AU640430B2

AU640430B2 - Im peptides

Info

Publication number: AU640430B2
Application number: AU67441/90A
Authority: AU
Inventors: Denis James Moss; Tom Brian Sculley
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 1989-11-24
Filing date: 1990-11-23
Publication date: 1993-08-26
Anticipated expiration: 2010-11-23
Also published as: AU6744190A

Description

TITLE: IM PEPTIDES

TECHNICAL FIELD

THIS INVENTION is directed to the diagnosis and treatment of herpes virus related diseases. In particular, it relates to the use of specific open reading frames (ORFs) within the Epstein-Barr virus

(EBV) which encode antigens recognized by EBV-specific antibodies raised during infectious mononucleosis (IM) and related diseases and the use of synthetic peptides based on the amino acid sequences encoded by these ORFs in a specific and reliable diagnostic test for, and treatment of, IM and related diseases.

BACKGROUND ART

Epstein-Barr virus (EBV) is a member of the herpes virus family and is present in all human populations. Primary infection usually occurs in early childhood and remains silent throughout a person's life. However, when uninfected adolescents and young adults are exposed to EBV, about 60% manifest infectious mononucleosis (IM).

The predominant laboratory test used to establish the diagnosis of IM has been the demonstration of heterophil antibodies. The rapid slide tests have become the most widely used method to detect these heterophil antibodies. In contrast, quantitative agglutination tests, such as the Paul-Bunnell-Davidsohn method, are more accurate but are also more tedious and time consuming.

The use of tests to measure heterophil antibodies have limitations, namely, only between.80-95% of IM patients produce these antibodies, these antibodies are absent in a large percentage of young children and the antibodies are produced in a variety of other diseases such as lymphoma, hepatitis and leukemia. The measurement of heterophil antibodies also does not give any indication of the severity of the disease and cannot be used to monitor the course of IM.

Immunofluoresence tests that measure antibodies to EBV can also be used in the diagnosis of heterophil- negative cases of IM, or patients with atypical manifestations.

These tests, however, are time consuming and require the use of trained personnel and specialized equipment which does not make them amenable to the routine analysis of large numbers of samples.

The diagnosis of an acute primary EBV infection can also be determined by an IgM response to EBV-viral capsid antigen (VCA) . The VCA is composed of a large number of different antigens. Components of VCA are defined by the fact that they are expressed late in the replicative cycle of the virus. Many VCA components have been mapped to specific open rea ing frames (ORF's) within the EBV genome though there are many ORF's, known to be expressed late in replication, to which specific VCA antigens have not yet been identified.

Genetic engineering and synthetic polypeptide technologies now enable the manufacture of large quantities of protein and polypeptide antigens. However, these techniques are only effective if the amino acid residue sequence of the native protein is known. The amino acid residue sequence of a natural protein can be determined by sequencing of the protein itself.

Alternatively, the DNA sequence that codes for the protein may also reveal the protein's amino acid residue sequence.

Antibodies can be used to determine whether an ORF present in a DNA sequence codes for a protein. This involves manufacturing an array of protein fragments or synthetic polypeptides whose amino acid residue sequences correspond to the hypothetical sequences obtained from the ORFs. The protein fragments or polypeptides to which naturally occurring antibodies immunoreact thereby identify the ORF as encoding a naturally occurring protein. The complete amino acid sequence of this protein could then be deduced from the DNA sequence of the ORF.

DISCLOSURE OF THE INVENTION

It is a general object of the present invention to overcome, or at least ameliorate, one or more of the above disadvantages, and to provide a specific and reliable test for the diagnosis for, and treatment of, IM and related diseases.

As the complete DNA sequence ^' for EBV has been identified, including the start and stop σodons which, prima facie, define potential ORFs for the transcription of the genetic code, the present inventors have synthesized peptides based on the predicted amino acid sequences encoded by these ORFs, even though the proteins to which these ORFs may relate were not first established as being produced by the virus, and have demonstrated that EBV-specific antibodies raised during IM react with the synthesized peptides.

Thus, according to a first aspect of the present invention, there is provided a peptide comprising a sequence which includes at least one segment which codes for an antigen recognized by EBV-specific antibodies raised during IM or a related disease.

As a second aspect, the present invention also includes within its scope a method of identifying a polypeptide suitable for use in the diagnosis of IM and related diseases, said method comprising:

(1) noting within EBV open reading frames transcribed late in the viral cycle for which a translation product may or may not have been established;

(2) synthesising one or more polypeptides each of which includes at least one segment wherein each segment comprises at least part of the amino acid sequence identified in that open reading frame; and

(3) determining whether said polypeptide is effective in the diagnosis of IM and related diseases.

