophylactically effective" means the ability to induce in a normalindividual an immune response sufficient to protectsaid patient for some period of time against 30 N. gonorrhoeae infection. 12 012315

As used herein, "peptide" means a linear or cyclic chain of amino acids, usually at least 4 and less than 50 amino acids in length.

As used herein, "peptide mimic" means apeptide which exhibits an immunological antibodybinding profile similar to that of a known epitope. PEPTIDE MIMICS AND THEIR USE INCOMPOSITIONS AND METHODS ACCORDING TO THIS INVENTION -

The présent invention is directed to peptidemimics that immunospecifically react with an antibodydirected to a conserved oligosaccharide epitope ofN. gonorrhoeae, which oligosaccharide epitope is notprésent in human blood group antigens. Such peptidemimics can be used in a manner similar to the anti-idiotypic antibodies described, for example in UnitedStates patents 5,476,784 and 6,099,839 (both incorporated herein by reference), as a surrogate antigen to elicit a T cell-dépendent immune responseagainst an oligosaccharide epitope of N. gonorrhoeae. -The peptide mimic may be administered touninfected individuals to induce a spécifie immuneresponse directed against gonococcal organisais or cellsbearing said oligosaccharide antigen. Such an immuneresponse can be immunoprophylactic in character, inthat it would prevent an infection should the récipientbe exposed to the gonococcal organisai or cells bearingsaid oligosaccharide antigen. A random peptide library may be screenedbased on antibody binding specificity in order toidentify candidate peptide mimics. The technology forsuch screening is known to those of skill in the art.

In one approach, a random peptide library expressed onE. coli flagella may be used to identify peptides that 13 0123)5 bind to a conserved oligosaccharide epitope ofN. gonorrhoeae, which oligosaccharide epitope is notprésent in human blood group antigens. For example,binding to mAb 2C7 may be assayed to identify candidatepeptide rnimics. Binding may be characterized bywestern blotting, flow cytométrie analysis orcompétition for binding of mAb 2C7 to LOS by solid-phase ELISA.

Antibody modeling may also be used to definean immunogenic site in the complementarity determiningrégions (CDRs) of an anti-idiotope corresponding to theepitope of interest. Such analysis may yield information about the three-dimensional conformation ofthe immunogenic site that is useful in the design of apeptide mimic of the immunogenic site.

Once a spécifie peptide mimic is identifiedand sequenced, it may be produced synthetically bymethods known in the art.

Peptide rnimics may also be modified to elicita greater immune response through the use of haptens,the use of adjuvants, linking the peptide mimic to acarrier protein, using a multiple antigen peptide,coupling the peptide mimic to a complément protein orthrough other methods known in the art.

The preferred pharmaceutical compositions ofthis invention are similar to those used forimmunization of humans with other peptides. Typi'cally,the peptide rnimics of the présent invention will besuspended in a stérile saline solution for therapeuticuses. The pharmaceutical compositions mayalternatively be formulated to control release of theactive ingrédients or to prolong their presence in apatient's System. Numerous suitable drug deliverySystems are known and include, e.g., implantable drug 012315 14 release Systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like.

The pharmaceutical compositions of this 5 invention may be administered by any suitable means such as orally, intranasally, subcutaneously,intramuscularly, intravenously, intra-arterially, orparenterally. Ordinarily, intravenous <i.v.) orparentéral administration will be preferred. io It will be apparent to those of ordinary skill in the art that the immunoprophylacticallyeffective amount of peptide mimics of this inventionwill dépend, inter alia, upon the administrationschedule, the unit dose of peptide mimic administered, 15 whether the peptide mimic is administered in combination with other therapeutic agents, the immunestatus and health of the patient, the therapeuticactivity of the peptide mimic administered and thejudgment of the treating physician. 20 In order that this invention may be better understood, the following examples are set forth.

These examples are for purposes of illustration only,and are not to be construed as limiting the scope ofthe invention in any manner.

