Virus Research, 23 (1992) l-12 0 1992 Elsevier Science Publishers

VIRUS

B.V. All rights reserved

016%1702/92/$05.00

00747

Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract Mary B. Mazanec 1,2,Michael E. Lamm ‘, Deborah Lyn 3, Allen Portner and John G. Nedrud ’

’

Departments of’ Pathology and 2 Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106, U.S.A. and 3 Department of Virology and Molecular Biology, St. Jude Childrens'Research Hospital, Memphis, TN 38101, U.S.A. (Received

17 September

1991; revision

received

and accepted

26 November

1991)

Summary

The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 ~1 ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the

Correspondence to: M.B. Mazanec, Pulmonary and Critical Care Division, Department University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.

of Medicine,

2

rationale for active mucosal immunization response. Parainfluenza

protocols designed to generate an IgA

virus; Mucosal immunity; IgA; Monoclonal antibody

Introduction

Respiratory viruses replicate and induce inflammation, resulting in clinical signs and symptoms at their site of entry, the respiratory mucosa. The mucosal immune system with its predominant effector, secretory IgA, forms the first line of immunologic defense at the interface between the pulmonary epithelial surface of the airways and the external environment. Thus, resistance to respiratory viral infections correlates with the presence of viral specific antibodies in local mucosal secretions (Clements et al., 1983; Liew et al., 1984; Mills et al., 1971; Smith et al., 1966). Secretory IgA constitutes the predominant antibody in the upper airway where most respiratory viruses invade the body. In the presence of a sufficient secretory IgA response, viral infection is confined to the upper pharynx, preventing virus spread into the lower respiratory tract and thus abrogating the complications associated with viral infection of the lower airway (Nedrud et al., 1987). The vast majority of the secretory IgA is produced locally by IgA committed plasma cells in the lamina propria underlying the respiratory epithelium (McDermott et al., 1982; Mazanec et al., 1989; Mestecky, 1987; Scicchitano et al., 1986). Once secreted, IgA oligomers diffuse to the basolateral surface of the overlying epithelial cells where they attach to the IgA receptor, secretory component, and are transported across the epithelium into the mucosal secretions (Brandtzaeg, 1985; Crago et al., 1978; Kuhn and Kraehenbuhl, 1979; Kuhn and Kraehenbuhl, 1982). As demonstrated previously, serum antibody does not significantly contribute to luminal antibody content in the absence of inflammation (McDermott et al., 1982; Mazanec et al., 1989; Scicchitano et al., 1986). Accordingly, the most effective way to protect the upper airway from viral invasion appears to be mucosal immunization to stimulate a secretory antibody response (Barber and Small, 1978; Mazanec et al., 1989; Nedrud et al., 1987). In addition, in the presence of an immature immune system, an immunocompromising illness or acute infection, passive administration of antibody could offer a feasible treatment modality. The different classes and subclasses of antibodies differ with respect to their inflammatory properties. IgA in general is less phlogistic than IgG, e.g., it is a weak activator of complement (Iida et al., 1976; Johnson et al., 1986; Pfaffenbach et al., 1982). Whether IgA’s lower inflammatory potential enhances or detracts from its ability to protect the airway against virus infection has not been determined. In previous passive immunization studies IgA protected the airway against subsequent viral challenge in rodent Sendai and influenza virus models (Mazanec et al., 1987; Renegar and Small, 1991a,b). However, the efficacy of IgA’s protection compared with IgG’s has not been investigated. The purpose of the present

3

study was to compare directly the protection afforded against subsequent viral challenge by passive immunization with either IgA or IgGl, IgG2a, or lgG2b anti-viral MAb. To investigate the relative efficacy of passive intranasal immunization with various isotypes of antibody, we utilized Sendai virus, a prototypical parainfluenza type I virus, which is a natural respiratory pathogen of mice that is related to the parainfluenza viruses that infect humans. The pathogenesis of infection, and the biochemistry and molecular biology of Sendai virus are well characterized, and the infection produced is typical of many moderate to severe viral respiratory infections of humans (Robinson et al., 1968; Ward, 1974; Ward et al., 1976). Sendai virus initially invades the upper airway, and if viral replication remains unchecked, infection spreads to the trachea and main bronchi. As with human viruses, disease can progress to bronchial pneumonia with secondary bacterial infection and death (Jakab, 1981; Robinson et al., 1968; Ward, 1974; Ward et al., 1976). Thus, murine Sendai virus infection is a good model to study passive immunization against respiratory viral infections. For these studies a panel of anti-Sendai virus MAb was generated. The advantage in using MAb rather than conventional antisera lies in the inherent homogeneity of MAb preparations and the ability to control for the antigenic specificity of the administered antibody. Use of MAb facilitates examination of the effect of antibody isotype on virus neutralization devoid of other immunologic elements. This is in marked contrast to passive immunization studies employing polyclonal sera which are inherently heterogeneous and from which it is difficult to obtain sufficiently pure fractions of individual antibody isotypes. The information derived from studies with MAb has the potential to provide insights that could be useful in devising strategies for prevention of human diseases.

