Microbial Pathogenesis 1990 ; 8 : 289-298
Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3 Burton J . Landau,' P . Susan Whittier,' Sydney D . Finkelstein, 2 * Barbara Alstein,' Janet B . Grun,'t Maggie Schultz' and Richard L . Crowell' Departments of 'Microbiology and Immunology, 'Pathology and Laboratory Medicine, Hahnemann University School of Medicine, Philadelphia, PA 19102, U .S.A . (Received November 11, 1989 ; accepted in revised form January 16, 1990)
Landau, B . J . (Dept of Microbiology and Immunology, Hahnemann University School of Medicine, Philadelphia, PA 19102, U .S .A .), P . S . Whittier, S . D . Finkelstein, B . Alstein, J . B . Grun, M . Schultz and R . L . Crowell . Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3 . Microbial Pathogenesis 1990 ; 8 : 289298 . Infection of adult male C3H/HeJ mice with a host range variant of Coxsackievirus B3 (CB3WRD) induced resistance in these mice to an otherwise lethal dose of Coxsackievirus B1 (CB1) . The protective effect induced by CB3W-RD was detectable as early as 1 day post-vaccination and was still present 10 weeks later . While untreated mice infected with CB1 died within 5 days because of massive hepatic necrosis, the liver was spared in mice immunized with CB3WRD and then challenged with CB1 . In general, CB1 titers in heart, liver, and pancreas of CB3WRD-vaccinated animals were lower than that found in unvaccinated animals . Virus neutralizing antibody was not a mediator of this heterotypic, virus-induced protective effect . In addition, the outcome of CB1 infection could be modified if superinfection with CB3W-RD took place within 1-4 days following CB1 infection . In this regard, maximum therapeutic efficacy was observed when CB1 infected mice were superinfected 2 days after CB1 infection . C131 -infected mice that survived as a result of treatment with CB3W-RD exhibited liver regeneration but did develop myocardial necrosis. Key words : Coxsackievirus B3 ; Coxsackievirus 131 ; heterotypic virus resistance ; cytokines; pancreatitis ; hepatitis ; liver regeneration ; heart muscle disease ; C3H/HeJ mice ; BALB/c mice; C57B1 /6 mice ; C57B1 /6-bgj /bg j mice .
Introduction Studies done to evaluate the pathogenesis of a variant of Coxsackievirus B3 (CB3WRD)' produced the initial observation that C3H/HeJ mice, when vaccinated with CB3W-RD, developed resistance to lethal disease following superinfection with a high dose of CB1 (J . B . Grun, unpublished data) . Generally, specific immunity to group B Coxsackieviruses is considered to be due to the development of neutralizing antibodies
2,3
with some reports suggesting that a heterotypic antibody response (usually IgM) may follow a given infection .` Other immunological factors that may affect the outcome 'Present address : Department of Pathology, Rhode Island Hospital, Providence, Rhode Island 02903, U .S .A . tPresent address : Quality Biologics Inc ., Copewood Street, Camden, New Jersey 08103, U .S .A . 0882-4010/90/040289+10 $03 .00/0
(c~ 1990 Academic Press Limited
290
B . J . Landau et al.
Table 1 Resistance of inbred mouse strains immunized with CB3W-RD to lethal disease following challenge with CB1 Mouse strain C3H/HeJ C57B1/6 BALB/c Beige
Number of Immunized mice tested with CB3W-RD 110 80 20 10 15 5 15 5
+° + + +
Survivors' (%) 100 0 90 0 80 0 100 0
'Eight-week-old male mice were immunized (+) by i .p . injection of 5x10 6 pfu/0 .5 ml of CB3W-RD and then challenged 14 days later by i .p . injection of 5x106 pfu/0 .5 ml of CB1 . 'As control, mice were sham immunized (-) with balanced salt solution supplemented with 3% calf serum (BSS-CaS) or dilutions of uninfected green monkey kidney cell lysate and then challenged 14 days later by i .p . injection of 5x10 6 pfu/0.5 ml of CB1 . `Immunized animals surviving for 21 days following CB1 challenge . Sham immunized animals died within 5 days following CB1 challenge .
.11
12,13
of Coxsackievirus infections include interferons, 6 9 NK cel ls, 10 T suppressor cel ls, production of anti-receptor antibody, t4 anti-idiotype antibody, 15 and other antiviral inhibitors . 16-20 We decided to study the phenomenon of heterotypic virus protection against CB1 since the results of preliminary experiments ruled out the production of neutralizing antibodies induced by CB3W-RD as mediating cross protection .
