HK1141034B - Peptide mimics of conserved gonococcal epitopes and methods and compositions using them - Google Patents

Description

Peptoids of conserved gonococcal epitopes and methods and compositions employing them

The present application is a divisional application of invention patent application No. 00817098.3 entitled "conserved gonococcal epitope peptide mimetics and methods and compositions for using them", filed on 27/10/2000.

Field of the invention

The present invention relates to peptidomimetics of conserved epitopes of Neisseria gonorrhoeae (Neisseria gonorrhoeae), which epitopes are not found on human blood group antigens. The invention also relates to methods and compositions for preventing gonorrhea infection using these peptidomimetics.

Background

The sexually transmitted disease, gonorrhea, poses a worldwide risk as one of the most commonly reported infectious diseases. Gonorrhea is caused by a gram-negative diplococcal neisseria gonorrhoeae. Although this pathogen primarily infects mucous membranes, it can invade tissues and evade host defenses. Neisseria gonorrhoeae is the causative agent of a variety of sequelae. Ranging from asymptomatic mucosal infections in men and women to overt disease syndromes. More severe syndromes include, for example, disseminated gonococcal infection ("DGI") in men and women, and salpingitis or pelvic inflammatory disease ("PID") in women. Salpingitis or PID itself can lead to long-term sequelae including ectopic pregnancy and infertility. Other important sequelae (sometimes requiring surgery) include recurrent infections, chronic pelvic pain, dyspareunia, intra-pelvic adhesions and other inflammatory residues.

It is estimated that the direct and indirect costs of treating PID and related ectopic pregnancy and infertility amount to $ 26 million in 1984 in the united states (53). In 1990, the total direct cost was estimated to be $ 21.8 million and the indirect cost to be $ 15.4 million. Assuming constant prevalence of inflation and PID, the total cost of the disease is expected to reach $ 80 billion in 2000 (9).

Despite the public health efforts in the united states to control gonococcal infections and the applicability of effective antibiotic therapy, nearly 315000 cases of gonorrhea have been reported by the annual centers for disease control ("CDC") (12). A significant proportion of all gonorrhoea cases occur in asymptomatic infected individuals who are the source of most new cases within the population (6). The increasing prevalence of antibiotic resistant strains has made the treatment of this infection more complex (10, 11, 52).

Neisseria gonorrhoeae has a variety of virulence factors. The surface components of the pathogen play an important role in attaching to and invading host cells and provide potential targets for host immune responses. Gonococcal infections elicit local and systemic humoral and cellular immune responses to several components that manifest as bacterial surface-exposed antigens, in particular pili, porin ("Por") or protein I ("PI"), haze-related proteins ("Opa") or protein II, Rmp or protein III, and lipooligosaccharides ("LOS") (7). Pili, Opa, Por and LOS are all associated with attachment and invasion of the host, and their surface exposed regions all show considerable variation (26, 45, 46). Intra-and inter-strain variation of gonococcal surface components has led to the hypothesis of tissue specificity and the potential for biological re-infection and sustained virulence at different sites.

Gonococcal infections have been shown to stimulate an increase in anti-gonococcal serum immunoglobulin levels in both symptomatic and asymptomatic patients. The peripheral humoral response is predominantly IgG (mostly subclass IgG3), with lesser amounts of IgM and IgA (13). Quantitatively, the antibody response is mainly directed against pili, Opa protein and LOS. Local antibodies are present in genital secretions, but in reduced amounts (48), possibly against different antigen targets than in serum (27). The main class of antibodies present in secretion is also IgG (mainly IgG3), rather than secretory IgA ("sIgA") (7). anti-LOS antibodies are also present, but in lesser amounts than antibodies against pili, Por and Opa. Although patients infected with neisseria gonorrhoeae may show antibody responses to a variety of gonococcal antigens, neisseria gonorrhoeae isolated from patients with disseminated infection (DGI) is resistant to bactericidal effects in normal human serum ("NHS") and in the sera of most convalescent patients (38). This serum-resistant phenotype, known as stable serum resistance ("SR"), allows organisms to evade local defenses, cross mucosal barriers, and spread through the bloodstream.

After subculture, killing of NHS by many gonococcal strains becomes phenotypically or serum sensitive (38). These organisms are referred to as serum sensitive ("SS") or labile serum resistant. These organisms are usually isolated from women with severe manifestations of local inflammation or clinically significant PID. Acute salpingitis, the pathologically opposite disease of PID (caused by SS gonococci), rarely progresses to bacteremia or DGI. This suggests that the intense local inflammatory response caused by SS gonococci may be sufficient to include infection and prevent bacteremia, despite the cost of damaging local tissues. SS gonococci produce significant quantities of complement-derived chemotactic peptide C5a over SR gonococci (16). This may result in a polymorphonuclear leukocyte ("PMN") mediated inflammatory response produced by SS gonococci.

The development of antibiotic resistant strains of neisseria gonorrhoeae has made the control of this infection increasingly difficult. The potential for inadequate treatment of gonococcal infections has accelerated the need for vaccines against gonococcal bacteria. Prevention of gonococcal infections, particularly serious complications of PID, has been the goal of many researchers. However, ongoing efforts to develop effective anti-gonococcal vaccines have met with a number of difficulties.

Attempts to use individual surface components of pathogens as targets for conventional vaccines have not been successful due to their antigenic variability. Pilus vaccines are only protective against infection by homologous strains (used to produce pilus vaccines), and Por vaccination has not been successful even in human experimental challenge. In addition, N.gonorrhoeae exhibits significant phenotypic heterogeneity, typically of 10³The frequency of > 1 in an organism shifts from one antigenic form to another (49, 50), which makes the surface of the organism a moving target for most vaccine protocols. Although vaccine candidate strains elicit antibody responses, the antibodies and immune responses produced are not broadly protective.

LOS is an important virulence determinant in Neisseria gonorrhoeae. There is considerable evidence to support the role of LOS as a primary target for bactericidal antibodies against the surface of neisseria gonorrhoeae (2, 16, 18, 37, 47). LOS antibodies have several important functions: bactericidal activity, complement activation by the classical or alternative complement pathways (2), and opsonin activity (16). In addition, LOS is considered to be the most potent gonococcal antigen that induces a functional antibody response to homologous and heterologous gonococci (51).

Monoclonal antibody ("mAb") 2C7(30) detected a LOS-derived oligosaccharide ("OS") epitope that appeared to be widely conserved and expressed in clinical isolates of gonococci. Carbohydrates are generally T cell independent antigens. When administered alone as immunogens, they typically elicit only primary antibody responses. In addition, oligosaccharides are small (< 10 saccharide units) (19), and may require additional biochemical derivatization to render them immunogenic. Thus, the use of these oligosaccharides as vaccine candidates is limited in several ways.

