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FEMS Immunology and Medical Microbiology 40 (2004) 21^26
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Bacteriophage-mediated nucleic acid immunisation
Jason R. Clark, John B. March
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
Received 28 August 2003 ; received in revised form 9 September 2003; accepted 10 September 2003
First published online 27 November 2003
Abstract
Whole bacteriophage V particles, containing reporter genes under the control of the cytomegalovirus promoter (PCMV ), have been used
as delivery vehicles for nucleic acid immunisation. Following intramuscular injection of mice with V-gt11 containing the gene for hepatitis
B surface antigen (HBsAg), anti-HBsAg responses in excess of 150 mIU ml31 were detected. When isolated peritoneal macrophages were
incubated with whole V particles containing the gene for green fluorescent protein (GFP) under the control of PCMV , GFP antigen was
detected on the macrophage surface 8 h later. Results suggested that direct targeting of antigen-presenting cells by bacteriophage
‘vaccines’ may occur, leading to enhanced immune responses compared to naked DNA delivery. Bacteriophage DNA vaccines offer
several advantages: they do not contain antibiotic resistance genes, they offer a large cloning capacity (V15 kb), the DNA is protected
from environmental degradation, they offer the potential for oral delivery, and large-scale production is cheap, easy and extremely rapid.
; 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords : DNA vaccination ; Bacteriophage; Hepatitis B; Vaccine delivery
1. Introduction
Nucleic acid immunisation consists of inoculating a target animal with puri¢ed DNA or RNA containing a
gene(s) under the control of an exogenous eukaryotic promoter, so that protective antigens are expressed within the
host [1,2]. Nucleic acid vaccines potentially have a number
of advantages over traditional attenuated, killed or subunit vaccines. They are cheaper and easier to produce than
recombinant protein vaccines, have fewer adverse side effects and induce both cellular (Th1) and humoral (Th2)
arms of the immune system. Studies in mice have demonstrated good antibody [2] and cellular [3] responses with
nucleic acid vaccines, and in some examples protection
against disease [4,5], but trials in larger animals, humans
and non-human primates have been plagued by a lack of
* Corresponding author. Tel. : +44 (131) 445 5111 ;
Fax : +44 (131) 445 6235.
E-mail address : [email protected] (J.B. March).
e⁄cacy when using naked DNA [6]. A number of methods
have been tested to improve responses against DNA vaccines and have been reviewed [7]. These include gene gun
delivery [5], association with liposomes [8] or other microparticles [9], or the inclusion of cytokines [10], chemokines
[11], or immunostimulatory CpG motifs [12] in the vaccine
vector or in co-injected nucleic acid. However, many of
these methods signi¢cantly increase the cost or ease of
production of DNA vaccines, while others can present
regulatory hurdles and alternative ways to improve responses are still being sought.
We have cloned a number of DNA vaccination cassettes
into standard bacteriophage V vectors and used these
whole phage particles, rather than puri¢ed DNA, as a
delivery vehicle for DNA vaccination. We have tested
antibody responses and/or antigen expression using ¢ve
di¡erent phage vaccines : single gene reporter constructs
containing the gene for green £uorescent protein (VGFP) or hepatitis B surface antigen (V-HBsAg), a whole
genome library of the bovine pathogen Mycoplasma mycoides subspecies mycoides small colony type (V-MmmSC),
a single immunodominant lipoprotein clone picked from
this library (V-B1) and a whole genomic library of the
caprine pathogen Mycoplasma capricolum subspecies capripneumoniae (V-Mccp). Typical results are presented here,
with more detailed information being available [13].
