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The Journal of Immunology, 2004, 172: 6304-6312.
Copyright © 2004 by The American Association of Immunologists

Dendritic Cells from Mice Neonatally Vaccinated with Modified Vaccinia Virus Ankara Transfer Resistance against Herpes Simplex Virus Type I to Naive One-Week-Old Mice1

Marco Franchini*, Hanspeter Hefti*, Sabine Vollstedt*, Bettina Glanzmann*, Matthias Riesen*, Mathias Ackermann*, Paul Chaplin{dagger}, Ken Shortman*,{ddagger} and Mark Suter2,*

* Institute of Virology, University of Zurich, Zurich, Switzerland; {dagger} Bavarian Nordic, Copenhagen, Denmark; and {ddagger} Walter and Eliza Hall Institute of Medical Research, Melbourne Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Modified vaccinia Ankara (MVA) is an attenuated virus. MVA induces the production of IFN and Flt3-L (FL), which results in the expansion of dendritic cells (DC) and enhanced resistance against viral infections. We report on the interplay among IFN, FL, and DC in the resistance against heterologous virus after injection of neonatal mice with MVA. The induction of serum FL was tested on day 2, and the expansion of DC was tested 1 wk after treatment with MVA. At this time point the resistance against infection with heterologous virus was also determined. After MVA treatment, serum FL was enhanced, and DC, including plasmacytoid cells in spleen, were increased in number. Mice that lacked functional IFN type I and II systems failed to increase both the concentration of FL and the number of DC. Treatment with MVA enhanced resistance against HSV-1 in wild-type animals 100-fold, but animals without a functional IFN system were not protected. Transfer of CD11c+ cells from MVA-treated mice into naive animals protected against lethal infection with HSV-1. Thus, although the increased resistance could be largely attributed to the increase in activation of IFN-producing plasmacytoid cells, this, in turn, depends on a complex interplay between the DC and T cell systems involving both FL and IFNs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neonatal mice are highly susceptible to intracellular pathogens. This may be due to poor reactivity of APCs to Ag and low number of crucial cells of the innate and specific immune systems (1, 2, 3). Some of these cell populations are strongly increased in number after application of Flt3 ligand (FL),3 a hemopoietic growth factor for progenitors of dendritic cells (DC), NK cells, and B lymphocytes in adult animals (4, 5). Adult as well as neonatal mice treated with FL have elevated numbers of DC, associated with increased resistance against infection with HSV-1 and L. monocytogenes (6, 7, 8).

Modified vaccinia Ankara (MVA), an orthopoxvirus, is an attenuated virus that was obtained after >570 serial passages of the Ankara strain of vaccinia virus on chicken fibroblast cells. Because MVA lost part of its genome, the viral replication is restricted to avian cells, and the virus is avirulent for animals (even chickens) and humans (9). This MVA strain has been used for human vaccination against smallpox in >120,000 individuals without negative effects (9). MVA does not produce soluble IFN receptors and TNF analogues (10, 11) and drives the immune system toward increased production of IFN-{alpha}{beta} (12), IL-2, and IL-12 (13). These cytokines favor the induction of NK cells, CTLs, and CD4 T cells important for the control of viral replication (14). Therefore, MVA can powerfully prime T cells of adults. Moreover, T lymphocytes of neonatal mice can also be activated by such avirulent or replication-controlled virus (15, 16, 17, 18).

Contact with pathogens induces maturation of DC and increases the efficiency of Ag presentation, leading to up-regulation of costimulatory molecules and expression of essential cytokines (19, 20, 21). Besides the control of pathogen spread, activated T cells secrete preformed FL (22, 23) necessary for the induction of increased numbers of DC (5). Two main DC subsets have been described: conventional DC (cDC) that are CD11chigh, MHC class IIhigh, CD45negative (24) and pre-DC with plasmacytoid morphology (pDC) that are CD11cint, MHC class IIlow, CD45RAhigh (25, 26, 27). After certain viral infections, pDC appear to be the first source of IFN-{alpha}{beta} (28). IFN-{alpha}{beta} (IFN type I) may coordinately activate hundreds or maybe even thousands of genes in different cells for immediate control of local viral replication (29). IFN type I is active in either an autocrine or a paracrine fashion both locally and at distant sites. Furthermore, IFN promotes the renewal and functional activation of cDC and pDC by activating the transcription factor IFN consensus sequence binding protein (IFN regulatory factor 8) in hemopoietic cells (30, 31), IL-12p40 production (32), and expression of CD8{alpha} (31, 33). Therefore, a small increase in the number of pDC or cDC may have a significant effect on the defense against viral infections.

