1 BAYER AG Pharmaceutical Division, Antiinfective Research, D-42096 Wuppertal, Germany
2 Institute of Medical Immunology, Humboldt University Berlin, Medical School (Charité), Campus Mitte, D-10098 Berlin, Germany
3 Zentrum für Molekulare Biologie (ZMBH), Ruprecht Karls University, D-69120 Heidelberg, Germany
4 BAYER AG Animal Health R&D/Bio, Leverkusen, Germany
5 Department of Microbiology, Virus Research Unit, University of Otago, Dunedin, New Zealand
Correspondence
Olaf Weber
Olaf.Weber.b{at}bayer.com
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Published ahead of print on 30 April 2003 as DOI 10.1099/vir.0.19138-0
Present address: Bayer Corporation, Pharmaceutical Division, Department of Cancer Research, 400 Morgan Lane, West Haven, CT 06516-4175, USA.
Present address: Mixis France, Faculté de Médicine Necker, 156, rue de Vaugirard, 75015 Paris, France.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Virus infections may modulate the clinical course of concomitant infections by other pathogens. It was demonstrated previously that virus infections of the liver abolish hepatocellular replication of human hepatitis B virus (HBV) in a non-cytolytic fashion mediated by inflammatory cytokines (Guidotti et al., 1996; Cavanaugh et al., 1998
). Based on this observation, it was postulated that virus clearance during human HBV infection is due primarily to this process rather than the destruction of infected cells (Guidotti et al., 1996
, 1999
). This paradigm shift in virus immunology may impact on future antiviral strategies. We wanted to utilize the paradigm for an immune-therapeutic approach using PPVO to combine immune evasion and immune stimulatory mechanisms. In this report, we demonstrate that inactivated PPVO induces a self-regulatory cytokine response that involves the upregulation of IL-12, IL-18, IFN-
and other T helper (Th) 1-type cytokines and their subsequent downregulation. Furthermore, we show that PPVO is effective in mice infected with herpes simplex virus type 1 (HSV-1), in a guinea pig model of recurrent genital herpes disease and in a transgenic mouse model of human HBV replication, without any signs of inflammation or other side effects. We conclude that induction of a self-regulatory cytokine response by an inactivated virus might have some advantages over existing immune therapies and that inactivated PPVO should be investigated in detail as a potential antiviral drug.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cytokine mRNA measurement.
Female BALB/cJ mice (4 weeks old, approximately 20 g body weight) were purchased from a commercial supplier (Bomholdtgard). Mice were divided into three treatment groups (n=20 animals per group): (i) PPVO strain D1701 (5x105 TCID50), diluted in 200 µl non-pyrogenic PBS (Seromed); (ii) complete Freund's adjuvant (CFA) (Sigma); and (iii) non-pyrogenic PBS (placebo). At 6, 12, 24 and 48 h after treatment, five mice were sacrificed and peritoneal cells, liver, axillar, gastric/epigastric/mesenteric lymph nodes and spleen were collected. Total RNA was prepared and cytokine gene expression was quantified using a competitive RT-PCR, as described previously (Siegling et al., 1994). Primer sequences used for competitive RT-PCR are provided in Table 1
. PCR products were subjected to agarose (1 %) gel electrophoresis and quantified using a video imaging system (Herolab) with the appropriate software.
|
Guinea pig model of genital herpes.
Female Hartley guinea pigs (Charles River Laboratories) were infected intravaginally with 2·5x105 p.f.u. HSV-2 strain MS. Clinical symptoms were scored as follows: 0, no lesion; 1, erythema; 2, vesicles; 3, confluent lesions; and 4, necrotizing vulvovaginitis. Animals with acute infection (score 3) were randomized and divided into three groups (n=10 animals per group). Treatment was started 10 days after healing of the disease (score 0). PPVO (1x106 TCID50) was administered i.p. every third day, five times in total. Acyclovir (Glaxo Wellcome) was administered twice daily for 10 consecutive days i.p. at a dosage of 25 mg kg-1 (per dose). Animals were examined daily for 40 consecutive days for herpes lesions; severity was scored on a scale of 04.
HBV transgenic mice.
