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Golovkin et al., Plant-based Smallpox Vaccine
For Classification: Plant Biology
Smallpox Subunit Vaccine Produced in Planta
Confers Protection in Mice
Maxim Golovkin, Sergei Spitsin, Vyacheslav Andrianov, Yuriy Smirnov, Yuhong Xiao I, Natalia Pogrebnyak, Karen
Markley, Robert Brodzik, Yuri Gleba II, Stuart N. IsaacsI and Hilary Koprowski*
Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, PA 19107-6799
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA;
Icon Genetics, Biozentrum Halle, Weinbergweg 22, D-06120 Halle (Saale), Germany
* to whom correspondence should be addressed:
Ph. 215-503-4761; Fax. 215-503-6795; E-mail: [email protected]
Number of text pages:
Number of figures:
Number of tables:
Number of words in the abstract:
Number of characters in the paper:
36 584 (with space)
Abbreviations: Vaccinia virus, VV, immunoglobulin G & A, IgG & IgA; cholera toxin, CT; plant-derived
B5 antigen, pB5;
Key words:
Plant biotechnology/ transgenic plants/ smallpox subunit vaccine/ B5 glycoprotein
/recombinant antigen/
Golovkin et al., Plant-based Smallpox Vaccine
We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain
(pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was
expressed using efficient transient and constitutive plant expression systems and tested by various
immunization routes in two animal models. Whereas oral administration in mice or the mini-pig with
collard-derived insoluble B5 did not generate an anti-B5 immune response, intranasal administration
of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a
strong anti-B5 immune response in both mice and the mini-pig. Mice immunized intramuscularly with
pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from
challenge with a lethal dose of VV. These results indicate the feasibility of producing safe and
inexpensive subunit vaccines utilizing plant production systems.
Golovkin et al., Plant-based Smallpox Vaccine
Plants have emerged as an excellent alternative to other expression systems for the production of
complex pharmaceutical proteins including recombinant subunit vaccines [1-6]. It was shown that
some plant-derived antigens can induce systemic and mucosal immune responses and, in some cases,
confer protection against challenge [1-3]. Plants provide the additional advantage of direct delivery
through oral or other mucosal routes [1, 6]. Despite some difficulties with the expression of certain
recombinant proteins, especially those of viral origin, plant biotechnology holds the promise of
producing medicinal proteins to be used in vaccine formulations.
Interest in a safe smallpox vaccine has been reawakened with the threat of bioterrorism [7, 8] and
continuous outbreaks of orthopoxvirus diseases [9, 10]. A live VV-based vaccine has been used to
eventually eradicate smallpox disease [11, 12] but does display side effects [13]. While one approach for
developing a safer vaccine is to use the highly attenuated live virus, recombinant protein-based
vaccines are likely to be safer. For orthopoxviruses, there are several candidate antigens that can
efficiently protect mice and non-human primates from lethal challenge [14-20]. The extracellular virus
(EV)-specific membrane glycoprotein encoded by the B5R gene [21-24] is the main target of EVneutralizing antibodies present in human-derived vaccinia gammaglobulin used to treat complications
arising from smallpox vaccination [25].
In this study we demonstrate that the VV B5 protein can be produced in two plant expression
systems. The use of the MagnIcon transient expression system [26-28] enabled rapid high-yield
production of soluble B5 as well as selection of optimal subcellular targeting signals to use in stable
plant transformation. Preparations of purified pB5 induced a strong immune response when
administered parenterally and intranasally, and mice vaccinated intramuscularly with pB5 were
protected from VV challenge.
