Host-dependent type 1 cytokine responses driven by inactivated viruses may fail to default in the absence of IL-12 or IFN-{alpha}/{beta}

Marel C. de Wit1,{dagger}, Marian C. Horzinek1, Bart L. Haagmans2 and Virgil E. J. C. Schijns3

1 Virology Unit, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, 3584 CL Utrecht, The Netherlands
2 Institute of Virology, Erasmus MC Rotterdam, 3015 GE Rotterdam, The Netherlands
3 Department of Vaccine Technology and Immunology, Intervet International BV, 5830 AA Boxmeer, The Netherlands

Correspondence
Marel C. de Wit
Marel.deWit{at}abbott.com


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Replicating viruses generally induce type 1 immune responses, with high interferon (IFN)-{gamma} levels and antibodies of the IgG2a isotype. In the present study we demonstrate the intrinsic ability of non-replicating virions to induce comparable immune responses in the notable absence of any adjuvant. Injection of inactivated pseudorabies virus, an alphaherpesvirus, by various routes into mice resulted in the generation of T helper (Th) 1 type immune response. Co-delivery of inactivated pseudorabies herpesvirus (iPRV) with protein redirected IgG1-dominated tetanus toxoid-specific responses towards an IgG1/IgG2a balanced response. Also inactivated preparations of viruses from the paramyxo- (Newcastle disease virus), rhabdo- (rabies virus), corona- (infectious bronchitis virus) and reovirus (avian reovirus) families led to IgG2a antibody responses; however, the genetic background of the host did result in considerable variation. Because disrupted virions also induced type 1 immune responses, we conclude that structural elements of virions inherently contribute to IFN-{gamma}-dependent isotype switching by inactivated viruses. Strikingly, immunizations in gene-disrupted mice showed that a functional IFN-{alpha}/{beta}, IFN-{gamma} or interleukin (IL)-12 pathway was not required for the generation of a polarized Th1 type immune response initiated by inactivated virus particles. These findings have a bearing on the understanding of immune responsiveness to virus structures and the design of vaccines containing virus components.

{dagger}Present address: Abbott BV, Siriusdreef 51, 2132 WT Hoofddorp, The Netherlands.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A hallmark of immune responses to virus infections is the activation of several interferon (IFN) genes, including expression of IFN-{gamma}. IFN-{gamma} can exert antivirus effects by directly inhibiting replication or through the stimulation of innate immune effector cells like natural killer (NK) cells, macrophages and neutrophils (Samuel, 2001). IFN-{gamma} also regulates adaptive immune responses by directing T helper (Th) cell polarization and antibody isotype switching (Coffman et al., 1991; Snapper & Paul, 1987). In mice it stimulates IgG2a, in contrast to IL-4, which is involved in IgE and IgG1 assembly (Coffman et al., 1986). Immune complexes containing IgG2a molecules activate the complement system and enhance phagocytosis by macrophages and antibody-dependent cell-mediated cytotoxicity; mechanisms involved in antivirus immunity (Kipps et al., 1985; Spiegelberg, 1974). Although antibodies are important in the early defence against e.g. vesicular stomatitis virus and polyoma virus (Bachmann et al., 1997; Szomolanyi-Tsuda & Welsh, 1996), their major role is in the protection against reinfections (Bachmann & Kopf, 1999). Consistent with the characteristic IFN-{gamma} production during virus infections, immunoglobulins of the IgG2a isotype predominate in virus-infected mice (Coutelier et al., 1987).

Production of IFN-{gamma} by NK cell and various T-cell populations is triggered either directly, through the recognition of infected cells, or indirectly, involving other cytokines. Especially, early interleukin (IL)-12 production is considered crucial for subsequent IFN-{gamma} synthesis (Hsieh et al., 1993; Magram et al., 1996). However, the mechanism underlying the preference for the Th1 pathway, including the induction of IgG2a antibodies, during virus infections has not been fully elucidated. In fact, this polarization of the T cell responses may occur independently from IL-12 function during virus infection (Oxenius et al., 1999; Schijns et al., 1998), in contrast to most parasitic and bacterial infections. This is plausible because IL-12 expression is inhibited by IFN-{alpha} (Cousens et al., 1997). IFN-{alpha} is induced both by virus replication intermediates like double-stranded RNA and by dendritic cells (DCs) pulsed with inactivated viruses (Milone & Fitzgerald-Bocarsly, 1998); also, it polarizes human T cells to the Th1 phenotype (Rogge et al., 1998). In mice, however, its regulatory role is unclear; although it fails to induce Th1 development (Wenner et al., 1996) IFN-{alpha} may stimulate IFN-{gamma} production during virus infections (Nguyen et al., 2002).

