WO1989006658A1

WO1989006658A1 - Immunogenic peptides

Info

Publication number: WO1989006658A1
Application number: PCT/US1989/000097
Authority: WO
Inventors: Stanley M. Lemon
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 1988-01-13
Filing date: 1989-01-13
Publication date: 1989-07-27
Also published as: AU3048689A

Abstract

The instant invention relates to immunogenic synthetic peptides comprising amino acid sequences corresponding to antigenic determinants of the capsid of HAV, advantageously, linked directly or through a spacer molecule to carrier molecules suitable for vaccination of mammals.

Description

IMMUNOGENIC PEPTIDES

BACKGROUND OF THE INVENTION Technical Field

The instant invention relates to vaccines for the prevention of hepatitis A viral (HAV) infection and, in particular, to vaccines comprising synthetic peptides having an amino acid sequence homologous to a portion of the amino acid sequence of A neutralization immunogenic site of the HAV capsid.

BACKGROUND INFORMATION

HAV accounts for 38% of the cases of viral hepatitis reported annually in the United States (D.P. Francis et al. Am. J. Med. 76:69-74 (1984)). On the basis of morphologic and biophysical characteristics, HAV has been classified among the Picornaviridae (I.D. Gust et al, Intervirology 20:1-7 (1983)). Like poliovirus, the virion is a naked icosahedron. The capsid contains at least three major structural proteins (VPl, VP2 and VP3), of which VPl was previously believed to be the dominant surface protein (A.G. Coulepis et al, Intervirology 18:107-127 (1982)). Only a single antigenic specificity has been associated with HAV, and significant antigenic variation has not been recognized among different HAV strains (S.M. Lemon et al. Infect. Immun. 42:418-420 (1983)). Denaturation of the virus leads to a loss of antigenicity, suggesting that the relevant antigenic sites are strictly conformationally dependent (J.V. Hughes et al, J. Virol. 52:465-473 (1984)). By covalently cross-linking monoclonal antibodies to intact HAV, one major antigenic site has been located on VPl (J.V. Hughes et al, J. Virol. 52:465- 473 (1984)). Analysis of mutants that are resistant to neutralizing monoclonal antibodies has proven useful for identifying immunogenic sites involved in the neutralization of other picornaviruses (D.C. Diamond et al. Science 229:1090-1093 (1985); P.D. Minor et al, J. Gen. Virol. 65:1159-1165 (1985); B. Sherry et al, J. Virol. 53:137-143 (1985)). Genomic nucleotide substitutions associated with neutralization resistance, when analyzed in the context of the three-dimensional structure of poliovirus type 1 (PVl) or human rhinovirus type 14 (HRV14), have identified multiple discrete sites on the virion surface which appear to function both as immunogens and as attachment sites for neutralizing monoclonal antibodies (J.M. Hogle et al. Science 229:1358-1365 (1985); M.G. Rossman et al. Nature (London) 317:145-153 (1985)). Four such neutralization immunogenic sites have been identified on both PVl and HRV14; they involve all three surface polypeptides (J.M. Hogle et al, Science 229:1358-1365 (1985); B. Sherry et al, J. Virol. 57:246-257 (1985)). In general, mutation occurring within one neutralization immunogenic site has conferred resistance to all monoclonal antibodies binding at that site, but not to those binding at alternate sites.

At present, no vaccine is available for prevention of infection with HAV. Because HAV closely resembles poliovirus and can be grown in cell cultures, intensive efforts over the past few years have been directed at the development of inactivated and/or live attenuated HAV vaccines. For a variety of technical reasons (including difficulties in achieving high yields of virus in cell cultures), a clinically useful vaccine for prevention of infection with this virus has not yet been developed.

SUMMARY OF THE INVENTION

It is a general object of the instant invention to provide synthetic peptides that are capable of inducing the production in mammals of neutralizing antibodies against HAV.

It is another object of the invention to provide vaccines comprising a synthetic peptide(s) having an amino acid sequence corresponding to an antigenic determinant of the HAV capsid that is capable of inducing protective immunity in mammals against HAV.

