zoster immunoglobulin preparations and the corresponding donor plasma

Jownal

of Biological Standardization

(1988) 16, 157-164

IgG subclass distribution among antibodies to varicella-zoster virus in human varicella/zoster immunoglobulin preparations and the corresponding donor plasma*

Kirsten M$yner”f and T. E. Micbaelsen$

IgG subclasses to varicella-zoster virus (VZV) were detected in plasma from different sources used for the production of varicella!zoster immunoglobulin (VZIG). IgG 1 and IgG3 were the principal virus antibodies in plasma from healthy donors as well as from convalescents after primary and reactivated disease. Anti-VZV IgG3 antibodies were predominant among varicella convalescents while IgGl antibodies dominated among zoster convalescents. IgG4 antibodies were present in roster convalescents and healthy donors but were rarely detected in varicella convalescents. Antiviral IgG2 antibodies were found only in a few cases. Studies of plasma samples collected from one varicella convalescent during a period of seven months following an outbreak of disease, demonstrated a rapid fall in antiviral IgG 1 and IgG3, while IgG4 increased to reach a maximum six months after the onset of symptoms. The relative distribution of VZV-specific subclasses in a plasma pool was conserved during a fractionation procedure combining polyethyleneglycol 6000 precipitation with ion exchange chromatography, thus suggesting that the protective efficacy is maintained in the resulting immunoglobin preparations.

INTRODUCTION The administration of varicella-toster immunoglobulin (WIG) is recommended for susceptible immunocompromised children when they are exposed to varicella-zoster * Received for publication 28 November 1987. t Unit for Vaccine Research and Supply and $ Dpt. of Immunology, Health, Geitmyrsvn. 75, 0462 Oslo 4, Norway. 0092-1157/88/020157+08

$03.00/O

@ 1988 The International

National

Association

Institute

of Biological

of Public

Srandardizatmn 157

K. MBYNER

AND

T. E. MICHAELSEN

virus (VZV). The WIG-preparations contain IgG antibodies which are purihed rrom human plasma with elevated serum levels of VZV-specific antibodies as determined by complement fixation tests. The plasma donors may be selected from convalescents with primary or reactivated VZV infections as well as from healthy individuals without any known recent VZV disease. Previous investigations suggest that the subclass distribution”* and epitope specificities’ of the VZV-specific antibodies may vary among individuals convalescing from primary disease (varicella) and disease caused by reactivation of VZV (zoster). However, it is not known which antibody qualities are relevant to the protective efficacy of a VZIG preparation. IgG subclasses differ with respect to biological properties and this suggests that the various subclasses may play different roles in the protection againsr disease.” The use of different sources of plasma donors, as well as different methods of plasma fractionation, may influence the virus-specific IgG subclass distribution in the resulting VZIG preparations. In the present study, an ELISA technique with polyclonal IgG subclass-specific antibodies was used to study the presence of WV-specific IgG subclasses in plasma from various groups of donors and in VZIG preparations produced by a method combining polyethyleneglycol (PEG) 6000 precipitation with ion exchange chromatography. MATERIALS

AND

METHODS

Plasma

Plasma was obtained from individuals with elevated serum levels of complementfixing antibodies to VZV. The plasma donors were selected from healthy blood donors and from individuals convalescing from varicella or zoster disease; the convalescent sera were collected 3-5 weeks after onset of symptoms. Plasma from different types of donor were pooled to constitute the starting material for each plasma fractionation. Each pool on average comprised plasma from 3 5 donors. Plasma samples were also collected from one varicella convalescent approximately once a month during a period of seven months following onset of disease. VZIG

The human VZIG preparations were produced at the National Institute of Public Health, Oslo, using a plasma fractionation method combining PEG 6000 precipitation with ion exchange chromatography, essentially as described by Falksveden & Lundblad. s In brief, the method included two precipitations with 11% PEG, ion exchange chromatography with CM C-50 cation exchanger and DEAE A-50 anion exchanger, and precipitation with 25% ethanol. Each VZIG preparation was made from the plasma from at least 100 individuals and the final IgG concentration was about 16%. Antigem

VZV antigen was prepared from the supernatant of a sonicate of VZV-infected cultures of human foetal diploid fibroblasts by the method described by Shehab & Brunell.’ Control antigen was prepared from an uninfected culture treated the same way as described for VZV antigen. Antisera

Rabbit antisera specific for human IgG subclasses were prepared by tolerance induction for IgG subclass cross-reacting epitopes at birth, followed by immunization at eight weeks of age as previously described by Michaelsen & Haug.’ Sheep antirabbit 158

