VIROLOGY
17554
1-49 (1990)
Antigenic
and Genetic Characterization of Sindbis Virus Monoclonal Antibody Mutants which Define a Pathogenesis Domain on Glycoprotein E2
Escape
DAVID F. PENCE, NANCY L. DAVIS, AND ROBERT E. JOHNSTON’ Department
of Microbiology
and Immunology,
University of North Carolina, Chapel Hi//, North Carolina 27599-7290
Received August 17, 1989; accepted November
6, 1989
The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SE), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by EZ-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each of 11 mutants were identified. SB/ R6, SB/R13, and SB-RURl3 mutants contained a mutation at either E2 codon 96 or 159. SB-RUR6 mutants contained changes at E2 codon 62,96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E~c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice. o 1990 Academic Press. Inc.
INTRODUCTION
tigenic sites, one on El (Chanas et a/., 1982; Schmaljohn et a/., 1983) and three independently mutable sites on E2, designated E2a, E2b, and E2c (Roehrig et a/., 1982; Schmaljohn et al., 1983; Stec and Schmaljohn, 1984; Stanley ef al., 1985; Stec et al., 1986; Olmsted et a/., 1986; Davis et al., 1987). On the basis of competitive binding studies, E2a and E2b appear to lie in relatively close proximity to one another, with E2c spatially separate. While E2a and E2c are present in all laboratory strains tested, E2b is strain-specific in nature, exhibiting alternate forms defined by two separate sets of MCAbs (Davis et al., 1987). Sequencing E2b neutralization escape mutants has shown that E2b specificity depends primarily on the identity of the amino acid at position 216 of E2 (Stec et al., 1986; Davis et al., 1987). Likewise, E2a-specific MCAb neutralization escape mutants map to E2 residue 190 (Strauss ef al., 1987). Elements of the E2c antigenic site have been related genetically to determinants of Sindbis virulence in experimental animals and to the ability of the virus to penetrate cultured cells (Olmsted et a/., 1986; Polo et al., 1988; Russell et al., 1989). Sindbis strain AR339 (SB) causes a lethal encephalitis in neonatal mice, with subcutaneous (SC) doses approaching 1 PFU per mouse causing 100% mortality within 7 days (Reinarz et al., 1971; Johnson et al., 1972). A rapidly penetrating mu-
Sindbis virus is the prototype member of the alphavirus genus of the family Togaviridae (for a review, see Schlesinger and Schlesinger, 1986). The singlestranded, message sense RNA genome is contained within an icosahedral viral core assembled from multiple copies of a single protomeric peptide, C (30K). Upon release from the infected cell by budding, the nucleocapsid acquires a cell-derived lipid envelope containing equimolar amounts of two transmembrane viral glycoproteins, El (50K) and E2 (45K) (Strauss et al., 1969; Schlesinger eta/., 1972; von Bonsdotff and Harrison, 1975). El and E2 exist as a heterodimeric spike (Rice and Strauss, 1982) on the virion surface, and directly interact with the external environment (Sefton et a/., 1978). The glycoprotein spikes are involved in several biological activities, including hemagglutination (Dalrymple et a/., 1976; Schmaljohn et a/., 1983) virus attachment and penetration (Olmsted et al., 1984, 1986; Flynn et a/., 1988) and interaction with elements of the host immune system including those responsible for the production of antibodies. Mapping of Sindbis virions with monoclonal antibodies (MCAbs) has identified at least four neutralizing an’ To whom requests for reprints should be addressed. 41
0042.6822/90
$3.00
CopyrIght 0 1990 by Academic Press. Inc All rights of reproduction I” any form resewed
PENCE, DAVIS, AND JOHNSTON
42
tant of SB, designated SB-RL (Baric et a/., 1980, 1981; Olmsted et al., 1984) is characterized by greater than 30% survival at SCdoses as high as 106. Although SB and SB-RL bind equivalent levels of E2c MCAbs, SBRL is more efficiently neutralized by these antibodies than is SB (Olmsted et al., 1986). Virulent revertants of SB-RL lose both the E2c neutralization sensitivity and rapid penetration phenotypes (Olmsted et a/., 1984, 1986). In contrast, variation at the E2b antigenic site has no demonstrated effect on virulence of SB in neonatal mice (Davis et al., 1987). Sequencing of the glycoprotein genes of SB, SB-RL, and virulent revertants of SB-RL suggested that substitution of arginine at E2 position 1 14 of SB-RL for serine in SB is responsible for the linked variation in penetration, virulence, and E2c neutralization efficiency (Davis et al., 1986). Examination of virus produced by transfection of RNAs transcribed from full-length cDNAs of Sindbis (Rice et a/., 1987; Polo et a/., 1988) confirmed this suggestion. Virus