The use of internal image (internal image) determinants (36) in vaccines has been proposed. Protective antibodies against epitopes of interest on pathogens can be produced using mAb technology (Ab 1). Specific antibodies (Ab1) can be purified and subsequently used as immunogens to generate anti-idiotypic antibodies (Ab2) that may be internal images of the original epitope on the pathogen.

Immunization with an anti-idiotypic antibody (Ab2) directed against the antigen-binding site of the primary antibody (Ab1) elicits a humoral immune response specific for the named antigen as predicted by Jerne's "network" theory (23). The anti-idiotype antibody (or Ab3) produced should be reactive with the original primary antigen. If the primary antigen is an oligosaccharide (and is therefore expected to elicit a T cell independent immune response), immunization with Ab2 (protein equivalent) may elicit a T cell dependent response.

The anti-idiotypic site of mAb2C7 has been shown to produce anti-LOS antibodies in mice and rabbits, which are gonococcal along with complement, and serum from animals immunized with this anti-idiotypic antibody also supports opsonophagocytosis by human PMNs (20).

Synthetic peptides that mimic the named antigen by binding to a specific antibody against the named antigen have also been shown to elicit immune responses against the named antigen (29, 24, 54).

There is a need for agents that can be used to prevent gonorrhea, with the aim of preventing gonococcal salpingitis, an infection that may be associated with aging and chronic pelvic pain, infertility and ectopic pregnancy (42). Another important objective is to prevent the spread of an organism from an infected, but asymptomatic, host to another immunological partner. This is important because a significant fraction of all gonorrhea cases in men and women are asymptomatic, while asymptomatic infected, sexually active people may be the major source of most new infections. Thus, a gonococcal vaccine that only reduces the severity of symptomatic gonorrhea can cause a higher proportion of asymptomatic/symptomatic cases, and therefore, such a vaccine may promote the spread of gonorrhea unless spread is also prevented (41).

Summary of The Invention

The present invention generally solves the above problems by providing peptoids of widely conserved oligosaccharide epitopes of neisseria gonorrhoeae which are not present in human blood group antigens. Also provided are methods of producing the peptidomimetics according to the invention.

The peptidomimetics according to the invention are useful in methods and compositions for preventing neisseria gonorrhoeae infection.

Brief Description of Drawings

FIG. 1 shows Western blot analysis of mAb2C7 binding to E.coli clones. 7 separate E.coli clones (PEP1-PEP7) (SEQ ID NOS: 1-7) were grown in IMC medium containing 100. mu.g/ml ampicillin and then induced to express the fusion proteins. Bacterial lysates from each clone were prepared and loaded on 14% SDS-PAGE gels. After electrophoresis, the proteins were transferred to Immobilon PVDF transfer membranes using a Biorad electrophoretic transfer device (Biorad, Hercules CA). The membrane was probed with mAb2C 7(a) or anti-thioredoxin antibody (B). One negative clone that was unable to bind mAb2C7 served as a control [ SEQ ID NO: 9].

FIG. 2 shows the peptidomimetic sequences from 7 E.coli clones that bind mAb2C 7.

FIG. 3 shows FACS analysis of mAb2C7 binding to E.coli clones expressing peptidomimetic fusions. Coli clones were grown in IMC medium containing 100. mu.g/ml ampicillin, and induced to express the fusion protein. Bacterial cells were fixed with 1% paraformaldehyde, then stained with mAb2C7, followed by FITC-conjugated anti-mouse IgG. One negative clone that was unable to bind mAb2C7 served as a control [ SEQ ID NO: 9]. The numbers below the E.coli clones represent the median fluorescence intensity of the populations bound to mAb2C7 compared to controls; the numbers in parentheses indicate the percentage of cells in the population (total population 100%).

FIG. 4 shows the inhibition of the binding of mAb2C7 to LOS by E.coli clones expressing the fusion peptides. Coli clones were grown in IMC medium containing 100. mu.g/ml ampicillin, and induced to express the fusion protein. Coli cells were incubated with mAb2C7 for 30 minutes before loading onto LOS coated plates. One negative clone that was unable to bind mAb2C7 served as a control [ SEQ ID NO: 9]. Data represent the mean of at least 2 experiments (double wells). Clone PEP1 showed maximum inhibition (66%) of mAb2C7 binding to LOS [ SEQ ID NO: 1]. PEP7, PEP3, PEP4, PEP2, PEP6, and PEP5 showed reduced binding inhibition respectively [ SEQ id no: 7, 3, 4, 2, 6 and 5 ].

Fig. 5 shows a polypeptide containing the consensus sequence (DE _ GLF) [ SEQ ID NO: 8] inhibition of binding of mAb2C7 to LOS. Data represent mean ± SE of 3 experiments (double wells). Peptide PEP1 inhibited mAb2C7 binding to LOS in a dose-responsive manner.

Fig. 6 shows the binding of mAb2C7 to multiple antigen peptide ("MAP") MAP 1.

Figure 7 shows the inhibition of mAb2C7 binding to LOS by multiple antigenic peptides.

FIG. 8 shows the IgG anti-LOS antibody response induced by octaMAP 1(octa-MAP1) in mice. (A)8 mice received a dose of 50 μ g of octamap 1 emulsified in freund's adjuvant on day 0 and again on day 21. (B)4 mice were immunized with purified LOS as a positive control. Mice were immunized with Freund's adjuvant (C) or an unrelated octaMAP control peptide (D) as a negative control.

FIG. 9 shows IgG anti-LOS antibody responses in all immunized mice. IgG anti-LOS antibody responses (mean. + -. SE) were shown for all mice, including animals that did not exhibit a response.

FIG. 10 shows only IgG anti-LOS antibody responses in responding mice. Antibody responses were defined as IgG anti-LOS (mean. + -. SE) above 0.4. mu.g/ml (4-fold above baseline IgG anti-LOS levels). Mice were immunized with octamap 1, LOS, freund's adjuvant alone, or an unrelated octamap control peptide. Induced IgG anti-LOS antibody levels are plotted as concentration versus time.

FIG. 11 shows only IgM anti-LOS antibody responses in responding mice. Mice were immunized with octamap 1, LOS, freund's adjuvant alone, or an unrelated octamap control peptide. Induced IgG anti-LOS antibody levels are plotted as concentration versus time.

