Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo.

JOURNAL OF VIROLOGY, Aug. 2003, p. 8249–8255 Vol. 77, No. 15
0022-538X/03/$08.00�0 DOI: 10.1128/JVI.77.15.8249–8255.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus
Genome Reconstituted In Vivo
Luka Cicin-Sain,1 Wolfram Brune,2 Ivan Bubic,3 Stipan Jonjic,3 and Ulrich H. Koszinowski1*
Max von Pettenkofer Institute, LMU, Munich,1 and Rudolf Virchow Center for Experimental Biomedicine,
University of Wu¨rzburg, Wu¨rzburg,2 Germany, and Histology and Embryology Department,
Rijeka Medical Faculty, Rijeka, Croatia3
Received 23 December 2002/Accepted 5 May 2003
Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either
proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in
vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus
bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with
fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid
with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural
mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC
in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show
that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium
as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute
a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only
in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV
antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced
into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel
concept for multivalent virus vaccines launched from bacterial vectors.
Live, attenuated bacteria have a potential to serve as vaccine
vectors for the oral delivery of foreign proteins (29) or DNA
(11, 13, 23). Oral vaccination procedures do not require edu-
cated personnel, and lyophilized bacterial preparations can be
kept and distributed at room temperature, without the costly
and difficult necessity of keeping the preparations cold. More-
over, oral vaccination can be performed simultaneously on
large numbers of subjects, which should make it an efficient
strategy for livestock vaccination.
Live antiviral vaccines are generally considered more effi-
cient than subunit or inactivated vaccines because they are
more likely to induce a broad range of immune responses to
the expressed gene products and provide a better protection.
Furthermore, since these vaccines replicate in the recipient,
the immunity they confer should be long lasting (39). There-
fore, a bacterial vaccination vector delivering an attenuated,
yet infectious virus may present the basis for efficient vaccines
that are easy to store, distribute, and administer.
Herpesviruses are important pathogens for humans and live-
stock. We have previously shown that the large genomes of
herpesviruses (up to 230 kb) can be cloned as bacterial artifi-
cial chromosomes (BACs) into Escherichia coli (1, 2, 24). Oth-
ers have demonstrated that BAC DNA encoding the genome
of a herpesvirus can induce specific protective immunity (35,
36). However, in these experiments, the BAC DNA was iso-
lated from bacteria by column chromatography and diluted in
phosphate-buffered saline (PBS) prior to injection into chick-
ens (36) or adsorbed to gold particles and injected into mice
with a gene gun (35).
For this study, we decided to test whether the infection of
animals with bacteria could lead to reconstitution of a replica-
tion competent virus from a bacterial vector in vivo and
whether this could lead to protective immunity.
The cytomegaloviruses (CMVs), ubiquitous members of the
betaherpesvirus subgroup, are marked by strict species specificity,
tropism for hematopoietic tissue and secretory glands, and slow
replication. The infection of mice with murine CMV (MCMV)
shares many aspects with human CMV infection and thus serves
as a biological model for experimental vaccine developments (16,
22, 26, 42). With double-stranded DNA genomes of 120 to 230
kbp and up to 220 genes, herpesviruses have some of the largest
genomes of viruses that infect mammals (25). Many herpesviral
genes are not essential for viral replication and could be ex-
changed for genes from unrelated species to generate recombi-
nant viruses. Therefore, the deletion of viral genes that are not
essential for the vaccination success and the insertion of genes
from other infectious agents are attractive concepts for the de-
velopment of live recombinant vaccines. For this purpose, we
introduced a gene from another virus into the MCMV genome
and determined the presence of the gene product in murine sera.
Here we show that infectious MCMV can be reconstituted in vivo
directly from bacteria that carry their genomes. We also show that
viral reconstitution leads to protective immunity and to strong
expression of additional transgenes inserted into the viral ge-
nome.
MATERIALS AND METHODS
Plasmids and bacterial strains. Plasmid pRep4-HBs was generated by insert-
ing the gene of the hepatitis B virus surface antigen (HBsAg) into pRep4
* Corresponding author. Mailing address: Max von Pettenkofer In-
stitute, 80446 Munich, Germany. Phone: 49 89 5160 5203. Fax: 49 89
5160 5227. E-mail: Koszinowski@m3401.mpk.med.uni-muenchen.de.
8249

(Invitrogen) as described (3). To obtain MCMV-HBs, the HBs gene bracketed
by a Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal
was excised from pRep4-HBs and inserted into the ie2 gene locus of the cloned
full-length MCMV BAC, pSM3fr (37), as previously described (3). Plasmid
pGB2�inv-hly (17) was kindly provided by C. Grillot-Courvalin (Unite´ des
Agents Antibacte´riens, Institut Pasteur, Paris, France).
Plasmid and BAC DNA was introduced by electroporation into the standard
E. coli BAC host, DH10B [genotype: F� mcrA �(mrr-hsdRMS-mcrBC)
�80dlacZ�M15 �lacX74 deoR recA1 araD139 �(ara leu)7697 galU galK rpsL
endA1 nupG]. MCMV-HBs was transferred by conjugation from DH10B to
serovar Typhimurium LT2 strains SA1970 (metA22 trpC2 hisF1009 rpsL120 xylR1
recA1 srl-202::Tn10), TT521 (recA1 rpsL srl-202::Tn10), and TT9081 [his-644(del:
OGDCBHAF) srl-202::Tn10 recA1]. The serovar Typhimurium strains were ob-
tained from K. E. Sanderson (Salmonella Genetic Stock Centre, University of
Calgary, Calgary, Canada).