When sera from patients who exhibited clinical symptoms of IM and related diseases were assayed using the peptides of the invention, a positive reaction was noted with a high correlation between this assay and known assays for identifying IM and related diseases. Therefore, according to a third aspect of the present invention, there is provided a method of diagnosis of infectious mononucleosis or a related disease, said method comprising assaying serum from a patient suspected of having infectious mononucleosis or a related disease with at least one peptide as hereinbefore defined.

The present invention also provides, as a fourth aspect, a kit for use in the diagnosis of IM or a related disease, said kit comprising:

(a) at least one peptide as hereinbefore defined; and

(b) a means for indicating the presence of a r e a c t i o n , p a r t i c u l a r l y a n immunoreaction, between said at least one peptide and another molecule(s), especially anti-EBV antibodies.

Since a patient with IM or a related disease contains antibodies to the peptides of the invention, it is likely that the peptides of the invention, when administered to the patient, would elicit anti-EBV antibodies.

Therefore, as a fifth aspect of the present invention, there is provided a vaccine that, when administered, is capable of inducing antibodies effective against EBV, said vaccine comprising:

(a) at least one peptide as hereinbefore defined; and

(b) a carrier and/or diluent and/or adjuvant. As used throughout the specification, the term "carrier or diluent" denotes an organic or inorganic, natural or synthetic material with which the active ingredient is combined in order to facilitate the administration of the vaccine of the invention. This carrier or diluent is, therefore, generally inert and it must be pharmaceutically acceptable. Similarly, the term "adjuvant" has the usual meaning in the art to describe a material which aids the operation of the active ingredient.

According to a sixth aspect of the present invention, there are also provided antibodies and substantially whole antibodies raised to - or induced by - the peptides of the invention as hereinbefore defined.

These molecules are collectively referred to as receptors and can be raised in animal hosts using the vaccine as hereinbefore defined.

Preferably, the peptide of the invention comprises at least one segment selected from the following sequences:

AHARDKAGAVMAMIL

ASLNSPKNGSNQLVI

ELESEPRPRPSRTPS QAM_.KI__DKVR_.SVD

SRSRGREAKKVQISD

LIKASLRKDRKLYAE

VSFSKTRRAIRESRA

CNYSAGEEDDQYHAN RPHRRPVSKRPTHKP

EITQEENRGEQRLGH

GALRARLDRPRPTAQ More preferably, the peptide of the invention comprises at least one segment selected from the following sequences:

NSPKNG KNGSNQ SNQLVI AHARDK RDKAGA VMAMIL SEPRPR PSRTPS

Most preferably, the peptide of the invention comprises at least one segment selected from the following sequences:

NSPKNGSNQAHARDKSEPRPR NSPKNGSNQRDKAGASEPRPR NSPKNGSNQSEPRPRKNGSNQ NSPKNGSNQLVISEPRPRPSRTPS NSPKNGSNQLVIPSRTPS

NSPKNGSNQAHARDKAGASEPRPR

A particularly preferred peptide of the invention comprises at least one segment containing the sequence:

NSPKNGSNQLVIPSRTPS

All amino acid residues identified throughout the specification are in the natural or L-configuration. In keeping with standard polypeptide nomenclature, abbreviations for amino acid residues are as follows:

SYMBOL AMINO ACID

Y L-tyrosine G glycine

F L-phenylalanine

M L-methionine

A L-alanine

S L-serine • I L-isoleucine

L L-leucine

T L-threonine

V L-valine P - L-proline K L-lysine

H L-histidine

Q L-glutamine

E L-glutamic acid

W L-tryptophan R L-arginine

D L-aspartic acid

N L-asparagine

C L-cysteine

DETAILED DESCRIPTION OF EMBODIMENTS

SUBJECTS. MATERIALS AND METHODS

Preparation And Use Of Synthetic Peptides

Peptides (15 aa each) were synthesized by the Multiple Simultaneous Peptide technique (MSPS) of Houghton, R.A. (1985, Proc. Natl. Acad: Sci. USA 82, 5131-5135). The synthetic peptides were linked to bovine serum albumin (BSA) with glutaraldehyde as described by Bulinski et al. (1983, Proc. Natl. Acad. Sci. USA 80, 1506-1510).

