Diagnosis and Treatment of Infections due to Streptococci and Enterococci

This invention concerns the diagnosis, prophylaxis and treatment of infections due to Streptococci and Enterococci, especially endocarditis and septicaemia.

Endocarditis is commonly caused by Streptococcal and Enterococcal infection. These are bacterial species which grow in the heart valves of an infected patient and cause damage thereto. Endocarditis is currently diagnosed by clinical features, echocardiogram and the presence of heart murmurs. The causative microorganism is usually identified by blood culture (culture-positive endocarditis). However, in approximately 10% of infective endocarditis patients the blood culture is negative. This may lead to a wrong diagnosis and/or delayed treatment.

Active infective endocarditis in which blood cultures are negative has been a recognised clinical entity since the beginning of the century. Etiological factors involved in such culture-negative endocarditis include (1) previous antibiotic therapy, (2) fastidious, slow growing bacteria and (3) non-bacterial organisms.

Patients with rheumatic fever, damaged heart valves or prosthetic valves are at risk of a secondary Streptococcal infection leading to endocarditis when having routine dental or gastrointestinal procedures.

Additionally, a growing problem in recent years has been the spread of

vancomycin-resistnt enterococci (VRE). The emergence of enterococci resistant to most or all licensed antibiotics leaves few treatment options and recent studies have shown that 36.6% of those patients with VRE in blood died as compared with 16.4% of those with vancomycin sensitive enterococci. Clearly a treatment for enterococci of all descriptions, and especially of VRE is extremely desirable.

One method of diagnosing endocarditis and, more particularly, culture-negative endocarditis, is by immunoblotting sera of such patients to reveal raised levels of antibodies against causative microorganisms and also a pattern on immunoblot which appears to be species specific (Clark & Burnie, J.Clin.Pathol. 1991, 4 , 152-156; Burnie et al, J. Clin. Pathol, 1987, 4Q: 1149-1158; Burnie & Clark, J. Immunol. Methods, 1989, 123: 217-225). However, although individual species of both Streptococcus and Enterococcus can be identified, there is some cross- reaction between species making absolute diagnosis sometimes difficult. Immunoblotting is also cumbersome as well as expensive.

Current therapy of both culture-positive and culture-negative endocarditis involves antibiotics, however, some of the antibiotics necessary to treat endocarditis are highly toxic, for example, vancomycin and gentamicin may be nephrotoxic and ototoxic. Additionally, it is difficult to assess patient response to antibiotic treatment since, although the organism may no longer be viable, fever may persist.

The present invention provides a method of diagnosis and treatment ("treatment" from herein being taken to include prophylaxis) of infections due to Streptocci and Enterococci which is specific to both culture-positive and culture-negative

endocarditis, thereby overcoming at least to some extent, the aforesaid problems.

Experiments were undertaken which resulted in the cloning, sequencing and characterisation of Streptococcal antigens, resulting in the determination of an antigen expressed during infection due to Streptococci and Enterococci, together with immunogenic fragments thereof which were found to be useful in the prophylaxis and treatment of infections due to both Streptococci and Enterococci.

According to the present invention there is provided a purified bacterial protein expressed during infection due to streptococci or enterococci and isolated from human sera having at least the sequence of formula (1):

NH- 10 30 50

EFTFYDENDQ PINFDNALLS VASLNREHNS IEMAKDYSGT FIKISGSSIG EKNGMIYATE

70 90 110

TLNFKQGQGG ARWTMYPNRQ PGSGWDSSDA PNSWYGAGAI S SGPTNHVT VGATSATNVM

130 150 170

SVAEMPQVPG RDNTEGKRPN I YSLNGKIR AVDVPKITKE KPTPPVAPTE PQAPTYEVEK

190 210 230

PLEPAPVAPS YENEPTPPVK TPDQPEPSKP EEPTYETEKP LEPAPVAPNY ENEPTPPVKT

250 270 290

PDQPDPSKPE EPNYETEKPL EPAPVAPSYE NEPTPPVKTP DQPEPSKPEE PNYDPLPTPP

310 330 350

LAPTPKQLPT PPAVPTVHFH YNRLFAQPQI NKEIKNEDGV DIDRTLVAKQ SWKFELKTE

370 390 410

ALTAGRPKTT SFVLVDPLPT GYQFDLEATK AASKGFETSY DKASHTVTFK ATEET AAFN

430 450 470

ADLTKSFETL YPTWGRVLN DGATY NNFT LTVNDATGVK SNIVRVTTPG KPNDPDNPNN

490 510 530

NYIKPLKVNK NKQGVNIDGK EVLAGSTNYY ELTWDLDQYK GDKSSKEAIQ NGFYYVDDYP

550 570 590

EEALTLQPEL VKIRDLEGNL VSGISVQQFD SLERAPKKVQ DLLKKANITV KGAFQLFSAD

610 NPAEF

or an immunogenic fragment thereof or an analogue thereof.

The single letters in formula (1) are each to be understood to represent a separate amino acid, and each is the conventional single letter symbol used for amino acids.

Bacterial protiens expressed during infection due to Streptococci or Enterococci according to the invention include those proteins wherein one or more amino acids in the sequence of formula (1) is replaced by another amino acid, providing that overall functionality of the protein is conserved.

A bacterial protein according to the invention may be further characterised by either one or both of the following features:-

(1) It is an immunodominant conserved antigen; and

(2) Recombinant human antibody in an animal model (mouse) protected against septicaemia infection;

A bacterial protein according to the invention may also be characterised in that it is involved in binding to heart valves.

The bacterial protein may be obtained from one of the group of Streptococcus oralis. Streptococcus sobrinus. Streptococcus gordonii. Streptoccocus sanguis. Streptococcus mutans. Streptococcus mitis. Streptococcus mitior. Streptococcus parasanguis. Streptococcus bovis. Enterococcus faecalis and Enterococcus faecium.

Additionally, the bacterial protein may be obtained from either one of the group of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium.

Particular fragments of a bacterial protein expressed during infection due to Streptococci or Enterococci include any peptide epitopes ("immunogenic fragments"), for example, a few amino acids or analogues thereof. Examples of such epitopes include YEVEKPLEPAPVAPS, SYENEPTPPVKTPD, KTPDQPEPSKPEEPT, EPAPVAPSYENEPTP, YEVEKELVDLPVEPS, KTPDQNIPDKPVEPT,

TMYPNRQPGSGWDSS and WYSLNGKIRAVDVPK. Peptides of this type may be synthesised using conventional liquid or solid phase peptide synthesis techniques.

In a further aspect the invention particularly provides a recombinant bacterial protein expressed during infection due to Streptococci or Enterococci having an amino acid sequence which includes at least the sequence of formula (1) or an immunogenic fragment thereof, or an analogue thereof.

As mentioned earlier, current treatment of endocarditis is by antibiotics and recovery is often difficult to assess as fever may persist after other symptoms have been relieved. Tests currently available to measure the efficiency of antibiotic treatment, for example, minimum inhibitory concentration, minimum bactericidal concentrations and back titrations, measure only organism sensitivity not actual organism death in the patient. We show for the first time, as detailed below, that in patients with endocarditis due to Streptococcus oralis. S. gordonii. S. sanguis and S. mitis and undergoing antibiotic therapy, resolution of the disease was accompanied by at least a 50% drop in IgM titre within two weeks. This provides a direct marker of successful antibiotic therapy showing for the first time a direct marker of pathogen kill. Since the IgM antibody is specific to endocarditis it can be used in the diagnosis of both culture-positive and, more particularly, culture-negative endocarditis.

Thus the present invention also provides a method of diagnosis of culture-positive and culture-negative endocarditis using IgM antibody to a bacterial protein expressed during infection due to Streptococci or Enterococci, said bacterial protein, and therefore IgM antibody raised thereto, falling by 50% within two weeks after commencement of antibiotic treatment, thereby acting as a marker of pathogen kill.

In another use the bacterial protein according to the present invention may be employed, using conventional techniques, for screening to obtain activity inhibiting agents for use in the prophylaxis and treatment due to Streptococci or Enterococci and in particular of culture-positive and culture-negative endocarditis-causing bacterial infection. Such screening methods forms a further aspect of the invention.

In a further use, the bacterial protein according to the invention is particularly well suited for the generation of antibodies. Thus according to a further aspect of the invention we provide a bacterial protein expressed during infection due to Streptococci or Enterococci having an amino acid sequence which includes at least the sequence of formula (1) or an immunogenic fragment thereof or an analogue thereof, for use as an immunogen.

Standard immunological techniques may be employed with the bacterial protein in order to use it as an immunogen. Thus, for example, any suitable host may be injected with the protein and the serum collected to yield the desired polyclonal anti-bacterial protein antibody after purification and/or concentration. Prior to injection of the host the bacterial protein may be formulated in a suitable vehicle and thus according to a further aspect of the invention we provide a composition comprising a bacterial protein expressed during infection due to Streptococci or Enterococci and having an amino acid sequence which includes at least the sequence of formula (1) or an analogue thereof together with a pharmaceutically acceptable carrier, diluent or excipient.

For purification of any anti-bacterial protein antibody, use may be made of affinity chromatography employing an immobilised bacterial protein of the present invention as the affinity medium. Thus according to another aspect of the invention we provide a bacterial protein expressed during infection due to Streptococci or Enterococci having an amino acid sequence which includes at least the sequence of formula (1), or an immunogenic fragment thereof or an analogue thereof, covalently bound to an insoluble support.

Various derivatives of the bacterial protein or fragment or analogue may be used to inhibit said protein, fragment or analogue. The use of the bacterial protein expressed during infection according to the invention as immunogens for the production of antibodies generates one type of inhibitor of the action of the protein. Generally, inhibitors of the bacterial protein are potentially useful in the diagnosis, and in particular the prevention and treatment, of infections due to Streptococci or Enterococci and in particular of both culture-positive and culture-negative endocarditis, and provide a further feature of the invention. Inhibitors include any antagonists of the action of the bacterial protein expressed during infection or agents which prevent their production, and in particular those which may be used in treatment of endocarditis-causing bacterial infections. Suitable inhibitors include, for example, pharmaceutical reagents, including antibodies, and chemical analogues of the bacterial protein expressed during infection to antagonise the action of the bacterial protein, and anti-sense RNA and DNA to prevent production of the bacterial protein. Suitable inhibitors may be determined using appropriate screens, for example, by measuring the ability of a potential inhibitor to antagonise the action of, or prevent the production of a bacterial protein expressed during infection due to Streptococci or Enterococci according to the invention or an immunogenic fragment thereof, or an analogue thereof, in a test model for example an animal model such as the mouse model.

It will also be appreciated that by suitable epitope mapping using conventional procedures[Geysen et al, J. Immunol. Methods, 102: 259-274 (1987); Hopp and Woods, PNAS USA, 7£(6): 3824-3828 (1981); Novotny et al, PNAS USA, 83: 226-230 (1986)], peptide fragments of the bacterial protein expressed during infection may be identified which can be chemically synthesised. Synthetic peptide

antigens of this type may be used to produce inhibitors e.g. to raise antibodies for use in diagnosis and/or therapy, as previously described, or to produce antiseras, e.g. non-specific polyclonal antisera, for use as a vaccine, and as discussed above form a further aspect of the invention.

According to a further aspect of the invention we provide a derivative of a bacterial protein expressed during infection due to Streptococci or Enterococci, said protein having an amino acid sequence which includes at least the sequence of formula (1) or an immunogenic fragment thereof or an analogue thereof, wherein the derivative inhibits said protein, fragment or analogue.

Such inhibitors may be used either alone or where appropriate in combination with other pharmaceutical agents, for example, other antibiotics.

One particularly useful group of inhibitors according to this aspect of the invention are antibodies capable of recognising and binding to the bacterial proteins.

Thus according to yet another aspect of the invention we provide isolated and purified antibody specific for one or more epitopes of a bacterial protein expressed during infection due to Streptococci or Enterococci having an amino acid sequence which includes at least the sequence of formula (1) or an immunogenic fragment thereof or an analogue thereof.

The antibody may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it

may be an immunoglobulin M antibody or, in particular, an immunoglobulin G antibody. The antibody or fragment may be of animal, for example, mammalian origin and may be for example of murine, rat or human origin. It may be a natural antibody or a fragment thereof, or, if desired, a recombinant antibody fragment, ie., an antibody or antibody fragment which has been produced using recombinant DNA techniques.

Particular recombinant antibodies or antibody fragments include human recombinant antibodies and in particular include (1) those having an antigen binding site at least part of which is derived from a different antibody, for example those in which the hypervariable or complementarity determining regions of one antibody have been grafted into the variable framework regions of a second, different antibody (as described in European Patent Specification No 239400); (2) recombinant antibodies or fragments wherein non-Fv sequences have been substituted by non-Fv sequences from other, different antibodies (as described in European Patent Specification Nos 171469, 173494 and 194276); or (3) recombinant antibodies or fragments possessing substantially the structure of a natural immunoglobulin but wherein the hinge region has a different number of cysteine residues from that found in the natural immunoglobulin but wherein one or more cysteine residues in a surface pocket of the recombinant antibody or fragment is in the place of another amino acid residue present in the natural immunoglobulin (as described in International Patent Application Nos PCT/GB88/00730 and PCT/GB88/00729).

The antibody or antibody fragment may be of polyclonal, or preferably, monoclonal origin. It may be specific for a single epitope or for a number of epitopes associated with the bacterial protein.

Antigen binding antibody fragments include, for example, fragments derived by proteolytic cleavage of a whole antibody, such as F(ab')2,Fab' or Fab fragments, or fragments obtained by recombinant DNA techniques, for example Fv fragments (as described in International Patent Application No PCT/GB88/0747).