In contrast to replicating viruses, inactivated viruses are usually adjuvanted in order to enhance the immune response, which results in an isotypic bias for IgG1 type antibodies in most cases (Brett et al., 1993; Katayama et al., 1999; Katz et al., 1991), even following co-administration in complete Freund's adjuvant. However, adjuvants may mask the intrinsic capacity of virus antigens to induce antibody responses of a certain quality. Remarkably, some inactivated viruses, when given without adjuvant, induce IgG2a antibodies comparable to live virus-induced responses (Brett et al., 1993; Hocart et al., 1989; Huang et al., 1993; Schijns et al., 1998). The present study has been set up to define host factors and conditions that play a role in the in vivo regulation of virus-specific IgG2a antibody responses induced by non-replicating virions. We used inactivated pseudorabies herpesvirus (iPRV) particles as immunizing antigens in order 1) to avoid virus persistence, 2) to allow standardization of antigenic load, 3) to exclude potential replication-associated immune escape mechanisms and 4) to prevent destruction of target cells by cytopathogenicity. For comparison we studied responses to live attenuated PRV.

We show that in addition to iPRV, inactivated viruses of different families characteristically induce IFN-{gamma}-dependent IgG2a antibody production. The quality of the antibody profile is influenced by the genetic background of the host, but is independent of the route of administration and endogenous IL-12 or IFN-{alpha}/{beta} function.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mice.
Wild-type (WT) and mutant 129/Sv mice, the latter deficient in their expression of IL-12 or IFN receptors, were bred at the Central Animal Laboratory, Utrecht University. IL-12-deficient (IL-12-/-), IFN{alpha}/{beta}-deficient (IFN{alpha}/{beta}-/-) and IFN-{gamma} receptor-deficient (IFN-{gamma}R-/-) mice were generated by gene targeting in murine embryonic stem cells (Huang et al., 1993; Magram et al., 1996; Muller et al., 1994). BALB/c and C57BL/6 mice were obtained from Harlan. All animals were housed in filtertop cages.

Unless indicated otherwise, 8- to 12-week-old female mice were immunized by intraperitoneal (i.p.) injection of 100 µl containing approximately 2x107 inactivated plaque-forming units (p.f.u.) of the respective virus preparations and boosted 4 weeks later by the same route. Mice were bled from the retro-orbital plexus at the indicated points in time. Approval for the animal experiments had been obtained from the Institutional Animal Welfare Committee.

Viruses and antigens.
Because of the high virulence of WT PRV in mice, an attenuated gE-, TK- mutant served as live replicating PRV control (1x106 p.f.u. per dose; Visser & Lütticken, 1989). A gE-, TK+ derivative of field isolate NIA-3 (Quint et al., 1987) was used to prepare iPRV. Inactivation of the virus was performed using a final concentration of 0·01 M 2-bromoethylamine hydrobromide (BEA) pH 7·4 and NaOH for 24 h at 37 °C. BEA was neutralized by adding 20 % sodium thiosulphate to a final concentration of 0·67 % (v/v). No residual live virus was found, as assessed by inoculation of BHK cell cultures (>1500 cm2 monolayer surface) with the preparation and checking for cytopathic effects (CPE). Disintegrated PRV particles were prepared by treatment of the inactivated virus with 1 % sodium deoxycholate (NaDOC) at 37 °C for 1 h (Balkovic et al., 1987). After dialysis against PBS, 10 µg and 50 µg of virus were used in the immunization studies.

Newcastle disease virus (NDV) clone 30 (Romer-Oberdorfer et al., 1999) and infectious bronchitis virus (IBV) strain M41 (Cavanagh, 1983) were grown in embryonated eggs and inactivated by incubation with formaldehyde (final concentration 0·075 %) at 4 °C for 21 days. No residual infectivity was found after inoculation of 0·1 ml of the preparations into the allantoic cavity of embryonated eggs and assessing embryo mortality and haemagglutination of chicken red blood cells after 6–7 days.

Reovirus (REO) strain 2408 (Rosenberger et al., 1989) grown in cultures of chicken embryo fibroblasts (CEF) was inactivated using formaldehyde (final concentration 0·2 %; incubation at 37 °C for 40 h). The preparation was neutralized using sodium metabisulphite and assayed for residual infectivity on a 500 cm2 CEF monolayer surface. After three to four passages, the cells were inspected for CPE. BHK (baby hamster kidney) cell-adapted rabies virus strain Pasteur (Consales et al., 1988) was inactivated by incubation with {beta}-propriolactone (final concentration 0·025 %) at 37 °C during 2 h.

BHK cell cultures were inoculated with the inactivated virus preparations and scrutinized for CPE for 14 days; also, mice were injected intracerebrally with 0·03 ml of the inactivated virus suspensions and observed for neurological symptoms for 21 days. No residual infectivity was found. Tetanus toxoid (TT; SVM) was administered at a concentration of 0·5 LF per dose.

Determination of antibody titres.
Antibody levels and subtypes were determined using ELISA as described by Schijns et al. (Schijns et al., 1994). Briefly, 96-well flat-bottom plates (Greiner) were incubated with 110 µl of the inactivated virus or TT diluted in carbonate buffer (0·05 M, pH 9·6) at 37 °C (or at 4 °C for TT) for 16 h and rinsed with tap water. Subsequently, two-fold serum dilutions (starting at 1/64) in 0·04 M PBS containing 0·1 % Tween 80 and 0·1 % BSA (Kordia, Leiden, The Netherlands) were added. After 1 h at 37 °C, the plates were rinsed again and incubated with IgG isotype-specific antibody/horseradish peroxidase conjugates (Southern Biotechnology) for 30 min. After another rinse, tetramethylbenzidine was allowed to react for 15 to 30 min at room temperature. The colour reaction was stopped with 2 M sulphuric acid and the result (absorbance) read at 450 nm. Antibody titres were defined as the reciprocal of the highest dilution with an absorbance 1·5 times that of the background, observed in naïve, non-immunized control groups; titres below a 2 log titre of 5 were regarded negative. No cross-reactivity between TT and PRV-specific antibodies was observed.