Essentially, 5mg of dry peptide were added to 0.5ml

(4mg/ml) BSA in lOOmM phosphate buffer, pH 7.3. To this were added 0.25ml of 0.25% (v/v) glutaraldehyde for each mg of dry peptide. The solution was left in the dark over night at 21°C to conjugate, after which the solution was extensively dialyzed against PBS containing

50mM glycine, pH 7.3. The conjugated peptides were then stored at -20°C until needed. Peptide sequences, deduced from 13 different ORFs transcribed late in viral replication, were synthesized. These peptides are identified in Table 1.

ORF

BCRF1 BCRF1 BDLF2 BDLF2 BDLF3 BDLF3 BKRF2 BILF2 BILF2 BSRF1 BGLF1 BGLF1 BALF1 BALF1 BBRF3 BBRF3 BXRF1 BXRF1 BORF1 BORF1 BLRF2 BLRF2 BMRF2

ELISA assay

The stock peptide-BSA conjugate was diluted 1/100 with DD H₂0 and 50 μl of the peptide-BSA conjugate were added to each well in 96 well microtiter plates (Flow Laboratories) and the plates were left overnight at 37°C to dry. To block the wells 200μl of a solution containing 5% BSA, 0.5M carbonate buffer pH 9.0 were added and left for 30 minutes at ambient temperature. The wells were then washed four times with 200 μl of 0.1% BSA-PBS/l%Tween. Human sera (diluted 1 in 100 in 5%BSA/2xPBS/l%Tween) were added and incubated at 21°C for lh. The plates were then washed six times with 0.1%BSA/2xPBS/0.1%Tween and 100 μl of peroxidase- labelled anti-human IgM (Tago, μ fraction) (diluted 1/5000 in 5%BSA/2xPBS/0.1%Tween) were added and then the plates incubated at 31°C for 30 minutes. The plates were again washed six times as above. The plates were given a rinse with distilled water (neutral pH) , and substrate ( 100 μl of ImM ABTS ( 2 , 2 -azino-bis ( 3- ethlybenzthiazoline-6-sulphonic acid) diammonium salt (Sigma, St. Louis, USA) in lOOmM phosphate-citrate buffer (pH 4.3) containing 0.004% (v/v) hydrogen peroxide) were added and the plates incubated at 37°C for 30 minutes. Finally the plates were read at 410nm.

Subjects and sera

Samples of sera (26) were obtained from patients with IM. These patients were diagnosed as having clinical symptoms of IM and were confirmed by immunofluoresence assays for IgM and IgG antibodies to VCA and by immunoblotting for antibodies to early antigens (EA) and absence of antibodies to EBV nuclear antigen 1 (EBNA1). Sera (14 were EBV seropositive and 8 EBV seronegative) were also obtained from healthy controls and their EBV status was determined by immunofluoresence assays to the EBV antigens. The normal controls were all negative for IgM antibodies to EBV. For the clinical trials, hundreds of samples of serum, from patients displaying IM-like symptoms, were collected.

Iπmunofluoresence assay for EBV antigens.

Anti-VCA titres were measured according to the method of Henle and Henle. (J Bacteriol. 91, 1248-1256).

Heterophil antibody assay

The Paul Bunnell test was used to measure heterophil antibody titres in serum.

RESULTS AND DISCUSSION

Screening of EBV peptides

The 23 synthetic peptides were initially screened against 26 samples of sera from confirmed IM patients, 8 EBV seronegative controls and 14 EBV seropositive controls. The results, presented in Fig. 1, illustrate that a number of peptides were reactive with IgM antibodies from IM patients while showing little reaction with the sera from either EBV seropositive or seronegative controls. Peptides # 2, 4, 6, 13, 16 and 18 were selected for further studies.

Reaction of IM and normal sera with selected EBV peptides.

Comparison of total Ig and IgM reactions with the peptides indicated that normal seropositive individuals lacked both IgM and IgG antibodies to these peptides (Fig. 2). However, it appeared that IM patients contained only IgM antibodies to these peptides. Measurement of IgG antibodies to the peptides, in both IM sera and sera from normal controls, confirmed the absence of these antibodies (results not shown).

These results indicated that measurement of either IgM or total Ig could be useful in identifying serum samples from IM patients.

Since only IgM antibodies to the peptides appear to be present in IM patients, there should not be a problem with rheumatoid factor or interference from IgG antibodies, both of which are usually a problem with indirect ELISA assays.

