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A Mechanism for the Inhibition of Fever by a Virus (Interleukin-118/Cytokine Receptor/Virus Pathogenesis/Vaccinia Virus) ANTONIO ALCAMI and GEOFFREY L

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A Mechanism for the Inhibition of Fever by a Virus (Interleukin-118/Cytokine Receptor/Virus Pathogenesis/Vaccinia Virus) ANTONIO ALCAMI and GEOFFREY L Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11029-11034, October 1996 Immunology A mechanism for the inhibition of fever by a virus (interleukin-118/cytokine receptor/virus pathogenesis/vaccinia virus) ANTONIO ALCAMI AND GEOFFREY L. SMITH* Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom Communicated by Bernard Moss, National Institute ofAllergy and Infectious Diseases, Bethesda, MD, July 19, 1996 (received for review December 1995) ABSTRACT Poxviruses encode proteins that block the cies of postvaccinial complications, but the reasons are un- activity of cytokines. Here we show that the study of such known (6). virulence factors can contribute to our understanding of not Poxviruses encode many proteins that interfere with host only virus pathogenesis but also the physiological role of immune functions (7, 8), such as soluble cytokine receptors cytokines. Fever is a nonspecific response to infection that that blockade cytokines and modulate virus virulence (9). The contributes to host defense. Several cytokines induce an VV IL-1,B receptor (vIL-103R) is encoded by gene B15R in the elevation ofbody temperature when injected into animals, but Western Reserve (WR) strain (10), and binds IL-1l3 with high in naturally occurring fever it has been difficult to show that affinity, but not IL-la or IL-1 receptor antagonist (11). any cytokine has a critical role. We describe the first example Inactivation of the B15R gene from VV WR decreases virus of the suppression of fever by a virus and the molecular virulence in mice after intracranial injection (12) but enhances mechanism leading to it. Several vaccinia virus strains in- virus virulence when administered intranasally (11). These cluding smallpox vaccines express soluble interleukin 1 (IL-1) contradictory results might be explained by either deletion receptors, which bind IL-1p but not IL-la. These viruses mutant virus having additional mutations affecting virus vir- prevent the febrile response in infected mice, whereas strains ulence or, alternatively, may reflect the properties of IL-ll3, a that naturally or through genetic engineering lack the recep- cytokine that induces beneficial effects but is detrimental when tor induce fever. Repair of the defective IL-18 inhibitor in the produced in large amounts (13). Since a natural route of virulent virus than the poxvirus infections is the respiratory tract (4), the vIL-lf3R smallpox vaccine Copenhagen, a more may function to prevent the adverse effects of excessive IL-1,B. widely used vaccine strains Wyeth and Lister, suppresses fever Here we report that the WR vIL-lf3R prevents the febrile and attenuates the disease. The vaccinia-induced fever was response in intranasally infected mice. In this model, smallpox inhibited with antibodies to IL-lp. These findings provide vaccine strains lacking the vIL-lf3R induced fever, and repair strong evidence that IL-118, and not other cytokines, is the of the nonfunctional vIL-l,BR in strain Copenhagen prevented major endogenous pyrogen in a poxvirus infection. fever and attenuated virus virulence. Fever is an ancient nonspecific systemic response to infection, which is conserved in both invertebrates and vertebrates (1). MATERIALS AND METHODS Temperature elevation during fever constitutes a component Cells and Viruses. The growth conditions for BS-C-1 and of host defense that increases host survival and enhances TK-143B cells and the sources of VV strains have been different arms of the inflammatory and immune responses, described (14). such as T-cell proliferation and differentiation, secretion of Reagents. Radioiodinated mouse or human recombinant interferons (IFNs), neutrophil migration, and the neutralizing IL-113 (80-180 ,uCi/,g) was obtained from DuPont/New capacity of antibodies (1-3). Fever may also have detrimental England Nuclear. In competition experiments with human effects in some circumstances (1, 2). 125I-IL-113 we used unlabeled human and mouse recombinant Fever induced after infection or inoculation of exogenous IL-1,B (2 x 108 units/mg) from R & D Systems. In competition pyrogens, such as lipopolysaccharide, is mediated by release of experiments with mouse 125I-IL-1f3 we used unlabeled mouse soluble proteins that act directly or indirectly on the central recombinant IL-la (8 x 106 units/mg) and IL-1lB (3.5 x 105 nervous system. Candidate endogenous pyrogens include the units/mg) from Genzyme. The hamster monoclonal antibody cytokines interleukin-la (IL-la), IL-1f3, tumor necrosis fac- B122 that neutralizes mouse IL-1,B was provided by D. Chaplin tor-a (TNF-a), TNF-,B (lymphotoxin-a), IL-6, IFN-a, and (Washington University School of Medicine, St. Louis) (15). IFN--y (1). These molecules induce body temperature elevation Construction of Recombinant Ws. The VV WR deletion when