The antibodies according to the invention may be prepared using well-known immunological techniques employing the bacterial protein expressed during infection as antigen. Thus, for example, any suitable host may be injected with the bacterial protein and the serum collected to yield the desired polyclonal antibody after appropriate purification and/or concentration (for example by affinity chromatography using the immobilised bacterial protein as the affinity medium). Alternatively, splenocytes or lymphocytes may be recovered from the bacterial protein-injected host and immortalised using for example the method of Kohler et al. , Eur. J. Immunol. ^: 511, 1976, the resulting cells being segregated to obtain a single genetic line producing monoclonal anti-streptococcal or -enterococcal bacterial protein antibodies. Antibody fragments may be produced using conventional techniques, for example, by enzymatic digestion with pepsin or papain. Where it is desired to produce recombinant antibodies according to the invention these may be produced using for example the methods described in European Patent Specification Nos 171469, 173494, 194276 and 239400.

Antibodies according to the invention may be labelled with a detectable label or may be conjugated with effector molecule for example a drug eg. an antibacterial agent or a toxin or an enzyme, using conventional procedures and the invention extends to such labelled antibodies or antibody conjugates.

The antibodies according to the invention have a diagnostic and/or preventative and/or therapeutic use. Thus for diagnostic use the antibodies may be employed to detect whether the bacterial protein is present in a host organism, to confirm whether the host has a particular Streptococcal or Enterococcal bacterial infection, and especially to test for the presence of such organisms in culture-negative endocarditis, and/or to monitor the progress of therapeutic treatment of such infections. Diagnostic methods of this type form a further aspect of the invention and may generally employ standard techniques, for example, immunological methods such as enzyme-linked immunosorbent methods, radioimmuno methods, latex agglutination methods or immunoblotting methods.

Antibodies according to the invention also have a therapeutic use in the treatment of bacterial infection due to Streptococci or Enterococci, for example, those just described and may be used alone or conjugated to an effector molecule, in the latter case to target the effector molecule, eg an antibacterial agent, to the infecting organism. For therapeutic use the antibody may be formulated in accordance with conventional procedures, for example, with a pharmaceutically acceptable carrier or excipient, eg., isotonic saline for the administration at an appropriate dosage, depending on the nature of the infection to be treated and the age of the patient.

If desired, mixtures of antibodies may be used for diagnosis and/or prevention and/or treatment, for example mixtures of two or more antibodies recognising different epitopes of a bacterial protein according to the invention, and/or mixtures of antibodies of a different class, eg., mixtures of IgG and IgM antibodies recognising the same or different epitope(s) of a bacterial protein of the invention.

The protein or any fragment, analogue, inhibitor, antibody or antigen binding fragment thereof according to the invention may be used in a method if treatment or diagnosis of the human or animal body.

The diagnostic test method may be selected from one of the group of enzyme-linked immunosorbent assay, radioimmunoassay, latex agglutination assay and immunoblot assay.

Such a protein, fragment, analogue, inhibitor, antibody or antigen binding fragment may form part of a composition for use in a method of diagnosis or treatment of the human or animal body together with a pharmaceutically acceptable carrier, diluent or excipient.

The bacterial proteins according to the invention may be prepared by a variety of processes, for example, by protein fractionation from appropriate bacterial cell extracts, using conventional separation techniques such as ion exchange and gel chromatography and electrophoresis, or by the use of recombinant DNA techniques, as more particularly described in the "Experiments" section hereinafter. The use of recombinant DNA techniques is particularly suitable for preparing substantially pure bacterial proteins according to the invention.

Thus according to a further aspect of the invention we provide a process for the production of a bacterial protein expressed during infection due to Streptococci or Enterococci having an amino acid sequence which includes at least the sequence of

formula (1) or a fragment or an analogue thereof, comprising the steps of (1) culturing a host organism transformed with a vector including a gene coding for a precursor of said protein and (2) recovering said protein.

Preferably the precursor cleaved in this aspect of the invention is a fusion protein comprising at least a portion of a protein produced in a transformed host organism and at least the amino acid sequence of formula (1). Such fusion proteins form a further aspect of the invention. Desirably the fusion protein includes a protein produced at a high level by a transformed host organism. Suitable such proteins include at least a portion of a chloramphenicol acetyltransferase (CAT) protein or, preferably at least a portion of the B-galactosidase protein.

According to a still further aspect of the invention we provide a DNA sequence coding for a bacterial protein or an immunogenic fragment or an analogue thereof expressed during infection due to Streptococci or Enterococci having substantially the nucleotide sequence of formula (2):

5" 10 30 50

GAATTCACCT TCTACGATGA AAATGACCAA CCAATTAATT TTGACAATGC TCTTCTTTCA

70 90 110

GTAGCCTCAC TTAACCGTGA GCATAACTCT ATTGAGATGG CTAAGGATTA TAGTGGTACT

130 150 170

TTTATTAAAA TCTCAGGTTC ATCCATCGGT GAAAAAAATG GCATGATTTA TGCCACAGAA

190 210 230

ACCCTGAACT TTAAACAAGG ACAGGGTGGA GCTCGCTGGA CAATGTATCC AAATCGTCAG

250 270 290

CCAGGTTCAG GTTGGGATTC ATCAGATGCA CCAAACTCTT GGTACGGTGC AGGGGCCATT

310 330 350

AGTATGTCCG GTCCTACGAA TCACGTTACA GTTGGTGCAA CATCTGCTAC CAATGTGATG

370 390 410

TCCGTAGCAG AAATGCCTCA AGTACCTGGA AGAGACAATA CTGAAGGTAA AAGACCAAAC

430 450 470

ATCTGGTACT CACTCAATGG TAAAATTCGT GCGGTTGACG TTCCGAAAAT TACAAAAGAA

490 510 530

AAACCAACTC CACCGGTAGC ACCAACTGAA CCACAAGCTC CTACCTATGA AGTGGAGAAA

550 570 590

CCACTGGAAC CGGCTCCAGT AGCACCAAGC TACGAAAATG AGCCAACTCC ACCAGTAAAA

610 630 650

ACTCCAGATC AACCGGAGCC ATCAAAACCA GAAGAGCCAA CATATGAGAC AGAGAAACCA

670 690 710

TTGGAACCAG CTCCAGTAGC ACCAAACTAC GAAAATGAGC CAACTCCACC AGTAAAAACT

730 750 770

CCAGATCAAC CAGACCCATC AAAACCGGAA GAGCCAAACT ATGAGACAGA GAAACCATTG

790 810 830

GAACCAGCTC CAGTAGCACC AAGCTATGAA AATGAGCCAA CTCCACCGGT AAAAACTCCA

850 870 890

GATCAACCAG AGCCATCAAA ACCAGAAGAG CCAAATTATG ATCCATTGCC AACTCCGCCG

910 930 950

CTAGCACCAA CTCCTAAGCA GTTGCCAACA CCACCAGCGG TGCCAACAGT TCACTTCCAT

970 990 1010

TACAATCGTC TATTTGCACA ACCTCAGATT AATAAAGAAA TTAAAAACGA GGATGGAGTA

1030 1050 1070

GATATTGATC GTACTCTAGT TGCTAAGCAG TCTGTAGTGA AGTTTGAGCT GAAAACAGAA

1090 1110 1130

GCTTTAACTG CTGGTCGTCC AAAAACAACT TCGTTTGTAT TGGTAGATCC ACTTCCAACT

1150 1170 1190

GGCTATCAGT TTGATTTGGA AGCAACCAAG GCTGCAAGCA AAGGTTTTGA AACAAGCTAT

1210 1230 1250

GACAAAGCTA GTCACACTGT AACCTTTAAG GCTACTGAGG AGACCTTAGC TGCTTTCAAT

1270 1290 1310

GCTGATTTGA CAAAATCCTT TGAGACTCTA TATCCAACTG TTGTTGGTCG TGTCTTGAAT

1330 1350 1370

GATGGGGCGA CTTATACGAA TAACTTTACA TTGACAGTCA ACGATGCTAC TGGTGTCAAG

1390 1410 1430

TCAAACATTG TTCGTGTAAC GACTCCAGGT AAACCAAATG ATCCTGACAA TCCAAATAAC

1450 1470 1490

AACTACATCA AGCCTTTGAA AGTTAACAAG AACAAGCAAG GTGTGAATAT TGATGGCAAA

1510 1530 1550

GAAGTTCTAG CTGGTTCAAC GAACTACTAT GAACTCACAT GGGATTTGGA TCAATACAAG

1570 1590 1610

GGAGATAAAT CTTCTAAAGA AGCGATTCAA AATGGTTTCT ACTATGTGGA TGATTATCCA

1630 1650 1670

GAAGAAGCTT TAACGCTTCA ACCAGAATTG GTTAAGATTC GTGATCTAGA GGGCAACCTT

1690 1710 1730

GTATCAGGTA TCAGTGTTCA ACAGTTTGAT AGTTTAGAAC GTGCGCCTAA GAAGGTTCAA

1750 1770 1790

GATCTGTTGA AGAAAGCAAA CATCACTGTT AAAGGTGCTT TCCAACTCTT CTCAGCTGAT

1810 AATCCAGCTG AATTC

and homologues thereof.

A DNA sequence according to the invention may be further characterised in that the bacterial protein for which it encodes may be characterised by either one or

both of the following features:-

(1) It is an immunodominant conserved antigen; and

(2) Recombinant human antibody in an animal model (mouse) protected against septicaemia infection;

A DNA sequence may also be further characterised in that the protein for which it encodes is involved in binding to heart valves.

DNA with this sequence may be obtained from bacterial genomic DNA as described in the "Experiments" section hereinafter.

The DNA sequence according to this aspect of the invention may be incorporated in an expression vector using conventional techniques. Thus in a further aspect of the invention we provide an expression vector including substantially a DNA sequence of formula (2) or a homologue thereof.

The vector may be adapted for use in a given host cell by the provision of suitable selectable markers, promoters and other control regions as appropriate. Host cells transformed with such vectors form a further aspect of the invention. Suitable host organisms include bacteria (eg. E.colD. and mammalian cells in tissue culture.

The DNA sequence of formula (2) may also be used to design DNA probes for use in identifying the presence of Streptococcal and Enterococcal bacteria in the infected state and the invention extends to such DNA probes. Such probes may also be of use for detecting circulating bacterial nucleic acids, for example using a polymerase

chain reaction, as a method of diagnosing such bacterial infections. The probe may also be synthesised using conventional techniques and may be immobilised on a solid phase, or may be labelled with a detectable label.

A DNA sequence, vector, probe or inhibitor according to the invention may be used in a method of treatment or diagnosis of the human or animal body.

It is known that Streptococci and Enterococci cause endocarditis by binding to heart valves and causing damage thereto. A possible mode of action of Streptococci in binding to heart valve tissue, or prosthetic valves, has been proposed to involve pre-binding of Steptococci to extracellular matrix proteins, such as fibronectin, which leads to the adherence and colonisation of bacteria to damaged valvular surfaces (Lowrance et al, J. Clin. Invest., 1990, £& 7-13). This suggests that fibronectin acts as a tissue receptor for the bacteria. However, we show for the first time that the prior art theory on the role of fibronectin in endocarditis is incorrect. In our studies, described below, antibody against fibronectin binds to PAc and is neutralised by prior crossabsorption with fibronectin indicating that PAc acts like fibronectin binding to damaged heart valves directly, and not via fibronectin. This molecular mimicry is the probable mode of action of PAc.

Thus the invention also provides fibronectin or an immunogenic fragment thereof or an analogue thereof or an antibody thereto or an antigen binding fragment thereof for use in a method of treatment or diagnosis of the human or animal body for infection due to Streptococci or Enterococci.

Additionally, as described below in the "Experiments" section, we have shown for the first time that serum from patients with endorcarditis due to S.oralis. S.gordonii and S.sanguis infection have antigen of approximately 85 kDa which reacts with a mouse monoclonal antibody specific to heat shock 90 molecules. We have also shown that in a mouse S. oralis infection model with death as an end point, antibody specific to HSP90 shows a statistically significant increase in survival. This shows that this antigen is in the HSP 90 group. Thus antibodies against HSP 90 may be used for the diagnosis and treatment of infections caused by S.oralis. S.gordonii and S.sanguis.

Thus the present invention provides antibodies specific to HSP 90 or immunogenic fragments or analogues thereof for use in a method of diagnosis or treatment of the human or animal body of infection due to streptococci or enterococci due to any one of the group of S.oralis. S.gordonii and S.sanguis.

The invention will be further apparant from the following "Experiments" section which exemplifies the invention.

Experiments

1. Characterisation of the Antibody Response

Sera was available from the following cases:

12 cases of septicaemia due to Streptococcus oralis.

14 cases of endocarditis due to Streptococcus gordonii.

2 cases of endocarditis due to Streptococcus oralis.

2 cases of endocarditis due to Streptococcus sanguis.

20 control sera from patients having no clinical evidence of endocarditis who were non-neutropenic, and

20 control sera from patients having no clinical evidence of endocarditis who were neutropenic.

2. Species Identification

Using the above sera, the causative organisms were identified, when available, according to the scheme of Beighton et al, J. Med. Microbiol, 1991, 35: 367-372. Specifically, isolates from 6 of the cases of S.oralis were available and identified as S.oralis by JM Hardie (personal communication). The others were aesculin- negative and raffinose-negative -viridans streptococci eliminating S.gordinii (100% aesculin positive), S. mitis (100% raffmose positive) and making it unlikely to be S.sanguis (75% aesculin positive and 75% raffmose positive). In the 14 cases of S.gordonii and 2 cases of S^sanguis endocarditis, isolates from 3 were positively identified as S.gordonii. All the other isolates were unobtainable but the original API

Streptococcal profile showed they were aesculin positive. This would eliminate S. mitis and make it unlikely that they were S.oralis (18% aesculin positive). They were then subdivided into either S.gordonii or S.sanguis according to whether the more dominant IgM response on immunoblot was against S.gordonii or S.sanguis. The cases of S.oralis endocarditis were identified according to Beighton et al 1991.