from clones containing an arginine codon at E2 114 is attenuated, rapidly penetrating, and efficiently neutralized by E2c MCAbs, while virus from otherwise isogenic clones containing a serine at E2 114 is virulent, slowly penetrating, and poorly neutralized by E2c antibodies. In this paper, we report the isolation, characterization, and sequencing of a panel of neutralization escape mutants of SB and SB-RL that are no longer able to bind E2c MCAbs R6 or R13. Sequence analysis of these isolates has identified mutations at three different, noncontiguous codons in the E2 gene that independently abolish E2c antibody binding. Some of these mutations also affect pathogenesis and penetration, depending on the identity of the encoded amino acid substitution and on the identity of the residue at E2 114. These results suggest that the E2c-specific MCAbs define a conformation-dependent antigenic site. This site may substantially overlap or be coincident with virion surface domains important for virulence in neonatal mice and penetration of cultured cells. MATERIALS AND METHODS Cell and virus culture, immunological reagents All virus stocks used in these experiments were produced by infection of baby hamster kidney (BHK) cells (ATCC CCL-l 0), in Eagle’s minimum essential medium containing 10% bovine calf serum (BCS), 10% tryptose phosphate broth (TPB), 50 units penicillin/ml, and 50 pg/ml streptomycin (complete medium). Cells were used in passages 53-64. The wild-type laboratory strain SB (Sindbis strain AR 339) was obtained originally from H. R. Bose (University of Texas, Austin). The
attenuated strain, SB-RL, was isolated by Baric et al. (1980, 1981) by selection for rapid growth of SB in BHK cells. All antibody escape mutants were derived from these strains. Procedures for production of virus seed and working stocks and for preparation of purified virus by ultracentrifugation have been described previously (Olmsted et al., 1986; Davis et al., 1986). The production and characterization of MCAbs R6, R13, R8, and Rl 0 (Olmsted et a/., 1986; Davis et al., 1987) also have been described. Antibody 49 was the gift of A. Schmaljohn, U.S. Army Research Institute of Infectious Diseases (Schmaljohn et a/., 1982). Selection of neutralization escape mutants Neutralization escape mutants were selected from parental stocks of SB and SB-RL. To obtain SB-RL variants resistant to MCAb R13 neutralization, 5 X 1O4PFU of SB-RL were included in a l-ml vol of phosphatebuffered saline with 1% BCS (PBS-l %) containing 20% (v/v) MCAb R13 ascites fluid and 5% guinea pig complement (Accurate Chemical Co.). The mixture was incubated for 30 min at 37”. The surviving virus was diluted to a maximum titer of 1000 PFU/ml and plated in 200-~1 aliquots on BHK cells in loo-mm tissue culture dishes. One hour later, the cells were overlaid with complete medium containing 0.7% (w/v) agarose. To obtain a neutralization estimate, a second parallel incubation of virus was performed in the same fashion, except that 20% normal mouse ascites fluid (NAF) was added instead of antibody. Well-isolated plaques were picked 24 hr later, and the agarose plugs were eluted for 12 hr at 4” in 300 ~1of complete medium. One hundred microliters of each eluate was then added to duplicate wells of a 96-well tissue culture plate containing 6 X 1O4 BHK cells per well in 100 ~1of complete medium. One well of each pair contained 1% MCAb R13 and the other, 1% NAF. The remaining 100 ~1of those isolates found to produce strong CPE in the presence of the selecting antibody was used to grow a virus seed stock, and from that a working stock of each isolate was obtained. Growth of these stocks was in the presence of MCAb R13. RLmc6-2 was isolated in a similar manner using MCAb R6, except five successive cycles of neutralization and amplification of survivors in tissue culture were conducted before plaque isolates were taken (Olmsted et al., 1986). The other variants of SB-RL selected with MCAb R6 and those of SB selected with MCAb R6 or R13 were plaqued directly from a single neutralization reaction in the same way as the SB-RUR13 variants described above, except that a goat anti-mouse IgG-specific second antibody (Sigma) was added to the neutralization
SINDBIS VIRUS MONOCLONAL
reactions to increase their efficiency. Plaque eluates were added directly to wells of a 96-well tissue culture plate containing BHK cells in 200 ~1 of complete medium containing 1% selecting antibody. Medium from wells showing strong CPE 24-36 hr later was assayed in a capture ELISA screen for loss of ability to bind the selecting antibody (Olmsted et al., 1986; Davis et al., 1987). Seed and working stocks were grown in the presence of the selecting antibody.
