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JOURNAL OF VIROLOGY, Aug. 2003, p. 8249–8255 0022-538X/03/$08.00ϩ0 DOI: 10.1128/JVI.77.15.8249–8255.2003Copyright 2003, American Society for Microbiology. All Rights Reserved.
Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus 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, ¨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 ens (36) or adsorbed to gold particles and injected into mice vectors for the oral delivery of foreign proteins (29) or DNA (11, 13, 23). Oral vaccination procedures do not require edu- For this study, we decided to test whether the infection of cated personnel, and lyophilized bacterial preparations can be animals with bacteria could lead to reconstitution of a replica- kept and distributed at room temperature, without the costly tion competent virus from a bacterial vector in vivo and and difficult necessity of keeping the preparations cold. More- whether this could lead to protective immunity.
over, oral vaccination can be performed simultaneously on The cytomegaloviruses (CMVs), ubiquitous members of the large numbers of subjects, which should make it an efficient betaherpesvirus subgroup, are marked by strict species specificity, tropism for hematopoietic tissue and secretory glands, and slow Live antiviral vaccines are generally considered more effi- replication. The infection of mice with murine CMV (MCMV) cient than subunit or inactivated vaccines because they are shares many aspects with human CMV infection and thus serves more likely to induce a broad range of immune responses to as a biological model for experimental vaccine developments (16, the expressed gene products and provide a better protection.
22, 26, 42). With double-stranded DNA genomes of 120 to 230 Furthermore, since these vaccines replicate in the recipient, kbp and up to 220 genes, herpesviruses have some of the largest the immunity they confer should be long lasting (39). There- genomes of viruses that infect mammals (25). Many herpesviral fore, a bacterial vaccination vector delivering an attenuated, genes are not essential for viral replication and could be ex- yet infectious virus may present the basis for efficient vaccines changed for genes from unrelated species to generate recombi- that are easy to store, distribute, and administer.
nant viruses. Therefore, the deletion of viral genes that are not Herpesviruses are important pathogens for humans and live- essential for the vaccination success and the insertion of genes stock. We have previously shown that the large genomes of from other infectious agents are attractive concepts for the de- herpesviruses (up to 230 kb) can be cloned as bacterial artifi- velopment of live recombinant vaccines. For this purpose, we cial chromosomes (BACs) into Escherichia coli (1, 2, 24). Oth- introduced a gene from another virus into the MCMV genome ers have demonstrated that BAC DNA encoding the genome and determined the presence of the gene product in murine sera.
of a herpesvirus can induce specific protective immunity (35, Here we show that infectious MCMV can be reconstituted in vivo 36). However, in these experiments, the BAC DNA was iso- directly from bacteria that carry their genomes. We also show that lated from bacteria by column chromatography and diluted in viral reconstitution leads to protective immunity and to strong phosphate-buffered saline (PBS) prior to injection into chick- expression of additional transgenes inserted into the viral ge-nome.
MATERIALS AND METHODS
* Corresponding author. Mailing address: Max von Pettenkofer In- stitute, 80446 Munich, Germany. Phone: 49 89 5160 5203. Fax: 49 89 Plasmids and bacterial strains. Plasmid pRep4-HBs was generated by insert-
5160 5227. E-mail: [email protected].
ing the gene of the hepatitis B virus surface antigen (HBsAg) into pRep4 TABLE 1. List of modified bacterial strains used in this study S. enterica serovar Typhimurium LT2, SA1970 S. enterica serovar Typhimurium LT2, TT521 S. enterica serovar Typhimurium LT2, TT9081 (Invitrogen) as described (3). To obtain MCMV-HBs, the HBs gene bracketed MCMV-infected MEFs as the antigen source, as previously described (20). In by a Rous sarcoma virus promoter and a simian virus 40 polyadenylation signal brief, microtiter plates were coated with lysates of either uninfected or MCMV- was excised from pRep4-HBs and inserted into the ie2 gene locus of the cloned infected MEFs. Serially diluted sera were applied in parallel to both kinds of full-length MCMV BAC, pSM3fr (37), as previously described (3). Plasmid plates, which was followed by wash steps and an incubation with peroxidase- pGB2⍀inv-hly (17) was kindly provided by C. Grillot-Courvalin (Unite´ des conjugated anti-mouse antibodies. Signals obtained from the plates coated with ´riens, Institut Pasteur, Paris, France).