FIG. 12 shows the survival of strain 15253 Neisseria gonorrhoeae and its lgG mutant (epitope negative to 2C 7) exposed to mouse immune serum (67% [ 100. mu.l serum in total reaction volume of 150. mu.l ] plus added human complement from normal human donor serum [ so that the final concentration of human complement is 17% by volume ]). Bacterial experiments were performed with (a) mAb2C7 mice against strain 15253 (positive control) and strain 15253 lgg (negative control) (4). mAb2C7 (100 μ l in 150 μ l total reaction mixture volume) at 25 μ g/ml mediated killing of 100% of 15253 strain, not 15253 lgg strain. (B) Normal mouse serum (pooled 20 mouse sera with an average concentration of IgG anti-LOS antibody of 0.1. mu.g/ml) failed to kill either strain. (C) Sera from single mice immunized with octamap 1 (containing 5.05 μ g/ml IgG anti-LOS antibody, pooled from blood taken at weeks 7-11) showed 92% kill (8% survival) against strain 15253, while strain 15253 lgg was fully viable. (D) Serum from a single mouse immunized with LOS (containing 21.98. mu.g/ml IgG anti-LOS antibody, combined with blood taken at weeks 7-11) did not kill both 15253 strain (179% survival) and 15253 lgG strain (133% survival). A single mouse immunized with negative control antigen (E) freund's adjuvant alone or (F) an unrelated octamap control peptide did not kill either strain. Figure 12 controls included complement sources without antibody (137.9% ± 1.0% survival (no killing) of strain 15253, 132.5% ± 14.3% survival (no killing) of 15253 lgg mutant).

FIG. 13 shows a graph of the killing rate of IgG anti-LOS antibody concentration against N.gonorrhoeae strain 15253. IgG anti-LOS antibody levels from each of the 3 mice immunized with octamap 1 were plotted against the percent killing of bacteria. Sera from mice containing 1.38, 2.50 and 5.05 μ g/ml anti-LOS antibody showed 31%, 74% and 92% killing of strain 15253, respectively. The killing rate of mAb2C7 was shown at 5 separate LOS antibody concentrations as a positive control.

Detailed Description

Definition of

As used herein, an "antibody" is an intact immunoglobulin molecule containing two immunoglobulin light and heavy chains each. Thus, antibodies include intact immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and subtypes thereof), wherein the light chains of the immunoglobulins may be of the kappa or lambda type.

As used herein, a "monoclonal antibody" is a monospecific antibody originally produced by an antibody-forming cell of a single clone.

As used herein, "immunoprophylactically effective" refers to the ability to induce an immune response in a normal individual sufficient to protect the patient from neisseria gonorrhoeae infection for a period of time.

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

As used herein, a "peptidomimetic" refers to a peptide that displays an immune antibody binding pattern similar to a known epitope.

Peptidomimetics and their use in compositions and methods according to the invention

The present invention relates to peptidomimetics that react immunospecifically with antibodies directed against conserved oligosaccharide epitopes of neisseria gonorrhoeae, which oligosaccharide epitopes are not present in human blood group antigens. These peptidomimetics can be used in a manner similar to the anti-idiotypic antibodies described in U.S. Pat. Nos. 5,476,784 and 6,099,839 (both incorporated herein by reference) as an alternative antigen to elicit a T cell dependent immune response against an epitope of an oligosaccharide of N.gonorrhoeae.

Such peptidomimetics can be administered to uninfected individuals to induce a specific immune response against gonococcal organisms or cells carrying the oligosaccharide antigens. This immune response may be immunoprophylactic in nature, since when the recipient comes into contact with the gonococcal organism or cells carrying the oligosaccharide antigen, it will prevent infection.

To identify candidate peptidomimetics, random peptide libraries can be screened for antibody binding specificity. Such screening techniques are well known to those skilled in the art. In one approach, a random peptide library expressed on E.coli flagella can be used to identify peptides that bind to a conserved oligosaccharide epitope of N.gonorrhoeae, which is not present in human blood group antigens. For example, binding to mAb2C7 can be assayed to identify candidate peptidomimetics. Binding can be characterized by Western blotting, flow cytometry or by competition of mAb2C7 for binding to LOS by solid phase ELISA.

Antibody modeling can also be used to determine immunogenic sites in the Complementarity Determining Regions (CDRs) against the unique sites that correspond to the epitope of interest. This analysis can yield information about the three-dimensional conformation of the immunogenic site, which is useful in the design of peptidomimetics for immunogenic sites.

Once a specific peptidomimetic is identified and sequenced, the peptidomimetic can be produced synthetically by methods well known in the art.

To elicit a stronger immune response, the peptidomimetics may also be modified using haptens, using adjuvants, linking the peptidomimetics to carrier proteins, using multiple antigenic peptides, coupling the peptidomimetics to complement proteins, or by other methods well known in the art.

Preferred pharmaceutical compositions of the invention are similar to those used to immunize humans with other peptides. The peptidomimetics of the present invention are typically suspended in a sterile saline solution for therapeutic use. Pharmaceutical compositions may be formulated in other ways to control the release of the active ingredient or to prolong its presence in the patient's system. Many suitable drug delivery systems are known, including, for example: implantable drug delivery systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like.

The pharmaceutical compositions of the invention may be administered by any suitable method, for example, orally, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, or parenterally. Intravenous (i.v.) or parenteral administration is generally preferred.

It will be appreciated by those skilled in the art that an immunoprophylactically effective amount of a peptidomimetic of the invention will depend, inter alia, on the schedule of administration, the unit dose of the peptidomimetic to be administered, whether the peptidomimetic is to be administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the peptidomimetic to be administered, and the judgment of the treating physician.

For a better understanding of the present invention, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

I. Identification of clones encoding peptides that specifically bind mAb2C7

A. Random peptide display

With FliTrx^TMRandom peptide library (Invitrogen, Carlsbad CA) random sequence peptides (12-mers) were expressed on the surface of E.coli. The DNA encoding the peptide library is inserted into a gene encoding the active loop of thioredoxin, which itself is inserted into a non-essential region of the flagellin gene. The expression of the fusion peptide is carried by a vector FliTrx^TMMiddle phage lambda major left promoter (P)_L) And (4) controlling. In this system, P is induced by addition of tryptophan_L. When induced, the fusion protein is exported and assembled as flagella on the bacterial cell surface in preparation for displaying the peptide.

B. Screening for peptides that bind mAb2C7

FliTrx^TMPeptide library (1.77X 10)⁸Primary clone) in IMC medium (0.2% w/v casamino acid, 0.5% w/v glucose, 42mM Na) containing 100. mu.g/ml ampicillin₂HPO₄、22mM KH₂PO₄、8.5mM NaCl、18.7mM NH₄Cl and 1mM MgCl₂) Grow overnight at medium 25 ℃. Expression of the fusion peptide was induced by addition of L-tryptophan to a final concentration of 100. mu.g/ml and the cultures were grown at 25 ℃ for 6 hours. The induced peptide fusion library was then incubated with 2C7 mAb-coated plates (20. mu.g/ml). After 1 hour incubation, the plates were washed 5 times with IMC medium containing 100. mu.g/ml ampicillin and 1% alpha-methyl mannoside. Bound E.coli was eluted by mechanical shearing or by competition with purified LOS prepared from gonococcal strain 15253 (mAb 2C7 epitope is known to be expressed in strain 15253) and then grown overnight at 25 ℃. After the 5 th panning, bound E.coli was eluted and plated on RMG agar (2% w/v casamino acid, 0.5% w/v glucose, 42mM Na) containing 100. mu.g/ml ampicillin at 25 ℃₂HPO₄、22mM KH₂PO₄、8.5mM NaCl、18.7mM NH₄Cl、1mM MgCl₂And 1.5% agar). Individual colonies were selected for binding to mAb2C7 by Western blot assay (a hybridoma cell line secreting mAb2C7 was deposited with the American type culture Collection [ "ATCC ]"]The deposit, assigned ATCC accession number HB-11859).

