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An isoelectric focusing study in herpes simplex virus encephalitis.

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An Isoelectric Focusing Study
in Herpes Srmpplex V m s lmcephahtx
Luigi M. E. Grimaldi, MD," Raymond P. Roos, MD," Roberto Manservigi, PhD,? Patricia G. Spear, PhD,t
Fred D. Lakeman, PhD,S and Richard J, Whitley, MDS
To establish an early reliable diagnostic test for herpes simplex virus (HSV) type 1 encephalitis (HSVE), we used
isoelectric focusing (IEF) and an IEF-overlay technique with radiolabeled HSV glycoprotein B (gB) to study 7 serum
and 12 cerebrospinal fluid (CSF) samples from 12 patients with presumed or biopsy-proved HSVE. Blood-brain barrier
damage and increased intra-blood-brain barrier IgG synthesis were detected in 5 of the 7 patients with HSVE. CSF
oligoclonal bands were found in 6 of 11patients. Using an IEF-overlay technique, we detected anti-gB antibody in all
serum (7 of 7) and in 10 of 12 CSF samples. Anti-gB antibody was found in 4 of 6 CSF specimens obtained within the
first week of disease (days 3 to 5) and in all samples collected later in the disease. The pH range of anti-gB antibody
activity was broad (4.5to 9.5), indicating a heterogeneous immune response to HSV. A hematogenous origin of the
CSF antibody was suggested because anti-gB antibody appeared in serum before matched CSF and because both serum
and matched CSF had a similar antibody IEF pattern. Local production of anti-gB antibody was suggested in some
cases because of a greater prominence of anti-gB antibody in CSF than in matched sera and because CSF oligoclonal
bands had anti-gB antibody activity. In contrast, only one of 6 CSF samples from patients with multiple sclerosis had
gB antibody activity; in this case, anti-gB antibody activity did not correspond in isoelectric point location to oligoclonal bands. The IEF-HSV-gB overlay technique may be a useful diagnostic test for HSVE and a valuable research
tool for studying qualitative aspects of the HSV humoral immune response.
Grimaldi LME, Roos RP, Manservigi R, Spear PG, Lakeman ED, Whitley RJ. An isoelectric focusing study
in herpes simplex virus encephalitis. Ann Neurol 1988;24:227-232
Since the first clinical description of herpes simplex
virus type 1 (HSV-1) encephalitis (HSVE), there have
been difficulties in establishing laboratory tests that are
consistently helpful in the early diagnosis of the disease. Th shortcomings of these tests are even more
apparent with the advent of antiviral drug5 that are
most effective when administered early in disease.
HSVE laboratory diagnostic tests have been frequently directed toward the identification of HSV and
viral proteins. The frequent inability to isolate HSV-1
from the cerebrospinal fluid (CSF) of patients with
HSVE led to recommendations of brain biopsy for
diagnostic purposes; however, the attendant risks and
invasiveness of the procedure have discouraged its use.
More recently, studies have involved the immunochemical identification of HSV proteins in the CSF
[l-31. Another direction of HSVE laboratory studies
is the identification of HSV-specific antibody within
the central nervous system (CNS) or CSF. Studies
have included tests for anti-HSV I&, A and M antibody, intrathecal anti-HSV antibody production 1471, and CSF oligoclonal Ig bands 14, 5, 8-10]. These
approaches have generally been suboptimal because
From the "Department of Neurology and Whe Marjorie B. Kovler
Viral Oncology Laboratories, The University of Chicago, Chicago,
IL; and the *Department of Pediatrics, University of Alabama, Birmingham, AL.
they have failed to generate diagnostically useful information sufficiently early to guide treatment with antiviral drugs.
