J. gen. Virol.
0979), 44, 81-87
81
Printed in Great Britain
Mouse Hybrid Cell Lines produce Antibodies
to Herpes Simplex Virus Type 1
By E. L. H O W E S * , E. A. C L A R K , E. S M I T H and N. A. M I T C H I S O N
I C R F Tumour Immunology Unit, Department of Zoology,
University College London, Gower Street, London W C I E 6BT, U.K.
(Accepted I4 December I978 )
SUMMARY
A solid-phase radioimmunoassay procedure has been devised for the assay of
antibodies produced in the mouse to herpes simplex virus type ~ (HSV-I). It is based
on the adsorption of virus to flexible micro-well plates and uses radio-iodine-labelled
rabbit antibody against mouse immunoglobulin to assess antibody binding. Using
this assay for screening, cell hybrids have been obtained which yield monoclonal
antibody to HSV-I. The hybrids are between spleen cells from hyperimmune mice
and an immunoglobulin-non-secreting, azaguanine resistant myeloma cell line
(NS- 0. From 48o hybrid cell lines initially examined, five stable cell lines were
obtained which released HSV-I-specific antibody in vitro and in vivo. Mice carrying
transplants of these cell lines yield binding titres in serum of up to 1/25ooo. Both
IgG and lgM antibodies were obtained in this way.
INTRODUCTION
The serological response to herpes simplex viruses (HSV) involves the formation of
multiple antibodies of different sub-class and different antigenic specificity (Hampar et al..
I968). Ten to 15 separate polypeptides, most of which are glycosylated, have been isolated
by different procedures from membranes of cells infected with HSV-I. Some of these are
not found in purified preparations of virus particles (Heine et al. I972; Honess & Watson,
~974; Glorioso & Smith, I977). It is by no means clear which of these virus-induced surface
proteins is most important in terms of immunological protection, and some proteins within
the virion are also of importance in this connection (Miyamoto et al. I97r ).
Recent successes at producing somatic cell hybrids which can form monospecific antibodies to, for example, influenza virus (Koprowski et aL I977), alloantigens (Barnstable
et al. I978), and haptens (Kohler & Milstein, t976 ) suggest that a similar approach may be
useful in studying the serological response to HSV-~ (see Melchers et al. I978). At a recent
workshop, Koprowski et al. (I 978) reported successful development of antibody-producing
cell lines to a number of virus antigens including those of HSV-I, but detailed results have
not been presented. A capacity to produce monospecific antibodies would be invaluable in
serological typing, in correlating neutralizing capacity with specific antibody production, in
the identification and purification of specific virus antigens, in defining more clearly potential
tumour associated minor antigens such as AG-4 (Aurelian & Strnad, ~976), and in evaluating
* Present address: Department of Pathology,San Francisco General Hospital, San Francisco,California
94I Io, U.S.A.
oo22-I3t7/79/0ooo-3516 $o2.oo (~ t979 SGM
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E.L.
HOWES
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antigens of importance in protection against herpes virus infection. The following study
represents an initial attempt at establishing stable somatic cell hybrids producing antibodies
to HSV-I.
METHODS
Preparation of virus. Herpes simplex virus type I, Kruger strain (HSV-~) was propagated
in cultures of established cell lines of rabbit kidney (RK-13), for immunization purposes,
and of baby hamster kidney (BHK-2D for radioimmunoassay. Growth was in Dulbecco's
modified Eagle's medium (E-4) supplemented with Io % calf serum. Virus was harvested
after 2 to 3 days incubation by gently shaking flasks to remove adherent cells, centrifuging
at 8oo rev/min and washing with serum-free medium, a process repeated three times. Cells
resuspended in a small volume were sonically disrupted, centrifuged at 35oo rev/min and
the supernatant passed through Millipore filters (I-2 to o.22/zm). Virus dilutions so prepared were stored at - I96 °C in liquid nitrogen at I to 2 × to 9 p.f.u./ml; I × Io 6 p.f.u, was
lethal in ~°Co-irradiated (25o to 40o R) C57 and BALB/c mice. Uninfected tissue culture
cell extracts similarly prepared served as a control in radioimmunoassays.
Solid phase radioimmunoassay ( RIA) for detection of antibody to HSV-I. An indirect solid
phase radioimmunoassay was used for the detection of virus antibodies (Rosenthal et al.
