FEMS Immunology and Medical Microbiology 27 (2000) 291^297
www.fems-microbiology.org
Chimeric animal and plant viruses expressing epitopes of outer
membrane protein F as a combined vaccine against
Pseudomonas aeruginosa lung infection
Harry E. Gilleland Jr. a; *, Linda B. Gilleland a , John Staczek a , Ronald N. Harty b ,
Adolfo Garc|¨a-Sastre c , Peter Palese c , Frank R. Brennan d , William D.O. Hamilton d ,
Mohammed Bendahmane e , Roger N. Beachy e
a
Department of Microbiology and Immunology, Louisiana State University Medical Center, School of Medicine in Shreveport, 1501 Kings Highway,
Shreveport, LA 71130-3932, USA
b
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6049, USA
c
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA
d
Axis Genetics plc, Babraham, Cambridge CB2 4AZ, UK
e
Donald Danforth Plant Science Center, 7425 Forsyth Blvd., P.O. Box 1098, St. Louis, MO 63105, USA
Received 19 July 1999; received in revised form 9 August 1999; accepted 9 August 1999
Abstract
Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety
of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa
261^274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305^318, NATAEGRAINRRVE), and 18 (aa 282^295,
NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a
vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective
epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B
site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the
mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A
chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMVPAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a
vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with
a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was
successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the
mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been
unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system.
Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of
P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the
chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the
immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge
with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual
chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein
F has vaccine potential warranting continued development into a vaccine for use in humans. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Outer membrane protein F; Epitope vaccine ; Chimeric virus vaccine ; In£uenza virus; Cowpea mosaic virus; Tobacco mosaic virus;
Pseudomonas aeruginosa
* Corresponding author. Tel. : +1 (318) 675-5767; Fax: +1 (318) 675-5764; E-mail : [email protected]
0928-8244 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 2 0 6 - 0
FEMSIM 1184 13-3-00
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1. Introduction
Table 1
Chimeric CPMVs constructed to date
We have traveled down a long path of discovery since
we ¢rst published in 1975 [1] the idea that certain outer
membrane (OM) proteins of Pseudomonas aeruginosa
might serve as a protective vaccine. Early on we surveyed
the protective abilities of a variety of puri¢ed OM proteins
(D2, E, F, G, and H), reaching the conclusion that protein
F had the greatest vaccine potential [2]. Protein F puri¢ed
from the OM of P. aeruginosa was shown to a¡ord signi¢cant protection in rodent models of systemic infection
[3], burned mice [4], and chronic pulmonary infection [5].
Recombinant protein F puri¢ed from Escherichia coli was
shown to retain vaccine e¤cacy in the burned mouse [2]
and the rat chronic pulmonary infection [6] models. Protein F is an excellent vaccine candidate in that it is present
in all strains of P. aeruginosa [7^9], is antigenically conserved in all strains [8,9], is surface-exposed [10^12], and is
essential to the bacterial cell [13]. The problems of obtaining su¤cient quantities of puri¢ed protein F and of having
to maintain this integral membrane protein in a detergent
environment led us to search for surface-exposed protective epitopes within protein F. Through use of synthetic
peptides conjugated to keyhole limpet hemocyanin as a
carrier, three surface-exposed linear B-cell epitopes (peptides 9, 10 and 18) [12,14] were identi¢ed. Peptide 9
(TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) had protective e¤cacy in models of both
chronic [9] and acute [15] pulmonary infections. Both epitopes 9 and 10 were conserved among the 59 strains of P.
aeruginosa surveyed [9]. Once these protective epitopes had
been identi¢ed, the question became how best to present
these epitopes as a vaccine. We chose to use a chimeric
viral vector for presentation of the P. aeruginosa epitopes.