HBV transgenic mice [Tg (HBV1.3 fsX-3'5')] that carry a frameshift mutation (GC) at position 2916/2917 (C. Kuhn & H. Schaller, unpublished; Weber et al., 2002) were used (n=16; 8 male and 8 female animals per group). PPVO was administered i.p. every third day, three times in total, unless indicated otherwise. Frozen tissue (50 mg) was minced and digested with proteinase K (Roche) over night at 56 °C. Nucleic acids were extracted using the phenol/chloroform procedure. Southern blot analysis was performed using 20 µg PstI-restricted genomic DNA. Before electrophoresis, DNA was digested with RNase A (Qiagen). Quantitative analysis of hepadnaviral nucleic acid was performed essentially as described recently (Weber et al., 2002
).
Histological and immunohistological analyses.
Analyses were performed as described recently (Weber et al., 2002). Briefly, liver specimens from one or two lobes were fixed in 4 % formaldehyde solution overnight at room temperature and embedded in paraffin. For immunohistochemical analysis, a polyclonal rabbit antibody against the HBV capsid antigen (HBcAg) (Dako) was used. Staining was carried out using the Vectastain ABC kit, as described by the manufacturer (Vector Laboratories).
Studies with human peripheral blood leukocytes.
Heperanized whole blood samples (100 µl) were diluted 1 : 10 with endotoxin-free culture medium and cultivated at 37 °C for 4 (TNF, IL-12) or 48 (IFN-) h in the presence or absence of low dose Concanavalin A (ConA, 12 µg ml-1) (Sigma), phytohaemagglutinin (PHA, 1 : 5000) (Sigma), mAb OKT-3 (1 µg ml-1) and different concentrations of PPVO. Supernatants were collected and measured by commercially available ELISA kits (IFN-
and IL-12 p70, Biosource; TNF-
, Immulite).
Statistical analysis.
Cytokine expression profiles were analysed using the parameter-free Wilcoxon test. HBV DNA reduction was analysed using variance analysis with subsequent post-hoc comparison of means. Survival analyses were performed using log rank analysis (STATISTICA, StatSoft).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
PPVO inhibits HBV replication in a transgenic mouse model
We have used HBV transgenic mice [strain Tg (HBV1.3 fsX-3'5')] that carry a frameshift mutation (GC) at position 2916/2917 (C. Kuhn & H. Schaller, unpublished; Weber et al., 2002). PPVO-treated mice showed approximately 7080 % less HBV DNA in the liver and 100 times less HBV DNA in the plasma in comparison to placebo-treated mice (P=0·04 and 0·002, respectively; variance analysis with post-hoc comparison of means) (Fig. 5
A, B). Significant effects on HBV replication were observed after a 3 week treatment using 30 mg 3TC kg-1 three times a day per os. PPVO activity was dose-dependent in plasma (Fig. 5C
) and in the liver (data not shown). The antiviral effect was maintained after repeated administrations (Fig. 5D
). Administration of a mAb against IFN-
reduced the antiviral effect significantly in the livers (Fig. 5E
) and plasma (data not shown) of HBV transgenic mice. Southern blot analysis was performed to compare the levels of replicative intermediates of HBV DNA in the livers. HBV DNA replication was unaffected in placebo-treated mice (Fig. 6
A) but was almost undetectable in the livers of mice treated with PPVO (Fig. 6B
). PPVO treatment has also led to almost undetectable levels of HBV-specific RNAs in the liver of these mice (data not shown). In addition, HBcAg, which is indicative of ongoing HBV replication, was markedly reduced in PPVO-treated mice (Fig. 6D
) but not affected in placebo-treated mice (Fig. 6C
). The intranuclear capsid antigen was also affected by PPVO treatment. No infiltration of lymphocytes or other cells has been detected histologically and liver enzymes were normal during and after treatment.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mechanisms of IFN- induction
Expression of the IFN--inducing cytokine IL-12 was elevated in mice treated with PPVO to levels that were also observed after CFA administration, although CFA induced much less IFN-
. Moreover, CFA was not able to protect the mice against virus infections. IL-18, a cytokine shown recently to be a powerful inducer of IFN-
(Micallef et al., 1996
), was induced only in PPVO-treated mice, an observation suggesting a role of IL-18 in PPVO-mediated biological effects in vivo. Upregulation of mRNA for both IFN-
-inducing cytokines could be found at the site of PPVO application only, whereas IFN-
upregulation was found both at the site of PPVO injection and in lymphoid organs. These data suggest that the IFN-
-inducing cytokines are induced locally by PPVO, circulate systemically (for cytokines they have an untypical long half-life) and upregulate IFN-