I973). This employed whole virus, candidate mouse antibody and a rabbit anti-mouse
immunoglobulin (RAMIG) labelled with l~Siodine by the chloramine-T method (Hunter &
Greenwood, t962). The RAMIG was prepared by affinity column chromatography (Porath
et al. t967) and was kindly supplied by N. Hogg.
Virus was diluted with E-4 medium and 5o #1 containing Io 6 p.f.u. HSV-1 was added to
wells in a flexible 'U' 96-well plate (Gibco Bio-Cult, Paisley) and incubated overnight at
room temperature. Medium alone or uninfected tissue culture extracts served as controls for
specific binding. Glutaraldehyde (o"5 %, I5o #1) was added to each well for ~o min and removed by flicking the inverted plate. The plates were washed thoroughly in four changes of
phosphate buffered saline (PBS), pH 7.2, a process repeated at each washing. To reduce nonspecific binding of proteins, too ~o foetal calf serum (FCS) was added for I h at 37 °C,
removed and the plates washed thoroughly.
Sera and tissue culture supernatants were centrifuged for 2 min at I 6 o o o g prior to
addition to wells. Determinations were usually performed in triplicate for serological studies
and in duplicate for tissue culture fluids. Fifty to too/zl of each dilution was added to each
series of wells, 2 to 3 wells with virus, 2 to 3 wells without virus, and incubated for 45 min at
room temperature. To determine antibody titres on sera, fourfold dilutions were made
beginning at i / i o or i / t o o dilutions of sera in PBS containing I o % FCS, and on tissue
culture supernatants in tenfold dilutions beginning with undiluted fluids. Initial screening
for in vitro antibody production was performed in duplicate on undiluted fluids.
Following incubation, removal of fluid and washing, RAMIG-lZsI was added to each well
and incubated for 45 min at loom temperature (50 #1 in 5° % foetal calf serum - P B S containing 3oooo to 5oooo ct/min/well). After washing, individual wells were cut out and
counted in a well type scintillation counter. Values were expressed for each dilution as a
binding ratio of mean counts from wells with virus to wells without virus. Control RIA
determinations were performed similarly with a purified Sendai virus preparation and
lyophilized commercial vaccinia virus.
Mouse antisera to HSV-I. C57 and BALB/c mice were hyperimmunized by intraperitoneal
injection of HSV-t, starting with 2 × I05 p.f.u, repeated three times at weekly intervals with
2 × 108 p.f.u, and were bled 2 weeks after the last injection. Pooled sera from groups of five
to six mice were also prepared at intervals following primary immunization.
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H S V - I antibody-producing hybrid cell lines
83
Cell hybridization. Spleen cells: hyper-immunized BALB/c mice were injected intravenously with I × io 4 p.f.u. HSV-I 2"5 months after the last boost and 3° h later the mice
were killed, spleens removed aseptically and cells milked from the spleen capsule. These
were suspended in RPMI I64o medium, centrifuged at 800 rev/min and RPMI replace d, a
process repeated three times. As a source of myeloma cells an immunoglobulin-non-secreting
8-azaguanine resistant mutant of MOPC-21 (BALB/c tumour), P3/NS IlI-Ag4-I (NS-Q,
kindly supplied by V. Oi and L. Herzenberg (Kohler & Miistein, I976), was grown in RPMI
and I5 % foetal calf serum at 37 °C + 5 % CO2. These cells were washed in serum-free media
just before use.
The hybridization technique followed the standard procedures using polyethylene glycol
(PEG; Galfre et al. I977; E. A. Clark et al. unpublished data) as modified by V. Oi and
L. Herzenberg (personal communication). Two × ~o8 immune spleen cells plus 2 to 4 × Io7
NS-I cells were centrifuged together and the supernatant removed. P E G (50 %, I.O ml) was
added to the pellet drop-wise with stirring over a I rain period and stirring continued with
gradual addition of medium to IO ml over 5 rain. The cells were then centrifuged and resuspended in RPMI I64o and I 5 % FCS; IOO #1 of the suspension (about ro 6 cells) were
added to each well of 96 fiat bottom weJ1 microplates (Falcon). One day later ioo/d of
hypoxanthine, aminopterin, and thymidine selective media (HAT; Littlefield, 2964) was
added to each well and on days 2, 3, 5, 8 and I I, half the medium was removed and replaced
with fresh HAT medium. On day I4, IOO/~1 of medium without aminopterin (HT) was used
instead, a process repeated every four days. After I month, normal RPMI medium was used.
