HLA-restricted hepatitis B virus CTL epitopes

Details HLA-restricted hepatitis B virus CTL epitopes HLA-restricted hepatitis B virus CTL epitopes

1995-06-05

2001-11-27

Wortman, Donna (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Amino acid sequence disclosed in whole or in part; or...

C424S193100, C424S196110, C424S227100

Reexamination Certificate

active

06322789

ABSTRACT:

BACKGROUND OF THE INVENTION
The hepatitis B virus (HBV) is not believed to be directly responsible for damage to hepatocytes, despite its predilection for infecting such cells. Rather, non-viral host factors are implicated in the pathogenesis of hepatitis. It is suspected that a variation in immune responsiveness to HBV infection may account for the wide diversity of syndromes associated with HBV infection.
Following an acute HBV infection, approximately 90% of affected adults recover without sequelae and develop immunity to the virus, although the clinical course of the infection during the acute phase can itself be quite variable. In 5-10% of infected adults, a chronic HBV infection becomes established. Chronic HBV infection can range from asymptomatic carrier state to continuous hepatocellular necrosis and inflammation, and in some instances may lead to hepatocellular carcinoma. Children exposed to HBV infection, particularly those less than one year old, often develop chronic infection and represent the major source of chronic infection. Worldwide, nearly 200 million people are chronically infected with HBV. And finally, in a small percentage of HBV infections (0.1-0.5%) a fulminant hepatitis results in such extreme cell death in the liver that fewer than one-fifth to one-third of these patients survive.
The immune response to hepatitis B virus is as complex as the disease. A variety of humoral and cellular responses have been identified to different regions of the HBV nucleocapsid core and surface antigens. T cell mediated immunity, particularly involving class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL), is believed to play an important role in resistance to hepatitis as well as several other viral infections. CTL recognize antigen in the form of small peptides in association with the class I histocompatibility molecules. The antigen-specific CTL, when stimulated, can secrete mediators which inhibit viral replication and eliminate infected cells, thereby contributing to an individual's recovery from the infection. Although studies suggest that the T cell repertoire of class I-restricted responses is focused on a limited number of discrete immunodominant epitopes of a viral protein (Braciale et al.,
Proc. Natl. Acad. Sci. USA
86:277-281 (1989)), for many viruses, including the hepatitis viruses and particularly HBV, few epitopes have been identified. See also Barnaba et al.,
Nature
345:258 (1990) have identified an All restricted epitope while Jin et al.,
J. Exp. Med.
168:293 (1988) have identified an A3 restricted epitope. Aichele et al.,
J. Exp. Med.
171:1815-1820 (1990), have demonstrated induction in vivo of an antiviral CTL response in an MHC class-I dependent fashion with a peptide from the nucleoprotein of lymphocytic choriomeningitis virus. Recently, Kast et al.,
Proc. Natl. Acad. Sci. USA
88:2283-2287 (1991), described in stimulation of Sendai virus-specific CTL in vivo using free synthetic peptide derived from the nucleoprotein to confer protection against subsequent viral challenge.
It has been suggested that hepatocyte injury during HBV infection may be mediated by an HLA class I-restricted CTL response to HBV antigen. In attempting to define the CTL response to HBV, it has been shown that peripheral blood lymphocytes from patients with acute and chronic HBV may be able to kill autologous hepatocytes in vitro, but the specificity of the cytolytic activity, its HLA restriction elements, and cellular phenotype were not established. See, Mondelli et al.,
J. Immunol.
129:2773 (1982) and Mondelli et al.,
Clin. Exp. Immunol.
6:311 (1987). More recently, Moriyama et al.,
Science
248:361-364 (1990), reported that the HBV major envelope antigen was expressed at the hepatocyte surface in a form recognizable by MHC class I-restricted, CD8
+