Generation of expression cassettes and transformation vectors. Plasmid pSI-80-10 [24] carrying a
fragment of the VV (strain WR) genome was used to amplify the full antigenic ectodomain portion of
B5 (aa 20 to 275) using the following primers: 5'-CCA TGG ATT GTA CTG TAC CCA CTA TGA ATA ACG3' and 5'-GCG GCC GCA TGA TAA GTT GCT TCT AAC GAT TCT A-3'. The PCR fragment was cloned
(NcoI-NotI) into a group of five intermediate pIV1.(1-5) vectors, thus fusing the gene to targeting signals
and 6x-Histidine (His6) and c-Myc tags at C-terminus [ImpactVector, Plant Research International,
Wageningen, Netherlands]. As determined by transient in planta expression (see below), the bestexpression apoplast-targeting [33] cassette, was subcloned (XbaI-SacI) into the stable plant
transformation binary vector pBIN-Plus [ImpactVector] with kanamycin selection, to generate pBIRubapopB5.
The pB5 NcoI-SacI fragments from the corresponding pIV-based plasmids were subcloned into the
carrier pro-vector plasmid pICH11599 [MagnIcon] and used for vacuum infiltration of wild type plants.
Golovkin et al., Plant-based Smallpox Vaccine
Insoluble (membrane-bound) form of pB5 protein was produced in transgenic collard plants by
fusing the B5 antigenic region amplified by two consecutive rounds of PCR with primers: F 5'-CTT
TTC TAT TTC -3'. The fragment was cloned into a binary vector pB002 [32] expression cassette driven
by CaMV-35S promoter and harboring a sub cellular anchor.
For bacterial expression the XbaI-SacI DNA fragments excised from the pIV-based constructs
were subcloned into the corresponding sites of the modified pGEX3 vector [Amersham Pharmacia Biotech,
Piscataway, NJ] in which the GST tag was deleted (pGEX3GST). Resulting constructs were transformed
into the E.coli Rosetta-2 (DE3) strain [Novagen Inc, Madison, WI] for expression.
Agrobacterium infiltration of plants using the MagnIcon system. For rapid transient expression of
recombinant B5 in planta we used the so-called “magnification” procedure [Icon Genetics, Haale (Saale),
Germany] as described [26-28] with minor modifications. GV3101 Agro-cultures carrying the B5
cassettes in pIV plasmids were each combined with equal volumes of cultures carrying the premanufactured helper plasmids supplying the sub cellular targeting signals and recombinase/integrase
complex. The mixture was diluted (1:50) in buffer (10mM MgSO4, 10mM MES (pH-5.6), 200uM
acetosyringon, and 0.02% Silwet L-77) and applied to mature Nicotiana benthamiana (Tobacco) and
Beta vulgaris var. cicla (Swiss chard) plants (6-8 weeks old). Plant tissues were harvested within 7-10
days and analyzed by Western blot and ELISA.
Generation of transgenic plants. Tobacco (Nicotiana tabacum cv Wisconsin) leaf discs were
transformed [34] with pBIRub-apopB5 and Collard (Brassica oleracea cv Morris Heading) explants
were transformed by our method [31] using the pB002-based construct. Kanymycin (tobacco) and
phosphinotricin (collard) resistant transgenic lines were selected and tested by PCR and Western
blotting for the presence of pB5.
pB5 protein expression analysis. Harvested and lyophilized plant tissues were homogenized and
ground to a fine powder in the presence of liquid nitrogen and/or dry ice in extraction buffer (50mM
sodium phosphate, pH 8.0; 0.3M NaCl; 0.2% Tween-20; 2.5mM -mercaptoethanol) supplemented
with complete protease inhibitor [Sigma, St. Louis, MO]. For SDS-PAGE, extracts of total soluble protein
were loaded at ~50ug per lane. In Western analysis proteins were detected using MYC 1-9E10.2 (IgG1)
monoclonal antibody (c-Myc-Mab) [ATCC, Manassas, VA] and/or B5-specific Mab 206C5-F12 [35], or
mouse/mini-pig pB5-specific serum antibodies (see below).