IgG SUBCLASSES

TO VARICELLA-ZOSTER

VIRUS

IgG was prepared by repeated injections of rabbit IgG ( 1OOpg) mixed with Freunds Complete Ad juvant . The rabbit-specific antibodies were isolated by passing the antiserum through individual columns containing IgG from different species, the rabbit IgG containing column being the last in the chain. The rabbit IgG column was separately eluted with 4 M guanidine in PBS pH 7.3, and the eluted antibodies subsequently renatured by passing through a column of Sephadex G-25 in PBS pH 7.1. Conjugate The sheep antirabbit antibodies were biotinylated by Biotin-X-NHS (Calbiochem) as described by Wofsy.8 Streptavidin alkaline phosphatase was prepared by conjugating O-5 mg of alkaline phosphatase (ALP) (Sigma, type VII) to 0.25 mg of streptavidin (Amersham) by the glutaraldehyde method described by Engvall and Perlmann.” ELlSA for VZV-specific IgG rubclasses

Individual wells of Immulon microelisa-plates M 129 B (Dynatech, Germany) were coated by adding VZV antigen or control antigen. The antigens were diluted 1:30() in 0.05 M carbonate-bicarbonate buffer, pH 9.6. The optimal dilution of antigen was determined by checkerboard titrations using a panel of positive and negative sera. The coating was performed at 4°C overnight, and the plates were then stored at 4°C. When performing the assay, 100 ,ul of reagent solution were added to the wells. All incubations were performed in a moist chamber for 120 min at 37”C, unless stated otherwise. Between each step, the microplate was washed four times with 0.01 h% phosphate buffered saline, pH 7.4, containing 0.05% Tween 20 and O.O2c/i sodiumazide (PBS-T). Plasma samples, plasma fractions, immunoglobulins, antisera and conjugate were diluted in PBS-T containing 0.05 M NaCl and 0.5% bovine serum albumin. The enzyme substrate was disodiump-nitrophenylphosphate (NPP) (Sigma, USA), diluted to 1 mg/ml in 10% diethanolamine buffer, pH 9.8, containing 10 mM MgCl. When tested for the presence of IgG2 and IgG4, the plasma samples and plasma pools were diluted 1:20, plasma fractions 1:lO and immunoglobulin preparations 1: 100. When tested for IgGl and IgG3, the samples were diluted ten times further. The samples to be tested were added to the antigen-coated wells. After being washed the individual wells were incubated with rabbit antiserum against each of the human IgG subclasses in the following dilutions: anti-IgGl 1: 1000, anti-IgG2 1: 1000, anti-IgG3 1:20 000 and anti-IgG4 1:5000. These dilutions were chosen to give the same OD-reading against wells coated with pools of myeloma proteins of a given subclass. The microplate was then incubated with biotinylated sheep antirabbit IgG, diluted 1:4000. The next incubation step contained streptavidin-ALP diluted 1:4000. NPP was then added and the microtiter plate was left at room temperature for one hour. The reaction was stopped by adding 50 ~1 of 1 N NaOH and the absorbance at 4 10 nm was recorded with an ELISA microplate reader MR 580 (Dynatech, Germany). The results were expressed as ODdlo nm (the average OD-value obtained in two VZV antigen-coated wells minus the value obtained in one well coated with control antigen). The control wells, which contained PBS, showed no reaction. IgG subclass specificity in the system was confirmed by inhibition experiments in which heterologous myeloma proteins showed less than 0.1% inhibition capacity compared with homologous myeloma proteins. Complement fixation

test (CFT)

CFTs were carried out essentially as described by Busby. “’ 159

K. MQ)YNER

AND

T. E. MICHAELSEN

RESULTS Subclass-spectjic anti-VZV

IgG dimibution

among plasma donors

In plasma samples collected from ten varicella convalescents during the first 3-5 weeks following onset of disease, IgG3 was found to be the predominant antiviral IgG subclass. Specific IgG 1 antibodies were also present, but at somewhat lower levels than IgG3. IgG4 antibodies were detected only in small amounts in a few of the plasma samples, and IgG2 antibodies were present only at border-line levels in some cases(Fig. la. Testing of plasma samples collected from one individual during the first seven months following the onset of varicella demonstrated time-dependent changes in the pattern of antiviral IgG subclass distribution (Fig. 2). The levels of virus-specific IgG 1 and IgG3 decreased rapidly during this period of time, with the most rapid decrease of IgG3 between the 2nd and 4th month. The serum level of antiviral IgG4 increased during the first months to reach a maximum approximately six months after onset of disease. Virus reactive IgG2 antibodies were detected only in small amounts in the plasma samples collected during the first five months. The complement fixation titres