3. Source of Strains for Immunoblotting

Using the protocol outlined by Burnie et al, J. Clin. Pathol., 1987, 40: 1149-1158, antigenic extracts were made from S.sanguis NCTC 7863, S.oralis NCTC 7864 and S.gordinii NCTC 7868.

4. Immunolblotting

Immunoblotting using the above strains was then carried out as described in Burnie et al, 1987.

The results of immumoblots of the patients sera described above is summarised in Table 1 (S.oralis). Table 2 (S.gordonii) and Table 3 (S.sanguis) and in Figures 1 to 5.

Figure 1 shows the immunoblot for S.oralis septicaemias. Paired IgMs and paired IgGs covering pre and post sera from 3 of the cases showing the antibody changes.

Figure 2 shows the immunoblot of S.gordonii endocarditis. Anti-fibronectin antibody

against S.gordonii NCTC 7868 in Track 1, crossabsorbed against fibronectin in Track 2. Tracks 3-8 show IgM and IgG respectively in 3 cases of S.gordonii endocarditis.

Figure 3 shows 5 further case ofS.gordonii endocarditis versus S.gordonii antigen Tracks 1/2, 3/4, 5/6, 9/10 and 11/12. Tracks 7/8 show pre-treatment of antibody levels of the same case as tracks 9/10 showing increased IgM to bands at approximately 85 KDa and IgG to bands at approximately 180, 58, 56 and 52 Kda.

Figure 4 shows the immunoblot of S.oralis endocarditis, Tracks 3/4 IgM and IgG, antifibronectin antibody crossreacting with 180 KDa band of S.oralis. Track 1 and effect of crossabsorption with fibronectin, Track 2.

Figure 5 shows the immunoblot of S.sanguis endocarditis, showing both IgM (track 3 and 5) and IgG (tracks 4 and 6) respectively of both cases, antifibronectin antibody crossreacting with the 120 KDa band of S.sanguis. Track 1 and the effect of crossabsorption with fibronectin, Track 2.

Comments

(a) S.oralis septaceamia

IgM and/or IgG against band at approximately 180 KDa when recovering from infection.

(b) S.gordonii endocarditis

Additional IgG was detected against bands at approximately 185 (1 case), 165 (2 cases), 155 (1 case), 140 (1 case), 132 (1 case), 110 (1 case), 94 ( 1 case), 61 (1

case), 50 (1 case) and 45 KDa (1 case). Additional IgM was detected against the 45 KDa band (1 case). The majority of cases has IgM and all IgG against the bands at 85 and 180 KDa from S.gordonii NCTC 7868. Other immunodominant bands included those at approximately 65 and 47 KDa.

(c) S.oralis endocarditis case 1: Had additional antibody as follows. IgM against bands at approximately 180, 140 and 65 KDa and IgG agaist bands at approximately 180, 140, 120, 58, 51, 46 and 35 KDa. Both cases had IgM and IgG against the bands at 85 and 180 KDa from S.oralis NCTC 7864.

(d) S.sanguis endocarditis

Both patients produced antibody (IgM and IgG) against bands at approximately 120 and 85 KDa from S.sanguis NCTC 7863.

(e) S .sobrinus/S .mutans endocarditis

Immunoblotting demonstrated that patients with endocarditis had antibody (IgM and IgG) against three bands at approximately 185, 200 and 220 KDa.

Additionally the bands at 85 KDa (S.oralis NCTC 7864, S.gordonii NCTC 7868 and S.sanguis NCTC 7863) all react with a mouse monoclonal specific to heat shock 90 molecules (versus LKVIRK, see our previous patent application Nos WO 92/01717, WO 91/00351 and GB 2270076) showing that this molecule is in the HSP 90 group (Figure 6), Track 1 £-J2 tis NCTC 7864, Track 2 S.gordonii NCTC 7868, Track 3 S.sanguis NCTC 7863, Track 4 clinical isolate of S.oralis.

5. Cloning of the immunodominant antigen of S.sobrinus and S.oralis

5.1 S.sobrinus cloning

(i) DNA isolation and lambda ZAPII library preparation.

Strain S.sobrinus Manchester University Collection of Bacteria No.263 (MUCOB 263), was used as the source of S.sobrinus. Initially, the MUCOB 263 organism was identified as an S. mutans. a fact reflected in the aboved mentioned publication. However, a more detailed testing of theis strain revealed that MUCOB 263 is biochemically more similar to S.sobrinus (Professor D. Bratthall, personal communication). To avoid future confusion, the organism has now been renamed S.sobrinus MUCOB 263. Bacterial cells were grown overnight at 37°C, with shaking, in brain-heart infusion broth containing 0.2% glucose and 40mM D,L-threonine (Sigma). The bacteria were harvested by spinning at 5,000 rpm for 10 mins before being washed, and resuspended in 12.5ml of 0.02 M Tris pH 8.2. Then 25 ml of 20 M polyethylene glycol (PEG) 24% in distilled water was added and mixed. Lysozyme was added at 34.6 mg/ml equivalent to lOOμg/unit obtained by measuring the OD600 of a tenfold dilution of bacterial cultures. After 1 hour at 37°C, the spheroblasts formed were spun down at 5,000rpm for 10 mins and thoroughly resuspended in 50 ml of 10 mM Tris-Cl pH 8.0, 1 mM EDTA (TE). The spheroblasts were lysed with 5.5 ml of 10% sodium dodecyl sulphate (SDS) at 60 °C for 15 mins. The DNA was purified by standard procedures (Maniatis et al, Molecular Cloning - A laboratory manual, 2nd edn. Cold Spring Harbour, New York, 1989), RNAase A (sigma) treated (after preboiling the RNAase at 100°C for 15 mins), proteinase K treated, phenol:chloroform:isoamylalcohol (25:24:1) extracted, and ethanol precipitated. Plasmid DNA was removed by ethanol precipitation in the presence of 0.3 M ammonium acetate pH 5.2. Further purification was obtained

by dialysis against TE over 36 hrs at 4°C.

The DNA was mechanically sheared, EcoRI linkers added, fractionated and inserts ligated into lambda ZAPII vector arms. The library had an insert size range of 2kb-7kb.

(ii) Antibody Screening

Serum was taken from a patient with endocarditis and used for antibody screening. Escherichia coli XL1 - Blue cells were infected at approximately 3,000 pfu/85mm on L broth agar (Bacto-tryptone 10 g/1, yeast extract 5 g/1, sodium chloride 10 g/1, maltose 2 g/1, bacto-agar 15 g/1). Plaques were transferred to nitrocellulose filters (0.45 μm pore size, Sartorius AG, Gottingen, Germany), impregnated with 10 mM isopropyl B-D-thiogalactopyranoside (IPTG), at 37°C for 2 hrs, after a 42 °C incubation for 3 hrs. These filters were blocked overnight at 4°C with 3% bovine serum albumin (BSA - Sigma) in buffered saline (150 mM NaCl, 10 mM Tris). Patient serum, diluted one hundredfold in 3% BSA, was added to the filters and incubated at room temperature for 2 hrs, the filters then being washed for 30 mins in washing solution (150 mM NaCl, Tween 20, 0.05%), before the second antibody, a thousandfold dilution of anti-human IgG alkaline phosphatase conjugate (Sigma) in BSA 3% was added. After 1 hr at room temperature, the filters were again washed and stained with equal volumes of naphthol ASMX phosphate (Sigma, 0.4 mg/ml in distilled water) and Fast Red TR salt (Sigma, 6 mg/ml in 0.2 M Tris pH 8.2) (the Fast Red stain). Positive plaques were transformed to 1.5 ml tubes containing 200 μ ϊf SM (100 mM sodium chloride, 50 mM Tris-Cl pH 7.5, 10 mM magnesium sulphate, gelatine, 0.0001%), and 2 to 3 drops of chloroform. Plaque

purification was performed by the above method.

(iii) Antigen-directed antibody selection

Two 100 μl aliquots of resuspended E.coli XL 1 -Blue cells were infected with a dilution of a high titre stock of purified positive phage, to give 5,000 pfu / 85 mm L broth agar plate. Phage adsorption occured at 37°C for 30 mins. The adsorption mixes were added to 2.5 ml aliquots of L broth agarose 0.8%, mixed, and poured onto L broth agar plates. These plates were incubated at 42 °C for 2.5 hrs, before nitrocellulose filters (0.45 μm pore size, Sartorius), presoaked in 10 mM IPTG and dried at room temperature for at least 1 hr, were added and incubated at 37 °C overnight. Each filter was then washed 3 times over 30 mins in Tris-buffered saline (150 mM sodium chloride, lOmM Tris-Cl pH 7.2), and blocked overnight at 4°C in 3% BSA. A tenfold dilution of patient serum in BSA 3% was added to the filters and incubated, with shaking, at room temperature for 3 hrs. This serum was removed, called the depleted serum and stored at 4°C. The filters were washed 5 times over 100 mins with Tween 20-Tris-buffered saline (150 mM sodium chloride, 10 mM Tris-Cl pH 7.2, Tween 20, 0.05%), and once with salt - Tween 20 solution (150 mM sodium chloride, Tween 20, 0.05%). The bound antibody was eluted by adding 5 ml of glycine saline buffer (150 mM sodium chloride, 200 mM glycine-Cl pH 2.8), and shaking at room temperature for 30 mins. The buffer was aspirated into a bijoux, containing Tris 0.04g per 100 μl, mixed, termed the eluted antibody and stored at 4°C. A Western blot of S.sobrinus MUCOB 263 was performed essentially by the immunoblot method above with the original serum, depleted serum and eluted antibody being used as the primary antibodies. The antigen was the supernatant of the strain following crushing in an X-press. The secondary antibody was a

thousandfold dilution of anti-human IgM and IgG alkaline phosphatase conjugates in 3% BSA, and the stain was the alkaline phosphatase stain.

Results

Six antibody positive clones were isolated and purified from the 60,000 pfu initially screened with culture-positive endocarditis patient serum. Antigen-directed antibody selection showed that the cloned, expressed sequence contained epitopes shared by S.sobrinus as the eluted antibody bound to the approximately 185 KDa antigen.

(iv) In vivo excision of DNA insert

A 10 ml overnight culture ofE.coli XLl-Blue cells grown at 37°C in L broth, containing tetracycline 125 μg/μl, was spun down at 2,000 rpm for 10 mins, resuspended in 4 ml of 10 mM magnesium sulphate, and stored at 4°C. In a 50 ml plastic conical tube, 200μl of the resuspended E.coli XLl-Blue cells, 180μl of high titre antibody-positive phage (1.78 x 108/85 mm plate approx) and 1 μl of R408 helper phage were combined and incubated at 37°C for 15 mins. (Helper phage and E.coli XLl-Blue cells from CLONTECH). Then, 5 ml of 2 x YT media (sodium chloride 5 g/1, yeast extract 10 g/1, bacto-tryptone 16 g/1 pH 7.0) was added to the dual-infected bacteria and incubated, with shaking, at 37 °C for 3 hrs. The cells were killed by heating at 70 °C for 20 mins and ruptured by vortexing for 5 mins. The cells were spun at 4,000 rpm for 5 mins, and the supernatant containing pBluescript SK(-) phagemid decanted and stored at 4°C. The phagemid was propagated by adding 10 μl of the phagemid solution to 200 μl of resuspended E.coli XLl-Blue cells, incubating the culture at 37 °C for 15 mins, and

plating 1 μl, 25 μl, 75 μl and 100 μl aliquots onto L broth agar plates, containing ampicillin 50 μg/ml.

(v) Plasmid DNA preparation and denaturation

The pBluescript SK(-) plasmid DNA was purified from the bacterial colonies with the Magic (RTM) Minipreps DNA purification System (Promega) and alkaline denatured by the method of Maniatis et al, 1989. Hence, 8 μl of solution, containing DNA 1.5 - 200 μg was added to 2 μl of 2 M sodium hydroxide, briefly vortexed and spun, and left at room temperature for 10 mins. Then 3 μl of 3 M sodium acetate (pH 4.8), 7 μl of distilled water, and 60 μl of -20°C absolute ethanol were added and the DNA precipitated at - 70°C for 30 mins. The precipitate was pelleted at 13,000 rpm for 1 min, washed with -20 °C ethanol 70%, and vacuum-dried. The dried pellets were stored at -20 °C.

(vi) DNA sequencing

DNA sequencing was performed by the two-step chain-termination method, with Sequenase (R) Version 2.0 (Cambridge Bioscience). The annealing step was carried out at 66 °C for 2 mins, the labelling at 22 °C for 4 mins, and the termination at 38°C for 5 mins. Areas containing secondary structure were resolved by the substitution of dITP for dGTG. Sequencing reactions were run on acrylamide gels 6% for 11, 8, 5 and 2 hrs at 46-51 °C.