25 ’ EXAMPLES I. Identification of Clones that Encode

Peptides that Specifically Bind to mAb 2C7 A. Random peptide display A FliTrx™ random peptide library (Invitrogen, 30 Carlsbad CA) was used to express peptides (12-mers) ofrandom seguence on the surface of E. coli. The DNAencoding this library of peptides is inserted within 15 012315 the gene encoding the active loop of thioredoxin whichis itself inserted into the nonessential région of theflagellin gene. Expression of the peptide fusion iscontrolled by the bactériophage lambda major leftwardpromoter (PJ in the vector pFliTrx™. In this system, PL is induced by the addition of tryptophan. Wheninduced, the fusion protein is exported and assembledinto flagella on the bacterial cell surface, allowing -for the display of the peptide. B· Screening of peptides that bind to mAb 2C7The FliTrx™ peptide library (1.77 x 10e primary clones) was grown overnight in IMC medium (0.2%w/v casamino acid, 0.5% w/v glucose, 42 mM Na2HPO4, 22mM KH2PO4, 8.5 mM NaCl, 18.7 mM NH4C1 and ImM MgCl2)containing 100 pg/ml ampicillin, at 25°C. Theexpression of fusion peptides was induced by adding L-tryptophan to a final concentration of 100 pg/ml, andthe culture was grown at 25°C for 6 h. The inducedpeptide fusion library was then incubated with a 2C7mAb-coated plate (20 ^g/ml) . After 1 h incubation, t-heplate was washed 5 times with IMC medium containing 100pg/ml ampicillin and 1% α-methyl mannoside. Bound E.coli were eluted by mechanical shearing or by compétition with purified LOS prepared from gonococcal‘ strain 15253 (the mAb 2C7 epitope is known to be expressed in strain 15253) , and then grown overnight at25°C. After the fifth round of panning, bound E. coliwere eluted and plated on RMG agar (2% w/v casaminoacid, 0.5% w/v glucose, 42 mM Na2HPO4, 22 mM KH2PO4, 8.5mM NaCl, 18.7 mM NH4CI, 1 mM MgCl2, and 1.5% agar)containing 100 Mg/ml ampicillin at 25°C. Individualbacterial colonies were chosen to assay binding to mAb2C7 by western blot (a hybridoma cell line secreting 012315 16 mAb 2C7 is deposited with the American Type CultureCollection ("ATCC"] and is assigned ATCC accessionnumber HB-11859).

The library was subjected to 5 rounds of5 positive sélection with mAb 2C7 coated on a 60 mm tissue culture plate or subjected to négative sélectionfor 1 h with irrelevant IgG3 (Sigma, St. Louis, MO)first, before proceeding to 5 rounds of positivesélection with mAb 2C7. io One hundred-seven colonies were randomly chosen and screened by western blot for the ability tobind mAb 2C7. Fourteen clones were identified thatbound to mAb 2C7. Plasmid DNA was then prepared f romthe positive clones and seguenced using primers that 15 bind to régions that are located at the 5 ' and 3 ' flanks of the inserted peptide's nucléotide sequence.Seven unique clones were identified, as shown inFigures 1 and 2 [SEQ ID NOS: 1-7] . C. Flow cytométrie analyses 20 Positive· E. coli clones were grown overnight in IMC media containing 100 ^g/ml ampicillin, at 25°Cand then induced to express the peptide fusions for 6h. E. coli cells were fixed with 0.5% paraformaldéhydeon ice for 10 min. Aliquots of 200-μΐ of fixed 25 organisms were spun at 2000 x g for 10 min.

Supernatants were discarded, and pellets wereresuspended in blocking buffer (IMC media containing100 ^g/ml ampicillin, 1% nonfat dry milk, 150 mM NaCland 1% α-methyl mannoside) containing mAb 2C7. 30 Suspensions were incubated at 37°C for 30 min beforespinning at 2000 x g for 10 min. Pellets were washedwith 100 μΐ of washing buffer (IMC media containinglOO/xg/ml ampicillin and 1% α-methyl mannoside) and then 012315 17 resuspended in 100 μΐ of blocking buffer containing FITC-conjugated anti-mouse IgG (Sigma, St. Louis, MO).

