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Cross-neutralizing and subclass characteristics of antibody from horses with equine infectious anemia virus
Veterinary Immunology and Immunopathology, 23 (1989) 41-49 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
41
Cross-neutralizing and Subclass Characteristics of Antibody from Horses with Equine Infectious A n e m i a Virus KATHERINE I. O'ROURKE, LANCE E. PERRYMAN and TRAVIS C. McGUIRE
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164 (U.S.A.) (Accepted 18 April 1989)
ABSTRACT O'Rourke, K.I., Perryman, L.E. and McGuire, T.C., 1989. Cross-neutralizing and subclass characteristics of antibody from horses with equine infectious anemia virus. Vet. Immunol, Immunopathol., 23: 41-49. Antibody responses in horses with equine infectious anemia virus (EIAV) were examined to determine their cross-neutralizing capacity. Antibodies induced by infection with any of six biologically cloned variants of EIAV cross-neutralized multiple variants from the group. Anti-EIAV antibody was found in both the IgG and IgG(T) subclasses in plasmas with virus-neutralizing activity and the majority of antiviral antibody was of the IgG (T) subclass. Depletion of IgG (T) did not increase the neutralization indexes of either neutralizing or non-neutralizing plasma samples.
INTRODUCTION
Equine infectious anemia virus (EIAV) is a lentivirus of horses which infects fixed and circulating cells of the monocyte lineage (McGuire et al., 1971a; Charman et al., 1976). EIAV shares genome sequence homology with other known lentiviruses, including human immunodeficiency virus (HIV), caprine arthritis-encephalitis virus (CAEV) and ovine visna virus (VV) ( Chiu et al., 1985; Stephens et al., 1986). These lentiviruses cause lifelong infection in the host with a variable pattern of clinical disease expression. Lentivirus infections are not eliminated in spite of the generation of humoral and cell-mediated antiviral immune responses. Cell-free viremia is temporarily cleared by immune responses during the first 35 days of infection with EIAV (Perryman et al., 1988). Relapsing disease is associated with the appearance of novel antigenic variants (Kono et al., 1973; Montelaro et al., 1984), suggesting that immune selection of rapidly evolving mutants represents one mechanism of viral 0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
42
K.I. O'ROURKE ET AL.
persistence. Most horses with EIAV become clinically normal within the first year after infection and remain asymptomatic and aviremic for years, although the virus can be transmitted by transfer of whole blood or washed leukocytes to susceptible horses (see review by Coggins, 1984). An understanding of the elements of the immune response which control disease in infected horses may contribute to development of an EIAV vaccine which prevents infection of horses under field conditions. We have previously described the antiglycoprotein and neutralizing antibody responses in four horses infected with a virulent EIAV isolate (O'Rourke et al., 1988). In this study, we extend those observations by describing an early cross-neutralizing response in a larger panel of horses infected with biologically cloned EIAV variants and by characterizing the relative contributions of antiviral antibody of the IgG and IgG(T) subclasses to the neutralizing response. MATERIALS AND METHODS
Virus isolates Antigenic variants of EIAV were generated by serial passage of the tissue culture adapted Wyoming strain of EIAV (EIAV-prototype) (Malmquist et al., 1973) through horses. The prototype strain was cloned sequentially three times by limiting dilution, propagated in equine dermal cells (ATCC CCL57) and serially passaged through three horses (H347, H309 and H354). All three horses developed clinical signs of EIAV infection, including periodic temperature increases, at which times virus was isolated. Virus isolates were made by incubation of 1 ml plasma, diluted 1:5 in minimal essential media (MEM), with a subconfluent monolayer of equine kidney (EK) cells in a 25-cm 2 tissue culture flask for 2 h at room temperature. Cells were maintained as described (O'Rourke et al., 1988) and screened at each passage for viral antigen by immunofluorescence (Crawford et al., 1971). Because there is no plaque assay for EIAV, a predominant isolate from each viremic period was selected by three serial limiting dilution clonings in EK cells. Between cloning rounds, virus from the endpoint wells was propagated in EK cells until the virus titer reached approximately 103 TDCIs0/ml. The stringency of triple endpoint cloning under these conditions makes it highly likely that each isolate represents a single virus variant. Isolates were numbered EIAV-WSU1 through EIAV-WSU5 following the proposal for naming human immunodeficiency lentiviruses (Coffin et al., 1986). EIA V infection Eight horses (A1948, A1950, A1951, A1955, A1907, A1908, A1924 and A1925) were infected by intravenous administration of 106 TCIDso EIAV. Plasma collected at day 0 and at intervals following infection was stored at - 7 0 °C for
CROSS-NEUTRALIZINGAND SUBCLASS CHARACTERISTICS OF ANTIBODY TO EIAV
43
subsequent virus isolation and at - 20 ° C for serologic assays. Rectal temperatures were recorded daily.
Enzyme linked immunosorbent assay (ELISA) Antiviral ELISA titers were determined using serial three-fold dilutions of plasma samples assayed on microELISA (Immulon I, Dynatech ) plates coated with 300 ng EIAV per well. Antiviral antibody was detected using rabbit antiserum specific for equine IgG or IgG (T) (McGuire and Crawford, 1972 ), goat anti-rabbit IgG-HRPO (Sigma) and 5-aminosalicylic acid as a chromogenic substrate. After approximately 2 h at room temperature, the optical density of each well was determined with an automated ELISA reader (Dynatech Laboratories). A well was scored positive if the A450was 2 standard deviations above the mean of six replicates of the corresponding dilution of normal horse plasma tested in the same assay. Each plasma dilution was tested in triplicate and the loglo endpoint titer was the highest dilution having two or more positive wells. Subclass specificity of each rabbit antiserum was verified by radioimmunoassay using [125I]-labelled purified equine IgG and IgG (T), and by ELISA on plates coated with purified equine IgG and IgG (T).