Analysis of cytokine production.
For the analysis of IL-4, IL-5 and IFN-{gamma} production, spleens were isolated 2 weeks after the booster immunization. Erythrocyte-depleted splenocytes (3x106 cells ml-1) were cultured in 24-well plates (Nunc) in the presence of 10 µg dialysed iPRV ml-1. Cell culture supernatants were harvested 2, 5 or 7 days afterwards and stored at -20 °C until use. Concentrations of IL-4, IL-5 and IFN-{gamma} were measured using sandwich ELISA with mass-calibrated standards (R&D Systems).

Statistical analysis.
If applicable, group means of cytokine responses and antibody titres were compared using Student's t-test. Values of P<0·05 were considered significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Inactivated PRV induces IgG2a antibody responses irrespective of the route of immunization
To assess the quality of specific antivirus antibodies we determined the ratios of isotype-specific serum antibody titres by ELISA in groups of immunized mice. When immunized i.p. with iPRV, 129/Sv mice were found to preferentially mount IgG2a antibody responses (Fig. 1). In contrast, immunization with tetanus toxoid (TT) resulted in responses merely of the IgG1 isotype (Fig. 2, left panel). Immunization routes have been shown to influence the polarization of immune responses (Rogers & Croft, 1999), which we could not confirm: intramuscular, intraperitoneal and intradermal vaccination of 129/Sv mice all resulted in dominant IgG2a production (Fig. 1). The quality of the antibody response following iPRV immunization therefore proved independent of the route of administration.



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Fig. 1. PRV-specific IgG antibody titres after immunization with iPRV via different routes. Sera from 129/Sv mice (n=5) were tested by ELISA 4 weeks after primary vaccination for IgG heavy and light (H+L) chains (grey columns), IgG1 (hatched columns) and IgG2a (solid columns), using PRV antigen. Mean titres±SEM are shown, the detection limit (D.L.) is indicated by the dotted line.

 


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Fig. 2. Influence of inactivated and live PRV on the antibody levels of IgG subclasses directed against TT. Groups of at least four 129/Sv mice were immunized i.p. with TT alone or in the presence of live or inactivated PRV. TT-specific IgG (H+L) (grey columns), IgG1 (hatched columns) and IgG2a (solid columns) antibody titres in sera taken 4 weeks after immunization were measured using ELISA. Mean titres±SEM are shown. Theoretical cross-reactivity between anti-PRV and anti-TT antibodies could be excluded because of negative reactions to each specific antigen in sera of animals immunized with only one of the two non-related antigens. The dotted line indicates the detection limit (D.L.).

 
Modulation of the antibody response directed against unrelated antigens
Virus infections may assist in the induction of IgG2a antibodies directed against co-administered non-virus antigens such as keyhole limpet haemocyanin and TT (Coutelier et al., 1988; Markine-Goriaynoff et al., 2000). To investigate the role of an inactivated virus, mice were immunized with TT in combination with live or iPRV, and with TT alone serving as a control. The total amount of antibodies directed against TT was not significantly influenced by co-administration of live or iPRV (Fig. 2), nor were the IgG1 titres affected. However, co-administration of iPRV enhanced TT-specific IgG2a levels (P=0·02), which indicates a shift of the TT-specific reaction towards a type 1 immune response. The antibody isotype profiles to PRV in these animals were similar to those after PRV administration alone; no cross-reactivity for TT or PRV antigens could be detected in sera of co-immunized animals (data not shown). These results indicate that iPRV can act as a type 1 subclass selective adjuvant.

Endogenous IFN-{gamma} but not IFN-{alpha}/{beta} or IL-12 is required for IgG2a isotype switching
To assess the contribution of IFN-{gamma} in isotype switching to IgG2a, antibody levels were determined in IFN-{gamma}R-/- mice after immunization with iPRV. As shown in Fig. 3(B), these animals attained lower antivirus IgG2a antibody levels than the controls (P=0·003), indicating that IFN-{gamma} is largely responsible for the preferential IgG2a isotype response. We then looked for IFN-{gamma} synthesis by PRV-specific T cells. Splenocytes isolated 14 days after the second immunization were stimulated in vitro with iPRV antigen. As shown in Fig. 4, high levels of IFN-{gamma} could indeed be demonstrated in the supernatant of these splenocytes, whereas IL-5 levels were below the detection limit, similar to levels of IL-4 (data not shown). In contrast to immunization with replicating attenuated PRV (Schijns et al., 1994) we noted that detectable cytokine levels (IFN-{gamma}, but no IL-4 or IL-5) could be measured only after 7 days of antigenic stimulation, indicating that the frequency of antigen-specific cytokine-producing cells was rather low using non-replicating PRV.



