Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-17
2002-10-29
Graser, Jennifer E. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024320, C536S024330, C536S024100, C424S250100, C435S243000, C435S252300, C435S320100, C435S069100, C435S069300
Reexamination Certificate
active
06472518
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to new genes isolated from
Neisseria meningitidis
. Isolated nucleic acids, probes, expression cassettes, polypeptides, antibodies, immunogenic compositions, antisense nucleic acids, amplification mixtures and new invasion deficient strains of
Neisseria meningitidis
. The invention also relates to methods of detecting Neisseria meningitidis and
Neisseria meningitidis
nucleic acids, and to methods of inhibiting the invasion of mammalian cells by
Neisseria meningitidis.
BACKGROUND OF THE INVENTION
Neisseria meningitidis
, a Gram-negative encapsulated diplococcus, is an obligate human pathogen and the causative agent of meningococcal meningitis, one of the most devastating forms of meningitis. These bacteria are isolated from humans worldwide and can cause sporadic and epidemic disease. Person-to-person transfer of
N. meningitidis
occurs mainly via the airborne route, and is particularly a problem in places where people are in close quarters, such as prisons, military camps, school class rooms, and day care centers. At any one time, between 2 and 10% of individuals in the population carry this organism asymptomatically (Greenfield, S., et al. (1971),
J. Infec. Dis
., 123:67-73; Moore, P. S., et al. (November 1994),
Scientific American
, p38-45; Romero, J. D. et al. (1994),
Clinical Microbiology Review
, 7:559-575). With such a high carrier rate, the threat or potential for outbreaks or epidemics is always present. Although significant advances have been made in the area of the pathogenesis of the organism, there is much to be learned about the genetics and cell biology of the host-parasite interaction.
Understanding the mechanism(s) of attachment and invasion is one of the most important aspects in
N. meningitidis
disease. In order to cause disease, meningacocci must survive and colonize the mucosa of the nasopharynx, pass through these tissue into the bloodstream replicate to large numbers in the blood, cross the blood-brain barrier and multiply in the cerebrospinal fluid (CFS) where they cause inflammation of the meninges. Various models have been used in order to mimic the events that take place during infection in humans. Mouse models (Miller, C. P. (1933),
Science
, 78:340-341; Holbein, B. E. (1981),
Can. J. Microbiol
., 27:738-741; Salit, I. E. (1984),
Can. J. Microbiol
., 30:1022-1029), human nasopharyngeal organ culture (Stephens, D. S., et al. (1991),
Rev Infect Dis
., 13:22-33), chick embryo (Buddingh, G. J. et al. (1987),
Science
, 86:20.21; Pine, L., et al.,
Micrbiol. Lett
., 130:37-44), and tissue culture monolayer and bilayer systems (Birkness, K. A., et al. (1995),
Infect. Immun
., 63:402-409) represent some of the models commonly used to study virulence of
N. meningitidis.
The organ culture system has been used successfully to assess the attachment and invasion properties of various
N. meningitidis
strains (Salit, I. E. (1984),
Can. J. Microbiol
., 30:1022-1029).
Designated by serogroup, serological classification of
N. meningitidis
is based on the capsular polysaccharide composition of the particular strain. Among the meningococci there are at least thirteen different serogroups: A, B, C, 29-E, H, I, X, L, W135, X, Y and Z. Of these serogroups, A, B and C comprise over 90% of the strains isolated from patients afflicted with meningococcal meningitis (Poolman, J. T., et al. (1995),
Infectious Agents and Disease
, 4:13-28). The nature of the capsule in serogroups A and C has led to the development of useful vaccines against these serogroups. However, the serogroup B capsular polysaccharide does not induce protection in humans. Many laboratories around the world are concentrating their efforts on the study and characterization of epitopes from various membrane and other extracellular factors for use as vaccine candidates. Some of the most common non-capsule factors in such studies include a number of outer membrane proteins (OMP) such as class 1 (Por A, a cation Specific porin), class 2 or 3 (Pot B, an anion specific protein) and to a lesser extent class 4 and class 5 OMPs (Rmp, and Opc and Opa opacity associated proteins, respectively). While class 5 Opc and Opa OMPs have been shown to play roles in the invasion of epithelial cells (Virji, M., et al. (1992),
Mol. Microbiol
., 6;2786-96) due to their antigenic and phase variability (Aho, E. L.; et al. (1991),
Mol. Microbiol
., 5:1429-37), they are not considered to be good vaccine candidates.