Single radial immunodiffusion (SRID) Plasma concentrations (mg/ml) of IgG and IgG(T) were determined by SRID using rabbit antisera specific for IgG and IgG (T) with a standard curve of equine serum of known IgG and IgG(T) concentrations (McGuire and Crawford, 1972).
Neutralization assay Neutralizations were performed by the constant plasma-diluted virus method. Serial three-fold dilutions of virus beginning with approximately 104 TCID~o/ ml were incubated with an equal volume of plasma for 30 min at 4 ° C. Three replicates of each mixture were added to subconfluent monolayers of EK cells and incubated at 4 °C for 2 h. Unabsorbed virus was aspirated and the cells maintained for 9 to 11 days in M E M with 3% calf serum. Infectivity of the plasma-treated virus was determined by direct immunofluorescence and fifty percent endpoints were calculated (Reed and Muench, 1938). Each neutralization assay consisted of pre-inoculation plasma samples from four horses and three replicates of each test sample. Neutralization was determined using a one-tailed unpaired t-test; positive neutralization was indicated if the mean of the test samples was significantly lower ( P < 0.01) than the mean of the four normal samples; weak neutralization was indicated if 0.01 < P < 0.05.
IgG(T) depletion To determine whether antiviral IgG (T) played a role in the neutralization of EIAV, plasma from foal A1907 was absorbed on a solid phase anti-IgG (T)
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K.I. O'ROURKEET AL.
column. Plasma collected at 0, 30 and 60 days post infection (dpi) was passed through a column of Sepharose 4B (Pharmacia) coupled to purified rabbit anti-IgG (T) and each sample was concentrated to its starting volume. Depletion of antiviral IgG(T) was monitored using the subclass-specific ELISA. Samples showing no IgG (T) activity were filter sterilized and used in the neutralization assay. A log neutralization index for each sample was expressed as the difference in lOglo titer between replicate virus samples pre-incubated with control media (MEM) or with plasma. A neutralization index of 0.97 is indicative of neutralization under our assay conditions (O'Rourke et al., 1988). RESULTS
The prototype strain of EIAV and each of five variants derived from serial horse passages of EIAV-prototype were biologically cloned three times to select individual virus variants (Table 1 ). The variants collected sequentially from each horse were demonstrated to be antigenic variants using a panel of equine and rabbit neutralizing antisera (data not shown). The four horses previously described (O'Rourke et al., 1988) were infected with EIAV-WSU5. Five more horses were infected, each with a single different variant. Plasma samples collected at 60 or 90 dpi were used in neutralization assays against each of the six variants (Table 2 ). Infection with the prototype strain did not result in clinical disease, and neutralizing antibody was not detected until 90 days post-infection. This plasma neutralized both the prototype strain and EIAV-WSU1. Plasma from the horses infected with EIAV-WSU1, WSU2, WSU3 and WSU4 showed a broad range of cross-reactivity against the horse-passaged variants, although none neutralized the prototype strain. The four horses infected with EIAV-WSU5 showed a more narrow range of cross neutralization at 60 dpi but only one, A1925, had an isolate specific response. Antiviral ELISA titers, adjusted for plasma concentration of IgG and IgG (T), TABLE1 Origin of EIAV variants Horse number
Infected with
H347 H309
EIAV-prototypea plasma from H347 27 dpi
H354
plasma from H309 40 dpi
Virus isolated from plasma dpi
Isolate
27 13
EIAV-WSU1 EIAV-WSU2
40 10
EIAV-WSU3 EIAV-WSU4
29
EIAV-WSU5
"10~TCIDso tissue culture adapted Wyoming strain EIAV (Malmquist et al., 1973 ).
45
CROSS-NEUTRALIZINGANDSUBCLASSCHARACTERISTICSOFANTIBODYTO EIAV TABLE 2 Neutralization of variants of EIAV by immune equine plasma Horse/Inoculum
dpi
Virus isolate Prototype
A1945/proto A1948/WSU1 A1950/WSU2 A1951/WSU3 A1955/WSU4 A1907/WSU5 A1908/WSU5 A1924/WSU5 A1925/WSU5
90 60 60 60 60 60 60 60 60
+~ -+ + + + .
WSU1
+ + + +
.
+ +b + + + . . .
WSU2 . + + + + + + + + + . .
WSU3 .
. + + + +
. . .
+ + + + . .
.
WSU4 . + + + + + + + -
WSU5
+ + + + + + +
+ + + + + + + +
a +indicates 0.01 < P < 0.05. b+ + indicates that the mean loglo titer of three replicate virus isolates preincubated with immune plasma, collected at the indicated number of days p.i., was significantly different (P < 0.01 ) from the mean titer of replicate samples preincubated with four normal plasma samples. TABLE 3 Antiviral IgG and IgG (T) titers of horses with EIAV Foal number
1907 1908 1924 1925
dpi
30 60 30 60 30 60 30 60
Adjusted ELISA log titer a IgG
IgG(T)
3.90 4.50 3.14 4.08 4.85 4.24 4.07 4.29
5.10 4.30 4.50 4.72 5.48 4.85 4.50 5.52
aLOgloof the reciprocal of the highest dilution giving positive results by ELISA using plates coated with EIAV, adjusted by dividing the ELISA titer by the plasma concentration of IgG or IgG (T) (mg/ml) determined by SRID. indicated that both subclasses contributed to the antiviral antibody response in the four horses infected with EIAV-WSU5 (Table 3). The mean antiviral I g G ( T ) t i t e r w a s s i g n i f i c a n t l y h i g h e r ( P < 0.01 ) t h a n t h e m e a n a n t i v i r a l I g G t i t e r i n t h e s a m p l e s c o l l e c t e d a t 30 a n d 60 d p i i n t h e s e h o r s e s . T h e r o l e o f IgG (T ) in n e u t r a l i z a t i o n was e x a m i n e d by selective a b s o r p t i o n of IgG ( T ) from t h e n o n - n e u t r a l i z i n g 30 d p i p l a s m a s a m p l e a n d t h e n e u t r a l i z i n g 60 d p i s a m p l e
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K.I.O'ROURKEET AL.