To detect anti-HSV antibody early in the disease
course, we studied HSVE CSF and serum samples
with an isoelectric focusing (1EF)-antigen overlay
technique using HSV glycoprotein B (gB), a major
immunodominant protein. CSF anti-gB antibody was
detected as early as 3 days after disease onset. We also
tested whether patients display a CSF anti-HSV gB
antibody IEF pattern different from that in the serum,
indicating a CNS-produced anti-HSV antibody, as one
might expect in HSVE. Our investigations showed that
the CSF and serum patterns were actually very similar,
suggesting a common origin of the anti-gB antibody.
The IEF-overlay technique can provide important information regarding qualitative aspects of the antiHSV antibody response.
Materials and Methods
Patients and Samples
All patients with HSVE had an acute febrile encephalopathy
accompanied by focal cerebral signs, with localization of disReceived Dec 14, 1987, and in revised form Feb 3 , 1988. Accepted
for publication Feb 17, 1988.
Address correspondence
D~ Roes, D~~~~~~~ of Neurology,
University of Chicago, 5841 South Maryland Avenue, Chicago, IL,
60637.
Copyright 0 1988 by the American Neurological Association 227
ease to the temporal or inferior frontal lobe as seen on an
imaging study, and CSF findings consistent with a viral infection. Paired CSF and serum samples were obtained from 6
patients who underwent brain biopsy with positive isolation
of HSV-1, and from 6 additional patients with HSVE who
did not undergo biopsy. In all patients the ratio of CSF to
serum HSV antibody suggested local synthesis of anti-HSV
antibody. In 5 of the 6 unbiopsied patients, serum was not
available for electrophoretical studies.
Specimens from 3 patients with viral encephalitis of unknown cause, 2 with herpes zoster with accompanying
neurological symptoms, and 3 with acute herpes labialis without CNS symptoms were used as control subjects. In the
patients with a diagnosis of viral encephalitis of unknown
cause, HSVE was tentatively excluded by a negative biopsy
and failure to isolate HSV from the biopsied tissue. Six patients with multiple sclerosis (MS) who had prominent CSF
oligoclonal bands were also included in the control group to
test for the possibility of nonspecific binding of the radiolabeled HSV gB to oligoclonal Ig bands blotted to the
nitrocellulose paper. Additional control specimens for the
IEF-overlay included mouse anti-HSV- 1 gB monoclonal
antibody, mouse anti-HSV- 1 gD monoclonal antibody, and
polyclonal rabbit anti-HSV-1 serum.
Quantitation Assays
IgG and albumin quantitation was performed by automated
nephelometry. The CSF/serum albumin ratio, IgG index
[I 17, and intra-blood-brain barrier (IBBB) IgG daily synthesis 112) were calculated and compared to normal published
values 1131.
HSV Antigen Preparation
Monolayers of HEp-2 cells growing in roller bottles were
infected with HSV-1 KOS virus at 10 pfu per cell in phosphate-buffered saline (PBS) containing 1% calf serum and
0.1% (wtlvol) glucose for 2 hours at 37" C. The virus was
then removed and Medium 199 was added. After 20 to 24
hours' incubation at 37" C, cells were harvested and pelleted
at low speed; virus was purified as described by Cassai and
co-workers 1147 and as modified by Johnson and associates
[I 51. The virus was then concentrated by centrifugation at
23,000 rpm in an SW27 rotor for 2 hours. The pelleted
virions were then extracted with Tris-saline (50 mM TrisHCI [pH 7.51, 100 mM NaC1)-50 mM octyl-B-D glucopyranoside (octyl-glucoside, Calbiochem-Behring, La Jolla,
CA)- 1 mM EDTA-1 mM phenylmethylsulfonyl fluoride200 U of aprotinin (Trasylol, Mobay Chemical, New York,
NY) per milliliter, sonicated 3 or 4 times for 30 seconds, and
then incubated for 8 to 14 hours on ice and resonicated
briefly. The lysate was centrifuged at 30,000 rpm in a 50Ti
rotor for 1 hour at 4" C. The supernatant was passed over
immunoaffinity columns prepared with purified mouse
monoclonal IgG 1-59 specific for HSV gB as previously described [ 157. The extract was passed over the column 5 times
at 4" C. The column was then washed for 5 hours with 20
mM Tris-HC1 (pH 7.5)-0.5M NaCI-O.l% Nonidet P40
(NP40, BDH Chemicals, Poole, England)-0.1 mM phenylmethylsulfonyl fluoride. The bound material was eluted off
with 3M potassium thiocyanate, mixed with 0.1% NP40,
228 Annals of Neurology
Vol 24 N o 2 August 1988
and dialyzed against Tris-saline containing 0.1% NP40 for
18 hours at 4" C. The gB obtained was resuspended at 1 mg/
ml in 0.4 M Tris-HCI-4 mM EDTA (pH 7.5) and labeled
with 1251 (Amersham, Arlington Heights, IL) by the chloramine T method. The final specific activity was -40 pCdpg.