Results
Induction of heterotypic resistance in mice Initially, experiments were designed to determine whether vaccination of a variety of inbred mouse strains with CB3W-RD would protect mice against infection with 5 x 106 pfu of CB1 . This dose of CB1 was 10 000 times more than that needed to kill all unprotected mice within 5 days . The data summarized in Table 1 show that most animals from four inbred strains of mice immunized with CB3W-RD survived for at least 21 days following challenge infection with CB1 . The survival of vaccinated C57B1 /6-bg'/bg' (beige) mice following CB1 challenge indicated that the protection phenomenon was not mediated by NK cells since these animals are NK cell-deficient . 21 The observation that several strains could be made resistant to an otherwise lethal dose of CB1 suggested that the phenomenon was not restricted to a particular H-2 haplotype . Control studies were also done to rule out neutralizing antibody to CB3W-RD as a mediator of protection of CB3W-RD vaccinated animals that had been challenged with CB1 . Thus, in vitro neutralization studies done by means of a plaque reduction assay in BGM cells showed that CB3W-RD was not neutralized by a 1 :2, 1 :20, or a 1 : 50 dilution of rabbit antibody to CB1 (neutralization titer = 1 : 50000) nor was CB1 neutralized by either a 1 : 2 or 1 : 20 dilution of rabbit antibody to CB3W (neutralization titer = 1 :40000) or a 1 : 10 dilution of mouse antibody (neutralization titer > 1 : 100) to CB3W-R D . Furthermore, virus neutralization studies done in C3H/HeJ mice revealed that mice inoculated i .p . with a mixture of 5 x 10 6 pfu of CB1 that had been previously
Heterotypic Coxsackievirus resistance in mice
291
mixed for 1 h at room temperature with a 1 :10 dilution of mouse anti-CB3W-RD (neutralization titer > 1 : 100) died within 5 days post-infection . Additionally, passive transfer by i .p . injection of 0 .5 ml of serum (neutralization titer >1 :100) from C3H/HeJ mice vaccinated with CB3W-RD 14 days earlier to immunologically naive C3H/HeJ mice, failed to protect the recipient mice against CB1 challenge 14 days after immunization . Thus, no evidence was found for cross neutralization between CB1 and CB3W-RD by plaque reduction or by mouse protection assays . Histologic studies revealed that the dose (5x10 6 pfu) of CB3W-RD used to immunize C3H/HeJ mice resulted in subtotal pancreatic acinar cell necrosis and dropout [Fig . 1 (a)] within 2-3 days post-immunization . No significant inflammatory process, however, was seen in the pancreas and the islet cells were spared . All other major organ systems in animals infected with CB3W-RD alone were histologically unremarkable . On the other hand, infection of unprotected C3H/HeJ mice with 5 x 10 6 pfu of CB1 resulted in total pancreatic acinar cell necrosis [Fig . 1 (b)] which was followed within a few days by diffuse coagulative hepatocellular necrosis and death [Fig . 2(a)] . In contrast, when CB3W-RD-vaccinated mice were challenged with CB1, no augmentation of existing pancreatic acinar cell damage occurred [Fig . 1 (c)] . Furthermore, CB1 failed to cause production of hepatic pathology in immunized animals [Fig . 2(b)] . Experiments were done to determine whether development of resistance of C3H/HeJ mice to lethal CB1 superinfection was influenced by the amount of CB3W-RD used for immunization . As shown in Table 2, an immunizing dose of 5x10 5 pfu was sufficient to protect all test animals against CB1 infection . However, only 40% of mice immunized with 5x10' pfu of CB3W-RD virus survived challenge infection . Histologic examination of C3H/HeJ mice immunized with 5x105 pfu of CB3W-RD and then challenged with CB1 revealed that the pancreas, liver and heart resembled those of immunized mice that had not been challenged with CB1 . Mice immunized with 5 x 10 3 pfu died when superinfected with CB1 . To determine the duration and rate at which resistance to CB1 developed following vaccination, C3H/HeJ mice were inoculated with 5 x 106 pfu CB3W-RD and challenged at intervals, with 5x106 pfu CB1 . The results are depicted in Fig . 3 and show that when mice were challenged as early as 24 h post-vaccination, all survived . This state of resistance lasted for c . 70 days at which time protection began to wane . That is, by 70 days post-immunization, two of three mice challenged with 5x10' pfu died and the remaining animal, being moribund, was killed . All animals (3/3) survived when challenged with 5x10 4 pfu of CB1 70 days post CB3W-RD immunization (data not shown) . Another set of experiments were performed to determine whether CB3W-RD could be used to modify or reduce the severity of disease caused by prior infection with CB1 . The data, summarized in Fig . 3, show that when C3H/HeJ mice were inoculated simultaneously with both CB1 and CB3W-RD, or were vaccinated with CB3W-RD 10 h after CB1 infection, none of the mice survived . In contrast, when mice were infected with CB1, and then superinfected with CB3W-RD 24 h later, 80% of the mice survived . Moreover, when CB1 infected mice were vaccinated with CB3W-RD 2 days after CB1 infection, all mice survived . The therapeutic efficacy of vaccinating CB1 -infected animals with CB3W-RD was reduced if vaccination was delayed until 3-4 days post-CB1 infection (only 60% of the vaccinated animals survived) . Histologic examination of tissues from mice that had been infected with CB1 and 2 days later had been vaccinated with CB3W-RD revealed total acinar necrosis [Fig . 4(a)] . This observation indicated that CB3W-RD treatment was unable to modify CB1 -induced pathology in the pancreas . The liver in these animals, however, appeared to regenerate
B . J . Landau et al.
