Vaccine xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Lambda display phage as a mucosal vaccine delivery vehicle for peptide

antigens
Patricia González Cano a, Lakshman N.A. Gamage a,1, Kristen Marciniuk a,c, Connie Hayes b, Scott Napper a,c,
Sidney Hayes b, Philip J. Griebel a,d,⇑
a
VIDO-InterVac, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
b
Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada
c
Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
d
School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 2Z4, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Bacteriophage are structurally stable in the gastro-intestinal tract and have favorable traits of safety, sta-
Received 1 July 2017 bility, ease of production, and immunogenicity. These attributes make them potential candidates as oral
Received in revised form 26 October 2017 vaccine delivery vehicles but little is known about their capacity to induce mucosal immune responses in
Accepted 7 November 2017
the small intestine. Whole body imaging of mice confirmed lambda bacteriophage (LP) were distributed
Available online xxxx
throughout the gastro-intestinal tract 24 h after oral delivery. In newborn calves, targeted delivery of LP
within the small intestine confirmed LP were immunogenic in a dose-dependent manner and were taken
Keywords:
up by Peyer’s patches. LP-specific IgA responses were induced within both Peyer’s patches and draining
IgA
Lambda display phage
mesenteric lymph nodes. A lambda display phage (LDP) was constructed to present three immunogenic
Mucosal immune responses disease specific epitopes (DSE) from cervid prion protein (amino acids 130–140 [YML]; 163–170 [YRR];
Peptide antigens and 171–178[YRR]) fused to phage capsid head protein D (LDP-DSE). Targeted delivery of purified
Peyer’s patches LDP-DSE to intestinal segments induced IgA responses to all three peptide epitopes. Further, delivery
of bacteria expressing soluble D-DSE also induced epitope-specific IgA responses in the targeted
Peyer’s patches. These are the first studies to report use of LDP to induce epitope-specific IgA responses
in the small intestine and confirm Peyer’s patches function as a site for LP uptake. Furthermore, IgA
responses to peptide epitopes on LDP were observed in the absence of a mucosal adjuvant. These obser-
vations confirm LDP have the capacity to function as a mucosal delivery vehicle with protein D as an
effective carrier for peptide epitopes.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Bacteriophage (phage) [1] are viruses [2] that infect bacteria
and phage are members of the microbiome [3,4] present in the oral
cavity, respiratory tract, [5] and digestive tract of humans [6–8]
and animals [9–11]. Phage have also been identified in the blood
of healthy human donors [12]. They play a role in shaping the
microbiome [11] and are considered a part of the normal micro-
flora [13]. Bacteriophage have been used for antimicrobial therapy
but also have potential as vaccine delivery vehicles [14–17].
Abbreviations: ASC, antibody secreting cells; PP, Peyer’s patch; LDP, Lambda
display phage; LP, lambda particles.
⇑ Corresponding author at: VIDO-InterVac, University of Saskatchewan, Saska-
toon, SK S7N 5E3, Canada.
E-mail address: philip.griebel@usask.ca (P.J. Griebel).
1
Current address: CFIA, 534-East Cordova St, Vancouver, BC V6A 1L7, Canada.
Since phage replicate in bacteria, they are considered safe for
eukaryotic hosts but as foreign antigens they interact with the
mammalian immune system [18]. Immune recognition of phage
may involve both mucosal and systemic immune systems since
phage have been isolated from blood and other tissues following
oral administration [19]. There was concern that phage may elicit
adverse local or systemic inflammatory immune responses but
phage and phage proteins failed to induce pro-inflammatory cyto-
kine production [20,21]. Also, as an oral vaccine delivery system
phage must survive the harsh conditions in the digestive tract. In
vitro experiments demonstrated phage could survive extremes of
pH with 68–77% recovery following exposure to a pH less than 2
[22].
Our previous studies confirmed lambda display phage (LDP)
could be employed for parenteral immunization [16]. We found
that 4 porcine Circovirus 2 (PCV2) coat protein (CAP) epitopes could

https://doi.org/10.1016/j.vaccine.2017.11.010
0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