secretion by antigen-triggered IL-12/IL-18-responsive T cells at the site of injection of PPVO as well as in peripheral immune organs such as lymph nodes and spleen. IFN-
mediates directly or indirectly its systemic antiviral efficacy (upregulation of MHC molecules, activation of CTL and NK cells, activation of macrophages and induction of opsonizing antibodies, etc.). Since administration of neutralizing antibodies against IFN-
, but not against IL-12 or IL-18, abolished antiviral activity against HSV and reduced activity against HBV, other scenarios of IFN-
induction are possible. Schijns et al. (1998)
demonstrated that, following an infection with mouse hepatitis virus, mice with a targeted disruption of the IL-12 p40 and/or p35 gene were still capable of producing a polarized Th1-type cytokine response, as evidenced by high IFN-
and non-detectable IL-4 production. Therefore, IL-12 and IL-18 may complement each other in PPVO-mediated IFN-
induction; it has been shown recently that IL-18 has antiviral activity against HBV (Kimura et al., 2002
). Since we have used an inactivated virus preparation, it is unlikely that de novo-synthesized viral proteins mediate cytokine induction. The effects we have observed were induced by treatment with preparations of whole PPVO but, using a vaccinia virus-based library of PPVO DNA, we have been able to identify PPVO candidate proteins that are responsible for the IFN-
-inducing activity (A. Friebe et al., unpublished data). Consistent with previous studies that were performed in vitro (Buettner et al., 1995
), vaccinia virus in these experiments did not possess antiviral activity in vivo.
In human cells, PPVO could also induce IFN- (together with a suboptimal T cell receptor stimulus) and this effect was blocked partially by anti-IL-12 and anti-IL-18 mAbs. When both mAbs were used together, IFN-
production was blocked almost completely (data not shown).
Possible advantages of inactivated PPVO over antiviral cytokine monotherapies
In addition to its IFN--stimulating activity, IL-18 has also pro-Th2 effects. It has been reported recently that IL-18 enhances IL-4 production by ligand-activated NK T lymphocytes (Leite-de-Moraes et al., 2000
). Therefore, IL-18 could also mediate the increase in IL-4. On the other hand, IL-4 has been demonstrated to downregulate the IL-18 receptor
chain, thereby negatively regulating IL-18 and IL-18-mediated effects (Smeltz et al., 2001
). We conclude that the PPVO-mediated IL-4 response might be part of cytokine networking and responsible for the downmodulation of the initial Th1 immune response. Further studies to address this question are in progress.
Our results are consistent with the finding that inflammatory cytokines are capable of abolishing HBV replication and HBV gene expression non-cytopathically (Guidotti & Chisari, 1996; Cavanaugh et al., 1998
; Guidotti et al., 1996
, 1999
). The therapeutic use of viruses that are not inactivated would have certain risks and could lead to uncontrollable effects. However, the application of therapeutic cytokines is limited. The half-life of recombinant IFN-
is low and the protein would have to have been administered at high dosages, which, in turn, would lead to serious side effects. In contrast to a single systemic application of recombinant IFN-
, PPVO appears to upregulate other effector cytokines also (TNF, etc.) and, in parallel, it induces regulatory cytokines, such as IL-4, detectable after 2448 h, in lymph nodes, and IL-10 in the liver. This may explain the high efficiency in virus clearing without significant evidence for harmful tissue destruction, particularly in transgenic mice. It has been shown that IL-12 administration is therapeutically useful in HBV transgenic mice (Cavanaugh et al., 1997
). Most of the antiviral activity of IL-12 is mediated via IFN-
induction, with the longer in vivo half-life of IL-12 explaining its higher efficacy as compared to IFN-
. Importantly, although we have observed a more pronounced Kupffer cell reaction in the livers of PPVO-treated HBV transgenic mice, no signs of toxicity or inflammation have been observed histologically and liver enzymes were found in a normal range upon and after treatment with PPVO (data not shown). IL-10, which was induced in the liver after PPVO administration, is known to downregulate T cell activation by antigen-presenting liver sinusoidal cells (Knolle et al., 1998
). We speculate that the lack of any inflammation in the livers of PPVO-treated mice might be related to the prolonged induction of IL-10 expression and the constant efficacy after repeated dosing by some of the unique immune escape mechanisms mediated by PPVO-encoded proteins (Haig & Mercer, 1998
; Haig & McInnes, 2002
; McKeever et al., 1988
; Haig et al., 1996
, 1997
; Kruse & Weber, 2001
).