uninfected MEF lysates were treated as unspecific background signal and sub- Plasmid and BAC DNA was introduced by electroporation into the standard tracted from the signals obtained from the plates coated with lysates of MCMV- E. coli BAC host, DH10B [genotype: FϪ mcrA ⌬(mrr-hsdRMS-mcrBC) infected MEFs. The protective effect of the anti-MCMV immunization was ␾80dlacZ⌬M15 ⌬lacX74 deoR recA1 araD139 ⌬(ara leu)7697 galU galK rpsL tested by challenge with lethal doses of SGD MCMV. Mice received i.p. injec- endA1 nupG]. MCMV-HBs was transferred by conjugation from DH10B to tions with 2 50% lethal doses (LD50) of SGD MCMV (i.e., 5 ϫ 104 PFU for 129 serovar Typhimurium LT2 strains SA1970 (metA22 trpC2 hisF1009 rpsL120 xylR1 IFN-␥R0/0 mice) on dpi 28. Animals were checked daily for survival for 2 weeks recA1 srl-202::Tn10), TT521 (recA1 rpsL srl-202::Tn10), and TT9081 [his-644(del: following the challenge. Survivors were monitored for an additional 3 months.
OGDCBHAF) srl-202::Tn10 recA1]. The serovar Typhimurium strains were ob- Each experiment was performed at least twice.
tained from K. E. Sanderson (Salmonella Genetic Stock Centre, University ofCalgary, 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.
Virus reconstitution in vivo by serovar Typhimurium carry-
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 ing the MCMV BAC. In our previous studies, the DH10B
mice were used. Stocks of tissue culture and salivary gland-derived (SGD) strain of E. coli served as the host for the MCMV BAC. As MCMV, derived from MW97.01 (37), were prepared as previously described (4).
DH10B is not commonly used as vector for DNA immuniza- Virus reconstitution. Fresh bacterial cultures were grown overnight in Luria-
tion, we introduced an MCMV BAC into recombinase A-de- Bertani medium supplied with antibiotics for selective growth. DNA transfer ficient (recA mutant) laboratory strains of Salmonella enterica from bacteria to mammalian cells was performed as previously described (5).
Two microliters of bacterial culture was used to inoculate NIH 3T3 fibroblasts serovar Typhimurium shown in Table 1. recA mutant bacterial grown in 96-well dishes without antibiotics. This corresponded to approximately strains were selected to ensure the stability of repetitive se- 300 bacteria per fibroblast. The cell culture dishes were spun for 5 min at 1,000 quences present in herpesvirus genomes (31). The rationale for ϫ g and incubated for 2 h at 37°C. The cell culture medium was subsequently the usage of serovar Typhimurium was their natural ability to replaced with fresh medium supplemented with ampicillin and gentamicin (100␮g/ml each). For infection of mice, bacterial cultures were centrifuged at 2,000 invade mammalian cells. Therefore, they should represent a ϫ g for 8 min at 4°C and serially diluted in cold sterile PBS, at concentrations better vehicle for DNA transfer in vivo than E. coli. The clones ranging from 2 ϫ 109 to 2 ϫ 106 CFU/ml (bacterial stocks were titrated in that received the MCMV BAC were selected by resistance to parallel on blood agar plates). Five hundred microliters of diluted bacteria chloramphenicol and tetracycline. To confirm that the clones (amounts between 106 to 109 CFU) were injected intraperitoneally (i.p.) into in question contained the complete MCMV BAC, BAC DNA each mouse. Lungs were obtained from mice infected with serovar Typhimuriumon 28th day postinfection (dpi) and tested for MCMV reconstitution by plaque was extracted and introduced into fibroblasts to give rise to assay on murine embryonic fibroblasts (MEFs) as previously described (32). The infectious virus as previously described (6). To check whether mice infected with E. coli were immunosuppressed with anti-CD4 and anti-CD8 bacteria could transfer viral DNA directly into mammalian antibodies (9) (1 mg each) on the 6th and 13th dpi. Mice were additionally cells in cell culture, we inoculated NIH 3T3 cells with WB241 immunosuppressed by intramuscular (i.m.) injection of 6.25 ␮g of hydrocortisoneon alternate days from day 13 postinfection onwards. Blood samples were col- bacteria. Five days after inoculation we observed the formation lected from tail veins of infected mice on days 12 and 20 postinfection. HBsAg of characteristic viral plaques, indicating direct reconstitution was detected in murine serum by use of a commercial microtiter enzyme-linked immunosorbent assay (ELISA) kit, containing wells coated with anti-HBs anti- recA mutant bacteria are attenuated in vivo because they bodies (catalog no. 931801; Ortho Diagnostic Systems) according to manufac-turer’s instructions. Salivary glands, lungs, and spleens from individual mice were cannot repair the DNA damage caused by reactive oxygen collected on dpi 21 and tested for MCMV reconstitution as previously described species (ROS) in endolysosomal compartments of professional (32). Each experiment was performed at least twice.