The library was subjected to 5 rounds of positive selection with mAb2C7 coated on 60mm tissue culture plates, or first negatively selected for 1 hour with irrelevant IgG3(Sigma, st. louis, MO), after which 5 rounds of positive selection were continued with mAb2C 7.

107 colonies were randomly selected and screened for their ability to bind mAb2C7 using Western blotting. 14 clones were identified that bound mAb2C 7. Plasmid DNA was then prepared from the positive clones and sequenced using primers that bind to regions located 5 'and 3' to the nucleotide sequence of the inserted peptide. 7 unique clones were identified as shown in FIGS. 1 and 2 [ SEQ ID NOS: 1-7].

C. Flow cytometry analysis

Positive E.coli clones were grown overnight at 25 ℃ in IMC medium containing 100. mu.g/ml ampicillin, and then induced to express the fusion peptide for 6 hours. Coli cells were fixed with 0.5% paraformaldehyde on ice for 10 min. A200. mu.l aliquot of the immobilized organisms was centrifuged at 2000Xg for 10 minutes. The supernatant was discarded and the pellet was resuspended in blocking buffer containing mAb2C7 (IMC medium containing 100. mu.g/ml ampicillin, 1% skim milk powder, 150mM NaCl and 1% alpha-methyl mannoside). The suspension was incubated at 37 ℃ for 30 minutes, after which it was centrifuged at 2000Xg for 10 minutes. The pellet was washed with 100. mu.l of washing buffer (IMC medium containing 100. mu.g/ml ampicillin and 1% α -methyl mannoside) and then resuspended in 100. mu.l of blocking buffer containing FITC-conjugated anti-mouse IgG (Sigma, St. Louis, Mo.). The mixture was incubated at 37 ℃ for 30 minutes, after which it was centrifuged at 2000Xg for 10 minutes. The supernatant was discarded, and the pellet was washed with 100. mu.l of washing buffer, followed by resuspension in 1ml of PBS. The suspensions were analyzed on FACS using CellQuest software (Becton Dickinson, Franklin Lakes NJ). One negative clone that failed to bind mAb2C7 served as a control.

The binding of e.coli cells to mAb2C7 was observed to be sequentially enhanced (according to median fluorescence intensity, "MFI") [ SEQ ID NOS: 3,4,6,5,2,7,1]. The e.coli clone PEP1 showed the greatest binding to mAb2C7 (MFI 19.81 compared to control MFI 4.91) as shown in figure 3 [ SEQ ID NO: 1].

D. Inhibition of ELISA

Positive E.coli clones were grown overnight at 25 ℃ in IMC medium containing 100. mu.g/ml ampicillin, and then induced to express the fusion peptide for 6 hours. Cultures were normalized to the same OD reading (OD)_600nm0.7), 1% skim milk powder, 150mM NaCl, and 1% α -methyl mannoside were added to block non-specific binding. 50 μ l aliquots of each culture broth were mixed withMu.l mAb2C7 (final concentration 20ng/ml) was incubated at 37 ℃ for 30 minutes, and then 100. mu.l of the mixture was loaded into wells of a microplate coated with purified LOS (80. mu.g/ml) prepared from strain 15253. The wells were incubated at 37 ℃ for 1 hour and then washed. After washing the wells, bound mAb2C7 was detected with anti-mouse IgG coupled to alkaline phosphatase. A negative clone that was unable to bind mAb2C7 served as a control.

Clone PEP1 showed maximum inhibition (66%) of mAb2C7 binding to LOS [ SEQ id no: 1]. PEP7, PEP3, PEP4, PEP2, PEP6, and PEP5 showed reduced binding inhibition, respectively, as shown in fig. 4 [ SEQ ID NOS: 7,3,4,2,6,5]. Inhibition ELISA results correlated with flow cytometric analysis results, as PEP1 also showed maximum binding to mAb2C 7. Binding of E.coli cells to mAb2C7 was roughly correlated with a decrease in the ability of E.coli clones to inhibit mAb2C7 from binding to LOS.

Synthetic peptidomimetics binding to mAb2C7

A synthetic peptide (PEP 1; IPVLDENGLFAP) was synthesized (Boston biologicals, MA) with a sequence corresponding to the consensus sequence "DE _ GLF" and comprising two cysteine flanking regions (CGP-and-GPC residues at the N-and C-termini, respectively) to assess specific binding to 2C7mAb by inhibition ELISA and to determine whether a peptidomimetic characterized as a thioredoxin-fusion protein retains antigenicity independent of the fusion content [ SEQ ID NO: 10].

Cysteine flanking regions were added to assess whether cyclization of the peptidomimetic affected antibody binding. In these peptidomimetics, cysteine residues allow for the formation of disulfide bonds between them, resulting in a circular peptidomimetic. These conformationally constrained peptides may be more similar to the epitope they mimic and therefore may be more immunogenic.

These peptides were diluted in blocking buffer (1% ovalbumin, 0.05% tween 20, 0.5M NaCl in PBS) to generate mixtures of different concentrations (0.1, 0.5, 1 mg/ml). A50. mu.l aliquot of each concentration was incubated with 50. mu.l mAb2C7 (stored at a concentration of 2. mu.g/ml, diluted in blocking buffer) for 1 hour at 37 ℃ and then 100. mu.l of the mixture was loaded into wells of a microplate coated with purified LOS (80. mu.g/ml) prepared from strain 15253. The wells were incubated at 37 ℃ for 1 hour and then washed. After washing the wells, bound mAb2C7 was detected with anti-mouse IgG coupled to alkaline phosphatase. Purified LOS prepared from gonococcus strain 15253 was used as a positive control. The non-reactive 15-mer peptide sequence generated by the random peptide library system described above served as a negative control peptide [ SEQ ID NO: 9].