(vii) Subcloning with the TA cloning (RTM) system

The phagemid clone did not carry the 5' end of the cloned gene, therefore it was decided to subclone the 5' end by Polymerase Chain Reaction (PCR), followed by TA cloning (RTM). The following PCR reagents were added to each of the 2 tubes containing 0.0 μg, 0.7 μg of S.sobrinus MUCOB 263 genomic DNA, with distilled water to give 64.3 μl total volume; 5.3 μl or 0.1 μg/μl Primer 1 (CAGTCTCCGTCCCAACGACTGCG), 4.4 μl of 0.1 μg/μl Primer 2 (GCTCCTCTTGTGACATGGTC), 10 μl of 10 x Taq buffer (Northumbria Biochemicals Ltd., Northumberland (nbl), and 16 μl of dNTP's (12.5 μl of each of dATP, dGTP, dCTP, dTTP (Promega), added to 950 μl of deionized water). Then, 60 μl of mineral oil was carefully layered on top of the PCR reactions, the DNA denatured at 95 °C for 10 mins, and 2.5 Weiss Units of Taq (nbl) added. The PCR conditions were 94 °C for 1.5 mins, 50°C for 1.5 mins, and 72°C for 3 mins for 30 cycles, before a final long extension of 72°C for 10 mins. The PCR products (30 μl) were run on a TBE gel 0.8%, at 50 volts for 2 hours, alongisde 4 μl of EcoRl - Hind III cut lambda DNA markers (nbl).

Insert DNA was subcloned with the TA Cloning (RTM) System Version 1.3 (InVitrogen Corporation, British Biotechnologies Ltd, Oxon) as specified in the manufacturers protocol. Overnight, 12 °C, ligation reactions containing 3.0 μl of a fourfold dilution, in distilled water, of the 0.7 μg PCR reaction, 4.7 μl of doubling dilution of the 0.7 μg PCR reaction, and a negative control were performed. In the transformation, white recombinant colonies were picked after overnight 37°C incubation, and twice replated on kanamycin (50μg/ml)/ 5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside (X-Gal-25 μl of 40 mg/ml solution/plate) to achieve pure and stable subclones. Plasmid DNA was-obtained, purified, denatured and sequenced. The origin of the cloned DNA from S.sobrinus was confirmed by Southern Blotting. The full

sequence obtained in shown in Table 4.

5.2 S.oralis cloning

This was done essentially by the methods described above. The differences were that the vector was lambda gtl 1 and the DNA source was a clinical isolate of Streptococcus oralis. The library was a genomic DNA library partially digested with EcoRl . Ten positive clones were identified and one of these subcloned by the TA Cloning (RTM) System. The origin of the clone from S.oralis was confirmed by Southern Blotting. Antigen directed antibody selection showed that it had epitopes which cross-reacted with the 180 KDa antigen of S.oralis (Figure 7). Tracks 1 and 3 show the original serum from two patients with antibody against S.oralis. and tracks 2 and 4 show the eluted subcomponent binding to the band at 180kDa. The sequence of the clone is given in Table 5.

Comparison of the Sequences

The sequence obtained from S.sobrinus had a 99.2% homology to the SpaA antigen of S.sobrinus and a 68.4% homology to the PAc antigen of S. mutans. The S.oralis peptide carried a 76.2% homology over 605 amino acids with the S. mutans PAc protein precursor and 73.8% homology over 606 amino acids with both the S.sobrinus SpaA protein precursor and the S. mutans surface antigen I/II precursor. Both sequences contain a three tandem repeat motif of 39 amino acids. Six peptides were derived from these and used to epitope map-this communal sera.

Epitope Mapping

Epitope mapping of the antigen cloned from S. oralis was carried out according to the protocol outlined by Geysen et al, J. Immunol. Methods, 102: 259-274 (1987) and references therein. In this epitope mapping, a complete set of overlapping nonapeptides was synthesised for the cloned S. oralis antigen. Peptide 1 covered residues 1-9, peptide 2 covered residues 2-10 and peptide 3 covered residues 3-11 etc. The epitopes were tested against various patient sera. All of the sera were examined at a 1 in 200 dilution for IgG and recording was stopped after 30 minutes. The sera examined were:

(1) Viridans endocarditis (n=8) (S. sanguis n=2, S. oralis n=2, S. gordonii n=4)

(2) S. oralis septicaemia n=5

(3) S. mutans endocarditis (n=2)

(4) E. faecalis endocarditis (n=2)

(5) Negative control (n=5)

Epitopes were defined as those peptides which:

(1) were positive in at least 3 wells

(2) had an optical density of each well which was at least double the negative control value and in the majority of wells greater than 0.8

This epitope mapping identified a total of 9 epitopes (see table 10) - NFKQGQG, RQPG, SWYGAG, GKIRAV, RLFAQPQ, AGRPK, PTGYQFD, YPTW and LLKKA.

After the initial epitope mapping, each individual serum was then reexamined for positivity with each well (see table 11).

6. Preparation of Immunodominant Epitopes from Cloned S.oralis

Of the synthetic peptides (see 5.2; "Comparison of the Sequences"), peptides 1-4 cover the sequence of S.oralis as short peptides of 15 amino acid length. Peptides 5 and 6 covered the S.sobrinus area which differed. These sequences are listed (Table 6) and illustrated (Figure 8).

The six synthetic peptides were produced by Cambridge Biochemicals Ltd., Nantwich and dissolved in a double-distilled water to give a final concentration of 2 mg ml. Aliquots, 0.5 ml, were stored at -70 °C and after one freeze-thaw cycle, stored at -20 ^β C.

The wells of six plastic microtitre plates (Falcon (R) 3912, Microtest (RTM) Flexible Assay Plate, 96 flat-bottom wells - Becton Dickinson & Co, Fred Baker, Liverpool), one for each peptide, were coated with 200 μl of the appropriate 10 μg/ml synthetic peptide solution diluted in phosphate buffered saline (one tablet dissolved in 100 ml distilled water, Oxoid, Unipath Ltd, Basingstoke; PBS). The peptides were added and left at 4°C overnight to coat the wells. Excess peptide was then removed by washing the microtitre plates five times in PBS with a Nunc-Immuno Wash (RTM) (InterMed, Denmark). The primary antibody, 200 μl of a 1/10 dilution in 3% BSA of sera was added and left at room temperature for 1 hour to allow antigen-antibody binding. After washing five times in PBS, the wells were filled with 200 μl of one of

i) a 1/1000 dilution of goat anti-human IgM peroxidase conjugate in 3%

BSA (Sigma), ii) a 1/1000 dilution of goat anti-human IgG peroxidase conjugate in 3%

BSA (Sigma), or

Hi) with PBS.

The conjugates were left to react at room temperature for 1 hour. The plates were then washed as before and 200 μl of ABTS stain was added to each well (three tablets of 2.2' am o-bis(3-ethylben--tfιiazoline-6- sulfonic acid); diammonium salt; Sigma) and 160 μl of 30% w/w hydrogen peroxide solution; Sigma, in 60 ml of 125 mM disodium hydrogen orthophosphate buffer brought to pH 4.0 with 1M citric acid). This ABTS stain was prepared just prior to use. The signal strength for each well was then read at 405 nm at 5, 10 and 15 mins after the start of the reaction with a microtitre plate reader (Titertek Muliskan (10) PLUS MKH, Labsystems, Finland) attached to an impact dot matrix printer (Panasonic KX-P1081, Panasonic Matsushita Electric Industrial Co Ltd, Osaka, Japan).

To determine which of the six peptides representing the S .sobrinus/S .oralis antigenic region which produced the most specific and strongest positive signal, each of the peptides was used in an indirect ELISA.

All peptides were screened against a panel of 5 sera (single sera from cases of S. mutans endocarditis, S.oralis endocarditis, S.oralis septicaemia, S.lactis endocarditis and S.aureus endorcarditis).

All 6 peptides were recognised by the sera from the cases of endocarditis due to S.mutans and S.oralis. They had much lower Optical Densities with the sera from the septicaemia due to S.oralis and the control sera from cases of endocarditis due to S.lactis and S.aureus. Peptide 1 showed the highest OD (IgG) in S.mutans endocarditis and was selected for further studies (Table 7).

Indirect ELISA

As a result of the epitope mapping and of the preparation of immunodominant epitopes from clones S. oralis (see above), three peptides were investigated further in indirect ELISA tests. These peptides were YEVEKPLEPAPVAP (Peptide 1), TMYPNRQPGSGWDSS (contains the epitope RQPG - epitope numbers 74- 79; Peptide 7) and WYSLNGKIRAVDVPK (contains the epitope GKIRAV - epitope numbers 144-147; Peptide 8).

Sera Tested

Sera from cases of endocarditis due to S.mutans (2), S.oralis (3), S.gordinii (10), S.sanguis (2), E.faecalis fi n. S.bovis (8), S.agalactiae (1), S.lactis (1), S.pneunoniae (4), Group G Streptococcus (2), S.aureus (2), Coagulase Negative Staphylococci (6), Candida albicans (1), Candida parapsilosis (2) and E.coli (1), septicaemias due to S.oralis (8), E.faecalis (7), E.faecium (2) and a brain abscess due to S.milleri. Further controls were sera from patients with SLE (3) and neutropenic leukaemic patients with no evidence of streptococcal infection (20).

The optical densities are given in Table 8 for Peptide 1, and in Table 8a for Peptides 1, 2 and 3.

Results

In summary, a raised IgG was specific to patients with Steptococcal endocarditis and a raised IgM specific to endocarditis due to Streptococcus oralis/gordonii/ sanguis/mitis.

If a cut off point of 0.6 is taken for the Optical Densitivity for IgG and 0.4 for IgM then all cases of S.mutans (patient 1), S.oralis (patient 4), S.gordonii (patients 6-14) and S.sanguis (patients 16 and 17) endocarditis fulfilled one or other of these criteria. All other sera were classified as negative. All controls were negative except for a raised IgG in cases of endocarditis due to E.faecalis. S.bovis and Group G streptococci (Table 9).

This data proves the value of the test in culture positive endocarditis. It could also be extended to cover culture negative cases.

One treatment of IgM fell in cases 6, 13 and 15 showing that a falling IgM was a marker of successful therapy.

An analysis of the overall results (see tables 12 and 13 below) showed that the tests performed using peptide 1 gave the most accurate and specific results (100% specificity), but were the least sensitive (50% sensitivity). Tests wth the other two

peptides were more sensitive, but less specific and gave more false positives. This suggests that although each of the peptides could be usefully used individually, a combination of tests using more than one peptide would allow for an overall test which was both highly sensitive and highly specific.

Table 12

Peptide 1 Peptide 7 Peptide 8

IgM IgG IgM IgG IgM IgG

True Positives 6 5 12 5 12 10

False Positives 0 1 28 15 25 21

True Negatives 42 41 14 27 17 21

False Negatives 6 7 0 7 0 2

(For raw data see table 8a)

Table 13

IgM Peptides IgG Peptides

1 7 8 1 7 8

Sensitivity 50% 100% 100% 41.5% 41.5% 83%

Specificity 100% 33% 40% 98% 64% 50%

Efficiency 89% 48% 53% 85% 59% 57%

7. Fibronectin Binding Studies _^

In order to test for fibronectin binding activity in the cloned proteins, each

of the peptides 1-6 was reacted in an indirect ELISA according to the previous protocol.

The indirect ELISA produced OD values greater than 2 for all six peptides regardless of whether fibronectin was added to the sandwich. This implied that the polyclonal serum (anti-fibronectin 0.5 mg/ml Sigma [F1509]) reacted with the peptides directly. This serum was then immunoblotted at a dilution of 1 in 40 and again after crossabsorbing, 100 μl of 1 mg/ml antibody, with 100 μl of 1 mg/ml of fibronectin (F2006 Sigma) at 37 °C for 30 mins. This showed that the antiserum reacted specifically with the 180 kDa antigen of S.oralis. 180 kDa antigen ofS.gordonii and 120 kDa antigen of S.sanguis. This is illustrated by Figure 2 tracks 1 and 2 (S.gordonii). Figure 4 tracks 1 and 2 (S.oralis) and Figure 5 tracks 1 and 2 (S.sanguis). Figure 9 shows the antibody crossreacting with the 185 kDa antigen of S.sobrinus (Track 1) and the 180 KDa antigen of S.oralis (clinical isolate) (Track 3) and the elimination of the crossreactivity by prior absoroption with fibronectin (Tracks 2 and 4).

This is with the previously identified immunodominant antigens of these microorganisms and implies that they act as a πiimic for fibronectin. This appears to be the mechanism by which these streptococci bind to heart valves. ■ A comparison of the sequences of the peptides with human fibronectin showed subtantial homology (GCG pogramme Gap; Sequence of fibronectin by Kornblitt et al. EMBO J. 4: 1755-1759, 1985). The most homologous region of the fibronectin molecule was a 69 residue sequence beginning at residue 750 of the fibronectin protein. The percentage similarity and identity values were 48.7% and 35.9% respectively (Figure 10).

8. Human Recombinant Antibodies

A library of immunoglobulin heavy and light chain variable (V) genes was prepared from the peripheral blood lymphocytes of a patient with infection due to a viridans streptococcus (blood culture positive) septicaemia following an oesophagectomy who recovered on amoxicillin and gentamicin. Screening of this library by immunoblot against S.oralis NCTC 7864 showed recombinant antibody against the band at 180 KDa (Figure 11, tracks 5 and 6). The pre and post IgM (Tracks 1 and 2) and IgG (Tracks 3 and 4) from a patient recovering from septicaemia due to S.oralis is shown for comparison.

The library was produced essentially as described by Marks et al (J. Mol. Biol., 1991, 222: 581-597) using the pCANTAB 5 vector, which is now commercially available as part of a kit from Pharmacia (Milton Keynes, UK). The heavy and light chain V genes, obtained from cDNA prepared from the mRNA of peripheral blood lymphocytes of a patient recovering from a viridans streptococcus (blood culture positive) septicaemia, were randomly combined and subcloned into Not I/Sfi I digested pCANTAB 5. The resulting single chain Fv fragments (ScFv), expressed on the surface of phage, were enriched by panning four times against the specific synthetic peptide epitopes YEVEKPLEPAPVAPS, TMYPNRQPGSGWDSS and WYSLNGKIRAVDVPK (Peptides 1, 7 and 8). Following the fourth panning, twenty clones (from each of the last pannings) were:

1. Bstl fingerprinted to establish the degree of focussing of the panning procedure; and

2. Examined in an indirect ELISA against the original peptide (as described

in detail previously). Conditions were:-

Recombinant antibody: neat

Phage specific monoclonal : 1 in 2,000

Antimouse Horse Radish Peroxidase 1 in 1,000

100ml of Peptide applied to well in PBS at lOug/ml and incubated overnight at 4°C.