cytotoxic T lymphocytes, and by envelope-specific antibodies. However, the HBV epitopic regions responsible for HBV-specific CTL activity were not identified.
The requirement for lympholines such as IL-2 in the generation of CD8+ CTL is well established, although the need for activation of CD4+ T helper cells to provide these lymphokines remains somewhat controversial. While the concept of linked T helper-B cell recognition for antibody production has been firmly defined, there is no compelling evidence for linked T helper-CTL recognition for the in vivo induction of CD8+ CTL. See, e.g., Buller et al.,
Nature
328:77-79 (1987); Sarobe et al.,
Eur. J. Immunol.
21:1555-1558 (1991); and Cassell and Forman,
Annals N.Y. Acad. Sci. :
51-60 (1991).
Individuals chronically infected with HBV are at risk of developing liver cirrhosis and/or hepatocellular carcinoma, and constitute an extremely large reservoir for spreading the disease. It would be desirable to stimulate the immune systems of those chronically infected to respond to appropriate HBV antigens and eliminate their infections, or to be able to prevent the evolution from an acute HBV infection to the chronic stage. Further, as the presently approved HBV vaccines provide only about 90% protection among these immunized, it is desirable to improve the existing vaccines by increasing or diversifying the immunogenicity of the vaccines to elicit a more effective immunity. Means are also needed for predicting which patients with acute HBV infection are likely to develop chronic HBV infection, so that appropriate treatment and precautions can be implemented earlier. Quite surprisingly, the present invention fulfills these and other related needs.
SUMMARY OF THE INVENTION
The present invention provides peptides which induce MHC class I-restricted CTL responses against HBV antigen. The peptides of interest are derived from sequences of the HBV surface antigen or nucleocapsid proteins. In certain embodiments the peptides comprise from six to about thirty amino acids and contain at least one epitope which is capable of inducing a MHC class I-restricted cytotoxic CTL response to HBV where the epitope(s) is within the HBV envelope antigen sequence designated 799.10 (HBenv
349-368
) [Seq. ID NO. 2] Leu-Ser-Pro-Thr-Val-Trp-Leu-Ser-Val-Ile-Trp-Met-Met-Trp-Tyr-Trp-Gly-Pro-Ser-Leu or 799.09 (HBenv
329-348
) [Seq. ID No. 7] Ala-Ser-Ala-Arg-Phe-Ser-Trp-Leu-Ser-Leu-Leu-Val-Pro-Phe-Val-Gln-Trp-Phe-Val-Gly (for subtypes ayw and adw). Other peptide embodiments comprise from six to thirty amino acids and have at least seven amino acids from the HBenv sequence 799.08 (HBenv
309-328
) [Seq. ID No. 1] Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser-Trp-Ala-Phe-Gly-Lys-Phe-Leu-Trp-Glu-Trp (for subtype ayw). In yet other embodiments a peptide is derived from HBV core antigen sequence region designated 802.03 (HBc
91-110
) [Seq. ID No. 4], having the sequence Thr-Asn-Met-Gly-Leu-Lys-Phe-Arg-Gln-Leu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe (subtype ayw). The peptides of the invention can be optionally flanked and/or modified at one or both of the N- and C-termini, as desired, and substitutions, deletions and additions may be made to the peptide as long as the ability of the peptide to stimulate HBV CTL activity or act as an analog antagonist is not substantially adversely affected.
In the various peptide embodiments it will be understood that the peptides can be covalently linked, e.g., via polymerization or conjugation, each to itself to form larger homopolymers, or with different peptides to form heteropolymers. In some instances peptides will be combined in a composition as an admixture and will not be linked. The CTL inducing peptide can also be linked to a lipid-containing molecule capable of enhancing a T lymphocyte response, or may be linked to a T helper peptide which induces a T-helper cell response, or may be linked to both a lipid-containing molecule and a T helper peptide, for example. Linkage to a lipid or a T-helper peptide may be either at the amino or carboxy termini.
Compositions are provided which comprise a peptide of the invention formulated with an additional peptide, a liposome, an

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