The labeled protein was aliquoted and stored at - 20" C until
used.
IEF and IEF-Antigen Overkay Technique
For IEF, 20 to 60 pl of unconcentrated CSF and a 1:20 to
1:100 dilution of serum in distilled water were applied to
5% polyacrylamide gels (LKB, Bromma, Sweden), p H range
3.5 to 9.5. In some gels, paired CSF and serum samples that
contained equal amounts of IgG were applied in adjacent
lanes. Proteins were focused for 2 hours at constant power
until a final voltage of 1,500 V was reached. Migrated proteins were either silver stained as previously described [l6]
or processed for IEF-antigen overlays [171. For the latter
study, focused proteins were transferred to nitrocellulose paper (Schleicher & Schuell, Keene, NH) with a Transblot
(LIU3) apparatus for 3 hours at 60 V or overnight at 30 V.
The paper was then cut, blocked for 6 hours in 3% bovine
serum albumin in PBS (BSA-PBS), and overlaid with 50,000
to 250,000 countdmidlane of purified '251-labeledgB in 3%
BSA-PBS for 6 to 12 hours. The paper was then washed 6
times for 10-minute intervals in 3% BSA-PBS, dried, and
exposed to X-Omat AR x-ray films (Eastman Kodak, Rochester, NY) at -70" C for autoradiography. Films were
developed 3 to 24 hours later.
Results
The Table summarizes clinical findings and laboratory
data from the patients with HSVE.
Quantitative and Qualitative I g Studies
of HSVE Specimens
IgG levels were elevated in 4 of 7 serum specimens
and in all 12 CSF samples from patients with HSVE.
CSF IgG levels were particularly elevated, in one sample reaching a value 75-fold higher than the upper
limit of normal. The elevated CSF IgG was partly a
result of BBB damage, as evidenced by CSF/serum
albumin ratio abnormalities in 5 of 7 tested patients,
and was partly a result of intrathecal IgG synthesis, as
indicated by the abnormalities in CSF IgG/albumin
ratios (8 of 12), IgG index ( 5 of 7), and IgG synthesis
rate (6 of 7). Oligoclonal IgG bands were present in 6
of 12 CSF samples, but in none of the serum specimens studied.
IEF-Antigen Overlay Studies
Anti-HSV gB antibody activity was detected by IEFantigen overlay in 5 of 7 paired CSF and 7 of 7 paired
serum samples from patients with HSVE, as well as in
all unpaired HSVE CSF samples. When the CSF specimens were grouped according to collection time after
onset of clinical disease, anti-gB antibody was found in
4 of 6 specimens obtained within the first week of
Clinical Data and Results of Patients with Herpes Simplex Virus Type 1 Encephalitis
Age
(yr)
Day
from
Onset
9
5
57
20
35
5
57
3
55
36
Biopsy
Sample
+
+
+
+
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
Serum
CSF
CSF
CSF
CSF
CSF
CSF
f
52
14
+
5
3
ND
23
23
38
31
58
19
3
4
25
10
ND
ND
ND
ND
ND
IgG
IgG/Albumin
(mg/dl)” Ratio (%)b
1,430
6.1
779
23.9
1,500
44.8
919
14.5
1,370
31.1
3,300
53.6
1,140
3.2
17.1
17.6
25.5
8.0
11.2
28
13
59
73
27
20
25
12
3
94
70
113
39
23
92
37
47
29
52
CSFISerum
Albumin Ratio
( x 10-3y
IgG
Indexd
9.35
(55.7)
24.85
(58.9)
40.7 7
(56.2)
22.67
(58.9)
7.31
(58.9)
10.04
(‘8.9)
3.8 1
(55.7)
0.54
0.60
1.24
75.15
0.73
86.05
0.69
24.85
3.18
128.85
1.61
182.90
0.74
1.86
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
I g G Daily
Synthesis
(mg)‘
ND
ND
ND
ND
ND
Oligoclonal
Bands
IEF-gb
Overlay
-
+
+
+
+
+
-
+
-
+
+
-
+
+
ND
?