Initial screening of supernatants was performed zo to 22 days after hybridization, and the
cells transferred to 24 well plates (Linbro), with gradually increasing medium as cells proliferated. The next transfer was to six well (Linbro) plates and then to 25 and 50 ml flasks.
Cells were then stored in liquid nitrogen in culture medium containing Io % DMSO. To
establish clones, cells were serially diluted in 2oo/d tissue culture medium in microtest plates,
and after 5 to 8 days wells were examined for the presence of individual clones. Fragments of
individual loci were removed by micromanipulation with a finely drawn Pasteur pipette.
Each clone fragment was then sub-cultured separately and after I week tested for antibody
production.
For passage in mice, cells were injected subcutaneously into BALB/c mice following 250
to 350 R (approx. 2 x lO6 cells per injection). Mice with tumours were bled at 2 weeks.
Neutralization assays. For the plaque forming assay, BHK-21 cells grown to confluence in
60 mm Petri dishes were overlaid with o'a5 to o.4o ml of virus dilution. For estimating virus
titres, tenfold dilutions of virus were added. Incubation for I h at 37 ° C + 5 % CO2 was
followed by withdrawal of culture fluid and the addition of 5 ml of medium containing 3 to
5 % pooled human serum. Incubation for two days was followed by fixation of the cells with
I °/o glutaraldehyde and staining with Giemsa stain prior to counting of plaques. All assays
were done in duplicate.
For the neutralization assay, pooled sera from mice bearing tumours and containing antibody to HSV-I by RIA, pooled hyperimmune sera and pooled normal sera were incubated
for I h at 37 °C, at different dilutions in equal vol. with 0"5 ml of a virus dilution containing
250 p.f.u, of HSV 1. The virus antibody mixture (o'4 ml) was then transferred to a confluent
microlayer of BHK 21 cells and a plaque forming assay performed. In some cases o.I ml of
a I : 2 dilution of guinea pig serum or inactivated guinea pig serum was also added to the
virus antibody mixture and volumes adjusted accordingly. Neutralization was defined as
the capacity of a given serum dilution to decrease the number of p.f.u, in controls without
antibody or with normal serum by 5o %.
Direct immunofluorescenee. Cells were washed, resuspended at 5 x io 5 cells/o-5 ml in PBS
containing o.o2 % sodium azide and incubated on ice for I h. After washing, cells were mixed
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E. L. H O W E S
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with IO #1 of fluorescein-labelled R A M I G specific f o r # chain, 71 chain or Y2 chain (Raffet al,
I976 ). These sera were kindly provided by M. Raft. After 3o rain incubation on ice and three
washings, cells were examined under a coverslip for surface immunofluorescence using a
Zeiss Ultraphot II microscope, equipped with phase contrast and incidence fluorescence
optics and an Osram HBo 2o0 mercury arc lamp. An lgM Thy-I.I secreting cell line
(TI2GI I; E. A. Clark et al. unpublished data) was tested as a positive control.
RESULTS
Efficacy of radioimmunoassay technique for assay of antibodies against HSV
Antibody titres at Io days following the injection of virus are given in Fig. I and show both
increased titres of antibody and increased binding ratios with greater quantities of injected
virus. Following the injection of ~o 6 p.f.u, of HSV-I, antibody titres were noted in increasing
quantities and increased binding ratios up to 20 days and then decreased somewhat at
40 days (Fig. 2). Normal sera consistently had a binding ratio of one and after multiple
determinations a binding ratio of greater than I'5:I was considered evidence for the
presence of antibody. Testing against uninfected tissue culture cell extracts from R H K and
B H K 2~ at no time showed specific binding. Hyper-immune sera (C57 and BALB/c) consistently had titres of i[4oooo; by absorbing these antisera with ro 7 p.f.u, of virus and
centrifuging at IOOOOOg for I h, antisera titres dropped to below t/[oo.
