Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1999-10-14
2004-03-30
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S190100, C424S192100, C530S350000
Reexamination Certificate
active
06713071
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention is in the field of animal health, and is directed to vaccines that protect swine against
Actinobacillus pleuropneumoniae
. More particularly, the present invention is directed to novel antigenic proteins shared by multiple serotypes of
A. pleuropneumoniae
, DNA molecules encoding the proteins, vaccines against
APP
comprising the proteins, and diagnostic reagents.
2. BACKGROUND OF THE INVENTION
A. pleuropneumoniae
(hereinafter referred to as “
APP
”) is a Gram negative coccobacillus recognized as one of the most important swine pneumonic pathogens (Shope, R. E., 1964, J. Exp. Med. 119:357-368; Sebunya, T. N. K. and Saunders, J. R., 1983, J. Am. Vet. Med. Assoc. 182:1331-1337). Twelve different serotypes have been recognized which vary in geographic distribution (Sebunya, T. N. K. and Saunders, J. R., 1983, above; Nielsen, R., 1985, Proc. Am. Assoc. Swine Pract. 18-22; Nielsen, R., 1986, Acta. Vet. Scand. 27:453-455). Immune responses to vaccination against
APP
have been mainly serotype-specific, suggesting that vaccine-induced immunity is directed to serotype-specific capsular antigens (MacInnes, J. I.). and Rosendal, S., 1988, Can. Vet. J. 29:572-574; Fedorka-Cray, P. J., et al., 1994, Comp. Cont. Educ. Pract. Vet. 16:117-125; Nielsen, R., 1979, Nord. Vet. Med. 31:407-413; Rosendal, S., et al., 1986, Vet. Microbiol. 12:229-240).
In contrast, natural immunity to any one serotype seems to confer significant protection from disease caused by other serotypes, suggesting that natural exposure induces cross-reactive immunity to shared antigens (Sebunya, T. N. K. and Saunders, J. R., 1983, above; Macinnes, J. I. and Rosendal, S., 1988, above; Fedorka-Cray, P. J., et al., 1994, above; Nielsen, R., 1979, above; and Rosendal, S., et al., 1986, above).
Virulence factors that might contribute to cross-protection have been proposed as possible vaccine candidates, including exotoxins (Apx) (Nakai, T., et al., 1983, Am. J. Vet. Res. 30 44:344-347; Frey, J., et al., 1993, J. Gen. Microbiol. 139:1723-1728; Fedorka-Cray, P. J., et al., 1993, Vet. Microbiol. 37:85-100); capsular antigens (Rosendal, S., et al., 1986, above); outer-membrane proteins (OMP) (Denee, H. and Potter, A., 1989, Infect. Immune 57:798-804; Niven, D. F., et al., 1989, Mol. Microbiol. 3:1083-1089; Gonzalez, G., et al., 1990, Mol. Microbiol. 4:1173-1179; Gerlach, G. F., et al., 1992, Infect. Immun. 60:3253-3261); and lipopolysaccharides (LPS) (Fenwick, B. W. and Osborn, B., 1986, Infect. Immun. 54:575-582). However, the patterns of cross-reactivity/cross-protection induced by such components do not cover all twelve
APP
serotypes. In addition, immunization with isolated individual components or combinations of individual components from
APP
have so far failed to confer protection from challenge with some heterologous serotypes (unpublished observations). Thus, it can be postulated that the cross-protective responses induced by natural infection are limited to specific serotype clusters.
Alternatively, it is possible that some of the antigens responsible for the cross-protection observed after natural infection have not yet been identified. Most studies regarding
APP
antigens have focused on the characterization of immunodominant antigens detected in convalescent serum using antibodies. Such an approach does not allow the identification of possible differences between the antibody specificities represented during primary versus secondary responses, nor the identification of dominant specificities at the infection site that are likely to be responsible for protection upon secondary encounter with the pathogen.