ELISA and neutralization assays Solid-phase and capture ELISA, as well as plaquereduction neutralization assays, were performed on virus according to the protocols described by Olmsted
et al. (1986). Sequencing Direct sequencing of the glycoprotein genes of the variants from purified viral RNA using reverse transcriptase was performed by oligonucleotide-primed dideoxynucleotide chain termination (Zimmern and Kaesberg, 1978) using primers and protocols as detailed by Davis et al. (1987). The E2 gene of each mutant was sequenced as well as the El genes of representative mutants: RLmc6-2, RLmcl3-1, RLmcl3-2, and RLmcl3-5.
Pathogenesis assay The parental strains and all antigenic variants were tested for virulence in litters of 18- to 36-hr-old CD-l mice (Charles River Labs). Neonatal mice were inoculated SCwith 100 PFU of virus in 50 ~1 of PBS-l %. Control animals received PBS-l % only. Litters were monitored for 14 days.
Estimation of penetration Virus penetration was measured as time-dependent acquisition of resistance to neutralization by polyclonal anti-SB antibody, in a manner similar to the method of Olmsted et a/. (1986). BHK cells in 60-mm dishes were inoculated with 0.2 ml of PBS-l% containing approximately 200 PFU of virus, and incubated at 37”. At 15 min postinfection, the inoculum was removed from replicate plates of each virus assayed. The plates were washed with 2 ml of PBS-l %, and 0.4 ml of a 1:2000 dilution of AR339-specific hyperimmune mouse ascites fluid (HMAF) in PBS-l % with 5% guinea pig complement (Accurate Chemical Co.) was added per plate. After incubation for 30 min at 37”, the antibody was removed. The plates were washed as before to remove residual antibody, and the cells were overlaid with complete media with agarose. A parallel set of plates
ANTIBODY
ESCAPE MUTANTS
43
were identically infected, and were overlaid after a 60min incubation at 37”. Penetration was estimated as the percentage of plaques arising after the antibody treatment, as compared with the controls which received no antibody. The penetration assay depends on the ability of HMAF to neutralize virions which have attached but not yet penetrated. To ensure that none of the escape mutations significantly reduced sensitivity to neutralization by HMAF, the efficiency of neutralization of attached virions by HMAF was monitored for each variant. Simultaneously with the above assay, two plates per isolate were cooled to 4” for 1 hr. The plates were then inoculated in the cold with the same virus dilutions used in the penetration assay. Virus attachment was allowed to proceed in the cold for an additional hour, after which the inoculum was removed, the plates washed, and antibody added in the same manner as described above. After 30 min at 4” the cells were warmed to 37” for 60 min to allow penetration of any unneutralized virions. The cells were then washed and overlaid. Percentage neutralization was calculated by comparison with the no-antibody control described above. This assay could be affected by alterations in the rate at which the variants adsorb. Therefore, adsorption was measured for those variants having significantly altered penetration as measured in the above assay. Virus was inoculated onto monolayers at 37”. Replicate cultures were removed at intervals, washed with PBSlo!, and overlaid with agarose for enumeration of attached PFU.