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rV Fig . 2 . (a) C3H/HeJ mouse infected with CB1 . Histologic section of liver taken 4 days after infection shows virtually total necrosis of hepatic parenchymal cells . Rounded necrotic cells resembling Councilman body (arrows) are seen . Hematoxylin-eosin, x120 . (b) C3H/HeJ mouse protected with CB3W-RD and challenged with CB1 . Histologic section of the liver 4 days after CB1 challenge shows no evidence of cell injury . Hematoxylin-eosin, x120 .
as evidenced by the presence of mitotic activity and absence of pathology [Fig . 4(b)] . This finding is in contrast to the irreversible injury seen in unvaccinated animals infected with CB1 alone [Fig . 2(a)] . Of interest was the finding that CB3W-RD treated animals recovering from CB1 -induced liver disease subsequently developed myocardial necrosis [Fig . 4(c)] . Development of heart muscle disease in this latter case, was in contrast to the absence of pathology in hearts of animals vaccinated with CB3W-RD prior to challenge with CB1 . Finally, groups of non-vaccinated and CB3W-RD vaccinated C3H/HeJ mice were each infected with 5x10 6 pfu CB1, and, at various times thereafter, the amount of CB1 in the heart, pancreas, and liver was determined . The results summarized in Table 3 revealed that in non-vaccinated animals, the titers of CB1 in the heart were c . 100100000 times greater than that found in vaccinated animals . Though not as marked as seen in the heart, the yield of CB1 in the pancreata of unprotected animals was 10 to c . 5000 times greater than recovered from pancreata in vaccinated animals . Given the fact that unvaccinated animals died of acute hepatocellular necrosis when Table 2 Effect of immunizing dose of CB3W-RD on the response of C3H/HeJ mice to challenge with CB 1 a Immunizing dose of CB3W-RD (pfu)
% Survivors post CB1 challenge'
5x106 5x105 5 x 10° 5x103
100 100 40 0
a Groups of four to five 8-week-old male mice in each of two experiments were immunized i .p . with varying doses of CB3W-RD and challenged 14 days later with 5x10' pfu of CB1 . 'Composite results of the percent of immunized mice surviving CB1 challenge for at least 14 days .
B . J . Landau et al.
2 94
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Day of CB3W-RD vaccination relative to CBI infection Fig . 3 . Duration and rate of development of resistance to Cal following vaccination with CB3W-RD (as shown to left of zero time) . Capacity of CB3W-RD to affect outcome of disease caused by pre-infection with Cal (as shown to right of zero time) . Composite results of two experiments involving three to 10 mice per time point .
challenged with CB1, it was surprising to note that differences in yield of CB1 from the liver of unvaccinated animals was not markedly different from that of unvaccinated animals .
Discussion and conclusions The experiments described above provide evidence for a previously unrecognized phenomenon in which vaccination with a host range variant of Coxsackievirus B3 produced protection of inbred mice against a massive and otherwise lethal dose of a heterotypic Coxsackievirus, CB1 . This protective effect was considered unusual and may involve more than one mechanism since it : (i) appeared within 24 h ; (ii) lasted for at least 70 days; (iii) involved protection of visceral organs, and (iv) resulted in reduced virus titers in organs such as the heart and pancreas, and to a lesser extent in the liver, and (v) was not mediated by either virus-specific neutralizing antibodies or NK cells . Also, our finding that protection could be demonstrated 70 days after vaccination led us to conclude that heterologous virus interference was not involved in mediating the protective effect found 2 weeks post-vaccination with CB3W-RD, since infectious CB3W-RD was cleared from the viscera within 14 days . However, during the first 2 weeks following vaccination with CB3W-RD the possibility that heterologous virus interference may play a role in protection against challenge with CB1 has not been ruled out . These observations, together with the data that the protective effect was mediated neither by virus-specific neutralizing antibodies, nor NK cells suggested that cytokines such as IL-6 22,23 and gamma interferon 24 might be involved . In this regard, it should be noted that the immunizing dose of CB3W-RD
Heterotypic Coxsackievirus resistance in mice
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Table 3 Recovery of CB1 from tissues at various times following infection of C3H/HeJ mice vaccinated with CB3W-RD' Amount of CB1 (pfu/g) ±SEM ° Days post CB1 infection Immunization with CB3W-RD
Organ
1
2
3
4
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6±2x109 6+1 x10'
+
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2+1x10 9 5+2x106 4+2x108 3+1x103
8++6x10 5 5+2x10 5 2+1x10 8 2+0x10 3
+
Liver Liver
5+1x10 5 4±1x10 5
3+1x107 2±2x107
1+0x108 1 ±0x108
1+6x10 8 4±1x10 6
+
'Twelve to 15 8-week-old male mice were either immunized with 5x10 6 pfu of CB3W-RD (+) or sham immunized with BSSCas (-) . Fourteen days later, mice in each group were inoculated i .p. with 5x106 pfu CB1 and the respective organs from three animals were pooled, homogenized and assayed for virus . 'Average of three to four experiments±standard error of the mean (SEM) .