Materials

and Methods

Animals

BALB/c mice obtained from The Jackson Laboratory, Bar Harbor, ME and Charles River VAF Facility, Portage, MI, both of which maintain animal colonies free from Sendai virus and other pathogenic microorganisms, were used both for production of MAb and passive immunization studies. Mice were maintained in microisolation cages in a laminar flow hood until used. Virus

Sendai virus strain 52 was obtained from the American Type Culture Collection (catalogue No. VR 105) Rockville, MD, through the Research Reference Reagents Branch of the National Institute of Allergy and Infectious Disease. A seed stock prepared in specific pathogen-free lo-day-old embryonated chicken eggs (SPAFAS, Norwich, CT) was used to prepare virus for immunization for MAb production and

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TABLE

1

Isotypes

of MAb against

the Sendai

virus HN glycoprotein

MAb

Isotype

ELISA titer

Neutralization titer

31 380 a,h 42/2 b

IgA IgA IgGl

10 7 10 6 10 65

10 4 10 3.5 10 35

IgG2a IgG2a IgG2b

10 h 10 ’ IO”

10 3 10 4.1 10 3.5

20 41/2 39/2

a ’

Directed ’ ELISA

against overlapping F’) or the same Ch) epitope on the HN glycoprotein and neutralization titers are rounded to the nearest half log.

(Lyn et al., 1991).

for airway challenge. Eggs were inoculated with 10’ plaque forming units (pfu) of virus, and allantoic fluid was harvested 2 to 3 days later. Virus was purified by differential centrifugation and stored at -70“ (Nedrud et al., 1987). M4b production and characterization IgA MAb were produced via a mucosal immunization protocol (Mazanec et al., 1987). IgG MAb were obtained after a conventional immunization procedure (Lyn et al., 1991). The isotypes of MAb were determined by ELISA with isotype specific reagents, with confirmation by Ouchterlony immunoprecipitation (Table 1). Epitope mapping was performed as described (Lyn et al., 1991). Gel filtration was used to separate IgA monomers from oligomers (Mazanec et al., 1989). Anti-Sendai virus titers of ascites containing the respective MAb were determined by a quantitative ELISA (Liang et al., 1988). Neutralization titers of ascites were measured by a plaque reduction assay (Mazanec et al., 19871. ELISA Anti-viral antibody titers were determined by ELISA (Nedrud et al., 1987; Portner, 1981). Briefly, Sendai virus was coated on polyvinylchloride plates overnight at 4°C pH 9.6, followed by blocking with 1% bovine serum albumin in PBS. Ascites fluid was serially diluted in 0.5 M NaCI, 0.01 M-phosphate, 1% bovine serum albumin, pH 7.2, and incubated on virus coated plates for 1 h. After washing, alkaline phosphatase-conjugated anti-mouse IgA or IgG was added for an additional hour. After washing, 1 mg/ml p-nitrophenol phosphate substrate solution was added and the OD,iO determined 60 min later with an automated microplate reader. Endpoints were calculated as the highest sample dilution yielding an OD of 0.05 above the conjugate control. Passive immunization In preliminary experiments, optimal timing of doses of MAb was determined. Antibody could be detected in bronchoalveolar lavage immediately after intranasal

5

administration under ether, but levels declined to nearly undetectable amounts 4-6 h post administration (unpublished observation). Therefore, in contrast to actively immunized animals where relatively steady state antibody levels persist, passively, topically immunized animals require repeated dosing in order to maintain significant antibody levels in the airway. For the final protocol, 50 ~1 dilutions of ascites containing equivalent anti-Sendai ELISA titers were administered intranasally to lightly etherized Sendai-free BALB/c mice 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Unless otherwise indicated, control animals were treated in the same manner with PBS. Three days after challenge, the animals were sacrificed by carbon dioxide asphyxiation, and virus titers of 10% (weight/volume in tissue culture media) lung homogenates were determined by plaque assay. Plaque assay