At the last meeting of this vaccine conference in 1996,
we reported that a chimeric in£uenza virus containing an
11-amino acid insert from the peptide 10 epitope displayed
within the antigenic B-site of the viral surface hemagglutinin [16] had e¤cacy as a protective vaccine in the chronic
pulmonary infection model [17]. Our plan was to construct
a chimeric in£uenza virus containing the peptide 9 epitope,
demonstrate its protective e¤cacy as a vaccine, and then
use the two chimeric viruses as a combined vaccine. The
importance of a combined vaccine that elicits protective
antibodies directed against two distinct epitopes within
protein F is paramount in the development of a successful
vaccine. The presence of the second epitope will prevent
the bacterium from being able to mutate perhaps a single
amino acid within one epitope and hence escape the protective antibodies elicited by the vaccine. Unfortunately,
despite multiple attempts to recover a chimeric in£uenza
virus containing a peptide 9 insert over the past three
years, no such chimera has been recovered.
By mid-1997 we were investigating the use of a second
viral vector system, termed EPICOAT0 , for presentation
of the Pseudomonas epitopes. The EPICOAT0 system is
CPMV-PAE-1:
CPMV-PAE-2:
CPMV-PAE-3:
CPMV-PAE-4:
CPMV-PAE-5:
CPMV-PAE-8:
CPMV-PAE-9:
CPMV-PAE-10:
CPMV-PAE-11:
epitope 9 on S subunit
epitope 10 on S subunit
epitope 18 on S subunit
epitopes 18 and 10 on S subunit
epitopes 18 and 10 on L subunit
epitopes 18 and 9 on L subunit
epitopes 9 and 18 on S subunit
epitopes 9 and 10 on S subunit
epitopes 9 and 10 on L subunit
based on the plant virus, cowpea mosaic virus (CPMV),
which can be modi¢ed to display foreign peptide inserts on
either its small (S) subunit capsid protein or its large (L)
subunit capsid protein. To date a number of chimeric
CPMVs have been constructed to contain the epitopes 9,
10, and 18 from OM protein F inserted either individually
or as tandem pairs (Table 1). CPMV-PAE-1, CPMV-PAE2, and CPMV-PAE-3, which had epitopes 9, 10, and 18
inserted individually into the S subunit, resulted in low
rates of plant infection and a low virus yield, a situation
that was remedied by coexpressing peptide 18 in tandem
with peptide 10. The 18-10 insert was placed into the S
subunit (CPMV-PAE-4) as was the usual procedure, as
well as into the L subunit (CPMV-PAE-5). The subunit
into which the peptide was inserted greatly in£uenced the
nature of the antibodies elicited [18]. CPMV-PAE-4 elicited antibodies that were directed toward the 18 epitope
quite predominantly (about 85%), with very little activity
directed toward the 10 epitope. CPMV-PAE-5, on the
other hand, elicited antibodies that reacted exclusively
with the 10 epitope, with little to no anti-18 activity. Furthermore, CPMV-PAE-5 elicited antibodies that had signi¢cantly greater opsonic activity against P. aeruginosa
than did anti-CPMV-PAE-4 antibodies [18]. The CPMVPAE-5 chimeric virus protects in the mouse model of
chronic pulmonary infection [19]. Attempts to construct
a chimeric CPMV with epitope 9 as the protective component have included the assembly of CPMV-PAE-10 and
CPMV-PAE-11 with the combined epitopes 9 and 10 in
tandem on the S or L subunit, respectively. These viruses
elicited antibodies that reacted with epitope 10 but lacked
anti-epitope 9 activity. We are purifying CPMV-PAE-8,
which has epitopes 18 plus 9 in tandem inserted in the L
subunit, and CPMV-PAE-9, which has a tandem 9 plus 18
insert in the S subunit, in an attempt to obtain a chimeric
CPMV that elicits anti-epitope-9 antibodies.
A third viral vector that we have recently begun using is
based on the plant virus, tobacco mosaic virus (TMV).