Isolation and purification of pB5 recombinant protein for immunization. Lyophilized and
powdered leaf tissue from MagnIcon-transformed N. benthamiana, Swiss chard and transgenic N.
tabacum plants was homogenized in extraction buffer. After centrifugation the supernatant was
Golovkin et al., Plant-based Smallpox Vaccine
filtered through Miracloth [IMD-biosciences, San-Diego, CA] and loaded on a Ni-Sepharose column [GEHealthcare, Piscataway, NJ].
Eluate was dialyzed against PBS with 0.1% Tween 20 and applied on a cMyc Mab-Sepharose column to be eluted with 0.1M glycine-HCl, pH 2.5. Batches were concentrated,
dialyzed against PBS + 0.1% Tween-20 and desalted on Sephadex-G50 spin-columns [Roche, Nutley,
N.J.]. Samples were brought to a concentration of 0.1ug/ul of pB5 with sterile PBS buffer and frozen in
liquid nitrogen as 10ug aliquots to be stored at -800C.
Preparation of plant material for oral immunization. Fresh collard leaves were harvested from
healthy transgenic plants. Collard-based pellets were prepared by mixing lyophilized and powdered
plant tissue in PBS with 0.25% (w/v) of cornstarch. Pellets were formed using plastic moulds,
lyophilized and stored at 40C in air-tight containers.
Immunological assessment of pB5 in mice. Six to 8-wk-old female BALB/c mice (5 or 10 mice per
group) were used in all experiments. For oral immunization each mouse was fed with 2-3g of fresh
tissue or 1g pellets (~100ug antigen) over a period of 6-8h. Control mice received wild type plant
material. In some groups plant material was supplemented with 10ug of CT [IMD-biosciences] as an
adjuvant. An additional group of mice received purified pB5 (2ug) with CT by gavage. For intranasal
immunization, 2ug of pB5 was administered in 10ul saline into both nostrils; in some groups, pB5 was
supplemented with CT (1ug). Mice were immunized 3 times at 2-week intervals. For parenteral
immunization, mice were injected 3 times at 2-week intervals with 2ug of purified pB5. First and
second immunizations were given subcutaneously with complete and incomplete Freund's adjuvant
respectively [Difco, Detroit, MI]. The third dose was administered interperitoneally in saline. Blood and
fecal matter was collected 10 days after each immunization. Proteins from fecal pellets were extracted
in PBS (10 v/w) supplemented with 1% BSA and protease inhibitors as described [36]. Mice were
sacrificed 10 days after the last immunization, bled by cardiac puncture, and sera were analyzed by
ELISA and Western blotting.
Immunizations for the challenge experiments were done using CpG oligodeoxynucleotides (ODN)
[sequence #1826: TCC ATG ACG TTC CTG ACG TT, Coley Pharmaceutical Group, Wellesley, MA] and alum
[Alhydrogel, Accurate Chemical, Westbury, NY] as an adjuvant (CpG/alum) at 50ug/mouse [30]. Mice
received three intramuscular vaccinations at 2-week intervals with 8ug of purified pB5 or 8ug of wild
type plant total soluble protein, or CpG/alum alone. Additional control groups included naive, nonvaccinated mice and mice that were vaccinated with VV by tail scarification. Mice were bled prior to
the third boost and 3 weeks later, immediately before intranasal challenge with VV.
Immunological assessment of pB5 in mini-pig. A single 8-month-old mini-pig was immunized
consecutively by the oral, intranasal and intramuscular routes. Oral immunization consisted of 2
feedings with 300g of transgenic collard leaves supplemented with 15ug of CT within 14 days. After 2
weeks, the mini-pig received three intranasal doses of pB5 (15ug) together with 2ug of CT (75-ul per
Golovkin et al., Plant-based Smallpox Vaccine
nostril in saline) at 2-week intervals. After additional 3 weeks, the mini-pig received three
intramuscular doses of 10ug of purified pB5 supplemented with 50ug CpG/alum. Blood, fecal matter,
saliva and vaginal secretion samples were obtained 10 days after each immunization and analyzed by
ELISA and Western blotting.