RESULTS Selection of E2c neutralization escape mutants of SB-RL The E2c antigenic site is one of at least three neutralizing antigenic sites on the E2 protein and is related to the attenuation and rapid penetration phenotypes of SB-RL. Therefore, we selected and characterized several E2c neutralization escape mutants in an effort to map this antigenic site on the primary amino acid sequence of the Sindbis glycoproteins and to determine the effect of mutations within this site on Sindbis virulence in animals and penetration in tissue culture. The results obtained from these experiments are summarized in Table 1. Detailed experimental results are found in Tables 2-5. The first panel of E2c escape mutants was derived from SB-RL after neutralization with MCAb R6 or R13 and growth of the surviving population of SB-RL in the presence of the selecting MCAb (see Materials and Methods). Included in this panel was mutant RLmc6-2
44
PENCE, DAVIS, AND JOHNSTON TABLE 1 SUMMARYOF E2c ESCAPEMUTANTS E2 codonb Strain8
62
96
SB-RL RLmc6-2 RLmcl3-1 RLmcl3-4 RLmcl3-2 RLmcl3-3 RLmcG-A6 RLmcl3-5 RLmcG-B2
Asn Asp
Tyr
SB SBmcl3-A6 SBmcG-A2 SBmcG-B3
Asn
His His His Phe
Tyr His
E2c MCAbd
114
159
Arg Au Arg Arg Arg Au Arg fw Arg
Virulence”
LYS Glu Glu
Asng
Ser Ser Ser Ser
LYS Asng Asng
Binding
Rapid penetratione
Neutralization
Att Vir Vir Vir Att Att Att Att Att
+ -
nd’ + nd nd + +
Vir Att Vir Vir
+
-
+ +
-
-
nd
a Variant name identifies the parent strain and the selecting monoclonal antibody, R6 or R13. b See Table 4 for detailed sequence data. c See Table 3 for detailed virulence results. Att, attenuated; Vir, virulent. d See Table 2 for detailed antigenic analysis. e See Table 5 for penetration analysis. ‘Not determined. g Creates a new site for N-linked glycosylation; gel migration of virion E2 proteins of these strains is retarded.
which was isolated and partially characterized previously (Olmsted et al., 1986). The SE-RL escape mutants are listed in the upper portion of Table 2. These
mutants were not neutralized effectively by the selecting antibody. Regardless of whether R6 or R13 was used in the selection, the resulting mutants were inca-
TABLE 2 REACTIVITYOF ANTIGENICVARIANTSWITHVIRUS-SPECIFICMONOCLONALANTIBODIESIN ELISA AND COMPLEMENT-MEDIATEDNEUTFZALIZATION ELISA reactivityb Strain
80% Neutralization end pointa
R6
R13
R8
RlO
R15
M49
+++ +++ +++ +++ +++ +++ +++
+++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
nd nd
nd +++
++++ nd nd nd
+++ +++ nd +++
SB-RL RLmcl3-1 RLmcl3-2 RLmcl3-3 RLmcl3-4 RLmc 13-5 RLmc6-2d RLmcG-A6 RLmcG-B2
1:1600
17554
1-49 (1990)
Antigenic
and Genetic Characterization of Sindbis Virus Monoclonal Antibody Mutants which Define a Pathogenesis Domain on Glycoprotein E2
Escape
DAVID F. PENCE, NANCY L. DAVIS, AND ROBERT E. JOHNSTON’ Department
of Microbiology
and Immunology,
University of North Carolina, Chapel Hi//, North Carolina 27599-7290
Received August 17, 1989; accepted November
6, 1989
The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SE), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by EZ-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each of 11 mutants were identified. SB/ R6, SB/R13, and SB-RURl3 mutants contained a mutation at either E2 codon 96 or 159. SB-RUR6 mutants contained changes at E2 codon 62,96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E~c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice. o 1990 Academic Press. Inc.
INTRODUCTION
tigenic sites, one on El (Chanas et a/., 1982; Schmaljohn et a/., 1983) and three independently mutable sites on E2, designated E2a, E2b, and E2c (Roehrig et a/., 1982; Schmaljohn et al., 1983; Stec and Schmaljohn, 1984; Stanley ef al., 1985; Stec et al., 1986; Olmsted et a/., 1986; Davis et al., 1987). On the basis of competitive binding studies, E2a and E2b appear to lie in relatively close proximity to one another, with E2c spatially separate. While E2a and E2c are present in all laboratory strains tested, E2b is strain-specific in nature, exhibiting alternate forms defined by two separate sets of MCAbs (Davis et al., 1987). Sequencing E2b neutralization escape mutants has shown that E2b specificity depends primarily on the identity of the amino acid at position 216 of E2 (Stec et al., 1986; Davis et al., 1987). Likewise, E2a-specific MCAb neutralization escape mutants map to E2 residue 190 (Strauss ef al., 1987). Elements of the E2c antigenic site have been related genetically to determinants of Sindbis virulence in experimental animals and to the ability of the virus to penetrate cultured cells (Olmsted et a/., 1986; Polo et al., 1988; Russell et al., 1989). Sindbis strain AR339 (SB) causes a lethal encephalitis in neonatal mice, with subcutaneous (SC) doses approaching 1 PFU per mouse causing 100% mortality within 7 days (Reinarz et al., 1971; Johnson et al., 1972). A rapidly penetrating mu-