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Fig. 3. Pseudorabies herpesvirus (PRV)-specific antibody responses in wild-type (WT) (A), IFN-{gamma} receptor-deficient (IFN-{gamma}R-/-)(B), IFN-{alpha}/{beta} receptor-deficient (IFN-{alpha}/{beta}R-/-) (C) and IL-12-deficient (IL-12-/-) (D)129/Sv mice. Sera of five animals per group, taken 4 weeks after a single i.p. immunization with iPRV, were analysed for specific IgG (H+L) (grey columns), IgG1 (hatched columns) and IgG2a (solid columns). Mean titres±SEM are shown. The dotted line point out the detection limit (D.L.).

 


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Fig. 4. Cytokine responses of splenocytes from wild-type (WT), IFN-{gamma} receptor-deficient (IFN-{gamma}R-/-) and IL-12-deficient (IL-12-/-) mice, isolated 2 weeks after a booster immunization with inactivated (i) PRV and restimulated with iPRV. After 7 days IL-5 (open columns) and IFN-{gamma} (solid columns) concentrations in the cell culture supernatant were measured using ELISA. Mean concentrations of five mice per group are shown±SEM. IFN-{gamma} levels in splenocytes cultures from naïve mice proved lower than 100 pg ml-1, while IL-5 levels were non-detectable (ND<30 pg ml-1).

 
Many inactivated viruses are potent inducers of IFN-{alpha}/{beta} (Isaacs & Lindenman, 1957), which may polarize T cells towards the type 1 phenotype. To address the role of these cytokines in IgG2a antibody regulation after immunization with non-replicating alphaherpesvirus particles, we injected iPRV into WT and IFN-{alpha}/{beta}R-/- mice and determined antivirus antibody subclass titres. As Fig. 3(C) shows, there were no conspicuous differences between the groups. It would appear therefore that class I IFNs do not play a key role in the polarization of the antibody response after vaccination with iPRV.

Surprisingly IL-12 seemed to be dispensable for Th1 development following iPRV immunization as well. IL-12-/- mice, like WT animals, responded with IgG2a-dominated antibody titres (Fig. 3D) as well as with IFN-{gamma} production upon restimulation of their splenocytes (Fig. 4).

Induction of responses after immunization with disrupted inactivated PRV particles
We needed to exclude trivial explanations for the immunogenicity of inactivated virions and the observed IgG2a responsiveness. Contamination of the preparations by endotoxin or by inflammatory substances comes to mind. Endotoxin was undetectable (<10 IU ml-1), as measured by the Limulus amebocyte lysate (LAL) test, and dialysed inactivated virus also induced type 1 antibody responses (not shown). Moreover, NaDOC disrupted virions also induced IgG2a-dominated antibody responses (Fig. 5A). This conclusion is corroborated by the results of the cytokine analysis after in vitro stimulation of splenocytes with PRV, depicted in Fig. 5(B).



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Fig. 5. PRV-specific immunoglobulin isotype (A) and cytokine responses (B) following immunization with iPRV particles or NaDOC disrupted virions. ELISA measured antibody titres in sera of five mice per group taken 4 weeks after primary immunization (A). Means±SEM above the detection limit (D.L.) of IgG (H+L) (grey columns), IgG1 (hatched columns) and IgG2a (solid columns) are shown. Splenocytes were isolated 2 weeks after booster immunization and restimulated with iPRV (B). Supernatants of 7-day cell cultures were tested using ELISA for IL-5 (open columns) and IFN-{gamma} (solid columns). Mean concentrations±SEM are shown. IFN-{gamma} levels in splenocytes cultures from naïve mice proved lower than 100 pg ml-1, and IL-5 levels were below the detection limit (<30 pg ml-1).

 
IgG2a antibody responses after immunization with other inactivated viruses
To test whether the observed phenomena are exclusive for iPRV, the study was expanded to include other viruses. Mice were immunized with inactivated preparations of enveloped and non-enveloped, RNA and DNA viruses (paramyxo-, rhabdo-, corona- and reovirus) and their sera assayed as before. In all cases, the IgG2a antibody subclass was most abundant (Fig. 6), although titres varied per virus. This indicates that structural virion differences are not essential and that the underlying mechanism may be of a general nature.



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Fig. 6. Serum levels of immunoglobulin isotypes 2 (NDV) or 4 (iPRV, rabies virus, IBV) weeks after primary i.p. immunization or 3 weeks after a booster vaccination [reovirus (REO)] with inactivated virus preparations. Groups of four to seven mice (129/Sv) were used, and their individual sera analysed by ELISA for virus-specific IgG (H+L) chains (grey columns), IgG1 (hatched columns) and IgG2a (solid columns). Antibody titres are expressed as group means±SEM. The detection limit (D.L.) is indicated by the dotted line.

