WO1995016704A1 - Hepatitis b virus peptides - Google Patents

Abstract

A synthetic peptide (or close analogue thereof) which is part of the natural sequence of hepatitis B surface antigen is chosen from the following (AA indicates amino acid number): AA 211-222, AA 316-333, AA 361-378. Also provided are derivatives of the above in which the peptide is conjugated to a hepatitis B virus T-cell epitope of forms part of a fusion protein or is conjugated to an non-immunogenic oligo- or polysaccharide or lipid. Vaccines comprising the above peptides or conjugates are also provided, together with a method of immunising or treating individuals suffering from viral disease.

Description

HEPATITIS B VIRUS PEPTIDES

The present invention relates to Hepatitis B antigens suitable for preparing vaccines against hepatitis B infection. The present invention also relates to novel formulations for therapeutic treatment of Hepatitis infections and to combination vaccine formulations including a Hepatitis B vaccine component.

Viral hepatitis, caused by the hepatitis B virus, is a very common viral illness which is responsible for many cases of liver cancer. Thus the development of effective vaccines is critical and, despite notable successes (for example a yeast derived recombinant vaccine known as Engerix -B made by SmithKline Beecham

Biologicals), is still an on-going task. A review on modern hepatitis vaccines, including a number of key references, may be found in the Lancet, May 12th 1990 at page 1142 ff (Prof A.L.W.F. Eddleston). See also 'Viral Hepatitis and Liver Disease' (Nyas, B.Ν., Dienstag, J.L., and Hoofnagle, J.H., eds, Grune and Stratton, Inc. (1984) and 'Viral Hepatitis and Liver Disease' (Proceedings of the 1990 International Symposium, eds F.B. Hollinger, S.M. Lemon and H. Margolis, published by

Williams and Wilkins). Hepatitis B virus (HBV) infection in humans is associated with the ocurrence in the serum of various structures carrying the hepatitis B surface antigen (HBsAg). In addition to infectious virions, filamentous and spherical particles of 22nm in diameter are present which are formed by association of host-derived lipids with the three hepatitis surface proteins: the major (S), middle (M) and large (L) proteins.

These proteins share the same sequence of 226 amino acids on the HBV genome, known as the S coding sequence. The entire amino acid coding sequence which immediately precedes the S protein coding sequence on the HBV genome is known as the pre-S coding sequence. The pre-S coding sequence codes for a 55 amino acid sequence which immediately precedes the S protein called the pre-S2 region and, depending on the virus subtype, either a 108 or 119 amino acid sequence which immediately precedes the pre-S2 region called the pre-S 1 region.

For the complete amino acid sequence of these regions, see European Patent

Application Publication Number 0414 374.

One approach to the development of synthetic vaccines for prevention and cure of the disease is to try to identify immunodominant T-cell epitopes. Ferrari et al

(Gastroenterology, 1992, 103, 255-263) have reported identification of such epitopes within the pre-S1 molecule, in particular amino acids 12 to 31. Ferrari et al did not carry out a study of T-cell epitopes within the S-sequence.

Work has also been carried out by Celis et al (1989) which has identified amino acids 193 to 207 as a T-cell epitope.

The present study was performed to examine which regions of the hepatitis B virus envelope proteins (HBV-env) can function as T-cell epitope. As described hereinbelow, the in vitro lymphoproliferative response of PBMC from 32 HBV-env vaccine recipients towards a series of 19 synthetic peptides, representing selected sequences of the preSl, preS2 and S region, was measured.

Surprisingly, we have found in the present study that T-cell epitopes exist which have not been identified by previous workers. The novel T-cell epitopes described herein in the S-sequence are of potential importance for the design of improved and more immunogenic vaccines.

The present invention provides a peptide which is part of the natural sequence of Hepatitis B surface antigen chosen from the following (AA indicates amino acid number):

AA 211 - 222

AA 316 - 333

AA 361 - 378

The amino acid sequences are given in the Examples hereinbelow.

The peptide of the invention may also be a close analogues of the above, for example a compound in which a limited number (for example one, two or three) amino acid insertions, deletions or mutations have been made. It is understood that this can include amino acid differences between serotypes. The peptide of the invention may also be glycosylated.

In a preferred aspect the invention provides AA 316 - 333 or a close analogue thereof having essentially the same function.

AA 316-333 was found to have at:

position 319: mutation site found in S

position 320: glycosylation site in S position 333: AA-difference between d and y serotypes

The peptides of the invention can be made by standard methodology. Compounds within the scope of the invention include derivatives of the peptides of the invention.

Such derivatives may be, for example: (a) Homo-conjugates, obtained by conjugating a peptide according to the invention with one or more other HBV T-cell epitopes. In one aspect, two or more of the peptides according to the invention may be conjugated with each other so as to form a molecule with two or more T-cell epitopes. Homo-conjugates as hereinabove defined may advantageously include known T-cell epitopes such as AA 12 - 31 and AA 193 -207. Known spacer groups may be used if appropriate and the conjugation may be carried out by methods known in the art.