Sindbis virus is the prototype member of the alphavirus genus of the family Togaviridae (for a review, see Schlesinger and Schlesinger, 1986). The singlestranded, message sense RNA genome is contained within an icosahedral viral core assembled from multiple copies of a single protomeric peptide, C (30K). Upon release from the infected cell by budding, the nucleocapsid acquires a cell-derived lipid envelope containing equimolar amounts of two transmembrane viral glycoproteins, El (50K) and E2 (45K) (Strauss et al., 1969; Schlesinger eta/., 1972; von Bonsdotff and Harrison, 1975). El and E2 exist as a heterodimeric spike (Rice and Strauss, 1982) on the virion surface, and directly interact with the external environment (Sefton et a/., 1978). The glycoprotein spikes are involved in several biological activities, including hemagglutination (Dalrymple et a/., 1976; Schmaljohn et a/., 1983) virus attachment and penetration (Olmsted et al., 1984, 1986; Flynn et a/., 1988) and interaction with elements of the host immune system including those responsible for the production of antibodies. Mapping of Sindbis virions with monoclonal antibodies (MCAbs) has identified at least four neutralizing an’ To whom requests for reprints should be addressed. 41
0042.6822/90
$3.00
CopyrIght 0 1990 by Academic Press. Inc All rights of reproduction I” any form resewed
PENCE, DAVIS, AND JOHNSTON
42
tant of SB, designated SB-RL (Baric et a/., 1980, 1981; Olmsted et al., 1984) is characterized by greater than 30% survival at SCdoses as high as 106. Although SB and SB-RL bind equivalent levels of E2c MCAbs, SBRL is more efficiently neutralized by these antibodies than is SB (Olmsted et al., 1986). Virulent revertants of SB-RL lose both the E2c neutralization sensitivity and rapid penetration phenotypes (Olmsted et a/., 1984, 1986). In contrast, variation at the E2b antigenic site has no demonstrated effect on virulence of SB in neonatal mice (Davis et al., 1987). Sequencing of the glycoprotein genes of SB, SB-RL, and virulent revertants of SB-RL suggested that substitution of arginine at E2 position 1 14 of SB-RL for serine in SB is responsible for the linked variation in penetration, virulence, and E2c neutralization efficiency (Davis et al., 1986). Examination of virus produced by transfection of RNAs transcribed from full-length cDNAs of Sindbis (Rice et a/., 1987; Polo et a/., 1988) confirmed this suggestion. Virus from clones containing an arginine codon at E2 114 is attenuated, rapidly penetrating, and efficiently neutralized by E2c MCAbs, while virus from otherwise isogenic clones containing a serine at E2 114 is virulent, slowly penetrating, and poorly neutralized by E2c antibodies. In this paper, we report the isolation, characterization, and sequencing of a panel of neutralization escape mutants of SB and SB-RL that are no longer able to bind E2c MCAbs R6 or R13. Sequence analysis of these isolates has identified mutations at three different, noncontiguous codons in the E2 gene that independently abolish E2c antibody binding. Some of these mutations also affect pathogenesis and penetration, depending on the identity of the encoded amino acid substitution and on the identity of the residue at E2 114. These results suggest that the E2c-specific MCAbs define a conformation-dependent antigenic site. This site may substantially overlap or be coincident with virion surface domains important for virulence in neonatal mice and penetration of cultured cells. MATERIALS AND METHODS Cell and virus culture, immunological reagents All virus stocks used in these experiments were produced by infection of baby hamster kidney (BHK) cells (ATCC CCL-l 0), in Eagle’s minimum essential medium containing 10% bovine calf serum (BCS), 10% tryptose phosphate broth (TPB), 50 units penicillin/ml, and 50 pg/ml streptomycin (complete medium). Cells were used in passages 53-64. The wild-type laboratory strain SB (Sindbis strain AR 339) was obtained originally from H. R. Bose (University of Texas, Austin). The