The mixtures were incubated at 37°C for 30 min before spinning at 2000 x g for 10 min. Supernatants were 5 removed, and pellets washed in 100 μΐ of washing bufferbefore resuspension in 1 ml of PBS. The suspensionswere analyzed on a FACS using CellQuest software(Becton Dickinson, Franklin Lakes NJ). A négativeclone that did not bind mAb 2C7 was used as a control. îo The binding of E. coli cells to mAb 2C7 was observed to increase from E. coli clone PEP3, PEP4, PEP6, PEP5, PEP2, PEP7 to PEP .1 (according to médianfluorescent intensity, "MFI") [SEQ ID NOS.-3, 4, 6, 5, 2, 7 and 1]. E. coli clone PEP1 showed the maximum 15 binding to mAb 2C7 (MFI = 19.81, compared to controlMFI = 4.91), as shown in Figure 3 [SEQ ID NO:lJ.

D. Inhibition ELISA

Positive E. coli clones were grown overnightin IMC media containing 100 gg/ml ampicillin at 25°C, 20 and then induced to express the peptide fusions for 6h. Cultures were normalized to the same OD reading(C©600nm ~ °·7)/ and 1% nonfat dry milk, 150 mM NaCl and1% α-methyl mannoside were added to block nonspecificbinding. 50 μΐ-aliquots of each culture were incubated 25 w'ith 50 μΐ of mAb 2C7 (final concentration 20 ng/ml) at37°C for 30 min, then 100 μΐ of the mixtures wereloaded into microtiter plate wells coated with purifiedLOS prepared from strain 15253 (80 μg/ml). The wellswere incubated at 37°C for 1 h, then washed. After the 30 wells were washed, bound mAb 2C7 was detected with anti-mouse IgG conjugated to alkaline phosphatase. Anégative clone that did not bind mAb 2C7 was used as acontrol. 012315 18 - PEPl clones showed the maximum inhibition ofraAb 2C7 binding to LOS (66%) [SEQ ID NO:1] , PEP7, PËF3, PEP4, PEP2, PEPS, and PEÇ5 showed respectivedecreases in inhibition of binding, as depicted in s Figure 4 (SEQ ID N0S:7, 3, 4, 2, 6 and S). Theinhibition ELISA résulté correlated with the flowcytométrie analysis résulta in that PEP1 also showedthe maximum binding to wAb 2C7. The binding of E. coli'celle to mAb 2C7 correlated approximately with îo decreases in inhibition of tnAb 2C7 binding to LOS by E. coli cloneB. II· Synthetic Peptide Mimic Binding to mRb 207 A synthetic peptide (PEP1, IPVLDENGLFAP) 15 whose seguence corresponds to the consensus seguence'’DE^GLF'' and includes two cysteine flanking régions(CGP- and -GPC residues at the K- and C- terminus,respectively) was synthesized (Boston Bioniolecules, MA)to assess spécifie binding to 2C7 mAb by inhibition 2o ELISA and to détermine whether peptide mirai ce characterized as thioredoxin-fusion proteins wouldretain the antigenicity indépendant of the fusioncontext [SEQ ID NO: 10] .

The cysteine flanking régions were added toas assess whether antibody binding is affected by cyclization of the peptide mimic. In these peptidesmimics, the cysteine residues allow for the formationof a disulfide bond befcween thera, resulting in a cyclicpeptide mimic. fîuch .conformationally constrained 30 peptides may more closely resemble the epitope thatthey mimic, and therefore may be more immunogenic.

Peptides were diluted in blocking buffer (1%ovalbumin, 0.05% tween-2 0, 0.5 M NaCl in PBS) to producemixtures of varying concentrations (0.1, 0.5 19 012315 and 1 mg/ml). 50 μΐ-aliquots from each of theconcentrations were incubated with 50 μΐ of mAb 2C7(stock concentration 2 μ9/πύ diluted in blockingbuffer) at 37°C for 1 h, then 100 μΐ of the mixtureswere loaded into microtiter plate wells coated withpurified LOS prepared from strain 15253 (80 μ9/τη1) .

The wells were incubated at 37°C for 1 h, then washed.After the wells were washed, bound mAb 2C7 was detected.with anti-mouse IgG conjugated to alkaline phosphatase.Purified LOS prepared from gonococcal strain 15253 wasused as a positive control. A non-reactive 15-merpeptide sequence generated by. the above describedrandom peptide library System was used as a négativecontrol peptide [SEQ ID NO:9]. PEP1 inhibited the binding of mAb 2C7 to LOSin a dose responsive manner (percentage inhibitionequalled 17, 77, and 91% with concentrations of 0.1, 0.5, and 1.0 mg/ml of PEP1, respectively) , as shown inFigure 5. The control 15-mer peptide was synthesizedas a cyclic peptide (*CKSNPIHIIKNRRNIPC*) [SEQ IDNO: 9] . This négative control peptide did not inhibitbinding of 2C7 mAb to the purified LOS coated plate.