+
+
-
+
+
+
+
+
+
+
+
+
+
+
”Normal: serum, 639-1,349 mgidl; CSF = 1-3 rngldl.
“Normal: 7-21%.
‘Age-adjusted normal values in parentheses.
“Normal: 5 0.7.
‘Normal: 5 0.85 mg.
CSF = cerebrospinal fluid; IEF
=
isoelectric focusing; ND = not done;
disease onset, 2 of 2 from the second week, and 4 of 4
from later than 2 weeks. The 2 patients with HSVE
without CSF anti-gB antibody had no evidence of intrathecal IgG production by the IgG index.
Anti-gB antibody activity was spread over a wide
pH range (4.5-9.5), indicating a heterogeneous, polyclonal anti-gB humoral immune response (Fig 1, lanes
h-k). The CSF anti-gB antibody IEF pattern was in
general remarkably similar to the pattern in paired
serum samples, suggesting that some of the CSF antibody may result from BBB damage. CSF specimens
collected a few days after the onset of clinical disease
tended to have no or relatively little anti-gB antibody
activity compared to the corresponding serum samples
(which contained an equivalent amount of IgG) (Fig 1,
lanes f, g); later in the disease course, other patients
showed an increase in CSF anti-gB antibody to a level
similar to (Fig 1, lanes h, i) or higher than (Fig 1, lanes
j , k) in the paired serum samples. The finding of more
prominent anti-gB activity in CSF than paired serum
specimens indicates that some of the anti-gB antibody
is locally produced, a conclusion supported by other
results (see below).
The IEF-antigen overlay technique was also used to
determine whether HSVE CSF silver-stained oligoclonal bands contained anti-gB activity. Figure 2 (lanes
+
= positive;
-
= negative
f-i) shows that the oligoclonal bands generally correspond in isoelectric point location to anti-gB activity;
these results suggest that some anti-gB antibody is locally produced within the CNS (since the silver-stained
oligoclonal bands were only visible in the CSF). However, as is clear from Figure l, lanes i and k, CSF antigB antibody activity was not confined to the bands, bur
rather diffusely distributed.
In contrast to the above findings, only one patient
with MS had evidence of anti-gB activity in the CSF,
despite prominent anti-gB antibody activity in two
serum samples (Fig 2, lanes j-m). Anti-gB antibody
activity in this one MS CSF sample did not correspond
in isoelectric point location with the cathodal oligoclonal IgG bands detected by silver staining (Fig 2,
lanes n, 0). This patient with MS was of special interest
because the patient had had recurrent HSV-2 cutaneous lesions for years and a recent history of cutaneous
lesions temporally associated with MS attacks. We presume that this CSF anti-gB antibody activity represents
a cross-reaction between HSV-1 and HSV-2 gB 1181.
These findings indicate that patients with inflammatory
CNS disease and serum anti-gB antibody activity do
not necessarily have detectable CSF anti-gB activity,
and that MS oligoclonal IgG bands are not directed
against HSV-gB.