TABLE 4 Neutralization indexesa of EIAV by IgG (T) depleted plasma dpi
Whole plasma
IgG (T) depleted plasmab
0 30 60
0.14 0.28 2.09
0.31 0.19 1.68
aDifference in log titer between viral samples pre-incubated with control media (MEM) or with plasma collected from horse A1907 at the indicated dpi. An index of 0.97 indicates neutralization, as described in Materials and Methods. bplasma was IgG (T) depleted by passage through a solid-phase immunosorbentcolumn ( Sepharose 4-B coupled to rabbit anti-IgG (T)).
from A1907. Absorption of IgG (T) from the non-neutralizing sample did not increase its neutralizing capacity. IgG (T) depletion of the 60 dpi neutralizing sample reduced its log neutralization index by 0.41 (Table 4). DISCUSSION
Sera collected late in infection from individuals with HIV or from horses with EIAV are broadly cross-reactive in neutralization assays against antigenic variants (Robert-Guroff et al., 1985; Weiss et al., 1986; Bolognesi, 1988; Kono, 1988 ). The frequent occurrence of group specific neutralizing antibody in horses early in infection has also been described (Kono, 1988). Our study provides confirmation of these data using a wider panel of independently derived biologically cloned variants. Because of the rapid mutation of EIAV (Payne et al., 1984) and the probable accumulation of multiple variants circulating simultaneously during lentivirus infection (Gibbons et al., 1988; Looney et al., 1988), studies of the mechanism of cross-neutralization using immune plasma are not definitive. Sequential serum samples taken after experimental infection of chimpanzees and after accidental infection of a laboratory worker with HIV indicate that group specific neutralizing antibody occurs months to years following appearance of type-specific neutralizing antibody (Bolognesi, 1988; Goudsmit et al., 1988). Immunization with synthetic peptides results in typespecific HIV neutralization (Palker et al., 1988). These observations suggest that cross-neutralization in HIV is due to recognition of a number of typespecific epitopes evolving slowly in infected individuals. In contrast, the nearly simultaneous appearance of type-specific and group-specific neutralizing antibody in the horses in this study suggests that additional mechanisms also contribute to EIAV cross-neutralization. The immune system in some horses may respond to conserved neutralization epitopes; alternatively, wide divergence of the inoculum strain may occur during or soon after the initial viremic
CROSS-NEUTRALIZING AND SUBCLASS CHARACTERISTICS OF ANTIBODY TO EIAV
47
peak. Only the use of non-replicating EIAV or subunit immunogens will permit evaluation of the heterologous cross-neutralizing effect. Variability in the crossneutralizing response among horses was demonstrated in the four animals infected with EIAV-WSU5. Factors including age, health, immunologic status and possibly genetics influence the course of disease in horses and may affect the neutralizing response as well. Among horses in this group, only animal A1925 had a variant-specific response. However, this animal's neutralizing titer against the homologous variant was only 2 (O'Rourke et al., 1988), suggesting that any response to heterologous isolates may have been below the detection limits of this assay. Low titered neutralizing antibody has been reported for this and other lentiviruses (Weiss et al., 1985; McGuire et al., 1988; O'Rourke et al., 1988). The factors inhibiting efficient neutralization are not understood. The neutralizing plasmas used in this study are characterized by a significant IgG (T) response to EIAV antigens. Equine IgG (T) has markedly restricted effector functions including failure of IgG (T ) -antigen complexes to fix complement by the classical pathway or to bind macrophage and neutrophil Fc receptors (McGuire et al., 1971b; Banks and McGuire, 1975 ). Further, precipitating complexes are formed only over a narrow zone of equivalence; in antibody excess, complexes remain soluble (McGuire et al., 1979). In this study, we investigated the role of IgG (T) in the low titered in vitro neutralizing activity of plasma from an infected horse. Selective removal of IgG(T) from a non-neutralizing plasma sample did not increase the neutralization index. This observation demonstrates that IgG (T) was not competing with IgG for binding to potential neutralization-sensitive epitopes under the conditions of this assay. However, in a complement-augmented system or an assay in which binding of antibodyvirus complexes to Fc receptors occurs, competitive binding of IgG and IgG (T) may affect neutralization. IgG (T) depletion of a neutralizing sample reduced the loglo neutralization index by 0.41, corresponding to a 60% decrease in the actual concentration of virus particles neutralizable with this sample. Since IgG (T) represented nearly half of the virus-reactive antibody in this sample, this observation indicates that IgG(T) may contribute to neutralization by this sample. At least four epitopes on EIAV gp90, of which only two contribute to neutralization, have been defined using murine monoclonal antibodies (Hussain et al., 1988). Subclass characterization of equine antibodies reactive with individual neutralization and non-neutralization epitopes remains to be considered in evaluating the role of subclass restriction in immunity to EIAV. ACKNOWLEDGEMENTS
Patrick Higgins, Patricia Mason, Jan Carlson and Clara Owen provided excellent technical assistance. Supported in part by N.I.H. grants AI24291 and
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K.I.O'ROURKEETAL.
AI24166, U.S.D.A. 86-CRSR 22850, the Grayson Foundation, the Morris Animal Foundation and the State of Washington Equine Research Program.