Because MVA could induce IFN and activate T cells, we treated newborn mice with this virus and determined FL induction, DC expansion, and activation in the presence and the absence of functional IFN. MVA-induced IFN was required for both increased FL levels and elevated numbers of DC. MVA-triggered immune stimulation induced 100-fold increased resistance against the heterologous virus HSV-1 in wild-type, but not IFN-deleted mice. Therefore, IFN was necessary for the up-regulation of FL and was required to efficiently control HSV-1 replication initiated by increased numbers of endogenous or transferred DC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 and 129Sv/Ev mice and animals with gene-targeted disruptions of IFN receptor type I (A129), type II (G129), or both (AG129) and recombination-activating gene 2 (RAG) were maintained and bred under specific pathogen-free-conditions at Institute for Laboratory Animal Science, University of Zurich (Zurich, Switzerland) (34, 35).

MVA

The Bavarian Nordic strain of MVA (MVA-BN; European Collection of Animal Cell Cultures, Salisbury, Wiltshire, U.K.; no. V000083008) was used for the vaccination experiments. The virus was produced in primary chicken embryo fibroblasts in a serum-free system. It was stored at –80°C in vials containing 1 ml of virus suspension, with a titer of 5 x 108 half-maximal tissue culture infective dose (TCID50)/ml, in 10 mM Tris, and 140 mM NaCl (pH 7.4). MV-BN was tested in highly immune-deficient mice with gene-deleted IFN and RAG and proved to be safe, as no virus replication occurred, and the mice survived for >100 days.

HSV-1

HSV-1F was originally obtained from Dr. B. Roizman (University of Chicago, Chicago, IL) and propagated on Vero cells. For challenge experiments virus was purified on a sucrose density gradient, and the titer was determined according to standard procedures (36). Challenge experiments were performed 1 wk after MVA-BN application. The LD50 in 8-day-old mice was calculated as previously described (37).

IFN-{alpha}

Recombinant human IFN{alpha} B/D hybrid was a gift from M. A. Horisberger (Novartis, Basel, Switzerland) (38) and was obtained from Prof. A. Metzler (Institute of Virology, Veterinary Faculty, University of Zurich, Zurich, Switzerland).

MVA-BN treatment

Mice were immunized within 12–24 h after birth with 50 µl of viral suspension i.p. For most experiments an inoculum of 2.5 x 106 TCID50/mouse was applied. Control animals were left untreated or were treated with UV-inactivated MVA-BN or physiological saline solution (0.9% NaCl and 308 mosmol/l).

FACS analysis

FACS analysis of blood and spleen cells was performed 7 days after MVA-BN application when not otherwise specified. Blood was collected after decapitation of mice and was drained in a 1.5-ml vial containing 50 µl of PBS solution with 6% Na2EDTA. Fifty microliters of this suspension was used for each stain. Spleen cells were harvested by mechanical dissociation on a steel mesh; 106 cells were used for each stain.

The following Abs were used: PE-anti-mouse CD8{alpha} (BD PharMingen, San Diego, CA; catalogue no. 01045B), PE-anti-mouse I-A/I-E (BD PharMingen; catalogue no. 06355A), FITC-anti-mouse CD19 (BD PharMingen; catalogue no. 553785), FITC-anti-mouse CD11b (BD PharMingen; catalogue no. 553310), PE-anti-mouse CD45R/B220 (BD PharMingen; catalogue no. 553090), PE-anti-mouse CD45RA (BD PharMingen; catalogue no. 553380), PE-anti-mouse Pan-NK cells-DX5 (BD PharMingen; catalogue no. 553858), FITC-anti-mouse CD11c (BD PharMingen; catalogue no. 09704D), FITC-anti CD4 (BD PharMingen; catalogue no. 01064D), and FITC-anti-mouse NK-1.1 (BD PharMingen; catalogue no. 553164).