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2 P.G. Cano et al. / Vaccine xxx (2017) xxx–xxx
be expressed as a single recombinant protein fused to lambda pro-
tein D (D-CAP) to generate LDP. Purified LDP-D-CAP, when injected
intra-dermally, induced both antibody and T-cell mediated
immune responses. Vaccine immunogenicity was confirmed by
PCV2 neutralization with sera from LDP-D-CAP vaccinated pigs
and a PCV2-specific DTH reaction [16]. The immunogenicity of
LDP, in the absence of an adjuvant, led us to hypothesize that puri-
fied LDP may also be used as a mucosal vaccine delivery vehicle.
LDP used in this investigation were designed to incorporate
multiple peptide epitopes previously optimized for the induction
of prion protein (PrP)-specific antibody responses [23,24]. Three
disease specific epitopes (DSE) from cervid PrP were fused to phage
capsid head protein D (D-DSE). We first investigated uptake of
lambda particles (LP) and LDP from the intestinal lumen and then
evaluated the capacity of D-DSE and LDP-DSE to induce mucosal
immune responses in the absence of an adjuvant. We provide evi-
dence that LDP are taken up from the small intestine by Peyer’s
patches (PPs) and that multiple B cell epitopes are immunogenic
when fused to lambda D-protein. These studies also confirm that
LDP were immunogenic and induced IgA antibody responses to
displayed peptide epitopes in the absence of a mucosal adjuvant.
2. Material and methods
2.1. Animal experiments
All animal experiments were conducted at the University of
Saskatchewan in accordance with guidelines approved by the
Canadian Council on Animal Care and endorsed by the University
of Saskatchewan Animal Care Committee (Protocol 2002105 for
intestinal surgeries; Protocol 19940212 for mouse imaging
studies).
2.1.1. Lambda phage labeling and imaging in mice
LP were labeled with AF750 (Alexa fluor 750Òsuccinimidyl
ester, (Thermo Fisher Scientific, Mississauga ON, Canada) as
described previously [25]. Briefly, 1  1012 purified LP particles
(1.13  1010 particles/lg phage DNA) were re-suspended in
300 mM NaHCO3, pH 8.6, containing AF750 at a final concentration
of 1 mg/mL and incubated for 1 h in the dark. Non-conjugated
AF750 was removed by dialyzing twice for 2.5 h each in a 0.5 mL
3.5 K MWCO Slide-A-LyzerTM mini dialysis device (ThermoFisher
Scientific), against 300 mM NaHCO3, pH 8.6. The concentration of
labeled phage was determined by DNA concentration and adjusted
to a final concentration of 1  1010 particles/ml in PBSA. Three
Balb/c mice were orally gavaged with 1  109 particles delivered
in 100 lL Ca++Mg++-free phosphate buffered saline (PBSA). Mice
were euthanized 24 h later and the gastrointestinal tract was
removed from the abdominal cavity before scanning with an IVISÒ
Lumina II in vivo Imaging System (PerkinElmer, Waltham MA,
USA).
2.1.2. Intestinal segments for targeted delivery of lambda phage
The surgical procedure used to prepare intestinal segments was
previously published [26]. For the current study jejunal segments
were prepared in 10–14 day old calves and subdivided with silk
ligatures into 10–15 cm long compartments that either contained
or excluded PPs. Details of intestinal segment preparation and
immunization protocols are provided with Supplementary Fig. 1.
2.2. Tissue collection and cell isolation
Calves were euthanized with 20 mL/45 kg Euthanyl (Bimeda-
MTC, Cambridge ON, Canada) and tissues were collected within
20 min and placed in ice-cold AIM V (Thermo Fisher Scientific.
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Single cell suspensions were isolated from PPs as previously
described ([27,28]). Single cell suspensions were prepared from
LNs by finely mincing tissues with a scalpel blade and passing cell
suspensions through a 40 mm nylon cell strainer (BD FalconTM,
Mississauga ON, Canada). Viable cell concentrations were adjusted
to 5  106 cells/mL for ELISPOT assays.
2.3. ELISPOT assay for IgA and IgG antibody-secreting cells (ASCs)
ELISPOT assays were performed as described previously [39]
with the following modifications. Multiscreen-HA ELISPOT
96-well plates (EMD Millipore, Oakville ON, Canada) were coated
with100 lL/well of LP (1  1010 pfu/mL) or 100 lL/well containing
8 lg/ml of either YYR, YML or RL peptide [23,29]. Bound
immunoglobulins were detected with either biotin-conjugated
anti-bovine IgG (AbDSerotec/Bio-Rad, Raleigh NC, USA) or anti-
bovine IgA (AbDSerotec). An image of each well was captured using
the AID Elispot Reader ELR07 (AutoimmunDiagnostika GmbH,
Strassberg, Germany) and the number of spots enumerated using
AID Elispot Reader Software V7.0 (AutoimmunDiagnostika GmbH).
Antigen-specific ASCs per million cells was calculated using the
formula: [(average number of ASCs for cells in triplicate wells with
an antigen – average number of ASCs for cells in triplicate wells
cultured with medium alone)  2].
2.4. D-DSE and D-His fusion protein purification
E. coli strain 594 = R594 lac3350 galK2 galT22 rpsL179
IN(rrnD-rrnE1 [30] was transformed with either the pcIpR-D-
[DSE]-mcs-timm or pcIpR-His-Dcoe-timm plasmids encoding the
D-fusion proteins as described in [31] and transformed bacteria
were used to analyze expression of the D-fusion proteins. D-His
protein was purified by following the manufacturer’s instructions
for Ni-NTA His binding Resin (Novagen, Madison WI, USA) and
eluted protein was purified with an ÄKTA Explorer FPLC System
(Amersham Pharmacia Biotech, Uppsala, Sweden). Protein was
loaded onto a size exclusion column (HiPrepSephacryl 100
xk26/65, GE Healthcare, Mississauga ON, Canada). Positive frac-
tions were pooled, concentrated with Amiconultra NMWL, 10
KDa (Millipore), and filtered through a 0.22 lm low protein bind-
ing filter (Millipore). Final protein concentration was determined
using the Pierce BCA protein assay kit (Thermo Fisher Scientific).
2.5. Gene expression plasmids for phage display
Genes corresponding to the three epitopes were synthesized to
enable either amino or carboxy-terminal fusion to the lambda D
protein and were ordered from Integrated DNA Technologies
(IDT; Coraville IA, USA). Genes were obtained either as insertions
cloned within their pIDTSMART-Amp plasmids or as synthetic gene
blocks. These were amplified by PCR, or cloned directly to produce
thermally inducible expression versions of ColE1-based pcIpR-D-
fusion-timm plasmids (Fig. 1A) and these were used to prepare
lambda display particles (LDP). Dcoe is a codon optimized (55
codon changes) version of the wild type gene, Dwt [31]. Polypep-
tides fused to D (Fig. 1B) were optimized for expression in E. coli
and to avoid problematic restriction sites. Construction of pcIpR-
D-fusion-timm plasmids is described in Supplemental Methods
File 1.
2.6. Preparation of phage lysates of LDP with D-fusions
LP displaying D-fusion proteins were prepared as described in
Sections 2.4 and 2.5 of [31], however, the infecting phage
kimm434(18,12)P22 replaced the previous kimm434cI lysate #5
which encodes the rep(O,P)k genes, since P expression can rapidly
 P.G. Cano et al. / Vaccine xxx (2017) xxx–xxx 3