Mice and viruses. BALB/c, C57BL/6, and 129SvEv gamma interferon recep-
tor-null (IFN-�R0/0) mice were bred under conventional barrier housing condi-
tions at the central breeding facility of the Rijeka Medical Faculty Animal house.
All animal experiments described here received approval by the Ethical Com-
mittee at the University of Rijeka. Sex- and strain-matched, 6- to 9-week-old
mice were used. Stocks of tissue culture and salivary gland-derived (SGD)
MCMV, derived from MW97.01 (37), were prepared as previously described (4).
Virus reconstitution. Fresh bacterial cultures were grown overnight in Luria-
Bertani medium supplied with antibiotics for selective growth. DNA transfer
from bacteria to mammalian cells was performed as previously described (5).
Two microliters of bacterial culture was used to inoculate NIH 3T3 fibroblasts
grown in 96-well dishes without antibiotics. This corresponded to approximately
300 bacteria per fibroblast. The cell culture dishes were spun for 5 min at 1,000
� g and incubated for 2 h at 37°C. The cell culture medium was subsequently
replaced with fresh medium supplemented with ampicillin and gentamicin (100
�g/ml each). For infection of mice, bacterial cultures were centrifuged at 2,000
� g for 8 min at 4°C and serially diluted in cold sterile PBS, at concentrations
ranging from 2 � 109 to 2 � 106 CFU/ml (bacterial stocks were titrated in
parallel on blood agar plates). Five hundred microliters of diluted bacteria
(amounts between 106 to 109 CFU) were injected intraperitoneally (i.p.) into
each mouse. Lungs were obtained from mice infected with serovar Typhimurium
on 28th day postinfection (dpi) and tested for MCMV reconstitution by plaque
assay on murine embryonic fibroblasts (MEFs) as previously described (32). The
mice infected with E. coli were immunosuppressed with anti-CD4 and anti-CD8
antibodies (9) (1 mg each) on the 6th and 13th dpi. Mice were additionally
immunosuppressed by intramuscular (i.m.) injection of 6.25 �g of hydrocortisone
on alternate days from day 13 postinfection onwards. Blood samples were col-
lected from tail veins of infected mice on days 12 and 20 postinfection. HBsAg
was detected in murine serum by use of a commercial microtiter enzyme-linked
immunosorbent assay (ELISA) kit, containing wells coated with anti-HBs anti-
bodies (catalog no. 931801; Ortho Diagnostic Systems) according to manufac-
turer’s instructions. Salivary glands, lungs, and spleens from individual mice were
collected on dpi 21 and tested for MCMV reconstitution as previously described
(32). Each experiment was performed at least twice.
Immunization and challenge procedures. Overnight bacterial cultures were
centrifuged and resuspended in cold sterile PBS at final concentrations of 5 �
109 CFU/ml. One hundred �l of bacterial suspension (5 � 108 CFU) was injected
either i.m. into gluteal muscles or subcutaneously (s.c.) into the soft tissue of the
dorsum. For i.p. injection bacteria were diluted 1:10 in sterile PBS and injected
in a volume of 500 �l ( 108 CFU). Positive-control mice were infected with 105
PFU of tissue culture-grown BAC-derived MCMV. No immunosuppressive pro-
cedure was applied. Blood samples were collected from the tail vein, and sera
from individual mice were serially diluted in PBS. The immunization against
MCMV and antibody production efficiency was tested by indirect ELISA using
MCMV-infected MEFs as the antigen source, as previously described (20). In
brief, microtiter plates were coated with lysates of either uninfected or MCMV-
infected MEFs. Serially diluted sera were applied in parallel to both kinds of
plates, which was followed by wash steps and an incubation with peroxidase-
conjugated anti-mouse antibodies. Signals obtained from the plates coated with
uninfected MEF lysates were treated as unspecific background signal and sub-
tracted from the signals obtained from the plates coated with lysates of MCMV-
infected MEFs. The protective effect of the anti-MCMV immunization was
tested by challenge with lethal doses of SGD MCMV. Mice received i.p. injec-
tions with 2 50% lethal doses (LD50) of SGD MCMV (i.e., 5 � 10
4 PFU for 129
IFN-�R0/0 mice) on dpi 28. Animals were checked daily for survival for 2 weeks
following the challenge. Survivors were monitored for an additional 3 months.
Each experiment was performed at least twice.
RESULTS
Virus reconstitution in vivo by serovar Typhimurium carry-
ing the MCMV BAC. In our previous studies, the DH10B
strain of E. coli served as the host for the MCMV BAC. As
DH10B is not commonly used as vector for DNA immuniza-
tion, we introduced an MCMV BAC into recombinase A-de-
ficient (recA mutant) laboratory strains of Salmonella enterica
serovar Typhimurium shown in Table 1. recA mutant bacterial
strains were selected to ensure the stability of repetitive se-
quences present in herpesvirus genomes (31). The rationale for
the usage of serovar Typhimurium was their natural ability to
invade mammalian cells. Therefore, they should represent a
better vehicle for DNA transfer in vivo than E. coli. The clones
that received the MCMV BAC were selected by resistance to
chloramphenicol and tetracycline. To confirm that the clones
in question contained the complete MCMV BAC, BAC DNA
was extracted and introduced into fibroblasts to give rise to
infectious virus as previously described (6). To check whether
bacteria could transfer viral DNA directly into mammalian
cells in cell culture, we inoculated NIH 3T3 cells with WB241
bacteria. Five days after inoculation we observed the formation
of characteristic viral plaques, indicating direct reconstitution
of MCMV from bacteria.
recA mutant bacteria are attenuated in vivo because they
cannot repair the DNA damage caused by reactive oxygen
species (ROS) in endolysosomal compartments of professional
phagocytes (7, 8). ROS are strongly induced in macrophages by
IFN-� (15, 27). As serovar Typhimurium has a preference to
infect macrophages in vivo (14, 33), this could lead to damage
of the viral DNA before the virus would initiate its replication.