Cyclic peptide mimics as describedimmediately above may further comprise one or more"tails" for coupling to a second agent, such as anadjuvant or a carrier protein, by methods known in theart. III. Increasinq The Immunogenicity Peptide Mimics

Although small peptides may be immunogenic,several studies hâve reported that certain smallpeptides may lack immunogenicity and resuit inineffective immune responses (particularly humoral 012315 20 responses) (3, 43). A number of strategies hâve beenused to increase the immunogenicity of small peptides.These include linking the peptide to a carrier protein(54, 28, 54), combining the peptide with an adjuvant(21, 22), using a multiple antigen peptide (MAP) toprovide a larger configurational structure that may bemore immunogenic (39) and coupling the peptide to acomplément protein to enhance the humoral immuneresponse (15). A. Multiple-antigen Peptide Synthesis

The multiple-antigen peptide (MAP) approachis a technique which associâtes the peptide mimic witha dendritic matrix of lysine residues (44, 8, 43).Peptides are attached to the amino groups of the lysinescaffold to yield a macromolecule that provides a highdensity of desired peptide epitopes on the surface ofthe complex. This approach has been shown to augmentthe immune response to peptides (3 9, 40) . A multiple antigen peptide of PEP1 and acontrol peptide were synthesized (Boston Biomolecules,MA) and binding to mAb 2C7 was assayed by direct andinhibition ELISA.

Solid phase ELISA was performed to assess thebinding of mAb 2C7 to multiple antigen peptides. Fordirect ELISA, Immulon 1 plates were coated overnightwith multiple antigen peptides (1 /ig/well) and reactedwith varying concentration of mAb 2C7. For inhibitionELISA, plates were coated with purified LOS preparedfrom N. gonorrhoeae strain 15253 (80 gg/ml) at 37°C for3 h. Peptides (linear or MAPs) were diluted inblocking buffer (1% ovalbumin, 0.05% tween-20, 0.5 MNaCl in PBS) to produce mixtures of varying 21 012315 concentrations. 50 μΐ-aliquots from each concentrationwere incubated with 50 μΐ of mAb 2C7 (stockconcentration 0.4 ^g/ml diluted in blocking buffer) at37°C for 1 h, then 100 μΐ of mixtures were loaded into 5 microtiter plate wells. The wells were incubated at37°C for 1 h, then washed. After the wells werewashed, bound mAb 2C7 was detected with anti-mouse IgGconjugated to alkaline phosphatase. Purified LOSprepared from gonococcal strain 15253 was used as a îo positive control in inhibition ELISA.

Multiple antigen peptide forms of PEP1 containing four linear PEP1 molécules ("Tetra-MAPl") oreight linear PEP1 molécules ("Octa-MAPl") showed strongbinding to mAb 2C7, whereas control MAP showed no 15 binding in direct ELISA, as depicted in Figure 6. BothTetra- and Octa-MAPl inhibit mAb 2C7 binding to LOSbetter than linear PEP1, as depicted in Figure 7. Halfmaximal inhibition (IC50) for both tetra- and octa-MAPlwas seen at 1.26 μΜ and 0.23 μΜ respectively. IC50 for 20 linear PEP1 55 μΜ. This may be due to increasedavidity of MAPI binding to mAb 2C7. Control MAPsshowed no significant inhibition.

Immunization with octa-MAPl induces an IgGanti-LOS antibody response in mice, as shown in Figure 25 8. The response profile seen in Figure 8 (A) , in which there is no significant IgG anti-LOS response until theboost at week 3, indicates that the Octa-MAPl eliciteda T-cell dépendent immune response in the respondingmice. These results demonstrate the promise of a 30 peptide mimic, such as Octa-MAPl, for immunizing humansagainst N. gonorrhoeae infection.