attenuated strain, SB-RL, was isolated by Baric et al. (1980, 1981) by selection for rapid growth of SB in BHK cells. All antibody escape mutants were derived from these strains. Procedures for production of virus seed and working stocks and for preparation of purified virus by ultracentrifugation have been described previously (Olmsted et al., 1986; Davis et al., 1986). The production and characterization of MCAbs R6, R13, R8, and Rl 0 (Olmsted et a/., 1986; Davis et al., 1987) also have been described. Antibody 49 was the gift of A. Schmaljohn, U.S. Army Research Institute of Infectious Diseases (Schmaljohn et a/., 1982). Selection of neutralization escape mutants Neutralization escape mutants were selected from parental stocks of SB and SB-RL. To obtain SB-RL variants resistant to MCAb R13 neutralization, 5 X 1O4PFU of SB-RL were included in a l-ml vol of phosphatebuffered saline with 1% BCS (PBS-l %) containing 20% (v/v) MCAb R13 ascites fluid and 5% guinea pig complement (Accurate Chemical Co.). The mixture was incubated for 30 min at 37”. The surviving virus was diluted to a maximum titer of 1000 PFU/ml and plated in 200-~1 aliquots on BHK cells in loo-mm tissue culture dishes. One hour later, the cells were overlaid with complete medium containing 0.7% (w/v) agarose. To obtain a neutralization estimate, a second parallel incubation of virus was performed in the same fashion, except that 20% normal mouse ascites fluid (NAF) was added instead of antibody. Well-isolated plaques were picked 24 hr later, and the agarose plugs were eluted for 12 hr at 4” in 300 ~1of complete medium. One hundred microliters of each eluate was then added to duplicate wells of a 96-well tissue culture plate containing 6 X 1O4 BHK cells per well in 100 ~1of complete medium. One well of each pair contained 1% MCAb R13 and the other, 1% NAF. The remaining 100 ~1of those isolates found to produce strong CPE in the presence of the selecting antibody was used to grow a virus seed stock, and from that a working stock of each isolate was obtained. Growth of these stocks was in the presence of MCAb R13. RLmc6-2 was isolated in a similar manner using MCAb R6, except five successive cycles of neutralization and amplification of survivors in tissue culture were conducted before plaque isolates were taken (Olmsted et al., 1986). The other variants of SB-RL selected with MCAb R6 and those of SB selected with MCAb R6 or R13 were plaqued directly from a single neutralization reaction in the same way as the SB-RUR13 variants described above, except that a goat anti-mouse IgG-specific second antibody (Sigma) was added to the neutralization
SINDBIS VIRUS MONOCLONAL
reactions to increase their efficiency. Plaque eluates were added directly to wells of a 96-well tissue culture plate containing BHK cells in 200 ~1 of complete medium containing 1% selecting antibody. Medium from wells showing strong CPE 24-36 hr later was assayed in a capture ELISA screen for loss of ability to bind the selecting antibody (Olmsted et al., 1986; Davis et al., 1987). Seed and working stocks were grown in the presence of the selecting antibody.
ELISA and neutralization assays Solid-phase and capture ELISA, as well as plaquereduction neutralization assays, were performed on virus according to the protocols described by Olmsted
et al. (1986). Sequencing Direct sequencing of the glycoprotein genes of the variants from purified viral RNA using reverse transcriptase was performed by oligonucleotide-primed dideoxynucleotide chain termination (Zimmern and Kaesberg, 1978) using primers and protocols as detailed by Davis et al. (1987). The E2 gene of each mutant was sequenced as well as the El genes of representative mutants: RLmc6-2, RLmcl3-1, RLmcl3-2, and RLmcl3-5.
Pathogenesis assay The parental strains and all antigenic variants were tested for virulence in litters of 18- to 36-hr-old CD-l mice (Charles River Labs). Neonatal mice were inoculated SCwith 100 PFU of virus in 50 ~1 of PBS-l %. Control animals received PBS-l % only. Litters were monitored for 14 days.
