Genetic modification of a vaginal strain of L actobacillus fermentum

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J. Med. Microbiol. -Vol. 41 (1994), 272-278
0 1994 The Pathological Society of Great Britain and Ireland
Genetic modification of a vaginal strain of L actobacillus
fermentum and its maintenance within the reproductive
tract after intravagina I adm i nistration
C. M. RUSH"?, L. M. HAFNER" and P. TIMMS"
* Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Brisbane,
Queensland, 4007 Australia and -/- Department of Molecular Biology, University of Siena, Siena, Italy
Summary. Many micro-organisms cause important diseases of the female genital tract.
Because systematic vaccination does not usually provide a good immune response at mucosal
sites, commensal lactobacilli from the female genital tract were developed as vehicles to
deliver continued doses of foreign antigen directly to the genital mucosal surface with the aim
of stimulating strong local mucosal immune responses. Lactobacilli were shown to be
common inhabitants of the genital tract of the animal model studied, the guinea-pig. One
species, Lactobacillus fermentum, was found in all guinea-pigs studied and was chosen for
genetic manipulation. Improved methods of electroporation were developed to enable the
routine transformation of L. fermentum B R l l strain with the broad host range plasmid
pNZ17. This recombinantly modified Lactobacillus strain was shown to possess good
segregational stability over 120 generations in the absence of antibiotic selection. When this
recombinant L. fermentum strain was administered to the vaginal tract of three guinea-pigs it
persisted for only 5 days. Despite the relatively short period of persistence in these initial
experiments, this novel vaccine approach could provide an effective means of stimulating
mucosal immunity in the female genital tract.
The female reproductive tract is susceptible to a
wide range of infections by bacteria and viruses
including Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum,
herpes simplex virus 11, human papilloma virus and
human immunodeficiency virus. Currently no vaccines
against any of these are available commercially. The
infections are usually sexually transmitted and many
are restricted to the superficial layers of the vaginal or
cervical epithelium. The reproductive tract mucosa
forms part of the common mucosal immune system,
which also includes the gastrointestinal, respiratory
and urinary tracts and the conjunctivae. Local secretion of antigen-specific IgA at all mucosal sites is
thought to be initiated locally in the mucosa-associated
lymphoid tissue, such as the Peyer's patches of the
small intestine and the lymphoid aggregates within the
respiratory tract.lP3Current approaches to vaccination
against mucosal pathogens have utilised the knowledge that antigenic stimulation at one mucosal site
Received 22 Dec. 1993; revised version accepted 28 April 1994.
may lead to an immune response and protection at
distant mucosal sites, via the dissemination of activated 1ymph0cytes.l~~
The most common approach to
date has been oral administration of antigen to activate
the gut-associated lymphoid tissue and produce an
immune response at distant mucosal sites, such as the
reproductive tract." However, several studies have
shown that while gut responses may be adequate, the
responses at more distant sites are often weak, and
that local antigen presentation may produce better
immune response^.^ Menge et ~ 1 showed
. ~
that a
vigorous local immune response to keyhole limpet
haemocyanin conjugated to cholera toxin B subunit
was induced after direct immunisation of the female
reproductive tract. Other investigators', have also
concluded that the level of specific antibody is influenced by the route of immunisation, with local
administration of antigen being the most potent
stimulator of specific IgA secretion.
Of the many factors that influence the level of
mucosal immunity produced, the frequency and dose
of the antigen are known to be very important.'
Repeated antigen doses often elicit stronger mucosal
immune responses.' Live vaccines with bacteria or
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viruses as vehicles may be the best method to vaccinate
against mucosal pathogens by providing a sustained
antigenic stimulus over many days. Our approach is to
try to use non-pathogenic indigenous bacteria, such as
members of the genus Lactobacillus, as vehicles for
delivering antigen to the vaginal mucosa. Lactobacilli
are the dominant bacterial species of the healthy
human vagina7 and are also routinely found as normal
flora in a wide variety of
Moreover, they
are harmless commensals, devoid of pathogenic potential.