In Figure 8(A), eight mice received a dose of50 μg of Octa-MAPl emulsified in Freund's adjuvant onday 0 and again on day· 21. Octa-MAPl, which mimics the 22 01231g 2C7 oligosaccharide epitope, induced IgG anti-LOSantibody in three of the eight mice. IgG anti-LOSresponses in these three mice rose significantly afterthe first boost at week 3, peaked at week 7 (the nexttime measured) and decreased thereafter. Figure 8(B)shows the positive control experiment in which fourmice were immunized with purified LOS. In these mice,

IgG anti-LOS titers increased minimally after the first.immunization and rose after boosting. Ail mice in theLOS group showed an anti-LOS antibody response. Fourmice immunized with either Freund's adjuvant (C) or anunrelated octa-MAP control peptide (D), both négativeControls, elicited weak or no IgG anti-LOS responses.

The mean IgG anti-LOS antibody responses from ailimmunized mice (from the experiments depicted in Figure8) are shown in Figure 9 (mean + SE, including animaisthat exhibited no response) .

IgG anti-LOS antibody responses for theresponder mice only (from the experiment s depicted inFigure 8) are shown in Figure 10. Antibody response isdefined as IgG anti-LOS (mean ± SE) greater than 0.4pg/ml (4 fold above baseline IgG anti-LOS levels). At7 and 10 weeks after primary immunization, respondermice immunized with Octa-MAPl elicited IgG anti-LOSantibody levels higher (p < 0.001) than antibody levelselicited by négative control antigens (Freund'sadjuvant alone or unrelated octa-MAP control peptide).

IgM anti-LOS antibody responses for respondermice only (from the experiments depicted in Figure 8)are shown in Figure 11. Mice immunized with Octa-MAPlthat had elicited IgG anti-LOS responses failed torespond with IgM anti-LOS levels higher than miceimmunized with négative control antigens. Immunizationwith LOS (positive control) elicited IgM anti-LOS 23 0123 15 antibody levels higher than animais immunized witheither Octa-MAPl or négative control antigens (Freund'sadjuvant alone or unrelated octa-MAP control peptide). Sérum from a mouse immunized with Octa-MAPl5 exhibited 2C7-specific complément-mediated bactericidal activity against N. gonorrhoeae strain 15253, as shownin Figure 12. Depicted in Figure 12 is a graph showingsurvival of N. gonorrhoeae strain 15253 and its lgtGmutant (2C7 epitope négative) (4) exposed to mouse îo immune sérum (67% final mouse immune sérum concentration by volume) plus added human complémentobtained from normal human donors (17% final humancomplément concentration by volume).

Strain 15253 exhibits the 2C7 epitope. 15 Strain 15253 lgtG contains a disrupted allele of lipooligosaccharide (LOS) glycosyl transferase G, whichtransfers glucose (via an a linkage) onto heptose-2 inthe core of LOS (4) . The disruption of the lgtG locusresults in the loss of 2C7 epitope expression. 20 A standard bactericidal assay was performed

to assess complément-mediated bactericidal activity inmouse sera (11) . In this assay, mouse sérum (67% finalvolume) (from various mice immunized or not asdescribed below) was incubated with approximately 2.5 X 25 I03 bacteria suspended in Morse A media (33) in the presence of human complément (17% final volume). Thereaction mixture was then shaken continuously at 37°Cfor 30 minutes. Aliquots of the reaction mixture werethen inoculated onto chocolaté agar plates at time 0 30 and 30 minutes. Survival was expressed as the percentincrease in colonies on the plate at 30 minutes,compared to those on the plate at 0 minutes. Greaterthan 100% survival in the assay indicates growth duringthe 30-minute incubation period. 24 012315 mAb 2C7 was used as a control, as it kills N.gonorrhoeae strain 15253 with added complément, butdoes not kill the 15253 lgtG mutant strain. As shownin Figure 12(A), mAb 2C7 possesses bactericidalactivity against 2C7 epitope-bearing gonococci. 25pg/ml of mAb 2C7 (100 μΐ in 150 μΐ of total volume ofreaction mixture) mediated 100% killing of strain15253, and no killing of strain 15253 lgtG. Sérum taken from a single mouse immunizedwith Octa-MAPl, containing 5.05 μg/ml of IgG anti-LOSantibody pooled from bleeds taken between weeks 7-11,showed 92% killing (8% survival) of strain 15253whereas strain 15253 lgtG survived fully, as depictedin Figure 12(C).