In IFN-�R0/0 mice (18), ROS production in macrophages is
not induced (21). Thus, we assumed that the use of IFN-�R0/0
mice would increase the chance to test the possibility of her-
pesvirus reconstitution from bacteria in vivo.
Serovar Typhimurium strain WB241 (Table 1) was grown
TABLE 1. List of modified bacterial strains used in this study
Bacterial vector Inserted plasmid
Name Species and strain MCMV-HBs BAC pRep4-HBs pGB2�inv-hly
Eco/M (WB231) E. coli K-12, DH10B � � �
Eco/M/inv (WB232) E. coli K-12, DH10B � � �
Eco/P/inv (WB233) E. coli K-12, DH10B � � �
WB240 S. enterica serovar Typhimurium LT2, SA1970 � � �
WB241 S. enterica serovar Typhimurium LT2, TT521 � � �
WB242 S. enterica serovar Typhimurium LT2, TT9081 � � �
8250 CICIN-SAIN ET AL. J. VIROL.

overnight as described in Materials and Methods and injected
i.p. into IFN-�R0/0 mice at doses indicated in the Fig. 1. Con-
trol mice were infected with 5 � 105 PFU of BAC-derived
MCMV (37). Mock-infected mice were used as negative con-
trols. The ability to mount a specific humoral response to
MCMV was taken as an indication of virus protein expression.
Mice infected s.c. with 108 or 109 CFU of bacteria displayed
elevated titers of anti-MCMV antibodies in their sera on the
28th dpi, whereas mice infected with 107 CFU or less showed
no anti-MCMV antibodies (Fig. 1A). Therefore, the infection
with 108 CFU of bacteria or more induced humoral immunity
against MCMV, whereas infection with 107 CFU or less did
not. The very same mice were challenged on dpi 28 with 105
PFU of SGD MCMV (4 LD50) and monitored for survival
(Fig. 1B). All mice infected with 109 CFU and 83% of mice
infected with 108 CFU of bacteria, as well as all of the virus-
infected controls, survived the challenge. Surprisingly, 83% of
the mice infected with 107 CFU survived the challenge as well,
despite the fact that only one mouse in this group serocon-
verted. (Fig. 1A). All mice infected with 106 CFU as well as the
uninfected control mice died by day 9 following challenge.
Therefore, the infection of mice with serovar Typhimurium
carrying MCMV BACs, induced specific antibody response
and protective immunity against MCMV.
In order to assess whether the immunization was caused by
the reconstitution of the infectious viral process or merely due
to expression of isolated viral genes from transferred DNA,
mice were infected according to the protocol described above,
and infectious viral titers in lungs were determined at dpi 28.
The animals in the groups infected with 108 or 109 CFU of
WB241 contained infectious virus in their organs as seen by
plaque assay on MEFs (Fig. 2). The specificity of these plaques
for MCMV was confirmed by staining with a monoclonal an-
tibody against the pp89 protein, the product of the IE1 MCMV
gene (not shown). In mice infected with 107 CFU or less we
could not detect virus. Therefore, the infection with 108 CFU
of serovar Typhimurium carrying the MCMV BAC leads to
reconstitution of live virus in vivo. Notably, the same number
of bacteria that were needed for specific antibody formation
was needed to initiate an active viral infection process. There-
fore, the ability to mount a specific antibody response could
serve as an indirect indicator for virus reconstitution in vivo.
Next, we tested whether bacteria carrying MCMV BACs
could induce the immunization of immunocompetent mice.
We infected groups of BALB/c mice with 108 CFU of WB241
bacteria and looked for infectious virus in lungs and spleen and
for specific antibody titers to MCMV in sera at 28 dpi. As
expected, mice did not show elevated antibody titers (data not
shown), nor could infectious virus be isolated from their or-
gans. Therefore, we concluded that the serovar Typhimurium
vectors used here could not effectively induce the reconstitu-
tion of the viral infectious process in IFN-�R�/� mice.
Virus reconstitution in vivo by E. coli carrying MCMV BAC.
In previous experiments, we have shown that the cloned
FIG. 1. 129SvEv IFN-�R0/0 mice were infected with the indicated
CFU of WB240 (six mice per group). Positive-control mice were in-
fected with 5 � 105 PFU of wild-type MCMV, and negative controls
were mock infected. (A) Sera were collected at dpi 28, serially diluted
up to 1:1,024, and assayed for anti-MCMV antibodies by ELISA.
Median and quartile absorbance values at 492 nm are displayed.
Pooled sera from a group of mice that received 5 � 105 PFU of
MCMV were used as positive controls. (B) On the same day, mice
were challenged with 2 LD50 of salivary gland-passaged MCMV and
monitored for survival. Survival curves for the first 10 days following
challenge are shown. Exclusively for presentation purposes, the sur-
vival curves are presented in two separate graphs. Error bars, quartiles.
FIG. 2. 129 SvEv IFN-�R0/0 mice were i.p. infected with the indi-
cated doses of WB240 or with 5 � 105 PFU of MCMV. Mice were
sacrificed on dpi 28, and lung homogenates were assayed on MEFs for
infectious MCMV titers. Circles represent virus titers in the organs of
individual mice. D.L., limit of detection.