FL assay

Sera were stored at –20°C until analysis. The FL concentration was measured in serum using a commercial ELISA system (Quantikine M; R&D Systems, Abingdon, U.K.).

IFN type I assay

This assay was performed using commercial available reagents as previously described (39).

DC isolation and transfer

Two different procedures for DC enrichment were used, namely positive selection for CD11c+ cells using paramagnetic beads (method 1) or density selection (method 2).

Method 1. After mechanical and enzymatic disruption of the spleens, CD11c+ cells were selected using MACS and superparamagnetic microbeads conjugated to monoclonal hamster anti-mouse CD11c Abs (Miltenyi Biotech, Bergisch Gladbach, Germany). The protocol proposed by the manufacturer was used, with LS+/VS+ selection columns. After two rounds of selection, the proportion of CD11c+ cells was >80% as determined by flow cytometry; 2 x 106 of these enriched cells were transferred to naive 7-day-old animals (40). The flow-through cells obtained after the selection procedure were used as the control. This cell population contained <0.1% CD11c+ cells. Mice in one group received 2 x 106 of these flow-through cells/mouse, and animals in second group received 50 x 106 cells/mouse; the latter corresponded to the number of cells from one spleen. A third group was left untreated.

Method 2. DC were enriched by a density centrifugation procedure described previously (41, 42). This led to two cell populations, one consisting of dense cells (non-DC) and one consisting of low density cells (DC), containing 45–50% CD11c+ cells. Challenge was performed i.p. 4 h after cell transfer with 5 x 104 PFU HSV-1F.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MVA-BN induces expansion in pDC with enhanced expression of MHC class II and CD8{alpha}

Previous experiments had indicated that vaccination at birth with a replication-controlled herpes virus (DISC HSV-1) induced T cell-based, viral-specific protection (18). In these experiments protection against lethal infection was observed as early as 7 days after vaccination. Protection at such a time point could be due to the innate immune system activated by the replication-restricted vaccine virus. Therefore, neonates were treated with MVA-BN to analyze the effect on the innate immune system as well as on the protection against challenge with the heterologous virus HSV-1.

At birth, groups of mice were treated with 2.5 x 107 TCID50 of MVA-BN or were given saline as a control. This viral dose was chosen because it was similar to that of DISC HSV-1 used for vaccination (18). One week later, the composition of immunologically relevant cell populations in blood and spleen was analyzed (Table I). In blood, CD8+ lymphocyte populations as well as the number of NK cells were increased. The number of CD11c+ cells was 2–3 times higher than that in controls, and the number of B cells was significantly decreased. In spleen, the absolute number of cells did not differ between MVA-BN-treated animals and controls (p < 0.105). In contrast to the blood, the spleen of MVA-BN-treated animals contained more CD4+ T lymphocytes than controls, but the number of NK cells was not increased. Similar to blood, the relative number of CD8+ lymphocytes was increased, and the number of B cells was decreased (Table I).


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  		Table I. Cell populations in blood and spleen of 7-day-old C57BL/6 mice treated at birth with 2.5 x 107 TCID50 of MVA-BNa

 
One week after viral treatment, the percentage of CD11c+ cells was higher than that in controls. A difference in the percentage of CD11c+ cells was first noted on day 5 after MVA-BN application when the number of these cells in the spleen of untreated controls was 3.6% and that in MVA-BN treated mice was 4.8%. Elevated numbers of CD11c+ cells were detectable for 2 wk after a single viral treatment at birth. Boosting the neonatally primed mice on day 7 with the same dose of virus further increased the number of these cells at 2 wk of age. UV-irradiated MVA-BN (UV-MVA-BN) injected into neonatal mice had no effect on the composition of any cell population analyzed (data not shown).