Results

Peptide 1

20 clones selected were identical on Bstl fingerprinting. Indirect ELISA varied from 0.25-0.30

(control 0.17). Two clones (PAC 1 and PAC 2 ) selected for animal work.

Peptide 7

16 clones produced 6 types. One type (Type A) was present in 7 out of the 16 clones and was the only type to produce a positive reading in the indirect ELISA (0.276-0.318) (control 0.17). One clone (Clone 3) selected for animal work (PAC 3).

Peptide 8

16 clones produced 10 types. Two of these, A and B were represented by 3 and 5 clones respectively. None of these clones produced a positive ELISA result. Five of the 8 clones produced a positive ELISA result (range 0.235-0.304 control 0.138) and a unique Bstl fingerprint. One of these (Clone 7) was selected for animal work (PAC 4).

In order to test the efficacy of the selected clones specific to Peptides 1-3 in treating S. oralis and vancomycin-resistant £. faecium infections, a set of experiments with PAC 1-4 and other controls was performed.

Experiment 1

This experiment was an acute streptococcal infection model using Balb/c mice with an end point of death. Conditions were:

S.oralis dose: 5.7 x IO ⁹

Phage dose: 5 x 10* pfu ml

Antibody given first, followed by S.oralis 2 hours later.

Table 14 (results of Experiment 1)

SURVIVORS

Antibody No. of mice 4 hours 24 hours 48 hours

Control: no antibody 15 7 3 1 M13K07 phage

DEPAGE 13 3 3 2

B3.7 13 6 6 6

B3.14 8 1 1 1

PAC 1 9 9 2 2

PAC 2 17 8 8 8

PAC 3 15 6 6 6

PAC 4 15 2 2 2

DEPAGE - antibody against a Candidal specific carboxy-end HSP90 antigen (sequence DEPAGE) which acts as an irrelevant phage and therefore a control. Fisher exact 2 tailed P value showed statistical significance for B3.7 at 48 hours (P 0.03), PAC 1 at 2 hours (P 0.0095) and PAC 2 at 48 hours (P 0.02). B3.7 is a recombinant

antibody specific to the HSP90 stress protein, suggesting that antibodies specific to the HSP90 protein may be used to diagnose and treat S. oralis and possibly other streptococcal and enterococcal infections.

Experiment 2

This was a chronic streptococcal infection model using CDl mice and a colony count of spleen and kidney was performed at the various end-points. Conditions were:

S.oralis dose 2.5 x IO ⁹

Phage dose 5 x 10 ¹⁰ pfu/ml

S.oralis given first, antibody given 24 hours later.

Positive organ count > 10 ⁴ /g/ml

S = Spleen, K = Kidney

S + K = combined spleen and kidney results

5 mice sacrificed on days 4, 7 and 12

Repeat antibody injection at day 10.

Table 15 (results of Experiment 2)

Day 4 Day 7 Day 12

S K S+K S K S+K S K S+K

Controhno antibody 4 4 8 0 0 0 1 0 1 Ml 3K07 phage

B3.14 1 2 3 0 0 0 0 0 0

PAC 2 0 1 1 1 1 2 1 0 1

PAC 3 1 2 3 0 0 0 0 0 0

No phage recovered after 24 hours post injection, all mice blood cultures negative.

Fisher exact 2 tailed P value showed statistical significance for PAC 2 (P = 0.005) when spleen and kidney results were combined from day 4.

Experiment 3

This was an infection model with CDl mice using high level vancomycin-resistant

E. faecium. Conditions were:

E. faecium dose: 3 x 10 ^10/ ml

Phage dose: 5 x 10 ¹⁰ pfu/ml

E. faecium given first, antibody given 24 hours later.

Positive > 10 ⁴ g/ml. S = spleen, K = Kidney

S + K = Spleen + Kidney.

Spontaneous deaths cultured at day 2

5 mice sacrificed on day 4

Remaining mice 3 for the M13K07 control and 5 for the PAC 2 antibody, sacrificed at day 7.

Table 16 (results of Experiment 3)

Day 2 Day 4 Day 7

(spontaneous (n=5) (n=3 control) deaths) (n=5 PAC 2)

S K S+K S K S+K

Control no antibody: 6 ^a 2 5 7 0 0 0 M13K07 phage

PAC 2 4 ^a 2 3 5 0 0 0

⁸ all spleens and kidneys cultured and positive > IO ⁴ /gl/ml Total positive Control : 19/22

PAC : 13/18

This produced by combing the spleen and kidney results obtained -rom the spontaneous deaths (day 2) with 5 mice (day 4) sacrificed assuming > IO ⁴ organisms /g/ml is positive. This suggests PAC2 may have some activity agianst vancomycin-resisitant Efaecium.

Experiment 4

This was an acute streptococcal infection model using Balb/c mice infected with vancomycin-resistant E. faecium. The end-point was death. Coonditions were:

Organism: high level (>256 mg/1) vancomycin-resistant E. faecium

E. faecium dose: 5 x 10 ¹⁰ /ml

Phage dose: 5 x 10 ¹⁰ pfu /ml

Antibody given first, followed by Efaecium 2 hours later.

Table 17 (results of experiment 4)

Survivors

Antibody No of mice 4hours 24hours 48hours

Control no antibody 15 10 5 1 M13K07 phage

PAC2 15 12 8 6

Fisher exact 2 tailed P value showed statistical significance for Pac 2 at 48 hours (p 0.02)

Table 1 Immunoblot testing of the S.oralis NCTC 7864 antigen against patient sera.

M.Wt. of Patient Sera S.oralis _ antigen Endocarditis Sequential sera Controls band S.oralis-. septicaemia

n = 12

S.oralis S.gordonii S.sanguis Constant Inc. x 2 Controls Controls Endo¬ Endo¬ Endo¬ Septicaemia or App. Neutropenic Non- carditis carditis carditis Neutropenic n = 2 n = 4 n = 2 n = 20 n = 20

IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG

192 1 1 2 10 1 2 1 3 1 5 4 6 2 1

185 1 4 1 1 1 1 3 2 3 1 2

180 2 2 4 13 1 2 2 9 10 1 3 4

155 1 9 1 4 2

140 1 2 2 6 3 8 1 2 2 8 1 2

105 1 1 5 1 1 2 2 1 1

85 2 2 4 11 1 2 8 1 1 3 3 1 1

76 1 1 3 1 1 5 2 1 2 2

65 1 3 1

51 1 6 3 1 11 2 1 11 6

46 2 3 1 1 1

35 1 1 1

Inc = Increased x 2 App = Appeared

Table 2 Immunoblot testing of the S.gordonii NCTC 7868 antigen against patient sera.

M.WI t. of ] Patient Sera S.ora Us antigc ;n Endocarditis Sequential sera Controls band S.oralis. septicaemia n = 12

S.oralis S.gordonii S.sangius Constant Inc. x 2 Controls Controls Endo¬ Endo¬ Endo¬ Septicaemia or App. Neutropenic Non- carditis carditis carditis Neutropenic n = 2 n = 4 n = 2 n = 20 n = 20

IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG

185 2 2 5 1 7

180 2 12 14 2 2 6 5 1 2

105 1 1 2 4 1 3 1

85 2 1 12 14 1 1 1 10 1 1

80 5 12 1 1 1 9 1 11 4

76 5 7 1 1 1 10 1 3

72 1 1 5 7 1 1 1 4 1 1

70 1 4 5 1 2 7

65 9 13 1 2 1 4 1 1

58 1 9

56 2 9 1

52 1 8 2

47 2 12 14 1 1 10 2 1 15 2 2

40 5 8 2 1 8 1 1

37 8 8 2 2

35 3 10 1 1 2 1

33 2 9

30 1 11 2 2

28 2 7 ^~

Inc = Increased App = Appeared

Table 3 Immunoblot testing of the S.sanguis NCTC 7863 antigen against patient sera.

M.Wt. of Patient Sera S.sanguis_ antigen band

Endocarditis Septicaemia

S. oralis S.gordonii S.sanguis S. oralis Controls Controls Endo¬ Endo¬ Endo¬ Septicaemia Neutropenic Non- carditis carditis carditis Neutropenic n = 2 n = 14 n = 2 n =9 n =20 n = 20

IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG

120 2 4 14 2 2 1 3 3 4

85 1 2 2 2 8 3 3 2 4

83 2 2

81 2 8 2 2

75 1 1 4 2 2 2 5 3 15 8

59 1 1 2 1 2 2

57 1 2 2 12 1 2 2

55 1 1 2 6 2 12

50 9 2

47 2 2 2 9 2 6

42 1 6 2 2 9

40 2 2 9 2

37 3 2 2

35 1

32

25 6 2

Table 4 Full sequence of cloned S.sobrinus gene

5' 10 30 50

GCTCCTCTTG TGACATGGTC ATAGTAACAG ATAATCTGTT TAATTTCAAG CAGATTTAAT

70 90 110

AGCCTCCAGG AAACTTGAAA.TAAAACTGAA ATAAAACTGA ATTTTTTATA AAGCCTAGAT

130 150 170

TAAGCAATCG TTTGCATTGA CAATCACTAG ATAAGTGTTA TTATAGATAG TATTGTAACG

190 210 230

AAACATTTCA GATGTTACAA AAATGTAAAT TGGAGGGAAT TATAATATGC AACGAAAAGA

M Q R K E

250 270 290

GACTTTTGGG TTTCGCAAAA GTAAAATCAG TAGGACCCTT TGTGGTGCCT TACTAGGAAC T F G F R K S K I S R T L C G A L L G T

310 330 350

TGCTATCTTA GCGTCTGTAA CAGGTCAAAA GGCGCTCGCT GAAGAAACAA GTACCACTTC A I L A S V T G Q K A L A E E T S T T S

370 390 410

AACTTCGGGG GTTAATACCG CAGTCGTTGG GACGGAGACT GGGAATCCCG CCACCAACCT T S G V N T A V V G T E T G N P A T N L

430 450 470

GCCTGACAAA CAGGACAATC CAAGTTCGCA AGCCGAGACA AGTCAGGCCC AAGCCGGTCA P D K Q D N P S S Q A E T S Q A Q A G Q

490 510 530

AAAGACAGGG GCAATGTCAG TAGATGTGTC TACAAGTGAG CTTGACGAAG CTGCTAAAAG K T G A M S V D V S T S E L D E A A K S

550 570 590

TGCCCAAGAA GCTGGTGTGA CCGTTTCGCA GGATGCTACC GTCGATAAAG GGACAGTAGA A Q E A G V T V S Q D A T V D K G T V E

610 630 650

AACTTCTGAC GAAGCTAACC AAAAAGAAAC CGAAATCAAG GATGACTACA GCAAGCAAGC T S D E A N Q K E T E I K D D Y S K Q A

670 690 710

AGCAGACATC CAAAAGACAA CAGAAGACTA CAAGGCAGCT GTGGCTCGTA ACCAAGCCGA A D I Q K T T E D Y K A A V A R N Q A E

730 750 770

AACAGACCGA ATCACTCAAG AAAACGCGGC TAAGAAGGCC CAATACGAAC AAGATTTGGC T D R I T Q E N A A K K A Q Y E Q D L A

790 810 830

GGCCAACAAG GCAGAAGTGG AACGCATTAC CAATGAGAAT GCGCAACGCA AGGCTGATTA A N K A E V E R I T N E N A Q R K A D Y

850 870- 890

CGAAGCTAAG CTGGCTCAAT ATCAAAAGGA CCTAGCAGCC GTTCAACAAG CTAATAATGA E A K L A Q Y Q K D L A A V Q Q {A N N D

Al 910 930 950

CAGTCAAGCA GCCTACGCTG CTGCCAAGGA AGCCTACGAC AAAGAATTGG CTCGGGTTCA S Q A A Y A A A K E A Y D K E L A R V Q

970 990 1010

AGCTGCTAAT GCCGCTGCTA AGAAAGAATA CGAAGAGGCT CTAGCTGCCA ACACCACTAA A A N A A A K K E Y E E A L A A N T T K

1030 1050 1070

GAATGAGCAA ATCAAGGCAG AAAACGCCGC TATCCAGCAA CGCAATGCCC AAGCTAAGGC N E Q I K A E N A A I Q Q R N A Q A K A

1090 1110 1130

AGATTACGAA GCCAAGTTGG CTCAATATGA AAAAGATTTA GCCGCAGCCC AGTCTGGTAA D Y E A K L A Q Y E K D L A A A Q S}{G N

A2 1150 1170 1190

TGCTACAAAT GAAGCGGACT ACCAAGCTAA GAAGGCAGCT TATGAACAAG AGTTAGCGCG A T N E A D Y Q A K K A A Y E Q E L A R

1210 1230 1250

CGTGCAAGCC GCTAATGCAG CTGCCAAGCA GGCCTACGAA CAAGCTCTAG CTGCCAACAC V Q A A N A A A K Q A Y* E Q A L A A N T

1270 1290 1310

GGCCAAGAAC GCCCAAATCA CGGCCGAAAA TGAGGCTATC CAGCAGCGCA ATGCGCAAGC A K N A Q I T A E N E A I Q Q R N A Q A

1330 1350 1370

TAAGGCTAAC TATGAAGCTA AATTAGCCCA ATATCAAAAG GATTTGGCCG CAGCTCAATC K A N Y E A K L A Q Y Q K D L A A A Q S}

1390 1410 1430

TGGTAACGCC GCTAATGAGG CAGACTACCA AGAAAAATTA GCAGCCTATG AAAAGGAACT

{G N A A N E A D Y Q E K L A A Y E K E L A3

1450 1470 1490

GGCTCGTGTG CAAGCAGCCA ATGCAGCTGC TAAGCAAGAA TATGAGCAGA AAGTTCAGGA A R V Q A A N A A A K Q E Y E Q K V Q E

1510 1530 1550

AGCTAATGCT AAAAATGCCG AAATTACGGA AGCCAACCGT GCTATCCGTG AACGCAATGC A N A K N A E I T E A N R A I R E R N A

1570 1590 1610

CAAGGCCAAG ACAGACTATG AACTCAAACT GTCTAAGTAC CAAGAAGAGC TTGCTCAGTA K A K T D Y E L K L S K Y Q E E L A Q Y

1630 1650 1670

CAAGAAGGAC CTAGCGGAAT ACCCAGCTAA ACTCCAAGCC TATCAAGATG AACAAGCCGC K K} D L A E Y P A K L Q A Y Q D E Q A A

1690 1710 ^* 1730

AATCAAGGCA GCTCTGGAAG AGTTGGAAAA GCACAAGAAT GAAGATTGGA ACCTCAGTGA I K A A L E E L E K H K N E D N L S E

1750 1770 1790

GCCCTCAGCC CAGAGTCTGG TCTATGACTT GGAGCCCAAT GCTCAGATTT CCCTAGTGAC P S A Q S L V Y D L E P N A Q I S L V T

1810 1830 1850

CGATTGGAAG CTACTGAAAG CCTCCTCCCT TGATGAATCC TTTAGCCACG ATACTGAACA D W K L L K A S S L D E S F S H D T E Q

1870 1890 1910

ATATAACAAA CACAACCTGC AGCCAGATAA TCTAAATATA ACCTATCTGG AGCAGGCTGA Y N K H N L Q P D N L N I T Y L E Q A D

1930 1950 1970

TGATGTGGCC TCCTCAGTAG AGCTCTTTGG TAATTTCGGT GATAAGGCTG GTTGGACAAC D V A S S V E L F G N F G D K A G T T

1990 2010 2030

CACTGTCAGC AATGGTTCAG AAGTTAAGTT TGCCTCTGTC CTCCTCAAGC GTGGCCAGAG T V S N G S E V K F A S V L L K R G Q S

2050 2070 2090

TGCTACAGCC ACCTATACCA ACCTGAAAAA CTCTTACTAC AATGGTAAGA AAATTTCTAA A T A T Y T N L K N S Y Y N G K K I S K

2110 2130 2150

GGTGGTCTAC AAGTATACGG TTGACCCTGA CTCCAAGTTT CAAAATCCTA CTGGTAACGT V V Y K Y T V D P D S K F Q N P T G N V

2170 2190 2210

TTGGTTAGGT ATCTTTACTG ACCCAACCCT AGGGGTCTTT GCCTCAGCCT ATACGGGTCA L G I F T D P T L G V F A S A Y T G Q

2230 2250 2270

AAACGAGAAG GATACCTCTA TCTTTATCAA GAATGAATTC ACCTTCTACG ATGAAGACGG N E K D T S I F I K N E F T F Y D E D G

2290 2310 2330

TAATCCCATC GACTTTGATA ATGCCCTCTT GTCAGTTGCC TCCCTTAACA GGGAACACAA N P I D F D N A L L S V A S L N R E H N

2350 2370 2390

TTCCATTGAG ATGGCCAAGG ACTACAGCGG TACCTTCGTT AAGATTTCTG GCTCATCCAT S I E M A K D Y S G T F V K I S G S S I

2410 2430 2450

TGGTGAAAAA AATGGCATGA TCTATGCGAC CGACACCCTC AACTTTAAAA AGGGTGAAGG G E K N G M I Y A T D T L N F K K G E G

2470 2490 2510

CGGTTCCCTT CACACCATGT ACACCAGAGC AAGTGAGCCT GGTTCAGGTT GGGACTCTGC G S L H T M Y T R A S E P G S G W D S A

2530 2550 2570

TGATGCTCCT AATTCTTGGT ATGGTGCTGG TGCTGTCAGA ATGTCCGGCC CAAACAACTA D A P N S W Y G A G A V R M S G P N N Y

2590 2610 2630

CATCACTTTG GGGGCAACCT CAGCGACCAA TGTCCTCAGC CTAGCTGAAA TGCCACAGGT I T L G A T S A T N V L S L A E M P Q V

2650 2670 2690

ACCTGGTAAA GATAATACTG CTGGTAAAAA ACCAAATATC TGGTATTCCC TTAATGGTAA P G K D N T A G K K P N I W Y S L N G K

2710 2730 2750

GATTCGGGCA GTCAATGTCC CTAAAGTGAC CAAGGAAAAA CCAACCCCAC CAGTTGAGCC I R A V N V P K V T K E K P T P P V E P

2770 2790 2810

AACCAAGCCA GACGAGCCAG TCTATGAAGT TGAGAAGGAA TTGGTAGATC TGCCAGTTGA T K P D E P V Y E V E K E L V D L P V E

2830 2850 2870

ACCAAGCTAC GAAAAGGAAC CAACCCCACC AAGCAAGACT CCAGACCAAA ATATCCCAGA P S Y E K {E P T P P S K T P D Q N I P D PI

2890 2910 2930

CAAACCAGTA GAGCCTACTT ATGAGGTTGA AAAGGAGCTG GAACCGGCAC CAGTTGAGCC K P V E P T Y E V E K E L E P A P V E P

2950 2970 2990

AAGCTACGAA AAGGAACCAA CGCCACCAAG CAAGACTCCG GATCAAGCGA TTCCAGACAA S Y E K}{E P T P P S K T P D Q A I P D K P2

3010 3030 3500

ACCGGTAGAG CCAACCTATG AGGTTGAAAA GGAGTTGGAA CCAGTACCTG TAGAAACAAA P V E P T Y E_V E K E L E P V P V E T N

3070 3090 3110

CTACGAAAAG GAACCAACCC CGCCTCAGTC AACCCCAGAC CAAGAAGAGC CCACCAAACC

Y E K} {E P T P P Q S T P D Q E E P T K P P3

3130 3150 3170

GGTGGAACCA AGCTACCAAA GCTTGCCAAC CCCACCAGTG GCACCGACTT ATGAAAAGGT

V E P S Y Q} S L P T P P V A P T Y E K V

3190 3210 3230

TCCTGGTCCT GTCAGTGTGC CAACGGTTCG GTACCACTAC TATAAACTAG CGGTCCAACC

P G P V S V P T V R Y H Y Y K L A V Q P

3250 3270 3290

CGGCGTCACC AAGAAAATCA AAAACCAGGA TGACCTGGAT ATTGACAAGA CCCTGGTGGC

G V T K K I K N Q D D L D I D K T L V A

3310 3330 3350

TAAGCAGTCG ACGGTTAAGT TCCAATTGAA GACAGCAGAC CTGCCAGCCG GTCGTCCAGA

K Q S T V K F Q L K T A D L P A G R P E

3370 3390 3410

AACGACCTCC TTTGTCTTGA TGGATCCTCT GCCAAGCGGT TACCAACTTA ATCTGGAAGC

T T S F V L M D P L P S G Y Q L N L E A

3430 ^• 3450 3470

TACCAAGGTC GCCAGCCCAG GCTTTGAAGC TAGCTATGAT GCCATGACCC ATACGGTAAC

T K V A S P G F E A S Y D A M T H T V T

3490 3510 3530

CTTCATCGCA ACCGCTGAGA CCTTGGCGGC GCTCAACCAG GATCTGACCA AGGCCGTGGC

F I A T A E T L A A L N Q D L T K A V A

3550 3570 3590

GACTATCTAC CCAACAGTTG TGGGACAAGT CCTCAACGAT GGCGCTACCT ACACCAATAA

T I Y P T V V G Q V L N D G A T Y T N N

3610 3630 3650

CTTCACCCTG ATGGTCAATG ATGCTTACGG TATTAAATCC AATATCGTTC GCGTGACCAC

F T L M V N D A Y G I K S N I V R V T T

3670 3690 3710

ACCAGGGAAA CCTAACGACC CAGACAACCC AAGCAACAAC TACATCACCC CGCACAAGGT

P G K P N D P D N P S N N Y I T P H K V

3730 3750 3770

CAAVAAGAAT GAAAACGGTG TGGTGATTGA TGGTAAGTCC GTCCTAGCTG GTACCACCAA

N K N E N G V V I D G K S V L A G T T N

3790 3810 3830

CTACTATGAA TTGACTTGGG ACCTGGACCA ATACAAGGGC GATAAATCGG CCAAGGAGAC

Y Y E L T W D L D Q Y K G D K S A K E T

3850 3870 3890

CATCCAAAAA GGCTTCTTCT ATGTGGATGA CTATCCTGAA GAAGCGCTGG ACTTGCGCAC I Q K G F F Y V D D Y P E E A L D L R T

3910 3930 3950

CGACCTGATT AAGCTGACCG ATGCCAACGG CAAGGCGGTC ACTGGTGTCA GCGTGGCTGA D L I K L T D A N G K A V A G V S V A D

3970 3990 4010

CTACGCCAGT CTGGAGGCCG CACCAGCAGC TGTTCAAGAC ATGCTCAAGA AGGCCAACAT Y A S L E A A P A A V Q D M L K K A N I

4030 4050 4070

TACCCCTAAG GGAGCCTTCC AAGTCTTTAC CGCTGACGAT CCTCAGGCCT TCTACGATGC T P K G A F Q V F T A D D P Q A F Y D A

4090 4110 4130

CTATGTGGTT ACCGGAACTG ACCTGACCAT CGTCACTCCA ATGACGGTCA AGGCTGAGAT Y V V T G T D L T I V T P M T V K A E M

4150 4170 4190

GGGCAAGATC GGTGGTAGCT ATGAAAACAA GGCCTACCAG ATTGACTTTG GTAATGGCTA G K I G G S Y E N K A Y Q I D F G N G Y

4210 4230 4250

TGAATCTAAT ATTGTGATTA ACAATGTGCC GCAAATCAAT CCTGAAAAGG ATGTGACCTT E S N I V I N N V P Q I N P E K D V T L

4270 4290 4310

GACCATGGAT CCAGCGGATA GTACCAATGT GGATGGACAG ACCATCGCCC TCAATCAGGT T M D P A D S T N V D G Q T I A L N Q V

4330 4350 4370

CTTTAACTAC CGTCTCATCG GTGGTATCAT TCCAGCGGAC CATGCCGAAG AGCTCTTTGA F N Y R L I G G I I P A D H A E E L F E

4390 4410 4430

GTACAGCTTT AGCGATGACT ATGACCAAAC TGGAGACCAG TACACGGGCC AATACAAGGC Y S F S D D Y D Q T G D Q Y T G Q Y K A

4450 4470 4490

CTTTGCCAAG GTTGACCTGA CCCTCAAGGA TGGTACAATC ATCAAGGCGG GTACTGACTT F A K V D L T L K D G T I I K A G T D L

4510 4530 4550

GACTTCATAT ACAGAAGCGC AAGTTGATGA AGCTAATGGC CAAATTGTTG TGACCTTCAA T S Y T E A Q V D E A N G Q I V V T F K

4570 4590 4610

GGAAGATTTC TTGCGGTCTG TGTCTGTAGA CTCGGCCTTC CAAGCGGAAG TCTACCTACA E D F L R S V S V D S A F Q A E V Y L Q

4630 4650 4670

GATGAAGCGG ATAGCCGTCG GGACCTTTGC CAATACCTAT GTCAATACGG TCAATGGAAT M K R I A V G T F A N T Y V N T V N G I

4690 4710 4730

TACCTATAGC TCTAATACGG TAAGGACCAG CACACCAGAG CCGAAGCAGC CAAGTCCAGT Y Y S S N T V R T S T P E P K Q P S P V

4750 4770 4790

GGTACCTAAG ACCACTACTA CGGTAGTCTT CCAGCCTCGT CAGGGTCAAG CTTATCAGCC V P K T T T T V V F Q P R Q G Q A Y Q P

4810 4830 4850

AGCGCCGCCA GCAGGAGCTC AATTGCCAGC CACAGGGGAT AGTAGCAATG CTTACCTGCC A P P A G A Q L P A T G D S S N A Y L P

4870 4890 4910

ACTTTTAGGC CTCGTAAGCC TGACTGCTGG CTTTAGCCTG TTAGGACTGC GCCGGAAGCA L L G L V S L T A G F S L L G L R R K Q

4930 4950 4970

GGACTAAAGA ATCCAACAAG AAAAAATGGG AAAGTTTGCC TTTCTCATTT TTTATATTCC D *

4990 5010 5030

CAGCTAGCTG AGTAGTCAAG AAGTACTCTT AGAAAACCCT AGAGAACATT AGCTAACTTT

5050 5070

TCCAAACCGA TAGACGTTTA TTTTAGTCTA AGTATGG

Table 5 The S.oralis DNA Sequence Encoding an Endocarditis - Specific Antigen Region