was introduced by intra-peritoneal injection, which increased the likelihood of interaction between virus and a variety of cell types including peritoneal macrophages ." One of the more surprising findings described above involved the demonstration that death due to CB1 infection of non-immune mice could be prevented if CB3WRD were injected i .p . within 1-4 days post-CB1 infection . In this experimental paradigm, it is probable that the lives of these animals were spared because liver regeneration occurred . However, the protective effect did not extend to the heart where myocardial necrosis occurred about 7-9 days post-CB1 infection . Perhaps IL-6 is associated with the initial host response to CB1 infection of the liver 22 while a growth factor such as transforming growth factor alpha may mediate liver regeneration . 26 On the other hand, it is possible that myocardial necrosis was not prevented because levels of CB3W-RD induced-cytokines such as IFN gamma were either produced too late or in insufficient quantities to inhibit CB1 replication in the myocardium . The apparent organ specificity of the protective effect was tested by challenge of CB3W-RD-immunized mice by intraperitoneal (i .p .) injection of another picornavirus, encephalomyocarditis (EMC) virus . Mice treated in this fashion died of encephalitis (data not shown) . Thus the role of the protective effect as a significant determinant in the pathogenesis of a specific viral infection may depend on the organ tropism of the virus . Finally, it is possible that the protective effect may have public health implications . This phenomenon may explain why poliovirus immunizations are not successful in ." those communities experiencing intercurrent infections with other enteroviruses Studies are in progress to determine the mechanism(s) responsible for this newly recognized heterotypic virus protection induced in animals following vaccination with a relatively avirulent Coxsackievirus strain .
Materials and methods Virus. An amyocarditic variant of CB3W, designated CB3W-RD, was obtained in our laboratory following serial blind passage of this virus through rhabdomyosarcoma (RD) cells . Stock virus pools of CB3W-RD were prepared and titrated as described elsewhere .' Coxsackievirus 131 was grown in monolayer cultures of HeLa cells and subsequently purified by CsCI density gradient techniques ."
Virologic and histologic studies. Virus plaque titrations 30 and virus neutralization studies
Heterotypic Coxsackievirus resistance in mice
297
involving a plaque reduction assay31 were done in green monkey kidney cells (BGM) obtained from Dr M . Menegus (University of Rochester) . For RD virus titrations, the serum-containing overlay medium was additionally supplemented with DEAE-Dextran (100 mg/ml) and 5 mm MgC1 2 . Histologic studies and isolation of virus from pancreas, liver and heart were done as described previously . 32
Mice . Seven-week-old male C3H/HeJ, C57B1/6, C57B1/6-bg j /bgj (beige) and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in a facility accredited by the American Association for Accreditation of Laboratory Animal Care . Periodically, mice were monitored for mouse hepatitis virus and found to be negative .
These studies were supported in part by grants from the National Institute of Allergy and Infectious Diseases (Al-03771), the Pennsylvania Affiliate of the American Heart Association and the Muscular Dystrophy Association .
References 1 . Reagan KJ, Goldberg B, Crowell RL . Altered receptor specificity of coxsackievirus B3 after growth in rhabdomyosarcoma cells . J Virol 1984 ; 49 : 635-40 . 2 . Melnick JL, Ledinko N . Immunological reactions of the Coxsackievirus . I . The neutralization test : techniques and applications . J Exp Med 1950 ; 92 : 463-82 . 3 . Melnick JL . Enteroviruses: polioviruses, Coxsackieviruses, echoviruses, and newer enteroviruses . In Fields BN, ed . Virology . New York : Raven Press, 1985 ; 739-94 . 4 . Minor TE, Helstron PB, Nelson DB, D'Alessio DJ . Counterimmunoelectrophoresis test for immunoglobulin M antibodies to group B Coxsackieviruses . J Clin Microbiol 1979 ; 9 : 503-6 . 5 . Dorries R, Ter Meulen V . Specificity of IgM antibodies in acute human Coxsackievirus B infections, analyzed by indirect solid phase enzyme immunoassay and immunoblot technique . J Gen Virol 1983 ; 64 :159-67 . 6 . Norris D, Loh PC . Coxsackievirus myocarditis : prophylaxis and therapy with an interferon stimulator . Proc Soc Exp Biol Med 1973 ; 142 :133-6 . 7 . Reyes MP, Lerner AM . Interferon and neutralizing antibody in sera of exercised mice with Coxsackievirus B-3 myocarditis . Proc Soc Biol Med 1976 ; 151 : 333-8 . 8 . Cho CT, Feng KK, McCarthy VP, Lenahan MF . Role of antiviral antibodies in resistance against Coxsackieviruses B3 infection . Interaction between preexisting antibody and an interferon inducer . Infect Immun 1982 ; 37 : 720-7 . 9 . Lutton CW, Gauntt CJ . Ameliorating effect of IFN-f and anti-IFN-fl on Coxsackievirus B3-induced myocarditis in mice. J Interferon Res 1985 ; 5 : 137-46 . 10 . Godeny EK, Gauntt CJ . Involvement of natural killer cells in Coxsackievirus B3-induced murine myocarditis. J Immunol 1986 ; 137 : 1-8 . 11 . Godeny EK, Gauntt CJ . Murine natural killer cells limit Coxsackievirus B3 replication . J Immunol 1987 ; 139 : 913-18 . 12 . Gauntt CJ, Paque RE, Trousdale MD et a/. Temperature-sensitive mutant of Coxsackievirus B3 establishes resistance in neonatal mice that protects them during adolescence against Coxsackievirus B3-induced myocarditis . Infect Immun 1983 ; 39 : 851-64 . 