Clinical study

In order to ascertain the viability of using the peptides to identify cases of IM, sera were obtained from patients who showed clinical symptoms of IM. These sera were assayed by ELISA, using the 6 selected synthetic peptides, by immunofluoresence for the presence of IgM antibodies to VCA and for heterophil antibodies. Patients were considered to have IM if they were both heterophil positive (titres of 1/16 or higher) and IgM positive (titres of 1/40, or higher) by immunofluorescence or heterophil negative but with atypical monnuclear cells and IgM positive by immunofluoresence. The ELISA based IgM assay using the synthetic peptides was considered to be positive when a serum reacted with four or more of the peptides (A⁴¹° of 0.40 or above). The results of the assays, obtained with all of the patients, are shown in Table 2. Patient

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50 TABLE 2 (cont)

Patient

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 TABLE 2 (cont)

Patient

100 101 102 103 104

The results of the statistical analysis of the correlation between the three assays systems are presented in Table 3.

TABLE 3 CORRELATIONS AND POLYCHORIC TEST STATISTICS

ELISA IF(IgM) HETEROPHIL ELISA 1.000 0.988 0. 901 IF(IgM) 1.000 0 . 964 HETEROPHIL 1. 000

ASYMPTOTIC VARIANCES OF ESTIMATED CORRELATIONS

ELISA-IF(IgM) = 0.00020 ELISA-HETEROPHIL = 0.00407 IF(IgM)-HETEROPHIL = 0.00094

The results obtained from the statistical analysis demonstrate a 96% correlation between the IF-IgM assay and the heterophil antibody assay which better the reported data. However, the correlation between the ELISA assay and IF-IgM was 99% indicating that this assay was more accurate at predicting patients with IM. There was one serum which was ELISA positive but IF-IgM negative and it is possible that this patient may have not. as yet developed a high enough titre of IgM antibodies to be detected by the IF-IgM test. Two sera samples were positive by IF-IgM and negative by the ELISA assay. Peptides # 2,4 and 6 were selected for further clinical trials, whereby 510 samples of serum, from patients displaying IM-like symptoms, were assayed for heterophil antibody using the Paul Bunnell test and for the presence of IgM antibodies to EBV by immunofluoresence as described above. These results were then correlated with the results obtained using the ELISA assay with the three synthetic EBV peptides and with the Monolert assay as marketed by Johnson and Johnson. The statistical analysis of these results are shown below.

C»____ATIONS AND TEST STATISTICS (PC=POLYCHORIC)

TEST OF MODEL TEST OF ZERO CORR. Correlat. Chi-squ.DF P-VALUE CHI-SQU. P-VALUE

ESTIMATED CORRELATION MATRIX

PB IFA ELISA MONO

PB 1.000 IFA 0.868 1.000 ELISA 0.882 0.897 1.000 MONO 0.718 0.595 0.614 1.000

PB - Paul Bunnell

IFA - Immunofluoresence assay for EBV-specific IgM

ELISA- ELISA assay using the synthetic EBV peptides of the invention

MONO - Monolert assay as marketed by Johnson and Johnson These results show a much higher correlation between the synthetic peptides of the invention and the IFA assay than did the PB test or the Monolert assay.

Rather than use three independent peptides in the assay system, longer peptides containing combinations of B- cell epitopes (ie the epitopes to which the antibodies bind) from each of the 15 amino acid peptides were constructed. To define the B-cell epitopes in each of the three peptides overlapping 6 amino acid synthetic peptides were prepared and assayed by ELISA using a pooled IM positive sera and a pooled normal sera.

ORIGINAL 15-mers PEPTIDES

#4 ASLNSPKNGSNQLVI

#2 AHARDKAGAVMAMIL #6 ELESEPRPRPSRTPS

SYNTHETIC OVERLAPPING PEPTIDES

PEPTIDE POSITIVE SERA NEGATIVE SERA

ASLNSPKNGSNQLVI

AHARDKAGAVMAMIL

ELESEPRPRPSRTPS

**

** - INDICATES THE PEPTIDES CONTAINING B-CELL EPITOPES, Having defined the B-cell epitopes contained within the original 15 aa peptides, different combinations of these epitopes were used in the synthesis of larger synthetic peptides. Examples of some of these combinations are shown below:

EPITOPE COMBINATIONS:

PEPTIDE A. NSPKNGSNQAHARDKSEPRPR

PEPTIDE B. NSPKNGSNQRDKAGASEPRPR

PEPTIDE C. NSPKNGSNQSEPRPRKNGSNQ PEPTIDE D. NSPKNGSNQLVISEPRPRPSRTPS

PEPTIDE E. NSPKNGSNQLVIPSRTPS

PEPTIDE F. NSPKNGSNQAHARDKAGASEPRPR

These 6 peptides were then tested for their reaction with sera from known IM patients and with sera from normal controls. The sera were all well characterized for the presence/absence of IgM antibodies to EBV by immunofluoresence. The results of that testing are presented in Table 4.