To be useful as vaccine vehicles, lactobacilli need to
carry and express foreign antigens. The genetic modification of lactobacilli only became a practical proposition in 1988 when electroporation was first used to
transform L. casei strains12 and, thereafter, was successful with several other Lactobacillus sp.13-17Genetic
modification of lactobacilli has usually been achieved
by the introduction of plasmids, either derived from
other naturally occurring lactobacillus plasmids15,l 7or
by utilising broad host range plasmid replicons from
other gram-positive bacteria.13~
The aims of the present study were: to determine if
lactobacilli could be isolated as common inhabitants
from the vagina of the chosen animal model, the
guinea-pig ; to isolate plasmids from these vaginal
lactobacilli ;to transform a vaginal Lactobacillus strain
with a plasmid (pNZ17) containing the broad host
range replicon from plasmid pSH71; to assess the
segregational stability of plasmid pNZ17 in the vaginal
Lactobacillus strain and; and to administer the transformed strain intravaginally and assess its maintenance within the vagina.
Materials and methods
Experimental animals
Sexually mature (i.e., older than 6 weeks) female
guinea-pigs were used.
Culture and enumeration of vaginal bacteria
The ano-genital area of each guinea-pig was cleansed with cotton-tipped swabs (Disposable Products
Pty Ltd, South Australia) moistened with phosphatebuffered saline (PBS). If the vaginal closure membrane
occluded the vaginal opening, the membrane was
punctured with a second swab. A third swab was
inserted into the vagina to a depth of 2.5 cm and
rotated. The swab was placed in 1 ml of sterile PBS,
3-mm sterile glass beads were added, and the liquid and
swab were thoroughly vortex mixed. Log,, dilutions
of the suspension were prepared in PBS, and 0-1 ml of
each dilution was plated on de Mann, Rogosa, Sharpe
agar (MRS; Unipath) and horse blood agar (HBA)
to determine total Lactobacillus and aerobic counts,
respectively. Plates were incubated at 37°C in air with
CO, 5 YOfor 48 h. For total anaerobic counts, dilutions
were plated on HBA and the plates were incubated for
48 h at 37°C in an anaerobic chamber (CO, 9.7 YO,H,
10.2%, N, 80.1 YO;Anaerobe Systems). MRS agar
plates containing chloramphenicol 10 pg/ml were used
for the isolation of chloramphenicol-resistant lactobacilli. Colony types were differentiated by morphological criteria, followed by Gram’s stain. Representative isolates were identified to the genus level
according to Bergey’s Manual of systematic bacterio1ogy.l8Lactobacillus spp. were identified by the use of
the Microbact 24AN identification system (Disposable
Products) and by reference to the VPI anaerobic
manua119 and Bergey’s Manual of systematic bacteriology.18
Lactobacillus strains and plasmids studied
The bacterial strains and plasmids used are shown in
table I. L. fermentum B R l l was a guinea-pig vaginal
isolate (obtained in this study) used for transformation, segregational and structural stability and colonisation studies. Other lactobacilli were isolated in
this study or were obtained from the American Type
Culture Collection as indicated in table I. The plasmid
pNZ717 (kindly donated by G. Simons, NIZO, The
Netherlands) is 5.7 kb in size and contains the broad
host range replicon of the Lactococcus lactis subsp.
lactis plasmid pSH7 1 and the chloramphenicol acetyltransferases (CAT) gene of pC194; L.fermentum CR1
refers to the L. fermentum B R l l isolate containing
Plasmid isolation from lactobacillus strains
Plasmids were isolated from vaginal and ATCC
lactobacilli by a modification of the method of
Anderson and McKay,,’ in which treatment with
lysozyme 10 mg/ml for 60 min replaced the original
step. Briefly, strains to be profiled were inoculated into
MRS broth and grown overnight at 37°C in air with
CO, 5 %. For plasmid screening, the broth culture was
diluted 1 in 50 and this was grown until the
was c. 0.5. Plasmid DNA was isolated, resuspended in
0.02 ml of distilled water and treated with RNAaseA
100 pg/ml for 60 min. Routinely, 0.01 ml of the DNA
solution was electrophoresed at 4.0 V/cm through
agarose 0-8YOin tris-borate-EDTA buffer containing
ethidium bromide 0.5 pg/ml incorporated into the
electrophoresis buffer. These electrophoresis conditions gave the optimal resolution of plasmid profiles.