13 . Estrin M, Smith C, Huber S . Antigen-specific suppressor T cells prevent cardiac injury in Balb/c mice infected with a nonmyocarditic variant of Coxsackievirus Group B, type 3 . Am J Path 1986 ; 125 : 578-84 . 14 . Axler DA, Crowell RL. Effect of anticellular serum on the attachment of enteroviruses to HeLa cells . J Virol 1968 ; 2 : 813-21 . 15 . Paque RE, Miller R . Monoclonal antibodies regulate the expression of virus-induced murine myocarditis . Infect Immun 1989 ; 57 : 2864-71 . 16 . Hughes TK, Blalock JE, McKerlie ML, Baron S . Cell-produced viral inhibitor: possible mechanism of action and chemical composition . Infect Immun 1981 ; 32 : 454-7 . 17 . Baron S, McKerlie ML . Broadly active inhibitor of viruses spontaneously produced by many cell types in culture. Infect Immun 1981 ; 32 : 449-53 . 18 . Coppenhaver DH, Baron JL, McKerlie ML, Sabados J, Baron S . Size and stability of a naturally occurring virus inhibitor . Anti Microb Agents Chemother 1984 ; 25 : 646-9 . 19 . Kumar S, McKerlie ML, Albrecht TB, Goldman AS, Baron S . A broadly active viral inhibitor in human and animal organ extracts and body fluids . Proc Soc Exp Biol Med 1984 ; 177 : 104-11 . 20 . Baron JL, Li J-L, McKerlie ML, Shabot JM, Coppenhaver DH . A new subtype of a natural viral inhibitor (CVI) that is stable in the gastrointestinal tract . Microbial Pathogenesis 1986 ; 1 : 241-7 . 21 . Roder J, Duwe A . The beige mutation in the mouse selectively impairs natural killer function . Nature 1979; 278 : 451-3 . 22 . Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H . Interferon fl2/B-cell stimulatory factor
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type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells . Proc Natl Acad Sci USA 1987 ; 84 : 7251-5 . 23 . Sehgal PB, Helfgott DC, Santhanam U et al. Regulation of the acute phase and immune responses in viral disease . Enhanced expression . J Exp Med 1988; 167 : 1951-6 . 24 . Maier K, Gabriel P, Koscielniak E et al. Human gamma interferon production by cytotoxic T lymphocytes sensitized during Hepatitis A infection . J Virol 1988 ; 62: 3756-63. 25 . Rager-Zisman B, Allison AC . The role of antibody and host cells in the resistance of mice against infection by Coxsackie B-3 virus . J Gen Virol 1973; 19 : 329-38 . 26 . Mead J, Fausto N . Transforming growth factor a may be a physiological regulator of liver regeneration by means of an autocrine mechanism . Proc Natl Acad Sci USA 1989 ; 86: 1558-62. 27 . Wheelock EF, Larke RPB, Caroline NL . Interference in human viral infections : present status and prospects for the future . In : J L Melnick, ed . Progress in medical virology 1968 ; 10 : 286-347 . 28 . Albert MJ . Failure of live, oral virus vaccines in developing countries . J Inf Dis 1987 ; 155 : 1350 . 29 . Crowell RL, Philipson L . Specific alterations of Coxsackievirus B3 eluted from HeLa cells . J Virol 1971 ; 8 : 509-15 . 30 . Crowell RL, Syverton JT. The mammalian cell-virus relationship . V I . Sustained infection of HeLa cells by Coxsackie B3 virus and effect on superinfection . J Exp Med 1961 ; 113 : 419-35 . 31 . Katze MG, Crowell RL . Indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Coxsackievirus Group B antibodies . J Gen Virol 1980 ; 48: 225-9 . 32 . Grun JB, Schultz M, Finkelstein SD, Crowell RL, Landau BJ . Pathogenesis of acute myocardial necrosis in inbred mice infected with Coxsackievirus B3 . Microbial Pathogenesis 1988; 4 : 417-30 .
Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3 Burton J . Landau,' P . Susan Whittier,' Sydney D . Finkelstein, 2 * Barbara Alstein,' Janet B . Grun,'t Maggie Schultz' and Richard L . Crowell' Departments of 'Microbiology and Immunology, 'Pathology and Laboratory Medicine, Hahnemann University School of Medicine, Philadelphia, PA 19102, U .S.A . (Received November 11, 1989 ; accepted in revised form January 16, 1990)
Landau, B . J . (Dept of Microbiology and Immunology, Hahnemann University School of Medicine, Philadelphia, PA 19102, U .S .A .), P . S . Whittier, S . D . Finkelstein, B . Alstein, J . B . Grun, M . Schultz and R . L . Crowell . Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3 . Microbial Pathogenesis 1990 ; 8 : 289298 . Infection of adult male C3H/HeJ mice with a host range variant of Coxsackievirus B3 (CB3WRD) induced resistance in these mice to an otherwise lethal dose of Coxsackievirus B1 (CB1) . The protective effect induced by CB3W-RD was detectable as early as 1 day post-vaccination and was still present 10 weeks later . While untreated mice infected with CB1 died within 5 days because of massive hepatic necrosis, the liver was spared in mice immunized with CB3WRD and then challenged with CB1 . In general, CB1 titers in heart, liver, and pancreas of CB3WRD-vaccinated animals were lower than that found in unvaccinated animals . Virus neutralizing antibody was not a mediator of this heterotypic, virus-induced protective effect . In addition, the outcome of CB1 infection could be modified if superinfection with CB3W-RD took place within 1-4 days following CB1 infection . In this regard, maximum therapeutic efficacy was observed when CB1 infected mice were superinfected 2 days after CB1 infection . C131 -infected mice that survived as a result of treatment with CB3W-RD exhibited liver regeneration but did develop myocardial necrosis. Key words : Coxsackievirus B3 ; Coxsackievirus 131 ; heterotypic virus resistance ; cytokines; pancreatitis ; hepatitis ; liver regeneration ; heart muscle disease ; C3H/HeJ mice ; BALB/c mice; C57B1 /6 mice ; C57B1 /6-bgj /bg j mice .