Also, we did not find inflammation in pathological examinations of HSV-infected guinea pigs. It has been described recently that IFN- is responsible for the clearance of virus infection from the CNS (Binder & Griffin, 2001
). Using the guinea pig model of recurrent genital herpes, we could answer three questions: (i) the effects of PPVO are not mouse specific; (ii) we are able to target infections even at immune-privileged sites such as the CNS; and (iii) this is possible without side effects.
Putative therapeutic options
Interestingly, PPVO induced the cytokine network in human blood cells. It stimulates TNF- and IL-12 secretion directly and, in pre-activated T cells, IFN-
. Blocking experiments with BPI demonstrated that this effect was not due to endotoxin contamination. Thus, PPVO might express similar effects in humans as in mice.
In summary, our data show that inactivated PPVO (strain D1701) has antiviral activity and that the induction of a cytokine cascade by inactivated PPVO might have advantages over existing immune therapies. More studies are needed to investigate the interaction of inactivated PPVO with the immune system of chronically infected animals. We conclude from our data that inactivated PPVO should be investigated further as a potential antiviral drug.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Binder, G. K. & Griffin, D. E. (2001). Interferon--mediated site-specific clearance of alphavirus from CNS neurons. Interferon-
-mediated site-specific clearance of alphavirus from CNS neurons. Science 293, 303306.
Büttner, M., Czerny, C. P., Lehner, K. H. & Wertz K. (1995). Interferon induction in peripheral blood mononuclear leukocytes of man and farm animals by poxvirus vector candidates and some poxvirus constructs. Vet Immunol Immunopathol 46, 237250.[CrossRef][Medline]
Cavanaugh, V. J., Guidotti, L. G. & Chisari, F. V. (1997). Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. J Virol 71, 32363243.[Abstract]
Cavanaugh, V. J., Guidotti, L. G. & Chisari, F. V. (1998). Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in transgenic mice. J Virol 72, 26302637.
Deane, D., McInnes, C. J., Percival, A. & 7 other authors (2000). Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2. J Virol 74, 13131320.
del Val, M., Hengel, H., Häcker, H., Hartlaub, U., Ruppert, T., Lucin, P. & Koszinowski, U. H. (1992). Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment. J Exp Med 176, 729738.[Abstract]
Fleming, S. B., McCaughan, C. A., Andrews, A. E., Nash, A. D. & Mercer, A. A. (1997). A homolog of interleukin-10 is encoded by the poxvirus orf virus. J Virol 71, 48574861.[Abstract]
Förster, R., Wolf, G. & Mayr, A. (1994). Highly attenuated poxviruses induce functional priming of neutrophils in vitro. Arch Virol 136, 219226.[Medline]
Grandvaux, N., tenOever, B. R., Servant, M. J. & Hiscott, J. (2002). The interferon antiviral response: from viral invasion to evasion. Curr Opin Infect Dis 1, 259267.
Guidotti, L. G. & Chisari, F. V. (1996). Current Opinion in Immunology, vol. 8, pp. 487483. Edited by R. Zinkernagel & B. Bloom. London: Current Biology.
Guidotti, L. G., Borrow, P., Hobbs, M. V., Matzke, B., Gresser, I., Oldstone, M. B. A. & Chisari, F. V. (1996). Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Natl Acad Sci U S A 93, 45894594.
Guidotti, L. G., Rochford, R., Chung, J., Shapiro, M., Purcell, R. & Chisari, F. V. (1999). Viral clearance without destruction of infected cells during acute HBV infection. Science 284, 825829.