REFERENCES Banks, K.L. and McGuire, T.C., 1975. Surface receptors on neutrophils and monocytes from immunodeficient and normal horses. Immunology, 28: 581-588. Bolognesi, D.P., 1988. Natural immunity to HIV and its possible relationship to vaccine strategies. Microbiol. Sci., 5 (8): 236-241. Charman, H.P., Bladen, S., Gilden, R.V. and Coggins, L., 1976. Equine infectious anemia virus: evidence favoring classification as a retravirus. J. Virol., 19 (2): 1073-1079. Chiu, I., Yaniv, A., Dahlberg, J.E., Gazit, A., Skuntz, S.F., Tronick, S.R. and Aaronson, S.A., 1985. Nucleotide sequence evidence for relationship of AIDS retrovirus to lentiviruses. Nature (London), 317: 366-368. Coffin, J., Haase, A., Levy, J.A., Montagnier, L., Oroszlan, S., Teich, N., Temin, H., Toyoshima, K., Varmus, H., Vogt, P. and Weiss, R., 1986. Human immunodeficiency viruses. Science, 232: 697-698. Coggins, L., 1984. Carriers of equine infectious anemia virus. J. Am. Vet. Med. Assoc., 184 (3): 279-281. Crawford, T.B., McGuire, T.C. and Henson, J.B., 1971. Detection of equine infectious anemia virus in vitro by immunofluorescence. Arch. Gesamte Virusforsch., 34: 332-339. Gibbons, J., Saag, M.S., Parks, E.S., Parks, W.P., Hahn, B.H. and Shaw, G.M., 1988. Genetic variation of human immunodeficiency virus type I (HIV-1) in vitro and in vivo. In: Human Retroviruses, Cancer and AIDS: Approaches to Prevention and Therapy. Alan R.Liss, New York, NY, pp. 101-114. Goudsmit, J., Thiriart, C., Smit, L., Bruck, C. and Gibbs, C.J., 1988. Temporal development of cross-neutralization between HTLV-III B and HTLV-III RF in experimentally infected chimpanzees. Vaccine, 6: 229-232. Hussain, K., Issel, C.J., Schnorr, K.L., Rwambo, P.M., West, M. and Montelaro, R.C., 1988. Antigenic mapping of the envelope proteins of equine infectious anemia virus: identification of a neutralization domain and a conserved region Qn glycoprotein 90. Arch. Virol., 98: 213-224. Kono, Y., 1988. Antigenic variation of equine infectious anemia virus as detected by virus neutralization. Arch. Virol., 98: 91-97. Kono, Y., Kobayashi, K. and Fukunaga, Y., 1973. Antigenic drift of equine infectious anemia virus in chronically infected horses. Arch. Virol., 41: 1-10. Looney, D.J., Fisher, A.G., Putney, S.D., Rusche, J.R., Redfield, R.R., Burke, D.S., Gallo, R.C. and Wong-Staal, F., 1988. Type-restricted neutralization of molecular clones of human immunodeficiency virus. Science, 241: 357-359. Malmquist, W.A., Barnett, D. and Becvar, C.S., 1973. Production of equine infectious anemia antigen in a persistently infected cell line. Arch. Gesamte Virusforsch., 42: 361-370. McGuire, T.C. and Crawford, T.B., 1972. Identification and quantitation of equine serum and secretory IgA. Infect. Immun., 6: 610-615. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1971a. Immunofluorescent localization of equine infectious anemia virus in tissue. Am. J. Pathol., 62 (2): 283-294. McGuire, T.C., Van Hoosier, G.L. and Henson, J.B., 1971b. The complement-fixation reaction in equine infectious anemia, demonstration of inhibition by IgG(T). J. Immunol., 107: 17381744. McGuire, T.C., Archer, B.G. and Crawford, T.B., 1979. Equine IgG and IgG(T) antibodies: de-
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pendence of precipitability on both antigen and antibody structure. Mol. Immunol., 16: 787790. McGuire, T.C., Norton, L.K., O'Rourke, K.I. and Cheevers, W.P., 1988. Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis. J. Virol., 62 (9): 3488-3492. Montelaro, R.C., Parekh, B., Orrego, A. and Issel, C.J., 1984. Antigenic variation during persistent infection by equine infectious anemia virus, a retrovirus, J. Biol. Chem., 259 (16): 10539-10544. O'Rourke, K.I., Perryman, L.E. and McGuire, T.C., 1988. Antiviral, anti-glycoprotein and neutralizing antibodies in foals with equine infectious anaemia virus. J. Gen. Virol., 69: 667-674. Palker, T.J., Clark, M.E., Langlois, A.J., Matthews, T.J., Weinhold, K.J., Randall, R.R., Bolognesi, D.P. and Haynes, B.F., 1988. Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc. Nat. Acad. Sci., U.S.A., 85: 1932-1936. Payne, S., Montelaro, R.C. and Issel, C.J., 1984. Genomic alterations associated with persistent infections by equine infectious anemia virus, a retrovirus. J. Gen. Virol., 65: 1395-1399. Perryman, L.E., O'Rourke, K.I. and McGuire, T.C., 1988. Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection. J. Virol., 62 (8): 3073-3076. Reed, L.J. and Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg., 27: 493-497. Robert-Guroff, M., Brown, M. and Gallo, R.C., 1985. HTLV-III neutralizing antibodies in patients with AIDS and AIDS-related complex. Nature (London), 316 (6023): 72-74. Stephens, R.M., Casey, J.W. and Rice, N.R., 1986. Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. Science, 231: 589-594. Weiss, R.A., Clapham, P.R., Cheingsong-Popov, R., Dalgleish, A.G., Carne, C.A., Weller, I.V.D. and Tedder, R.S., 1985. Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature (London), 316: 69-72. Weiss, R.A., Clapham, P.R., Weber, J.N., Dalgleish, A.G., Lasky, L.A. and Berman, P.W., 1986. Variable and conserved neutralization antigens of human immunodeficiency virus. Nature (London), 324: 572-575.