It is an additional object of the invention to provide a method of detecting the presence of HAV antibodies in biological test samples.

These, and other objects which will be clear to those skilled in the art from the following detailed description, have been accomplished by providing synthetic peptides useful in producing an immunogenic response to the viral causative agents of HAV.

The invention relates to immunogenic preparations and vaccines made therefrom. Vaccines of the instant invention comprise a synthetic peptide(s) having an amino acid sequence corresponding to an antigenic determinant of the VP3 structural protein of the capsid of HAV, advantageously, linked to a suitable carrier molecule. In one embodiment, the instant invention comprises a synthetic peptide having an amino acid sequence homologous to a portion of the amino acid sequence of a neutralization immunogenic site of the VP3 structural protein of the capsid of HΛV, which peptide is capable of inducing in a mammal neutralizing antibodies against HAV. The synthetic peptide(s) of the instant invention corresponds to the region of VP3 containing the aspartate (asp)-70 residue.

In another embodiment, the instant invention comprises a DNA segment encoding the above-described peptides.

In a further embodiment, the instant invention comprises an immunogenic conjugate capable of inducing in a mammal neutralizing antibodies against HAV. The conjugate comprises a synthetic peptide having an amino acid sequence corresponding to an antigenic determinant of the VP3 structural protein of the capsid of HAV.

In yet another embodiment, the instant invention comprises a method of producing immunity to HAV comprising administering the above-described conjugate to a mammal. In another embodiment, the instant invention comprises a method of detectiπ-j the presence of HAV antibodies in biological test samples.

BRIEF DESCRIPTION OF THE DRAWING

FIGURES 1A and IB. Nucleotide sequence of VPl of HM175 of HAV. FI_GURES 2_A and 2B.Nucleotide sequence of VP3 of HM175 of HAV.

FIGURE 3. Competition between monoclonal antibodies for attachment to HAV fixed to a solid- phase support.

FIGURE 4. Map of genomic regions from various HAV neutralization-escape mutants sequenced by the dideoxynucleotide method.

FIGURE 5. Resistance of mutant S3U to neutralization by 9 different monoclonal antibodies to HAV.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to synthetic peptides, and DNA segments encoding same, corresponding to immunogenic epitopes of HΛV and vaccines made therefrom. These novel immunogenic agents are prepared by synthesizing, either chemically or by recombinant techniques, peptides having an amino acid sequence corresponding to, or homologous with, B-cell epitopes present on the VP3 structural protein of the capsid of HΛV. The peptides of the instant invention correspond to the region of VP3 containing the asp residue at position 70. The peptides, advantageously, linked to an appropriate carrier molecule, evoke the production in mammals of high titers of neutralizing antibodies against HAV. Vaccines thus formed are useful for immunization against HAV infection when adminis ered to mammals, for example, intramuscularly, subcutaneously or intradermally, advantageously, intradermally. To topologically map immunogenic sites on HAV which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition i munoassays employing radiolabelled monoclonal antibodies and HM-175 virus according to procedures described in J. Virol., Vol. 61, No. 2, Feb. 1987, pp. 491-498 (the entire contents of which reference is hereby incorporated by reference and relied upon). Whereas two antibodies (K3-4C8 and K3-2F2) bound to overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8—K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodomin- ant in humans.

