VIROLOGY
252, 373±375 (1998)
VY989462
ARTICLE NO.
Monoclonal Antibody-Resistant Mutants Selected with a Respiratory Syncytial
Virus-Neutralizing Human Antibody Fab Fragment (Fab 19) Define a
Unique Epitope on the Fusion (F) Glycoprotein
James E. Crowe, Jr.,*,1 Cai-Yen Firestone,* Roberta Crim,*,2 Judy A. Beeler,*,2 Kathleen L. Coelingh,*,3
Carlos F. Barbas III,² Dennis R. Burton,² Robert M. Chanock,* and Brian R. Murphy*
*Respiratory Viruses Section, Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0720; and
²The Scripps Research Institute, La Jolla, California 92037
Received June 4, 1998; returned to author for revision July 17, 1998; accepted October 8, 1998
A recombinant human antibody fragment, designated RSV Fab 19, efficiently neutralizes respiratory syncytial virus (RSV).
Here we report the results of our sequence analysis of antibody escape mutants that identified F glycoprotein amino acids
critical for binding of human or murine RSV F-neutralizing antibodies. © 1998 Academic Press
activity of such mAbs (Crowe et al., 1994). It is likely that
two neutralizing mAbs that bind to distinct antigenic sites
on the F glycoprotein of RSV will be required for passive
antibody immunoprophylaxis or immunotherapy of RSV
disease to prevent the emergence in vivo of antibody
escape mutants. We have developed a human RSV antibody fragment, Fab 19, that has high therapeutic RSVspecific activity in mice (Crowe et al., 1994). This Fab or
a recombinant bivalent IgG molecule that incorporates
this Fab can be used as a component in an RSV antibody
cocktail consisting of two or more mAbs that recognize
distinct antigenic sites. The current study defines an
amino acid residue on the RSV F glycoprotein that is
essential to its binding to the human anti-RSV antibody
fragment Fab 19. Similar information was obtained for a
panel of murine RSV-neutralizing mAbs that had been
isolated previously (Arbiza et al., 1992; Beeler and van
Wyke Coelingh, 1989).
INTRODUCTION
Antibodies specific for respiratory syncytial virus (RSV)
are of importance because of the urgent need to prevent
or treat the severe disease caused by this paramyxovirus
in infants or in persons of any age with underlying cardiopulmonary disease or immunodeficiency. RSV is the
major cause of serious viral lower respiratory tract illness in infants and young children throughout the world
(Collins et al., 1996). Experimental studies in rodents and
nonhuman primates have demonstrated that RSV-specific polyclonal or monoclonal-neutralizing antibodies
can prevent virus infection in the lungs when administered before exposure to virus and can effect rapid resolution of infection when administered at the height of
infection (Crowe et al., 1994; Hemming et al., 1985; Prince
et al., 1985; Walsh et al., 1984). Recent clinical trials in
high-risk infants and children using pooled human immunoglobulin G that contained a high titer of RSV-neutralizing antibodies (RSVIG) for prophylaxis against severe disease have confirmed these experimental observations (Groothuis et al., 1993). We are developing RSVspecific neutralizing human monoclonal antibodies
(mAbs) for topical or intramuscular administration that
might overcome the need for intravenous administration
by virtue of the high level of RSV-specific neutralizing
RESULTS AND DISCUSSION
The predicted amino acid changes encoded by the F
gene nucleotide sequence of the murine MARMs that
were neutralized by Fab 19 are shown in Table 1. F gene
nucleotide sequences derived for all three Fab 19
MARMS (designated RSV 4B, RSV 5A, and RSV 5B) were
identical. These viruses contained a single nucleotide
change of A to G at position 6458 in the RSV genome
(position determined in the message sense, from the 39
end of the virus). This nucleotide change is predicted to
encode an amino acid change at position 266 in the F
glycoprotein of isoleucine to methionine. The binding
epitope of RSV Fab 19 most likely includes amino acid
position 266 on the F glycoprotein; however, there is an
alternative possibility that the change at position 266
1
To whom reprint requests should be addressed at Division of
Pediatric Infectious Diseases, Department of Pediatrics, Vanderbilt
University Medical Center, D-7235 MCN, 1161 21st Avenue South,
Nashville, TN 37232-2581. Fax: (615) 343-9723. E-mail: james.e.crowe@
vanderbilt.edu.
2
Current address: Food and Drug Administration, Bethesda, MD
20892.
3
Current address: Aviron, Mountain View, CA.
373
0042-6822/98 $25.00
Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
374
CROWE ET AL.
TABLE 1
RSV Monoclonal Antibody-Resistant Mutants (MARMs) Selected with the Human Recombinant Fab 19 Contain a Predicted Amino Acid
Change from the Wild-Type Sequence that Differs from Changes Found in Mutants Selected with Murine mAbs
Mutation in RSV F glycoprotein
Nucleotide
Antibody used to
select MARM
Murine
1237
1214
1129
151
1200
1142
MARM selected with
indicated mAbsa
v1237
v1214
v1129
v151
v1200
v1142
1153
1269
1308F
v1153
v1269
v1308F
1302A
v1302A
RSV 19b
C4848
Human
Fab 19
Fab 19
Fab 19
4B
5A
5B
Amino acid
Position(s)
Codon change wild-type
A2 3 MARM
Position(s)
AA change wild-type
A2 3 MARM
839
839
837
829
829
108
829
798
1179
735
1275
735
1275
1298c
AAC 3 TAC
AAC 3 TAC
TCC 3 TTC
AAG 3 AAT
AAG 3 AAT
TTT 3 TCT
AAG 3 AAT
AAT 3 AGT
CCC 3 CAC
GCA 3 GTA
AAA 3 ACA
GCA 3 GTA
AAA 3 AGA
CGT 3 AGT
276
276
275
272
272
32
272
262
389
241
421
241
421
429
Asn 3 Tyr
Asn 3 Tyr
Ser 3 Phe
Lys 3 Asn
Lys 3 Asn
Phe 3 Ser
Lys 3 Asn
Asn 3 Ser
Pro 3 His
Ala 3 Val
Lys 3 Thr
Ala 3 Val
Lys 3 Arg
Arg 3 Ser
[
[
[
811
811
811
[
[
[
ATA 3 ATG
ATA 3 ATG
ATA 3 ATG
[
[
[
266
266
266
[
[
[
Ile 3 Met
Ile 3 Met
Ile 3 Met
a
All mutants were derived from the RSV wild-type strain A2.