0928-8244 / 03 / $22.00 ; 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/S0928-8244(03)00344-4
FEMSIM 1652 15-12-03
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2. Materials and methods
2.3. Immunisation of mice
2.1. Construction of V vaccines
Mice were vaccinated twice at weeks 0 and 3 with either
HBsAg phage vaccine constructs, control phage, or
HBsAg protein. The eight groups used were as follows :
(a) 5U1011 plaque-forming units (pfu) negative control
V-gt11 given i.m. in 50 Wl saline bu¡er ; (b) 25 Wg control
pCMV-HBs plasmid DNA (5U1012 copies) given i.m. in
50 Wl saline; (c) 1011 freeze-dried phage particles delivered
subcutaneously by Helios gene gun (Bio-Rad); (d) 5U1011
pfu V-HBsAg complexed with liposomes (Transfectam,
Promega, cat. no. E1232), given i.m. in 50 Wl saline bu¡er;
(e) 5U1011 V-HBsAg in saline/Montanide ISA 206 oil adjuvant given i.m. in a total volume of 50 Wl; (f) 5U1011
pfu V-HBsAg given i.m. in 50 Wl saline bu¡er ; (g) 5U1011
V-HBsAg complexed with chitosan (Fluka) in saline bu¡er, administered in 5 Wl per nostril; (h) 1 Wg of recombinant HBsAg (Aldeveron) delivered i.m. in 50 Wl endotoxin-free saline. Ten mice were used per group.
Whole genome libraries of Mccp and MmmSC were
made by cloning Tsp509I partial digests into the EcoRI
site of the VZAP express vector (Stratagene). The V-B1
clone was selected from the MmmSC library by performing plaque lifts and Western blotting with bovine convalescent and rabbit hyperimmune serum raised against
MmmSC. Plaques which showed good responses against
both sera were picked, ampli¢ed and stored. This selection
was possible since insert genes cloned into the VZAP Express vector are under the control of both a prokaryotic
(lacZ) and a eukaryotic (cytomegalovirus, CMV) promoter.
V-EGFP was prepared by linearising plasmid pEGFPC1 (Clontech) with EcoRI, and cloning the whole plasmid into EcoRI-digested V-gt11 DNA. V-HBsAg was
prepared by linearising plasmid prcCMV-HBs(S) (Aldeveron) with MfeI (which gives EcoRI-compatible ends)
and cloning the whole plasmid into the EcoRI site of
V-gt11.
2.2. In vitro uptake of bacteriophage by macrophage
Prior to the experiment, two mice were inoculated in the
peritoneal cavity with 1.5 ml 3% thioglycollate broth.
After 3^4 days the mice were killed and the peritoneal
cavities washed with 6 ml phosphate-bu¡ered saline
(PBS). The peritoneal exudate (containing macrophages)
was spun at 250Ug and 4‡C and the pellet re-suspended in
0.3% saline to lyse red blood cells. The macrophage suspension was again spun and the pellet re-suspended in 1 ml
of serum-free RPMI 1640, counted and added to 24-well
tissue culture plates at a concentration of 5U106 cells per
well in a volume of 1 ml. Macrophages were incubated at
37‡C for 30 min, before the supernatant was removed and
wells washed once with serum-free RPMI to remove nonadherent cells. 1 ml RPMI+10% foetal bovine serum was
added to each well along with bacteriophage (1011 per
well) or naked DNA (25 Wg per well). Plates were incubated overnight at 37‡C. After incubation, macrophages
were detached by placing plates at 4‡C for 1 h and the
cells re-suspended with a wide-bore pipette. 10 ml PBS was
added to the cells which were centrifuged at 250Ug and
4‡C for 10 min, before washing twice. Cells were then spun
onto charged microscope slides at a concentration of
5U105 cells per slide. Macrophages were stained immunohistochemically using a Vectastain Elite Universal ABC kit
(PK-6200) as per the manufacturer’s instructions, with
speci¢c anti-GFP rabbit polyclonal primary antiserum (Invitrogen, cat. no. R970-01). After immunostaining, cells
were visualised by counter-staining with haematoxylin, before dehydration and mounting.