Introduction Studies done to evaluate the pathogenesis of a variant of Coxsackievirus B3 (CB3WRD)' produced the initial observation that C3H/HeJ mice, when vaccinated with CB3W-RD, developed resistance to lethal disease following superinfection with a high dose of CB1 (J . B . Grun, unpublished data) . Generally, specific immunity to group B Coxsackieviruses is considered to be due to the development of neutralizing antibodies
2,3
with some reports suggesting that a heterotypic antibody response (usually IgM) may follow a given infection .` Other immunological factors that may affect the outcome 'Present address : Department of Pathology, Rhode Island Hospital, Providence, Rhode Island 02903, U .S .A . tPresent address : Quality Biologics Inc ., Copewood Street, Camden, New Jersey 08103, U .S .A . 0882-4010/90/040289+10 $03 .00/0
(c~ 1990 Academic Press Limited
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B . J . Landau et al.
Table 1 Resistance of inbred mouse strains immunized with CB3W-RD to lethal disease following challenge with CB1 Mouse strain C3H/HeJ C57B1/6 BALB/c Beige
Number of Immunized mice tested with CB3W-RD 110 80 20 10 15 5 15 5
+° + + +
Survivors' (%) 100 0 90 0 80 0 100 0
'Eight-week-old male mice were immunized (+) by i .p . injection of 5x10 6 pfu/0 .5 ml of CB3W-RD and then challenged 14 days later by i .p . injection of 5x106 pfu/0 .5 ml of CB1 . 'As control, mice were sham immunized (-) with balanced salt solution supplemented with 3% calf serum (BSS-CaS) or dilutions of uninfected green monkey kidney cell lysate and then challenged 14 days later by i .p . injection of 5x10 6 pfu/0.5 ml of CB1 . `Immunized animals surviving for 21 days following CB1 challenge . Sham immunized animals died within 5 days following CB1 challenge .
.11
12,13
of Coxsackievirus infections include interferons, 6 9 NK cel ls, 10 T suppressor cel ls, production of anti-receptor antibody, t4 anti-idiotype antibody, 15 and other antiviral inhibitors . 16-20 We decided to study the phenomenon of heterotypic virus protection against CB1 since the results of preliminary experiments ruled out the production of neutralizing antibodies induced by CB3W-RD as mediating cross protection .
Results
Induction of heterotypic resistance in mice Initially, experiments were designed to determine whether vaccination of a variety of inbred mouse strains with CB3W-RD would protect mice against infection with 5 x 106 pfu of CB1 . This dose of CB1 was 10 000 times more than that needed to kill all unprotected mice within 5 days . The data summarized in Table 1 show that most animals from four inbred strains of mice immunized with CB3W-RD survived for at least 21 days following challenge infection with CB1 . The survival of vaccinated C57B1 /6-bg'/bg' (beige) mice following CB1 challenge indicated that the protection phenomenon was not mediated by NK cells since these animals are NK cell-deficient . 21 The observation that several strains could be made resistant to an otherwise lethal dose of CB1 suggested that the phenomenon was not restricted to a particular H-2 haplotype . Control studies were also done to rule out neutralizing antibody to CB3W-RD as a mediator of protection of CB3W-RD vaccinated animals that had been challenged with CB1 . Thus, in vitro neutralization studies done by means of a plaque reduction assay in BGM cells showed that CB3W-RD was not neutralized by a 1 :2, 1 :20, or a 1 : 50 dilution of rabbit antibody to CB1 (neutralization titer = 1 : 50000) nor was CB1 neutralized by either a 1 : 2 or 1 : 20 dilution of rabbit antibody to CB3W (neutralization titer = 1 :40000) or a 1 : 10 dilution of mouse antibody (neutralization titer > 1 : 100) to CB3W-R D . Furthermore, virus neutralization studies done in C3H/HeJ mice revealed that mice inoculated i .p . with a mixture of 5 x 10 6 pfu of CB1 that had been previously
Heterotypic Coxsackievirus resistance in mice
291
mixed for 1 h at room temperature with a 1 :10 dilution of mouse anti-CB3W-RD (neutralization titer > 1 : 100) died within 5 days post-infection . Additionally, passive transfer by i .p . injection of 0 .5 ml of serum (neutralization titer >1 :100) from C3H/HeJ mice vaccinated with CB3W-RD 14 days earlier to immunologically naive C3H/HeJ mice, failed to protect the recipient mice against CB1 challenge 14 days after immunization . Thus, no evidence was found for cross neutralization between CB1 and CB3W-RD by plaque reduction or by mouse protection assays . Histologic studies revealed that the dose (5x10 6 pfu) of CB3W-RD used to immunize C3H/HeJ mice resulted in subtotal pancreatic acinar cell necrosis and dropout [Fig . 1 (a)] within 2-3 days post-immunization . No significant inflammatory process, however, was seen in the pancreas and the islet cells were spared . All other major organ systems in animals infected with CB3W-RD alone were histologically unremarkable . On the other hand, infection of unprotected C3H/HeJ mice with 5 x 10 6 pfu of CB1 resulted in total pancreatic acinar cell necrosis [Fig . 