phagocytes (7, 8). ROS are strongly induced in macrophages by Immunization and challenge procedures. Overnight bacterial cultures were
IFN-␥ (15, 27). As serovar Typhimurium has a preference to centrifuged and resuspended in cold sterile PBS at final concentrations of 5 ϫ infect macrophages in vivo (14, 33), this could lead to damage 109 CFU/ml. One hundred ␮l of bacterial suspension (5 ϫ 108 CFU) was injectedeither i.m. into gluteal muscles or subcutaneously (s.c.) into the soft tissue of the of the viral DNA before the virus would initiate its replication.
dorsum. For i.p. injection bacteria were diluted 1:10 in sterile PBS and injected In IFN-␥R0/0 mice (18), ROS production in macrophages is in a volume of 500 ␮l (ϳ108 CFU). Positive-control mice were infected with 105 not induced (21). Thus, we assumed that the use of IFN-␥R0/0 PFU of tissue culture-grown BAC-derived MCMV. No immunosuppressive pro- mice would increase the chance to test the possibility of her- cedure was applied. Blood samples were collected from the tail vein, and serafrom individual mice were serially diluted in PBS. The immunization against pesvirus reconstitution from bacteria in vivo.
MCMV and antibody production efficiency was tested by indirect ELISA using Serovar Typhimurium strain WB241 (Table 1) was grown RECONSTITUTION OF VIRUSES FROM BACs IN VIVO FIG. 2. 129 SvEv IFN-␥R0/0 mice were i.p. infected with the indi- cated doses of WB240 or with 5 ϫ 105 PFU of MCMV. Mice weresacrificed on dpi 28, and lung homogenates were assayed on MEFs forinfectious MCMV titers. Circles represent virus titers in the organs ofindividual mice. D.L., limit of detection.
not. The very same mice were challenged on dpi 28 with 105PFU of SGD MCMV (4 LD ) and monitored for survival (Fig. 1B). All mice infected with 109 CFU and 83% of miceinfected with 108 CFU of bacteria, as well as all of the virus-infected controls, survived the challenge. Surprisingly, 83% ofthe 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 theuninfected control mice died by day 9 following challenge.
Therefore, the infection of mice with serovar Typhimuriumcarrying MCMV BACs, induced specific antibody responseand protective immunity against MCMV.
In order to assess whether the immunization was caused by the reconstitution of the infectious viral process or merely dueto 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 FIG. 1. 129SvEv IFN-␥R0/0 mice were infected with the indicated WB241 contained infectious virus in their organs as seen by CFU of WB240 (six mice per group). Positive-control mice were in- plaque assay on MEFs (Fig. 2). The specificity of these plaques fected with 5 ϫ 105 PFU of wild-type MCMV, and negative controls for MCMV was confirmed by staining with a monoclonal an- were mock infected. (A) Sera were collected at dpi 28, serially diluted tibody against the pp89 protein, the product of the IE1 MCMV up to 1:1,024, and assayed for anti-MCMV antibodies by ELISA.
gene (not shown). In mice infected with 107 CFU or less we Median and quartile absorbance values at 492 nm are displayed.