Normal mouse sérum representing a pool of 20mouse sera with a mean concentration of IgG anti-LOSantibody of 0.1 μg/ml failed to kill either strain, asshown in Figure 12 (B). Control mouse sérum withoutcomplément showed 116.1% + 4.7% survival (no killing)for strain 15253, and 123.1% ± 3.5% survival (nokilling) for the lgtG mutant of 15253. The complémentsource without antibody exhibited 137.9% ± 1.0%survival (no killing) for strain 15253, and 132.5% ±14.3% survival (no killing) for the lgtG mutant of15253 . Sérum taken from a single mouse immunizedwith LOS (containing 21.98 μg/ml of IgG anti-LOSantibodies, pooled from bleeds taken between weeks 7-

11) effected no killing of strain 15253 (179% survival)and strain 15253 lgtG (133% survival), as shown inFigure 12 (D) . Sérum taken from single mice immunizedwith Freund's adjuvant alone or unrelated Octa-MAP 25 012315 control peptide, as négative control antigens, did notkill either strain, as shown in Figures 12 (E) and 12 (F)respectively.

IgG anti-LOS antisera obtained from mice5 immunized with Octa-MAPl exhibited concentration- dependent killing of N. gonorrhoeae strain 15253, asshown in Figure 13.

Figure 13 shows a plot of IgG anti-LOSantibody concentration versus killing of N. gonorrhoeae 10 strain 15253. When IgG anti-LOS antisera levels fromeach of three mice immunized with Octa-MAPl wereplotted against bacterial killing, a dose-responseprofile resulted (mouse sera containing 1.38, 2.50 and5.05 μg/ml of anti-LOS antibodies showed 31, 74 and 92 15 % killing, respectively, of strain 15253) . Killing by mAb 2C7 was also shown at 5 separate LOS antibodyconcentrations as a positive control. B. Coupling A Peptide MimicTo Complément Protein C3d 20 It is expected that the immunogenicity of peptide mimics of gonococcal epitopes, such as Octa-MAPl described herein, can be further enhanced throughcoupling with complément factor C3d.

Numerous studies hâve demonstrated an important 25 rôle of complément protein C3 in the induction of humoral immune responses (1, 5, 14, 17, 25, 32, 34 and35). C3-depleted mice show diminished antibodyresponses to T-cell dépendent protein antigen, such askeyhole limpet haemocyanin ("KLH") (34, 35). 30 Complément receptor 1-(CRI or CD35) and complémentreceptor 2-(CR2 or CD21) déficient mice hâve animpaired T-cell dépendent antibody response (1, 14, 012315 26 32) . It has further been shown that C3d covalentlylinked to hen egg lysozyme ("HEL") resulted in anenhanced antibody response to the HEL antigen (15) .

Mice immunized with a fusion protein that consisted of 5 three copies of C3d and 1 copy of HEL elicited aΙΟ,ΟΟΟ-fold increase in anti-HEL antibody response,compared to antibody responses in mice immunized withHEL alone. Anti-HEL antibody responses induced by the -fusion protein were approximately 100- fold higher than 10 those induced by HEL emulsified in Freund's adjuvant.

Octa-MAPl can be coupled to C3d by cloning an octa-MAPl DNA sequence into a.. C3d fusion proteincassette and transforming this construct into anexpression System. The OctaMAPl-C3d fusion protein can 15 then be expressed, purified and used as an immunogen.Alternatively, the OctaMAPl-C3d gene fusion, in the .form of DNA, can be used as a DNA vaccine according tomethods known in the art. A hybridoma producing anti-idiotypie 20 antibodies exhibiting immunological reactivity similarto the peptide mimics of the instant invention isexemplified by a cell culture deposited in the ATCC(10801 University Boulevard, Manassas, Va. 20110-2209U. S. A.) on March 26, 1993 and assigned ATCC accession 25 number HB 11311.

Hybridoma 2C7 secreting the mAb 2C7 exhibiting immunological reactivity similar to thepeptide mimics of the instant invention is exemplifiedby a cell culture designated as 2C7 and deposited in 30 the ATCC on March 9, 1995. This culture was assignedATCC accession number HB-11859.