(b) Recombinant polypeptides, made by well known recombinant DNA techniques in which the sequence of one or more of the peptides according to the invention has been 'built in' to a longer amino acid sequence. Advantageously the molecule will incorporate more than one T-cell epitope. It will be understood that the recombinant product does not correspond to the natural HBsAg molecule but is a synthetic molecule designed to present T-cell epitopes in an advantageous manner. (c) Hetero-conjugates in which a peptide or homo-conjugate according to the invention is conjugated to a non-immunogenic peptide or oligo- or polysaccharide, for example the Hib polysaccharide. The T-cell epitope(s) in such a molecule advantageously functions as a 'carrier' to improve or induce a humoral response against another antigen. Appropriate spacer groups and standard methods for carrying out the coupling chemistry may be employed.

In certain circumstances it may be desirable to conjugate the peptide of the invention or derivative thereof as hereinabove defined with other molecules, for example lipids and/or proteins, to improve delivery. Conjugates of this type are also within the scope of the invention.

The invention also provides a vaccine composition comprising an effective amount of the peptide of the invention or derivative or conjugate thereof. The advantage of the above vaccines is that they have both an immunoprotective and therapeutic potential. It is an accepted fact that an optimal vaccine needs to stimulate not only neutralising antibody but also needs to stimulate as effectively as possible cellular immunity mediated through T-cells. The vaccines of the invention may be used to provide protection against primary infection and stimulate advantageously both specific humoral (neutralising antibodies) and also effector cell mediated (DTH) immune responses.

Accordingly there is further provided a method of immunising or treating patients by administering to a human in need thereof an effective dose of the vaccine of the invention.

The vaccine of the invention comprises the peptide, derivative thereof or conjugate of the invention admixed with a suitable carrier. In one aspect the vaccine of the invention may comprise a potent adjuvant, for example 3-O deacylated

monophosphoryl lipid A (or 3 De- O-acylated monophosphoryl lipid A), available from Ribi Immunochem, Montana, USA. For the preparation of this material see GB 2 220 211 A. The carrier may be an oil in water emulsion, a lipid vehicle, or alum (aluminium salt).

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalene and an emulsifier such as Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.

Advantagously the vaccine of the invention comprises one or more other antigens in addition to the peptide, derivative or conjugate of the invention so that it is effective in the treatment or prophylaxis of additional bacterial, viral or fungal infections. Preferably the vaccine comprises at least one other component selected from non-hepatitis antigens which are known in the art to afford protection against one or more of the following: diphtheria, tetanus, pertussis, Haemophilus influenzae b (Hib), and polio. Such combination vaccines may advantageously include a component which is protective against Hepatitis A, especially the killed attenuated strain derived from the HM-175 strain as is present in Havrix (SmithKline Beecham Biologicals).

Suitable components for use in combination vaccines are already commercially available and details may be obtained from the World Health Organisation. For example the IPV component may be the Salk inactivated polio vaccine. The pertussis component may comprise a whole cell or acellular product. In one aspect the vaccine according to the invention is a paediatric vaccine.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, US Patent 4,372,945 and by Armor et al., US Patent 4,474,757.

The amount of antigen in each vaccine dose is selected as an amount which induces an immunoprotective or therapeutic response without significant, adverse side effects in typical vaccinees. Such amount will vary depending on which specific agents are employed. Generally it is expected that each dose will comprise 1-1000ug of total antigen, preferably 2-100ug, most preferably 4-40ug. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

The following examples describe the identification of T-cell epitopes according to the invention. Examples

Materials and methods

Vaccinees: PBMC were isolated from subjects previously immunised with the Engerix B (PRS003-study), SL^* (PRS003-study) or Hevac B vaccines( table 1). The selected vaccine recipients were cellular (lymphoproliferative response to HBsAg in vitro) and humoral (HBsAb in vivo) high responders to the different hep. B vaccines. Since all subjects had diverse human leucocyte antigen (HLA) backgrounds (outbred population) they were HLA class II typed, using serological (DR and DQ) and DNA (DR and DP)-typing techniques.

Antigens: The amino acid sequence and position in the HBV-env protein of each of the 19 synthetic peptides (subtype ad) used in this study, are represented in figures 1 and 2.

T-cell proliferation assays: Assays were performed in 200 μl of RPMI 1640 supplemented with 25 mM Hepes, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 5.10^-5 M β-mercaptoethanol, 5.10^-5 M indomethacine and containing 10 % heat-inactivated human AB⁺ serum. 4.10⁵ PBMC were cultured in 96- well round-bottomed culture plates containing various concentrations of the HB-env peptides (3, 10 or 30 μg/ml final concentration) or HBsAg particles (3 μg/ml). After 5 days of incubation (37°C in 5% CO₂), ³H-thymidine (0.5 μCi) was added to the wells. 20h later the cells were harvested with an automated harvesting device and thymidine incorporated into the DNA was determined by liquid scintillation counting. The stimulation index (S.I.) is the ratio between the mean cpm obtained in the presence of antigen and those obtained in the absence of antigen.