IaGl

I.0

IgG2

IgG3

1964

(b)

t

s

0.0 _

0.0

IgGl

IgGl

IgG2

IgG2

IgG;

lgG3

ldl-mJl IgG4

IgG4

Fig. 1. Distribution of VZV-specific IgG subclasses among varicella convalescents (a), zoster convalescents (b), and normal blood donors wirhout any known recent disease(c). Plasma samples from ten individuals in each group were tested and the results are denoted in the same order of individuals for all subclasses. The samples were diluted 1:20 in tests for IgG2 and IgG4 and 1:200 in tests for IgGl and IgG3.

160

IgG SUBCLASSES

lgG3 e \ \

+ 0

VIRUS

\

.W2 5

TO VARICELLA-ZOSTER

IO

1*-*-c--*-*15 20

Weeks after

outbreak

25

30

35

of vorlcello

Fig. 2 VZV-specific IgG subclasses in plasma samples collected from a varicella convalescent in the seven months following an outbreak of the disease. The plasma samples were diluted I:20 in tests for IgG(+), IgG4(W and I:200 in tests for IgGl(A), IgG3(.).

obtained in these plasma samples showed a fall during the first months after infection parallel to that seen for IgG 1 and IgG3. In zoster convalescents, the specific IgG 1 levels were considerably increased compared with varicella convalescents (Fig. lb). In the case of antiviral IgG3, great individual variations were observed among the zoster convalescents. IgG4 antibodies were present in most of these samples, but IgG2 antibodies were only rarely found. Among healthy blood donors without any known recent VZV infection or reactivation, variable IgG subclass patterns of the antibodies were seen (Fig. lc). However, plasma samples collected from one normal donor during a period of 14 months, demonstrated that the relative distribution of antiviral subclasses was constant during that period.

o-0’

’ancaet 1.’ ’ ! 1.’ IgGl

-.abcdef IgG2

a bcdef

IgG3

abcdef IQG4

Fig. 3. VZV IgG subclasses in six different plasma pools (a, b, c, d, e, f) used for the production of varicella roster immunoglobulin. The plasma samples were diluted 1:20 in tests for IgG2, IgG4 and I:200 in tests for IgGl, IgG3.

161

K. M@YNER

AND

T. E. MICHAELSEN

Antivirul IgG &class preparations

dirtridution

among plasma pooh, plasma fktions

and VZIG

Six different plasma pools constituting the starting material of VZIG preparations were tested with repect to VZV IgG subclasses. The subclass patterns were similar in all plasma pools tested with IgG1 being the predominant subclass (Fig. 3). Samples collected from the JgG-fraction of one plasma pool at different steps during the fractionation procedure demonstrated that the subclass pattern in the starting material was retained during the procedure (Fig. 4). The relative distribution of antiviral IgG subclasses in three different VZIG-preparations was also comparable with the distribution observed in the starting materials.

0.0

1234

1234

I234

A

0

C

I234 D

I234

1234

E

F

Fig. 4. VZV IgG subclass distribution during the purification procedure. Samples were collected from the starting material (A), the extract from first precipitation with 11% PEG (B), eluate from CM C-50 cation exchange column (C), extract from second precipitation with 11% PEG (D), unbound material from DEAE A-50 anion (E), and extract from precipitation with 25% ethanol (F). The various subclasses are denoted 1, 2, 3 and 4, respectively. The material was diluted 1: 10 in tests for IgG2, IgG4, and 1: 100 in tests for IgGl, IgG3.

DISCUSSION The variable patterns of VZV-specific IgG observed among the three different groups of plasma donors suggest that the choice of donors may have consequences for the relative subclass distribution of these antibodies in the resulting immunoglobulin preparations. Among individuals convalescing from varicella or zoster, the plasma for immunoglobulin production is collected 3-5 weeks after the onset of symptoms, as the anti-VZV complement fixing antibodies are usually most elevated at this time. Studies of samples collected from one individual during the seven months after a varicella injection confirmed that plasma collected during the first 3-5 weeks contained the highest levels of VZV-specific IgGl and IgG3 antibodies, whereafter they declined. The level of antiviral IgG4 antibodies, however, did not reach a maximum until six months after the onset of symptoms, suggesting that these antibodies need a more prolonged stimulation time as reported by Aalberse eta/. , ” or are directed against viral epitopes formed late in the infection. The predominance of antiviral IgG3 among varicella convalescents suggests that IgG3 is the main subclass stimulated by primary VZV infection. This is in accordance with the study by Beck12 suggesting that IgG3 antibodies may play a major role in the protection against virus infections. However, VZV-specific IgG 1 antibodies were also present in all serum samples collected from the varicella convalescents and may also be of importance for the protection against 162