The coat protein of TMV can accept foreign peptides inserted between amino acids serine 154 and glycine 155,
and in the assembled virion the inserted epitope is displayed on the surface of the chimeric virion [20^23]. Initial
attempts to construct chimeric TMVs used the 9 and 10
epitopes individually. A chimeric virus for the 9 epitope
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was successfully recovered, and a supply of chimeric virus
was prepared. However, in these studies, the 10 epitope
could not be inserted successfully to yield the desired chimeric virus. Further attempts to isolate a chimeric TMV
carrying epitope 10 are currently ongoing. The chimeric
virus with the epitope 9 insert is protective in our model
system (manuscript in preparation).
At last, we have both epitopes 9 and 10 of OM protein
F of P. aeruginosa expressed individually in a chimeric
virus vector, with each having protective e¤cacy when
used individually as a vaccine in the mouse model of
chronic pulmonary infection. This allows us now to use
the two viruses as a combined vaccine to determine
whether such a combined vaccine can elicit antibodies directed toward each epitope, with the protective e¤cacy of
the combined vaccine remaining at a level comparable to
or surpassing that provided by the individual chimeric
virus vaccines.
2. Materials and methods
2.1. Viruses used
The in£uenza viruses used in this study included the
wild-type A/WSN/33 (WSN) virus and the HG10-11 chimeric virus constructed by ribonucleoprotein transfection
as reported previously [16,17]. HG10-11 contains the 11amino acid sequence AEGRAINRRVE from the P. aeruginosa OM protein F peptide 10 epitope inserted into site
293
B of the hemagglutinin between amino acids 158 and 159
(HA1 numbering). The TMV strains used included the
wild-type TMV-U1 strain and the chimeric TMV-9-14 virus, whose construction will be reported elsewhere (manuscript in preparation). TMV-9-14 contains the 14-amino
acid sequence TDAYNQKLSERRAN of the protein F
peptide 9 epitope inserted into the TMV coat protein between amino acids 154 and 155. Stocks of the two in£uenza viruses were grown in Madin-Darby canine kidney
cells. Stocks of the TMV strains were puri¢ed from
the leaves of systemically infected Nicotiana tabacum
Xanthi-nn plants.
2.2. Bacterial strains and culture conditions
The strains of P. aeruginosa used included the following: the PAO strain (Difco 0-5; Difco Laboratories, Detroit, MI, USA) that corresponds to a Fisher-Devlin immunotype 7, ATCC 27584 (Difco 0-6; Fisher-Devlin
immunotype 1), ATCC 27313 (Difco 0-11; Fisher-Devlin
immunotype 2), ATCC 27314 (Difco 0-2; Fisher-Devlin
immunotype 3), ATCC 27315 (Difco 0-1; Fisher-Devlin
immunotype 4), ATCC 27585 (Difco 0-10; Fisher-Devlin
immunotype 5), ATCC 27579 (Difco 0-7,8; Fisher-Devlin
immunotype 6), and the KG1077 strain which is a protein
F-de¢cient mutant of the PAO strain [13] obtained from
N. Gotoh (Kyoto, Japan). All ATCC strains were obtained from the American Type Culture Collection, Rockville, MD, USA. All strains were grown at 30³C with
shaking in nutrient broth or on nutrient agar plates.
