Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1994-12-02
2001-03-20
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S261100, C424S200100, C424S184100, C424S203100, C424S252100, C435S069300, C435S909000, C435S243000, C435S252300
Reexamination Certificate
active
06203799
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of invention is
Vibrio cholerae
vaccines.
After more than 100 years of research on cholerae, there remains a need for an effective cholerae vaccine. There have been six pandemics of this disease caused by strains of
V. cholerae
belonging to the “Classical” biotype. The etiological agents of the current (seventh) pandemic belong to the “El Tor” biotype. Recently the seventh pandemic has extended to a new locale, that of South America. Beginning in January of 1991, an epidemic of cholerae resulted in greater than 250,000 cases and over 2,000 deaths in Peru, Ecuador, Columbia, and Chile. In November of 1992, an antigenically distinct, non-01 form of
V. cholerae
emerged in India and Bangladesh and within eight months caused an estimated 500,000 cases and 6,000 deaths. The pandemic potential of this new strain, designated serogroup 0139 synonym “Bengal”, seems assured and is a new cause of concern throughout the developing world. These recent experiences underline the need for effective cholera vaccines against disease due to 01 serogroup El Tor biotype of
V. cholerae
and Bengal 0139 serogroup of
V. cholerae.
The major issues which must be overcome to produce effective cholerae vaccines are safety, stability and a high degree of antigenicity. Because natural infection by and recovery from cholerae induces immunity lasting at least 3 years, much effort has been made to produce live, attenuated cholerae vaccines that, when administered orally, would mimic the disease in its immunization properties, but would not cause adverse symptoms or reactions in the immunized individual (i.e., vaccines which display low reactogenicity). Vaccines of this type involve deletion mutations that inactivate the gene encoding the A subunit of cholerae toxin, a protein which is responsible for most of the diarrhea seen in this disease. See, for example, Mekalanos, U.S. Pat. Nos. 5,098,998 and 4,882,278, and Kaper et al., U.S. Pat. No. 4,935,364, hereby incorporated by reference. While both oral, killed whole cell vaccines and several live, attenuated cholerae vaccines have been developed, the most promising of these provide little protection against the El Tor biotype of
V. cholerae
and probably no protection against the 0139 serotype.
V. cholerae
only causes disease when colonization of the small bowel occurs. This colonization is also required for the induction of a localized immune response, an important aspect of development of effective vaccines. It is thought that interaction and uptake of bacteria by Peyers patches is the essential step in the localized immune response pathway. Thus, colonization of the intestine can be divided into two distinct steps: 1) interaction with Peyers patches and subsequent immune responses; and 2) interaction with enterocytes and subsequent disease processes (reactogenicity). Although the factors affecting colonization are not well understood, they are believed to include the TcpA pili and motility.
SUMMARY OF THE INVENTION
The invention features nontoxigenic genetically stable mutant strains of
V. cholerae
which are useful as a live, oral vaccines for inducing immunological protection against cholerae. The mutant strains are genetically engineered mutants which lack DNA encoding a functional ctxA subunit and which also have genetic defects causing them to be soft agar penetration-defective. We have found that such mutants have extremely low reactogenicity in both clinical and laboratory tests, yet elicit a strong immune response. As a result, the soft agar penetration-defective strains have the necessary and desirable characteristics of a human vaccine to
V. cholerae.
By parental strain is meant any strain from which the mutant strain descends. Any number of mutations may be added to the parental strain prior to or subsequent to the introduction of the soft agar penetration-defective mutation.
By soft agar penetration-defective strain is meant a strain lacking the ability to penetrate a media of high viscosity as measured in vitro by swarming on and within agar media which is between 0.25 and 0.45% agar. Mutants which fail to penetrate soft agar are those which will not spread beyond a diameter of 2 mM, most preferably 1 mM, when stabbed or plated by dilution onto soft agar and incubated overnight at 300. Soft agar penetration-defective mutants may be filamentous, motility defective (Mot
−
), lacking flagella (Fla
−
), and/or show a decreased ability to bind HEp-2 cells (HEp-2
−
). The most preferable strains are Mot
−
Fla
+
HEp-2
−
, or filamentous.