IgG SUBCLASSES

TO VARICELLA-ZOSTER

VIRUS

varicella disease. The relative increase in virus-specific IgGl antibodies observed among taster convalescents compared with varicella convalescents, is consistent with the results obtained with monoclonal subclass antibodies in the study by Sundqvist et al. 2 and may suggest that the synthesis of specific IgGl antibodies is selectively stimulated by virus reactivation. The relative increase in specific IgG4 antibodies among zoster convalescents is consistent with the results from the varicella convalescents, suggesting that the IgG4 response is delayed compared with the other subclasses. The present study also revealed greater individual variations with regard to specific IgG3 antibodies in roster convalescents than previously observed by Sundqvist et al.* These discrepancies may be explained by differences in specificity between the polyclonal and monoclonal antibodies used in the two investigations or arbitrary differences among plasma donors. As Weigle & Grose3 have reported, roster convalescents seem to have serum antibodies directed against a much broader spectrum of antigens that the varicella convalescents. The elevated level of VZV-specific IgGl antibodies observed among zoster convalescents in the present study may comprise antibodies directed against other virul antigens than the antibodies stimulated by primary infection, or they may be caused by a general switch in the IgG response from IgG3 to IgGl which is located downstream in the IgG locus. I3 Studies

of the subclass distribution

among

plasma

pools suggest

that individual

differences between plasma units from different types of donors were equalized when the plasma units were pooled. The IgG subclass distribution at various steps during the fractionation

process demonstrated

that the procedure

combining

PEG-precipitation

with ion exchange chromatography was a mild process with respect to preserving all of the four IgG subclasses, giving a product with a IgG subclass distribution corresponding to the starting material. Normal

plasma

that showed

high

complement

consuming

activities

had relative

subclass distributions compatible with a compromise between varicella and zoster convalescents, thus justifying the use of such donors as the source for immunoglobulin production. Acknowledgements

The authors thank Dr. D. Wiger for the preparation of VZV antigen. The skilful technical assistance of Mr. S. R. Andersen and Ms. T. Lonnes Urdal is gratefully acknowledged.

REFERENCES 1. Leonard LL, Schmidt NJ, Lennette EH. Demonstration of viral antibody activity in two immunoglobulin G subclasses in patients with varicella-zoster virus infection. J Immunol 1970; 101: 23-27. 2. Sundqvist VA, Linde A, Wahren B. Virus-specific immunoglobulin G subclasses in herpes simplex and varicella-zoster virus infections. J Clin Microbial 1984; 20: 94-98. 3. Weigle KA, Grose C. Molecular dissection of the humoral immune response to individual varicella-zoster viral proteins during chickenpox, quiescence, reinfection, and reactivation. J Infect Dis 1984; 149: 741-749. 4. Shakib F. Basic and clinical aspects of IgG subclasses. Monographs in Allergy 1986; 19. 5. Falksveden LG, Lundblad G. Ion exchange and polyethylene glycol precipitation of Immunoglubulin G. In: Curling JM, ed. Methods of plasma protein fractionation, London New York Toronto Sydney San Fransisco: Academic Press. 1980; 93-105. 163

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AND T. E. MICHAELSEN

6. Shehab Z, Brunell PA. Enzyme-linked immunosorbent assay for susceptibility to varicella. J Infect 1983; 148: 472476. 7. Michaelsen TE, Haug E. Human IgG subclass-specific rabbit antisera suitable for immunoprecipitation in gel, ELISA and multilayer haemagglutination techniques. J. Immunol Meth 1985; 84: 203-220. 8. Wofsy L. Methods and applications of hapten-sandwich labeling. Meth in Enzymol 198 3; 92: 472-749. 9. Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J Immunol 1972; 109: 129-135. 10. Busby DWG. Direct complement fixation test. In: Busby DWG, ed. Virological technique. London: J. & A. Churchill Ltd. 1964; 136-143. 11. Aalberse RC, van der Gaag R, van Leeuwen J. Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4-restricted response. J Immunol 1983; 130: 722-726. 12. Beck OE. Distribution of virus antibody activity among human IgG subclasses. Clin Immunol 1981; 43: 626-632. 13. Flanagan JG, Rabbits TH. Arrangement of human immunoglobulin heavy chain constant region genes implies evolutionary duplication of a segment containing, y, E and &genes. Nature 1982; 300: 709-713.

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