cure ColE1 plasmids [34]. This is not observed when (O-P)k is
replaced by rep(18,12) from phage P22 (Hayes, unpublished).
Non-permissive 594 cells transformed with pcIpR-Dcoe-YML-
timm were thermally induced and infected with kimm434
Dam123 to prepare LDP-YML phage particles by complementation.
2.7. Western blot
Bacterial cell pellets were re-suspended in 3 mL lysis buffer
(50 mMTris, 300 mMNaCl, 0.1% NP40, 10 mM Imidazole, 0.02%
NaN3 and 10% glycerol (pH = 8.0) per g wet weight. The sample
was vortexed and 10 lg/mL lysozyme (Sigma Aldrich) added
before incubating samples for 20 min at 4 °C. Sample were soni-
cated (Vibra-Cell) on ice with 6 pulses of 10 s before centrifuging
at 13,000 rpm at 4 °C for 15 min. Protein concentration in the
supernatant was determined with the Pierce BCA protein assay
kit (Thermo Fisher Scientific). For each protein sample, 15 lg was
run in a 12% SDS-PAGE and Kaleidoscope Precision plus Protein
markers (BioRad; 10–250 KDa) were included in the first lane.
Proteins were transferred to a 0.2 lm PVDF membrane (Bio-Rad).
D-His and D-SJH proteins were detected using murine anti-LP
serum and goat anti-mouse IgG conjugated with HRP (Santa Cruz,