Pooled control IM serum

** Pooled control normal serum

IM Infectious mononucleosis

NOR Normal

These results demonstrate that any of these epitope combinations could be used to identify serum from IM patients. However some of the peptides gave reasonably strong reactions with sera from normal individuals (peptides A,B and F in particular). Of those peptides which had low reactions with the normal sera peptide E gave the lowest values and so was chosen for further studies.

Peptide E was assayed with a larger contingent of sera from patients with IM and normal controls. Control wells, containing only BSA, were also included in the assay to check for sera which may react with the carrier protein. The results of that assay are presented in Table 5.

TABLE 5 ELISA ASSAY OF PEPTIDE E.

These results demonstrate that peptide E could be used to reliably detect cases of IM while showing little reaction with sera from normal individuals.

The clinical and other data obtained indicates that the ELISA assay using the peptides of the present invention is a specific and reliable test for the diagnosis of infectious mononucleosis and related diseases. The present invention should also find use in the treatment of such diseases.

Those skilled in the art will appreciate that the above embodiments are given by way of exemplification of the invention only, and that changes may be made to the details set out therein without departing from the scope of the invention as defined in the following claims.

Claims

1. A peptide comprising a sequence which includes at least one segment which codes for an antigen recognized by EBV-specific antibodies raised during IM or a related disease.

2. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence AHARDKAGAVMAMIL.

3. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence

ASLNSPKNGSNQLVI.

4. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence ELESEPRPRPSRTPS.

5. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence QAMKKIEDKVRKSVD.

6. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence LIIASLRKDRKLYAE.

7. A peptide as defined in Claim 1, wherein said at .least one segment comprises the sequence VSFSKTRRAIRESRA.

8. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence

CNYSAGEEDDQYHA .

9. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence RPHRRPVSKRPTHKP.

10. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence EITQEENRGEQRLGH.

11. A peptide as defined in Claim 1, wherein said at least one segment. comprises the sequence

GALRARLDRPRPTAQ.

12. A peptide as defined in Claim 1. wherein said at least one segment comprises the sequence SRSRGREAKKVQISD.

13. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNG.

14. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence KNGSNQ.

15. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence SNQLVI.

16. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence AHARDK.

17. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence RDKAGA.

18. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence VMAMIL.

19. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence SEPRPR.

20. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence PSRTPS.

21. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNGSNQAHARDKSEPRPR.

22. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence

NSPKNGSNQRDKAGASEPRP .

23. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNGSNQSEPRPRKNGSNQ.

24. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNGSNQLVISEPRPRPSRTPS.

25. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNGSNQLVIPSRTPS.

26. A peptide as defined in Claim 1, wherein said at least one segment comprises the sequence NSPKNGSNQAHARDKAGASEPRPR.

27. A peptide as defined in any one of Claims 1 to 26 in association with any suitable carrier and/or diluent and/or adjuvant.

28. A method of identifying a polypeptide suitable for use in the diagnosis of IM and related diseases, said method comprising:

(1) noting within EBV open reading frames transcribed late in the viral cycle for which a translation product may or may not have been established; (2) synthesising one or more polypeptides each of which includes at least one segment wherein each segment comprises at least part of the amino acid sequence identified in that open reading frame; and

29. A method of diagnosis of infectious mononucleosis or a related disease, said method comprising assaying serum from a patient suspected of having infectious mononucleosis or a related disease with at least one peptide as defined in any one of Claims 1 to 27.

30. A kit for use in the diagnosis of IM or a related disease, said kit comprising:

(a) at least one peptide as defined in any one of Claims 1 to 27; and

(b) a means for indicating the presence of an immunoreaction between said peptide and anti-EBV antibodies.

31. A kit for use in the diagnosis of IM or a related disease, said kit comprising:

(a) at least one peptide as defined in any one of Claims 1 to 27; and

(b) a means for indicating the presence of a reaction between said peptide and another molecule.

32. A vaccine that, when administered, is capable of inducing antibodies effective against infection by EBV, said vaccine comprising:

(a) at least one peptide as defined in any one of Claims 1 to 26; and

(b) a carrier and/or diluent and/or adjuvant.

33. Antibodies and substantially whole antibodies raised to - or induced by - at least one peptide as defined in any one of Claims 1 to 27.

34. A peptide as defined in Claim 1 substantially as described with reference to the Examples.

35. A method as defined in Claim 28 substantially as described with reference to the Examples.