Virus mutants resistant to monoclonal antibody-mediated neutralization have proven helpful in identifying immunogenic sites on other picornaviruses (D.M.A. Evans et al, Nature 304:459- 462 (1983); P.D. Minor et al, J. Gen. Virol. 67:1283-1291 (1986); B. Sherry et al, J. Virol. 53:137-143 (1985)), but the application of this approach to characterization of HAV antigenic sites has been hampered by slow and inefficient virus replication ln vitro. Most HAV isolates are noncytopathic, highly cell-associated, and demonstrate a substantial nonneutralizable fraction in vitro (S. M. Lemon, N. Engl. J. Med. 313:1059- 1067 (1985); J.R. Ticehurst, Seminars in Liver Pis. 6:46-55 (1986)). To overcome these problems, a radioimmunofocuε assay was developed for HAV. The assay was based on the immune autoradiographic detection of virus replication foci developing beneath agarose overlays. The method was modified for clonal isolation of virus variants (S.M. Lemon et al, J. Clin. Microbiol. 17:834-839 (1983); S.M. Lemon et al, J. Virol. Methods 11:171-176 (1985)). A quantitative neutralization assay, based on the radioimmunofocus assay (S.M. Lemon et al, J. Infect. Pis. 148:1033-1039 (1983)), demonstrated that extraction of virus with lipid solvents substantially reduces the nonneutralizable virus fraction (S.M. Lemon et al, J. Gen. Virol. 66:2501- 2505 (1985)). Nonetheless, most clonally isolated virus variants recovered from agarose overlays following stringent monoclonal antibody-mediated neutralization reactions demonstrated continuing neutralization susceptibility. The selection of spontaneous neutralization-escape mutants of HAV required a process of repeated neutralization and passage in the presence of antibody, followed by clonal isolation from agarose overlays (J.T. Stapleton et al, J. Virol. 61:491-498 (1987)).

Neutralization resistance of such mutants is associated with reduced binding of antibody to the virion (J.T. Stapleton et al, J. Virol. 61:491- 498 (1987)). To identify capsid protein amino acid mutations associated with neutralization resistance, and by inference sites involved in antibody attachment on the virion surface, methods for RNA sequencing (P.M.A. Evans et al, Nature 304:459-462 (1983); F. Sanger et al, Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977)) were modified to permit the sequencing of as little as 2 to 10 ng of template RNA.

Mutant S30, selected by neutralization and passage in the presence of monoclonal antibody K2- 4F2, showed no decrease in titer when incubated with this antibody (J.T. Stapleton et al, J. Virol. 61:491-498 (1987); see Table 1). The entire capsid- encoding region of S30 RNA was sequenced by primer- extension (see Example 2). Its sequence was then compared with that obtained from cPNA clones of parental virus with normal neutralization phenotype, and with the sequence of S32 virus. This variant was selected against monoclonal antibody B5-B3 but had substantially lost neutralization resistance during amplification in the absence of antibody.

The only mutation identified in the capsid- encoding region of the neutralization-resistant S30 virus was a G to C substitution at base 1677, pre¬ dicting a change in asp-70 of capsid protein VP3 to his. This substitution also was present in two other mutants selected against K2-4F2, S18 and S27, but absent in mutant S20 which had reverted to neu¬ tralization susceptibility during final large volume cell culture passage (see Table 1). Because of the method of mutant selection (J.T. Stapleton et al, J. Virol. 61:491-498 (1987)), these viruses are be¬ lieved to represent sibling clones derived from a single neutralization-resistant parent. Asp-70 was unchanged in S32, and also in SI, S33, S34 and S57. The SI, S33, S34 and S57 mutants were initially selected for resistance to other antibodies but had reverted to neutralization susceptibility during passage in the absence of antibody. In variant S32, a C to T mutation was evident at base position 2512, predicting a change in ser-102 of VPl to leu. S32 demonstrated significant neutralization suscepti¬ bility after ampli ication in the absence of antibody (see Table 1).

Table 1 Neutralization Resistance of HAV Variants

Control Cognate Neutralization Neutralization Antibod Mutant Index¹ Index²

Neutralization index = log^_Q reduction in virus titer following neutralization with the cognate antibody, determined by radioimmuno- focus reduction assay (S.M. Lemon et al, J. Infect. Pis. 148:1033-1039 (1983))

Neutralization index of parent HM175 virus in same assay. 658 1°

To determine whether the substitution at asp-70 of VP3 conferred resistance to other monoclonal antibodies (see Example 1), S3ϋ was tested for neutralization resistance to nine other monoclonal antibodies, including B5-B3 (see Example 3). Although the degree of neutralization varied with different antibodies, S30 was relatively resistant to each. Neutralization resistance was most evident with monoclonal antibodies having activities similar to the cognate K2-4F2 in competition studies (813, 6A5, 10.09 and 1B9; see Example 1), but S30 was at least partially resistant to each of the antibodies tested.