Screening for antibodies produced by somatic ceil hybrids
At 2I days after hybridization, out of 48o initial wells, 17 had supernatant with a binding
ratio greater than 5:I, and another 15 greater than 2:I (8.8 %). These 41 initially positive
isolates were transferred to 24-well plates and rescreened at 35 days after initial hybridization. Fifteen wells had a binding ratio of greater than z'5: I and I4 wells a ratio of greater
than I'5:I. Supernatants from five wells had lost activity and eight wells had been lost
through contamination.
A third screening was performed at a time when cells from the original wells were growing
in z5 ml flasks. At this stage, supernatants from lo wells had lost activity, an additional three
had become contaminated, and three did not grow. The 13 remaining positive cell lines
were frozen down and were also injected subcutaneously into irradiated (300R) BALB[c
mice (106 to l0 T cells/injection site). All mice injected grew tumours and sera were screened
two weeks after cell injection. Five cell lines produced antibody, but eight did not. These
eight cell lines also lost activity in continuous cell cultures. The five antibody-producing
cell lines were injected into 5 to lo irradiated BALB/c mice and after 2 weeks, sera were
collected and pooled. The cell lines were also cultured in RPMI + 2 % foetal calf serum for
z 4 h. The pooled sera and supernatants were analysed by RIA for the presence of antibody
against HSV-t (Table I). Radioimmunoassays for activity against a Sendai and vaccinia
virus did not show activity.
Each of the five stable positive cell lines was cloned by limiting dilution and RIA performed on sub-cultured clones. RIA was repeated on 8 to I I clones from each antibody producing cell line; the incidence of positive sub-cultured clones correlated well with the initial
titres of cell line supernatants (Table I).
Capacity of cell lines to neutralize HSV-I in plaque forming assay
Neutralizing activity of pooled sera from mice bearing tumours from each of the original
cell lines (see Table I for comparison oftitres by RIA) was tested in a plaque forming assay
at I : 5 dilution. Neither individually nor when all five pooled sera were combined was there
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H S V - I antibody-producing hybrM cell lines
7" 107 p.f.u. 106 p.f.u.
6
12-
5
13-
o 4
8-
3
6lO4
~2
1
4-
i ~ i 0
-/.- ....
days
-..fd s \ \
2"
N1.
N 1. sera
|
|
|
N= 1 2
4
6
8
10
;2
Fourfold dilution of antibody ( 1/ 10 × 2N)
Fig. I
N=
1~0
112
2
4I
6i
8~
Fourfold dilution of antibody ( 1/ 10 X 2N)
Fig. 2
Fig. t. Detection of serum antibody to HSV-I using a solid-phase radioimmunoassay (RIA). Mice
were inoculated i.p. with different doses of HSV-I, bled IO days later and serum antibody titres
measured. The results are expressed as well binding ratios = mean count/min of wells with virus
(V + )[ mean counts/min of wells without virus ( V - ) . Antibody titres and well binding ratios both
increased as the amount of infective virus was increased.
Fig. 2. Kinetics of the primary serum antibody response to HSV-I. Mice were inoculated i.p. with to 6
p.f.u. HSV- i, bled at different times after immunization, and antibody titres assessed. Antibody levels
increased up to 20 days after virus infection and then waned by 4o days. Results are expressed as
well binding ratios ( V + / V - ) as in Fig. I.
T a b l e i.
Antibody titres to HSV-I somatic cell hybrids
r
Wells producing
antibody
Antibody activity (titres)
Limiting dilution
~"
~ antibody forming clones
Tissue culture Pooled sera*
Total no. of clones
supernatants
from original wells
HIE8
I/IOO
HIG4
I/IO
HzAI
H2B4
H5B7
Sera from hyperimmunized mice
C57
BALB/c
I/IOO
< I/I0
I/lOO
1/256oo
1/64oo
1/256oo
I/t600
1/6400
Antibody titres
9/IO
2/9
7/to
I/II
4/8
I/IO24O
1/4o96o
* Pooled sera from five to ten tumour-bearing mice.
e v i d e n c e o f n e u t r a l i z i n g activity. T h i s was t r u e w h e t h e r o r n o t c o m p l e m e n t was a d d e d d u r i n g
n e u t r a l i z a t i o n . By c o n t r a s t , p o o l e d sera f r o m C57 a n d B A L B / c m i c e h y p e r i m m u n i z e d
a g a i n s t H S V - I h a d n e u t r a l i z i n g titres o f I / 8 O a n d I/6O, respectively. T h e p r e s e n c e o f
c o m p l e m e n t i n c r e a s e d t h e c a p a c i t y o f B A L B / c a n t i s e r a to n e u t r a l i z e to 1 / 6 4 o w h e r e a s
i n a c t i v a t e d c o m p l e m e n t s h o w e d a titre o f I/8O.