VOL. 77, 2003 RECONSTITUTION OF VIRUSES FROM BACs IN VIVO 8251

MCMV genome can be transferred directly from its E. coli host
to cultured fibroblasts to initiate the production of infectious
virus in these cells (5). This requires the introduction of the
plasmid pGB2�inv-hly (17), which directs the expression of
the invasin gene of Yersinia pseudotuberculosis and the hemo-
lysin O gene from Listeria monocytogenes in E. coli, and the
presence of
1-integrin molecules on the surface of the recip-
ient fibroblasts. We wanted to test to which extent these bac-
teria could also be used as vectors to deliver the MCMV
genome to somatic cells of a mouse and to initiate the infec-
tious cycle in vivo. The rationale for this was that the in vivo
infection of nonphagocytic cells like fibroblasts, which do not
express ROS, would give an opportunity to recA mutant bac-
terial vectors to reconstitute viral infection in IFN-�R�/� an-
imals.
Moreover, we wanted to analyze the efficiency of the expres-
sion of a transgene from a recombinant herpesvirus. Thus, we
used a recombinant MCMV BAC, MCMV-HBs, which con-
tains the HBsAg gene driven by a Rous sarcoma virus pro-
moter (3). In a previous study we have shown that levels in
serum of a secreted virus-encoded protein such as the HBsAg
reflect MCMV productivity in vivo and thus can be used to
monitor the course of infection over time. Bacteria containing
a high-copy-number plasmid with the identical eukaryotic HBs
expression cassette were used to control for possible HBsAg
expression in the absence of viral replication and to compare
virus reconstitution by bacteria with plasmid DNA delivery by
bacteria.
In order to assay for viral reconstitution, BALB/c mice were
infected i.p. with 108 CFU of either Eco/M/inv or Eco/P/inv
bacteria (Table 1). T lymphocytes were depleted on dpi 6 and
13, and hydrocortisone was applied on alternate days from the
13th dpi onwards in order to suppress the immune response
and to increase the chances of establishing a productive viral
infection. Lungs, salivary glands, and spleens were collected on
the 21st dpi, and organ homogenates were tested by plaque
assay on fibroblasts for the presence of infectious MCMV.
Only one out of eight mice infected with Eco/M/inv showed
reconstitution of infectious virus in the lungs and spleen (Fig.
3A). In repeated experiments in which we used BALB/c or
C57BL/6 mice, we did not detect viral reconstitution (data not
shown).
In a separate experiment, we infected groups of IFN-�R0/0
mice with graded amounts of either Eco/M/inv or Eco/P/inv
using the same infection and immune suppression protocol. All
mice that received 108 CFU of Eco/M/inv were positive for
MCMV plaques in their lungs and salivary glands (Fig. 3B).
The mice infected with 107 CFU of Eco/M/inv were negative
for MCMV as confirmed by plaque assay. As expected, Eco/
P/inv-infected mice showed no evidence for MCMV infection.
Therefore, the infection with E. coli carrying the MCMV BAC
leads to in vivo reconstitution of infectious virus in IFN-�R0/0
mice but not in IFN-�R�/� mice. There was no significant
difference in the number of bacteria required to launch the
infectious process either by serovar Typhimurium or by E. coli.
IFN-�R0/0 mice are more sensitive to virus infection than
normal mice. To clarify whether the antiviral activity of IFN-�
alone could explain the lack of reconstitution in IFN-�R�/�
mice, we tried to reconstitute infectious virus from bacteria
using B-cell-deficient �MT/�MT mice, which were T cell de-
pleted before infection with bacteria. Mice lacking both B and
T cells cannot control MCMV infection (38), whereas IFN-
�R0/0 mice can (30). Thus, this system should allow the detec-
tion of very low initial amounts of reconstituted virus. How-
ever, at dpi 21 we could not detect any virus out of organs of
�MT/�MT mice infected with 108 CFU of Eco/M/inv E. coli
(data not shown). Therefore, mice very sensitive to MCMV,
yet capable of IFN-� signaling, could not reconstitute infec-
tious virus. This finding argues for an important role of the
IFN-� signaling pathway during the early phase of virus recon-
stitution in vivo.
Sera were collected from the very same IFN-�R0/0 mice
whose organs were assayed for virus titer and shown in Fig. 3,
on dpi 12 and 20 and assayed for the product of the introduced
viral transgene. The presence of the viral protein HBsAg was
determined by direct ELISA. At day 12, no HBsAg could be
detected in any of the groups (Fig. 4). At day 20, four out of
five mice infected with Eco/M/inv but none of the mice in-
fected with Eco/P/inv showed detectable amounts of HBsAg in
their sera (Fig. 4). Notably, the amount of HBsAg correlated
with the viral yield in the salivary gland in individual mice,
which was determined in parallel (r � 0.91). Therefore, the
MCMV-HBs reconstitution is associated with a considerable
HBsAg expression in the sera of animals. Animals that re-
ceived the HBs gene alone as a multicopy plasmid did not
produce measurable HBsAg concentrations. The absence of
HBsAg at dpi 12 indicates that HBsAg was expressed to de-
tectable levels only upon virus reconstitution. In fact, no infec-
tious virus was detected when mice infected under identical
conditions with Eco/M/inv were sacrificed at dpi 14 (data not
shown).
FIG. 3. (A) BALB/c or (B) 129 SvEv IFN-�R�/� mice were i.p.
infected with 108 CFU of WB232 (E) or WB233 (F). T lymphocytes
were depleted on dpi 6 and 13, and hydrocortisone was applied on
alternate days from dpi 13 onwards. Mice were sacrificed on dpi 21,
and organ homogenates were assayed on MEFs for infectious MCMV
titers. Circles represent virus titer in organs of individual mice. D.L.,
detection limit.