The similarity in numerical designation of Fab 19 is fortuitous; the murine mAb 19 is not related to the human Fab 19.
c
Previously determined sequence change (Arbiza et al., 1992).
b
causes a conformational alteration of the protein that
abolishes binding of the Fab at a different site that does
not include the amino acid 266. The region of the F
glycoprotein in which amino acid 266 is located is quite
interesting in that all of the MARMs derived by selection
with site A neutralizing murine mAbs were determined to
contain an amino acid substitution between residues
262 and 276. However, MARMs selected by murine
mAbs did not contain a predicted amino acid change at
position 266. Interestingly, Fab 19 neutralizes MARMs
containing a change at nearby amino acid position 262,
272, 275, or 276, but competition analysis had suggested
previously that Fab 19 was directed at a distinct antigenic site (Barbas et al., 1992). It is possible that the lack
of competition of the human Fab fragments against
whole mouse immunoglobulin molecules in the competition binding assay is a function of the smaller size of the
Fab.
Several nucleotide substitutions could result in an
amino acid change at position 266; however, the same
A-to-G substitution was found in three instances. The
reason for this finding is not clear. One possibility is that
a mutant virus was present in the wild-type parent virus
pool at an undetectable level and virus passages in the
presence of Fab enriched for the growth of this mutant,
rather than independently selecting three mutants de
novo during antibody-treated passages.
In summary, we have identified an amino acid residue
on the RSV F glycoprotein that is essential to binding for
the first isolated RSV-neutralizing human mAb. This residue is near other residues important for binding a panel
of murine RSV mAbs. The Fab 19 holds promise as an
important component of an antibody preparation for immunoprophylaxis or immunotherapy of RSV disease in
humans (Crowe et al., 1994).
MATERIALS AND METHODS
The human Fab 19, previously cloned from a combinatorial phage display library, was expressed in Escherichia coli and then purified from crude cell lysates by
immunoaffinity chromatography using an anti-human
Fab antibody as described previously (Barbas et al.,
1992; Crowe et al., 1994). Purified RSV Fab 19 was analyzed for its ability to neutralize the RSV A2/HEK-7 wildtype virus (Connors et al., 1995) and each of 11 viruses in
a panel of previously described MARMs selected for
resistance to neutralization by the various murine mAbs
listed in Table 1 (Arbiza et al., 1992; Beeler and van Wyke
Coelingh, 1989). The Fab 19 neutralized the wild-type
RSV FUSION GP NEUTRALIZATION AB EPITOPE
parent and each of the MARMS with a high level of
specific activity (,1 mg/ml).
Three independently derived MARMs were isolated
from the RSV A2 wild-type virus, each by a separate
passage series in which virus was selected two times by
growth in the presence of the human Fab 19 at a concentration of 30 mg/ml, and then amplified by growth in
the absence of Fab 19. Each of three independently
derived virus suspensions derived in this manner was
biologically cloned by three plaque passages. The
MARMs so selected were shown to resist complete
neutralization with Fab 19 in cell culture. For example,
the titer of the RSV A2 wild-type virus was reduced by 5.7
log10 PFU/ml after incubation with 150 mg/ml Fab compared with PBS control, whereas the titer of each of the
Fab 19-selected MARMs was reduced by ,10-fold. The
MARMs were reduced in plaque titer in repeated tests
on average by 3- to 4-fold after incubation with Fab 19
compared with incubation with PBS. It is not known
whether this represents a virus specific effect, but this
reduction falls within the expected range of variability of
the plaque assay.
Viral RNA from each of the MARMs was prepared from
virus-infected LLC-MK2 or HEp-2 cell monolayer cultures
as described (Connors et al., 1995). The RNA was reversed transcribed into cDNA and the F protein gene
was amplified by PCR using gene-specific primers,
cloned into a plasmid vector or, in some cases, the
replicative form of phage M13mp19, and sequenced by
the dideoxynucleotide method using either plasmid or
RSV F gene-specific sequencing primers and T7 polymerase (Sequenase; U.S. Biochemical, Cleveland, OH)
as described previously (Connors et al., 1995). Consensus sequences were determined by sequencing cDNA
clones derived from at least two independent PCRs.
Differences between the observed sequences and that
of the previously determined parental virus RSV A2/
HEK-7 were confirmed by sequencing two or more
clones in both forward and reverse directions. The previously described RSV antibody escape mutant C4848
(also designated v324 in the previous publication), which
contains a single nucleotide change in the F glycoprotein
375
that encodes a predicted amino acid change at amino
acid position 429 from the N-terminus (Arbiza et al.,
1992), was kindly supplied by Dr. Geraldine Taylor (AFRC
Institute for Animal Health, Compton, U.K.).
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