Table 2
Immunization protocol
Immunization group
First immunizing dose (day 0)
Second immunizing dose (day 14)
Third immunizing dose (day 28)
wt WSN
102 p.f.u. of WSN in 50 Wl PBSa ,
given INb
105 p.f.u. of WSN given with alum
in 100 Wl PBS, given IM in right hip
105 p.f.u. of WSN in 100 Wl PBS,
given IM in left hip
HG10-11
103 p.f.u. of HG10-11 in 50 Wl PBS,
given IN
105 p.f.u. of HG10-11 given with alum
in 100 Wl PBS, given IM in right hip
105 p.f.u. of HG10-11 in 100 Wl PBS,
given IM in left hip
wt TMV-U1
50 Wg of TMV-U1 given with alumc in
100 Wl PBS, given IMb in right hip
10 Wg of TMV-U1 given with alum in
100 Wl PBS, given IM in left hip
10 Wg of TMV-U1 in 100Wl PBS,
given IM in right hip
TMV-9-14
50 Wg of TMV-9-14 given with alum
in 100 Wl PBS, given IM in right hip
10 Wg of TMV-9-14 given with alum in
100 Wl PBS, given IM in left hip
10 Wg of TMV-9-14 in 100 Wl PBS,
given IM in right hip
wt WSN/wt TMV-U1
102 p.f.u. of WSN in 50 Wl PBS, given
IN plus 50 Wg of TMV-U1 given with
alum in 100 Wl PBS, given IM in right
hip
105 p.f.u. of WSN given with alum in
100 Wl PBS, given IM in left hip plus
10 Wg of TMV-U1 given with alum in
100 Wl PBS, given IM in right hip
105 p.f.u. of WSN in 100 Wl PBS,
given IM in left hip plus 10 Wg of
TMV-U1 in 100 Wl PBS, given IM
in right hip
HG10-11/TMV-9-14
103 p.f.u. of HG10-11 in 50 Wl PBS,
given IN plus 50 Wg of TMV-9-14
given with alum in 100 Wl PBS, given
IM in right hip
105 p.f.u. of HG10-11 given with alum
in 100 Wl PBS, given IM in left hip plus
10 Wg of TMV-9-14 given with alum in
100 Wl PBS, given IM in right hip
105 p.f.u. of HG10-11 in 100 Wl PBS,
given IM in left hip plus 10 Wg of
TMV-9-14 in 100 Wl PBS, given IM
in right hip
a
Phosphate bu¡ered saline, pH 7.3.
Routes of inoculation were either intranasal (IN) or intramuscular (IM).
c
Aluminum hydroxide (1:1) used as adjuvant.
b
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2.3. Immunization procedures
Five-week-old, speci¢c-pathogen-free ICR mice (Harlan
Sprague Dawley, Indianapolis, IN, USA) were used. Six
immunization groups were included, consisting of mice
immunized with the wild-type WSN in£uenza virus alone,
the chimeric HG10-11 in£uenza virus alone, the wild-type
TMV-U1 virus alone, the chimeric TMV-9-14 virus alone,
the combined wild-type in£uenza WSN virus plus the
wild-type TMV-U1 virus, and the combined chimeric viruses HG10-11 in£uenza plus TMV-9-14. All groups received three immunizations at 2-week intervals as detailed
in Table 2. Two weeks after the ¢nal immunization (day
42), ¢ve mice from each group were killed by cardiac
puncture under sodium pentobarbital anesthesia, and the
sera obtained were pooled for each group of mice. The
remaining mice were challenged using the chronic pulmonary infection model (see below). All mice used in this
study were handled in accordance with the guidelines of
the Louisiana State University Medical Center-Shreveport
Animal Care and Use Committee.
2.4. Monitoring the antibody response
The pooled sera obtained from each group of mice on
day 42 were used in enzyme-linked immunosorbent assays
(ELISA) to determine the titer of immunoglobulin G
(IgG) antibodies to various antigens. The ELISA was performed as described previously [4]. For one assay [14],
synthetic peptides 9 or 10 were used as the antigen. Flatbottomed, 96-well Immulon 1 microtiter plates were irradiated [24] face-up under a 15-W UV lamp for 20 min at
ambient temperature. Each well of the UV-irradiated
plates was immediately coated with 100 Wl of a 25-Wg
ml31 peptide 9 or peptide 10 solution in PBS, pH 7.2.
The plates were incubated at 37³C overnight prior to
use. A second ELISA used whole cells of the various
strains of P. aeruginosa as the antigen [9]. Each well of
the microtiter plate was coated with 100 Wl of a bacterial
suspension consisting of approximately 108 cells per ml in
PBS, pH 7.2. For all the bacterial strains, cells were harvested by centrifugation during exponential growth phase
from nutrient broth, and the pelleted cells were resuspended in PBS. Prior to coating the wells, the bacterial
suspension was incubated at 56³C for 30 min. The plates
with the coated wells were incubated overnight at 37³C.