 
Influence of host genotype on IgG2a titres
The experiments described thus far have been performed in WT and gene knockout mice on a 129/Sv background. To examine the influence of the host genotype, live and iPRV preparations were used to immunize mice of the inbred strains 129/Sv, C57BL/6 and BALB/c. Immunization with TT, an inducer of IgG1-dominated humoral responses, was used as a control. Live PRV immunization was found to induce type 1 responses in all mouse strains, although variations in the IgG2a/IgG1 ratios were observed (Fig. 7A). Upon immunization with iPRV, IgG2a antibody responses were lower in C57BL/6 (P<0·0001) and BALB/c (P=0·0003) mice than in the 129/Sv strain (Fig. 7B). Th2 isotype immune responses with relatively low levels of IgG2a were observed in the TT-immunized mice of all strains tested (Fig. 7C). Hence, host genetic factors seem to control the phenotype of immune responses after immunization with inactivated virus more noticeably than after infection. We finally asked the question whether the genetic background of the host is important also for viruses other than PRV, and immunized 129/Sv, C57BL/6 and BALB/c mice with inactivated rabies virus and NDV. C57BL/6 mice produced IgG2a antibodies against both viruses, although the ratios with respect to IgG1 were lower than that observed in 129/Sv mice (Fig. 8). On the other hand, BALB/c mice responded to NDV with a dominant IgG2a antibody reaction, with higher IgG2a/IgG1 ratios when compared to the TT response. This finding illustrates that in addition to the host's genetic background, viruses differ in their capacity to induce IgG2a antibodies.



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Fig. 7. Antibody titres in mice with different genetic backgrounds. Mice (n=5) of the 129/Sv, C57BL/6 and BALB/c strains were injected by the i.p. route with live PRV (A), iPRV (B) or TT (C). Group means±SEM above the detection limit (D.L.) of specific IgG (H+L) (grey columns), IgG1 (hatched columns) and IgG2a (solid columns) antibodies detected in serum 3 weeks after secondary immunization are shown. Secondary responses are depicted because of low primary antibodies in C57BL/6 and BALB/c mice, which, however, showed a similar profile.

 


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Fig. 8. Influence of the genetic background on the antibody response induced by inactivated rabies virus (A) or NDV (B). Serum levels of IgG subtypes 2 weeks after primary (NDV) or booster (rabies) vaccination were measured using specific ELISA systems and are shown as means of five mice±SEM. The secondary responses to rabies virus are depicted because of low primary antibodies in C57BL/6 and BALB/c mice, which, however, showed a similar profile. Grey columns represent IgG (H+L), hatched columns IgG1 and solid columns IgG2a. The detection limit (D.L.) is indicated by the dotted line.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virions may fulfil the structural requirements for IgM antibody responses in the absence of an adjuvant. Highly ordered, densely packed surface epitopes found on virions are assumed to cross-link surface Ig receptors on B cells and thereby evoke IgM responses, often in the absence of T cells (Fehr et al., 1996; Ochsenbein et al., 2000). In the presence of activated T cells, IgM-producing B cells may readily switch to the production of IgG. Immunizations with inactivated preparations like mouse adenovirus or lactate dehydrogenase virus (Markine-Goriaynoff et al., 2000) evoke IgG responses, often showing an IgG1 isotypic bias, even when complete Freund's adjuvant was co-administered (Oxenius et al., 1999). In our study, immunizations with iPRV, a herpes simplex-related {alpha}-herpesvirus, led to IgG2a-dominated immune responses, especially in 129/Sv mice. In C57BL/6 and BALB/c mice a more balanced IgG2a/IgG1 ratio was noted. Virions of other virus families, including the enveloped rhabdo-, corona- and paramyxoviridae, as well as the non-enveloped reoviruses, equally exhibit this Th1 polarization. Also, the observation that iPRV, similar to replicating PRV, enhances IgG2a production to the co-administered non-virus TT antigen, suggests involvement of non-cognate, bystander immune modulation by IFN-{gamma} in 129/Sv mice. This phenomenon has been noted for replicating viruses before (Coutelier et al., 1988; Monteyne et al., 1993), but not for inactivated virions.

Disintegrated PRV particles were also found to evoke IgG2a-dominated responses, although the IgG2a/IgG1 ratio was somewhat lower than when intact PRV virions had been used. This may indicate that Th1 type immune responses are not entirely dependent on the particulate form of the antigen, though it contributes to the isotype bias. Other virus features, like the genomic material, can also influence the outcome of an immune reaction. The double stranded DNA present in herpes virions, is able to induce the expression of co-stimulatory molecules and cytokine genes upon transfection in non-immune thyroid cells (Suzuki et al., 1999). Such signal molecules could provide the critical immune activating and response directing molecules necessary for Th1 proliferation and down-stream IgG2a production.

Although modest IgG2a synthesis occurs in the absence of IFN-{gamma} function (Markine-Goriaynoff et al., 2000; Muller et al., 1994), our studies demonstrate that the functional presence of IFN-{gamma} substantially increases IgG2a isotype synthesis in response to iPRV. Notably, restimulation of IFN-{gamma}R-/- splenocytes with virus antigen resulted in high IFN-{gamma} and no detectable IL-5 production. This suggests that IFN-{gamma} function is not essential to drive virion-induced IFN-{gamma} production by T cells. Similar to earlier studies (Schijns et al., 1994) IFN-{gamma}R-/- splenocytes produced higher levels of IFN-{gamma} than WT splenocytes. We speculate on less IFN-{gamma}-mediated feedback inhibition or less cytokine consumption in the absence of a functional IFN-{gamma} receptor.