Virus in lung homogenates was assayed by serially diluting samples into Hanks’ balanced salt solution and plating 0.2 ml aliquots into 35mm tissue culture wells containing Hanks’ balanced salt solution-washed confluent monolayers of LLCMK, monkey kidney cells. After 60-90 min at 37°C the inoculum was removed and the cells were overlaid with 2 ml of Medium 199 containing no serum, 0.4% Bact-Agar (Difco Laboratories, Detroit, MI), and 2.5 pg/ml trypsin (GIBCO, Grand Island, NY). After 3 days, the agar was removed by vigorous shaking and plaques were visualized by hemadsorption with a 0.1% suspension of freshly washed guinea pig red blood cells. Samples were run in duplicate. Statistical analysis

Comparisons among groups in each experiment were evaluated by one-way analysis of variance with Fisher’s protected t-test. Calculations were done on an Apple Macintosh SE-30 computer (Cupertino, CA) with Statview 512 + software (Brain Power, Inc., Calabasas, CA).

Results

In the first experiment, the protection afforded by anti-Sendai virus IgA and IgG MAb was compared. Ascites containing MOPC 315, an irrelevant IgA MAb, and PBS served as controls. Mice were passively intranasally immunized with 50 ~1 of ascites containing equivalent ELISA titers and neutralization titers of anti-Sendai virus MAb IgA 37 or IgG2a 20, 1 h before and 4 and 24 h following intranasal challenge with Sendai virus. Both these MAbs are specific for the Sendai HN protein, a surface glycoprotein of the virus responsible for attachment to susceptible cells (Merz et al., 1981; Portner, 1981). As shown in Fig. 1, animals receiving either of these virus specific MAb had a 10,000 to 100,000 fold reduction in lung virus titer 3 days after challenge as compared to controls (p = 0.0001). Animals

n IgAcoNTRoc PBS

1

0

IgA 37 (HN)

0

IgG 20 (HN)

2

EXPERIMENT

NUMBER

Fig. 1. Passive protection by MAb. Mice (7/group in Experiment 1 and S/group in Experiment 2) were passively immunized intranasally with 50 ~1 of ascites fluid containing either MAb IgA 37 or MAb IgG2a 20 anti-Sendai virus antibody 1 h before and 4 and 24 h following intranasal challenge with 10” pfu Sendai virus (Experiment 1) or 2.5 x 10’ pfu Sendai virus (Experiment 2). Control animals received either PBS or MOPC 315, an irrelevant IgA MAb. The concentrations of antibodies were normalized for ELISA titer. * All animals had virus titers equal to or less than 10 pfu, the limit of detection in this assay.

treated with IgG MAb in one experiment had slightly lower lung virus titers compared to those inoculated with IgA MAb (p = 0.03). In a second experiment with these two MAbs, mice were challenged with a higher dose of virus. Again, mice immunized with either anti-Sendai virus MAb IgA 37 or IgG2a 20 had significantly reduced virus titers in their lung homogenates when compared to control animals (p = 0.007 for IgA; p = 0.015 for IgG). In this experiment, both viral specific MAb provided equivalent protection. Since it is conceivable that a difference between the ability of different antibody isotypes to limit viral infection may be negated at high antibody concentrations, an experiment was designed to examine the neutralizing potential of IgA versus IgG over a range of concentrations (Fig. 2). Animals were intranasally passively

r

i 1 : 10 YONOCLONAL

1 : 100 ANTIBODY

il 1

0

IgA37

n

IgG20

: 1000

DILUTION

Fig. 2. Effect of anti-HN MAb concentration on passive protection. Mice (6/group) were intranasally immunized with 50 ~1 of 1 : 10, 1 : 100 or 1 : 1000 dilutions of ascites fluid containing anti-HN MAb IgA 37 or MAb IgG2a 20.