Transformation of vaginal L. fermentum B R l l
L. fermentum B R l l was rendered resistant to
chloramphenicol and kanamycin by the introduction
of plasmid pNZ17. Bacterial cells were cultivated in
MRS broth overnight at 37°C in air with CO, 5 YOand
then a 1 in 50 dilution in 10ml of MRS broth was
reached 0.3. At this point,
incubated until the ODsOOmm
penicillin G (Sigma) was added to give a final con-
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Table I. Lactobacillus strains and plasmids studied.
Strains and plasmid
Relevant characteristics
Guinea-pig vaginal isolate, this study
L. fermentum BR 1 I
Guinea-pig vaginal isolate, this study
L. acidophilus BR 9
Guinea-pig vaginal isolate, this study
L. sp. BR 10
Guinea-pig vaginal isolate, this study
L. sp. BR 3
Guinea-pig vaginal isolate, this study
L. sp. wc 2
ATCC isolate
L. casei ATCC 393
L. plantarum ATCC 14917 ATCC isolate
L..fermentum BRl 1 containing
L..fermentum CRl
pNZ 17
Lactococcus IactislE. coli shuttle
Plasmid pNZ 17
vector, 5.7 kb; pSH71 replicon;
chloramphenicol acetyl transferase
gene from pC194
phenicol 10 pg/ml until the cells had reached late
logarithmic phase (ODGOOmm
= 3.0). Bacterial cells
were harvested by centrifugation at 4000 g for 5 min at
4"C, washed and resuspended in PBS to a concentration of loll cfu/ml. The ano-genital area of each
guinea-pig was cleansed and 0.2 ml of the L.fermentum
strain CR1 suspension was flushed into the vagina
with a smoothed glass pipette with a rubber suction
bulb attached. In initial experiments, vaginal swabs
were taken from four guinea pigs 1,2, 3,4 and 5 days
after inoculation, and cultured to enumerate chloramphenicol-resistant bacteria. In subsequent experiments, the inoculation procedure was modified: 0.2 ml
of the suspension were inoculated daily into the vagina
of guinea-pigs for 5 days. After the final intravaginal
inoculation, animals were swabbed daily for 7 days to
assess whether vaginal colonisation by L. fermenturn
strain CRl had occurred.
centration of 10 pg/ml and growth was continued until
the ODGOOmm
reached 0.6-0-7. Cells from the 10-ml
culture were pelleted and washed three times in 0.5 M
sucrose before being resuspended in a final volume of
0.5 ml of ice-cold 0.5 M sucrose. Cells were either
used immediately or stored at -80°C for later use.
Electrotransformation of lactobacilli was done with
the BioRad Gene Pulser apparatus and chilled cuvettes
with a 0.2-cm inter-electrode gap. pNZ17 plasmid
DNA (0.5 pg) was added to 0.1 ml of a chilled
suspension of L. fermenturn cells and the mixture was
held on ice for at least 5 min. Electrotransformation
parameters included a field strength of 12.5 kV/cm
with a parallel 200 ohm resistance and a capacitance of
25 pF. After the pulse, the cell-DNA suspension was
returned to ice for 10 min and subsequently diluted 10fold in MRS broth supplemented with 25 mM sucrose.
Cell suspensions were incubated at 37°C in air with
CO, 5 % for 2 h to allow the cells to recover.
Chloramphenicol was then added to the suspension to
a final concentration of 0.1 pg/ml to induce expression
of the CAT gene. Transformants were selected by
plating 0.1 ml of the suspension on MRS agar plates
containing chloramphenicol 10 pg/ml. Plates were
incubated at 37°C for 48 h and transformants were
confirmed by plasmid isolation.
The vaginal flora of all guinea-pigs examined consisted almost exclusively of gram-positive bacteria ;
gram-negative bacteria were isolated only occasionally
(table 11). The dominant aerobic genera isolated were
Corynebacteriurn and Enterococcus ; the dominant
anaerobic genus isolated was Propionibacterium. Bifidobacteriurn spp. and Bacteroides spp. were also
isolated from some animals. Lactobacilli were isolated
consistently from all guinea-pigs and, although not
numerically the most prevalent organisms, they always
represented a significant component of the microbial
flora (102-103 cfu/swab). L. fermenturn was isolated
from all guinea-pigs examined. Several other species of
Lactobacillus were isolated routinely but could not be
definitively identified to species level. The most common gram-negative organism isolated in this study
was Proteus sp.-presumably faecal contaminants. No
attempts were made to classify organisms other than
lactobacilli to species level. One strain of L.fermentum,
Segregational stability of pNZl7 in L. fermentum
Table 11. Culture and enumeration of guinea-pig vaginal
The segregational stability of pNZ17 in the vaginal
isolate L.fermentum CRl was determined by repeated
subculture in the absence of chloramphenicol; 0.1 ml
of culture was diluted and plated on to MRS agar with
and without chloramphenicol 10 pg/ml. After incubation for 24 h, the number of colonies in the presence
and absence of chloramphenicol was counted. Cultures were passaged 22 times, which represents c. 120
generations for this strain.