It is generally accepted that lymphocytes are educated during primary infections so that when there is secondary exposure to a pathogen the host is better able to prevent disease (MacKay, C. R., 1993, Adv. Immunol. 53:217-240). Memory cells responsible for this activity (antigen-experienced T and B lymphocytes) persist for long periods of time, and are capable of reactivation following an appropriate subsequent encounter with the antigen. In contrast to naive cells, they generally show a faster response time, specialized tissue localization, and more effective antigen recognition and effector functions (MacKay, C. R., 1993, above; Linton, P. and Klinman, N. R., 1992, Sem. Immun. 4:3-9; Meeusen, E. N. T., et al., 1991, Eur. J. Immunol. 21:2269-2272).
During the generation of a secondary response, the frequency of precursor cells capable of responding to the particular antigen is higher than that present during the primary response. Trafficking patterns of memory cell subsets following secondary responses are also different from those of naive cells. Naive cells migrate relatively homogeneously to secondary lymphoid tissues, but they home poorly to non-lymphoid tissues. By contrast, memory cells display heterogeneous trafficking and, in some instances, migration has been shown to be restricted to certain secondary lymphoid tissues and non-lymphoid sites (MacKay, C. R., 1993, above; Gray, D., 1993, Ann. Rev. Immunol. 11:49-77; Picker, L. S., et al., 1993, J. Immunol. 150:1122-1136). Studies in both rodents and sheep have indicated that lymphocytes from the gut preferentially migrate back to the gut, whereas cells draining from the skin or from lymph nodes preferentially migrate back to the skin or lymph nodes (Gray, D., 1993, above; Picker, L. S., et al., 1993, above). Thus, upon secondary encounter with a pathogen, specific effector cells for cell-mediated immunity and antibody secretion can home to infection sites and local lymph nodes more effectively (Meeusen, E. N. T., et al., 1991, above). As a result, infiltrating lymphocytes will rapidly proliferate and their specificities will predominate during early stages of re-infection.
Recovery of local B cells from tissues and draining lymph nodes early after re-infection has allowed some researchers to obtain antibodies with a narrower specificity range (Meeusen, E. N. T. and Brandon, M., 1994, J. Immunol. Meth. 172:71-76). Such antibodies have been successfully used to identify potential protective antigens to several pathogens (Meeusen, E. N. T. and Brandon, M., 1994, above; Meeusen, E. N. T. and Brandon, M., 1994, Eur. J. Immunol. 24:469-474; Bowles, V. M., et al., 1995, Immunol. 84:669-674). The invention disclosed herein below is based on a modification of this approach, in which antibody-secreting cell (ASC) probes were recovered that were associated with local memory responses elicited after homologous and heterologous
APP
challenge. Antibodies obtained from bronchial lymph node (BLN) cultures after heterologous challenge recognized four previously unrecognized proteins present in all twelve
APP
serotypes. Partial amino acid sequences for each protein were obtained and used to generate PCR primers that allowed the identification of five novel
APP
proteins and polynucleotide molecules that encode them.
3. SUMMARY OF THE INVENTION
The present invention provides five novel, low molecular weight proteins isolated from
APP
, which are designated herein, respectively, as “Omp20,” “OmpW,” “Omp27,” “OmpA1,” and “OmpA2”. These “
APP
proteins” and the polynucleotide molecules that encode them are useful either as antigenic components in a vaccine to protect swine against
APP
, or as diagnostic reagents to identify swine that are, or have been, infected with
APP
, or that have been vaccinated with a vaccine of the present invention.
The amino acid sequence of Omp20 is encoded by the Omp20-encoding ORF of plasmid pER416 which is present in host cells of strain Pz416 (ATCC 98926), and its deduced amino acid sequence is presented as SEQ ID NO:2, which comprises a signal sequence from amino acid residues 1 to 19. The amino acid sequence of OmpW is encoded by the OmpW-encoding ORF of plasmid pER418 which is present in host cells of strain Pz418 (ATCC 98928), and its deduced amino acid sequence is presented as SEQ ID NO:4, which comprises a signal sequence from amino acid residues 1 to 21. The amino acid sequence o
Ankenbauer Robert G.
Baarsch Mary Jo
Campos Manuel
Keich Robin
Rosey Everett
Pfizer Inc.
Portner Ginny Allen
Scully Scott Murphy & Presser
Smith Lynette R. F.
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