Class 1 OMPs appear to be good candidates for vaccine studies since these proteins have been shown to induce protective immunity. Evaluation of various non-capsular antigens as potential vaccine candidates in in vitro bactericidal assays and an infant rat model revealed that class 1 OMP had the highest protective capacity compared to factors such as LPS and class 2/3 OMPs (Saukkonen, K., et al. (1989),
Vaccine
, 7:325-328). However, preliminary data from vaccine trial studies suggests that these factors do not elicit a complete immune response, especially in children (Romero. J. D. et al. (1994),
Clinical Microbiology Review
, 7:559-575; Poolman, J. T., et al. (1995),
Infectious Agents and Disease
, 4:13-28). The development of fusion or hybrid genes containing epitopes from class 1 OMP show great promise as vaccine candidates (Van der Ley, P., et al. (1992),
Infect. Immun
., 60:3156-3161; Van der Ley, P., et al. (1993),
Infect. Immun
., 61:4217-4224). However, these hybrids do not elicit protection in infants, and the immunity induced is type specific and very short-lived (Poolman, J. T., et al: (1995),
Infectious Agents and Disease
, 4:13-28). Far these and other reasons, it is or importance to identify alternative serogroup B vaccine antigens. Initial attachment and invasion by the pathogen is critical to the disease process. If mucosal immunity can be derived against these bacterial factors, the disease process and the carrier state can be prevented. The present invention provides these and other features.
SUMMARY OF THE INVENTION
The invention provides nucleic acids and encoded polypeptides associated with invasion of
Neisseria meningitidis
. The polypeptides are used as diagnostic reagents as immunogenic reagents; and as components of vaccines. The nucleic acids are used as diagnostic reagents, as components of vectors and vaccines, and to encode the polypeptides of the invention. The invention also provides strains of
Neisseria meningitidis
which have an invasion deficient phenotype.
In one embodiment, the invention provides isolated nucleic acids encoding the polypeptides of the invention, including ORF 1 (SEQ ID NO:2), ORF 2 (ORF2 a (SEQ ID NO:4) and ORF2b (SEQ ID NO:5), two separate embodiments depending on alternate start sites for the ORF2 polypeptide), ORF 3 (SEQ ID NO:7) and, conservatively modified variations of each of the polypeptides. Exemplar nucleic acids include Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), and Seq 3 (SEQ ID NO:7) (see,
FIGS. 5
,
6
, and
7
respectively). Other nucleic acids encoding the same polypeptides include those with silent codon substitutions relative to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3) for Seq 3 (SEQ ID NO:6); as well as conservatively modified variations thereof.
Isolated nucleic acids which hybridize under stringent conditions to the exemplar nucleic acids Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) are also provided. For example, a complementary nucleic acid to a sequence provided by Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) hybridizes to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively. Nucleic acids which include substantial subsequences complementary to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6) also hybridize to Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), or Seq 3 (SEQ ID NO:6), respectively.
Isolated nucleic acids which hybridize under stringent conditions to Seq 4 (SEQ ID NO:8) are provided. Seq 4 (SEQ ID NO:8) is a genomic sequence which encodes Seq 1 (SEQ ID NO:1), Seq 2 (SEQ ID NO:3), an
Quinn Frederick D.
Raymond Nigel
Ribot Efrain M.
Stephens David S.
Centers for Disease Control and Prevention, as represented by th
Graser Jennifer E.
Needle & Roseberg, P.C.
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