Sensitive analytic ELISAs for subclass herpes virus IgG

Journal

of Virological

Methods,

203

10 (1985) 203-214

Elsevier JVM 00374

SENSITIVE

ANALYTIC

ELISAs

G. GILLJAMi.

V.-A. SUNDQVIST’.

FOR SUBCLASS

A. LINDE’,

HERPES

P. PIHLSTEDT2.

VIRUS

A.E. EKLUND’

IgG

and

B. WAHREN’ ‘Department

of Virology,

Virology, Karolinska sion Service, S-182

higher

IgGl

frequency

reactivated

of antiviral

appeared

and ‘Department

of Surgery,

to three herpes viruses was studied

by monoclonal

to be formed

of virus-specific

IgG3

in response

antibodies

and Department

of

Danderyd

Hospital.

to HSV-1 werecommon.

cell-related

reactivities

disease had a near-normal

varicella-zoster

virus

subclass

in a population

led to the identification

of

of certain viral

to almost all CMV, HSV-I and VZV infections.

to CMV and HSV-1

more often than VZV. The presence

while IgG4responses

cytomegalovirus

01 Stockholm.

antibodies

Subclassification

to CMV showed

and Crohn’s

S-104

S-IOS 21 Stockholm,

of ClinicalImmunologyandBloodTransfi-

1984)

subclinically

relationship, antibody

Hospital,

distribution

blood donors.

IgG patterns.

National

Sweden

29 October

The subclass healthy

Laboratory, 2Deparrment

Karolinska

88 Danderyd,

(Accepted

Bacteriological

Institute. S-104 01 Stockholm.

suggested

A

may be

of CMV and VZV IgG4 showed a familial

Persons with IgG4as

in a high frequency. pattern

that these infections

Patients

the only subclass-reactive with leukemias,

myelomas

to the herpes viruses.

herpes simplex

virus

INTRODUCTION

IgG subclass responses of IgGl alone or with IgG3 have been described in connection with virus infections (Linde et al., 1983; Morel1 et al., 1983; Skvaril, 1983; Vartdal and Vandvik, 1983; Sundqvist et al., 1984; Linde, 1985). Humoral reactivity to herpes viruses appears to be restricted, mainly to the subclasses IgGl and IgG3 (Linde et al., 1983; Sundqvist et al., 1984). In certain cases we found that IgG4 may occur, though evidence was sometimes difficult to obtain because reactivity to IgG4 is poor in conventional serological reagents. Since the subclasses differ in their biological properties, it is important to know the subclass herpes virus responses of a common population of blood donors. For latent viruses, accurate identification of completely seronegative donors is likewise important. Four hundred and seventy healthy blood donors were analysed for their total specific IgGs and the subclass distribution to cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). In addition, patients with inflammatory and malignant blood diseases were studied. The findings should assist 0166-0934/85/%03.30

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

204

in the selection of hyperimmunoglobulin donors and true seronegative also indicate genetic relationships in viral immune responses. MATERIALS

AND

donors.

They

METHODS

Human sera

Four hundred and seventy sera from healthy blood donors were used (Tables 1 and 2). Most, but not all sera could be used for all assays. The mean age of the blood donors was 39 yr and the sex ratio (male : female) was 65 : 35. Sixty additional sera from healthy persons were used as concomitant controls in one study. Thirty-one patients had Crohn’s disease, 12 in an active phase as judged by the clinical picture and the electrophoretic serum pattern. Thirty patients had malignant blood diseases: 17 with acute non-lymphoblastic leukemia, 10 with acute lymphocytic leukemia (ALL), 2 with chronic myelocytic leukemia and one with myelofibrosis. A follow-up of these patients has been described (Wahren et al., 1984). Six patients had myelomas, 2 each of IgGI and IgG2 subclasses, one each with IgG3 and IgG4 myeloma. ELISA for total viral IgG, IgM and virus-specific

IgG subclasses

Viral Ig assays.