Whole cells of the PAO strain, the KG1077 protein Fde¢cient strain, and the strains representing Fisher-Devlin
immunotypes 1^6 were used in these assays as antigens.
The ELISAs were performed 2 or 3 times with each of the
pooled antisera.
2.5. Challenge in the chronic pulmonary infection model
Mice were challenged by using a model of chronic pulmonary infection with P. aeruginosa [5,17,25]. Two weeks
after the ¢nal immunization, the mice were challenged
with agar beads containing P. aeruginosa cells of the FD
immunotype 4 strain. The mice were ¢rst anesthetized with
an intraperitoneal injection of sodium pentobarbital and
then inoculated via a tracheal incision with 50 Wl of an
agar bead slurry encasing approximately 5U103 c.f.u. of
P. aeruginosa. A beaded-tip 22-gauge needle was gently
guided to favor inoculation of the left lung. The incision
was closed with sterile wound clips. Eight days after the
challenge, protection a¡orded to immunized mice by the
chimeric viruses was assessed by two methods. First, the
lungs were examined macroscopically for the presence of
lesions [17]. Lesions were scored as follows : 0, absence of
any macroscopic lesion; 1‡ , presence of one or two small
lesions not exceeding 1 mm in diameter; 2‡ , presence of
three or more small lesions not exceeding 1 mm in diameter; 3‡ , presence of a medium lesion 2^5 mm in diameter ;
4‡ , presence of a large lesion exceeding 5 mm in diameter.
Mice were placed into one of two groups based upon
the lung lesion score, either those with essentially
normal lungs (0 or 1‡ ) or those with signi¢cant lesions
(2‡ ^4‡ ). Scoring of the pulmonary lesions was performed
by an investigator with more than 12 years of experience
in macroscopic lung lesion scoring. Second, bacterial
quantitation of the number of P. aeruginosa c.f.u. present
in the lungs was performed as described previously
[5,6].
Statistical analyses of the di¡erences between control
wild-type virus immunized mice and chimeric virus-immunized mice upon scoring of lung lesions and quantitation
of the bacteria present in the lungs were performed with
the IBM EpiStat Basic Statistics Program, and P values
were calculated by the Fisher exact test. P values of 9 0.05
were considered to be signi¢cant.
Table 3
Representative titers of IgG antibodies in pooled antisera from the various immunization groups against various antigens by ELISA
Immunization group
wt WSN
HG10-11
wt TMV-U1
TMV-9-14
wt WSN/wt TMV-U1
HG10-11/TMV-9-14
Titer determined by ELISA with following as coating antigen:
FD2 whole cells
FD4 whole cells
KG1077 whole cells
peptide 9
peptide 10
50
1 600
100
6 400
100
12 800
50
1 600
50
12 800
50
12 800
0
50
50
400
50
400
50
50
200
12 800
100
12 800
50
800
100
200
50
3 200
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295
Table 4
Scoring of macroscopic lung lesions following challenge with FD4 immunotype P. aeruginosa in a chronic pulmonary infection model
Immunization group
No. of mice having lesions scored as 2‡ ^4‡ /total no. of mice (%)
P value
wt WSN
HG10-11
wt TMV-U1
TMV-9-14
wt WSN/wt TMV-U1
HG10-11/TMV-9-14
22/29 (75.9%)
9/29 (31.0%)
24/30 (80%)
12/28 (42.9%)
22/26 (84.6%)
13/28 (46.4%)
^
0.0007
^
0.004
^
0.004
P values were determined by Fisher's exact test
3. Results
The IgG antibody response elicited by each of the viral
preparations gave a similar reaction to the PAO (FD7)
strain and to each of the heterologous FD1-6 strains, as
illustrated in Table 3 with the FD2 and 4 strains. The
wild-type WSN and TMV-U1 viruses used individually
or in combination elicited no signi¢cant antibody reactivity against whole cells of the FD immunotype strains, the
protein F-de¢cient KG1077 strain, or to peptides 9 or 10.