Fig. 1. Design of a thermally inducible expression plasmids for the expression of peptide epitopes from cervid prion protein fused with lambda phage protein D. (A) pcIpR
plasmid expression vector [31–33] where gene expression from the lambda pR promoter is regulated by the encoded temperature sensitive CI repressor. (B) Synthetic
versions of D, or D-fusion inserts prepared, mainly by cloning between the BamHI and AscI restriction endonuclease sites. The cervid PrP sequence varies [47] and the
sequence shown represents GenBank: ABS87878.1 [Cervus elaphus] submitted, 2008 by M. Perucchini et al., unpublished.

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Dallas TX, USA). The YML epitope in D-DSE was detected with
affinity purified sheep anti-YML IgG [29] and HRP-conjugated rab-
bit anti-sheep Ig (LSBio, Seattle WA, USA).
2.8. Production of mouse anti-lambda phage antisera
Mouse anti-LP serum was produced by injecting C75BL6 mice
subcutaneously with 1  1010 pfu LP formulated in PBS with 30%
EmulsigenÒ D (MVP Technologies, Omaha NB, USA). Mice were
immunized three times at 4 week intervals and exsanguinated
for serum collection 14 days after the last immunization.
2.9. Statistical analyses
Statistical analyses were performed using GraphPad 6.00 pro-
gram (GraphPad Software Inc. San Diego CA, USA). Shapiro-Wilk
normality test was performed to determine whether parametric
or non-parametric analyses were appropriate. One-way ANOVA
with Tukey’s Multiple Comparison Test was used to compare ASC
data from multiple tissues or treatment groups.
3. Results
3.1. Design of lambda phage displaying immunogenic peptide epitopes
was reacted with lysate from transformed cells prior to heat induc-
tion (Fig. 2B; Lane 3).
3.3. Lambda phage passage through the gastro-intestinal tract (GIT)
Bacteriophage have been shown to survive passage through the
GIT of chickens [35] and cattle [36] but the LP we selected was a
hybrid with immunity genes from phage 434, a replication region
from P22, and the remainder, including all the capsid genes were
from LP. We examined the capacity of LP to survive passage
through the stomach and distribute throughout the GIT by imaging
Alexa fluor 750 labeled LP following oral administration to mice.
Imaging 24 h after oral gavage, confirmed LP was located primarily
in the stomach (Fig. 3; red-yellow color) but LP were also visible
throughout the large intestine. Fluorescent LP were visible in the
caecum (blue color) and colon (blue color) and LP in the colon con-
centrated within fecal pellets. Passage of LP through the GIT was
also confirmed by traces of blue fluorescence at the base of the tail
and external to the anus (Fig. 3). Thus, imaging of fluorescently
labeled LP confirmed orally delivered LP was distributed through-
out the GIT but no visible fluorescence localize to either PPs in the
small intestine or the mesenteric lymph nodes (LNs). A more sen-
sitive assay system was required to determine whether LP could be
taken up by gut-associated lymphoid tissues.