The total CMV, HSV-1 and VZV IgG and IgM ELISAs have been described previously (Sundqvist and Wahren, 1981; Sundqvist, 1983; Sundqvist et al., 1984). CMV antigen was prepared from ultrasonicated nuclei of CMV Ad169-infected human fibroblasts, strain MRC-5. It was used for coating of microplates in the CMV IgG assay and labelled with horseradish peroxidase (HRP) for the CMV IgM assay. HSV-1 antigen was prepared from ultrasonicated whole HSV-1 strain F9004infected green monkey kidney cells (National Bacteriological Laboratory, Stockholm, Sweden). VZV antigen was from whole human lung (HL) fibroblasts infected with VZV (Strain ULF; National Bacteriological Laboratory, Stockholm, Sweden) and ultrasonicated. Non-specific reactivity was excluded by the differential pattern of sera to the different viral antigens and in a few cases by titration with control antigen prepared from uninfected cells. Sheep anti-human IgG conjugated to alkaline phosphatase (ALP) (batch 53 F-8830) for the IgG assay was obtained from Sigma (St. Louis, MO). Hundred ul were used in a dilution of 1 : 1200. The substrate was p-nitrophenylphosphat’e (AdlO”,,,). Repeated sampling (2-10 samples) from I5 healthy persons without any known disease showed the natural variation in total viral IgG antibody titers to be 15% for CMV, 21% for VZV and 20% for HSV. The u-capture technique was used for the CMV and VZV IgM assays with HRP-labelled virus antigens (Sundqvist, 1983). HSV IgM was analysed on antigen-coated plates and identified by anti-human IgM conjugated to ALP. Subclass

essentially

assays.

The CMV, HSV-1 and VZV IgG subclass assays were performed as described (Linde et al., 1983; Sundqvist et al., 1984). Microplates (Nunc

205

Immunoplates

I, Nunc, Aarhus,

For CMV antibodies, coated with an antigen own plates, results

were used in parallel

were identical

Denmark)

were coated with viral antigen

(see above).

CMV plates (Pharmacia Diagnostics, Uppsala, Sweden) from CMV-infected HL nuclei, prepared similarly to our for total viral IgG and subclasses

to those for our own plates. Human

of 50 sera. The

sera to be examined

were

diluted IO-fold from 10-l to 10m5,and 100 ~1 were added to the plates. After washing, 100 pl of ascitic fluids with monoclonal antibodies against IgG l-4 (Lowe et al., 1982) were added. The fluids were from Seward Laboratories (London, U.K.) and used in the following dilutions: for IgGl clone BAM 15 (1 : 2,000), for IgG2 clone BAM 14 (1 : 200-400), for IgG3 clone BAM 08 (1 : 2,000) and for IgG4 clone BAM ll(1: 800). In addition, for IgG2 the clone HP6992 (Center for Disease Control, Atlanta, GA) was used in a dilution of 1 : 50,000. This clone was a kind gift from Dr. C. Reimer, CDC. In the subclass assay, dilutions of monoclonal anti-IgGs were added so that each serum dilution was examined for all four subclasses. As a control, the monoclonal antibodies were also added to antigen-coated serum-free wells. HRP-labelled rabbit anti-mouse IgG (Dakopatts, Copenhagen, Denmark) was diluted 1 : 1,500 and 100 ~1 were added for subclass assays. The incubation time for each step was 105 min at 37°C. The substrate was orthophenylenediamine (OPD, Fluka AG, Chemische Fabrik, Buchs, Switzerland) activated by H,O,. After incubation for 60 min in the dark at room temperature, the reaction was terminated by 100 pi of 2.5 M H,SO, and absorbance read at Abg2”,,,. All assays were read in a Dynatech MR 600 (Arlington, Virginia). The background reactivity of monoclonal antibodies to human IgGl-4 was established using7- 13 seronegative sera diluted 10e2.These backgrounds varied with each clone and each viral antigen. The levels used were the X f 3 SD absorbance values 492nm). With IgGl they were 0.28 for CMV, 0.18 for HSV and 0.22 for VZV. With (A IgG2 they were 0.25 for CMV, HSV and VZV, with IgG3 they were 0.32 for CMV, 0.32 for HSV and 0.31 for VZV. With IgG4 they were 0.43 for CMV, 0.40 for HSV and 0.42 for VZV. Values obtained above these levels were regarded as positive. ELISA for total IgG4 Microplates were coated with sheep anti-human IgG4; 100 pi/well of an 1 : 800 dilution (Central Laboratory of The Netherlands, Red Cross Transfusion Service, Amsterdam, The Netherlands). Human sera were added in dilutions and thereafter the clone BAM 11 anti-IgG4 and HRP-labelled rabbit anti-mouse as above. The WHO serum 67/97 (0.35 mg IgG4/ml) was used as a standard, and the amount of total IgG4 in each serum was calculated. Presentation of ELISA results Titers of total viral IgG were presented as serum dilutions at which the antibody titration curve crosses A,,, nm = 0.20 (Sundqvist and Wahren, 1981). For the subclassspecific ELISAs, titration curves are shown for some sera. For the material as a whole