PEP1 inhibited mAb2C7 binding to LOS in a dose-responsive manner (percent inhibition equal to 17%, 77%, 91% for PEP1 at concentrations of 0.1, 0.5, 1.0 mg/ml), as shown in figure 5. Control 15-mer peptides were synthesized as cyclic peptides (× CKSNPIHIIKNRRNIPC) [ SEQ id no: 9]. This negative control peptide did not inhibit the binding of 2C7mAb to the purified LOS-coated plate.

The cyclic peptidomimetics described above may further comprise one or more "tails" for coupling to a second agent, such as an adjuvant or carrier protein, using methods well known in the art.

Enhancing the immunogenicity of peptidomimetics

Although small peptides may be immunogenic, several studies have reported that some small peptides may lack immunogenicity and elicit an ineffective immune response (particularly a humoral response) (3, 43). Many strategies have been applied to increase the immunogenicity of small peptides. Comprising linking a peptide to a carrier protein (54, 28, 54), combining the peptide with an adjuvant (21, 22), providing a larger configuration of structures (39) that may be more immunogenic using a Multiple Antigen Peptide (MAP), coupling the peptide to a complement protein to enhance the humoral immune response (15).

A. Multiple antigen peptide synthesis

Multiple Antigen Peptide (MAP) is a technique that binds a peptidomimetic to a lysine residue of a dendritic matrix (44, 8, 43). The peptide is attached to the amino group of the lysine framework (scaffold) to create a macromolecule that provides a high density of the desired peptide epitope on the surface of the complex. This approach can enhance the immune response to the peptide (39, 40).

Multiple antigenic peptides of PEP1 and a control peptide (Boston biologicals, MA) were synthesized and binding to mAb2C7 was determined by direct and inhibition ELISA.

Solid phase ELISA was performed to assess the binding of mAb2C7 to the multiple antigen peptide. For direct ELISA, Immulon 1 plates were coated overnight with multiple antigen peptides (1 μ g/well) and reacted with different concentrations of mAb2C 7. For inhibition ELISA, plates were coated with purified LOS (80. mu.g/ml) prepared from strain 15253 of Neisseria gonorrhoeae for 3 hours at 37 ℃. Peptides (linear or MAP) were diluted with blocking buffer (1% ovalbumin, 0.05% tween 20, 0.5M NaCl in PBS) to generate mixtures of different concentrations. A50. mu.l aliquot of each concentration was incubated with 50. mu.l mAb2C7 (stock concentration 0.4. mu.g/ml, diluted in blocking buffer) for 1 hour at 37 ℃ and then 100. mu.l of the mixture was loaded into the wells of the microplate. The wells were incubated at 37 ℃ for 1 hour and then washed. After washing the wells, bound mAb2C7 was detected with anti-mouse IgG coupled to alkaline phosphatase. Purified LOS prepared from gonococcal strain 15253 was used as a positive control in an inhibition ELISA.

Multiple antigenic peptide versions of PEP1 containing either 4 linear PEP1 molecules ("quad MAP 1") or 8 linear PEP1 molecules ("octamap 1") showed strong binding to mAb2C7, while control MAP showed no binding in direct ELISA, as shown in figure 6. Both tetramap 1 and octamap 1 inhibited mAb2C7 binding to LOS more strongly than linear PEP1, as shown in fig. 7. Half maximal Inhibition (IC) of both quad MAP1 and octamap 1₅₀) Observed at 1.26. mu.M and 0.23. mu.M, respectively. IC for Linear PEP1₅₀At 55. mu.M. This is probably due to the increased affinity of MAP1 for binding to mAb2C 7. Control MAP showed no significant inhibition.

Immunization with octamap 1 induced an IgG anti-LOS antibody response in mice, as shown in figure 8. The response pattern is shown in fig. 8(a), where there is no significant IgG anti-LOS antibody response, which is boosted until week 3, indicating that octamap 1 elicits a T cell-dependent immune response in responding mice. These results demonstrate the predictive prognosis of immunising humans against neisseria gonorrhoeae infection with a peptidomimetic (e.g. octamap 1).

In fig. 8(a), 8 mice received a dose of 50 μ g of octamap 1 emulsified in freund's adjuvant on day 0 and again on day 21. Octamap 1 mimicking the epitope of 2C7 oligosaccharide induced IgG anti-LOS antibodies in 3 of 8 mice. IgG anti-LOS responses in these 3 mice were significantly elevated after the first boost at week 3, peaking at week 7 (the next time measured), and then decreasing. FIG. 8(B) shows a positive control experiment in which 4 mice were immunized with purified LOS. In these mice, IgG anti-LOS titers were minimally increased after the first immunization and increased after the boost. 4 mice (all negative controls) immunized with Freund's adjuvant (C) or an unrelated octaMAP control peptide (D) elicited a weak or no IgG anti-LOS response. Figure 9 shows the mean IgG anti-LOS antibody response (mean ± SE, including animals that did not exhibit a response) for all immunized mice (from the experiment shown in figure 8).

FIG. 10 shows only IgG anti-LOS antibody responses in responding mice (from the experiment shown in FIG. 8). Antibody responses were defined as IgG anti-LOS (mean. + -. SE) above 0.4. mu.g/ml (4-fold above baseline IgG anti-LOS levels). Responding mice immunized with octamap 1 developed IgG anti-LOS antibody levels higher (p < 0.001) than those induced by negative control antigen (freund's adjuvant alone or unrelated octamap control peptide) at weeks 7 and 10 after the initial immunization.

FIG. 11 shows only IgM anti-LOS antibody responses in responding mice (from the experiment shown in FIG. 8). Mice immunized with octamap 1 that had developed an IgG anti-LOS response failed to respond to higher IgM anti-LOS levels than mice immunized with the negative control antigen. Immunization with LOS (positive control) resulted in higher IgM anti-LOS antibody levels than animals immunized with octamap 1 or the negative control antigen (freund's adjuvant alone or an unrelated octamap control peptide). Sera from mice immunized with octamap 1 showed 2C 7-specific complement-mediated bactericidal activity against neisseria gonorrhoeae strain 15253, as shown in figure 12. FIG. 12 shows a graph showing the survival of strain 15253 Neisseria gonorrhoeae and its lgG mutant (epitope negative 2C 7) exposed to mouse immune serum (67% volume percent final concentration of mouse immune serum) plus added human complement obtained from normal human donors (17% volume percent final concentration of human complement).

Strain 15253 displays the 2C7 epitope. The 15253 lgg strain contains a disrupted Lipooligosaccharide (LOS) glucosyltransferase G allele, which transfers glucose (via the alpha bond) to heptose-2 in the LOS core (4). Disruption of the lgtG locus results in loss of expression of the 2C7 epitope.