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Cross-neutralizing and Subclass Characteristics of Antibody from Horses with Equine Infectious A n e m i a Virus KATHERINE I. O'ROURKE, LANCE E. PERRYMAN and TRAVIS C. McGUIRE
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164 (U.S.A.) (Accepted 18 April 1989)
ABSTRACT O'Rourke, K.I., Perryman, L.E. and McGuire, T.C., 1989. Cross-neutralizing and subclass characteristics of antibody from horses with equine infectious anemia virus. Vet. Immunol, Immunopathol., 23: 41-49. Antibody responses in horses with equine infectious anemia virus (EIAV) were examined to determine their cross-neutralizing capacity. Antibodies induced by infection with any of six biologically cloned variants of EIAV cross-neutralized multiple variants from the group. Anti-EIAV antibody was found in both the IgG and IgG(T) subclasses in plasmas with virus-neutralizing activity and the majority of antiviral antibody was of the IgG (T) subclass. Depletion of IgG (T) did not increase the neutralization indexes of either neutralizing or non-neutralizing plasma samples.
INTRODUCTION
Equine infectious anemia virus (EIAV) is a lentivirus of horses which infects fixed and circulating cells of the monocyte lineage (McGuire et al., 1971a; Charman et al., 1976). EIAV shares genome sequence homology with other known lentiviruses, including human immunodeficiency virus (HIV), caprine arthritis-encephalitis virus (CAEV) and ovine visna virus (VV) ( Chiu et al., 1985; Stephens et al., 1986). These lentiviruses cause lifelong infection in the host with a variable pattern of clinical disease expression. Lentivirus infections are not eliminated in spite of the generation of humoral and cell-mediated antiviral immune responses. Cell-free viremia is temporarily cleared by immune responses during the first 35 days of infection with EIAV (Perryman et al., 1988). Relapsing disease is associated with the appearance of novel antigenic variants (Kono et al., 1973; Montelaro et al., 1984), suggesting that immune selection of rapidly evolving mutants represents one mechanism of viral 0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
42
K.I. O'ROURKE ET AL.
persistence. Most horses with EIAV become clinically normal within the first year after infection and remain asymptomatic and aviremic for years, although the virus can be transmitted by transfer of whole blood or washed leukocytes to susceptible horses (see review by Coggins, 1984). An understanding of the elements of the immune response which control disease in infected horses may contribute to development of an EIAV vaccine which prevents infection of horses under field conditions. We have previously described the antiglycoprotein and neutralizing antibody responses in four horses infected with a virulent EIAV isolate (O'Rourke et al., 1988). In this study, we extend those observations by describing an early cross-neutralizing response in a larger panel of horses infected with biologically cloned EIAV variants and by characterizing the relative contributions of antiviral antibody of the IgG and IgG(T) subclasses to the neutralizing response. MATERIALS AND METHODS
Virus isolates Antigenic variants of EIAV were generated by serial passage of the tissue culture adapted Wyoming strain of EIAV (EIAV-prototype) (Malmquist et al., 1973) through horses. The prototype strain was cloned sequentially three times by limiting dilution, propagated in equine dermal cells (ATCC CCL57) and serially passaged through three horses (H347, H309 and H354). All three horses developed clinical signs of EIAV infection, including periodic temperature increases, at which times virus was isolated. Virus isolates were made by incubation of 1 ml plasma, diluted 1:5 in minimal essential media (MEM), with a subconfluent monolayer of equine kidney (EK) cells in a 25-cm 2 tissue culture flask for 2 h at room temperature. Cells were maintained as described (O'Rourke et al., 1988) and screened at each passage for viral antigen by immunofluorescence (Crawford et al., 1971). Because there is no plaque assay for EIAV, a predominant isolate from each viremic period was selected by three serial limiting dilution clonings in EK cells. Between cloning rounds, virus from the endpoint wells was propagated in EK cells until the virus titer reached approximately 103 TDCIs0/ml. The stringency of triple endpoint cloning under these conditions makes it highly likely that each isolate represents a single virus variant. Isolates were numbered EIAV-WSU1 through EIAV-WSU5 following the proposal for naming human immunodeficiency lentiviruses (Coffin et al., 1986). EIA V infection Eight horses (A1948, A1950, A1951, A1955, A1907, A1908, A1924 and A1925) were infected by intravenous administration of 106 TCIDso EIAV. Plasma collected at day 0 and at intervals following infection was stored at - 7 0 °C for
CROSS-NEUTRALIZINGAND SUBCLASS CHARACTERISTICS OF ANTIBODY TO EIAV
43
subsequent virus isolation and at - 20 ° C for serologic assays. Rectal temperatures were recorded daily.
Enzyme linked immunosorbent assay (ELISA) Antiviral ELISA titers were determined using serial three-fold dilutions of plasma samples assayed on microELISA (Immulon I, Dynatech ) plates coated with 300 ng EIAV per well. Antiviral antibody was detected using rabbit antiserum specific for equine IgG or IgG (T) (McGuire and Crawford, 1972 ), goat anti-rabbit IgG-HRPO (Sigma) and 5-aminosalicylic acid as a chromogenic substrate. After approximately 2 h at room temperature, the optical density of each well was determined with an automated ELISA reader (Dynatech Laboratories). A well was scored positive if the A450was 2 standard deviations above the mean of six replicates of the corresponding dilution of normal horse plasma tested in the same assay. Each plasma dilution was tested in triplicate and the loglo endpoint titer was the highest dilution having two or more positive wells. Subclass specificity of each rabbit antiserum was verified by radioimmunoassay using [125I]-labelled purified equine IgG and IgG (T), and by ELISA on plates coated with purified equine IgG and IgG (T).
Single radial immunodiffusion (SRID) Plasma concentrations (mg/ml) of IgG and IgG(T) were determined by SRID using rabbit antisera specific for IgG and IgG (T) with a standard curve of equine serum of known IgG and IgG(T) concentrations (McGuire and Crawford, 1972).