1 (b)] which was followed within a few days by diffuse coagulative hepatocellular necrosis and death [Fig . 2(a)] . In contrast, when CB3W-RD-vaccinated mice were challenged with CB1, no augmentation of existing pancreatic acinar cell damage occurred [Fig . 1 (c)] . Furthermore, CB1 failed to cause production of hepatic pathology in immunized animals [Fig . 2(b)] . Experiments were done to determine whether development of resistance of C3H/HeJ mice to lethal CB1 superinfection was influenced by the amount of CB3W-RD used for immunization . As shown in Table 2, an immunizing dose of 5x10 5 pfu was sufficient to protect all test animals against CB1 infection . However, only 40% of mice immunized with 5x10' pfu of CB3W-RD virus survived challenge infection . Histologic examination of C3H/HeJ mice immunized with 5x105 pfu of CB3W-RD and then challenged with CB1 revealed that the pancreas, liver and heart resembled those of immunized mice that had not been challenged with CB1 . Mice immunized with 5 x 10 3 pfu died when superinfected with CB1 . To determine the duration and rate at which resistance to CB1 developed following vaccination, C3H/HeJ mice were inoculated with 5 x 106 pfu CB3W-RD and challenged at intervals, with 5x106 pfu CB1 . The results are depicted in Fig . 3 and show that when mice were challenged as early as 24 h post-vaccination, all survived . This state of resistance lasted for c . 70 days at which time protection began to wane . That is, by 70 days post-immunization, two of three mice challenged with 5x10' pfu died and the remaining animal, being moribund, was killed . All animals (3/3) survived when challenged with 5x10 4 pfu of CB1 70 days post CB3W-RD immunization (data not shown) . Another set of experiments were performed to determine whether CB3W-RD could be used to modify or reduce the severity of disease caused by prior infection with CB1 . The data, summarized in Fig . 3, show that when C3H/HeJ mice were inoculated simultaneously with both CB1 and CB3W-RD, or were vaccinated with CB3W-RD 10 h after CB1 infection, none of the mice survived . In contrast, when mice were infected with CB1, and then superinfected with CB3W-RD 24 h later, 80% of the mice survived . Moreover, when CB1 infected mice were vaccinated with CB3W-RD 2 days after CB1 infection, all mice survived . The therapeutic efficacy of vaccinating CB1 -infected animals with CB3W-RD was reduced if vaccination was delayed until 3-4 days post-CB1 infection (only 60% of the vaccinated animals survived) . Histologic examination of tissues from mice that had been infected with CB1 and 2 days later had been vaccinated with CB3W-RD revealed total acinar necrosis [Fig . 4(a)] . This observation indicated that CB3W-RD treatment was unable to modify CB1 -induced pathology in the pancreas . The liver in these animals, however, appeared to regenerate
B . J . Landau et al.
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rV Fig . 2 . (a) C3H/HeJ mouse infected with CB1 . Histologic section of liver taken 4 days after infection shows virtually total necrosis of hepatic parenchymal cells . Rounded necrotic cells resembling Councilman body (arrows) are seen . Hematoxylin-eosin, x120 . (b) C3H/HeJ mouse protected with CB3W-RD and challenged with CB1 . Histologic section of the liver 4 days after CB1 challenge shows no evidence of cell injury . Hematoxylin-eosin, x120 .
as evidenced by the presence of mitotic activity and absence of pathology [Fig . 4(b)] . This finding is in contrast to the irreversible injury seen in unvaccinated animals infected with CB1 alone [Fig . 2(a)] . Of interest was the finding that CB3W-RD treated animals recovering from CB1 -induced liver disease subsequently developed myocardial necrosis [Fig . 4(c)] . Development of heart muscle disease in this latter case, was in contrast to the absence of pathology in hearts of animals vaccinated with CB3W-RD prior to challenge with CB1 . Finally, groups of non-vaccinated and CB3W-RD vaccinated C3H/HeJ mice were each infected with 5x10 6 pfu CB1, and, at various times thereafter, the amount of CB1 in the heart, pancreas, and liver was determined . The results summarized in Table 3 revealed that in non-vaccinated animals, the titers of CB1 in the heart were c . 100100000 times greater than that found in vaccinated animals . Though not as marked as seen in the heart, the yield of CB1 in the pancreata of unprotected animals was 10 to c . 5000 times greater than recovered from pancreata in vaccinated animals . Given the fact that unvaccinated animals died of acute hepatocellular necrosis when Table 2 Effect of immunizing dose of CB3W-RD on the response of C3H/HeJ mice to challenge with CB 1 a Immunizing dose of CB3W-RD (pfu)
% Survivors post CB1 challenge'
5x106 5x105 5 x 10° 5x103
100 100 40 0
a Groups of four to five 8-week-old male mice in each of two experiments were immunized i .p . with varying doses of CB3W-RD and challenged 14 days later with 5x10' pfu of CB1 . 'Composite results of the percent of immunized mice surviving CB1 challenge for at least 14 days .