inoculated into experimental animals but their contri- mutant lacking B15R (vA&Bl5R) has been described (11). A bution to fever under physiological conditions has been more plaque-purified wild-type virus vAA8 (vBl5Rwt) and a rever- difficult to establish, partly due to the lack of experimental tant virus vAA9 (vBl5Rrev) were constructed by transient models for naturally occurring fever. Dissecting the role of dominant selection (16, 17). Plasmid pAA20 was derived from cytokines in fever is further complicated because they have pSJH7 (18) by insertion of a 2854-bp SalI-BamHI DNA pleiotropic effects, interact with each other at multiple levels, fragment excised from pAA1 (11) and containing B15R and and can increase pathology following their overproduction. flanking regions. Plasmid pAA20 was used for reinsertion of the WR B15R gene into the endogenous locus of vAB15R and Poxviruses are large cytoplasmic DNA viruses that normally locus of the strain. Following produce an acute infection in the host (4, 5). Vaccinia virus the corresponding Copenhagen the transient dominant selection procedure, a plaque-purified was the vaccine used to eradicate smallpox, a devastating (VV) virus (vAA10 or vCOP-vIL-lBR-) and a Copen- human disease produced by variola virus (6). Different vaccine Copenhagen strains induced different reactions to vaccination and frequen- Abbreviations: dpi, days postinfection; IFN, interferon; IL, interleu- kin; pfu, plaque forming units; TNF, tumor necrosis factor; vIL-1,BR, The publication costs of this article were defrayed in part by page charge vaccinia virus IL-113 receptor; VV, vaccinia virus; WR, Western payment. This article must therefore be hereby marked "advertisement" in Reserve. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To-whom reprint requests should be addressed. 11029 Downloaded by guest on September 30, 2021 11030 Immunology: Alcami and Smith Proc. Natl. Acad. Sci. USA 93 (1996) hagen strain expressing the B15R protein (vAA11 or vCOP- closely related to vAB15R, was used here. In addition, a virus vIL-1f3R+) were isolated. The genomic structure of the re- revertant (vBlSRrev) was constructed by reinsertion of B15R combinant viruses was confirmed by Southern blotting and into vAB15R by transient dominant selection. The expression PCR analysis. of vIL-lI3Rs by these viruses was as predicted (not shown). Animals. Female BALB/c mice (5-6 weeks old) were The pathogenicity of these viruses was tested in intranasally housed at 20-22°C with light from 8 a.m. to 8 p.m. Mice were infected BALB/c mice, which produces an extensive respira- infected intranasally with purified virus (11). Rectal temper- tory infection and virus dissemination to other organs. Infec- ature was determined each day (9-11 a.m.) with a mouse rectal tion of groups of 10 mice wiih doses ranging from 5 x 103 to probe (Harvard Apparatus) and a digital thermometer. The 107 plaque forming units (pfu) of WR, vB15Rwt, vAB15R, and change in body temperature was calculated by subtracting the vBlSRrev produced similar numbers of mortalities, and ani- baseline temperature of each group, determined during 6-8 mals infected with vAB15R showed accelerated weight loss and days before infection, from each subsequent time point. Body signs of illness (not shown), confirming previous observations temperature was also determined with the Electronic Labo- (11). To investigate this further, we measured the body tem- ratory Animal Monitoring System from BioMedic Data Sys- perature of infected mice, since fever is one of the systemic tems (Maywood, NJ). Microchip battery-free transponders (14 responses affected by IL-1 (Fig. 1). Surprisingly, intranasal mm x 2.2 mm, 120 mg) were implanted subcutaneously in the inoculation with 2.5 x 103 or 7.5 x 103 pfu of VV wild-type dorsal thoracic area with a sterile needle. Temperature read- (vBl5Rwt) did not induce fever but reduced body temperature ings were collected with a DAS-5004 Pocket Scanner. Mice by 0.5-1°C. By 6 or 7 days postinfection (dpi), the body were also weighed daily and monitored for signs of illness temperature decreased dramatically, with temperatures as low (ruffled fur, arched back, and reduced mobility). Virus infec- as 26-30°C at high virus doses, which coincided with severe tivity in blood (collected by intracardiac puncture) and various signs of illness and weight loss. After day 10, surviving animals organs was determined by plaque assay on TK- 143B cells (19). started to recover and body temperature and weight returned Assays for Soluble IL-l13Rs and IL-1p3. Binding assays for to normal levels by 22 dpi. In contrast, animals infected with soluble IL-l13Rs were performed with 125I-IL-1,f as described vAB15R developed fever during the first 5-6 dpi (Fig. 1). (11). Plasma and organ homogenates (50 ,lA) were centrifuged Later, these animals also developed hypothermia and recov- at 13,000 rpm for 5 min in a microcentrifuge before assay. ered from infection at the same rate as those infected with Levels of mouse plasma IL-103 were determined with the vBl5Rwt. Reinsertion of the B15R gene into vAB15R InterTest-113X mouse IL-1p3 ELISA kit (Genzyme).
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