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E. L. H O W E S
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Class of cell surface immunoglobulin on hybrid cell lines
By direct immunofluorescence following sodium azide treatment to prevent capping, two
cell lines HIE8, H5B7 and control IgM-producer TI2GI I, were found to have surface IgM
and one, HIG4, stained faintly with anti-72. The other two lines had no detectable surface
immunoglobulin by this method.
DISCUSSION
A somatic cell hybridization between a non-secreting mouse myeloma cell line (NS-I,
BALB/c) and spleen cells from BALB/c mice, hyperimmunized with HSV-I resulted in the
production of five stable cell lines producing antibody to HSV-x as detected in a sensitive
solid-phase radioimmunoassay. Two of these lines were shown to have surface IgM by
direct immunofluorescence. None of the lines produced antibody able to neutralize virus in a
plaque forming assay, although three of the lines produced antibody detectable at dilutions
of I/IOO in tissue culture supernatants and greater than 1/64oo in pooled sera from tumour
bearing mice.
The solid state radioimmunoassay using whole virus proved a reproducible and sensitive
method of antibody screening. The antibody response in immunized mice could be followed
easily and both IgG and IgM binding antibodies could be detected (unpublished data).
Saturation of non-specific binding sites by foetal calf serum made the use of flexible plastic
wells feasible and easier to use than polystyrene balls in order to reduce non-specific binding
(Kalino et al. I977a , b).
Approximately 9 % of wells growing cells gave evidence of antibody production 2~ days
after the cell fusion, a figure similar to that obtained with SRBC (Kohler & Milstein, I976).
But a combination of (i) loss of antibody formation, (ii) failure of cells to grow, and to a
lesser extent, (iii) fungal contamination, resulted in a final yield of less than ~% of stable
cell lines, i.e. lines that were stable in culture for more than six months, that could be grown
as tumours in mice, stored in liquid nitrogen and remain active in forming antibody. Limited
dilution (and selection of cell clones by micromanipulation) showed that the two stable cell
lineswith the highest antibodyproduction contained a majorityofclones producing antibody,
whereas those stable cell lines with the lower antibody production had fewer clones producing antibody. This suggests that the loss of capacity of wells to form antibody at an early
stage of the tissue culture was due, at least in part, to cell-overgrowth. In order to obtain the
greatest number of antibody-forming clones for analysis of virus antigens, it would be best to
isolate specific antibody forming clones as early as possible. The one day interval between
booster injection and collection of spleen cells used in this study may not have been optimal.
Other data on murine antibody responses to the Thy-1 antigen (E. A. Clark et al. unpublished
data) indicate that the yield of producer clones accurately reflects the frequency of antibody
forming cells present during the fusion. The anti-Thy-I plaque forming cell response peaks
at 3 to 4 days post boost.
The antibodies produced by the hybrid cell lines appear to be specific for HSV-I determinants. They did not cross react with other viruses by R1A; nor did they react with uninfected extracts of the cell lines in which HSV-I was grown. However, none of the five
monoclonal antisera tested was capable of neutralizing HSV-I virus, even when all five
antibodies were combined. Whether neutralization is a multi- or single-hit phenomenon
(Della-Porter & Westaway, I978 ) could not be answered in the present study. It has been
shown that a neutralizing antibody can be produced by animal immunization with an
isolated glycoprotein of HSV-1 (Powell et al. I974) and the failure to neutralize in the present
study may well be due to a lack of that particular antibody.
Our efforts at defining the molecules with which these clonal products react have so far
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HSV-I
a n t i b o d y - p r o d u c i n g h y b r i d cell lines
87
been unsuccessful, owing to non-specific binding of virus components to the staphylococcal
protein A used in the radioimmune precipitation process.
E. A . C. w a s t h e r e c i p i e n t o f a n E d n a A . O l d M e m o r i a l F e l l o w s h i p o f t h e C a n c e r R e s e a r c h
Institute of New York.
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