Surprisingly, iPRV-induced responses fail to default to type 2 immune reactions in the absence of IFN-{alpha}/{beta} or IL-12 gene function. IFN-{alpha}/{beta}, historically discovered using inactivated influenza virus (Isaacs & Lindenman, 1957), is characteristically produced by plasmacytoid DCs (Hochrein et al., 2001). Like IL-12, it is a key regulator of innate and adaptive immune responses after infections (Finkelman et al., 1991). IFN-{alpha}/{beta}, IL-18 and IL-12-independent type 1 immune responses have been described in particular for virus infections (Grob et al., 1999; Oxenius et al., 1999; Schijns et al., 1998; van den Broek et al., 1995; Xing et al., 2000), but not for immune responses following exposure to non-replicating virus particles. We can however not rule out that in the absence of one of these two cytokines the other, or alternative Th1-associated cytokines, like IL-18, IL-23 or IL-27, compensates the deficiency (Okamura et al., 1995; Oppmann et al., 2000; Pflanz et al., 2002; Xing et al., 2000). Double depletion experiments should answer this possibility.

Apart from typical cytokines correlated with Th1 development, the virion-associated IFN-{gamma}-driven IgG2a production may be triggered by very early recognition events in T cell polarizing DCs or B cells. However, limited information is available on the interaction between virions and immune cells. Recently, a few virus-related pathogen-recognition receptors have been defined, such as for the G protein of respiratory syncytial virus, which is involved in Toll-like receptor (TLR) 4-dependent responses (Haynes et al., 2001; Kurt-Jones et al., 2000). Immune-stimulatory activity has been noted also for the rabies virus nucleocapsid protein (Lafon et al., 1992), without knowledge of its putative recognition receptor. It can therefore not be excluded that the induction of Th1 development upon recognition of inactivated viruses depends on a pathogen-recognition receptor, as shown for an increasing number of non-virus microbial antigens (Jankovic et al., 2002).

Since the genetic background of the host did influence the outcome of the immune reaction (Hocart et al., 1989; Raj et al., 1992), we are led to conclude that the observed Th1 pathway may be activated differently in genetically different hosts. This could be the result of expression differences of pattern-recognition receptors on immune cells, or down-stream response modifying genes; a topic for further investigation.


   ACKNOWLEDGEMENTS
 
We thank Dr M. Aguet (ETH, Zurich, Switzerland) for providing us with the IFN-{alpha}/{beta} and IFN-{gamma}R-/- mice, and Dr H. Mossmann (Max-Planck Institut, Freiburg, Germany) and Dr G. Alber (University of Leipzig, Leipzig, Germany) for the provision of IL-12-/- mice. We also acknowledge Karel van Stokkom for his assistance in performing the animal experiments, Hanneke van Zuilekom and Nicolette Scholtes for their excellent technical help and Winfried Degen for helpful discussions.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bachmann, M. F. & Kopf, M. (1999). The role of B cells in acute and chronic infections. Curr Opin Immunol 11, 332–339.[CrossRef][Medline]

Bachmann, M. F., Kalinke, U., Althage, A., Freer, G., Burkhart, C., Roost, H., Aguet, M., Hengartner, H. & Zinkernagel, R. M. (1997). The role of antibody concentration and avidity in antivirus protection. Science 276, 2024–2027.[Abstract/Free Full Text]

Balkovic, E. S., Florack, J. A. & Six, H. R. (1987). Immunoglobulin G subclass antibody responses of mice to influenza virus antigens given in different forms. Antiviral Res 8, 151–160.[CrossRef][Medline]

Brett, S. J., Dunlop, L., Liew, F. Y. & Tite, J. P. (1993). Influence of the antigen delivery system on immunoglobulin isotype selection and cytokine production in response to influenza A nucleoprotein. Immunology 80, 306–312.[Medline]

Cavanagh, D. (1983). Coronavirus IBV: further evidence that the surface projections are associated with two glycopolypeptides. J Gen Virol 64, 1787–1791.[Abstract]

Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnik, A. & Paul, W. E. (1986). B cell stimulatory factor-1 enhances the IgE response of lipopolysaccharide-activated B cells. J Immunol 136, 4538–4541.[Abstract/Free Full Text]

Coffman, R. L., Varkila, K., Scott, P. & Chatelain, R. (1991). Role of cytokines in the differentiation of CD4+ T-cell subsets in vivo. Immunol Rev 123, 189–207.[Medline]

Consales, C. A., Valentini, J. G., Albas, A., Mendonca, R. M., Fuches, R. M., Soares, M. A. & Pereira, C. A. (1988). The preparation of cultured rabies virus and the production of antiserum for human use. J Biol Stand 16, 27–32.[Medline]

Cousens, L. P., Orange, J. S., Su, H. C. & Biron, C. A. (1997). Interferon-alpha/beta inhibition of interleukin 12 and interferon-gamma production in vitro and endogenously during viral infection. Proc Natl Acad Sci U S A 94, 634–639.[Abstract/Free Full Text]

Coutelier, J. P., van der Logt, J. T., Heessen, F. W., Warnier, G. & Van Snick, J. (1987). IgG2a restriction of murine antibodies elicited by viral infections. J Exp Med 165, 64–69.[Abstract]

Coutelier, J. P., van der Logt, J. T., Heessen, F. W., Vink, A. & Van Snick, J. (1988). Virally induced modulation of murine IgG antibody subclasses. J Exp Med 168, 2373–2378.[Abstract]