206

the results background

are presented

Immunojborescence

Indirect

as the presence

levels at a dilution

of virus-specific

of 1 : 100. Total

subclass

antibody

above

IgG4 is given as mg/ml.

(IF)

IF (Wahren

and oberg,

1979) for CMV IgGl-4

was performed

on slides

with acetone-fixed CMV Ad169-infected human fibroblasts. A multiplicity of infection of 1 .O gave around 70% of infected cells containing CMV late antigens (LA) with nuclear inclusion bodies 7 days after culture at 37’C. Human sera were added to the fixed cells in a dilution of 1 : 2 and incubated in a humid atmosphere at 37°C for 30 min. After rinsing in phosphate-buffered saline, the slides were incubated with mouse monoclonal antibodies to IgGl, IgG2, IgG3 or IgG4, all diluted 1 : 5, followed by fluorescein isothiocyanate-labelled sheep anti-mouse IgG diluted 1 : 20 (National Bacteriological Laboratory, Stockholm). Known positive and negative human sera served as controls. CMV IF or complement fixation titers in a seropositive population usually range between 5 and 1,280 with a mean of 80 (Wahren et al., 1969). After washing and mounting in glycerol, the appearance of nuclear inclusion bodies was read in a Zeiss fluorescence microscope. Special care was taken not tocount cells with cytoplasmic Fc-receptor staining only. Such Fc-receptors are produced in the late phase of CMV infection and react also with non-specific IgG (Wahren and bberg, 1979). RESULTS

Total virus-specific

IgG

The distribution of total virus-specific IgG is shown for HSV, CMV and VZV (Table 1). Sera with very high antibody levels are of special interest when selecting donors for hyperimmunoglobulin. When the upper 5% of the seropositive population

TABLE

1

Total ELISA

antiviral

IgG distribution

of 470 sera from healthy

blood

donors,

% Sera with

Median

Titer limit

reactiwty”

titer of

for upper

in

positive

dilutions

wa
10-50

250

CMV

15

13

72

3,000

16,000

HSV-I

17

IO

73

8.000

30,000

9x

3.500

20.000

WY

mean ape 39 yr

0.5

“ A ~,” nm > 0.2 at dilutions

1.5 10. 50 or hlpher.

5%

207

is selected,

the total IgG titer is over 16,000 for CMV, 30,000 for HSV-1 and 20,000

for VZV as measured

with our assays. Sera with titers above these limits were assayed

for specific IgM. With CMV, 4% ofsera with IgG titers over 16,000 had low IgM titers of 50-500. With HSV, 5% of persons with IgG titers over 30,000 had specific IgM and with VZ,V none with high IgG titers had antiviral

IgM.

Antiviral IgG subclasses in IgG-seropositive sera All sera considered positive for total virus specific IgG were positive for one or several subclasses to the virus in question (Fig. 1). IgG 1, alone or together with IgG3, occurred most frequently to all three viruses (Fig. 1, Table 2). IgG 1 occurred in the highest levels to CMV, HSV-1 and VZV (Fig. 2a). IgG2 was demonstrable in 30% of sera to CMV, but in very few cases to HSV-I or VZV (Table 2). IgG3 was common to CMV and HSV-1 (Table 2). Mean IgG3 levels to CMV and HSV-1 were higher than those to VZV (Fig. 2b). IgG4 occurred occasionally to CMV and VZV, more often to HSV-1 (Fig. 2c, Table 2). Some typical titration curves are shown in Fig. 3. When subclass antibodies were also calculated as titers (dilution giving A492nm = 0.2), the subclass’ IgGl titers were rather well related to total viral IgG titer (r = 0.55-0.75 for IgGl to CMV, HSV-1 or VZV). For IgG4 the relation was less apparent (r = 0.15-0.44). IgG3 occurs early in primary disease (Sundqvist et al., 1984) and we therefore determined the specific IgM of patients with high IgG3 levels: only one of 7

2

100

iz ‘i ;

HSV

1

50

a 100

LIL-1

vzv

‘“!L__ llll--- 3_ 43 ;

2

9

i

208 TABLE

2

Distribution

of IgG subclasses

to herpes viruses

in seropositive

and seronegative

Total No.