Intravaginal administration of L. fermentum C&l
Lactobacilli were grown in the presence of chloram-
Ecological studies of the guinea-pig vagina
Aerobes and facultative anaerobes
Corynebacterium sp. 1
Corynebacterium sp. 2
Staphylococcus sp.
Enterococcus sp.
L . fermentum
L . acidophilus
Lactobacillus sp.
Streptococcus sp.
Obligate anaerobes
Propionibacterium sp.
BiJfidobacterium sp.
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Fig. 1. Agarose gel of plasmid preparations of vaginal and culture collection lactobacilli. Lane 1, mol. wt markers; 2, L. fermentum BR 1 1;
3, L. fermentum BRll containing pNZ17 (= L. fermentum CR 1); 4, L. casei ATCC 393; 5, L. plantarum ATCC 14917; 6 , L. sp. WC 2 ; 7,
Lactobacillus sp. BR 10; 8, E. coli containing pNZ17.+indicates chromosomal DNA; indicates plasmid DNA bands.
120 110 -
100 -
70 -
30 20
10 -
Number of generations
Fig. 2. Segregational stability of pNZ17 in L.fermentum strain CR1 after repeated subculture with (-)
sensitive to chloramphenicol 5 pglml, was designated
B R l l and chosen for further study.
Plasmid isolation from vaginal lactobacilli
Plasmid profiles of several vaginal and culture
collection lactobacilli are shown in fig. 1. Extrachromosomal DNA was detected in most of the
lactobacilli examined (L. fermentum BRl 1, L . casei
ATCC 393, L. plantarum ATCC 14917, Lactobacillus
sp WC2). All preparations still contained some evi-
or without (---) chloramphenicol.
dence of chromosomal DNA contamination (+ in fig.
1) despite efforts to use optimal procedures for plasmid
isolation. The high mol. wt plasmid migrating above
the contaminating chromosomal DNA in most lanes
possibly represents the lactose fermentation plasmid
which is a common feature of many lactic acid bacteria.
Furthermore, several strains contained plasmids of
lower mol. wt (L. fermentum BRl1, L . plantarum
ATCC 14917). Plasmid preparations of L . casei ATCC
393 and L . plantarum ATCC 14917 were made to
assess the reliability of the modified Anderson and
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Fig. 3. The persistence of L. fermenturn CR1 in the guinea-pig vagina after intravaginal inoculation; ---, number of Cm' CR1 bacteria on
day 0; -,
number of Cm' bacteria removed from the vagina following inoculation of 10" CR1 bacteria on five successive administrations
as indicated by (T).
McKay method. L. casei ATCC 393 is known
from previous reports to contain a large 27-kb Lac+
plasmid (as shown in fig. 1, lane 4), and L. plantarum
ATCC 14917 to contain an 8-kb plasmid (as shown in
fig. 1, lane 5). Similar plasmid profiles were obtained
repeatedly in this work, confirming the suitability of
the modified procedure.
Electrotransformation of vaginal strain L. fermenturn
B R l l with p N Z l 7
L.fermenturn BRll was regularly transformed with
plasmid pNZl7 by the electroporation protocol described, with transformation efficiencies up to 2 x lo3
transformants/pg of pNZ 17. Culture collection lactobacilli gave higher transformation efficiencies of 1O4
transformants/pg or better (data not shown). Transformation of L. fermentum BR11 with pNZ17 was
confirmed by agarose gel electrophoresis (fig. 1, lane
3). Restriction endonuclease digestion of this plasmid
preparation with Pstl, which attacks a single site in
pNZ17, gave a 5.7-kb linear plasmid band identical in
size to Pst 1-digested pNZ17 from Escherichia coli
(data not shown).