Pooled sera from a group of mice that received 5 ϫ 105 PFU of could not detect virus. Therefore, the infection with 108 CFU MCMV were used as positive controls. (B) On the same day, mice of serovar Typhimurium carrying the MCMV BAC leads to reconstitution of live virus in vivo. Notably, the same number monitored for survival. Survival curves for the first 10 days following of bacteria that were needed for specific antibody formation challenge are shown. Exclusively for presentation purposes, the sur-vival curves are presented in two separate graphs. Error bars, quartiles.
was needed to initiate an active viral infection process. There-fore, the ability to mount a specific antibody response couldserve as an indirect indicator for virus reconstitution in vivo.
overnight as described in Materials and Methods and injected Next, we tested whether bacteria carrying MCMV BACs i.p. into IFN-␥R0/0 mice at doses indicated in the Fig. 1. Con- could induce the immunization of immunocompetent mice.
trol mice were infected with 5 ϫ 105 PFU of BAC-derived We infected groups of BALB/c mice with 108 CFU of WB241 MCMV (37). Mock-infected mice were used as negative con- bacteria and looked for infectious virus in lungs and spleen and trols. The ability to mount a specific humoral response to for specific antibody titers to MCMV in sera at 28 dpi. As MCMV was taken as an indication of virus protein expression.
expected, mice did not show elevated antibody titers (data not Mice infected s.c. with 108 or 109 CFU of bacteria displayed shown), nor could infectious virus be isolated from their or- elevated titers of anti-MCMV antibodies in their sera on the gans. Therefore, we concluded that the serovar Typhimurium 28th dpi, whereas mice infected with 107 CFU or less showed vectors used here could not effectively induce the reconstitu- no anti-MCMV antibodies (Fig. 1A). Therefore, the infection tion of the viral infectious process in IFN-␥Rϩ/ϩ mice.
with 108 CFU of bacteria or more induced humoral immunity Virus reconstitution in vivo by E. coli carrying MCMV BAC.
against MCMV, whereas infection with 107 CFU or less did In previous experiments, we have shown that the cloned MCMV genome can be transferred directly from its E. coli hostto cultured fibroblasts to initiate the production of infectiousvirus in these cells (5). This requires the introduction of theplasmid pGB2⍀inv-hly (17), which directs the expression ofthe invasin gene of Yersinia pseudotuberculosis and the hemo-lysin O gene from Listeria monocytogenes in E. coli, and thepresence 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 MCMVgenome to somatic cells of a mouse and to initiate the infec-tious cycle in vivo. The rationale for this was that the in vivoinfection of nonphagocytic cells like fibroblasts, which do notexpress ROS, would give an opportunity to recA mutant bac-terial vectors to reconstitute viral infection in IFN-␥Rϩ/ϩ an-imals.
Moreover, we wanted to analyze the efficiency of the expres- sion of a transgene from a recombinant herpesvirus. Thus, weused 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 inserum of a secreted virus-encoded protein such as the HBsAg FIG. 3. (A) BALB/c or (B) 129 SvEv IFN-␥RϪ/Ϫ mice were i.p.
reflect MCMV productivity in vivo and thus can be used to infected with 108 CFU of WB232 (E) or WB233 (F). T lymphocyteswere depleted on dpi 6 and 13, and hydrocortisone was applied on monitor the course of infection over time. Bacteria containing alternate days from dpi 13 onwards. Mice were sacrificed on dpi 21, a high-copy-number plasmid with the identical eukaryotic HBs and organ homogenates were assayed on MEFs for infectious MCMV expression cassette were used to control for possible HBsAg titers. Circles represent virus titer in organs of individual mice. D.L., expression in the absence of viral replication and to compare virus reconstitution by bacteria with plasmid DNA delivery bybacteria.
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 pleted before infection with bacteria. Mice lacking both B and bacteria (Table 1). T lymphocytes were depleted on dpi 6 and T cells cannot control MCMV infection (38), whereas IFN- 13, and hydrocortisone was applied on alternate days from the ␥R0/0 mice can (30). Thus, this system should allow the detec- 13th dpi onwards in order to suppress the immune response tion of very low initial amounts of reconstituted virus. How- and to increase the chances of establishing a productive viral ever, at dpi 21 we could not detect any virus out of organs of infection. Lungs, salivary glands, and spleens were collected on ␮MT/␮MT mice infected with 108 CFU of Eco/M/inv E. coli the 21st dpi, and organ homogenates were tested by plaque (data not shown). Therefore, mice very sensitive to MCMV, assay on fibroblasts for the presence of infectious MCMV.
yet capable of IFN-␥ signaling, could not reconstitute infec- Only one out of eight mice infected with Eco/M/inv showed tious virus. This finding argues for an important role of the reconstitution of infectious virus in the lungs and spleen (Fig.