Grimaldi et al: IEF in HSV Encephalitis 229
PH
3.5
6.5
9.5
d e
o b c
L+
-+,
,
f g
h i
Ldoy3
day5
HL
Controls
j
I m
k
dayl2,
HSVE
HZV
_
_
_
I
_
_
F i g 1 Autoradzogramr ,hototng anti-herpes simplex zlvuj
(HSV)-gB antibod) a i t i i i t ) i n monotlonal antibodzer (rnAb),
cerum (ser), and ierehroJptnal&iid (CSF) aJ detected ti] an
i,oelectrrcfocurinR-gB dntigeri oterluy technique (we text) (Lane
d Positiie control mouse anti-FhV gB mAh tb) Negalite control mouie anti-HSV gD nAh (intentionally ofJerexpojedt o demonrtrate the latk of reactrrity ugain\t HSV gBI
Paritwe ton-
s s s
pH
,
s 10
I
Controls
s
I0
s I0
CSF, I CSF,
HSVE # I
HSVE 442
I s e r
Fig 2 Serum (serfand terebrospivial~i~zd
(CSF) samples
rilvir itainrng
rtudied by iboelritric focucirig (iEF)folloz~~edby
(S) o r a hrrpeb wriple*L itru\ (HSVI-gB IEF oterlay (10) show
i n g the relationship of itlter-sturried oligoclonal b a d t o anti-gB
antihod> act11 ity (Lane ai Normalserum ib) Normul CSF (c)
Subarute sclerosing panencephalitiJ CSF Serum cd ei and CSF
(f.gI from Patient 1 with HSV-1 encephalrtis (HSVE), CSF
(h.1)from HSVE Patient 2 , Jerum Q,kj andCSF ~l.mifrom a
repreJentatwe patient 2~ ith multiple sc/erorzs (MS 6 I), CSF
(n,ol from a patient with MS and HSV-2 cutaneous lesions (MS
230 Annals of Neurology Vol 24
-______-- -
tml rabbit gB antiserum id,e) Sera from 2 patients uvth hcrJjes
labialis (HL) Serum and paired C\F tampler from 3 patientr
collected on days 3 if;gi. 5 (h,t),and 12 O.ki afer thr onret o/
clinical HSV-1 encephalitis (HSVE) (equaf amountj o/ I g z( ere
applied in the case of paired sumpler) Serum (1) and CYF (nil
from a patzent wzth herpes zoster (l-IZV) u ith neurologttitl tnvolvement
No 2 August 1988
s
10
s
I ser
ro s
CSF,
MS # I
10
,CSF,
MS #2
#2). Szlver-stained ohgoclonal band are seen i n Ianer c.J,hI,
and n above a relatzziei) unzjom stuznzng pattern. maznly i n the
cathodal area of the el A w m ~haw been placed in the 10 lanrr
t o indicate antz-g8 antibody u t i t tzy thut ltnes up uvrh the ci1ver-stained ohgoclonal bands of the HSVE CSF None of the
cathodalb locatedstlzer-stained band i n lanr MS CSF (lane n i
correspond to anti-gB antibody uctiwty (lane 01, ewn utter prolonged exposure ofthe autoradiogram A r noted, the upper part
of the figure corresponds to p H 3 5 ianode,, the louier purt cowesponds to pH 9 5 (cathode)
Anti-HSV gB antibody activity was detected in
serum specimens from 2 patients with HSV-1 herpes
labialis (Fig 1, lanes d,e). Anti-gB antibody was also
present in CSF and serum from a patient with herpes
zoster (Fig 1, lanes l,m), reflecting an antigenic similarity between HSV-1 gB and herpes zoster glycoproteins C191. None of the serum or CSF samples from 4
patients with viral encephalitis of unknown cause had
anti-gB activity. As expected, anti-gB monoclonal antibody had reactivity against gB (Fig 1, lane a), whereas
anti-gD monoclonal antibody had none (Fig 1, lane b).