Solid-phase ELISA. The assay [37] was performed in 96-well MaxiSorp plates [Nalgen-Nunc, Rochester,
NY] coated overnight at 40C with E.coli purified B5 at 1µg/ml in PBS. Antigen-specific antibodies
were detected using the following antibodies: goat anti-mouse IgG [BD Biosciences, San Jose, CA] and
anti-mouse IgA [Sigma, St. Louis MO] or Goat anti-pig IgG/IgA [Bethyl Lab. Montgomery, TX]. Results are
given as mean ±SD.
Comet inhibition assay. The in vitro assay was essentially performed as described in [30]. Briefly, a
confluent monolayer of BSC-1 cells grown in 12-well plates was infected with VV strain IHDJ at
~5pfu/well for 2h at 370C. The inoculum was removed and 1ml of fresh MEM medium containing
2.5% fetal calf serum and 25ul (1:40 dilution) of pooled sera obtained from mice 3 weeks after the last
boost vaccination (just prior to challenge).were added. Plates were incubated for ~36h at 370C, and
stained with 0.2% crystal violet in 4% ethanol, and wells were photographed.
Vaccinia virus challenge. Three weeks after the last protein vaccination, mice were intranasally
challenged with 1.2 x 106pfu of VV strain WR in 20ul of sterile PBS. Mice were weighed and
monitored each day. Animals that appeared morbid or had lost >30% of their initial body weight were
sacrificed in accordance with the institutional guidelines for animal welfare [38].
Expression cassette design and cloning strategy. The smallpox B5 glycoprotein (42 kDa)
encoded by the VV (strain WR) B5R gene [21] was chosen for the production in planta of the full
extracellular antigenic domain (aa 20-275) [Fig. 1A], which contains the major neutralization epitopes
[14, 29-30]. Expression cassettes containing the His6- and c-Myc-tags from pIV vectors [ImpactVector]
[Fig. 1B] were subcloned into the MagnIcon [IconGenetic] plasmid pICH11599 [Fig. 1B] and used for
empirical testing to determine the best expression in planta in conjunction with helper plasmid(s)
supplying various plant intracellular signals in trans [26-28] This revealed the apoplast secretion signal
to be superior. Thus, the apoplast-targeting cassette from pIV-based vector under the rbcS promoter
was subcloned into the binary plant transformation vector pBIN-Plus [ImpactVector] [Fig. 1B] generating
pBIRub-apopB5 for stable tobacco transformation. The fusion of the full extracellular antigenic domain
with an intracellular membrane anchor was used to generate transgenic collard plants producing
insoluble pB5 in abundant vegetative biomass suitable for oral feeding [31] [Fig. 3B, C].
Production of B5 protein in plants. The MagnIcon system [IconGenetics] provided a robust and rapid
method [26-28] to express recombinant pB5 in wild type plants [Fig. 2A]. The pB5 protein was readily
Golovkin et al., Plant-based Smallpox Vaccine
detected at 6-8 days post-infection in the leaf tissues transfected with the B5 and apoplast-targeting
module [Fig. 2B]. The pB5 antigen remained stable in lyophilized plant tissues for several months at
~100 mg/kg level when stored in air-tight containers at ambient temperatures or 40C. Powdered
material was used in a two-step affinity purification (Ni- and c-Myc Mab affinity columns) to obtain
standardized pB5 samples at >50% purity [Fig. 2B, C] suitable for intranasal and parenteral
Stable transgenic tobacco plants expressing soluble B5 protein were generated [Fig. 3A, B] using the
pBIRub-apopB5. Several independent transgenic lines revealed the presence of pB5 by Western
blotting [Fig. 3A; right, bottom] and showed no morphological abnormalities [Fig. 3A, left].