Asp-70 of VP3 is, therefore, involved in the antigenic site recognized by each of these monoclonal antibodies, and is thus part of an immunσdominant antigenic site of HAV. While the mutation at asp-70 of VP3 of HAV might hypo- thetically have resulted in capsid conformational changes preventing neutralization by antibodies binding at distant sites, such an interpretation conflicts with crystallographic evidence that mutations conferring neutralization resistance in poliovirus and rhinσvirus cluster at specific surface sites most likely representing the relevant epitopes (M.D. Rossman et al. Nature 317:145-153 (1985); J.M. Hogle et al. Science 229:1358-1365 (1985)).

Given in FIGURES 1A and IB is the nucleoti e sequence of VPl of HM175 strain HΛV (circled residue is ser-102 of predicted amino acid sequence). Figures 2A and 2B show the nucleotide sequence of VP3 of HM175 strain HAV (circled residue is asp-70 of predicted amino acid sequence).

SUBSTITUTE SHEET Synthetic peptides of the instant invention derived from the asp-70 region of VP3 comprise, for example, an amino acid sequence that includes IPTLAAQFPFNASDSVGQ, DSVGQQ, SDSVGQ, ASPSVG, NASDSV, FNASPS, PFNASP or a portion of any one of these sequences that is capable of inducing neutralizing antibodies.

Peptides of the invention can be synthesized either chemically (R.B. Merrifield, J.A.C.S. , Vol. 83, 1963, pp. 2149-2154) or using recombinant techniques (B.E. Clarke et al, Nature, Vol. 330, 1987, pp. 381-384).

Carrier molecules to which the synthetic peptides of the instant invention can be covalently linked (conjugated) are, advantageously, non-toxic, pharmaceutically acceptable and of a size sufficient to produce an immune response in mammals. An example of a suitable carrier molecule is the hepatitis B core protein (B.E. Clarke et al, Nature, 330:381-384 (1987)). Other suitable carrier molecules well known in the art can also be used.

The synthetic peptides can also be administered with a pharmaceutically acceptable adjuvant, for example, alum. Linkage of a carrier molecule to a synthetic peptide of the invention can be direct or through a spacer molecule. Spacer molecules are, advantageously, non-toxic and reactive. Suitable spacer molecules include those well known in the art.

Linkage of the carrier molecule to the synthetic peptide can be accomplished using a coupling agent. Advantageously, the heterofunctional coupling agent M-maleimidobenzoyl- N-hydroxysuccinimide ester or the water soluble compound m-maleimido-benzoylsulfosuccinimide ester is used (Green et al. Cell 28:477 (1982)).

The present invention also relates to a vaccine against HAV infection comprising, in addition to the above-described synthetic peptides having an amino acid sequence corresponding to the region of VP3 containing the asp-70 residue (examples of such sequences being given above), an additional immunogenic sequence(s) from the HAV capsid which, advantageously, is a synthetic peptide having an amino acid sequence corresponding to the region of VPl containing the ser-102 residue. Such synthetic peptides comprise, for example, a sequence of amino acids that includes TFTFNS, FTFNSN, TFNSNN, FNSNNK, NSNNKE, SNNKEY, or a portion of any one of these sequences.

The present invention also relates to a method of producing immunity to HAV in a mammal comprising administering to a mammal at least one of the above-described conjugates in an amount sufficient to induce the production of neutralizing antibodies against HAV.