Neutralization assay Neutralizations were performed by the constant plasma-diluted virus method. Serial three-fold dilutions of virus beginning with approximately 104 TCID~o/ ml were incubated with an equal volume of plasma for 30 min at 4 ° C. Three replicates of each mixture were added to subconfluent monolayers of EK cells and incubated at 4 °C for 2 h. Unabsorbed virus was aspirated and the cells maintained for 9 to 11 days in M E M with 3% calf serum. Infectivity of the plasma-treated virus was determined by direct immunofluorescence and fifty percent endpoints were calculated (Reed and Muench, 1938). Each neutralization assay consisted of pre-inoculation plasma samples from four horses and three replicates of each test sample. Neutralization was determined using a one-tailed unpaired t-test; positive neutralization was indicated if the mean of the test samples was significantly lower ( P < 0.01) than the mean of the four normal samples; weak neutralization was indicated if 0.01 < P < 0.05.
IgG(T) depletion To determine whether antiviral IgG (T) played a role in the neutralization of EIAV, plasma from foal A1907 was absorbed on a solid phase anti-IgG (T)
44
K.I. O'ROURKEET AL.
column. Plasma collected at 0, 30 and 60 days post infection (dpi) was passed through a column of Sepharose 4B (Pharmacia) coupled to purified rabbit anti-IgG (T) and each sample was concentrated to its starting volume. Depletion of antiviral IgG(T) was monitored using the subclass-specific ELISA. Samples showing no IgG (T) activity were filter sterilized and used in the neutralization assay. A log neutralization index for each sample was expressed as the difference in lOglo titer between replicate virus samples pre-incubated with control media (MEM) or with plasma. A neutralization index of 0.97 is indicative of neutralization under our assay conditions (O'Rourke et al., 1988). RESULTS
The prototype strain of EIAV and each of five variants derived from serial horse passages of EIAV-prototype were biologically cloned three times to select individual virus variants (Table 1 ). The variants collected sequentially from each horse were demonstrated to be antigenic variants using a panel of equine and rabbit neutralizing antisera (data not shown). The four horses previously described (O'Rourke et al., 1988) were infected with EIAV-WSU5. Five more horses were infected, each with a single different variant. Plasma samples collected at 60 or 90 dpi were used in neutralization assays against each of the six variants (Table 2 ). Infection with the prototype strain did not result in clinical disease, and neutralizing antibody was not detected until 90 days post-infection. This plasma neutralized both the prototype strain and EIAV-WSU1. Plasma from the horses infected with EIAV-WSU1, WSU2, WSU3 and WSU4 showed a broad range of cross-reactivity against the horse-passaged variants, although none neutralized the prototype strain. The four horses infected with EIAV-WSU5 showed a more narrow range of cross neutralization at 60 dpi but only one, A1925, had an isolate specific response. Antiviral ELISA titers, adjusted for plasma concentration of IgG and IgG (T), TABLE1 Origin of EIAV variants Horse number
Infected with
H347 H309
EIAV-prototypea plasma from H347 27 dpi
H354
plasma from H309 40 dpi
Virus isolated from plasma dpi
Isolate
27 13
EIAV-WSU1 EIAV-WSU2
40 10
EIAV-WSU3 EIAV-WSU4
29
EIAV-WSU5
"10~TCIDso tissue culture adapted Wyoming strain EIAV (Malmquist et al., 1973 ).
45
CROSS-NEUTRALIZINGANDSUBCLASSCHARACTERISTICSOFANTIBODYTO EIAV TABLE 2 Neutralization of variants of EIAV by immune equine plasma Horse/Inoculum
dpi
Virus isolate Prototype
A1945/proto A1948/WSU1 A1950/WSU2 A1951/WSU3 A1955/WSU4 A1907/WSU5 A1908/WSU5 A1924/WSU5 A1925/WSU5
90 60 60 60 60 60 60 60 60
+~ -+ + + + .
WSU1
+ + + +
.
+ +b + + + . . .
WSU2 . + + + + + + + + + . .
WSU3 .
. + + + +
. . .
+ + + + . .
.
WSU4 . + + + + + + + -
WSU5
+ + + + + + +
+ + + + + + + +
a +indicates 0.01 < P < 0.05. b+ + indicates that the mean loglo titer of three replicate virus isolates preincubated with immune plasma, collected at the indicated number of days p.i., was significantly different (P < 0.01 ) from the mean titer of replicate samples preincubated with four normal plasma samples. TABLE 3 Antiviral IgG and IgG (T) titers of horses with EIAV Foal number
1907 1908 1924 1925
dpi
30 60 30 60 30 60 30 60
Adjusted ELISA log titer a IgG
IgG(T)
3.90 4.50 3.14 4.08 4.85 4.24 4.07 4.29
5.10 4.30 4.50 4.72 5.48 4.85 4.50 5.52
aLOgloof the reciprocal of the highest dilution giving positive results by ELISA using plates coated with EIAV, adjusted by dividing the ELISA titer by the plasma concentration of IgG or IgG (T) (mg/ml) determined by SRID. indicated that both subclasses contributed to the antiviral antibody response in the four horses infected with EIAV-WSU5 (Table 3). The mean antiviral I g G ( T ) t i t e r w a s s i g n i f i c a n t l y h i g h e r ( P < 0.01 ) t h a n t h e m e a n a n t i v i r a l I g G t i t e r i n t h e s a m p l e s c o l l e c t e d a t 30 a n d 60 d p i i n t h e s e h o r s e s . T h e r o l e o f IgG (T ) in n e u t r a l i z a t i o n was e x a m i n e d by selective a b s o r p t i o n of IgG ( T ) from t h e n o n - n e u t r a l i z i n g 30 d p i p l a s m a s a m p l e a n d t h e n e u t r a l i z i n g 60 d p i s a m p l e
46
K.I.O'ROURKEET AL.