Fehr, T., Bachmann, M. F., Bluethmann, H., Kikutani, H., Hengartner, H. & Zinkernagel, R. M. (1996). T-independent activation of B cells by vesicular stomatitis virus: no evidence for the need of a second signal. Cell Immunol 168, 184–192.[CrossRef][Medline]

Finkelman, F. D., Svetic, A., Gresser, I., Snapper, C., Holmes, J., Trotta, P. P., Katona, I. M. & Gause, W. C. (1991). Regulation by interferon alpha of immunoglobulin isotype selection and lymphokine production in mice. J Exp Med 174, 1179–1188.[Abstract]

Grob, P., Schijns, V. E., van den Broek, M. F., Cox, S. P., Ackermann, M. & Suter, M. (1999). Role of the individual interferon systems and specific immunity in mice in controlling systemic dissemination of attenuated pseudorabies virus infection. J Virol 73, 4748–4754.[Abstract/Free Full Text]

Haynes, L. M., Moore, D. D., Kurt-Jones, E. A., Finberg, R. W., Anderson, L. J. & Tripp, R. A. (2001). Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 75, 10730–10737.[Abstract/Free Full Text]

Hocart, M. J., Mackenzie, J. S. & Stewart, G. A. (1989). The immunoglobulin G subclass responses of mice to influenza A virus: the effect of mouse strain, and the neutralizing abilities of individual protein A-purified subclass antibodies. J Gen Virol 70, 2439–2448.[Abstract]

Hochrein, H., Shortman, K., Vremic, D., Scott, B., Hertzog, P. & O'Keeffe, M. (2001). Differential production of IL-12, IFN-{alpha} and IFN-{gamma} by mouse dendritic cell subsets. J Immunol 166, 5448–5455.[Abstract/Free Full Text]

Hsieh, C. S., Macatonia, S. E., Tripp, C. S., Wolf, S. F., O'Garra, A. & Murphy, K. M. (1993). Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260, 547–549.[Medline]

Huang, S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann, H., Kamijo, R., Vilcek, J., Zinkernagel, R. M. & Aguet, M. (1993). Immune response in mice that lack the interferon-gamma receptor. Science 259, 1742–1745.[Medline]

Isaacs, A. & Lindenman, J. (1957). Virus interference. II: Some properties of interferon. Proc R Soc 147, 268–273.

Jankovic, D., Kullberg, M. C., Hieny, M., Caspar, P., Collazo, C. M. & Sher, A. (2002). In the absence of IL-12, CD4+ T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10-/- setting. Immunity 16, 429–439.[Medline]

Katayama, S., Oda, K., Ohgitani, T., Hirahara, T. & Shimizu, Y. (1999). Influence of antigenic forms and adjuvants on the IgG subclass antibody response to Aujeszky's disease virus in mice. Vaccine 17, 2733–2739.[CrossRef][Medline]

Katz, D., Lehrer, S., Galan, O., Lachmi, B. E. & Cohen, S. (1991). Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice. FEMS Microbiol Immunol 3, 305–320.[Medline]

Kipps, T. J., Parham, P., Punt, J. & Herzenberg, L. A. (1985). Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies. J Exp Med 161, 1–17.[Abstract]

Kurt-Jones, E. A., Popova, L., Kwinn, L. & 8 other authors (2000). Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1, 398–401.[CrossRef][Medline]

Lafon, M., Lafage, M., Martinez-Arends, A., Ramirez, R., Vuillier, F., Charron, D., Lotteau, V. & Scott-Algara, D. (1992). Evidence for a viral superantigen in humans. Nature 358, 507–510.[CrossRef][Medline]

Magram, J., Connaughton, S. E., Warrier, R. R. & 7 other authors (1996). IL-12-deficient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 4, 471–481.[Medline]

Markine-Goriaynoff, D., van der Logt, J. T., Truyens, C., Nguyen, T. D., Heessen, F. W., Bigaignon, G., Carlier, Y. & Coutelier, J. P. (2000). IFN-gamma-independent IgG2a production in mice infected with viruses and parasites. Int Immunol 12, 223–230.[Abstract/Free Full Text]

Milone, M. C. & Fitzgerald-Bocarsly, P. (1998). The mannose receptor mediates induction of IFN-alpha in peripheral blood dendritic cells by enveloped RNA and DNA viruses. J Immunol 161, 2391–2399.[Abstract/Free Full Text]

Monteyne, P., Van Broeck, J., Van Snick, J. & Coutelier, J. P. (1993). Inhibition by lactate dehydrogenase-elevating virus of in vivo interleukin 4 production during immunization with keyhole limpet haemocyanin. Cytokine 5, 394–397.[Medline]

Muller, U., Steinhoff, U., Reis, L. F., Hemmi, S., Pavlovic, J., Zinkernagel, R. M. & Aguet, M. (1994). Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921.[Medline]

Nguyen, K. B., Watford, W. T., Salomon, R., Hofmann, S. R., Pien, G. C., Morinobu, A., Gadina, M., O'Shea, J. J. & Biron, C. A. (2002). Critical role for STAT4 activation by type 1 interferons in the interferon-gamma response to viral infection. Science 297, 2063–2066.[Abstract/Free Full Text]