Total No.

9/c Positive

of sub-

of sera

each subclass

class-posi-

in each

tive sera

category

CMV

All

326

HSV-I

All

331

vzv

All

458

100

IgGl

sera

sera with reactivity

within

IgG2d

IgG3

IgG4

96

30

87

25

100

5

71

62

I5

21

21

I9

Total viral IgG 250”

Total viral IgG IO-50b CMV

17

63

5

0

6

HSV-I

13

47

34

0

4

6

1

7

I4

0

0

0

4

vzv Total viral IgG<

10

CMV HSV-1 vzv For subclass

7

68

4

0

I5

81

I9

0

4

3

0

2

0

0

0

0

combinations,

see Fig. I.

IgG3 was the only subclass

to CMV in 4 sera.

IgG4 was the only subclass

to CMV in I I sera.

IgG3 was the only subclass

to HSV-1 in 1 serum.

IgG4 was the only subclass

to HSV-I

IgG3 was the only subclass

to CMV in 2 sera.

IgG4 was the only subclass

to CMV in 4 sera.

IgGl

to HSV-I

was the only subclass

Figures in table are with the anti-IgG2

in I serum.

in 4 sera. clone HP 6992. With clone I4 the figures with total viral IgG 350

for CMV IgG2 were lower.

patients with the highe$ IgG3 anti-CMV also had specific CMV IgM; 2/7 patients with high IgG3 to HSV-I had specific IgM and none of 7 patients with high IgG3 to VZV had specific IgM. IgG subclass distribution in seronegative

sera and sera with very low total viral IgG

titers

It has been our policy to evaluate sera in the ELISA for viral IgG antibodies at a serum dilution of 1 : 50. When, in addition, all sera with total viral IgG below 50 were subclassified, we demonstrated that several sera contained virus specific IgG 1, IgG3 or IgG4 (Table 2). Over the total IgG range of IO-50,5% (3 sera out of 63) contained

209

b

looi

CMV IgG3

492 rm

Fig. 2. 3 I Distribution

of virus-specific

Tlrc mcdlan .4,,? ,,,,, for wra diluted virus-specific

lgG3

virus-specific

I$?4

AJVZn,,, valws A4iilnn

values

lgGl AJYzn,,~vatuesfnrCMV, I : I.000 were for CMV

of CMV,

HSV-I

of C’MV, HSV-1

and VZV and WV

HSV-I

1.1, HSV-1

and VZVinseradiluCed 1.X VZV

in sera diluted in scra diluted

1: 100.

0.9. b) Distribution

of

I : 100. c) Distribution

of

1: 100.

210

E C

5 cr

2.0

1.0

CMV

,

J

CMV

Q.; -\

\

a

b+---

HSV

vzv

&-i&6%--

C

serum dilution Fig. 3. Titration

curves of antibodies

of subclasses

IgCi 1, 3 and 4 to CMV. b) A serum with CMV I&4 typical antibody negative

pattern

to VZV with mainly IgGl.

IgCi

I (e), IgG3 (3) and IgG4 (0). a) A typical serum with

only. c) A common

finding of HSV IgG 1 and lgG4. d) A

The IgG3 reactivity

is low and would be evaluated

as

here.

CMV-specific IgGl, 6% CMV IgG3 and 19% CMV IgG4. Sera with HSV-I IgGl, IgC3 or lgG4 were also found, as well as IgGl to VZV. Some sera which were considered entirely negative (total viral IgG< 10 in ELISA) contained CMV or HSV-1 IgGl, IgG3 or IgG4 (Table 2). In this latter group 7/68 sera (10%) were unexpectedly positive for CMV, mostly due to IgG4; 15/81 (19%) unexpectedly had HSV-1 antibodies, mostly due to IgGl. Virus specific IgG4 and total IgG4