Segregational stability of plasmid p N Z l 7 in L.
.fermenturn strain C R l
Even in the absence of the selective agent chloramphenicol, plasmid pNZ17 was maintained relatively
stably in L. fermenturn strain CRl for at least 120
generations (fig. 2). During the first 50 generations
there was virtually no loss of plasmid; there was a
30% loss of plasmid during the next 70 generations.
Intravaginal administration of L. fermentum strain
After intravaginal inoculation of L. fermenturn
strain CR1 into three guinea-pigs, its maintenance in
the vagina was assessed by the ability to recover
chloramphenicol-resistant organisms (fig. 3). In initial
experiments, L.fermenturn strain CR1 cells were given
once only on day 0. In all experimental animals, the
number of chloramphenicol-resist ant bacteria (Cm')
recovered from the vaginal tract decreased daily from
lo4on day 1, to < 10 by day 7. The modified procedure
of inoculation daily for 5 days did not significantly
increase the persistence of Cm' bacteria: 3 x lo5
organisms were recovered by vaginal swabbing 1 day
after the fifth inoculation and the numbers had fallen
to 3 x lo2organisms by day 7. Thereafter, Cm' bacteria
were below the detection level of lo1.
Systemic immunisation has generally proved to be
ineffective against mucosal pathogens, primarily because, although it induces strong systemic antibody
responses, it usually fails to induce a significant
response at the appropriate mucosal surface.2 In
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contrast, direct mucosal immunisation often stimulates a more effective mucosal respon~e.l-~
most of these studies have focused on either the
gastrointestinal or respiratory tracts as the mucosal
sites of administration. Very few studies have focused
on the female reproductive tract, despite the importance of local infections at this site.5 However, limited
studies have shown that administration of antigens
intravaginally does have the potential for the induction
of a strong local immune respon~e.~
In a mouse model,
the presentation of intraluminal antigen to either
dendritic or follicular cells within the vaginal epithelium or stroma, has been shown to result in the
migration of these antigen-presenting cells to local
lymph nodes, either inguinal or those ventral to the
vagina.21These activated cells presumably then go on
to secrete specific antibody back into the vaginal
One of the requirements for effective mucosal
immunity, typified by the production of sIgA (and
perhaps IgG originating from the circulation), is that
22 Live
the antigen provides a sustained
micro-organisms can provide this sustained stimulus
and thereby induce stronger mucosal responses. Therefore, our approach was to utilise non-pathogenic
bacteria that are normal residents of the female
reproductive tract as vehicles for the delivery of
antigens locally and hence the induction of a local
immune response. Lactobacilli are an excellent choice
as the delivery vehicle for several reasons ;lactobacilli
are generally non-pathogenic organisms ; they are
present in large numbers at several mucosal surface^;^
and they have a natural ability to stimulate the immune
While the present results indicate that lactobacilli
are not the dominant bacterial genera of the chosen
animal model, the guinea-pig, as they are in man,
they did constitute a constant and significant part of
the vaginal microflora in all guinea-pigs examined.
Other gram-positive species, including corynebacteria
and enterococci were found to be the dominant aerobic
genera and perhaps these micro-organisms also could
be used as vaccine delivery vehicles. These two genera
also are found as a substantial component of the
indigenous microflora of mice, rats, monkeys and
However, the fact that lactobacilli
are not the most prevalent micro-organisms is not of
great concern, because the main requirement is for the
vaccine organism to be able to colonise the vagina and
to be able to compete with the established and more
numerous resident micro-organisms. The fact that
lactobacilli were present in significant numbers in all
guinea-pigs examined, indicates that they are able to
satisfy this requirement.
Plasmids are commonly used for the introduction of
foreign DNA into bacterial cells. Plasmids, cryptic or
coding for phenotypically defined characteristics, such
as carbohydrate fermentation, have been shown to be
ubiquitous in naturally occurring Lactobacillus strains.