Treatment with a high dose of 2.5 x 107 TCID50 of MVA-BN at birth significantly changed the cellular composition of blood and spleen. To determine the virus dose needed for maximal induction of CD11c+ cells, we injected graded doses of MVA-BN. Maximal numbers of CD11c+ cells were detected after treatment with 2.5 x 106 TCID50 of virus (Table II). Doses below and above this were less effective. Therefore, 2.5 x 106 TCID50 of MVA-BN was selected for additional experiments.


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  		Table II. CD11c+ cells in spleen of 7-day-old C57BL/6 mice treated at birth with different doses of MVA-BN

 
Spleen cells from mice treated at birth with MVA-BN or UV-MVA-BN and from untreated controls were analyzed at 7 days for the expression of CD11c, CD45RA, and CD8{alpha}+ (Table III). After viral treatment, CD11c+, CD11c+/CD45+, and CD11c+/CD8{alpha}+ were increased in number compared with those in UV-MVA-BN-treated mice or controls. The proportion of these cells in spleen further increased when the mice were treated a second time at 1 wk of age and analyzed 2 wk after birth (data not shown).


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  		Table III. Subpopulations of CD11c+ cells in spleens from 7-day-old C57BL/6 mice treated at birth with 2.5 x 106 TCID50 of MVA-BN or UV-inactivated MVA-BN (UV-MVA-BN)

 
For a more detailed analysis of DC, spleen cells from MVA-BN-treated, UV-MVA-BN-treated, and control mice were enriched by density fractionation (see Materials and Methods), and the cell population was analyzed by flow cytometry. CD11chigh cells that did not express CD45RA or CD45R were considered cDC, whereas CD11clow cells that also expressed CD45RA or CD45R were considered pDC (25, 26, 43). The percentage of pDC was significantly higher in MVA-BN-treated mice than in the other two groups (Fig. 1A and Table III). The proportion of cDC was unchanged.



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  		FIGURE 1. Comparison of the surface phenotype of enriched DC from spleen cells of MVA-BN-treated, UV-MVA-BN-treated, and untreated control animals. A, Groups of C57BL/6 mice were treated with 2.5 x 106 TCID50 of MVA-BN or UV-inactivated virus (UV-MVA-BN) on day 0 or were left untreated (control). On day 7 single-cell suspensions from spleen were enriched for DC (see Materials and Methods) and stained with CD11c and CD45RA. Gated CD11cint/CD45RA+ (pDC) and CD11chigh/CD45RA– (cDC) are shown. B, Gated pDC and cDC (A) were further analyzed for the expression of MHC class II and CD8{alpha}, and the relative number of positive cells for the marker is indicated. The dotted line gives the background of equal cell numbers lacking the mAb of interest.

 
Treatment of mice with MVA-BN, but not UV-treated virus, lead to an increased expression of MHC class II and CD8{alpha} on pDC, but not on cDC (Table III and Fig. 1B). Thus, treatment with MVA-BN increased the number of pDC and their expression of MHC II and CD8{alpha}.

MVA-BN treated mice have an increased concentration of FL in serum that depends on intact IFN

As FL is a hemopoietic growth factor for DC lineage cells, we measured its concentration in serum after treatment of mice with MVA-BN at birth. An increase in FL concentration within 24 h was observed (Fig. 2A). Maximal serum FL concentrations were detected within 48 h of viral treatment, and elevated levels were still present on day 7. To confirm and extend this observation, groups of 10-day-old mice were also treated with MVA-BN, and serum FL was determined at different time points (Fig. 2B). The kinetics of FL production were similar to those in newborns (Fig. 2A). In 10-day-old mice FL serum levels peaked on day 2, but the decline was faster than in newborns (Fig. 2). Therefore, in both age groups a maximal relative increase in FL was observed 48 h after MVA-BN treatment.



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  		FIGURE 2. MVA-BN induces serum FL in C57BL/6 mice. The FL concentrations in the sera of C57BL/6 mice injected with 2.5 x 107 TCID50 of MVA-BN at 1 (A; •) or 10 (B; {blacksquare}) days of age were compared with data from age-matched untreated controls ({circ} and {square}). The p values were determined by Mann-Whitney U test: *, p < 0.05; * *, p < 0.01.