8252 CICIN-SAIN ET AL. J. VIROL.

Virus reconstitution in vivo by bacteria carrying the MCMV
BAC is independent of bacterial invasion genes. Virus recon-
stitution from E. coli was restricted to IFN-�R0/0 mice, as it was
the case with serovar Typhimurium vectors. Therefore, we
assumed that this process was occurring in professional phago-
cyte cells. The additional genes supplied for active bacterial
invasion and virus reconstitution in vitro might therefore be
dispensable for virus reconstitution in vivo. In order to test this
hypothesis, bacteria carrying the MCMV BAC, but not the
pGB2�inv-hly plasmid, were tested for DNA transfer and viral
reconstitution.
Groups of IFN-�R0/0 mice infected with 108 CFU of either
Eco/M/inv, Eco/M, or Eco/P/inv bacteria were monitored for
MCMV specific antibody production by IFN-�R0/0 mice over
time. We observed that five of five of the mice infected with
Eco/M and four of five mice infected with Eco/M/inv bacteria
mounted an antibody response (Fig. 5). The MCMV-specific
antibody titer was only slightly elevated over the values from
control mice at dpi 14 but rose by dpi 28 and at dpi 42 was
comparable to titers obtained from MCMV infected mice at
dpi 70. Control mice infected with Eco/P/inv did not mount a
specific immune response. Eco/M bacteria, which lack the
pGB2�inv-hly invasion plasmid, induced a strong MCMV-
specific antibody response. The presence of pGB2�inv-hly did
not enhance the immunization rate. This was in accordance
with the observation that infectious MCMV could be isolated
from organs of Eco/M-infected mice (not shown). Taken to-
gether, these data indicate that successful virus reconstitution
in vivo was independent of the genes encoded by pGB2�inv-
hly, whereas the pGB2�inv-hly plasmid was required for BAC
transfer and virus reconstitution in cell culture.
Next, we tested whether the immunization procedure by E.
coli was also protective against a challenge with a lethal dose of
MCMV. Groups of 10 IFN-�R0/0 mice were immunized by
inoculation with 109 CFU of either Eco/M or Eco/M/inv. Con-
trol mice were inoculated with Eco/P/inv. Half of each group
received the bacteria i.m. and the other half received them s.c.
On the 28th dpi, seroconversion occurred in most of the Eco/M
or Eco/M/inv infected mice (8 of 10 in each group), but in none
of the controls, as detected by ELISA (data not shown). On the
same day mice were challenged with 2 LD50 of SGD MCMV
and monitored for survival. All five mice infected i.m. with
Eco/M/inv and four of five of mice infected s.c. with Eco/M/inv,
as well as all animals infected with Eco/M (10 of 10) survived
the challenge (Fig. 6). All mice from the control groups died by
dpi 8 or 9. The results indicate that mice immunized by bac-
teria carrying the MCMV BAC were protected against a chal-
lenge with lethal doses of virus.
DISCUSSION
The cloning of herpesvirus genomes as bacterial artificial
chromosomes allows fast and accurate mutagenesis procedures
of basically any viral gene in the prokaryotic E. coli host (6, 24).
The biological properties of the viral mutants are analyzed
upon the reconstitution of the virus in eukaryotic cells. The
application of random transposon mutagenesis procedures led
to the establishment of libraries of mutant genomes (6). In
order to test the biological properties of random mutant librar-
ies, virus reconstitution was achieved by direct inoculation of
host cells in vitro by invasive bacteria carrying BACs (5).
Whether these bacteria would also allow virus reconstitution
in vivo was the next logical question. Direct in vivo reconsti-
tution of a virus infection from viral DNA is not a new concept.
However, successful approaches had been restricted so far to
viruses carrying much smaller genomes (around 10 kb), and to
the direct injection of DNA (12, 40, 41). Here we show with the
example of the 230-kb genome of MCMV that there should be,
in principle, no viral genome size limit for virus reconstitution
from DNA in vivo.
Suter et al. have shown that injection of herpesvirus BAC
DNA can lead to protective immunity in the absence of viral
FIG. 4. 129 SvEv IFN-�R0/0 mice were i.p. infected with 108 CFU
of either Eco/M/inv or Eco/P/inv. T lymphocytes were depleted on dpi
6 and 13, and hydrocortisone was applied on alternate days from day
13 onwards. Sera were collected at days 12 and 20 and assayed for the
presence of HBsAg by direct ELISA. At day 12, no HBsAg could be
detected in any of the groups. At day 20, four out of five mice infected
with Eco/M/inv and none of the mice infected with Eco/P/inv showed
detectable amounts of HBs in their sera.
FIG. 5. 129 SvEv IFN-�R0/0 mice were infected i.p. with 108 CFU
of indicated bacteria (five mice per group). Sera were obtained at 14,
24, and 42 dpi, serially diluted in PBS, and assayed for anti-MCMV
antibodies by ELISA. Arithmetic means and standard deviations of
absorbance values at 492 nm for the 1:64 dilution step are shown. Sera
from mice infected with 105 PFU of MCMV were obtained at dpi 70
and used as positive controls (Œ). Sera from uninfected mice (‚) were
used as negative controls.
VOL. 77, 2003 RECONSTITUTION OF VIRUSES FROM BACs IN VIVO 8253

replication (35). Another recent report has shown that the
inoculation of Marek’s disease virus BAC DNA diluted in PBS
into chicken could lead to protective immunity (36). Our data
show for the first time that a herpesvirus infection can be
launched directly from bacteria carrying the infectious virus
genome, omitting the need for technical preparatory work to
isolate BAC DNA. We have also demonstrated that additional
genes introduced into the virus are effectively expressed (3).
