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Short Communication |
1 Institute of Veterinary Virology, University of Bern, Switzerland
2 Institute of Animal Genetics, Nutrition and Housing, University of Bern, Switzerland
3 Clinical Research, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Switzerland
Correspondence
Giuseppe Bertoni
bertoni{at}ivv.unibe.ch
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Present address: UMR754 INRA-ENVL-UCBL, ‘Rétrovirus et Pathologie Comparée’, IFR128, Université Claude Bernard Lyon 1, 50 avenue Tony Garnier, 69007 Lyon, France. 
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). The quality, rather than the magnitude alone, of this cellular response determines the effect on virus replication, i.e. restriction or enhancement. Indeed, vaccination trials against lentiviruses have shown that vaccines might have deleterious effects, increasing the susceptibility to infection instead of conferring protection. Enhancing antibodies induced by vaccination account only for a proportion of these vaccine side effects and strong evidence has been found that helper T cells might also favour virus replication in vaccinated animals (Raabe et al., 1998; Richardson et al., 1997, 2002; Staprans et al., 2004). In these experiments, challenge infection of vaccinated animals resulted in more rapid increase or higher peaks of viral load, an increased proportion of infected animals and, finally, an increased severity and accelerated progression of virus-associated lesions. These studies point to potential danger for human immunodeficiency virus (HIV) vaccination trials and offer an opportunity to identify the correlates of protection or enhancement through the functional characterization of the cellular immune response induced by vaccination.
Small ruminant lentiviruses (SRLVs) include caprine arthritis encephalitis virus (CAEV) and maedi–visna virus (MVV). CAEV causes a persistent infection in goats, typically characterized by slowly progressing arthritis of the carpal joints and indurative mastitis, whereas MVV causes progressive pneumonia, mastitis and, rarely, encephalitis in infected sheep. SRLV does not infect T cells and the infected animals remain immunocompetent throughout the infection. Monocytes are the principal cellular targets of SRLV, and virus replication in these cells is restricted until their maturation to macrophages in tissues. In spite of their resistance to SRLV infection, CD4+ T cells have been shown to be crucial in MVV infection, as the number of infected monocytes in the draining lymph nodes of challenged animals is drastically reduced in sheep depleted of CD4+ T cells (Eriksson et al., 1999). To test the effect of inducing a CAEV-specific T-helper-cell response on virus replication in vivo, we immunized goats with a CAEV synthetic peptide capable of priming a strong CD4+ T-cell response and subsequently challenged the animals with CAEV. The peptide used was part of the Gag structural protein of CAEV, which shows a strong structural and sequence homology with the homologous peptide of HIV and other retroviruses and carries an immunodominant T-helper epitope capable of inducing a strong immune response in vaccinated goats (Fluri et al., 2006). In the original study describing this peptide, we showed that the caprine leukocyte antigen (CLA) haplotype influenced the immunological response to the Gag peptide in vaccinated animals (Fluri et al., 2006). The CLA type is also associated with the incidence of clinical arthritis in goats naturally infected with CAEV (Ruff & Lazary, 1988; Ruff et al., 1993), a situation analogous to that observed in HIV, where specific HLA alleles relate to delayed or accelerated progression to AIDS (Martin & Carrington, 2005). The goats included in our experiment carried a CLA associated with arthritis in CAEV-infected goats and a slow and low response to the Gag peptide (Be1-D5), a haplotype associated with the absence of clinical lesions and a rapid and high response to the immunizing peptide (Be10-D2), or were heterozygous (Ruff & Lazary, 1988; Ruff et al., 1993). The experimental set-up was chosen: (i) to assess the role of the CLA background in the quality of the immune response induced by the virus challenge in non-vaccinated animals, (ii) to determine the consequences of these potential differences in the type of immune response for the capacity to control the viral load, and (iii) to evaluate the effects of stimulating a specific immune response to the Gag peptide on the equilibrium between virus and host following a CAEV challenge. The strength and quality of the T-cell responses and the viral load of the animals were monitored by T-cell proliferation assays, real-time RT-PCR for the mRNA of several cytokines, and viral RNA and real-time PCR for the proviral load (Ravazzolo et al., 2006). Nineteen Saanen goats were included in the experiment and were divided in two groups according to their CLA type to assure an equal distribution of CLA haplotypes (Be1-D5, Be10-D2 or heterozygous) between the vaccine and the control groups. Nine goats were immunized intramuscularly with 100 µg synthetic Gag peptide emulsified in Freund’s incomplete adjuvant and received a booster shot 21 days later. All goats were challenged intravenously with 5x105 TCID50 of molecularly cloned CAEV strain CO. Proviral DNA and viral mRNA were quantified in peripheral blood mononuclear cells (PBMCs) collected at different time points after challenge. At 55 days post-infection (p.i.), Gag-immunized goats carried a significantly higher concentration of provirus in their blood than the control group, pointing to vaccine-mediated enhancement of virus replication, whereas, at a later stage, both groups controlled the infection with similar efficiency (Fig. 1). Detection of viral Env and Rev transcripts, as indicators of local ongoing virus replication, succeeded only in the PBMCs of a few animals and only for the last two time points examined (results not shown), supporting previous observations that blood is not a preferred site of CAEV replication (Ravazzolo et al., 2006).