The sequence of each DSE was previously optimized for induc-
tion of antibody responses when expressed as a fusion with the
leukotoxin protein from Mannheimia haemolytica [24,29]. Design
of the heat-inducible D-fusion plasmids along with sequence and
configurational details of the DSE epitopes displayed on LDP-D-
DSE are presented in Fig. 1.
3.2. Expression of peptide epitopes in D-fusion
Expression of D-fusion protein in cells transformed with pcIpR-
D-fusion-timm plasmids was confirmed by probing lysate of heat-
induced cells with mouse anti-LP serum. Specificity of the antisera
for D protein was confirmed with purified lambda His-D (Fig. 2A;
Lane 1). The antisera also reacted with an approximately 22 kDa
protein present in lysate from pcIpR-D-fusion-timm transformed
cells following heat-induction (Fig. 2A; Lane 3). The presence of
DSE in the D-fusion protein was also confirmed by reaction with
sheep anti-YML IgG (Fig. 2B; Lane 2). Specificity of this reaction
was confirmed by the absence of a visible band when antisera
3.4. LP induce mucosal immune responses in gut-associated lymphoid
tissues (GALT)
Surgically isolated intestinal segments provide a system for
monitoring M-cell uptake of particulate material [37] and assaying
the induction of mucosal immune responses in GALT [38,39]. Sur-
gically isolated segments of jejunum were prepared in young
calves to determine whether LP were taken up by PPs and induced
a local mucosal immune response. A dose titration experiment
with purified LP revealed that a significant (P < .05) dose-
dependent, LP-specific ASC response was induced in PPs (Fig. 4A).
This response consisted primarily of IgA ASCs but also included
some IgG ASCs. LP-specific ASC responses were detected in PPs fol-
lowing injection of 1  108 LP but increasing the dose to 1  1012
LP/compartment significantly (P < .01) increased the frequency of
IgA ASCs (Fig. 4A). IgA and IgG ASCs were also detected in the MLNs
draining intestinal compartments injected with LP (Fig. 4B) but sig-
nificantly (P < .05) more IgG and IgA ASC were detected in PPs than
MLNs following the injection of 1  1012 LP (Fig. 4).

Fig. 2. Expression of YYR, YML, and RL epitopes in D-fusion protein was confirmed by Western blot. (A) Expression of D-fusion in transformed cells was confirmed by probing
Western blots with a polyclonal mouse anti-lambda phage antisera: Lane 1- Purified D-His protein (Known MW is 12.5 kDa); Lane 2- Molecular weight markers; and Lane 3-
Lysate from cells transformed with pcIpR-D-DSE-timm following induction to express D-fusion protein (Approximate MW is 22 kDa). (B) Expression of prion protein peptide
epitopes was confirmed by probing Western blots with affinity purified sheep anti-YML IgG: Lane 1- molecular weight markers: Lane 2- lysate from cells transformed with
pcIpR-D-DSE-timm following induction to express D-fusion protein; and Lane 3- lysate from cells transformed with pcIpR-D-DSE-timm prior to heat induction.

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 P.G. Cano et al. / Vaccine xxx (2017) xxx–xxx 5

Stomach

Small
Colon
Intesne

Caecum

Fig. 3. Whole body imaging of mouse and isolated gastrointestinal tract at 24 h

after oral administration of 1  109Alexa Fluor 750 labeled lambda phage. The
highest concentration of phage (red-yellow coloration) was visible in the stomach
with lower concentrations (green-blue) of phage visible in the caecum and within
fecal pellets distributed throughout the colon and rectum. Red arrows indicate a
low level of phage (blue-purple) distributed on the outside of the body (anus and
tail) and phage was transferred to subcutaneous tissue during removal of the
gastrointestinal tract. Image presented is representative of results from three mice.
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)

3.5. Mucosal uptake of LP particles

Bacteriophage are particulate antigens [40] and may be transcy-

tosed by mucosal epithelial cells, internalized by DC transepithelial
dendrites (TEDs), or transcytosed by M-cells located within the fol-
licle associated epithelium (FAE) overlying PPs [41]. To determine
the route by which LP are taken up, intestinal compartments were
prepared in separate intestinal segments. In one segment, two
compartments included PPs (Supplementary Fig. 1) and in the sec-
ond segment the two compartments lacked a visible PP. The com-
partments lacking PPs provided mucosal epithelial cells and DC
TEDs for LP uptake but M-cells present in PPs would be absent.
LP-specific IgA ASCs were detected only in PPs from compartments
injected with LP (Fig. 5; PP- LP) but not from compartments
Fig. 4. Dose-dependent antibody responses to LP in Peyer’s patches (A) and
mesenteric lymph nodes (B) draining surgically isolated intestinal segments. Each
dose of LP was injected into a separate intestinal compartment, each containing a
jejunal Peyer‘s patch (JPP), within a single mid-jejunal segment of intestine. One
month after injecting LP, the JPPs and mesenteric lymph nodes adjacent to each
compartment were collected. Single cell suspensions prepared from these tissues
were used to determine the frequency of LP-specific antibody-secreting cells (ASCs)
in ELISPOT assays measuring IgA and IgG ASCs. Data presented are the mean and
1SD of values from three animals with three replicate ELISPOT wells analyzed for
each tissue sample. Significantly (P < .05) more LP-specific IgA and IgG ASCs were
detected in JPPs exposed to 1  1012 LP particles than lower doses of LP.