Thus, the use of BAC-derived viruses has a potential as vaccine
and vaccine vector, and this is also the case when the viral
genome is launched from bacteria.
Interestingly, no infectious virus could be isolated from or-
gans before day 21 after injection of bacteria. Since the infec-
tion with 105 PFU MCMV leads to detectable viral titers in
organs within few days, this implied that the peak of productive
viral replication was delayed for at least 2 weeks. This finding
was not unexpected, as we have already reported a time lag in
the production of infectious virus already upon transfection of
cultured cells with MCMV BAC DNA (37). Therefore, at dpi
28, lung virus titers after virus reconstitution from serovar
Typhimurium are still high, whereas in mice infected with
MCMV, the virus infection was already contained by the im-
mune system. The other evidence for this delay, are the kinet-
ics of anti-MCMV antibody titers. Titers were still low at 14
days postinfection, rose by dpi 28 and even more by dpi 42.
Therefore, Direct comparison of antibody titers at dpi 28
shows a marked difference between mice that were infected by
bacteria or by the virus (Fig. 1A). However, at dpi 70, antibody
titers from vaccinated mice were undistinguishable from mice
infected with MCMV (data not shown).
It was surprising that infection with 107 CFU of serovar
Typhimurium did not induce a detectable antibody response or
production of infectious virus yet protected mice against a
lethal challenge with SGD MCMV. Low-frequency DNA
transfer events leading to expression of viral genes together
with an abortive infection could explain this observation. In
that case, the expression of viral genes might induce a protec-
tive cellular immunity, but not a strong humoral immune re-
sponse. Indeed, DNA immunization against the MCMV by a
plasmid encoding for an immunodominant viral gene has al-
ready been shown to induce protective T-cell immunity in the
absence of an antibody response (16). Moreover, it was dem-
onstrated that an additional injection of formalin inactivated
viruses could induce a specific humoral immune response (26).
Another unexpected observation was that virus reconstitu-
tion in vivo was not linked to the ability of E. coli to invade
cells, as opposed to the situation in vitro. This suggested that
the bacteria were not actively entering host cells. Therefore,
the probable target cells, into which the viral genomes entered
and started the viral replication, could be phagocytic cells that
actively took up bacteria. The half-life of granulocytes is only a
few hours (19), which is too short to allow MCMV reconstitu-
tion and replication. Thus, the properties of professional
phagocytic cells, i.e., macrophages and dendritic cells, probably
dictate the success of the transfer, rather than the invasive
properties of the bacteria. Therefore, it was not surprising that
viral reconstitution could be achieved only in IFN-�R0/0 mice
that are deficient for ROS production. Professional phagocytes
in these mice lack activating signals from IFN-� pathways (21)
and therefore can eliminate intracellular pathogens only at a
lower rate (10, 18). This indicates that strategies for the im-
provement of the virus reconstitution efficiency in vivo should
focus on the survival of bacteria in phagocytic cells.
To maintain the stability of the herpesvirus genome, the
BACs are usually propagated in recA mutant bacterial strains.
Originally, we developed the herpesvirus BAC system for the
mutagenesis of viral genomes. Recombination-deficient (recA
mutant) bacteria are preferred as hosts for the MCMV BAC
because the 21 chi sites (34) present in the MCMV genome, as
well as repeated viral sequences within the MCMV genome
(31), could potentially serve as hot spots for spontaneous re-
combination (34).
The requirement for recA mutant bacterial strains for prop-
agation of herpesvirus BACs at present appears to be the
major obstacle for further development. Since parenteral ad-
ministration of 108 CFU of recA mutant bacteria was required
for successful reconstitution in IFN-�R0/0 mice, the amounts of
bacteria required for efficient transfer in fully immunocompe-
tent mice may need to be even higher. Additionally, these
strains have not been designed for oral administration and
DNA delivery via mucosal routes. On the other hand, recA�
strains of serovar Typhimurium have already been used as
vectors for oral DNA vaccination (11, 28). Can recA� strains
be used to increase transfer efficiency? We have seen that viral
BAC DNA can be maintained in the recA� E. coli strain CBTS
or GS500, at least for short periods of time, without losing the
potential to start a productive infection (5, 24). However, our
attempts to introduce an intact MCMV BAC into recA� strains
of serovar Typhimurium have so far not met with success.
Perhaps the genome repeats and the chi sequences need to be
deleted or modified, in order to increase the stability of the
viral genomes in a recA� host. Careful design of viral muta-
tions could allow the propagation of stable viral mutant ge-
FIG. 6. 129 SvEv IFN-�R0/0 mice were i.m. or s.c. infected with 109
CFU of Eco/M or Eco/M/inv bacteria. Control mice were infected with
Eco/P/inv. At 28 dpi, mice were challenged with 2 LD50 of SGD
MCMV and monitored for survival. Survival curves for the first 14 days
following challenge are shown.
8254 CICIN-SAIN ET AL. J. VIROL.

nomes in recA� bacterial strains. If recA� bacterial strains
could be used for oral delivery of viral DNA, this would have
a huge potential for livestock vaccination and perhaps even for
human vaccines. The observation that a herpesvirus infection
can be reconstituted from bacteria in vivo presents the first
step in this direction.
ACKNOWLEDGMENTS
This work was supported by DFG SPP “New vaccination strategies,”
Bayerische Forschungsstiftung “Forimmun,” and Br1730/2.
We thank Torsten Sacher, Markus Wagner, and Zsolt Ruzsics for
useful discussions; Tanja Saulig, Mijo Golemac, and Miljana Kricka for
technical assistance; and Astrid Krmpotic and Milena Hasan for tech-
nical advice.