Transgenic collard plants expressing insoluble pB5 were generated by our method [31] using the
pB002-based binary vector [32] carrying the pB5-anchor fusion expression cassette [Fig. 1B]. Several
transgenic lines revealed an antigen-specific band(s) in leaf tissues by Western analysis [Fig. 3B, right].
The accumulation of pB5 in collard was higher when compared to transgenic tobacco expressing
soluble pB5. The presence of additional amino acids at the C-terminal end (anchor) is probably
responsible for the differences in migration of soluble and insoluble forms of pB5 on SDS-PAGE and
the B5-specific double-banding [Fig. 3B; right, bottom]. Transgenic collard plants grew large rosettes
exceeding 30 cm in diameter [Fig. 3B, left] and weighed ~1.5kg. Transgenic collard leaves were directly
used for oral immunization in either fresh form and/or standardized 1g pellets [Fig. 3C].
Immunogenicity of pB5 protein in mice. Sera from mice immunized orally, intranasally, or
parenterally with pB5 were tested for the presence of B5-specific antibodies by ELISA [Fig. 4]. No
immune response was detected in serum of mice fed with transgenic collard plant material (fresh
leaves, pellets) as well as with purified pB5 administered by gavage [ Fig. 4A]. Supplementation of
plant material with cholera toxin (CT) did not invoke a response, though resulted in detection of antiCT responses present in sera (IgG) and in fecal pellet extracts (IgA) [data not shown]. Intranasal
immunization with purified pB5 without any adjuvant did not produce antibodies in mice, though
when supplemented with CT, led to a distinct anti-B5 immune response [Fig. 4B]. The strongest antiB5 immune response was detected in sera of mice immunized parenterally [ Fig. 4C]. Western analysis
of protein extracts from plant- and bacterially expressed B5 [Fig. 4D] confirmed the B5 specificity of
the mouse serum antibodies.
Immunogenicity of pB5 protein in the mini-pig. Analysis of the immune response in the mini-pig,
revealed a response pattern similar to that observed in mice [Fig. 5]. After feeding no anti-B5-specific
antibodies were detected in the serum [Fig. 5A], saliva or vaginal secretions [data not shown]. However,
after intranasal immunization together with CT, the serum showed an increase in B5-specific IgG [Fig.
5B]. No B5-specific IgA was present in either saliva or vaginal secretions, while a strong anti-CT
response was detected in both the serum (IgG) and saliva (IgA). Furthermore, no anti-CT response
was detected in fecal matter or vaginal washes [data not shown]. As shown in Fig. 5C a sustained increase
Golovkin et al., Plant-based Smallpox Vaccine
in B5-specific serum IgG was detected after intramuscular immunization with purified pB5 in
combination with CpG-ODN and alum (CpG/alum).
Protection of pB5-vaccinated mice against lethal challenge with VV. The level of protection in
immunized mice was determined by analyzes of sera for the presence of B5-specific antibodies:
measuring the in vitro functional anti-VV activity and challenging the mice with the lethal dose of VV
[Fig. 6]. Mice were inoculated with VV by tail scarification and with plant-derived B5 (in CpG/alum)
intramuscularly; control mice were injected with extracts of total soluble protein (in CpG/alum) or left
non-inoculated. Three weeks after the second boost vaccination, sera were tested by ELISA [ Fig. 6A]
and in the comet inhibition assay [Fig. 6B] as a qualitative measure of anti-EV antibody activity. We
note that the serum immune response in mice was equally pronounced when using either CpG/alum or
Freund’s as an adjuvant [compare Fig. 6A and Fig. 4C]. As shown in Fig. 6B, sera from pB5-vaccinated
mice altered comet formation, while sera from VV-vaccinated mice inhibited comet formation
completely (left); control mouse sera did not alter comet formation (right).