injected with PBSA (Fig. 5; PP-PBS). Similarly, LP-specific IgA ASCs

were only detected in MLNs draining intestinal compartments
injected with LP and when the compartment contained a PP
(Fig. 5; MLN-PP-LP). Thus, PP functioned as an immune induction
site for LP-specific IgA ASC responses and were also required for
the induction of LP-specific ASC responses in the draining MLN.
We also analyzed LP-specific ASC responses in lymph nodes drain-
ing a variety of tissues to determine if sufficient LP were absorbed
from the intestine to induce a systemic immune response. LP-
specific IgA ASC responses were not detected in LNs associated
with either the liver (hepatic LN) or lung (mediastinal LNs) but a
low number of LP-specific ASCs were detected in the spleen of
two animals (Fig. 5; Non-intestinal sites).
3.6. LDP induce mucosal antibody responses to multiple peptide
epitopes
Bacteria possess a variety of pathogen-associated molecular
pattern molecules (PAMPs) and E. coli expressing recombinant
antigens have been used in vivo and in vitro to induce immune
response [42]. Therefore, the immunogenicity of D-DSE expressed
in E. coli was compared with purified LDP-DSE to determine if par-
ticulate LDP were essential for uptake by MALT and induction of
antibody responses to the peptide epitopes displayed on D protein.
IgA ASC responses specific to the three DSE and LP were not
detected with cells isolated from PPs in compartments injected
with PBSA (Fig. 6A; PBS). In contrast, IgA ASC responses specific
to DSE were detected with cells from PPs in compartments injected
with purified LDP (Fig. 6A; Purified LDP-DSE). IgA ASC responses
specific to DSE were also detected with cells isolated from PPs
located in compartments injected with either heat-induced
pcIpR-D-DSE-timm transformed E. coli cells or lysate of heat-
induced pcIpR-D-DSE-timm transformed E. coli cells (Fig. 6 A;
Bacteria D-DSE and Soluble D-DSE). Immunization with purified
LDP-DSE or bacteria expressing D fusion protein also induced
LP-specific IgA ASCs (Fig. 6A: Lambda). The frequency of DSE-
specific IgA ASCs in PPs varied significantly (P < .05) when

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Fig. 5. LP uptake from the small intestine requires mucosa-associated lymphoid tissue. Intestinal compartments were constructed that either contained or excluded (No PP) a
visible Peyer‘s patch (PP). Compartments were injected with either PBS or 1  1012 LP re-suspended in PBS and one month later, PPs, draining mesenteric lymph nodes (MLN)
and peripheral lymphoid organs (spleen, mediastinal LN in the thorax, and hepatic lymph nodes) were collected to assay LP-specific IgA ASCs. LP-specific IgA ASCs were only
detected in PPs and associated draining MLN of intestinal compartments that contained visible PP and were injected with LP. LP-specific IgA ASCs were not detected in hepatic
and mediastinal LNs but were detected in the spleen of two of three animals. Data presented are values from individual animals and represent the mean value from triplicate
wells in an ELISPOT assay.

Fig. 6. IgA antibody secreting cell (ASC) responses to peptide epitopes (YYR, YML, and RL) displayed as a fusion with lambda capsid protein D (D-DSE). Duplicate
compartments, each containing a jejunal Peyer’s patch (JPP), were prepared in two separate intestinal segments within each calf (n = 3). One compartments in the first
segment were injected with PBS and the second compartment was injected with purified lambda phage displaying D-DSE (Purified LDP-DSE). One compartment in the second
segment was injected with E. coli cells transformed with pcIpR-D-DSE-timm and heat-induced for three h and the second compartment was injected with the lysate from an
equivalent number of E. coli cells. One month after immunization the JPP (A) and mesenteric lymph nodes (B) were collected from the intestinal segments and ELISPOT assays
were performed to determine the frequency of IgA ASCs specific for YYR, YML, and RL peptides and LP. The frequency of peptide-specific IgA ASCs was compared among PPs
and significant (P < .05) differences are indicated by different letters (a, b) above columns. The frequency of peptide-specific IgA ASCs was compared between mesenteric
lymph nodes draining each intestinal segment and significant (P < .05) differences are indicated by different letters (a, b) above columns. Data presented are the mean and
1SD of values from three animals.

