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
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ABSTRACT |
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Present address: Abbott BV, Siriusdreef 51, 2132 WT Hoofddorp, The Netherlands.
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INTRODUCTION |
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Production of IFN- 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-
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-
(Cousens et al., 1997
). IFN-
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-
may stimulate IFN-
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--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-
/
function.
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METHODS |
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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 67 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
-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- 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-
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.
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RESULTS |
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Endogenous IFN- but not IFN-
/
or IL-12 is required for IgG2a isotype switching
To assess the contribution of IFN- in isotype switching to IgG2a, antibody levels were determined in IFN-
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-
is largely responsible for the preferential IgG2a isotype response. We then looked for IFN-
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-
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-
, 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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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-
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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DISCUSSION |
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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- function (Markine-Goriaynoff et al., 2000
; Muller et al., 1994
), our studies demonstrate that the functional presence of IFN-
substantially increases IgG2a isotype synthesis in response to iPRV. Notably, restimulation of IFN-
R-/- splenocytes with virus antigen resulted in high IFN-
and no detectable IL-5 production. This suggests that IFN-
function is not essential to drive virion-induced IFN-
production by T cells. Similar to earlier studies (Schijns et al., 1994
) IFN-
R-/- splenocytes produced higher levels of IFN-
than WT splenocytes. We speculate on less IFN-
-mediated feedback inhibition or less cytokine consumption in the absence of a functional IFN-
receptor.
Surprisingly, iPRV-induced responses fail to default to type 2 immune reactions in the absence of IFN-/
or IL-12 gene function. IFN-
/
, 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-
/
, 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--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.
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ACKNOWLEDGEMENTS |
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Received 27 August 2003;
accepted 10 December 2003.
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