5' 10 30 50

GAATTCACCT TCTACGATGA AAATGACCAA CCAATTAATT TTGACAATGC TCTTCTTTCA E F T F Y D E N D Q P I N F D N A L L S

70 9θ ^' 110

GTAGCCTCAC TTAACCGTGA GCATAACTCT ATTGAGATGG CTAAGGATTA TAGTGGTACT V A S L N R E H N S I E M A K D Y S G T

130 150 170

TTTATTAAAA TCTCAGGTTC ATCCATCGGT GAAAAAAATG GCATGATTTA TGCCACAGAA F I K I S G S S I G E K N G M I Y A T E

190 210 230

ACCCTGAACT TTAAACAAGG ACAGGGTGGA GCTCGCTGGA CAATGTATCC AAATCGTCAG T L N F K Q G Q G G A R W T M Y P N R Q

250 270 290

CCAGGTTCAG GTTGGGATTC ATCAGATGCA CCAAACTCTT GGTACGGTGC AGGGGCCATT P G S G W D S S D A P N S W Y G A G A I

310 330 350

AGTATGTCCG GTCCTACGAA TCACGTTACA GTTGGTGCAA CATCTGCTAC CAATGTGATG S M S G P T N H V T V G A T S A T N V M

370 390 410

TCCGTAGCAG AAATGCCTCA AGTACCTGGA AGAGACAATA CTGAAGGTAA AAGACCAAAC S V A E M P Q V P G R D N T E G K R P N

430 450 470

ATCTGGTACT CACTCAATGG TAAAATTCGT GCGGTTGACG TTCCGAAAAT TACAAAAGAA I W Y S L N G K I R A V D V P K I T K E

490 510 530

AAACCAACTC CACCGGTAGC ACCAACTGAA CCACAAGCTC CTACCTATGA AGTGGAGAAA K P T P P V A P T E P Q A P T Y E V E K

550 570 590

CCACTGGAAC CGGCTCCAGT AGCACCAAGC TACGAAAATG AGCCAACTCC ACCAGTAAAA P L E P A P V A P S Y E N {E P T P P V K

PI

610 630 650

ACTCCAGATC AACCGGAGCC ATCAAAACCA GAAGAGCCAA CATATGAGAC AGAGAAACCA T P D Q P E P --S- K P E E P T Y E T E K P

670 690 710

TTGGAACCAG CTCCAGTAGC ACCAAACTAC GAAAATGAGC CAACTCCACC AGTAAAAACT L E P A P V A P N Y E N}{E P T P P V K T

P2

730 750 770

CCAGATCAAC CAGACCCATC AAAACCGGAA GAGCCAAACT ATGAGACAGA GAAACCATTG P D Q P D P S K P E E P N Y E T E K P L

790 810 830

GAACCAGCTC CAGTAGCACC AAGCTATGAA AATGAGCCAA CTCCACCGGT AAAAACTCCA E P A P V A P S Y E NJ{E P T P P V K T P

P3

850 870 890

GATCAACCAG AGCCATCAAA ACCAGAAGAG CCAAATTATG ATCCATTGCC AACTCCGCCG D Q P E P S K P E E P N Y D} P L P T P P

910 930 950

CTAGCACCAA CTCCTAAGCA GTTGCCAACA CCACCAGCGG TGCCAACAGT TCACTTCCAT L A P T P K Q L P T P P A V P T V H F H

970 990 1010

TACAATCGTC TATTTGCACA ACCTCAGATT AATAAAGAAA TTAAAAACGA GGATGGAGTA Y N R L F A Q P Q I N K E I K N E D G V

1030 1050 1070

GATATTGATC GTACTCTAGT TGCTAAGCAG TCTGTAGTGA AGTTTGAGCT GAAAACAGAA D I D R T L V A K Q S V V K F E L K T E

1090 1110 1130

GCTTTAACTG CTGGTCGTCC AAAAACAACT TCGTTTGTAT TGGTAGATCC ACTTCCAACT A L T A G R P K T T S F V L V D P L P T

1150 1170 1190

GGCTATCAGT TTGATTTGGA AGCAACCAAG GCTGCAAGCA AAGGTTTTGA AACAAGCTAT G Y Q F D L E A T K A A S K G F E T S Y

1210 1230 1250

GACAAAGCTA GTCACACTGT AACCTTTAAG GCTACTGAGG AGACCTTAGC TGCTTTCAAT D K A S H T V T F K A T E E T L A A F N

1270 1290 1310

GCTGATTTGA CAAAATCCTT TGAGACTCTA TATCCAACTG TTGTTGGTCG TGTCTTGAAT A D L T K S F E T L Y P T V V G R V L N

1330 1350 1370

GATGGGGCGA CTTATACGAA TAACTTTACA TTGACAGTCA ACGATGCTAC TGGTGTCAAG D G A T Y T N N F T L T V N D A T G V K

1390 1410 1430

TCAAACATTG TTCGTGTAAC GACTCCAGGT AAACCAAATG ATCCTGACAA TCCAAATAAC S N I V R V T T P G K P N D P D N P N N

1450 1470 1490

AACTACATCA AGCCTTTGAA AGTTAACAAG ^" AACAAGCAAG GTGTGAATAT TGATGGCAAA N Y I K P L K V N K N K Q G V N I D G K

1510 1530 1550

GAAGTTCTAG CTGGTTCAAC GAACTACTAT GAACTCACAT GGGATTTGGA TCAATACAAG E V L A G S T N Y Y E L T W D L D Q Y K

1570 1590 1610

GGAGATAAAT CTTCTAAAGA AGCGATTCAA AATGGTTTCT ACTATGTGGA TGATTATCCA G D K S S K E A I Q N G F Y Y V D D Y P

1630 1650 1670

GAAGAAGCTT TAACGCTTCA ACCAGAATTG GTTAAGATTC GTGATCTAGA GGGCAACCTT E E A L T L Q P E L V K I R D L E G N L

1690 1710 1730

GTATCAGGTA TCAGTGTTCA ACAGTTTGAT AGTTTAGAAC GTGCGCCTAA GAAGGTTCAA V S G I S V Q Q F D S L E R A P K K V Q

1750 1770 1790

GATCTGTTGA AGAAAGCAAA CATCACTGTT AAAGGTGCTT TCCAACTCTT CTCAGCTGAT D L L K K A N I T V K G A F Q L F S A D

1810

AATCCAGCTG AATTC

N P A E F

This 1.81 kb DNA fragment carried the three tandem repeat proline (PI - 3) common to the family of antigen 1/11 proteins important in dental caries. The repeat proline rich region of this amino acid sequence was bracketed ({}) as above.

Table 6

Peptide Number Amino Acid Sequence

1 YEVEKPLEPAPVAPS

2 SYENEPTPPVKTPDC

3 KTPDQPEPSKPEEPT

4 EPAPVAPSYENEPTP

5 YEVEKELVDLPVEPS

6 KTPDQNIPDKPVEPT

Table 7

Clinical History of Serum

Peptide Number

1 2 3 4 5 6

M G M G M G M G M G M G

S.mutans 0.341 1.506 0.331 0.381 0.338 1.365 0.445 1.225 0.261 1.491 0.241 0.869 endocarditis

S.oralis 0.442 0.902 0.561 0.891 0.491 0.768 0.5670.936 0.653 0.784 0.373 0.873 endocarditis

S.oralis 0.024 0.118 0.036 0.215 0.019 0.123 0.065 0.168 0.043 0.241 0.117 0.218 septicaemia

S.lactis 0.306 0.589 0.319 0.528 0.309 0.492 0.3770.415 0.3370.421 0.2440.449 endocarditis

S.aurevs 0.075 0.177 0.145 0.579 0.068 0.153 0.015 0.127 0.101 0.267 0.063 0.217 endocarditis

ELISA readings for peptides 1-6

Table 8

Indirect ELISA results for Peptide 1

Concentration of peptide: 200μl at lOμg/ml Primary antibody 1/100

Patient Sera Mean ^a Mean Sample O.D. ^b Standard Deviation No. Date Blank O.D. IgM IgG IgM IgG

S.mutans Endocarditis

1 16.03.88 0.131 0.117 0.698 0.001 0.015

2 17.09.87 0.193 0.012 0.742 0.003 0.0017

S.oralis Endocarditis (n=3)

3 23.05.78 0.141 0.462 0.960 0.018 0.002

4 12.12.81 0.197 0.447 0.902 0.023 0.031

5 23.02.84 0.121 0.043 0.038 0.001 0.008 30.08.84 0.131 0.153 0.699 0.004 0.007

S.gordonii Endocarditis (n=14)

6 1.07.86 0.131 0.422 0.936 0.001 0.002

15.08.86 0.131 0.339 0.855 0.003 0.004

22.09.86 0.131 0.228 0.81 0.010 0.005

6.10.96 0.131 0.173 1.06 0.003 0.002

7 8.01.87 0.131 0.487 0.685 0.010 0.012

8 18.12.86 0.131 0.059 0.662 0.004 0.006 21.01.87 0.131 0.270 0.715 0.005 0.009

9 10.08.86 0.131 0.43 0.41 0.007 0.004

10 17.07.86 0.131 0.568 0.601 0.010 0.012

11 14.07.93 0.131 0.095 0.471 0.012 0.003

12 20.03.93 0.158 0.164 0.404 0.001 0.03 22.03.93 0.158 0.168 0.475 0.003 0.03

13 06.05.87 0.12T 0.438 0.079 0.023 0.003 12.08.87 0.121 0.274 0.579 0.021 0.016

14 7.06.86 0.131 0.174 0.459 0.006 0.024

9.06.86 0.151 0.144 0.469 0.004 0.013

16.06.86 0.153 0.151 0.587 0.005 0.011

19.06.86 0.193 0.134 0.706 0.006 0.008

23.06.86 0.151 0.149 0.562 0.007 0.011

26.06.86 0.153 0.106 0.475 0.005 0.017

15 2.12.87 0.012 0.301 1.391 0.006 0.011 30.12.87 0.131 0.217 1.083 0.005 0.007

S.sanguis Endocarditis (n=2)

16 21.07.86 0.121 0.46 0.543 0.006 0.011 7.08.86 0.121 0.487 0.685 0.001 0.011

17 14.07.93 0.121 0.416 0.401 0.049 0.022

S.oralis Septicaemics (n=8)

18 23.11.88 0.133 0.046 0.112 0.017 0.001 6.12.88 0.131 0.024 0.118 0.001 0.004

19 22.08.89 0.131 0.023 0.194 0.001 0.003 6.09.89 0.144 0.034 0.205 0.0016 0.001 17.10.89 0.121 0.006 0.042 0.003 0.004

20 11.03.88 0.197 0.007 0.048 0.018 0.008

1.04.88 0.133 0.043 0.102 0.003 0.011

10.05.88 0.197 0.021 0.014 0.001 0.001

21 27.06.89 0.197 0.058 0.031 0.015 0.021 1.80.89 0.151 0.003 0.095 0.002 0.001

22 2.08.88 0.144 0.017 0.082 0.001 0.014

23 4.04.89 0.135 0.044 0.061 0.002 0.002

24 22.03.93 0.193 0.029 0.022 0.001 0.001 22.04.93 0.158 0.032 0.041 0.003 0.001 26.04.93 0.139 0.034 0.044 0.011 0.021 27.04.93 0.158 0.042 0.036 0.001 0.009

25 21.07.93 0.139 0.089 0.027 0.003 0.009 21.07.93 0.139 0.113 0.031 0.009 0.005 4.08.93 0.139 0.129 0.032 0.001 0.008 17.08.93 0.139 0.095 0.103 0.003 0.019

E.faecalis Endocarditis

26 25.02.93 0.141 0.027 0.075 0.001 0.014 25.02.93 0.141 0.024 0.063 0.001 0.003 29.02.93 0.141 0.026 0.071 0.003 0.001

27 15.06.93 0.147 0.085 0.248 0.003 0.007 16.06.93 0.147 0.071 0.275 0.002 0.013

28 20.01.92 0.141 0.041 0.069 0.004 0.001

29 24.06.88 0.153 0.085 0.283 0.003 0,017

30 10.06.86 0.135 0.054 0.361 0.003 0.012

31 19.11.87 0.133 0.033 0.652 0.009 0.006

32 29.01.89 0.131 0.081 0.564 0.011 0.016 13.10.89 0.133 0.024 0.281 0.003 0.022 16.11.89 0.197 0.016 0.195 0.006 0.005 8.01.90 0.133 0.043 0.371 0.008 0.018

33 10.05.88 0.197 0.096 1.079 0.003 0.018 15.05.88 0.131 0.088 1.011 0.003 0.018 21.05.88 0.131 0.048 0.531 0.002 0.015 28.08.88 0.131 0.156 0.674 0.003 0.013

34 6.11.86 0.141 0.086 0.082 0.013 0.001

35 12.01.87 0.144 0.039 0.387 0.008 0.022

36 5.01.87 0.135 0.011 0.559 0.001 0.015

Efaecal is Septicaem ics

37 30.12.87 0.133 0.029 0.371 0.001 0.007

38 10.10.85 0.193 0.018 0.175 0.004 0.025

39 24.08.89 0.193 0.033 0.002 0.003 0.002 11.10.85 0.151 0.052 0.266 0.001 0.008 11.10.85 0.153 0.049 0.218 0.011 0.014