In contrast, the chimeric HG10-11 in£uenza virus containing the epitope 10 insert elicited IgG antibodies that reacted at a titer of 1600 to whole cells of the FD immunotype strains and of 800 to peptide 10. As expected, HG1011 did not elicit antibodies reactive with protein F-de¢cient cells or peptide 9. The chimeric TMV-9-14 containing the epitope 9 insert elicited antibodies that reacted at
high titer (6400^12 800) with whole cells of the FD immunotype strains and to peptide 9, but not with peptide 10 or
with the protein F-de¢cient strain. The chimeric viruses
given as a combined vaccine elicited antibodies that reacted at high titer (12 800) with whole cells of the FD
immunotype strains but not with cells of the protein Fde¢cient KG1077 cells. Most importantly, the combined
vaccine elicited antibodies that reacted at high titer with
both epitopes 9 and 10.
Both the chimeric HG10-11 and TMV-9-14 viruses
when used individually as a vaccine a¡orded signi¢cant
protection against subsequent challenge with P. aeruginosa
in the chronic pulmonary infection model in mice. This
was evidenced by both the reduction in the number of
2‡ ^4‡ lung lesions in mice immunized with either of the
chimeric viruses compared to mice immunized with its
appropriate wild-type virus (Table 4), as well as the increase in the number of mice having no P. aeruginosa
remaining in the lungs 8 days after challenge (Table 5).
More importantly, when the two chimeric viruses were
used as a combined vaccine, the combination retained
protective e¤cacy at the level provided by the individual
chimeric viral vaccines. The percentage of mice having 2‡ ^
4‡ lesions in mice immunized with HG10-11 alone, TMV9-14 alone, or HG10-11 plus TMV-9-14 was 31%, 43%,
and 46%, respectively (Table 4). Likewise, the percentage
of mice having no P. aeruginosa present in the lungs after
8 days was 70%, 61%, and 65% in mice immunized with
HG10-11 alone, TMV-9-14 alone, or HG10-11 plus TMV9-14, respectively (Table 5). No increase in protective e¤cacy was apparent for the combined vaccine over that
provided by the individual chimeric viruses.
4. Discussion
P. aeruginosa is an opportunistic pathogen that causes
only minor infections in healthy individuals, but it can
cause severe to life-threatening infections in compromised
patients, such as patients with severe burns or advanced
cancer, the elderly, the immunosuppressed, and children
with cystic ¢brosis (CF). P. aeruginosa remains the leading
cause of morbidity and mortality in CF patients due to its
ability to chronically colonize the CF lung. No means
presently exists to block the colonization of the CF lung
by P. aeruginosa. We envision one major clinical use of
our epitope vaccine would be to immunize CF children in
order to block their chronic pulmonary colonization with
P. aeruginosa and thus avoid the subsequent pulmonary
Table 5
Quantitation of P. aeruginosa present in the lungs of mice following challenge with FD4 immunotype P. aeruginosa in a chronic pulmonary infection
model
Immunization group
No. of mice with lungs yielding no growth/total no. of mice (%)
P value
wt WSN
HG10-11
wt TMV-U1
TMV-9-14
wt WSN/wt TMV-U1
HG10-11/TMV-9-14
2/19 (10.5%)
14/20 (70%)
6/20 (30%)
11/18 (61.1%)
6/17 (35.3%)
13/20 (65%)
^
0.0002
^
0.05
^
0.07
P values were determined by Fisher's exact test
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pathology that usually follows. Immunization will most
likely require a series of inoculations early in infancy,
such as at 2, 4, and 6 months of age, followed by boosters
annually to maintain protective levels of antibodies
throughout their lifetime. Use of a protective epitope vaccine instead of a puri¢ed OM protein vaccine will lessen
the likelihood of developing detrimental allergic-type reactions to the Pseudomonas immunogen itself. Should unwanted reactions directed against the viral vector develop,
then the booster inoculations could be continued with the
Pseudomonas epitopes inserted into a di¡erent viral vector.