TABLE 4 Neutralization indexesa of EIAV by IgG (T) depleted plasma dpi
Whole plasma
IgG (T) depleted plasmab
0 30 60
0.14 0.28 2.09
0.31 0.19 1.68
aDifference in log titer between viral samples pre-incubated with control media (MEM) or with plasma collected from horse A1907 at the indicated dpi. An index of 0.97 indicates neutralization, as described in Materials and Methods. bplasma was IgG (T) depleted by passage through a solid-phase immunosorbentcolumn ( Sepharose 4-B coupled to rabbit anti-IgG (T)).
from A1907. Absorption of IgG (T) from the non-neutralizing sample did not increase its neutralizing capacity. IgG (T) depletion of the 60 dpi neutralizing sample reduced its log neutralization index by 0.41 (Table 4). DISCUSSION
Sera collected late in infection from individuals with HIV or from horses with EIAV are broadly cross-reactive in neutralization assays against antigenic variants (Robert-Guroff et al., 1985; Weiss et al., 1986; Bolognesi, 1988; Kono, 1988 ). The frequent occurrence of group specific neutralizing antibody in horses early in infection has also been described (Kono, 1988). Our study provides confirmation of these data using a wider panel of independently derived biologically cloned variants. Because of the rapid mutation of EIAV (Payne et al., 1984) and the probable accumulation of multiple variants circulating simultaneously during lentivirus infection (Gibbons et al., 1988; Looney et al., 1988), studies of the mechanism of cross-neutralization using immune plasma are not definitive. Sequential serum samples taken after experimental infection of chimpanzees and after accidental infection of a laboratory worker with HIV indicate that group specific neutralizing antibody occurs months to years following appearance of type-specific neutralizing antibody (Bolognesi, 1988; Goudsmit et al., 1988). Immunization with synthetic peptides results in typespecific HIV neutralization (Palker et al., 1988). These observations suggest that cross-neutralization in HIV is due to recognition of a number of typespecific epitopes evolving slowly in infected individuals. In contrast, the nearly simultaneous appearance of type-specific and group-specific neutralizing antibody in the horses in this study suggests that additional mechanisms also contribute to EIAV cross-neutralization. The immune system in some horses may respond to conserved neutralization epitopes; alternatively, wide divergence of the inoculum strain may occur during or soon after the initial viremic
CROSS-NEUTRALIZING AND SUBCLASS CHARACTERISTICS OF ANTIBODY TO EIAV
47
peak. Only the use of non-replicating EIAV or subunit immunogens will permit evaluation of the heterologous cross-neutralizing effect. Variability in the crossneutralizing response among horses was demonstrated in the four animals infected with EIAV-WSU5. Factors including age, health, immunologic status and possibly genetics influence the course of disease in horses and may affect the neutralizing response as well. Among horses in this group, only animal A1925 had a variant-specific response. However, this animal's neutralizing titer against the homologous variant was only 2 (O'Rourke et al., 1988), suggesting that any response to heterologous isolates may have been below the detection limits of this assay. Low titered neutralizing antibody has been reported for this and other lentiviruses (Weiss et al., 1985; McGuire et al., 1988; O'Rourke et al., 1988). The factors inhibiting efficient neutralization are not understood. The neutralizing plasmas used in this study are characterized by a significant IgG (T) response to EIAV antigens. Equine IgG (T) has markedly restricted effector functions including failure of IgG (T ) -antigen complexes to fix complement by the classical pathway or to bind macrophage and neutrophil Fc receptors (McGuire et al., 1971b; Banks and McGuire, 1975 ). Further, precipitating complexes are formed only over a narrow zone of equivalence; in antibody excess, complexes remain soluble (McGuire et al., 1979). In this study, we investigated the role of IgG (T) in the low titered in vitro neutralizing activity of plasma from an infected horse. Selective removal of IgG(T) from a non-neutralizing plasma sample did not increase the neutralization index. This observation demonstrates that IgG (T) was not competing with IgG for binding to potential neutralization-sensitive epitopes under the conditions of this assay. However, in a complement-augmented system or an assay in which binding of antibodyvirus complexes to Fc receptors occurs, competitive binding of IgG and IgG (T) may affect neutralization. IgG (T) depletion of a neutralizing sample reduced the loglo neutralization index by 0.41, corresponding to a 60% decrease in the actual concentration of virus particles neutralizable with this sample. Since IgG (T) represented nearly half of the virus-reactive antibody in this sample, this observation indicates that IgG(T) may contribute to neutralization by this sample. At least four epitopes on EIAV gp90, of which only two contribute to neutralization, have been defined using murine monoclonal antibodies (Hussain et al., 1988). Subclass characterization of equine antibodies reactive with individual neutralization and non-neutralization epitopes remains to be considered in evaluating the role of subclass restriction in immunity to EIAV. ACKNOWLEDGEMENTS
Patrick Higgins, Patricia Mason, Jan Carlson and Clara Owen provided excellent technical assistance. Supported in part by N.I.H. grants AI24291 and
48
K.I.O'ROURKEETAL.
AI24166, U.S.D.A. 86-CRSR 22850, the Grayson Foundation, the Morris Animal Foundation and the State of Washington Equine Research Program.