Another important characteristic of many of the
lactobacillus plasmids investigated previously has
been the broad host range of their replication control
elements. The use of resident plasmid replicons as the
basis for the construction of cloning vectors is quite
~ o m m o n . l l~7, In
~ ~ the
* present study, all vaginal
lactobacilli showed evidence of plasmid DNA, and,
therefore, the construction of cloning vectors based on
their replicons would be feasible. However, we have
not pursued this approach at this stage. Instead, we
chose to base our cloning vehicles on plasmids with a
broad host range among gram-positive bacteria. Plasmids belonging to this family of small plasmids
replicate by rolling circle replication and are dispersed
throughout the gram-positive genera. The plasmid
chosen contained the replicon of pSH71, a cryptic
plasmid of Lactococcus lactis subsp. lactis, which has
been shown to function in several industrial lactobacilli.14,15 In the present study, plasmid pNZ17
readily transformed the vaginal L. fermenturn BRl 1
isolate at efficiencies of 2 x lo3. Penicillin G added to
culture media of logarithmically replicating cells has
been shown by several groups, including our own
(data not shown) to increase the transformability of
gram-positive bacteria, as have other cell-wall modifiers, such as glycine and 1ys0zyme.l~Although the
transformation efficiency of our vaginal L. fermenturn
strain was lower than that of the highly transformable
L. casei ATCC 393, the transformation protocol was
found to be simple and reliable.
If a recombinant vector is to be used to deliver
foreign antigen to the reproductive tract, it is desirable
that it is stably maintained in the host strain under
non-selective conditions. Bates et a1.16 reported variable stabilities of recombinant plasmids in different
Lactobacillus strains, perhaps due to incompatibility
of the introduced plasmid with the resident lactobacillus plasmid. Posno et a l l 5 showed that the
plasmid pGK12, which contains the replicon of the
Lactococcus lactis subsp. cremoris pWV0 1, a plasmid
very closely related to the pSH71 replicon used in the
present work, was stable in a L. plantarum strain, but
was highly unstable in L. pentosus. The presence of a
resident plasmid in their host strain was not mentioned. The high-level stability of pNZl7 in the vaginal
isolate L. fermenturn CR1 in the present study may be
explained by compatibility with the resident plasmids
of the host L. fermenturn BRll strain. The large
plasmids naturally occurring in this strain may not be
of the ssDNA plasmid family and, therefore, plasmid
incompatibility was not a significant problem. The
segregational and structural stability of vaccine plasmids may alter when foreign genes are inserted, and
this needs to be investigated further.
Under the conditions used in this work, the transformed vaginal L . fermenturn CRl strain did not
persist for long periods in the vagina after intravaginal
administration. Previous
24 have indicated
that, to achieve effective colonisation of the female
reproductive tract by lactobacilli, it is important to
control a range of factors including the growth phase
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of the inoculated cells, the nature and pH of the cell
suspension media and the cell concentration of the
inoculum. Although these factors were considered in
the design of the present experiments, the intravaginal
administration of L. fermenturn strain CR1 failed to
establish it within the vagina of the guinea-pigs.
Recolonisation of the vaginal ecosystem by a recombinantly modified bacterium is obviously complex,
and other factors that may have contributed to the
short-term persistence include : in-vivo segregational
instability; change in the adhesive properties of the
vaginal isolate due to in-vitro manipulation ; colonisation resistance of the native vaginal microflora; and
removal of the recombinant lactobacilli by the daily
swabbing routine before they had a chance to become
established. Factors which promote efficient colonisation by the modified micro-organism will need to be
investigated further.
This study demonstrated that naturally occurring
isolates of vaginal lactobacilli are amenable to genetic
modification. Previous studies on genetic manipulation and colonisation with lactobacilli have focused
on culture collection or dairy strains. Lactobacillus
strains often demonstrate host specificities and thus
the use of a naturally occurring vaginal isolate to
deliver antigen within the genital tract is desirable.
However, further experiments are required to find
optimal conditions for the delivery of these strains to
the female reproductive tract. While it is not yet fully
proven that colonisation of the reproductive tract with
recombinantly modified lactobacilli will lead to production of sufficient titre of antibodies to be protective
against invasion by the pathogen, this approach to
vaccination should result in antibodies of the IgA
class, at the correct tissue location, the vaginal mucosa.
Therefore, we believe that the use of genetically
modified commensal micro-organisms, such as lactobacilli, has great potential in the development of
vaccines against a range of diseases of the female
reproductive tract.
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This work was supported by grants from the National Health and
Medical Research Council of Australia and the QUT Research and
Development Scheme.
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