Haig, D. M. & Mercer, A. A. (1998). Ovine disease. Orf. Vet Res 29, 311326.[Medline]
Haig, D. M. & McInnes, C. J. (2002). Immunity and counter-immunity during infection with the parapoxvirus orf virus. Virus Res 88, 316.[CrossRef][Medline]
Haig, D., McInnes, C., Deane, D. & 8 other authors (1996). Cytokines and their inhibitors in orf virus infection. Vet Immunol Immunopathol 54, 261267.[CrossRef][Medline]
Haig, D., McInnes, C. J., Deane, D., Reid, H. W. & Mercer, A. A. (1997). The immune and inflammatory response to orf virus. Comp Immunol Microbiol Infect Dis 20, 197204.[CrossRef][Medline]
Haig, D. M., McInnes, C. J., Thomson, J., Wood, A., Bunyan, K. & Mercer, A. A. (1998). The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double stranded RNA-dependent kinase. Immunology 93, 335340.[CrossRef][Medline]
Kimura, K., Kakimi, K., Wieland, S., Guidotti, L. G. & Chisari, F. V. (2002). Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 76, 1070210707.
Knolle, P. A., Uhrig, A., Hegenbarth, S., Loser, E., Schmitt, E., Gerken, G. & Lohse, A. W. (1998). IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules. Clin Exp Immunol 114, 427433.[CrossRef][Medline]
Kruse, N. & Weber, O. (2001). Selective induction of apoptosis in antigen-presenting cells in mice by Parapoxvirus ovis. J Virol 75, 46994704.
Lane, H. C., Depper, J. M., Greene, W. C., Whalen, G., Waldmann, T. A. & Fauci, A. S. (1985). Qualitative analysis of immune function in patients with the acquired immunodeficiency syndrome. Evidence for a selective defect in soluble antigen recognition. New Engl J Med 2, 7984.
Leite-de-Moraes, M. C., Hameg, A., Pacilio, M., Koezuka, Y., Taniguchi, M., Van Kaer, L., Schneider, E., Dy, M. & Herbelin A. (2000). IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells. J Immunol 166, 945951.
Marsig, E. & Stickl, H. (1988). Highly attenuated poxviruses induce functional priming of neutrophils in vitro. Zentralbl Veterinarmed B 35, 601609 (in German).[Medline]
Mayr, A., Büttner, M., Wolf, G., Meyer, H. & Czerny, C. (1989). Experimental detection of the paraspecific effects of purified and inactivated poxviruses. Zentralbl Veterinarmed B 36, 8199 (in German).[Medline]
McKeever, D. J., Jenkinson, D. M., Hutchinson, G. & Reid, H. W. (1988). Studies on the pathogenesis of orf virus infection in sheep. J Comp Pathol 99, 317328.[Medline]
Micallef, M. J., Ohtsuki, T., Kohno, K. & 9 other authors (1996). Interferon--inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-
production. Eur J Immunol 26, 16471651.[Medline]
Mocarski, E. S., Jr (2002). Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol 10, 332339.[CrossRef][Medline]
Mossman, K. L. (2002). Activation and inhibition of virus and interferon: the herpesvirus story. Viral Immunol 15, 315.[CrossRef][Medline]
Schijns, V. E. C. J., Haagmans, B. L., Wierda, C. M. H., Kruithof, B., Heijnen, I. A. F. M., Alber, G. & Horzinek, M. C. (1998). Mice lacking IL-12 develop polarized Th1 cells during viral infection. J Immunol 160, 39583964.
Siegling, A., Lehmann, M., Platzer, C., Emmrich, F. & Volk, H. D. (1994). A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. J Immunol Methods 177, 2328.[CrossRef][Medline]
Smeltz, R. B., Chen, J., Hu-Li, J. & Shevach, E. M. (2001). Regulation of interleukin (IL)-18 receptor chain expression on CD4+ T cells during T helper (Th)1/Th2 differentiation: critical downregulatory role of IL-4. J Exp Med 194, 143153.
Weber, O., Schlemmer, K. H., Hartmann, E. & 10 other authors (2002). Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res 54, 6975.[CrossRef][Medline]
Received 31 January 2003;
accepted 5 April 2003.