Thus, we believe that a vaccine based on protective, surface-exposed epitopes of OM protein F of P. aeruginosa
has great potential for successful use in this clinical situation.
If an epitope vaccine contained a single epitope, it might
be possible for the bacterium to escape the protective antibodies elicited by altering a single critical amino acid residue within that epitope. However, escape from protective
antibodies is much less likely if the bacterium is required
to mutate two separate epitopes within the protein. For
this reason, we consider it vital that our epitope vaccine be
a combined vaccine consisting of chimeric viruses containing inserts for both protein F epitopes 9 and 10. It is also
crucial to the development of our epitope vaccine that
epitopes 9 and 10 not act in an overtly antagonistic manner when combined as a vaccine so as to result in signi¢cantly less protective e¤cacy than a¡orded by the chimeric
viruses upon use as individual vaccines. It would be ideal
if an enhanced, synergistic e¡ect was observed upon combining the two epitopes into a vaccine. It is also highly
desirable that both epitopes 9 and 10 be expressed as inserts in the same viral vector for use as the combined
vaccine for a number of reasons, including ease of vaccine
production, ability to switch viral vectors if vector-related
immunological problems develop upon repeated immunization, as well as patent and licensing considerations.
We have studied the use of three di¡erent viruses as
vectors for our Pseudomonas epitopes. In none of the three
viruses have both epitopes 9 and 10 been successfully inserted in a manner that will elicit antibodies reactive with
each epitope. We have constructed a chimeric in£uenza
virus with an 11-amino acid insert of epitope 10 in the B
site of the viral hemagglutinin that elicits protective antibodies directed toward epitope 10. We have constructed a
CPMV chimera with an insert of epitopes 18 and 10 in
tandem within the L subunit protein of the virus that
elicits protective antibodies directed solely toward epitope
10. We have constructed a chimeric TMV with a 14-amino
acid epitope 9 insert within its coat protein that elicits
protective antibodies directed toward epitope 9. E¡orts
are continuing with each virus system to obtain a chimeric
virus with the remaining epitope inserted in such a manner
that protective antibodies directed toward that epitope
are elicited. In the meantime, we used the available
chimeric viruses to test whether a combined vaccine con-
taining both epitopes 9 and 10 of protein F possesses
vaccine e¤cacy and to determine the level of protection
a¡orded.
Upon use of the chimeric HG10-11 in£uenza virus given
with TMV-9-14 as a combined vaccine, the vaccine successfully elicited high titers of IgG antibodies reactive with
whole cells of various FD immunotype strains of P. aeruginosa (Table 3). Most importantly, the combined vaccine
elicited antibodies reactive at high titers with each of epitopes 9 and 10. Furthermore, the combined vaccine afforded protection in the chronic pulmonary infection model upon challenge with P. aeruginosa at approximately the
same level of protection provided by the vaccines individually (Tables 4 and 5) [17], indicating that no signi¢cant
antagonistic e¡ect interferes with the use of the two epitopes together in a combined vaccine. These results indicate
that a combined chimeric viral vaccine containing epitopes
9 and 10 of OM protein F has potential for successful use
as a vaccine to prevent pulmonary infection with P. aeruginosa. Once we successfully obtain the two desired chimeric viruses in one of the three viral vector systems under
investigation, we should be able to proceed to a clinical
trial of our chimeric viral epitope vaccine.
Acknowledgements
This research was funded in part by Grant RO1AI44424 from the National Institutes of Health (NIH)
and by contracts from Axis Genetics plc to J.S. and
H.E.G. R.N.B. and M.B. thank Jean and Keith Kellogg,
The Seaver Foundation, Aline and Sam Skaggs, and the
NIH (Grant AI 27161) for support.
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