While we hâve hereinbefore described a numberof embodiments of this invention, it is apparent thatour basic constructions can be altered to provide other 27 012315 embodiments which utilize the processes andcompositions of this invention. Therefore, it will beappreciated that the scope of this invention is to bedefined by the daims appended hereto rather than by 5 the spécifie embodiments which hâve been presentedhereinbefore by way of example. 28 ο 123 ! 5 1. 2. 10 3 . 15 4 . 20 5. 25 6. 7 . 30 8 . 9. 35

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Claims (28)

1. 0123 15 34 CLAIMS 'We claim:

   1. A peptide mimic of a conserved gonococcalepitope not found on human blood group antigens,wherein said peptide mimic is capable of inducing ina mammal an immune response against said conservedgonococcal epitope.
2. 2. The peptide mimic according to claim 1,wherein the amino acid sequence of the peptide mimiccomprises the sequence DE_GLF.
3. 3. The peptide mimic according to claim 1,wherein the immune response is T-cell dépendent.
4. 4. The peptide mimic according to claim 1 or 2,wherein the amino acid sequence of the peptide mimiccomprises cysteine residues at each terminus.
5. 5. The peptide mimic according to claim 4,wherein a cyclic peptide is formed through disulfidebridges between the cysteine residues at eachterminus of said sequence.
6. 6. The peptide mimic according to claim 5,wherein the peptide mimic further comprises at leastone tail for coupling to a second agent.
7. 7. The peptide mimic according to claim 6,wherein the second agent is an adjuvant. 012315 35
8. 8. The peptide mimic according to claim 1 or 2,wherein the peptide mimic further comprises anadjuvant or a carrier protein.
9. 9. The peptide mimic according to claim 1 or 2,wherein the peptide mimic is part of a multipleantigen peptide.
10. 10. The peptide mimic according to claim 1 or 2,wherein said peptide mimic competes with gonococcalLOS for binding to monoclonal antibody 2C7.
11. 11. A peptide mimic which immunospecificallybinds to an antibody that binds to an oligosaccharideepitope of N. gonorrhoeae, which oligosaccharideepitope is not présent in human blood group antigens.
12. 12. The peptide mimic according to claim 11,wherein the peptide mimic binds to monoclonalantibody 2C7.
13. 13. The peptide mimic according to claim 11,wherein the peptide mimic binds to a monoclônalantibody produced by immunizing a mammal with ananti-idiotypie monoclonal antibody, or fragmentthereof, produced by a hybridoma cell line having thecharacteristics of HB 11311 as deposited with theATCC.
14. 14. The peptide mimic according to' claim 11,wherein the peptide mimic is part of a multipleantigen peptide. 36 Ο 12315
15. 15. A composition for immunizing againstN. gonorrhoeae infection comprising animmunoprophylactically effective amount of amimic according to any one of daims 1-3, 5- 5 11-14.
16. 16. A composition for immunizing againstN. gonorrhoeae infection comprising animmunoprophylactically effective amount of amimic comprising the peptide sequence of SEQ 10 17. A composition for immunizing against N. gonorrhoeae infection comprising animmunoprophylactically effective amount of amimic comprising the peptide sequence of SEQ
17. 18. A composition for immunizing against *15 W. gonorrhoeae infection comprising an immunoprophylactically effective amount of amimic comprising the peptide sequence of. SEQ
18. 19. A composition for immunizing againstN. gonorrhoeae infection comprising an 20 immunoprophylactically effective amount of amimic comprising the peptide sequence of SEQ
19. 20. A composition for immunizing againstN. gonorrhoeae infection comprising animmunoprophylactically effective amount of a 25 mimic comprising the peptide sequence of SEQ
20. 21. A composition for immunizing againstN. gonorrhoeae infection comprising an peptide, 9 or peptideID NO:1 peptideID NO:2 peptideID NO:3 peptideID NO:4 peptideID NO:5 i 012315 3* immunoprophylactically effective amount of a peptidemimic comprising the peptide sequence of SEQ ID NO: 6.