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T-cell responses to vaccination and infection were tested by monitoring the proliferative response of T cells following stimulation with the Gag peptide and with a whole-virus preparation, as described previously (Fluri et al., 2006). As predicted, at 5 and 12 days p.i., we found a significant difference in Gag peptide-induced T-cell proliferation between the vaccinated and the control animals (P=0.017 and P=0.001, respectively; one-sided Wilcoxon rank sum test). At these two time points, only one animal of the control group showed a proliferative activity with a stimulation index (SI) of 5.85 and 12.5, respectively, whereas cells from five (day 5) and seven (day 12) vaccinated goats proliferated with an SI as high as 96. This statistically significant difference was lost in the later time points, due to the increased response to the Gag peptide in the infected control animals. No significant difference between vaccinated and control animals was observed following stimulation with whole virus. To test the hypothesis that functionally inappropriate help provided by CD4+ T lymphocytes might impair the control of CAEV replication, we characterized the transcription of cytokine mRNA genes in PBMCs collected at 12, 55 and 480 days p.i., stimulated in vitro either with whole-virus preparation or with the Gag peptide. Previous studies have shown that a CD4+ T-helper response skewed towards high levels of interleukin (IL)-4 transcription and a lower gamma interferon (IFN-
) response in PBMCs stimulated in vitro with viral antigen is predictive for disease (Cheevers et al., 1997). No correlations were observed for samples collected at 12 or 480 days p.i. In contrast, at 55 days p.i., the proviral load in PBMCs of vaccinated goats correlated strongly with IFN- mRNA expression by PBMCs stimulated in vitro with whole-virus preparation, and even more strongly with IL-4 mRNA expression (Fig. 2a, b). No correlation was shown for PBMCs stimulated in vitro with the Gag peptide (data not shown). We observed a highly significant correlation between IL-4 and IFN- mRNA following stimulation with either Gag peptide or whole-virus preparation (Fig. 2c, d). IFN- production was confirmed using a commercial ELISA (Bovigam; Biocor Animal Health, Omaha, NE, USA) on the supernatants of in vitro-stimulated PBMCs. We detected a correlation (P=0.04, data not shown) between the proviral load at 55 days p.i. and IFN- protein for PBMCs of vaccinated goats following stimulation with whole-virus preparation. No statistical correlation was found for the control group. We therefore concluded that the inefficient control of CAEV replication in vaccinated goats did not depend on either IFN- or IL-4, although both cytokines could be seen as markers of in vitro activation of CD4+ T cells. Granulocyte–macrophage colony-stimulating factor (GM-CSF) induces monocyte to macrophage maturation, stimulates MVV replication in vitro and has been shown to be overexpressed in alveolar macrophages of sheep naturally infected with MVV (Zhang et al., 2002). Furthermore, GM-CSF treatment of cells transfected with a CAEV plasmid has been shown to activate virus replication (Murphy et al., 2006). Hence, we measured GM-CSF mRNA expression in the PBMCs of both groups, stimulated in vitro with Gag peptide or whole-virus preparation. With both stimuli, we observed a significant correlation between the proviral load and GM-CSF mRNA in vaccinated goats (Fig. 3) and between GM-CSF and either IFN- or IL-4 mRNA, at 55 days p.i. (data not shown). At day 12 p.i., the PBMCs of the vaccinated animals stimulated with the Gag peptide expressed a higher level of GM-CSF mRNA than the control animals (P=0.01; one-sided Wilcoxon rank sum test). No significant correlation between GM-CSF mRNA and either proviral load or IFN- and IL-4 mRNA was observed at the other two time points examined. In light of the biological activity of this cytokine on SRLV, these results suggest GM-CSF as a key cytokine driving virus replication in the early phase of infection. The enhanced expression of this cytokine by Gag peptide-primed CD4 T cells may explain the higher viral load measured in the vaccinated animals. To confirm the role of GM-CSF in the chronic phase of the infection, we analysed the expression of GM-CSF in different tissues of five goats that had been experimentally infected with CAEV for 8 years (Ravazzolo et al., 2006). To our surprise, we found that GM-CSF was more strongly expressed where the virus replication levels were lower (data not shown). This suggests that at a time when the virus had reached its set point and a stable balance with its host, the expression of GM-CSF was no longer the crucial factor controlling the viral load. This may be a logical consequence of the maturation of the immune response, whose efficiency, paradoxically, may be bolstered by the expression of GM-CSF (Robinson et al., 2006).
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). Compared with the control animals, the range of viral loads measured in the vaccinated animals was narrower and appeared to be synchronized over time. Eventually, the immune system of both groups of animals succeeded in controlling the viral load quite efficiently. This notwithstanding, the enhancement of infection observed at day 55 p.i., although restricted to an early time point of infection, indicates that great care should be used when designing vaccine candidates against lentiviruses. It also points to new directions aiming at the identification of correlates of immune protection or enhancement. In particular, it needs to be clarified whether, in addition to GM-CSF, other factors facilitating CAEV replication act in a paracrine fashion, exclusively in the context of a close cognate type of contact between the infected macrophage and the virus-specific CD4+ T cell. Indeed, CAEV might have developed an as-yet-unknown strategy to manipulate the immunological response of CD4+ T cells either directly or by manipulating the expression of immunomodulators in infected antigen-presenting cells, as already shown for in vitro-cultured macrophages (Lechner et al., 1997). We are currently evaluating these possibilities. Finally, as CAEV does not infect T cells, it may represent a better model than other lentiviruses to study the effect of a T-cell response on enhancement of virus replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Eriksson, K., McInnes, E., Ryan, S., Tonks, P., McConnell, I. & Blacklaws, B. A. (1999). CD4+ T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo. Virology 258, 355–364.[CrossRef][Medline]
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Zhang, Z., Harkiss, G. D., Hopkins, J. & Woodall, C. J. (2002). Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro. Clin Exp Immunol 129, 240–246.[CrossRef][Medline]
Received 17 December 2006;
accepted 28 January 2007.
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