Standard bacterial assays were performed to assess complement-mediated bactericidal activity in mouse sera (11). In this assay, mouse sera (67% final volume) (from different mice immunized or not immunized as described below) were compared to approximately 2.5X 10 suspended in MorseA medium (33) in the presence of human complement (17% final volume)³The bacteria were incubated. The reaction mixture was then shaken continuously at 37 ℃ for 30 minutes. Aliquots of the reaction mixture were inoculated onto chocolate agar plates at time 0 and 30 minutes. Viability was expressed as the percentage increase in colonies on the plate at 30 minutes over 0 minutes. Survival above 100% in the assay indicates growth during 30 min incubation.

mAb2C7 was used as a control because it together with the added complement killed neisseria gonorrhoeae strain 15253 but not the 15253 lgg mutant. As shown in fig. 12(a), mAb2C7 had bactericidal activity against gonococci carrying the 2C7 epitope. 25 μ g/ml mAb2C7 (100 μ l in a total volume of 150 μ l reaction mixture) mediated 100% killing of strain 15253, but did not kill strain 15253 lgG.

Serum from a single mouse immunized with octamap 1 (containing 5.05 μ g/ml IgG anti-LOS antibody, combined with blood taken at weeks 7-11) showed 92% killing (8% survival) of strain 15253, while strain 15253 lgg was all viable, as shown in fig. 12 (C).

Normal mouse serum (average concentration of IgG anti-LOS antibody 0.1. mu.g/ml) representing a collection of 20 mouse sera failed to kill either strain, as shown in FIG. 12 (B). Sera from control mice without complement showed 116.1% ± 4.7% survival (no killing) for strain 15253 and 123.1% ± 3.5% survival (no killing) for the 15253 lgg mutant. Complement sources without antibody showed a survival rate of 137.9% ± 1.0% for strain 15253 (no killing) and 132.5% ± 14.3% for the 15253 lgg mutant (no killing).

Serum from a single mouse immunized with LOS (containing 21.98. mu.g/ml IgG anti-LOS antibody, combined with blood taken at weeks 7-11) did not kill both 15253 strain (179% survival) and 15253 lgG strain (133% survival), as shown in FIG. 12 (D). Sera collected from a single mouse immunized with freund's adjuvant alone or an unrelated octamap control peptide as a negative control antigen did not kill either strain as shown in fig. 12(E) and 12(F), respectively.

IgG anti-LOS antiserum obtained from mice immunized with octamap 1 showed concentration-dependent killing of neisseria gonorrhoeae strain 15253, as shown in figure 13.

FIG. 13 shows a graph of the killing rate of IgG anti-LOS antibody concentration against N.gonorrhoeae strain 15253. When IgG anti-LOS antiserum levels from each of the 3 mice immunized with octamap 1 were plotted against bacterial kill rates, dose-response plots were obtained (mouse sera containing 1.38, 2.50, and 5.05 μ g/ml anti-LOS antibody showed 31%, 74%, and 92% kill rates against strain 15253, respectively). Killing by mAb2C7 was also shown at 5 separate LOS antibody concentrations as a positive control.

B. Coupling of A peptidomimetics with complement protein C3d

It is expected that the immunogenicity of peptoids of gonococcal epitopes (octamap 1 as described herein) could be further enhanced by coupling with complement factor C3 d.

Numerous studies have demonstrated the important role of complement protein C3 in the induction of humoral immune responses (1, 5, 14, 17, 25, 32, 34 and 35). C3 deficient mice show reduced antibody responses to T cell dependent protein antigens such as keyhole limpet hemocyanin ("KLH") (34, 35). Mice deficient in complement receptor 1- (CR1 or CD35) and complement receptor 2- (CR2 or CD21) have reduced T-cell dependent antibody responses (1, 14, 32). It was further shown that C3d covalently linked to hen egg lysozyme ("HEL") elicited an enhanced antibody response to the HEL antigen (15). Mice immunized with a fusion protein consisting of 3 copies of C3d and 1 copy of HEL resulted in a 10000-fold increase in anti-HEL antibody response over mice immunized with HEL alone. The fusion protein induced an anti-HEL antibody response approximately 100-fold higher than that induced by HEL emulsified in freund's adjuvant.

Octamap 1 was coupled to C3d by cloning the octamap 1 DNA sequence into the C3d fusion protein cassette and transforming the expression system with this construct. The octamap 1-C3d fusion protein can then be expressed, purified and used as an immunogen. In addition, the octamap 1-C3d gene fusion can also be used as a DNA vaccine in the form of DNA, according to methods well known in the art.

An example of a hybridoma producing an anti-idiotype antibody that shows similar immunoreactivity to the peptoid of the invention is the cell culture deposited at ATCC (10801 university boulevard, Manassas, va.20110-2209 u.s.a.) on day 3, 26, 1993, assigned ATCC accession No. HB 11311.

An example of hybridoma 2C7 secreting mAb2C7 (which shows similar immunoreactivity as the peptoids of the present invention) is the cell culture designated 2C7 deposited at the ATCC on 3/9 of 1995. The culture was assigned ATCC accession number HB-11859.

While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic structure can be varied to provide other embodiments employing the methods and compositions of this invention. It is, therefore, to be understood that the scope of the invention is to be defined by the appended claims rather than by the specific embodiments described by way of example hereinabove.

Cited documents

Disruption of Ahearn, j.m., m.b. fischer, d.croix, s.goerg, m.ma, j.xia, x.zhou, r.g.howard, t.l.rothstein and m.c.carroll.1996.cr2 loci leads to B-1a cell depletion and a reduction in B cell response to T-dependent antigens. Immunity 4: 251.

apicella, m.a., m.western, s.a.morse, h.schneider, p.a.rice and j.m.griffiss.1986. bacterial antibody response of normal human serum to lipooligosaccharide neisseria gonorrhoeae. J.infect.dis.153: 520-526.

Synthetic vaccines Arnon, r, m.shapira and c.o.jacob.1983. Methods 61: 261-273.

Identification of the gene (IgtG) encoding the lipooligosaccharide beta chain that synthesizes glucosyltransferase in Neisseria gonorrhoeae A., R.Wang, S.N.Uljon, P.A.Rice and E.C.Gotschlich.1998. Proc.natl.acad.sci.usas 95: 10872.

5 Bottger, E.C. and D.bitter-Suermann.1987 modulation of complement and humoral immune responses. Birth 8: 261.

gonococcal infection, Britigan, b.e., m.s.cohen and p.f.sparling.1985: a model of molecular pathogenesis. N.eng.j.med.312: 1683-1694.

Brooks, G.F. and C.J.Lammel, 1989 humoral immune responses to gonococcal infection. Chn.micro.rev.2s: S5-S10.

Filamentous phage display of burrit, j.b., c.w.bond, k.w.doss and a.j.jesiatis.1996. oligopeptide libraries. Anal. biochem.338: 1-13.

CDC/NIH conference of pelvic inflammatory disease: the prevention, management and research directions in the nineties of the twentieth century. Month 9, 1990.