Ochsenbein, A. F., Pinschewer, D. D., Odermatt, B., Ciurea, A., Hengartner, H. & Zinkernagel, R. M. (2000). Correlation of T cell independence of antibody responses with antigen dose reaching secondary lymphoid organs: implications for splenectomized patients and vaccine design. J Immunol 164, 6296–6302.[Abstract/Free Full Text]

Okamura, H., Tsutsi, H., Komatsu, T. & other authors (1995). Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378, 88–91.[CrossRef][Medline]

Oppmann, B., Lesley, R., Blom, B. & 22 other authors (2000). Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725.[Medline]

Oxenius, A., Karrer, U., Zinkernagel, R. M. & Hengartner, H. (1999). IL-12 is not required for induction of type 1 cytokine responses in viral infections. J Immunol 162, 965–973.[Abstract/Free Full Text]

Pflanz, S., Timans, J. C., Cheung, J. & 18 other authors (2002). IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells. Immunity 16, 779–790.[Medline]

Quint, W., Gielkens, A., van Oirschot, J., Berns, A. & Cuypers, H. T. (1987). Construction and characterization of deletion mutants of pseudorabies virus: a new generation of ‘live’ vaccines. J Gen Virol 68, 523–534.[Abstract]

Raj, N. B., Cheung, S. C., Rosztoczy, I. & Pitha, P. M. (1992). Mouse genotype affects inducible expression of cytokine genes. J Immunol 148, 1934–1940.[Abstract/Free Full Text]

Rogers, P. R. & Croft, M. (1999). Peptide dose, affinity, and time of differentiation can contribute to the Th1/Th2 cytokine balance. J Immunol 163, 1205–1213.[Abstract/Free Full Text]

Rogge, L., D'Ambrosio, D., Biffi, M., Penna, G., Minetti, L. J., Presky, D. H., Adorini, L. & Sinigaglia, F. (1998). The role of Stat4 in species-specific regulation of Th cell development by type I IFNs. J Immunol, 6567–6574.

Romer-Oberdorfer, A., Mundt, E., Mebatsion, T., Buchholz, U. J. & Mettenleiter, T. C. (1999). Generation of recombinant lentogenic Newcastle disease virus from cDNA. J Gen Virol 80, 2987–2995.[Abstract/Free Full Text]

Rosenberger, J. K., Sterner, F. J., Botts, S., Lee, K. P. & Margolin, A. (1989). In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates. Avian Dis 33, 535–544.[Medline]

Samuel, C. E. (2001). Antiviral actions of interferons. Clin Microbiol Rev 14, 778–809.[Abstract/Free Full Text]

Schijns, V. E., Haagmans, B. L., Rijke, E. O., Huang, S., Aguet, M. & Horzinek, M. C. (1994). IFN-gamma receptor-deficient mice generate antiviral Th1-characteristic cytokine profiles but altered antibody responses. J Immunol 153, 2029–2037.[Abstract/Free Full Text]

Schijns, V. E., Haagmans, B. L., Wierda, C. M., Kruithof, B., Heijnen, I. A., Alber, G. & Horzinek, M. C. (1998). Mice lacking IL-12 develop polarized Th1 cells during viral infection. J Immunol 160, 3958–3964.[Abstract/Free Full Text]

Snapper, C. M. & Paul, W. E. (1987). Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236, 944–947.[Medline]

Spiegelberg, H. L. (1974). Biological activities of immunoglobulins of different classes and subclasses. Adv Immunol 19, 259–294.[Medline]

Suzuki, K., Mori, A., Ishii, K. J., Saito, J., Singer, D. S., Klinman, D. M., Krause, P. R. & Kohn, L. D. (1999). Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. Proc Natl Acad Sci U S A 96, 2285–2290.[Abstract/Free Full Text]

Szomolanyi-Tsuda, E. & Welsh, R. M. (1996). T cell-independent antibody-mediated clearance of polyoma virus in T cell-deficient mice. J Exp Med 183, 403–411.[Abstract]

van den Broek, M. F., Muller, U., Huang, S., Aguet, M. & Zinkernagel, R. M. (1995). Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors. J Virol 69, 4792–4796.[Abstract]

Visser, N. & Lütticken, D. (1989). Experiences with a gI-/TK- modified live pseudorabies virus vaccine: strain Begonia. In Vaccinatation and Control of Aujeszky's Disease, pp. 37–44. Edited by J. T. Van Oirschot.

Wenner, C. A., Guler, M. L., Macatonia, S. E., O'Garra, A. & Murphy, K. M. (1996). Roles of IFN-gamma and IFN-alpha in IL-12-induced T helper cell-1 development. J Immunol 156, 1442–1447.[Abstract]

Xing, Z., Zganiacz, A., Wang, J., Divangahi, M. & Nawaz, F. (2000). IL-12 independent Th1-type immune responses to respiratory viral infection: requirement of IL-18 for IFN-{gamma} release in the lung but not for differentiation of viral-reactive Th1-type lymphocytes. J Immunol 164, 2575–2584.[Abstract/Free Full Text]

Received 27 August 2003; accepted 10 December 2003.



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