REFERENCES
1. Adler, H., M. Messerle, M. Wagner, and U. H. Koszinowski. 2000. Cloning
and mutagenesis of the murine gammaherpesvirus 68 genome as an infec-
tious bacterial artificial chromosome. J. Virol. 74:6964–6974.
2. Borst, E. M., G. Hahn, U. H. Koszinowski, and M. Messerle. 1999. Cloning
of the human cytomegalovirus (HCMV) genome as an infectious bacterial
artificial chromosome in Escherichia coli: a new approach for construction of
HCMV mutants. J. Virol. 73:8320–8329.
3. Brune, W., M. Hasan, M. Krych, I. Bubic, S. Jonjic, and U. H. Koszinowski.
2001. Secreted virus-encoded proteins reflect murine cytomegalovirus pro-
ductivity in organs. J. Infect. Dis. 184:1320–1324.
4. Brune, W., H. Hengel, and U. H. Koszinowski. 1999. A mouse model for
cytomegalovirus infection, p. 19.7.1–19.7.13. In J. Coligan, A. Kruisbeen, D.
Marguiles, E. Shevach, and W. Strober (ed.), Current protocols in immu-
nology. John Wiley, New York, N.Y.
5. Brune, W., C. Menard, J. Heesemann, and U. H. Koszinowski. 2001. A
ribonucleotide reductase homolog of cytomegalovirus and endothelial cell
tropism. Science 291:303–305.
6. Brune, W., C. Menard, U. Hobom, S. Odenbreit, M. Messerle, and U. H.
Koszinowski. 1999. Rapid identification of essential and nonessential her-
pesvirus genes by direct transposon mutagenesis. Nat. Biotechnol. 17:360–
364.
7. Buchmeier, N. A., C. J. Lipps, M. Y. So, and F. Heffron. 1993. Recombina-
tion-deficient mutants of Salmonella typhimurium are avirulent and sensitive
to the oxidative burst of macrophages. Mol. Microbiol. 7:933–936.
8. Carlsson, J., and V. S. Carpenter. 1980. The recA� gene product is more
important than catalase and superoxide dismutase in protecting Escherichia
coli against hydrogen peroxide toxicity. J. Bacteriol. 142:319–321.
9. Cobbold, S. P., A. Jayasuriya, A. Nash, T. D. Prospero, and H. Waldmann.
1984. Therapy with monoclonal antibodies by elimination of T-cell subsets in
vivo. Nature 312:548–551.
10. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, and T. A.
Stewart. 1993. Multiple defects of immune cell function in mice with dis-
rupted interferon-gamma genes. Science 259:1739–1742.
11. Darji, A., C. A. Guzman, B. Gerstel, P. Wachholz, K. N. Timmis, J. Wehland,
T. Chakraborty, and S. Weiss. 1997. Oral somatic transgene vaccination
using attenuated S. enterica serovar Typhimurium. Cell 91:765–775.
12. Dubensky, T. W., B. A. Campbell, and L. P. Villarreal. 1984. Direct trans-
fection of viral and plasmid DNA into the liver or spleen of mice. Proc. Natl.
Acad. Sci. USA 81:7529–7533.
13. Fennelly, G. J., S. A. Khan, M. A. Abadi, T. F. Wild, and B. R. Bloom. 1999.
Mucosal DNA vaccine immunization against measles with a highly attenu-
ated Shigella flexneri vector. J. Immunol. 162:1603–1610.
14. Garcia-del Portillo, F. 2001. Salmonella intracellular proliferation: where,
when and how? Microbes Infect. 3:1305–1311.
15. Goldberg, M., L. S. Belkowski, and B. R. Bloom. 1990. Regulation of mac-
rophage function by interferon-gamma. Somatic cell genetic approaches in
murine macrophage cell lines to mechanisms of growth inhibition, the oxi-
dative burst, and expression of the chronic granulomatous disease gene.
J. Clin. Investig. 85:563–569.
16. Gonzalez-Armas, J. C., C. S. Morello, L. D. Cranmer, and D. H. Spector.
1996. DNA immunization confers protection against murine cytomegalovi-
rus infection. J. Virol. 70:7921–7928.
17. Grillot-Courvalin, C., S. Goussard, F. Huetz, D. M. Ojcius, and P. Courva-
lin. 1998. Functional gene transfer from intracellular bacteria to mammalian
cells. Nat. Biotechnol. 16:862–866.
18. Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo,
J. Vilcek, R. M. Zinkernagel, and M. Aguet. 1993. Immune response in mice
that lack the interferon-gamma receptor. Science 259:1742–1745.
19. Janeway, C. A., and P. Travers. 1997. Immunobiology: the immune system in
health and disease, 3rd ed. Current Biology, Ltd./Garland Publisihng, Inc.,
New York, N.Y.
20. Jonjic, S., M. del Val, G. M. Keil, M. J. Reddehase, and U. H. Koszinowski.
1988. A nonstructural viral protein expressed by a recombinant vaccinia virus
protects against lethal cytomegalovirus infection. J. Virol. 62:1653–1658.
21. Kamijo, R., D. Shapiro, J. Le, S. Huang, M. Aguet, and J. Vilcek. 1993.
Generation of nitric oxide and induction of major histocompatibility complex
class II antigen in macrophages from mice lacking the interferon gamma
receptor. Proc. Natl. Acad. Sci. USA 90:6626–6630.