Treatment guidelines for Sexually Transmitted Diseases (STDs) 10.CDC.1982. MMWR.31: suppl.2: S37-S42.

Chromosome-mediated resistance to neisseria gonorrhoeae-usa. MMWR.33: 408-410.

CDC website 2000.http://www.cdc.qov/ncidod/dastlr/crcdir/Resist/crisp.html

Cohen, i.r., d.s.kellogg and l.c.norins.1969. test serum antibody responses in human gonorrhea: immunoglobulin G, A and m.br.j.yen.dis.45: 325-327.

Croix, d.a., j.m. ahearn, a.m. rosengard, s.han, g.kelsoe, m.ma and m.c. carroll.1996. Exp.med.183: 1857.

dempsey, p.w., m.e.d.alison, s.akkaraju, c.c.goodnow and d.t.fearon.1996 complement C3d as molecular adjuvants: linking innate and adaptive immunity. Sciences 271: 348.

specificity of anti-neisseria gonorrhoeae antibodies stimulating neutrophil chemotaxis, Densen, p, s. Clin, invest.80: 78-87.

Fischer, m.b., m.ma, s.goerg, x.zhou, j.xia, x.zhou, r.g.howard, t.l.rothstein, e.kremmer, f.s.rosen, and m.c.carroll.1996. J.immunol.157: 549.

the nature and heterogeneity of neisseria gonorrhoeae antigens involved in bacterial responses, Glynn, a.a. and m.e.ward.1970. Infect.immun.2: 162-168.

Griffiss, h.m., j.p.o' Brien, r.yamasaki, g.d.williams, p.a.rice and h.schneider.1987 the physical heterogeneity of neisserial lipooligosaccharides reflects oligosaccharides that differ in apparent molecular weight, chemical composition and antigen expression. Infect.immun.55: 1792-1800.

Gulati, S., D.P.McQuillen, J.Sharon and P.A.Rice.1996 Experimental immunization with monoclonal anti-idiotypic antibodies mimicking the N.gonorrhoeae lipooligosaccharide epitope 2C 7. J.infect.dis.174: 1238-48.

Gupta, R.K. and G.R.Siber.1995. adjuvants for human vaccines-current, problem and prospect. Vaccine 13: 1263-1276.

Gupta, R.K. and G.R.Siber.1995. methods for quantifying IgG subclass antibodies in mouse sera using enzyme-linked immunosorbent assays. Methods 181: 75-81.

Jerne, n.k.1974. network theory of the immune system. Inst. pasteur. immun.125c: 373-389.

Peptide mimotopes of the carbohydrate antigen, Kieber-Emmons T.1998. Immunol.res.17: 95-108.

Production of memory cells I, Klaus g.g.b. and j.h.humphey.1977. A role for C3 in B memory cell production. Immunology 33: 31.

identification and isolation of novel pili types produced by neisseria gonorrhoeae P9 variants according to in vivo selection, Lambden, p.p., j.e.heckels, h.mcbride and p.j.watt.1981. Fems. microbiol.lett.10: 339-341.

Antibody-antigen specificity in the immune response to neisseria gonorrhoeae infection, Lammel, c.j., r.l.sweet, p.a.rice, j.s.knapp, g.k.schoolnik, d.c.heilbron and g.f.brooks.1985. J.infect.dis.152: 990-1001.

Lowell, g.h., w.r.ballou, l.f.smith, r.a.wirtz, w.d.zollinger and w.t.hockmeyer.1988 proteosome-lipopeptide vaccine: increased immunogenicity of malaria CS peptide. Science 240: 800-802.

Antigens and immunomimics of peptidomimetic positions of Luo P., M.Agadjjanyan, J.Qiu, M.A.Westernink, Z.Steplewski and T.Kieber-Emmons.1998.Lewis carbohydrate antigens. Mol.immunol.35: 865-879.

Andrell, r.e., h.schneider, m.a.apicella, w.d.zollinger, p.a.rice and j.m.griffiss.1986. Infect.immun.54: 63-69.

Complement-mediated bactericidal experiments, McQuilien d.p., s.gulti and p.a.rice.1994. Methods enzymol.236: 137.

strikingly reduced humoral responses in Molina, h., v.m.holers, b.li, y. -f.fang, s.mariathasan, j.goellner, j.strauss-Schoenberger, r.w.karr and d.d.chaplin.1996. complement receptor 1 and 2 deficient mice. Proc.natl.acad.sci.usa 93: 3357.

glucose metabolism in Morse s.a., s.stein and j.hins.1974. J.Bact.120: 702.

the role of complement in allergy induction. Nature [ New Biol ] 273: 157.

the role of complement in inducing antibody production in vivo 35.Pepys, m.b.1974. Exp.med.140: 126.

characterization of gonococcal antigens that produce gonococcal bactericidal antibodies in infection, Rice, p.a. and d.l.kasper.1977. Clin, invest.60: 1149-1158.

Characterization of serum resistance of transmitted neisseria gonorrhoeae, Rice, p.a. and d.l.kasper.1982. Clin, invest.70: 157-167.

In vitro interaction of meningococci from group b with rabbit polymorphonuclear leukocytes in Roberts, r.b.1967.b. Exp.med.126: 795-817.

Several T helper cell epitopes of circumsporozoite protein of Plasmodium grisea (Plasmodium berghei), Romero, p.j., j.p, Tam, d.schlesinger, p.clavjo, p.j.barr, r.s.nussenzweig, v.nussenzweig and f.zavala.1988. Eur.j.immunol.18: 1951-1957.

Schaaper, w.m., Lu, y.a., Tam, j.p., and r.h.meloen.1990, page 765, in: [ peptide: chemistry, structure and biology, Rivier, i.e., and g.r.marshall (eds.). ESCOM Science Publishers, Leiden.

Schoolnik, g.k. and z.a.mcgee.1985. gonococcal vaccine development strategy: the general description of the vaccine group establishment in the national institutes of health, page 329-. At the following stage: schoolnik, g.f.brooks, s.falhow, c.e.frasch, j.s.knapp, j.a.mccutchen and s.a.morse.

Schreiber, j.r., m.patarawan, m.tosi, j.lennon and g.b.pier.1990. anti-idiotype induced lipooligosaccharide specific antibody response to pseudomonas aeruginosa (pseudomonas aeruginosa). J.immun.144: 1023-1029.

Shinnick, T.M., J.G.Sutcliff, N.Green and R.Lerner.1983. synthetic peptide immunogens as vaccines. Annu, rev, microbiol.37: 425-446.

Libraries of peptides and proteins displayed on filamentous phages at Smith, g.p. and j.k.scott.1993. Methods enzymol.217: 228-257.

45.Swanson, J.1982. colony turbidity and protein II composition of gonococci. Infect.immun.37: 359-368.