IFN-␥ signaling pathway during the early phase of virus recon- 3A). In repeated experiments in which we used BALB/c or C57BL/6 mice, we did not detect viral reconstitution (data not Sera were collected from the very same IFN-␥R0/0 mice whose organs were assayed for virus titer and shown in Fig. 3, In a separate experiment, we infected groups of IFN-␥R0/0 on dpi 12 and 20 and assayed for the product of the introduced mice with graded amounts of either Eco/M/inv or Eco/P/inv viral transgene. The presence of the viral protein HBsAg was using the same infection and immune suppression protocol. All determined by direct ELISA. At day 12, no HBsAg could be mice that received 108 CFU of Eco/M/inv were positive for detected in any of the groups (Fig. 4). At day 20, four out of MCMV plaques in their lungs and salivary glands (Fig. 3B).
five mice infected with Eco/M/inv but none of the mice in- The mice infected with 107 CFU of Eco/M/inv were negative fected with Eco/P/inv showed detectable amounts of HBsAg in for MCMV as confirmed by plaque assay. As expected, Eco/ their sera (Fig. 4). Notably, the amount of HBsAg correlated P/inv-infected mice showed no evidence for MCMV infection.
with the viral yield in the salivary gland in individual mice, Therefore, the infection with E. coli carrying the MCMV BAC which was determined in parallel (r ϭ 0.91). Therefore, the leads to in vivo reconstitution of infectious virus in IFN-␥R0/0 MCMV-HBs reconstitution is associated with a considerable mice but not in IFN-␥Rϩ/ϩ mice. There was no significant HBsAg expression in the sera of animals. Animals that re- difference in the number of bacteria required to launch the ceived the HBs gene alone as a multicopy plasmid did not infectious process either by serovar Typhimurium or by E. coli.
produce measurable HBsAg concentrations. The absence of IFN-␥R0/0 mice are more sensitive to virus infection than HBsAg at dpi 12 indicates that HBsAg was expressed to de- normal mice. To clarify whether the antiviral activity of IFN-␥ tectable levels only upon virus reconstitution. In fact, no infec- alone could explain the lack of reconstitution in IFN-␥Rϩ/ϩ tious virus was detected when mice infected under identical mice, we tried to reconstitute infectious virus from bacteria conditions with Eco/M/inv were sacrificed at dpi 14 (data not using B-cell-deficient ␮MT/␮MT mice, which were T cell de- RECONSTITUTION OF VIRUSES FROM BACs IN VIVO 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-MCMVantibodies by ELISA. Arithmetic means and standard deviations ofabsorbance values at 492 nm for the 1:64 dilution step are shown. Serafrom mice infected with 105 PFU of MCMV were obtained at dpi 70and used as positive controls (Œ). Sera from uninfected mice (‚) wereused as negative controls.
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 dpi6 and 13, and hydrocortisone was applied on alternate days from day13 onwards. Sera were collected at days 12 and 20 and assayed for the MCMV. Groups of 10 IFN-␥R0/0 mice were immunized by presence of HBsAg by direct ELISA. At day 12, no HBsAg could be inoculation with 109 CFU of either Eco/M or Eco/M/inv. Con- detected in any of the groups. At day 20, four out of five mice infected trol mice were inoculated with Eco/P/inv. Half of each group with Eco/M/inv and none of the mice infected with Eco/P/inv showed received the bacteria i.m. and the other half received them s.c.
detectable amounts of HBs in their sera.