 
A potentially important source of FL is activated lymphocytes (22). Therefore, we tested whether MVA-BN was able to induce FL in the absence of functional T and B lymphocytes. For technical reasons, 10-day-old mice were used for these experiments, and the concentration of serum FL was determined at a single time point 2 days after viral treatment (Fig. 3). FL up-regulation in 12-day-old RAG mice treated with MV-BN (treated, 921 ± 31 pg/ml; controls, 475 ± 7 pg/ml) was comparable to that inwildtype mice (treated, 969 ± 70 pg/ml; controls, 518 ± 32 pg/ml; Fig. 3). Thus, in the absence of functional RAG, MVA-BN stimulated an increased production of FL.



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  		FIGURE 3. MVA-BN does not induce FL in mice without functional IFN. C57BL/6, AG129 mice without a functional IFN system and RAG–/– mice were injected with 2.5 x 107 TCID50 of MVA-BN at 10 days of age, and the serum FL concentration was measured 2 days later.

 
To test the role of IFNs in FL regulation, we injected MVA-BN into mice with either one or both IFN systems inactivated. In general, mice with a disrupted IFN type I or II system reacted to MVA-BN application with a slight, but significant, increase in FL concentration (data not shown). In contrast, AG129 mice, without an intact IFN system, did not respond with elevated production of FL after administration of virus (Fig. 3).

MVA-BN treatment at birth induces increased resistance against HSV-1 in 8-day-old mice

We had previously observed that administration of FL during the first 7 days of life induced a state of increased resistance against HSV-1 (8). As MVA-BN induced FL up-regulation, we tested MVA-BN-treated mice for increased resistance against HSV-1 (Table IV). For the challenge experiments, mice were treated at birth with MVA-BN (2.5 x 106 TCID50/mouse). On day 8, a challenge with either 103 or 105 PFU of HSV-1 was given. After MVA-BN treatment, 65% of the mice survived a viral dose that killed 100% of the control mice (100 LD50), and 90% survived a dose that killed 45.5% of the controls (1 LD50). In additional experiments, groups of mice treated with UV-inactivated MVA-BN were infected with HSV-1. This treatment had no effect (Fig. 4). Therefore, mice treated with live, but not UV-inactivated, MVA-BN acquired increased resistance against HSV-1.


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  		Table IV. Challenge of 8-day-old C57BL/6 mice with virulent HSV-1 after treatment with 2.5 x 106 TCID50 of MVA-BN at birth

 


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  		FIGURE 4. MVA-BN induces resistance against infection with HSV-1. Groups of mice were treated at birth with 2.5 x 106 TCID50 of MVA-BN (solid line) or with an equal dose of UV-inactivated MVA-BN (dotted line) or were left as untreated controls (dashed line) and infected with HSV-1 on day 8. The data from the surviving animals after challenge are shown.

 
It was possible that increased resistance to HSV-1 was due to efficient priming of cross-reacting CTLs induced by MVA-BN (44). Therefore, we tested resistance induction in 8-day-old RAG mice vaccinated with 2.5 x 106 TCID50 of MVA-BN. Based on earlier experiments, we used a challenge dose of 5 x 102 PFU of HSV-1 (8). Both treated and control mice died after HSV-1 challenge, but the onset of the disease was significantly delayed in MVA-BN-treated mice. Indeed, all controls died between days 4 and 6 after the challenge, whereas the MVA-BN-treated littermates died between days 6 and 13. Thus, MVA-BN-treated RAG mice have increased innate protection compared with controls (Fig. 5), but specific immunity present in C57BL/6 mice appears to play a significant role in protection (Fig. 4 and Table IV).



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  		FIGURE 5. MVA-BN induces increased innate resistance against infection with HSV-1 in RAG–/– mice. Groups of RAG–/– mice were treated with 2.5 x 106 TCID50 of MVA-BN (solid line) at birth or were left as untreated controls (dashed line) and infected with HSV-1 on day 8. The pooled data from the surviving animals after challenge from two separate experiments are shown.