FEMSIM 1652 15-12-03
2.4. Measurement of antibody responses
Antibody responses against recombinant HBsAg or bacteriophage V coat proteins were measured by indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were taken every 2 weeks from week 0 onwards. Mice
were killed and bled out at di¡erent stages throughout the
experiment: for mice numbers 1^2 in each group, four
bleeds were tested (weeks 0^6), for mice 3^6, ¢ve bleeds
were tested (weeks 0^8), and for mice 7^10, six bleeds were
tested (weeks 0^10). ELISA plates were coated overnight
in 0.05 M sodium carbonate bu¡er at pH 9.2 with either
100 ng of puri¢ed HBsAg (Aldeveron) or 109 bacteriophage per well. Coating bu¡er was then removed and
200 Wl per well blocking bu¡er (5% Marvel dry skimmed
milk in PBS-Tween) was added for 30 min at 37‡C. Blocking bu¡er was then removed and primary antibody was
added at a dilution of 1:100 in blocking bu¡er at 100 Wl
per well and plates were incubated for 3 h at 37‡C. Plates
were then washed ¢ve times in PBS-Tween and horseradish peroxidase secondary antibody (Dako) added for 1 h
at 37‡C at the manufacturer’s recommended dilution.
Plates were then washed ¢ve times in PBS-Tween and
200 ml per well substrate (Sigma Fast-OPD tablets) added
and the plates developed for 10 min in the dark. The
reaction was stopped by the addition of 50 ml per well
of 3 M H2 SO4 and the optical density read at 492 nm.
Selected serum samples had the anti-HBsAg response
quanti¢ed by comparison to international standards
(Bio-Rad Monolisa Standards, cat. no. 72399).
2.5. Statistical analysis
A one-way analysis of variance using Tukey’s multiple
comparison test was used.
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Fig. 1. Expression of cloned inserts in APCs. Mouse peritoneal macrophage were cultured overnight in the presence of (a) V-GFP or (b) unmodi¢ed control V-gt11 and immunohistochemically stained for the
presence of expressed protein. The large panel shows macrophage counterstained with haematoxylin at U40 magni¢cation. The smaller circled
panel shows uncounterstained macrophage at U1 magni¢cation. Macrophage cultured with V-GFP showed signi¢cantly more staining than
those cultured with control phage.
3. Results and discussion
Being a particulate antigen, bacteriophage are rapidly
taken up by antigen-presenting cells (APCs) [14], and following injection they are targeted to the spleen and liver
Kup¡er cells (where they persist for several days) [15]. To
demonstrate the uptake of phage particles and expression
of cloned inserts by APCs in vitro, reporter phage
(V-GFP) were added to activated mouse peritoneal macrophages, and GFP expression on the cell surface was demonstrated by immunohistological staining (Fig. 1). Similar
results were seen with macrophage cultured with V-B1
(data not shown), suggesting that direct immune priming
by professional APCs should occur in mice following injection with phage ‘DNA’ vaccines. This is in agreement
with earlier research where whole adenovirus particles
containing a L-galactosidase reporter gene under the control of a eukaryotic promoter were used to transduce macrophages. Subsequent staining of transduced macrophages
with X-gal showed expression of the reporter protein [16].
However, since V phage (containing inserts under the control of a eukaryotic promoter) have also been shown to be
capable of directly transducing mammalian cells in culture
[17], it is possible that indirect priming via non-speci¢c
cellular uptake of the phage may also be occurring.
To study antibody responses following ‘phage vaccination’ in vivo, BALB/c mice were administered 1011 pfu
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23
V-HBsAg phage i.m. in either saline bu¡er, oil adjuvant
or in association with liposomes. Recombinant phage were
also administered nasally after association with the mucosal adjuvant chitosan [18] and by gene gun after freeze
drying. Control mice were immunised with unmodi¢ed
V-gt11 phage (i.m. in bu¡er), HBsAg protein (1 Wg i.m.
in bu¡er) and naked DNA (plasmid prcCMV-HBs(S), i.m.
in bu¡er). Ten mice were used per group and antibody
responses were measured against bacteriophage coat proteins and against recombinant HBsAg (with a control
monoclonal antibody used to allow comparison between
plates) by ELISA. Summarised results are shown in Fig. 2.