On the 28th dpi, seroconversion occurred in most of the Eco/Mor Eco/M/inv infected mice (8 of 10 in each group), but in none Virus reconstitution in vivo by bacteria carrying the MCMV
of the controls, as detected by ELISA (data not shown). On the BAC is independent of bacterial invasion genes. Virus recon-
stitution from E. coli was restricted to IFN-␥R0/0 mice, as it was and monitored for survival. All five mice infected i.m. with the case with serovar Typhimurium vectors. Therefore, we Eco/M/inv and four of five of mice infected s.c. with Eco/M/inv, assumed that this process was occurring in professional phago- as well as all animals infected with Eco/M (10 of 10) survived cyte cells. The additional genes supplied for active bacterial the challenge (Fig. 6). All mice from the control groups died by invasion and virus reconstitution in vitro might therefore be dpi 8 or 9. The results indicate that mice immunized by bac- dispensable for virus reconstitution in vivo. In order to test this teria carrying the MCMV BAC were protected against a chal- hypothesis, bacteria carrying the MCMV BAC, but not the pGB2⍀inv-hly plasmid, were tested for DNA transfer and viralreconstitution.
DISCUSSION
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 The cloning of herpesvirus genomes as bacterial artificial MCMV specific antibody production by IFN-␥R0/0 mice over chromosomes allows fast and accurate mutagenesis procedures time. We observed that five of five of the mice infected with of basically any viral gene in the prokaryotic E. coli host (6, 24).
Eco/M and four of five mice infected with Eco/M/inv bacteria The biological properties of the viral mutants are analyzed mounted an antibody response (Fig. 5). The MCMV-specific upon the reconstitution of the virus in eukaryotic cells. The antibody titer was only slightly elevated over the values from application of random transposon mutagenesis procedures led control mice at dpi 14 but rose by dpi 28 and at dpi 42 was to the establishment of libraries of mutant genomes (6). In comparable to titers obtained from MCMV infected mice at order to test the biological properties of random mutant librar- dpi 70. Control mice infected with Eco/P/inv did not mount a ies, virus reconstitution was achieved by direct inoculation of specific immune response. Eco/M bacteria, which lack the host cells in vitro by invasive bacteria carrying BACs (5).
pGB2⍀inv-hly invasion plasmid, induced a strong MCMV- Whether these bacteria would also allow virus reconstitution specific antibody response. The presence of pGB2⍀inv-hly did in vivo was the next logical question. Direct in vivo reconsti- not enhance the immunization rate. This was in accordance tution of a virus infection from viral DNA is not a new concept.
with the observation that infectious MCMV could be isolated However, successful approaches had been restricted so far to from organs of Eco/M-infected mice (not shown). Taken to- viruses carrying much smaller genomes (around 10 kb), and to gether, these data indicate that successful virus reconstitution the direct injection of DNA (12, 40, 41). Here we show with the in vivo was independent of the genes encoded by pGB2⍀inv- example of the 230-kb genome of MCMV that there should be, hly, whereas the pGB2⍀inv-hly plasmid was required for BAC in principle, no viral genome size limit for virus reconstitution transfer and virus reconstitution in cell culture.
Next, we tested whether the immunization procedure by E. Suter et al. have shown that injection of herpesvirus BAC coli was also protective against a challenge with a lethal dose of DNA can lead to protective immunity in the absence of viral lethal challenge with SGD MCMV. Low-frequency DNAtransfer events leading to expression of viral genes togetherwith an abortive infection could explain this observation. Inthat 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 aplasmid encoding for an immunodominant viral gene has al-ready been shown to induce protective T-cell immunity in theabsence of an antibody response (16). Moreover, it was dem-onstrated that an additional injection of formalin inactivatedviruses 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 invadecells, as opposed to the situation in vitro. This suggested thatthe bacteria were not actively entering host cells. Therefore,the probable target cells, into which the viral genomes enteredand started the viral replication, could be phagocytic cells thatactively took up bacteria. The half-life of granulocytes is only afew hours (19), which is too short to allow MCMV reconstitu-tion and replication. Thus, the properties of professionalphagocytic cells, i.e., macrophages and dendritic cells, probably FIG. 6. 129 SvEv IFN-␥R0/0 mice were i.m. or s.c. infected with 109 dictate the success of the transfer, rather than the invasive CFU of Eco/M or Eco/M/inv bacteria. Control mice were infected withEco/P/inv. At 28 dpi, mice were challenged with 2 LD properties of the bacteria. Therefore, it was not surprising that MCMV and monitored for survival. Survival curves for the first 14 days viral reconstitution could be achieved only in IFN-␥R0/0 mice that are deficient for ROS production. Professional phagocytesin these mice lack activating signals from IFN-␥ pathways (21)and therefore can eliminate intracellular pathogens only at a replication (35). Another recent report has shown that the lower rate (10, 18). This indicates that strategies for the im- inoculation of Marek’s disease virus BAC DNA diluted in PBS provement of the virus reconstitution efficiency in vivo should into chicken could lead to protective immunity (36). Our data focus on the survival of bacteria in phagocytic cells.