In addition to being used as a vaccine or a component of a vaccine, the synthetic peptides of the instant invention can also be used for diagnostic purposes. Peptides of the instant invention can be used, for example, in standard enzyme linked immunosorbent assays or radioimmuno- assays to detect the presence of HAV antibodies. The following nonlimiting examples illustrate the invention in more detail. Exampl e 1

COMPETITION BETWEEN MONOCLONAL ANTIBODIES FOR ATTACHMENT TO HAV FIXED TO A SOLID-PHASE SUPPORT

Gradient-purified HM175 strain HΛV was bound to polyclonal human convalescent antibody coating wells of a polyvinylchloride microtiter plate (J.T. Stapleton et al, J. Virol. 61:491-498 (1987)) Dilutions of ten different urine monoclonal antibodies were added to the virus-coated wells prior to the addition of much smaller quantities of [¹²⁵I .-labelled monoclonal antibody K3-4C8 or B5-B3 (see toiid ana stippled columns, respectively, in Figure 3). The quantity of radiolabelled antibody bound to virus was compared with the quantity bound in the absence of any competing antibody (see Figure 3). Results obtained with radiolabelled K3-4C8 were similar to those obtained with radiolabelled K2-4F2. The source of the monoclonal antibodies was as follows: K3-4C8, K3-2F2 and K2-4F2, Commonwealth Serum Institute,

Melbourne, Australia (A. MacGregor et al, J. Clin. Microbiol. 18:1237-1243 (1983)); 6A5, 1B9, 2D2 and 3E1, J. Hughes and E. Emini, Merck Institute of Therapeutic Research, West Point, PA, USΛ (J.V. Hughes et al, J. Virol. 52:465-473 (1984)); 813 and 10.09, D. Crevat and E. Deloince, Clonatec, Paris, France; and B5-B3, R. Tedder, Middlesex Hospital, London, U.K.

^SL'E^ST^fTUTE Example _2

MAP OF GENOMIC REGIONS

The map of genomic regions from various HAV neutralization-escape mutants was sequenced by the dideoxynucleotide method (D.M.A. Evans et al, Nature 304:459-462 (1983); F. Sanger et al, Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977)). The genomic region encoding the capsid proteins of HM175 strain HAV is depicted at the top of Figure 4, with nucleotide positions and putative peptide cleavage sites displayed according to Cohen et al (J.I. Cohen et al, J. Virol. 61:50-59 (1987)). At the right of Figure 4, the various HM175 variants included in this study (SI through S57) are listed along with the parent virus HM175, while to the left are shown the antibodies against which these variants were selected. JC was polyclonal human convalescent antibody, while the other antibodies were murine monoclonal ascitic fluids. Solid lines indicate RNA regions sequenced, while vertical arrows indicate the location of identified nucleotide base substitutions. Neutralization-escape variants of HM175 virus were isolated as described previously (J.T. Stapleton et al, J. Virol. 61:491-498 (1987)), and amplified in 850 cm² roller bottle cultures of

BS-C-1 cells in the absence of antibody (to conserve antibody) . Virus was assessed for continued neutralization-resistance or reversion to neutralization susceptibility at two weeks by radioimmunofocus reduction assay (see Table 1; J.T. Stapleton et al, J. Virol. 61:491-498 (1987); S.M. Lemon et al, J. Infect. Pis. 148:1033-1039 (1983)) and harvested by freeze-thawing of cells at three weeks. After purification of virus on hybrid sucrose-cesium chloride gradients (J. de Chastonay et al, Virology 157:268-275 (1987)), virus was treated with proteinase K/sodium dodecyl sulfate (J.R. Ticehurst et al, Proc. Nat. Acad. Sci. USA 80:5885-5889 (1983)) and viral RNA was extracted with phenol-chloroform. 2-10 ng purified viral RNA was annealed with 13.5 ng HAV-specific oligonucleotide primers and sequenced by reverse transcriptase-mediated primer-extension (D.M.A.

Evans et al, Nature 304:459-462 (1983); F. Sanger et al, Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977)) in the presence of dideoxynuclotides and high specific activity [ S]-labelled deoxynucleotides (1000-1500 Ci/mole each). Reaction products were separated on 8% acrylamide, 7.6 M urea, Tris-borate- EPTA gradient gels. The sequence was compared with that derived from cPNA clones of cell culture- adapted HM175 virus.