comparing among the three types of immunogens injected into
intestinal compartments but interpretation of these results is lim-
ited by the fact that equivalent amounts of D-DSE protein was
probably not delivered by purified LDP-DSE, E. coli cells and lysate
of the of heat-induced pcIpR-D-DSE-timm transformed E. coli cells.
The MLN is a continuous chain adjacent to the small intestine
(Supplementary Fig. 1) and it is not possible to determine which
part of a MLN collects lymph and DCs from individual compart-
ments within an intestinal segment. Therefore, IgA ASCs responses
in the MLN were analyzed for each separate intestinal segments.
For the first intestinal segment, DSE-specific IgA ASC responses
reflect immunization with purified LDP-DSE since no detectable
DSE-specific ASC response was detected with PP cells isolated from
the compartment injected with PBSA (Fig. 6A). For the second
intestinal segment, IgA ASC responses may reflect uptake of D-
DSE from either intact E. coli cells or bacterial lysate since both
preparations induced DSE-specific ASC responses in the PPs. Anal-
ysis of ASC responses in MLNs draining the two intestinal segments
confirmed responses were induced to all three peptide epitopes
and LP (Fig. 6B). The frequency of these IgA ASCs was similar in
both the PPs and draining MLN for intestinal segments injected
with purified LDP-DSE (Fig. 6B). The frequency of DSE-specific ASCs
was, however, significantly (P < .05) lower in MLN collected from
the intestinal segment injected with both intact and lysed trans-
formed E. coli cells than MLN draining the intestinal segments
injected with purified LDP-DSE (Fig. 6B).