40 8.01.88 0.135 0.041 0.286 0.019 0.001

41 27.11.87 0.197 0.034 0.354 0.001 0.004

42 20.04.85 0.144 0.02 0.132 0.003 0.006

43 27.06.88 0.144 0.034 0.152 0.013 0.011

E.faeciu m Septicaem ics

44 19.07.85 0.197 0.135 0.436 0.015 0.026

Group G Streptococcal endocarditis

60 29.10.86 0.131 0.131 0.651 0.009 0.019

61 27.03.85 0.197 0.111 0.789 0.017 0.015

NON NEUTROPENIC CONTROLS

Staphylococcus aureus Endocarditis

62 8.02.88 0.149 0.087 0.077 0.029 0.018

63 28.02.88 0.149 0.106 0.037 0.004 0.008

Coagulase Negative Staphylococcus Endocarditis

64 30.09.89 0.139 0.054 0.062 0.007 0.001

65 16.06.89 0.153 0.051 0.215 0.013 0.007 1.07.86 0.131 0.146 0.472 0.003 0.015

66 24.02.87 0.139 0.243 0.083 0.001 0.025 5.03.87 0.121 0.387 0.098 0.008 0.006

67 2.01.88 0.144 0.016 0.192 0.006 0.019

68 10.08.86 0.144 0.029 0.287 0.012 0.019 25.10.86 0.139 0.067 0.071 0.008 0.006

69 15.02.88 0.133 0.075 0.177 0.011 0.008

Endocarditis due to Candida albicans

70 24.10.86 0.147 0.244 0.159 0.004 0.003

Endocarditis due to Candida parapsilosis

71 1.02.88 0.153 0.077 0.192 0.006 0.008

11.06.88 0.139 0.121 0.165 0.005 0.006

18.06.88 0.158 0.258 0.216 0.007 0.004

72 8.05.86 0.139 0.087 0.081 0.008 0.007

Endocarditis due to Escherichia coli

73 1.07.88 0.135 0.038 0.358 0.003 0.018 2.07.88 0.133 0.022 0.379 0.011 0.011 5.07.88 0.197 0.073 0.344 0.008 0.002

SLE

74 13.06.93 0.158 0.327 0.167 0.004 0.011 16.06.93 0.147 0.322 0.245 0.008 0.022

75 2.05.88 0.144 0.023 0.285 0.009 0.004

76 18.04.89 0.193 0.015 0.304 0.004 0.003

Brain abscess due to Streptococcus milleri

11 17.10.86 0.153 0.182 0.312 0.006 0.022 30.10.86 0.151 0.342 0.353 0.011 0.001 6.11.86 0.193 0.011 0.185 0.003 0.013

NEUTROPENIC CONTROLS

78 7.08.88 0.193 0.019 0.183 0.004 0.019

79 11.11.86 0.151 0.101 0.251 0.013 0.009

80 21.10.86 0.149 0.101 0.078 0.015 0.018

81 25.07.91 0.139 0.024 0.045 0.003 0.001

82 9.02.89 0.012 0.151 0.147 0.003 0.003

83 15.06.93 0.151 0.021 0.177 0.004 0.009

84 23.06.92 0.193 0.016 0.176 0.025 0.015

85 19.01.88 0.149 0.108 0.033 0.015 0.001

86 25.10.88 0.193 0.007 0.035 0.005 0.004

87 21.03.86 0.158 0.026 0.045 0.006 0.008

88 18.02.86 0.139 0.037 0.081 0.001 0.008

89 6.08.91 0.144 0.034 0.421 0.005 0.014

90 24.04.87 0.133 0.017 0.023 0.003 0.003

91 12.12.89 0.144 0.013 0.047 0.001 0.004

92 30.10.86 0.139 0.076 0.125 0.009 0.014

93 2.07.86 0.139 0.125 0.094 0.007 0.003

94 5.02.87 0.149 0.119 0.032 0.001 0.003

95 5.09.98 0.135 0.017 0.021 0.004 0.003

96 5.08.93 0.153 0.055 0.152 0.014 0.026

97 19.08.93 0.158 0.022 0.058 0.001 0.004 1.09.93 0.158- 0.044 0.088 0.009 0.006 14.09.93 0.158 0.026 0.144 0.003 0.022

98 3.05.87 0.141 0.131 0.252 0.004 0.014

Mean blank O.D., this value was obtained for each microtitre plate by calculation of the mean O.D. for the six wells containing stain only. This mean blank O.D. was subtracted from the each of the sample O.D. measurements to produce the corrected mean sample O.D. value.

Mean sample O.D., this was the value obtained after subtraction of the mean blank O.D. value from the mean of the three O.D. measurements taken from each of the sample wells.

Table 8a

Indirect ELISA results

Concentration of peptides: 200ml at lOug/ml

Primary antibody 1/100 ^a Peptide numbers 1,7 and 8 as before.

Patient No Mean Sample Optical Density

IgM IgG l ^a _? a 8 ^a l ^a 7 ^a 8 ^a

S.mutans Endocarditis

1 0.117 0.636 0.862 0.698 0.602 0.733

S.oralis Endocarditis

4 0.447 0.584 1.236 0.902 0.433 0.678

S.gordonii Endocarditis

6 0.422 0.742 1.443 0.936 0.524 1.694

8 0.27 0.582 0.891 0.715 0.319 1.01

9 0.43 1.103 0.95 0.41 0.388 0.19

10 0.568 0.700 0.824 0.601 0.319 0.505

11 0.095 0.641 1.396 0.471 0.685 0.889

12 0.164 0.988 1.399 0.404 0.828 0.859

13 0.274 1.098 1.605 0.579 0.841 0.799

14 0.174 0.991 1.600 0.459 0.542 0.64

S.sanguis Endocarditis

16 0.46 1.335 1.366 0.543 1.802 2.267

17 0.417 1.126 1.07 0.401 0.335 0.632

S.oralis Septicaemias

18 0.046 0.136 0.243 0.112 0.324 0.536

19 0.023 0.324 0.314 0.194 0.294 0.586

20 0.007 0.228 0.295 0.048 0.272 0.607

21 0.058 0.206 0.280 0.031 0.289 0.476

22 0.017 0.316 0.879 0.982 0.384 0.824

24 0.029 0.911 1.327 0.022 0.475 0.754

25 0.089 0.348 0.720 0.027 0.238 0.369

E.faecalis Endocarditis

26 0.027 0.344 0.390 0.075 0.376 0.636

32 0.081 0.226 0.317 0.564 0.388 0.756

35 0.039 1.133 1.228 0.387 0.906 1.086

36 0.011 0.549 0.926 0.559 0.591 1.176

Ejaecalis Septicaemias

38 0.018 0.784 1.344 0.175 0.39 0.796

39 0.033 0.590 0.78 0.002 0.432 0.87

E.faecium Septicaemias

45 0.144 0.47 1.228 0.269 0.588 1.086

S.oralis Endocarditis

51 0.203 1.063 1.075 0.823 0.410 0.802

S.aureus Endocarditis

62 0.087 0.333 0.266 0.077 0.435 0.401

63 0.106 0.356 0.805 0.037 0.484 0.646

Coagulase negative

Staphylococcus endocarditis

64 0.054 0.596 0.879 0.062 0.612 0.829

68 0.029 1.502 1.205 0.287 1.446 1.521

SLE

74 0.327 0.697 1.509 0.167 0.542 1.839

Brain abscess S.milleri

11 0.182 1.421 2.162 0.312 0.500 0.781

Neutropenic Controls

78 0.193 0.570 0.551 0.183 0.804 0.428

79 0.151 0.579 0.491 0.251 0.726 0.428

80 0.149 0.744 0.628 0.078 0.525 0.512

81 0.139 0.422 0.414 0.045 0.530 0.412

82 0.012 0.406 0.382 0.147 0.931 0.809

83 0.151 0.328 0.394 0.177 0.233 0.396

84 0.193 0.601 0.533 0.176 0.950 0.683

85 0.149 0.581 0.34 0.033 0.372 0.60

86 0.193 0.456 0.325 0.035 0.616 0.313

87 0.158 0.610 0.597 0.045 0.542 0.377

88 0.139 0.559 0.475 0.081 0.682 0.497

89 0.144 0.628 0.130 0.421 0.477 0.166

90 0.133 0.142 0.182 0.023 0.280 0.730

91 0.144 0.394 0.305 0.047 0.636 0.317

92 0.139 0.403 0.323 0.125 0.616 0.588

93 0.139 1.021 0.23 0.094 0.858 0.57

94 0.149 0.42 0.47 0.032 0.53 0.432

95 0.135 0.463 0.436 0.021 0.711 0.674

96 0.153 0.568 0.539 0.152 0.760 0.587

97 0.158 0.743 0.650 0.058 0.914 0.535

98 0.141 0.366 0.384 0.252 0.528 0.366

Table 9

IgG fcθ.6 IgM *0.4 IgM -.0.4 and IgG hθ.6

S.mutans endocarditis 100% 0% 100%

S.oralis endocarditis 100% 66% 100%

S.gordonii endocardits 60% 50% 80%

S.sanguis endocarditis 50% 100% 100%

S.oralis septicaemias 0% 0% 0%

E.faecalis endocarditis 18% 0% 18%

E.faecalis septicaemia 0% 0% 0%

Efaecium septicaemia 0% 0% 0%

S.bovis endocardits 12.5% 12.5% 25%

S.agalactiae endocarditis 0% 0% 0%

S.lactis endocarditis 0% 0% 0%

S.pneumoniae endocarditis 0% 0% 0%

Group G streptococcal 100% 0% 100% endocarditis

Staphylococcus aureus 0% 0% 0% endocardits

Coagulase Negative 0% 0% 0% endocarditis

Candida albicans 0% 0% 0% endocarditis

Candida parapsilosis 0% 0% 0% endocarditis

E. coli endocarditis 0% 0% 0%

SLE 0% 0% 0%

S.milleri 0% 0% 0%

Neutropenic controls 0% 0% 0%

Table 10

Epitope Epitope Negative Viridans S. oralis S. mutans E. faecalis Number Sequence Control Endocarditis Septicaemia Endocarditis Endocarditis

n=3 n=8 n=5 n=2 n=2

O.D. S.D O.D. S.D. O.D. S.D. O.D. S.D. O.D. S.D.

61 0.400 0.027 1.039 0.353 0.429 0.200 0.833 0.125 0.588 0.054 62 NFKQGQG 0.388 0.034 0.972 0.347 0.419 0.230 0.808 0.122 0.508 0.027 63 0.444 0.058 0.992 0.329 0.430 0.215 0.888 0.195 0.540 0.013

74 0.341 0.019 0.902 0.319 0.322 0.211 0.839 0.004 0.407 0.120 75 0.283 0.033 1.038 0.470 0.351 0.180 0.891 0.011 0:450 0.119 76 RQPG 0.282 0.034 1.053 0.474 0.455 0.180 0.917 0.029 0.493 0.140 77 0.350 0.045 0.981 0.358 0.309 0.070 1.041 0.043 0.472 0.130 78 0.280 0.023 0.918 0.425 0.307 0.080 1.142 0.405 0.423 0.096 79 0.282 0.015 0.781 0.312 0.271 0.091 1.005 0.418 0.373 0.084

90 0.308 0.054 0.852 0.318 0.287 0.070 1.027 0.192 0.378 0.088 91 SWYGAG 0.316 0.069 0.924 0.340 0.328 0.080 1.064 0.138 0.428 0.136 92 0.344 0.076 0.917 0.429 0.298 0.060 0.914 0.033 0.377 0.115 93 0.306 0.007 0.957 0.378 0.444 0.140 1.083 0.225 0.483 0.131

144 0.306 0.072 0.824 0.318 0.348 0.180 0.779 0.129 0.389 0.115 145 GKIRAV 0.384 0.071 1.028 0.309 0.437 0.210 0.971 0.199 0.515 0.120 148 0.303 0.042 0.852 0.261 0.348 0.150 0.881 0.240 0.438 0.115 147 0.395 0.035 0.933 0.285 0.397 0.180 0.944 0.258 0.483 0.131

320 0.392 0.031 1.147 0.657 0.491 0.180 1.010 0.151 0.494 0.115 321 RLFAQPQ 0.453 0.049 1.209 0.417 0.548 0.230 1.284 0.300 0.649 0.171

322 0.467 0.124 1.145 0.356 0.531 0.190 1.289 0.346 0.593 0.116

359 0.395 0.043 0.921 0.295 0.401 0.200 0.749 0.177 0.480 0.040 360 0.492 0.065 1.049 0.374 0.428 0.240 0.775 0.030 0.491 0.062 361 AGRPK 0.407 0.032 0.907 0.318 0.382 0.190 0.657 0.094 0.0462 0.067 362 0.430 0.008 1.054 0.325 0.423 0.230 0.753 0.091 0.525 0.082 363 0.421 0.042 0.946 0.279 0.391 0.021 0.665 0.088 0.477 0.083

376 0.277 0.035 0.791 0.479 0.428 0.080 0.762 0.190 0.339 0.095 377 PTGYQFD 0.415 0.039 1.045 0.427 0.359 0.140 1.548 0.728 0.487 0.155 378 0.351 0.062 0.802 0.290 0.291 0.140 1.274 0.793 0.380 0.151

426 0.309 0.081 0.852 0.391 0.269 0.100 1.275 0.617 0.410 0.147 427 0.215 0.084 0.473 0.161 0.225 0.050 0.525 0.117 0.289 0.030 428 YPTW 0.325 0.013 1.170 0.457 0.450 0.180 1.143 0.125 0.561 0.194 429 0.339 0.046 0.964 0.336 0.413 0.150 1.050 0.179 0.488 0.165 430 0.349 0.088 0.976 0.328 0.465 0.140 1.002 0.198 0.458 0.139

577 0.279 0.042 0.863 0.302 0.353 0.190 0.681 0.142 0.466 0.081 578 0.349 0.019 0.907 0.260 0.410 0.210 0.808 0.270 0.557 0.103 579 LLKKA 0.351 0.051 0.937 0.254 0.415 0.210 0.882 0.221 0.549 0.098 580 0.395 0.077- 1.035 0.267 0.423 0.210 0.898 0.183 0.587 0.124 581 0.331 0.029 0.847 0.261 0.581 0.150 0.843 0.153 0.482 0.141