show for the first time that a herpesvirus infection can be To maintain the stability of the herpesvirus genome, the launched directly from bacteria carrying the infectious virus BACs are usually propagated in recA mutant bacterial strains.
genome, omitting the need for technical preparatory work to Originally, we developed the herpesvirus BAC system for the isolate BAC DNA. We have also demonstrated that additional mutagenesis of viral genomes. Recombination-deficient (recA genes introduced into the virus are effectively expressed (3).
mutant) bacteria are preferred as hosts for the MCMV BAC Thus, the use of BAC-derived viruses has a potential as vaccine because the 21 chi sites (34) present in the MCMV genome, as and vaccine vector, and this is also the case when the viral well as repeated viral sequences within the MCMV genome (31), could potentially serve as hot spots for spontaneous re- Interestingly, no infectious virus could be isolated from or- gans before day 21 after injection of bacteria. Since the infec- The requirement for recA mutant bacterial strains for prop- tion with 105 PFU MCMV leads to detectable viral titers in agation of herpesvirus BACs at present appears to be the organs within few days, this implied that the peak of productive major obstacle for further development. Since parenteral ad- viral replication was delayed for at least 2 weeks. This finding ministration of 108 CFU of recA mutant bacteria was required was not unexpected, as we have already reported a time lag in for successful reconstitution in IFN-␥R0/0 mice, the amounts of the production of infectious virus already upon transfection of bacteria required for efficient transfer in fully immunocompe- cultured cells with MCMV BAC DNA (37). Therefore, at dpi tent mice may need to be even higher. Additionally, these 28, lung virus titers after virus reconstitution from serovar strains have not been designed for oral administration and Typhimurium are still high, whereas in mice infected with DNA delivery via mucosal routes. On the other hand, recAϩ MCMV, the virus infection was already contained by the im- strains of serovar Typhimurium have already been used as mune system. The other evidence for this delay, are the kinet- vectors for oral DNA vaccination (11, 28). Can recAϩ strains ics of anti-MCMV antibody titers. Titers were still low at 14 be used to increase transfer efficiency? We have seen that viral days postinfection, rose by dpi 28 and even more by dpi 42.
BAC DNA can be maintained in the recAϩ E. coli strain CBTS Therefore, Direct comparison of antibody titers at dpi 28 or GS500, at least for short periods of time, without losing the shows a marked difference between mice that were infected by potential to start a productive infection (5, 24). However, our bacteria or by the virus (Fig. 1A). However, at dpi 70, antibody attempts to introduce an intact MCMV BAC into recAϩ strains titers from vaccinated mice were undistinguishable from mice of serovar Typhimurium have so far not met with success.
infected with MCMV (data not shown).
Perhaps the genome repeats and the chi sequences need to be It was surprising that infection with 107 CFU of serovar deleted or modified, in order to increase the stability of the Typhimurium did not induce a detectable antibody response or viral genomes in a recAϩ host. Careful design of viral muta- production of infectious virus yet protected mice against a tions could allow the propagation of stable viral mutant ge- RECONSTITUTION OF VIRUSES FROM BACs IN VIVO nomes in recAϩ bacterial strains. If recAϩ bacterial strains 20. Jonjic, S., M. del Val, G. M. Keil, M. J. Reddehase, and U. H. Koszinowski.
could be used for oral delivery of viral DNA, this would have 1988. A nonstructural viral protein expressed by a recombinant vaccinia virus
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ACKNOWLEDGMENTS
murine cytomegalovirus (MCMV) infection by using an attenuated MCMV
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Bayerische Forschungsstiftung “Forimmun,” and Br1730/2.
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