IgG4 has the lowest total content

in human

sera. However,

herpes virus IgG4 was

clearly measurable in 21-62% of the cases (Table 2). The lowest frequency was seen with CMV and VZV. We therefore analysed the co-variation of IgG4-specific viral antibodies (Table 3). Among individuals with CMVor VZV IgG4 antibodies, 83-88% also had HSV-1 IgG4. When IgG4 to CMV or VZV was absent, it was also likely to be absent to the other virus (86-89%). IgG4 to HSV occurred more commonly (62% of all seropositive sera, Table 2). It is therefore not surprising that persons with HSV-1 IgG4 did not always have CMV or VZV IgG4 (Table 3). It is interesting to note, however, that in the absence of HSV IgG4, very few persons (8%) had demonstrable IgG4 to CMV or VZV.

211 TABLE

3

Relationship

between subclass IgC4

antibodies

to herpes viruses

First, second

“r Reactive and non-reactive

variable”

sera

Both IgG4

Both lgG4

positive

negative

CMV.

HSV

8@

42

WV.

HSV

83

42

CMV,

VZV

55

89

VZV,

CMV

63

86

HSV,

CMV

34

g

HSV.

VZV

27

92

” The sera had to be seropositive for the two viral antigens analysed on each occasion. For instance. CMV IgG4-positive

sera were studied for the presence of HSV

studied for the presence of CMV

IgG4

IgG4 (first line); HSV

IgG4 sera were then

(fifth line) etc. The number of sera in each group ranged from

58-257. h Values differinp

from expected (x2-test P
are underlined.

On the assumption that IgG4 antibody production might have a genetic background, we compared sera taken from relatives of persons with high HSV-1 or CMV IgG4 to relatives of randomly selected persons (Table 4). A significant difference was found between these two groups of family members (P
TABLE

4

Distribution

of virus-specific

IgG4

in families

No. of

No. of

‘;; Persons with IgG4

families

family

antlbodirs

to

members” CMV

HSV-I

vzv

48

71

51

P
P
P
45

21

47

21

326

25

62

21

CP

52

9’ _d

,’ Close relatl\es groups

1 and

were studied: parents, sibhnps and children. The distribution 2.

h Index cases had high lgG4

’

to HSV,

Index cases were chosen at random

” Blood donor\.

see Table

2.

CMV

or both.

(laboratory

personnel).

of relatives was similar in

212

HSV-1 IgG4 compared (P
to the whole population

and VZV (P
was significant

but not HSV-1 (P
in the two groups

(1.0 i

with respect to CMV The mean

1.1 and 1.2 f 1.1 mg/ml

total

IgG4

respectively).

The persons with elevated CMV IgG4 had a higher mean total IgG4 (2.4 compared with 0.8 mg/ml). This was so also for VZV IgG4 but not for HSV-1 IgG4. Altogether 231457 persons had anti-CMV antibody of the IgG4 subclass only. The measurable total antiviral IgG titer in these sera was low. These sera had a reactivity also with control cell antigen in ELISA and Fc-receptor reactivity but no reactivity with nuclei of uninfected cells (ANF) in IF. Nine of the 23 sera (39%) stained intranuclear inclusion bodies in CMV-infected cells with the anti-IgG4 as a second antibody and therefore must be considered to contain both CMV IgG4 and an anticellular reactivity of the IgG4 subclass but of unknown specificity. Inflammatory

and other diseases of unknown etiology

Three groups of patients with known diseases, where a subclass antibody deviation might be seen, were studied. Here, reactivity to CMV was measured (Table 5). In patients with Crohn’s disease we found a low frequency of CMV IgG4. Total IgG4 in the patients with Crohn’s disease was 2.1 f 1.8 mg/ml. In 6 patients with myeloma of subclasses IgGl-4, antiviral antibodies of the same types as in healthy donors were found.

