Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-28
2003-11-25
Low, Christopher S. F. (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S300000, C530S328000, C530S350000, C435S320100, C435S091200, C424S184100, C424S200100, C514S04400A
Reexamination Certificate
active
06653461
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to nine amino acid-long peptides of the major outer membrane protein (“MOMP”) from
Chlamydia trachomatis
(“Ct”). These peptides activate human cytotoxic T-lymphocytes (“CTLs”).
Ct is an intracellular bacterium that is the leading cause of preventable infectious blindness (ocular trachoma) in the developing world and of sexually transmitted disease (“STD”) in the United States and certain other parts of the developed world. The estimated annual incidence of Ct-caused STD is in the millions. While most Ct-caused disease can be treated with antibiotics, untreated or inadequately treated infections result in hundreds of thousands of cases of pelvic inflammatory disease each year in the United States, alone.
Adverse outcomes of pregnancy, ectopic pregnancy and tubal infertility are among the consequences of genital tract infections with Ct. Moreover, apparent clearance of infection by a given serovar (serologically distinct strain of Ct) can be followed by the infection becoming latent and prolonged or by re-infection. This is important because much Ct-caused pathology results from tissue-damaging inflammatory responses of the immune system that are triggered by repeated or prolonged exposures to the whole organism. Therefore, there is a need for improved means to prevent primary infections.
A great deal of effort has been put into developing a vaccine against diseases caused by Ct infections. While whole inactivated organisms are often used as a vaccine to immunize humans, such a vaccine is not desirable in the case of Ct because certain proteins expressed by Ct, such as chlamydial heat shock proteins, induce pathological immune responses rather than protective immune responses and, thus, contribute to disease. As a result, much vaccine-related activity in chlamydial research is centered on developing a “subunit vaccine” that consists only of Ct protein antigens or specific parts of the proteins that elicit protective immune responses in vaccinees. The fact that B-cell responses (neutralizing antibody) to Ct MOMP protect mice from Ct-caused disease has led to a prevailing theory that MOMP, when used to vaccinate humans, might also induce protective B- and T-cell responses.
However, using whole MOMP as a vaccine is not a good solution. Whole MOMP is too difficult to isolate from natural Ct cultures in large quantities that are sufficiently pure for use in mass vaccination. Larger quantities of recombinant MOMP could theoretically be produced in
E. coli
, but the chemical properties (e.g. insolubility except in detergents) impede its large scale preparation as a non-toxic vaccine. Furthermore, use of whole MOMP has too much risk of adverse side effects.
Consequently, emphasis has been given to developing a subunit vaccine that contains multiple B- and T-cell “epitopes” in MOMP, i.e. short antigenic MOMP peptides that are recognized by B and T cells. To achieve that goal, it is critical to identify which MOMP peptides are recognized by B and T cells of infected people. To date, there have been a number of reports regarding attempts to develop vaccines based on single or multiple MOMP peptide fragments, where the focus is on raising Th—cell and/or B-cell responses (mostly in mice, but in some cases, in humans). See H. Su et al., 172 J. Exp. Med. 203-212 (1990) (serovar A); J. Allen et al., 147 J. Immunol. 674-679 (1991) (serovar B); M. Ishizaki et al., 60 Infect. & Immun. 3714-3718 (1992) (serovars B, C); G. Zhong et al., 151 J. Immunol. 3728-3736 (1993) (serovar B). L. Ortiz et al. 157 J. Immunol. 4554-4567 (1996) (serovar E) and U.S. Pat. No. 6,001,372 (serovar E). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth herein.
The 371 amino acid sequence of mature MOMP of Ct serovar E, a common cause of genital tract infections, is also disclosed in L. Ortiz et al. 157 J. Immunol. 4554-4567 (1996). The naturally occurring DNA coding sequence of serovar E-MOMP is disclosed in E. Peterson et al., 18 Nuc. Acids. Res. 3414 (1990) (SEQ ID NO:9). MOMP is a transmembrane protein and comprises more than 60% of all outer membrane proteins of Ct.
Sequence analysis of MOMP from various sources has revealed that differences in amino acid sequence confined to surface-exposed “variable segments” (VSs) of MOMP account for the serological specificity of different serovars and, also, for differences in Th-cell responses to different isolates. See M. Ishizaki et al., 60 Infect. & Immun. 3714-3718 (1992). On the other hand, membrane-embedded regions of MOMP contain amino acid sequences that are conserved among different Ct serovars, and hence the name, “constant segments (CSs)”. Notably, a majority of Th-cell epitopes is located in MOMP CSs in contrast to B-cell epitopes, which are exclusively located in MOMP VSs.
T cells recognize their peptide epitopes only when they are presented on the surface of other cells in association with a particular kind of HLA (human leukocyte antigen; human MHC (major histocompatibility complex)) molecules. Different kinds of HLA molecules present different peptide epitopes. A complicating matter in vaccination of humans is the fact that HLA genes are extremely polymorphic in the human population. That is, different individuals express different HLA types, and a T-cell epitope that elicits immune responses in some individuals may not do so in others. This is a particularly troubling problem for those seeking to develop vaccines for the human population in general. Consequently, there remains a need to identify many different peptide epitopes presented by diverse HLA allotypes that elicit immune responses in the majority of population. Such a set of epitopes can then be used to create a “cocktail” type sub-unit vaccine containing multiple T-cell epitopes as well as B-cell epitopes.
Recently there has been a description in M. Holland et al., 107 Clin. Exp. Immunol. 44-49 (1997) of two MOMP peptides that stimulated limited CTL responses in HLA-B8+ or HLA-B35+ individuals who had experienced trachoma, an eye infection with Ct. However, only two of twelve HLA-B8+ subjects responded to the peptide therefor, and only one of thirteen HLA-B35+ subjects responded to the peptide therefor. These CTLs showed low lytic activity against targets incubated with the peptides and ability of the CTLs to lyse Ct-infected target cells was not examined.
Apart from vaccine utility, it is desirable to find CTL epitopes that can be used as components of diagnostic tests (e.g. to confirm the presence of the disease once a positive test result has been obtained using conventional tests).
In summary, the identification of human CTL epitopes is needed to design a sub-unit vaccine, and is of interest in developing diagnostic tests.
BRIEF SUMMARY OF THE INVENTION
Our invention concerns a specific type of T cell responses, i.e. cytotoxic T lymphocyte (CTL) responses, in human genital tract infections with Ct. CTLs have been well documented as critical players in providing protection against infections with intracellular pathogens, including viruses, bacteria, fungi and parasites. CTLs exert their protective effector function by specifically recognizing an infected cell and secreting cytotoxic molecules that lead to the lysis and death of the infected cell, as well as the pathogens residing inside the cell. CTL recognition of an infected cell requires presentation on the cell surface of short peptide epitopes derived from proteins of the pathogens in association with HLA class I molecules.
Such peptides are generated by “the antigen processing machinery” of the infected cell, which includes cytosolic proteases and the transporter molecules that move the peptides into a cellular compartment where they can bind to HLA class I molecules. It should be noted that only a fraction of peptides generated by a cell are indeed capable of activating CTLs. This is because each CTL expresses at the cell surface T-cell receptors that are specific for a sing
DeMars Robert I.
Kim Seon-Kyeong
Kam Chih-Min
Low Christopher S. F.
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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