Tramont, E.C., J.C.Sadoff and M.S.arteminstein.1974. Cross-reactivity of Neisseria gonorrhoeae and Neisseria meningitidis (Neisseria meningitidis), and the nature of the antigen involved in the bactericidal reaction. J.Infect.Dis.130: 240-247.

Anti-gonococcal antibodies in genital secretions, pages 274-278, Tramont, E.C. and J.Ciak.1978: g.f.brooks, e.c.gotschlich, w.d.sawyer and f.e.young (eds.) "immunobiology of neisseria gonorrhoeae" Washington dc.asm.

Tramont, e.c., j.w.boslego, r.chung, d.mccansney, j.ciak, j.sadoff, m.piziak, c.c.brinton, s.wood and j.bryan.1985. parenteral gonococcal pilus vaccine, p 316-. In the following steps: schoolnik, g.f.brooks, s.falhow, c.e.frasch, j.s.knapp, j.a.mccutchen and s.a.morse.

Tramont, e.c.1989 gonococcal vaccine. Clin.Micro.Rev.2S: S74-S77.

Binding activity of all components of single immunoglobulin variable domains secreted by e.coli, Ward, e.s., d.gushow, a.d.griffiths, p.t.jones and g.winter.1989. Nature 341: 544-546.

Surface properties of neisseria gonorrhoeae, Ward, m.e., p.r.lambden, j.e.heckels and p.j.ward.1978: determinants of antibody complement killing sensitivity. Gen.micro.108: 205-212.

Ward, m.m., r.e.ward, j.h.huang and h.kohler.1984. idiotypic vaccines against Streptococcus pneumoniae (Streptococcus pneumonia), pre-studied. J.immunol.139: 2775-2780.

53.Washington, A.E.1982 progress in treatment recommendations for gonococcal infections. Rev. infect. dis.4s: S758-S771.

Peptidomimetics of the meningococcal group C capsular polysaccharide, western, m.a., p.c. giardina, m.a. apicella and t.kieber-emmons.1995. Proc.natl.acad.sci.usa.92: 4021-4025.

The principle of developing synthetic vaccines against malaria Plasmodium falciparum (Plasmodium falciparum) malaria, Zavala, f. Science 228: 1436-1440.

Sequence listing

<110>Rice，Peter

<120> peptidomimetics of conserved gonococcal epitopes and methods and compositions employing them

<130>BOS-3

<140> to be distributed

<141>2000-10-27

<150>60/162491

<151>1999-10-29

<160>10

<170>PatentIn version 3.0

<210>1

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>1

Ile Pro Val Leu Asp Glu Asn Gly Leu Phe Ala Pro

1 5 10

<210>2

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>2

Trp Gly Leu Asp Tyr Glu Arg Gly Asn Tyr Glu Glu

1 5 10

<210>3

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>3

Asp Ala Leu Ala Val Asp Gln Met Gly Arg Phe Gly

1 5 10

<210>4

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>4

Val Leu Val Gly Glu Lys Gly Leu Phe Glu Gly Gly

1 5 10

<210>5

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>5

Glu Ala Leu Val Leu Asp Thr Asn Gly Leu Met Ser

1 5 10

<210>6

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>6

Ala Asp Arg Thr Gln Gly Leu Gly Trp Gly Ala Ser

1 5 10

<210>7

<211>12

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(12)

<223> synthetic construct

<400>7

Glu Glu Val Gly Ser Ile Leu Tyr Gly Leu Gly Gly

1 5 10

<210>8

<211>6

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(3)..(3)

<223> X ═ any amino acid

<220>

<221> peptides

<222>(1)..(6)

<223> synthetic construct

<400>8

Asp Glu Xaa Gly Leu Phe

1 5

<210>9

<211>17

<212>PRT

<213> Artificial/unknown

<220>

<221> peptides

<222>(1)..(17)

<223> synthetic construct

<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> peptides

<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

Claims (18)

1.A peptide mimic of a conserved gonococcal epitope not found on human blood group antigens, wherein said peptide mimic is capable of inducing an immune response in a mammal to said conserved gonococcal epitope, wherein said peptide mimic has an amino acid sequence as set forth in SEQ ID NO: as shown in figure 7, the first and second,

optionally wherein the peptidomimetic is combined with an adjuvant,

the peptidomimetic is linked to a carrier protein,

the peptoid is coupled to a complement protein;

the peptidomimetics are provided as multiple antigenic peptides, or

The peptidomimetic is further linked to at least one tail for coupling to a second agent.

2. The peptidomimetic according to claim 1, wherein the immune response is T cell dependent.

3. The peptide mimic according to claim 1, wherein the peptide mimic is further linked to at least one tail for coupling to a second agent.

4. The peptide mimic according to claim 3, wherein the second agent is an adjuvant.

5. The peptide mimic according to claim 1 or 2, wherein the peptide mimic is combined with an adjuvant or linked to a carrier protein.

6. The peptide mimic according to claim 1 or 2, wherein the peptide mimic is part of a multiple antigen peptide.

7. The peptide mimic according to claim 1 or 2, wherein the peptide mimic competes with gonococcal LOS for binding to monoclonal antibody 2C7 produced by the hybridoma deposited with accession number HB-11859.

8. The peptide mimic according to claim 1, wherein the peptide mimic binds to monoclonal antibody 2C7 produced by the hybridoma deposited as accession number HB-11859.

9. The peptide mimic according to claim 1, wherein the peptide mimic binds to a monoclonal antibody, or fragment thereof, produced by immunizing an animal with an anti-idiotype monoclonal antibody, wherein said monoclonal antibody is produced by the hybridoma cell line having ATCC accession No. HB 11311.

10. The peptide mimic according to claim 1, wherein the peptide mimic is part of a multiple antigen peptide.

11. The peptide mimic according to claim 1, wherein the peptide mimic is coupled to a complement protein.

12. The peptide mimic according to claim 11, wherein the peptide mimic is coupled to complement protein C3 d.

13. A composition for immunization against neisseria gonorrhoeae infection comprising an immunoprophylactically effective amount of a peptidomimetic according to any one of claims 1 to 12.

14. A composition for immunizing against neisseria gonorrhoeae infection comprising an immunoprophylactically effective amount of a peptidomimetic consisting of the amino acid sequence of SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

15. Use of the peptidomimetic of claim 1 for the preparation of a medicament for immunizing a mammal against neisseria gonorrhoeae infection.

16. Use of the peptidomimetic of claim 12 for the preparation of a medicament for immunizing a mammal against neisseria gonorrhoeae infection.

17. A method of increasing the antigenicity of a peptidomimetic according to claim 1 comprising the step of coupling the peptidomimetic to a complement protein.

18. The method according to claim 17, wherein the complement protein is C3 d.