All three mouse groups vaccinated i.m. with V-HBsAg
showed signi¢cantly higher HBsAg antibody responses
than groups vaccinated with plasmid prcCMV-HBs(S) or
unmodi¢ed V-gt11 (P 6 0.01, comparison of ¢nal bleeds).
Anti-HBsAg responses in excess of 150 mIU ml31 were
observed in mice injected i.m. with V-HBsAg (Fig. 2d,
animal 4, ¢nal bleed), whereas the internationally recognised level for protection is 10 mIU ml31 [19]. All 30
mice vaccinated i.m. with V-HBsAg showed signi¢cant
(P 6 0.01) anti-HBsAg responses compared to the pre-immunisation signal (Fig. 2d^f). There was no signi¢cant
di¡erence in the anti-HBsAg responses between these three
groups, indicating that phage-mediated DNA vaccination
is as e¡ective when delivered in bu¡er as in adjuvant or
liposomes. In contrast, plasmid-based DNA vaccination is
reported to be signi¢cantly more e¡ective when administered with liposomes [20]. In certain animals in which
phage were co-administered with adjuvant, some transient
trauma was seen at the site of injection, however, no adverse reactions were noted in the other groups. Gene gun
immunisation gave poor responses against both HBsAg
(Fig. 2c) and phage coat proteins (Fig. 3b), suggesting
ine⁄cient delivery of phage particles by this route. In
our test, freeze-dried phage particles were applied directly
without prior attachment to gold beads. This could explain the variable responses to both HBsAg and phage
coat proteins seen with this group (i.e. poor epithelial penetration) and suggests that with modi¢cation, gene gun
delivery of phage DNA vaccines could be a viable proposition. With standard gene gun immunisation, DNA is
bound to gold particles before in vivo delivery using
high pressure helium. In addition, subsequent research in
our laboratory (unpublished results) has shown that bacteriophage V displays a 1^2 log10 drop in titre when freezedried in the absence of any stabilisers, which may also
have contributed to the lower anti-HBsAg response following vaccination by this route. Mice vaccinated nasally
with V-HBsAg in combination with chitosan showed no
response against HBsAg (Figs. 2g and 3a for group average responses) but did show a response against the phage
coat proteins (Fig. 3b). This indicated that phage antigens
were successfully delivered, but that the insert was not
being expressed, either because the mechanism of phage
uptake was di¡erent following mucosal immunisation, or
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Fig. 2. HBsAg antibody responses in mice inoculated with phage vaccines measured by ELISA. a: 5U1011 pfu negative control V-gt11 given i.m. in 50
Wl saline bu¡er. b: 25 Wg control pCMV-HBs plasmid DNA (5U1012 copies) given i.m. in 50 Wl saline. c : 1011 freeze-dried phage particles delivered
subcutaneously by Helios gene gun (Bio-Rad). d: 5U1011 pfu V-HBsAg complexed with liposomes (Transfectam, Promega, cat. no. E1232), given i.m.
in 50 Wl saline bu¡er. Arrow indicates response of 150 mIU when response was compared to international standards (Bio-Rad Monolisa standards, cat.
no. 72399). e: 5U1011 V-HBsAg in saline/Montanide ISA 206 oil adjuvant given i.m. in a total volume of 50 Wl. f: 5U1011 pfu V-HBsAg given i.m. in
50 Wl saline bu¡er. g: 5U1011 V-HBsAg complexed with chitosan in saline bu¡er, administered in 5 Wl per nostril. h: 1 Wg of recombinant HBsAg delivered i.m. in 50 Wl endotoxin-free saline. Ten mice were used per group. Black bars are control (pre-vaccination) bleeds and each grey bar represents one
bleed per mouse. Mice were killed at di¡erent stages throughout the experiment, so not all animals were bled at every time point. Final bleeds occurred
6^10 weeks after vaccination and identically numbered mice are always comparable between groups.
because the phage particles had been compromised during
complexing with chitosan.