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challenged with CB1, it was surprising to note that differences in yield of CB1 from the liver of unvaccinated animals was not markedly different from that of unvaccinated animals .
Discussion and conclusions The experiments described above provide evidence for a previously unrecognized phenomenon in which vaccination with a host range variant of Coxsackievirus B3 produced protection of inbred mice against a massive and otherwise lethal dose of a heterotypic Coxsackievirus, CB1 . This protective effect was considered unusual and may involve more than one mechanism since it : (i) appeared within 24 h ; (ii) lasted for at least 70 days; (iii) involved protection of visceral organs, and (iv) resulted in reduced virus titers in organs such as the heart and pancreas, and to a lesser extent in the liver, and (v) was not mediated by either virus-specific neutralizing antibodies or NK cells . Also, our finding that protection could be demonstrated 70 days after vaccination led us to conclude that heterologous virus interference was not involved in mediating the protective effect found 2 weeks post-vaccination with CB3W-RD, since infectious CB3W-RD was cleared from the viscera within 14 days . However, during the first 2 weeks following vaccination with CB3W-RD the possibility that heterologous virus interference may play a role in protection against challenge with CB1 has not been ruled out . These observations, together with the data that the protective effect was mediated neither by virus-specific neutralizing antibodies, nor NK cells suggested that cytokines such as IL-6 22,23 and gamma interferon 24 might be involved . In this regard, it should be noted that the immunizing dose of CB3W-RD
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Table 3 Recovery of CB1 from tissues at various times following infection of C3H/HeJ mice vaccinated with CB3W-RD' Amount of CB1 (pfu/g) ±SEM ° Days post CB1 infection Immunization with CB3W-RD
Organ
1
2
3
4
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+
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+
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5+1x10 5 4±1x10 5
3+1x107 2±2x107
1+0x108 1 ±0x108
1+6x10 8 4±1x10 6
+
'Twelve to 15 8-week-old male mice were either immunized with 5x10 6 pfu of CB3W-RD (+) or sham immunized with BSSCas (-) . Fourteen days later, mice in each group were inoculated i .p. with 5x106 pfu CB1 and the respective organs from three animals were pooled, homogenized and assayed for virus . 'Average of three to four experiments±standard error of the mean (SEM) .
was introduced by intra-peritoneal injection, which increased the likelihood of interaction between virus and a variety of cell types including peritoneal macrophages ." One of the more surprising findings described above involved the demonstration that death due to CB1 infection of non-immune mice could be prevented if CB3WRD were injected i .p . within 1-4 days post-CB1 infection . In this experimental paradigm, it is probable that the lives of these animals were spared because liver regeneration occurred . However, the protective effect did not extend to the heart where myocardial necrosis occurred about 7-9 days post-CB1 infection . Perhaps IL-6 is associated with the initial host response to CB1 infection of the liver 22 while a growth factor such as transforming growth factor alpha may mediate liver regeneration . 26 On the other hand, it is possible that myocardial necrosis was not prevented because levels of CB3W-RD induced-cytokines such as IFN gamma were either produced too late or in insufficient quantities to inhibit CB1 replication in the myocardium . The apparent organ specificity of the protective effect was tested by challenge of CB3W-RD-immunized mice by intraperitoneal (i .p .) injection of another picornavirus, encephalomyocarditis (EMC) virus . Mice treated in this fashion died of encephalitis (data not shown) . Thus the role of the protective effect as a significant determinant in the pathogenesis of a specific viral infection may depend on the organ tropism of the virus . Finally, it is possible that the protective effect may have public health implications . This phenomenon may explain why poliovirus immunizations are not successful in ." those communities experiencing intercurrent infections with other enteroviruses Studies are in progress to determine the mechanism(s) responsible for this newly recognized heterotypic virus protection induced in animals following vaccination with a relatively avirulent Coxsackievirus strain .
Materials and methods Virus. An amyocarditic variant of CB3W, designated CB3W-RD, was obtained in our laboratory following serial blind passage of this virus through rhabdomyosarcoma (RD) cells . Stock virus pools of CB3W-RD were prepared and titrated as described elsewhere .' Coxsackievirus 131 was grown in monolayer cultures of HeLa cells and subsequently purified by CsCI density gradient techniques ."
Virologic and histologic studies. Virus plaque titrations 30 and virus neutralization studies
Heterotypic Coxsackievirus resistance in mice
297
involving a plaque reduction assay31 were done in green monkey kidney cells (BGM) obtained from Dr M . Menegus (University of Rochester) . For RD virus titrations, the serum-containing overlay medium was additionally supplemented with DEAE-Dextran (100 mg/ml) and 5 mm MgC1 2 . Histologic studies and isolation of virus from pancreas, liver and heart were done as described previously . 32
Mice . Seven-week-old male C3H/HeJ, C57B1/6, C57B1/6-bg j /bgj (beige) and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in a facility accredited by the American Association for Accreditation of Laboratory Animal Care . Periodically, mice were monitored for mouse hepatitis virus and found to be negative .
These studies were supported in part by grants from the National Institute of Allergy and Infectious Diseases (Al-03771), the Pennsylvania Affiliate of the American Heart Association and the Muscular Dystrophy Association .
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