Results

A) PBMC responses to 19 synthetic HB-env peptides

Table 1 represents the PBMC responses to different concentrations of the HB-env synthetic peptides in 32 HB-env vaccinees. The proliferative response induced by 3 μg/ml HBsAg is also indicated. Results are expressed as stimulation indices. Stimulation indices higher than 2 were considered as significant values of

proliferative response. No significant levels of PBMC proliferation to HB-env peptides were observed in 11 non-vaccinated control persons (results not shown) Figure 3 represents the % of subjects with a positive proliferative response to each of the peptides tested. A person was considered to recognize a given peptide if his/her S.I. to that peptide was > 2 for at least two different antigen concentrations. These values are underlined in table 1. All vaccinees were responsive to HBsAg (100 % of the subjects tested). Five peptides elicited a T-cell response in≥ 30 % of the subjects examined. One T-cell epitope, recognized by 12 out of 17 SL^* vaccinees tested, was located in the preS1 sequence (R452; AA 12-31). Four other T-cell epitopes were located in the S-sequence: R443 (AA 193-207), 2 (AA 211-222), 6 (AA 316-333) and 9 (AA 361-378). They were recognized by 53 % (17/32), 33 % (10/30), 55 % (17/31) and 30 % (9/30) of the subjects studied (table 2).

B) HLA-restriction of peptide presentation

In humans, T helper responses are restricted by products of the HLA DR, DP and DQ genes of the MHC. To investigate the HLA-restriction of the peptide presentation, we analyzed the correlation between an individuals' HLA molecules and the recognition of a peptide by that individual. To perform these deductions each peptide was considered in detail. The HLA phenotype of all subjects recognizing a certain peptide was listed and some potential HLA-restriction molecules were withheld. This was analysed for the 5 epitopes recognized by≥ 30 % of the subjects tested. The methodology used is clarified in tables 3 to 7. An example is described for peptide 6 (table 6). If peptide 6-recognition occured in more than 50 % of the individuals bearing a particular HLA-molecule, than this HLA-molecule was considered as a potential HLA-restriction determinant in the presentation (recognition) of peptide 6. By doing so, 4 potential HLA-restriction molecules for peptide 6 were determined: DR2, DPw2, DPw3 and DQw1. In a next step each potential HLA-restriction molecule was analyzed in more detail. An example is given for DR2. Peptide 6 -recognition occured in 13 out of 14 DR2⁺ individuals tested (> 50 %). One DR2⁺ individual was unable to recognize peptide 6. Four non-DR2⁺ individuals also recognized peptide 6. The same listing was done for every potential HLA-restriction determinant. Considering all results, it is most likely that the DR2-molecule is the restriction determinant for peptide 6-presentation (or at least one of the determinants).

The same deduction was done for the four other epitopes (tables 3, 4, 5 and 7). Peptide R443 (AA 193-207) was most probably presented by DPw4, and peptide R452 (AA 12-31) by DQw1 or DPw4. No clear HLA-restriction pattern could be demonstrated for peptides 2 and 9. Perhaps these are promiscuous peptides that bind in more than one HLA-molecule.

A summary of the proposed restriction determinants is given in table 8.

We are trying to consolidate the proposed HLA-restriction with blocking experiments using moAb's to DR,DP and DQ. The restriction pattern of peptide presentation is also studied in peptide-specific T-cell lines. Conclusion

We report the identification of five immunodominant T-cell epitopes within the hepatitis B envelope proteins which could be helpful for the design of improved and more immunogenic hepatitis B vaccines. Our data were the result of a population (n=32)- based analysis and not of a clone-based analysis. This makes our data more relevant.

Claims

Claims

1. A peptide which is part of the natural sequence of Hepatitis B surface antigen chosen from the following (AA is amino acid number):

AA 211 - 222

AA 316 - 333

AA 361 - 378 or a close analogue of the above sequences in which there are up to three insertions, deletions or mutations.

2. A peptide according to Claim 1 which is glycosylated.

3. A derivative of a peptide according to claim 1 or claim 2 in which the peptide has been conjugated with one or more HBV T-cell epitopes.

4. A derivative of a peptide according to claim 1 or claim 2 in which the said peptide is fused to an amino acid sequence, with the proviso that the derivative does not correspond to natural hepatitis B surface antigen.

5. A derivative according to claim 4 which comprises more than one T-cell epitope.

6. A peptide according to claim 1 or claim 2 or a derivative according to any one of claims 3 to 5 which is conjugated to a non-immunogenic peptide, oligo- or polysaccharide, or lipid.

7. A vaccine composition comprising a peptide or derivative according to any preceding claim in combination with a suitable adjuvant or carrier.

8. A vaccine composition according to claim 7 in which the adjuvant is 3-O-deacylated monophosphoryl lipid A.

9.. A combination vaccine comprising a vaccine according to claim 7 or 8 admixed with at least one other component effective in the treatment or prophylaxis of additional bacterial, viral or fungal infections.

10. A method of immunising or treating a human patient by administering to the patient an effective amount of the vaccine according to any one of claims 7 to 9.

11. The use of a peptide or derivative according to any one of claims 1 to 6 in the

manufacture of a vaccine for the prophylaxis or treatment of viral infections.

12. A diagnostic test kit or reagent comprising a protein or derivative thereof according to any one of claims 1 to 6.