 
Role of MVA-BN-induced type I IFN in the resistance against challenge with HSV-1

As MVA-BN induces type I IFN in PBMC in vitro (12), and virus-stimulated DC from adult mice are able to produce high amounts of IFN (25, 39), we tested whether MVA-BN was able to induce IFN type I in newborn mice. We were unable to detect elevated levels of serum IFN-{alpha} in neonatal or 12-day-old mice 2 days after exposure to MVA-BN. In contrast, cultures of pDC from 1-wk-old mice infected in vitro with MVA-BN at a multiplicity of infection of 1 secreted ~1000–1500 U of IFN-{alpha}/105 cells. In uninfected cultures no IFN-{alpha} was detected. Thus MVA-BN was able to induce secretion of IFN-{alpha} in vitro and expansion of DC in vivo.

Because we could not detect serum IFN-{alpha} in MVA-BN-treated mice, we tested resistance against HSV-1 in mice lacking IFN type I. MVA-BN was injected into 1-day-old A129 mice. Challenge with 10 PFU of HSV-1 was performed on day 8. In both MVA-BN-treated and controls, <50% of the mice survived. This confirms that the increase in resistance induced by MVA-BN depends on an intact IFN type I system, as previously shown for FL-treated mice (8).

This raised the question of whether IFN type I that was induced after MVA-BN application increased protection. Therefore, we injected 105 U of rIFN{alpha} B/D either once or twice into naive animals and tested the effect by challenge with HSV-1, as shown in a representative experiment (Fig. 6). Five days after infection, all seven untreated controls were dead. By contrast, six of the mice that had received only one treatment and all seven mice that received two rIFN treatments were still alive at this time point. Three of the seven animals treated twice with rIFN survived the challenge.



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  		FIGURE 6. rIFN-{alpha} B/D induces resistance against infection with HSV-1. Groups of seven C57BL/6 mice were treated once with 105 U of rIFN-{alpha} at birth (dashed line) or with an additional dose of 105 U the following day (solid line) or were left as untreated controls (dotted line) and infected with HSV-1 on day 8. Data from the surviving animals after challenge from an experiment of two are shown.

 
DC are able to protect naive mice from HSV-1

We next determined whether the protective effect was due to the DC induced by MVA-BN treatment. These DC were transferred into naive mice and tested for resistance against HSV-1. Naive, 8-day-old mice were challenged with 5 x 104 PFU of HSV-1 4 h after transfer of cells from MVA-treated mice. In a first experiment, splenocytes from 8-day-old mice treated at birth with MVA-BN were separated into DC-rich (low density) and DC-poor (high density) fractions (45). Mice receiving 5 x 106 cells from the DC-rich fraction (2.5 x 106 CD11c+) survived the challenge to 50%, whereas all the mice receiving 10 times less DC or untreated control mice died within 5 days (Fig. 7A).



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  		FIGURE 7. Transfer of DC from MVA-BN-treated C57BL/6 mice into naive littermates confers resistance against infection with HSV-1. Groups of C57BL/6 mice were treated with 2.5 x 106 TCID50 of MVA-BN at birth, spleen cells were prepared on day 7, DC were isolated by negative selection and density fractionation (see Materials and Methods) or by positive selection with magneto beads, and the cells were transferred to naive littermates, followed by challenge with HSV-1. A, Transfer of negatively selected DC. The light density cell fraction that contains ~50% of CD11c+ cells (solid line, 5 x 106 cells; dashed line, 5 x 105 cells) and the dense cell fraction that contains <1% CD11c+ cells (dotted line, 5 x 106 cells) are shown. B, Transfer of DC selected by magneto beads. Positively selected CD11c+ cells (solid line, 2 x 106), cells from the CD11c negative fraction (dotted line, 2 x 106 cells; long-dashed line, 50 x 106 cells), and cells from untreated controls (short-dashed line) are shown. Data from the surviving animals after challenge from an experiment of two are shown.