Vaccinated mice were then challenged intranasally with a lethal dose of VV and monitored daily
for weight loss and morbidity [Fig. 6C]. Only the mice vaccinated with either VV or the pB5 survived
the challenge. Control naïve, CpG/alum and non-transgenic plant material vaccinated mice all died,
indicating that survival is B5-dependent. While 100% of the pB5-vaccinated mice survived, they did
experience a greater weight loss than the VV-vaccinated mice [Fig. 6C, bottom]. These showed a
maximal weight loss of ~5% on day-3 post-challenge while pB5-vaccinated mice underwent a loss
close to a 30% on day-8 post-challenge.
Recombinant protein production is a well-established technology that utilizes efficient strategies
for the generation of subunit vaccines [39, 40, 41]. Recently developed plant biotechnology offers
additional advantages in production scale, economy, product safety and ways of delivery [1-6]. At
present, the main goal is to increase the overall expression yield of functional plant-derived proteins,
especially those of viral origin, which sometimes seriously impair transgenic plant growth and
development [42-44].
To overcome the impediment in the case of VV B5, we utilized two plant expression systems. The
transient system MagnIcon was used to optimize expression cassette arrangements for the antigen and
expedite the production of B5 protein in soluble form, which is more amenable for extraction and
purification [45]. We note, that from several plant-specific targeting signals used to produce soluble
form of pB5, the apoplast signal led to the highest expression of recombinant protein. This
information aided in efficient production of soluble pB5 in transgenic plants. The use of C-terminal
tags facilitated the process of protein purification of greater than 50% purity. Although the expression
levels were lower than those obtained by MagnIcon, our transgenic plants are readily available for
inexpensive up-scaled production.
Golovkin et al., Plant-based Smallpox Vaccine
An obvious advantage of producing recombinant medicinal proteins in plants is the possibility of
oral administration [1, 6]. Considering the inevitable protein degradation in the gastrointestinal tract,
the antigen must be present at higher levels as to induce an adequate immune response. In addition,
plant tissues might serve to protect subunit vaccines against digestion in the gut. To test this, we
constitutively expressed B5 in transgenic collard displaying a large vegetative biomass [31]. We
confirmed the plant retaining a normal morphology while insoluble antigen was expressed at high
levels suitable for direct feeding experiments. This strategy we previously implemented to express
rabies viral G-protein in Arabidposis as a fusion protein rendering it membrane-bound and facing the
cytosol [Golovkin et al, in prep], thus ensuring for correct posttranslational modifications (especially
glycosylation) and proper folding of the viral glycoproteins. The goal was to increase the likelihood of
increased antigen expression of immunologically functional quality.
Mice and the mini-pig fed with transgenic collard and CT exhibited no detectable pB5 immune
response, although a clear CT-specific IgG and IgA response was observed. This response was
induced in both animal models to a lower dose of CT than the overall amount of pB5 in the feed.
Moreover, no B5-specific antibody response was detected in mice immunized with soluble and
purified pB5 plus CT by gavage. It is plausible to speculate that an antigenic protein able to induce an
immune response after oral administration must naturally be taken up through the oral route [6].
Intranasal administration of soluble plant-derived antigen together with CT led to a steady increase in
antibody titers after each immunization in both mice and the mini-pig. The titers in the mini-pig were
lower than in mice. Perhaps this is due to a suboptimal dose of pB5 and/or the use of non-optimal
adjuvants. Intranasal administration of antigen leads to the appearance of IgA in stool, saliva and
vaginal secretions [46]; however, we did not detect any. Nevertheless, a CT-specific IgA was present in
the saliva of the immunized mini-pig and not in stool or vaginal samples.