Filamentous strains are defined as those which appear elongated by microscopic examination, i.e. 25% or more cells appear to be greater than 15 nM in length under conditions of logarithmic growth.
Mot
−
Fla
+
strains are defined as those strains which have complete flagellum when inspected by electron microscopy, yet remain soft agar penetration-defective and show decreased or non-existent swimming behavior relative to the parent strain when observed in liquid medium. Useful Mot
−
Fla
+
strains include strains which have disruptions of the motB gene described below or
V. cholerae
homolog of the motA gene from
E. coli
. Most preferably, the soft agar phenotype is caused by a disruption in the motB gene, which causes complete loss of swimming behavior in liquid media, but strains with partial swimming behavior defects may also be useful as vaccines. Mot
−
Fla
+
strains are generally preferable soft agar penetration-defective strains for vaccines because they are penetration defective, yet present all the flagellar antigens as immunogens.
Fla
−
strains are defined as those strains which have defective flagellum and are, therefore, soft agar penetration-defective. Useful soft agar penetration-defective strains which are Fla
−
may be detected by the observation of incomplete, defective or nonexistent flagellum when evaluated by electron microscope, and by their decreased or non-existent swimming behavior relative to the parent strain in addition to their soft agar penetration-defective phenotype. Useful Fla
−
phenotypes may be obtained by the disruption of the
V. cholerae
homologs of the fliC, fljB, flhC, flhD, fliA, flgM, fliS, flit, fliD, fljA, flhA, fliH, fliI, flgA, flgD, fliK, fliB, flig, flim, flin, flIF, fliE, flgB, flgC, flgF, flgG, flgI, flgH, flgE, flgK, flgL, fliD, figj, flhB, flhE, fliJ, flil, fliO, fliP, fliQ, fliR of
E. coli, S. typhimurium
, species Bacillus,
V. parahaemolyticus
, species Helicobacter,
C. crescentus, P. mirabilif
, and
B. Pertussis
(listed in order of preference, see Table 1, below), for example. Most preferably, the disruption is a disruption of the fliC gene of
V. cholerae
described herein a disruption of the motility gene or genes which are disrupted or the disruptions present in the Bengal-15, Peru-14, Peru-15, Bah-15, and Bang-15 strains.
TABLE 1
Flagellar and motility gene products of
S.
typhimurium
and
E. coli
and their known or
suspected functions
Gene product
Function/location
Regulatory proteins
FlhC, FlhD
Master regulators of the flagellar regulon
acting on class 2 operons.
Transcription initiation (&sgr;) factors?
FliA
Transcription initiation (&sgr;) factor for
class 3a and 3b operons.
FlgM
Anti-FliA (anti-&sgr;) factor. Also known as
RflB. Active only when flagellar
assembly has not proceeded through
completion of the hook.
FliS, FliT, FliD?
Repressor of class 3a and 3b operons (RflA
activity)
FljA
Repressor of fliC operon.
Hin
Site-specific recombinase, affecting fljB
promoter.
Proteins involved in
the assembly process
FlhA, FliH, FliI
Export of flagellar proteins? FlhA is
homolog of various virulence factors.
FliI is homolog of the catalytic
subunit on the F
0
F
1
ATPase.
FlgA
Assembly of basal-body periplasmic P ring.
FlgD
Initiation of hook assembly.
FliK
Control of hook length.
FliB
Methylation of lysine residues on the
filament protein, flagellin; function
of this modification unknown.
Flagel
Camilli Andrew
Gardel Claudette L.
Mekalanos John J.
Bieker-Brady Kristina
Clark & Elbing LLP
Minnifield Nita
Presidents and Fellows of Harvard College
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