Discussion
W e were able to detect CSF anti-gB antibody activity
in 10 of 12 patients with HSVE, including one patient
on the third day of disease and 3 others during the first
week. We failed to detect CSF anti-gB antibody in 2
other patients at days 3 and 5 of disease. Our results
generally agree with those of Kahlon and co-workers
C20), who found anti-gB antibody by immunoblot and
radioimmunoassay within the first week of disease;
however, in contrast to our findings, they failed to
detect antibody within the first 3 to 4 days. The results
are also in agreement with studies of Lakeman and
associates C31, who found that 62% of patients with
HSVE had evidence of CSF gB antigen during the first
week of disease.
We used the IEF-antigen overlay technique to test
for CSF anti-gB activity at different isoelectric points
from those in paired serum samples indicating locally
produced anti-HSV antibody. In Theiler’s virus-infected mice, for example, there is anodally located
anti-Theiler’s virus antibody activity in the CSF that is
not present in the serum (17); in contrast, patients
with HSVE have a similar broad isoelectric point distribution of anti-gB antibody activity in both CSF and
paired serum specimens. Anti-HSV-gB antibody tends
to appear initially in serum and shortly after, with a
similar pattern, in the CSF (Fig 1, lanes f-k), suggesting a hematogenous origin for the CSF antibody. Perhaps there is an initial systemic immunization in HSVE
or a systemic immunization concomitant with one
present in the CNS, compared to the primary Theiler’s
virus CNS disease that follows intracerebral inoculation.
Although HSVE CSF had a similar anti-gB antibody
IEF pattern to serum, we were able to demonstrate
locally synthesized anti-HSV antibody activity in a limited number of samples because the CSF had more
prominent anti-gB antibody activity than serum (which
contained an equal amount of IgG) and because the
CSF had silver-stained oligoclonal IgG bands that were
not present in the serum with anti-gB activity. Vandvik
and co-workers [S] also found that CSF oligoclonal
IgG bands generally corresponded to anti-HSV antibody activity using an imprint immunofuration tech-
nique. The lower incidence of oligoclonal IgG bands in
our study (50%) compared to other studies [4]may
reflect a more notable BBB damage in our patients.
What is the role of the IEF-antigen overlay technique in the diagnosis of HSVE and how can the test
be improved? In contrast to other antibody detection
methods, the IEF-overlay technique provides information regarding qualitative aspects of the HSV immune response. These data may be important not only
in the diagnosis of HSVE, but also in evaluating the
intactness of the humoral immune response after HSV
immunization and during viral latency.
The use of antigens other than gB in the IEFoverlay may increase the test’s usefulness. We chose
gB as an overlay because the anti-gB antibody response is one of the earliest and strongest responses
after HSV infection and because it is involved in the
neutralization response [211. Recent studies have suggested that antibody directed against p40, a nonsurface
nucleocapsid protein, may be an even earlier response
in primary HSV-1 infections E223. The IEF-overlay
technique could conceivably be used with radiolabeled
purified p40 or another HSV protein to improve sensitivity, and perhaps even demonstrate an HSV protein
antibody IEF pattern unique to the CSF of patients
with HSVE. HSV proteins could be combined in a
cocktail and expression vectors constructed to contain
epitopes unique to HSV-1, thereby gaining more specificity than with the IEF-gB overlay. The absence of a
“sandwich” antibody in our technique also avoids some
potential cross-reactions present in other assay systems
r31.
The IEF-overlay technique could also be used to
confirm results obtained from other tests such as enzyme-linked immunosorbent assay, which has a high
false-positive rate. The concentration of IgG into a
restricted p H zone and the use of radiolabeled antigen
to a high specific activity allow results to be available in
less than a day, as in our study.
Supported in part by the Brain Research Foundation (an affiliate of
the University of Chicago) and from Cons~glioNazionale Ricerche
Special Project “Controllo Malattie da Infezione.”
The authors thank Bonita DuPont for her guidance, Renata Vanakojis for technical assistance, and Mary Witt for assistance in preparing
the manuscript.
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