The highest serum antibody response was found to be in parenterally immunized animals together
with various adjuvants. Intramuscular vaccination of mice with purified pB5 in CpG/alum generated
an antibody response which showed an in vitro and in vivo activity against VV. Sera from vaccinated
animals, but not from control groups, were able to alter virus spread in the comet inhibition assay. The
greater comet-inhibition activity of sera from vaccinia-vaccinated mice in comparison to that from
pB5-vaccinated mice likely reflects the reaction to multiple EV targets after VV vaccination. The pB5
immunized mice were found to be protected from lethal challenge with VV and all mice survived the
challenge. These however experienced a greater weight loss when compared to the VV-vaccinated
mice. This was expected, given that optimal protection to the challenge is provided by the multicomponent vaccine [17, 18]. Therefore, several potent antigens are under further investigation for the
development of a highly efficient, multi-component subunit smallpox vaccine made entirely in planta.
In conclusion, the efficient production of the B5 antigenic domain in planta was analyzed for its
ability to induce protective immunity. The antigen was produced in soluble and insoluble forms upon
transient and stable plant transformation. The soluble pB5 was purified to be used for immunization
Golovkin et al., Plant-based Smallpox Vaccine
by gavage, intranasal and parenteral routes. Transgenic collard with large edible biomass was
primarily utilized for direct oral administration. A B5-specific response was detected after intranasal
immunization, while the strongest response was observed after parenteral administrations. Moreover,
intramuscularly immunized mice generated neutralizing antibodies that inhibited the spread of VV in
vitro and provided 100% protection against lethal challenge.
Our study presents a major step toward the efficient production of viral proteins in plants that are
functionally potent and ready for use in subunit vaccine formulations to counter infectious diseases
such as smallpox.
We thank TJU and KCC research and animal facilities for their support; G. Golovin and R. Egolf
for technical help and greenhouse work; Dr. P. Kozlowski and Dr. M. Neutra for advice on mucosal
adjuvants; and Dr. T. Kohl for helpful comments on the manuscript. This work was supported by a
grant from Commonwealth of Pennsylvania Department of Health to Biotechnology Foundation
Laboratories (H.K.). Y.X. and S.N.I. were supported by the NIH NIAID Middle Atlantic Regional
Center of Excellence grant U54 AI057168.
Golovkin et al., Plant-based Smallpox Vaccine
Fig. 1. Vaccinia virus EV B5 glycoprotein: expression cassettes and cloning strategy. (A)
Schematic diagram of the full-length B5 protein comprised of a signal peptide (SP; aa 1-19) four modular short
consensus repeat (SCR) domains (aa 20-237), stalk region (aa 238-275), putative transmembrane domain
(TM) and cytoplasmic tail (CT). White triangles indicate neutralizing antibody epitopes; arrowhead indicates
the B5-specific Mab site (B5 Mab) within the stalk region; and pin-heads indicate the three N-linked
glycosylation sites within SCR2. (B) The B5 antigenic ectodomain (aa 20-275) was cloned into various
expression cassettes with C-terminus-specific tags (His6 and/or c-Myc), an intracellular targeting signal, such as
apoplast (Apo), or the subcellular outer membrane anchor. The pICH115999 provector was used for in planta
transient transformation. For stable transformation, the pBIN-Plus (soluble pB5) and pB002-based (insoluble
pB5) vectors under the rbcS (black arrow) and CaMV-35S (grey arrow) promoters, respectively, were used. For
bacterial expression, the B5 cassettes were subcloned into the modified pGEX3 vector (pGEX3GST).
Fig. 2. Transient expression and purification of soluble pB5. (A) Six-week-old N. benthamiana (left)
or Swiss chard (right) plants were used for MagnIcon-based expression. (B) Major purification steps of soluble
pB5 visualized by SDS-PAGE staining (top) and Western blot analysis with c-Myc Mab (bottom). pB5 in total
soluble protein (TSP) extract (Extract) detected as a single band (Lane 1). Lanes 2-5 represent consecutive Nicolumn elutions (Step I), and lanes 6-9 represent fractions eluted from the c-Myc-Mab-affinity column (Step
II). Lane 1, TSP extract; 2, extract washed with 5mM imidazole; 3, extract washed with 20mM imidazole; 4,
elution in 80mM imidazole; 5, consequent elution in 120mM imidazole; 6, dialyses of eluate for c-Myc Mabaffinity column application (c-Myc-column); 7, flow-through in PBS buffer; 8, elution with c-Myc peptide; 9,
consequent elution in acid glycine buffer (pH 2.5), and 10, marker. (C) Visual evaluation of concentrated
recombinant pB5 from separate purified batches against a BSA titration curve.