REFERENCES Banks, K.L. and McGuire, T.C., 1975. Surface receptors on neutrophils and monocytes from immunodeficient and normal horses. Immunology, 28: 581-588. Bolognesi, D.P., 1988. Natural immunity to HIV and its possible relationship to vaccine strategies. Microbiol. Sci., 5 (8): 236-241. Charman, H.P., Bladen, S., Gilden, R.V. and Coggins, L., 1976. Equine infectious anemia virus: evidence favoring classification as a retravirus. J. Virol., 19 (2): 1073-1079. Chiu, I., Yaniv, A., Dahlberg, J.E., Gazit, A., Skuntz, S.F., Tronick, S.R. and Aaronson, S.A., 1985. Nucleotide sequence evidence for relationship of AIDS retrovirus to lentiviruses. Nature (London), 317: 366-368. Coffin, J., Haase, A., Levy, J.A., Montagnier, L., Oroszlan, S., Teich, N., Temin, H., Toyoshima, K., Varmus, H., Vogt, P. and Weiss, R., 1986. Human immunodeficiency viruses. Science, 232: 697-698. Coggins, L., 1984. Carriers of equine infectious anemia virus. J. Am. Vet. Med. Assoc., 184 (3): 279-281. Crawford, T.B., McGuire, T.C. and Henson, J.B., 1971. Detection of equine infectious anemia virus in vitro by immunofluorescence. Arch. Gesamte Virusforsch., 34: 332-339. Gibbons, J., Saag, M.S., Parks, E.S., Parks, W.P., Hahn, B.H. and Shaw, G.M., 1988. Genetic variation of human immunodeficiency virus type I (HIV-1) in vitro and in vivo. In: Human Retroviruses, Cancer and AIDS: Approaches to Prevention and Therapy. Alan R.Liss, New York, NY, pp. 101-114. Goudsmit, J., Thiriart, C., Smit, L., Bruck, C. and Gibbs, C.J., 1988. Temporal development of cross-neutralization between HTLV-III B and HTLV-III RF in experimentally infected chimpanzees. Vaccine, 6: 229-232. Hussain, K., Issel, C.J., Schnorr, K.L., Rwambo, P.M., West, M. and Montelaro, R.C., 1988. Antigenic mapping of the envelope proteins of equine infectious anemia virus: identification of a neutralization domain and a conserved region Qn glycoprotein 90. Arch. Virol., 98: 213-224. Kono, Y., 1988. Antigenic variation of equine infectious anemia virus as detected by virus neutralization. Arch. Virol., 98: 91-97. Kono, Y., Kobayashi, K. and Fukunaga, Y., 1973. Antigenic drift of equine infectious anemia virus in chronically infected horses. Arch. Virol., 41: 1-10. Looney, D.J., Fisher, A.G., Putney, S.D., Rusche, J.R., Redfield, R.R., Burke, D.S., Gallo, R.C. and Wong-Staal, F., 1988. Type-restricted neutralization of molecular clones of human immunodeficiency virus. Science, 241: 357-359. Malmquist, W.A., Barnett, D. and Becvar, C.S., 1973. Production of equine infectious anemia antigen in a persistently infected cell line. Arch. Gesamte Virusforsch., 42: 361-370. McGuire, T.C. and Crawford, T.B., 1972. Identification and quantitation of equine serum and secretory IgA. Infect. Immun., 6: 610-615. McGuire, T.C., Crawford, T.B. and Henson, J.B., 1971a. Immunofluorescent localization of equine infectious anemia virus in tissue. Am. J. Pathol., 62 (2): 283-294. McGuire, T.C., Van Hoosier, G.L. and Henson, J.B., 1971b. The complement-fixation reaction in equine infectious anemia, demonstration of inhibition by IgG(T). J. Immunol., 107: 17381744. McGuire, T.C., Archer, B.G. and Crawford, T.B., 1979. Equine IgG and IgG(T) antibodies: de-
CROSS-NEUTRALIZINGANDSUBCLASSCHARACTERISTICSOF ANTIBODYTO EIAV
49
pendence of precipitability on both antigen and antibody structure. Mol. Immunol., 16: 787790. McGuire, T.C., Norton, L.K., O'Rourke, K.I. and Cheevers, W.P., 1988. Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis. J. Virol., 62 (9): 3488-3492. Montelaro, R.C., Parekh, B., Orrego, A. and Issel, C.J., 1984. Antigenic variation during persistent infection by equine infectious anemia virus, a retrovirus, J. Biol. Chem., 259 (16): 10539-10544. O'Rourke, K.I., Perryman, L.E. and McGuire, T.C., 1988. Antiviral, anti-glycoprotein and neutralizing antibodies in foals with equine infectious anaemia virus. J. Gen. Virol., 69: 667-674. Palker, T.J., Clark, M.E., Langlois, A.J., Matthews, T.J., Weinhold, K.J., Randall, R.R., Bolognesi, D.P. and Haynes, B.F., 1988. Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides. Proc. Nat. Acad. Sci., U.S.A., 85: 1932-1936. Payne, S., Montelaro, R.C. and Issel, C.J., 1984. Genomic alterations associated with persistent infections by equine infectious anemia virus, a retrovirus. J. Gen. Virol., 65: 1395-1399. Perryman, L.E., O'Rourke, K.I. and McGuire, T.C., 1988. Immune responses are required to terminate viremia in equine infectious anemia lentivirus infection. J. Virol., 62 (8): 3073-3076. Reed, L.J. and Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg., 27: 493-497. Robert-Guroff, M., Brown, M. and Gallo, R.C., 1985. HTLV-III neutralizing antibodies in patients with AIDS and AIDS-related complex. Nature (London), 316 (6023): 72-74. Stephens, R.M., Casey, J.W. and Rice, N.R., 1986. Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. Science, 231: 589-594. Weiss, R.A., Clapham, P.R., Cheingsong-Popov, R., Dalgleish, A.G., Carne, C.A., Weller, I.V.D. and Tedder, R.S., 1985. Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature (London), 316: 69-72. Weiss, R.A., Clapham, P.R., Weber, J.N., Dalgleish, A.G., Lasky, L.A. and Berman, P.W., 1986. Variable and conserved neutralization antigens of human immunodeficiency virus. Nature (London), 324: 572-575.