 
A second approach involved the transfer of positively isolated CD11c+ cells from 8-day-old mice treated at birth with MVA-BN to naive age-matched mice. A suspension of 2 x 106 splenocytes containing >80% CD11c+ cells protected naive mice from HSV-1 infection (Fig. 7B). By contrast, mice receiving either 2 x 106 or 50 x 106 cells from the CD11c-negative fraction did not show increased resistance against HSV-1. Therefore, transfer of CD11c+ cells that include cDC and pDC into naive mice, but not other cells, including MVA-BN-primed T cells, is able to protect against lethal infection with HSV-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Newborn mice have an immature immune system. The relative numbers of T and B lymphocytes are low, and APCs are inefficient in T cell activation (2, 17, 46). However, T cell-specific immune responses can be induced in these animals with strong immunogens, in particular replication-controlled or attenuated virus (17, 18, 47). In this study we determined the effects of the attenuated virus, MVA-BN, on the resistance in newborn mice. We analyzed pathways initiated by the contact of the virus with the immune system resulting in a 100-fold increase in resistance against a nonrelated virus, HSV-1. Three principle elements participated in conferring stronger resistance: IFN, FL, and DCs.

Requirement of IFN for induction of endogenous FL

Administration of exogenous FL during the first 7 days of life induces increased resistance in 7-day-old mice against HSV-1 (8). Hence, the production of endogenous FL was analyzed in this study. Maximal levels of FL were detected within 48 h after exposure to MVA-BN (Figs. 2 and 3). T cells are an important source of FL. In activated human T cells, preformed FL is translocated from intracellular stores to the cell surface. FL is then released within 24–96 h (22, 23). Vaccinia virus is a potent activator of CD4+, CD8+, and {gamma}{delta} T cells both in adult as well as neonatal individuals, and thus increased levels of FL may be induced in these cells (17, 48, 49, 50). Moreover, activation of virus-specific CD8+ T cells in adult mice requires ~30 h (51) or even less time for T cells of newborns (52). Therefore, the time frame of 48 h for maximal levels of serum FL parallels that of T cell activation. However, we need to include other sources of FL than T cells (53), as indicated from our studies in RAG mice (Fig. 3).

In the absence of functional IFN type I and II receptors, no increased production of FL was observed (Fig. 3). These data were confirmed in AGR129 mice that have no functional IFN and RAG (35) (data not shown). However, in the presence of either of the IFN systems, enhanced levels of FL could be induced by MVA-BN.

Effects of endogenous FL and IFN on DC development

Treatment of adult mice with exogenous FL leads to an increased number of NK cells, B cells, and DC (5, 54, 55). By contrast, in neonatal mice treated with FL, DC lineage cells were the only cells increased in number (8). In blood of MVA-BN-treated mice, NK cells were increased, whereas in blood and spleen the number of CD11c+ cells was increased, and that of CD19/B220+ cells (B cells) was decreased (Tables I and II). Therefore, increased production of FL in MVA-treated animals was associated with up-regulation of DC in different compartments.

Besides the requirements for endogenous FL regulation, IFNs are likely to influence the development of FL-stimulated bone marrow cells (39, 56, 57). MVA-BN is a potent inducer of type I IFN, as shown in this study with pDC and in other studies using a variety of cells from different species (10, 12). In addition, MVA-BN does not produce soluble IFN receptor analogues able to neutralize the protective effects of IFN (11, 58). Flow cytometric analysis of isolated spleen cells revealed that the CD11c+ gated cells had increased levels of CD45RA and CD8{alpha} (Table III). Data from cell density-enriched DC revealed that only pDC were increased in number, and their levels of CD8{alpha} and MHC class II were up-regulated (Fig. 1 and Table III).

Recent observations indicate that the transcription factor IFN consensus sequence binding protein (IFN regulatory factor 8), which can be activated by IFN, is needed for the development of pDC and the expression of CD8{alpha} on these cells (31, 33). The number of DC in 7-day-old mice lacking a functional IFN system (AG129 mice) was similar to that in C57BL/6 mice, and FL was present in the sera of both mouse strains (Fig. 3) (8). However, treatment of AG129 mice with MVA-BN did not increase the production of FL or DC up-regulation even though expansion of DC in these mice by exogenous FL was possible (8).