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4. Discussion
There is increasing interest in using bacteriophage as an oral
vaccine delivery vehicle due to their safety and inherent immuno-
genicity [40]. Our imaging studies confirmed LP can pass through
the stomach and within 24 h be distributed throughout the gas-
trointestinal tract. This distribution provides an opportunity for
LP uptake by MALT in the small and large intestine but imaging
of fluorescent LP did not reveal LP localization to MALT. Therefore,
we used an intestinal surgery model [38,39] to target LP to MALT
and determine if uptake was sufficient to induce mucosal immune
responses. The current study provides evidence that LP and LDP are
taken up by PPs in quantities sufficient to induce IgA antibody
responses. These observations are consistent with previous reports
that LDP were immunogenic when injected parenterally and
induced antibody responses when delivered without an adjuvant
[16]. The current observations (Fig. 6) confirm that LDP are also
immunogenic when delivered mucosally in the absence of an adju-
vant. This is observation is important since the lack of effective
mucosal adjuvants remains a major barrier to mucosal vaccine
development.
Viable phage have been recovered from blood, urine and multi-
ple organs following oral delivery to mice, rabbits and humans
(reviewed in [43]). Neither the site nor mechanism of phage
absorption from the GIT has been determined, but it is was
assumed that uptake can occur through mucosal epithelial cells.
M-cells present in the FAE of MALT also provide an important route
for uptake of particulate material from the intestinal lumen [41].
M-cell endocytosis of particulate antigens is influenced by a num-
ber of physical parameters [44] but the efficiency of M-cell uptake
is 15–250 fold higher for 100 nm diameter particles than larger
microparticles [45]. LP have an isosahedral head that is approxi-
mately 60 nm in diameter and a noncontractile tail of approxi-
mately 150 nm in length [46], a size consistent with M-cell
uptake but formal proof of LP transcytosis by M-cells will require
further histological analysis. Targeted delivery of LP to intestinal
compartments, either with or without PPs, provided evidence that
LP uptake by MALT was sufficient to induce LP-specific IgA
responses (Figs. 4 and 5). If LP transcytosis by mucosal epithelium
or intraepithelial DCs occurred then the quantity was not sufficient
to induce detectable ASC responses in lymph nodes draining
intestinal compartments that lacked a PP (Fig. 5). The presence of
a low number of LP-specific ASCs in the spleen (Fig. 5) may have
resulted from either systemic circulation of a low number of LP
absorbed from the intestine or trafficking of plasma cells induced
in MALT. The current data confirm, however, that PP provide an
effective route for LP uptake from the small intestine and this
implicates M-cells in the process of LP uptake.
Lambda D gene was used to display immunogenic peptide epi-
topes since it was previously confirmed that peptide epitopes dis-
played on D, when incorporated into LDP, could induce antibody
responses [16]. These previous results also indicated that phage
proteins provided T cell epitopes which are required for the induc-
tion of antibody response to small peptide epitopes. It was not
known, however, whether the D protein by itself provided suffi-
cient T cell epitopes to function as a carrier protein for peptide epi-
topes. To address this question we analyzed DSE-specific antibody
responses following delivery of D-DSE fusion protein, not assem-
bled into LDP (Fig. 6). D-DSE expressed in bacteria induced ASC
responses to all three peptide epitopes. This observation confirms
D protein can function as an effective carrier protein for B cell pep-
tide epitopes. It is interesting, however, that significantly (P < .05)
greater ASC responses were observed for two of the three epitopes
when PPs were exposed to intact bacteria versus bacterial lysate.
This observation may be consistent with M-cells being more
Please cite this article in press as: Cano PG et al. Lambda display phage as a mucosal vaccine delivery vehicle for peptide antigens. Vaccine (2017), https://
doi.org/10.1016/j.vaccine.2017.11.010
7
efficient in the up-take of particulate versus soluble antigens.
Intact bacterial cells may also increase the magnitude of immune
responses through an adjuvant effect associated with co-delivery
of D-DSE with bacterial PAMPS.
MLNs amplify the production of mucosal effector cells, such as
IgA plasma cells, due to their extensive distribution adjacent to the
small intestine (Supplementary Fig. 1). The frequency of DSE-
specific IgA ASCs was significantly (P < .05) greater in MLNs drain-
ing intestinal segments injected with purified LDP versus segments
injected with bacterial cells expressing D-DSE (Fig. 6B). This obser-
vation supports the conclusion that LDP can induce mucosal
immune responses in both MALT and the draining LNs. The intesti-
nal segment model used in our studies facilitated an analysis of
both LDP uptake and the immunogenicity of peptide epitopes dis-
played on LDP. This model does not, however, address the critical
issue whether LDP survival in the stomach and transit through
the intestine (Fig. 3) will be sufficient to induce mucosal immune
responses in the GIT. Dose titration studies (Fig. 4) clearly indicate
that induction of mucosal immune responses is dependent on ade-
quate LP uptake by PPs. Induction of IgA responses to LP may pro-
vide an important mechanism by which uptake of LDP can be
further enhanced following multiple immunizations. Recent stud-
ies demonstrated that secretory IgA, bound to soluble proteins or
particulate antigens, can enhance intestinal M-cell uptake of these
immune complexes [48]. Therefore, future studies are required to
determine the dose of LDP and the number of oral immunizations
required to induce detectable IgA ASC responses in PPs and IgA in
feces. The dose of purified LDP required to achieve these results
will have a significant impact on the practicality of using purified
LDP as an oral vaccine delivery platform.
Acknowledgements
This research was funded by Alberta Livestock and Meat Agency
(ALMA), Saskatchewan Agriculture Development Fund (ADF) and
the Natural Sciences and Engineering Research Council of Canada
(NSERC). Dr. Philip Griebel is funded by a Tier I Canada Research
Chair (CRC) in Neonatal Mucosal Immunology provided by Canada
Institutes for Health Research (CIHR). The authors declare that they
have no conflict of interest. We would also like to thank Karthic
Rajamanickam for assistance in the construction of single epitope
gene expression plasmids for phage display. We also acknowledge
the excellent technical assistance of Dr. Steward Walker and the
VIDO-Intervac Animal Care staff for performing surgeries and
post-operative animal care. This manuscript is published with per-
mission of the Director of VIDO as journal series #807.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.vaccine.2017.11.
010.
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