Example 3

RESISTANCE OF MUTANT S30 TO NEUTRALIZATION BY NINE PIFFERENT MONOCLONAL ANTIBOPIES TO HAV

The HAV variant S30 was selected by repeated neutralization and passage in the presence of antibody K2-4F2 (J.T. Stapleton et al, J. Virol. 61:491-498 (1987)) and has a mutation at base position 1677 leading to substitution of asp-70 of VP3 with his. Neutralization resistance was assessed by the radioimmunofocus reduction method (S.M. Lemon et al, J. Infect. Pis. 148:1033-1039 (1983)) using a chloroform-extracted, 0.1% sodium lauryl sarcosinate-treated virus inoculum of 58

16

approximately 4000 radioimmunofocus-forming units/ml and a 10 —i or 10 — "_? dilution of antibody. Results are presented as log^_n reductions in virus titer and are shown for S30 virus as stippled columns, and for diagqnally striped plaque-puπf led parent HM175 vi rus as -ε-θl-_-α- columns

(see Figure 5). Previous studies demonstrated that

S30 virus was also resistant to neutralization by

K3-2F2 (J.T. Stapleton et al, J. Virol. 61:491-498

(1987)). The foregoing invention has been described in some detail by way of examples for purposes of clarity and understanding. It will be obvious to those skilled in the art from a reading of the disclosure that the synthetic peptides of the instant invention may differ slightly in amino acid sequence without departing from the scope of tin? invention. It will also be obvious that various combinations in form and detail can be made without departing from the scope of the invention.

' HEE

Claims

WHAT IS CLAIMEP IS:

1. A synthetic peptide capable of producing an immunogenic response to the viral causative agent of hepatitis A comprising a chain of amino acids having a sequence homologous to a portion of the amino acid sequence of a neutral¬ ization immunogenic site of the VP3 structural protein of the HAV capsid, which peptide is capable of inducing in a mammal neutralizing antibodies against HAV.

2. The peptide according to claim 1, wherein said peptide comprises an amino acid sequence corresponding to a region of VP3 containing asp-70.

3. The peptide according to claim 2, which peptide comprises the sequence PSVGQQ.

4. The peptide according to claim 2, which peptide comprises the sequence SPSVGQ.

5. The peptide according to claim 2, which peptide comprises the sequence ASPVGV.

6. The peptide according to claim 2, which peptide comprises the sequence NASDSV.

7. The peptide according to claim 2, which peptide comprises the sequence FNASPS.

8. The peptide according to claim 2, which peptide comprises the sequence PFNASD.

9. The peptide according to claim 2, which peptide comprises the sequence IPTLAAQFPFNASPSVGQ.

10. An immunogenic conjugate capable of inducing in a mammal the production of high titers of neutralizing antibodies against HAV, said conjugate comprising:

(i) a carrier molecule covalently linked to (ii) a synthetic peptide consisting essentially of a chain of amino acids having a sequence homologous to a portion of the amino acid sequence of a neutralization immunogenic site of the VP3 structural protein of the HAV capsid containing asp-70.

11. The conjugate according to claim 10, further comprising a synthetic peptide having an amino acid sequence homologous to a portion of the amino acid sequence of an immunogenic site of the VPl structural protein of the HAV capsid containing ser-102.

12. The conjugate according to claim 10, wherein said carrier molecule is hepatitis B core protein.

13. The conjugate according to claim 10, wherein said carrier molecule is covalently linked to said peptide through at least one spacer molecule.

14. A method of producing immunity to HAV in a mammal comprising administering to said mammal at least one conjugate according to claim 10 in an amount sufficient to induce the production of neutralizing antibodies against HAV.

15. A method of determining the presence and titers in mammalian serum of neutralizing antibodies against HAV comprising the steps of:

(i) contacting a synthetic peptide consisting essentially of a chain of amino acids having a sequence homologous to a portion of the amino acid sequence of a neutralization immunogenic site of the VP3 structural protein of the HAV capsid containing asp-70, with antibodies from mammalian serum; and

(ii) measuring the formation of complexes between said peptide and said antibody.

16. A PNA segment encoding a sequence of amino acids homologous to a portion of the amino acid sequence of a neutratization immunogenic site of the VP3 structural protein of the HAV capsid, which pepide is capable of inducing in a mammal neutralizing antibodies against HAV.