22. MacDonald, M. R., X. Y. Li, R. M. Stenberg, A. E. Campbell, and H. W. t.
Virgin. 1998. Mucosal and parenteral vaccination against acute and latent
murine cytomegalovirus (MCMV) infection by using an attenuated MCMV
mutant. J. Virol. 72:442–451.
23. Medina, E., and C. A. Guzman. 2001. Use of live bacterial vaccine vectors for
antigen delivery: potential and limitations. Vaccine 19:1573–1580.
24. Messerle, M., I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Ko-
szinowski. 1997. Cloning and mutagenesis of a herpesvirus genome as an
infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 94:
14759–14763.
25. Mocarski, E. S., and C. T. Courcelle. 2001. Cytomegaloviruses and their
replication, p. 2629–2673. In D. Knipe and P. Howley (ed.), Fields virology,
4th ed. Lippincott-Raven, Philadelphia, Pa.
26. Morello, C. S., M. Ye, and D. H. Spector. 2002. Development of a vaccine
against murine cytomegalovirus (MCMV), consisting of plasmid DNA and
formalin-inactivated MCMV, that provides long-term, complete protection
against viral replication. J. Virol. 76:4822–4835.
27. Nathan, C. F., H. W. Murray, M. E. Wiebe, and B. Y. Rubin. 1983. Identi-
fication of interferon-gamma as the lymphokine that activates human mac-
rophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:
670–689.
28. Paglia, P., E. Medina, I. Arioli, C. A. Guzman, and M. P. Colombo. 1998.
Gene transfer in dendritic cells, induced by oral DNA vaccination with
Salmonella typhimurium, results in protective immunity against a murine
fibrosarcoma. Blood 92:3172–3176.
29. Poirier, T. P., M. A. Kehoe, and E. H. Beachey. 1988. Protective immunity
evoked by oral administration of attenuated aroA Salmonella typhimurium
expressing cloned streptococcal M protein. J. Exp. Med. 168:25–32.
30. Presti, R. M., J. L. Pollock, A. J. Dal-Canto, A. K. O’Guin, and H. W. Virgin.
1998. Interferon gamma regulates acute and latent murine cytomegalovirus
infection and chronic disease of the great vessels. J. Exp. Med. 188:577–588.
31. Rawlinson, W. D., H. E. Farrell, and B. G. Barrell. 1996. Analysis of the
complete DNA sequence of murine cytomegalovirus. J. Virol. 70:8833–8849.
32. Reddehase, M. J., F. Weiland, K. Munch, S. Jonjic, A. Luske, and U. H.
Koszinowski. 1985. Interstitial murine cytomegalovirus pneumonia after ir-
radiation: characterization of cells that limit viral replication during estab-
lished infection of the lungs. J. Virol. 55:264–273.
33. Richter-Dahlfors, A., A. M. Buchan, and B. B. Finlay. 1997. Murine salmo-
nellosis studied by confocal microscopy: Salmonella typhimurium resides
intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes
in vivo. J. Exp. Med. 186:569–580.
34. Smith, G. R., S. M. Kunes, D. W. Schultz, A. Taylor, and K. L. Triman. 1981.
Structure of chi hotspots of generalized recombination. Cell 24:429–436.
35. Suter, M., A. M. Lew, P. Grob, G. J. Adema, M. Ackermann, K. Shortman,
and C. Fraefel. 1999. BAC-VAC, a novel generation of (DNA) vaccines: a
bacterial artificial chromosome (BAC) containing a replication-competent,
packaging-defective virus genome induces protective immunity against her-
pes simplex virus 1. Proc. Natl. Acad. Sci. USA 96:12697–12702.
36. Tischer, B. K., D. Schumacher, M. Beer, J. Beyer, J. P. Teifke, K. Oster-
rieder, K. Wink, V. Zelnik, F. Fehler, and N. Osterrieder. 2002. A DNA
vaccine containing an infectious Marek’s disease virus genome can confer
protection against tumorigenic Marek’s disease in chickens. J. Gen. Virol.
83:2367–2376.
37. Wagner, M., S. Jonjic, U. H. Koszinowski, and M. Messerle. 1999. Syst.
excision of vector sequences from the BAC-cloned herpesvirus genome
during virus reconstitution. J. Virol. 73:7056–7060.
38. Welsh, R. M., J. O. Brubaker, M. Vargas-Cortes, and C. L. O’Donnell. 1991.
Natural killer (NK) cell response to virus infections in mice with severe
combined immunodeficiency. The stimulation of NK cells and the NK cell-
dependent control of virus infections occur independently of T and B cell
function. J. Exp. Med. 173:1053–1063.
39. Whitley, R. J., and B. Roizman. 2002. Herpes simplex viruses: is a vaccine
tenable? J. Clin. Investig. 110:145–151.
40. Will, H., R. Cattaneo, G. Darai, F. Deinhardt, H. Schellekens, and H.
Schaller. 1985. Infectious hepatitis B virus from cloned DNA of known
nucleotide sequence. Proc. Natl. Acad. Sci. USA 82:891–895.
41. Will, H., R. Cattaneo, H. G. Koch, G. Darai, H. Schaller, H. Schellekens,
P. M. van Eerd, and F. Deinhardt. 1982. Cloned HBV DNA causes hepatitis
in chimpanzees. Nature 299:740–742.
42. Ye, M., C. S. Morello, and D. H. Spector. 2002. Strong CD8 T-cell responses
following coimmunization with plasmids expressing the dominant pp89 and
subdominant M84 antigens of murine cytomegalovirus correlate with long-
term protection against subsequent viral challenge. J. Virol. 76:2100–2112.
VOL. 77, 2003 RECONSTITUTION OF VIRUSES FROM BACs IN VIVO 8255