Fig. 3. Constitutive expression of B5 in tobacco and collard. (A) Transgenic tobacco expressing
soluble pB5 grown to maturity (left) and used for protein gel analysis (right). Equal amounts of TSP were
loaded onto the SDS-PAGE (top) and analyzed by Western blotting using c-Myc Mab (bottom). Bands of
expected size were detected in transgenic lines. Bacterial-expressed B5 used as a positive control (+); nontransgenic TSP extract used as negative control (wt). (B) Transgenic collard was grown to maturity (left), leaf
tissue was collected and used for protein analysis (right). Equal amounts of TSP extracts were loaded onto an
SDS-PAGE and stained (top) or used for Western blotting with B5-specific MAb (bottom). Band(s) of expected
size (bottom panel, pB5) were detected in several transgenic lines. (C) For oral feeding plant material was used
in fresh or lyophilized form (left). 1g tablets (right) containing a standardized quantity of recombinant
(intracellular, membrane-bound, insoluble) pB5 antigen were prepared in plastic molds (middle).
Fig. 4. Serum antibody response to pB5 in mice. IgG titers in sera of mice immunized with pB5 by three
different routes. Each group was immunized as described in the Materials and Methods. (A) Anti-B5 response
after oral immunization of mice fed 3 times with fresh leaves or pellets supplemented with CT. (B) B5-specific
Golovkin et al., Plant-based Smallpox Vaccine
immune response after intranasal application of purified pB5 alone (1st and 2nd) and together with CT adjuvant
(3rd and 4th). (C) Anti-B5 response after parenteral immunization with purified pB5 applied with Freund’s
adjuvant. (D) Western blot analysis of sera from mice immunized parenterally (panel C: 3rd i.p samples).
Sequential dilutions of total soluble protein extracts from transgenic plants (left) and E.coli (right) expressing
B5 probed with c-Myc Mab or sera from mice that received parenteral immunization with pB5. Mobility
difference of B5 from plant versus E.coli is indicated by arrowheads.
Fig. 5. Serum antibody response to pB5 protein in mini-pig. The mini-pig was used for all
immunizations as described in the Materials and Methods. IgG titers in serum are given as the mean (±SD) of
triplicate determinations. (A) Oral immunization with transgenic plant material. (B) Intranasal immunization
with purified pB5. (C) Intramuscular immunization with pB5 in CpG/alum. (D) Western blot analysis of sera
from the mini-pig injected with pB5 (3rd injection). Mobility difference of B5 from plant versus E.coli is
indicated by arrowheads.
Fig. 6. pB5-specific antibody response protects from lethal challenge. (A) IgG titers detected in sera
from mice immunized with live VV or with pB5 in Cpg/alum (left). Control groups were injected with adjuvant
alone (CpG/alum), plant TSP extracts, or left non-vaccinated (right). (B) Characteristic comets that form with
VV are altered by the B5 antibodies present in sera from mice immunized with pB5 (left); comet formation was
not altered in negative control groups: adjuvant only (CpG/alum), TSP vaccinated, non-vaccinated (right).
Comet formation was completely inhibited by sera vaccinated with VV (far left). (C) Survival (top) and weight
loss (bottom) after lethal intranasal challenge with VV three weeks after the last intramuscular vaccination (or 7
weeks after the single vaccinia-vaccination by tail scarification). Mice were challenged intranasally with live
VV and monitored for weight loss. Those with a >30% initial weight loss (or morbid) were sacrificed. Symbols
and colors for groups are the same as in (A).
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