21. 22. A composition for immunizing againstN. gonorrhoeae infection comprising an 5 immunoprophylactically effective amount of a peptide mimic comprising the peptide sequence of SEQ ID NO : 7.
22. 23. A composition for immunizing againstN. gonorrhoeae infection comprising animmunoprophylactically effective amount of a peptidemimic comprising the peptide sequence of SEQ ID NO:10.
23. 24. The use of a compound containing animmunoprophylactically effective amount of apeptide mimic according to any one of daims 1-3and a pharmaceutically acceptable carrier in the 15 manufacture of a médicament for use in a method of immunizing a mammal against N. gonorrhoeaeinfection.
24. 25, The use of a compound containing animmunoprophylactically effective amount of a 20 peptide mimic according to any one of daims 11- 14 and a pharmaceutically acceptable carrier inthe manufacture of a médicament for use in amethod of immunizing a mammal against N.gonorrhoeae infection. 2S. The peptide mimic according to claim 1 or 2.1,vzherein the peptide mimic is coupled to a complémentorotsin. 25 - 35 012315
25. 27. The peptide mimic according to claim'2»., -,,-.75:5^ ras peptide mimic as ccupisà co complémentprotein C3û,
26. 28. The use of a compound containing an5 immunoprophylactically effective amount of a peptide mimic according to claim 27 and apharmaceutically acceptable carrier in themanufacture of a médicament for use in a methodof immunizing a mammal against N. gonorrhoeae 10 infection.
27. 29. A composition for immunizing againstir» gonorrhoeae infection comprising animmunoprophylactically effective amount of a peptidemimic according to claim 27. 15 50· A method. for increasing the antigenicity of a t, peptide mimic according to claim 1 or 11 comprising’ the step of coupiing said peptide mimic to acomplément protein.
28. 31. The method according to claim 30, wherein thecomplément protein is-C3d. 20 SEQUENCE LISTING <110> Rice, Peter <120> Peptide Mimics Of Conserved Gonococcal EpitopesAnd Methods And Compositions Using Them <130> BOS-3 <140> Not Yet Assigned <141> 2000-10-27 <150> 60/162491 <151> 1999-10-29 <160> 10 <170> Patentln version 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct <400> 1 0)2315 Ile Pro Val Leu Asp Glu Asn Gly Leu Phe Ala Pro15 10 <210> 2 <211> 12 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1) · . (12) <223> synthetic construct <400> 2 Trp Gly Leu Asp Tyr Glu Arg Gly Asn Tyr Glu Glu15 10 <210> 3 <211> 12 <212> PRT <213> Artificial/Unknown Page 1 012315 <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct <400> 3 Asp Ala Leu Ala Val Asp Gin Met Gly Arg Phe Gly15 10 <210> 4 <211> 12 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct <400> 4 Val Leu Val Gly Glu Lys Gly Leu Phe Glu Gly Gly15 10 <210> 5 <211> 12 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct <400> 5 Glu Ala Leu Val Leu Asp Thr Asn Gly Leu Met Ser15 10 <210> 6 <211> 12 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct Page 2 U1231S <400> 6 Ala Asp Arg Thr Gin Gly Leu Gly Trp Gly Ala Ser15 10 <210> 7 <211> 12 <212> PRT <213> Artificial/ünknown <220> <221> PEPTIDE <222> (1)..(12) <223> synthetic construct <400> 7 Glu Glu Val Gly Ser Ile Leu Tyr Gly Leu Gly Gly1 5 10 <210> 8 <211> 6 <212> PRT <213> Artificial/ünknown <220> <221> PEPTIDE <222> (3)..(3) <223> X = any amino acid <220> <221> PEPTIDE <222> (1)..(6) <223> synthetic construct <400> 8 Asp Glu Xaa Gly Leu Phe1 5 <210> 9 <211> 17 <212> PRT <213> Artificial/ünknown <220> <221> PEPTIDE <222> (1) .. (17) <223> synthetic construct Page 3 012315 <400> 9 Cys Lys Ser Asn Pro Ile His Ile Ile Lys Asn Arg Arg Asn Ile Pro 1 5 10 15 Cys <210> 10 <211> 15 <212> PRT <213> Artificial/Unknown <220> <221> PEPTIDE <222> (1) · · (15) <223> synthetic construct <400> 10 Cys Gly Pro Ile Pro Val Leu Glu Asn Gly Leu Phe Gly Pro Cys 1 5 10 15 Page 4