Estimation of penetration Virus penetration was measured as time-dependent acquisition of resistance to neutralization by polyclonal anti-SB antibody, in a manner similar to the method of Olmsted et a/. (1986). BHK cells in 60-mm dishes were inoculated with 0.2 ml of PBS-l% containing approximately 200 PFU of virus, and incubated at 37”. At 15 min postinfection, the inoculum was removed from replicate plates of each virus assayed. The plates were washed with 2 ml of PBS-l %, and 0.4 ml of a 1:2000 dilution of AR339-specific hyperimmune mouse ascites fluid (HMAF) in PBS-l % with 5% guinea pig complement (Accurate Chemical Co.) was added per plate. After incubation for 30 min at 37”, the antibody was removed. The plates were washed as before to remove residual antibody, and the cells were overlaid with complete media with agarose. A parallel set of plates
ANTIBODY
ESCAPE MUTANTS
43
were identically infected, and were overlaid after a 60min incubation at 37”. Penetration was estimated as the percentage of plaques arising after the antibody treatment, as compared with the controls which received no antibody. The penetration assay depends on the ability of HMAF to neutralize virions which have attached but not yet penetrated. To ensure that none of the escape mutations significantly reduced sensitivity to neutralization by HMAF, the efficiency of neutralization of attached virions by HMAF was monitored for each variant. Simultaneously with the above assay, two plates per isolate were cooled to 4” for 1 hr. The plates were then inoculated in the cold with the same virus dilutions used in the penetration assay. Virus attachment was allowed to proceed in the cold for an additional hour, after which the inoculum was removed, the plates washed, and antibody added in the same manner as described above. After 30 min at 4” the cells were warmed to 37” for 60 min to allow penetration of any unneutralized virions. The cells were then washed and overlaid. Percentage neutralization was calculated by comparison with the no-antibody control described above. This assay could be affected by alterations in the rate at which the variants adsorb. Therefore, adsorption was measured for those variants having significantly altered penetration as measured in the above assay. Virus was inoculated onto monolayers at 37”. Replicate cultures were removed at intervals, washed with PBSlo!, and overlaid with agarose for enumeration of attached PFU.
RESULTS Selection of E2c neutralization escape mutants of SB-RL The E2c antigenic site is one of at least three neutralizing antigenic sites on the E2 protein and is related to the attenuation and rapid penetration phenotypes of SB-RL. Therefore, we selected and characterized several E2c neutralization escape mutants in an effort to map this antigenic site on the primary amino acid sequence of the Sindbis glycoproteins and to determine the effect of mutations within this site on Sindbis virulence in animals and penetration in tissue culture. The results obtained from these experiments are summarized in Table 1. Detailed experimental results are found in Tables 2-5. The first panel of E2c escape mutants was derived from SB-RL after neutralization with MCAb R6 or R13 and growth of the surviving population of SB-RL in the presence of the selecting MCAb (see Materials and Methods). Included in this panel was mutant RLmc6-2
44
PENCE, DAVIS, AND JOHNSTON TABLE 1 SUMMARYOF E2c ESCAPEMUTANTS E2 codonb Strain8
62
96
SB-RL RLmc6-2 RLmcl3-1 RLmcl3-4 RLmcl3-2 RLmcl3-3 RLmcG-A6 RLmcl3-5 RLmcG-B2
Asn Asp
Tyr
SB SBmcl3-A6 SBmcG-A2 SBmcG-B3
Asn
His His His Phe
Tyr His
E2c MCAbd
114
159
Arg Au Arg Arg Arg Au Arg fw Arg
Virulence”
LYS Glu Glu
Asng
Ser Ser Ser Ser
LYS Asng Asng
Binding
Rapid penetratione
Neutralization
Att Vir Vir Vir Att Att Att Att Att
+ -
nd’ + nd nd + +
Vir Att Vir Vir
+
-
+ +
-
-
nd
a Variant name identifies the parent strain and the selecting monoclonal antibody, R6 or R13. b See Table 4 for detailed sequence data. c See Table 3 for detailed virulence results. Att, attenuated; Vir, virulent. d See Table 2 for detailed antigenic analysis. e See Table 5 for penetration analysis. ‘Not determined. g Creates a new site for N-linked glycosylation; gel migration of virion E2 proteins of these strains is retarded.
which was isolated and partially characterized previously (Olmsted et al., 1986). The SE-RL escape mutants are listed in the upper portion of Table 2. These
mutants were not neutralized effectively by the selecting antibody. Regardless of whether R6 or R13 was used in the selection, the resulting mutants were inca-
TABLE 2 REACTIVITYOF ANTIGENICVARIANTSWITHVIRUS-SPECIFICMONOCLONALANTIBODIESIN ELISA AND COMPLEMENT-MEDIATEDNEUTFZALIZATION ELISA reactivityb Strain
80% Neutralization end pointa
R6
R13
R8
RlO
R15
M49
+++ +++ +++ +++ +++ +++ +++
+++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
nd nd
nd +++
++++ nd nd nd
+++ +++ nd +++
SB-RL RLmcl3-1 RLmcl3-2 RLmcl3-3 RLmcl3-4 RLmc 13-5 RLmc6-2d RLmcG-A6 RLmcG-B2
1:1600