TABLE

5

CMV IgG subclass

distribution

in certain

diseases

‘;i CMV

CMV lgG subclass

srroposltive

in seropositive

distribution

indiwduals

(% )

lrrra IgGl

IgG2

IgG3

81

100

0

90

9

(3.900) 73

100

0

58

19

68

98

3

77

33

(4,100) 72

96

3

87

25

(median Crohn’s

disease (n = 31)

age 17-64; median 38 Hematolopical malipnancy

(leukemias)

(n = 30)

age l-38; median I5 Healthy concomitant. controls (n = 60) Blood donors, see Table 2 (n = 326)

titer)

lgG4

(5.100)

(3,000)

DISCUSSION

The distribution healthy population antibodies appears

of subclass

antiviral

IgG to herpes viruses was studied

in a large,

of blood donors. The combination of IgGl and IgG3 subclass to be most common to CMV and HSV-1, while IgGl is most

common to VZV. It is not surprising that IgGl, which is the major IgG subclass, occurs in the high frequency found here and previously (Linde et al., 1983; Sundqvist et al., 1984). A small proportion of persons had no detectable virus-specific IgGl but only Ig,G3 or IgG4 anti-CMV. The reason for the unresponsiveness of the IgGl subclasses in certain persons is unknown. IgG2 is the second IgG subclass in total quantity. It is commonly produced with polysac:charides and in bacterial disease (Sieber et al., 1980). It was found in a low frequency and low amounts in the herpes virus antibody populations. This may be due to the prolific occurrence of proteins or glycoproteins in most viruses. It is also conceivable, however, that some viral antigen components, poorly exposed in our ELISA assay, under other circumstances may give an IgG2 response. IgG3 differs functionally from the other subclasses, i.e. it does not adhere to Staphylococcus A, it does not react with the rheumatoid factor, it has the most rapid turnover, and it is the most active subclass in activating the Cl component of complement (Ishizaka et al., 1967). The large hinge region that is typical of IgG3 (Michaelsen et al., 1977) may be important for accommodating antigens with determinants far apart. Since IgG3 appears to have a complement-dependent neutralizing activity (own unpublished findings), it is possible that prompt functional neutralization capacity favours its production in acute infections. Previous studies have shown that IgG3 also is frequent after VZV recurrences (Sundqvist et al., 1984). Virus-specific IgG3 has also been shown to appear in the acute phase of rubella (Linde, 1985). The low frequency of virus-specific IgM and the very low IgM titers in sera with high viral IgG suggested that no, or very few persons had recent clinical infections. According to the high frequency and levels of IgG3 found here, it is thus possible

that both CMV and HSV are subclinically

reactivated

more often than VZV.

In several cases we clearly detected antiviral IgG in sera considered negative or with very low total antiviral IgG. In 19% of so-called seronegative sera, HSV subclass antibodies were detected, mainly of the IgGl subclass. Since we evaluated subclass antibodies at a dilution of 1 : 100 and also had a stringent background cut-off (X + 3 SD for negative sera), this is more likely to be an under- than an overestimation of the frequency of persons who have experienced a herpes infection, but who would be considered seronegative by conventional methods. This can partly be explained by the high sensitivity of the subclass assays. In sera with CMV IgG4 only, we detected both IgG4 reactions to normal cells and in 9 out of 23 sera specific CMV inclusion staining. This phenomenon will have to be studied further. These persons should not be used as donors of seronegative blood products without further control, since several may have had the disease in question (see below).

214

Monoclonal

antibody

IgG4 antibodies. related.

ELISA

Our analyses

However,

indicated

the only satisfactory

that IgG4 production

it has also been suggested

may give rise to antibodies not be detected

is probably

that prolonged

of the IgG4 subclass

by complement

fixation,

way of detecting may be genetically

exposure

(Aalberse

to an antigen

et al., 1983). IgG4 would

since this subclass

does not bind comple-

ment. High total IgG4 has been suggested to be related to allergic disorders (Gwynn et al., 1978) or acquired respiratory disease (Van Nieuwkoop et al., 1982). Our previous studies suggest that patients with herpes infection and with high antiviral IgG4 also have higher total IgG4 levels (Linde et al., 1983). The possibility that the specific IgC4 rise is due to a high total IgG4 is of interest, since allergic diseases seem to have a genetic component, though not much is known about this. Crohn’s disease, too, has been ascribed to congenital factors. In this disease we found a low frequency of CMV IgG4, normal total IgG4 and normal frequencies of other subclasses. Total CMV antibody titers to CMV in Crohn’s disease were previously shown to be similar to those in healthy persons (Bernades et al., 1980). We found no apparent defects in subclass antibody patterns in blood diseases, such as leukemias or myelomas of various monoclonal origins. REFERENCES Aalbcrse. Alvine,

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