DC develop from myeloid as well as lymphoid precursors (59, 60). Some DC populations are absent in mice that do not express genes essential for lymphoid development (61, 62). Common developmental pathways between DC and B cells have been shown (63, 64). In FL-treated animals the development of B cells was not affected (8). By contrast, MVA-BN-treated animals had significantly lower numbers of B cells than controls, whereas the numbers of CD4+ and CD8+ T cells were increased (Table I). Therefore, the cytokines induced by the replicating virus (65) may not only influence the maturation of DC lineage cells, but may deviate B cell development to DC. Common DC and B cell pathways may exist, because a certain proportion of DC have rearranged Ig genes (64).

FL induced by virus replication required functional IFN systems. Indeed, within the DC populations the relative expression of MHC class II and CD8{alpha} was similar on cells of MVA-BN-treated and control AG129 mice that lack functional IFN (data not shown). These data confirm previous reports that IFN induce and activate DC, in particular pDC (31, 33, 57).

DC induce protection against challenge with HSV-1

In neonatal mice both intact IFN type I and FL are required for increased antiviral resistance (8). As MVA-BN induces both FL and IFN type I, we tested the resistance of 1-wk-old mice that were treated with MVA-BN at birth. We found a 100-fold increased resistance against infection with HSV-1 (Table IV and Fig. 4). Thus, the three elements, IFN, FL, and DC, were required to establish increased antiviral resistance in neonatal mice (Figs. 3, 4, and 6 and Table IV). To gain more insight into the effector phase of the immune response, DC from MVA-BN-treated animals were transferred to naive 8-day-old animals. DC, enriched by two different methods, induced increased protection (Fig. 7). The role of DC is further supported by our recent findings that DC derived from bone marrow cells cultured with FL (66) also induced protection against HSV-I infection (data not shown). In the future this system will allow a closer examination of the nature and activation state of the DC involved. Therefore, in the effector phase increased numbers of DC appear sufficient to induce increased resistance to viral infection in naive C57BL/6 animals. Although it is clear that increased numbers of DC alone are sufficient to induce increased resistance, it is also clear that more efficient protection is obtained if specific adaptive immunity is involved. Comparison of C57BL/6 mice with RAG-deficient mice lacking specific lymphocytes shows that specific immunity boosts resistance 103- to 104-fold above the innate, IFN-dependant resistance alone (Fig. 5) (8).

A 2- to 3-fold increase in pDC in blood and spleen of MVA-BN-treated animals may not appear sufficient to explain the impressive biological effect in preventing deadly infection with a heterologous virus. However, approximately one spleen equivalent of 2 x 106 MVA-BN-induced DC transferred to naive mice was sufficient to confer protection against an otherwise lethal virus challenge. After viral challenge, pDC may secrete IFN locally and exert its multiple effects. IFN has direct and indirect antiviral activities. DC, in concert with IFN and other cytokines, induce innate and specific immunities crucial for efficient protection. Clearly, although IFN and DC are central to the development of resistance to virus infection, the complex interplay between all interacting systems and the activation state of DC requires further unraveling.


   Acknowledgments
 
We thank Bogdana Salathé for the excellent and competent work with the laboratory animals. We highly appreciate the virus production team at Bavarian Nordic for providing virus and reanalysis after infection.


   Footnotes
 
1 This work was supported by Bavarian Nordic (Munich, Germany), the Swiss National Science Foundation, and the Kanton of Zurich. Back

2 Address correspondence and reprint requests to Dr. Mark Suter, University of Zurich, Institute of Virology, Winterthurerstrasse 266a, 8057 Zurich, Switzerland. E-mail address: msuter{at}vetvir.unizh.ch Back

3 Abbreviations used in this paper: FL Flt3 ligand; cDC, conventional DC; DC, dendritic cell; MVA, modified vaccinia Ankara; pDC, pre-DC with plasmacytoid morphology; RAG, recombination-activating gene; TCID50, half-maximal tissue culture infective dose. Back

Received for publication August 22, 2003. Accepted for publication March 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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