In general, DNA vaccines do not raise high antibody
responses but prime the immune system, protecting
against subsequent infection. In a mouse experiment where
vaccination with V-Mccp was followed by inoculation with
whole M. capricolum subsp. capripneumoniae, the ratio of
IgG1 to IgM was s 5:1 in serum extracted after challenge,
indicative of a secondary immune response and successful
priming by the phage vaccine, whereas serum from a
mouse which had been vaccinated with a control phage
only gave a ratio of 1.35:1, indicating a primary response
[13].
Our study has shown that bacteriophage-mediated
DNA vaccination consistently gave better antibody responses when compared to naked DNA, although plasmid
copy numbers were 50^200 times higher than phage copy
numbers. Although a strong antibody response was also
seen against the carrier phage V coat proteins, this may
actually enhance the e⁄ciency of the delivery system, since
the formation of immune complexes should more e¡ectively target APCs following boosting. In all experiments
to date we have observed greatly increased ‘vaccine’ anti-
FEMSIM 1652 15-12-03
body titres following a second injection (sometimes up to
8 weeks after the primary immunisation; data not shown).
This would suggest that an initial priming of the immune
system against the phage carrier may be important, and
speci¢c experiments to investigate the in£uence of the antiphage response on the e⁄ciency of the system are under
way. Further work is also under way on the characterisation of cellular immune responses and in more detailed
challenge studies.
Compared to standard naked DNA technology, the system o¡ers several advantages: vaccine nucleic acid is contained within a stable matrix which appears to both target
APCs and protect it from nuclease degradation. Expression constructs of up to 15^20 kb in size can be used in a
standard bacteriophage V vector, raising the possibility
that multiple vaccines can be delivered within a single
phage (including cytokine ‘adjuvants’), or that large eukaryotic intron-containing genes can be used directly as
nucleic acid vaccines. Multiple gene constructs should be
particularly useful against diseases such as malaria or trypanosomiasis, in which parasites with complex life cycles
or antigen switching are involved. In addition, simultaneous vaccination against several disease serotypes using
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J.R. Clark, J.B. March / FEMS Immunology and Medical Microbiology 40 (2004) 21^26
25
Acknowledgements
We thank Catherine Jepson, Ray McInnes and Louise
Gibbard for technical support and expertise and Jill Sales
of BIOSS for statistical analysis. This work was funded by
the Scottish Enterprise Proof of Concept fund and the
Edinburgh Technology Fund.
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Fig. 3. Comparison of 10 averaged ¢nal bleeds from each group against
(a) HBsAg protein and (b) unmodi¢ed V-gt11 bacteriophage by ELISA.
Results are normalised against a standard positive control on each ELISA plate to allow an accurate comparison between groups. For HBsAg
plates a monoclonal antibody (clone NF5, Aldeveron) was used at a dilution of 1:50 000, while a mouse polyclonal antiserum (1:500) was used
for the phage-coated plates. The signal from each test sample was normalised to the signal from this standard to allow the relative signal to
be compared between di¡erent plates. Mice vaccinated with V-HBsAg
showed signi¢cantly higher responses against HBsAg compared to
V-gt11 alone, when phage are given in liposomes, Montanide and saline.
*P = 0.01, **P = 0.001.
a single construct should be possible. Nucleic acid vaccines
prepared using this technology can be easily and inexpensively prepared to a high yield (su⁄cient phage to deliver
millions of doses could be grown up in a few days, an
important consideration bearing in mind the current threat
of bio-terrorism). Compared to viruses of eukaryotic organisms, there should be no possibility of vaccine replication in host tissue, and phage do not carry antibiotic resistance genes, thus removing signi¢cant potential barriers
to regulatory approval. Bacteriophages have been used in
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spleen and liver following oral administration, suggesting
that parenteral delivery may not be necessary for the effective use of phage vaccines. A simple vaccination strategy involving oral delivery of phage tablets or liquid suspensions may be all that is required.
FEMSIM 1652 15-12-03
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