CoNlRx

1:10

MONOCLONAL

1:100 ANTIBODY

0

IgA380

n

IgG41/2

1:1000 DILUTION

Fig. 3. Passive immunization with MAb against overlapping HN epitopes. Ascites containing equivalent ELBA titers of MAb IgA 380 and IgG2a 41/2, both of which are directed against overlapping epitopes on the HN viral glycoprotein, were used to immunize animals (6/group) intranasally.

immunized with 50 ~1 of a 1: 10, 1: 100 or 1: 1000 dilution of ascites containing IgA MAb 37 or IgG2a MAb 20, both of which are directed against the HN viral glycoprotein. Passive immunization with either the IgA or the IgG MAb significantly reduced lung virus titers 1000 to l,OOO,OOO fold compared to control animals at the 1: 10 dilution and 100 to 10,000 fold at the 1: 100 dilution (p < 0.001). IgG MAb afforded greater anti-viral protection than IgA at both the 1: 10 and 1: 100 dilutions (p < 0.01). Although the results of this experiment suggest that the neutralization potential of IgG may be greater than that of IgA at high antibody concentrations, this result was not uniformly observed (Fig. 3). In the next experiment, an IgA (380) and an IgG2a (41/2) MAb directed against overlapping epitopes on the HN viral glycoprotein were compared. Epitope mapping was performed as described (Lyn et al., 1991). Animals were intranasally passively immunized 1 h before and 4 and 24 h following intranasal inoculation of 2.5 x lo4 pfu Sendai virus. Animals receiving IgA MAb at 1: 10 or 1: 100 or IgG MAb at all dilutions had 10 to 1000 fold reductions in lung virus titers compared to control animals (p I 0.051 (Fig. 31. There was no significant difference in protection afforded by IgA versus IgG MAb, as compared to the previous experiment where IgG appeared to neutralize the virus more effectively. Since the antibodies used in the Fig. 2 experiment were against different epitopes while MAb in the Fig. 3 experiment reacted with overlapping epitopes, the results suggest that certain epitopes on viral proteins may be more critical to viral infectivity and that antibodies reactive with these sites may be more effective in preventing viral infection. The various subclasses of IgG differ in their physicochemical and functional properties (Dangl et al., 1988; Neuberger and Rajewsky, 1981). In the previous experiments, the IgG MAb tested were of the IgG2a subclass and the relative protection afforded by passive intranasal immunization with IgA MAb was comparable to that afforded by the IgG2a MAb. In the next experiment, the relative efficacy of passive intranasal immunization with IgA was compared to that of two other subclasses of IgG. All anti-viral MAb were directed against the same epitope

n I!#=0 0 IgGZb39/2 H

CoNmx

1:10

MONOCLONAL

1:100 ANTIBODY

IgGl

42/Z

1:1000 DILUTION

Fig. 4. Passive immunization with IgA vs IgG subclasses against the same HN epitope. Mice (g/group) were intranasally immunized with MAb IgA 380, IgG2b 39/2 or IgGl 42/2. All MAb were specific for the same epitope on the HN virus glycoprotein.

on the HN viral glycoprotein. All MAb were normalized to equivalent ELISA titer, which also gave equal neutralization titer. Mice were intranasally immunized with IgA MAb 380, IgG2b MAb 39/2 or IgGl MAb 42/2 1 h before and 4 and 24 h after inoculation with 2.5 x lo4 pfu Sendai virus. At all dilutions lung viral titers in animals receiving viral specific MAb were significantly reduced when compared to control animals treated with PBS (p < 0.05) (Fig. 4). Both IgG subclasses evaluated in this experiment provided equivalent anti-viral protection. In addition, in all instances IgA MAb achieved levels of anti-viral prophylaxis similar to either IgGl 42/2 or IgG2b 39/2 except for IgA vs IgG2b at 1: 10 dilution, where p = 0.04. Secretory IgA is an oligomeric, mainly dimeric, antibody molecule whereas serum IgA may include significant amounts of monomer, as in humans (Kilian et al., 1988). The next experiment was accordingly designed to determine whether there is a protective advantage intrinsic to the oligomeric configuration. MAb IgA 380 was separated into monomeric and oligomeric fractions by gel filtration. Mice were passively intranasally immunized with equivalent ELISA titer doses of either monomeric or oligomeric MAb 380. The monomeric and oligomeric fractions were also determined to have equivalent in vitro neutralization activity. Fig. 5 shows that both experimental groups, receiving either IgA monomer or oligomer, demonstrated significant reduction in lung viral titers compared to the PBS control group (p < 0.01). However, there was no significant difference in virus recovery from the lungs of animals treated with IgA monomer or oligomer.

Discussion

The morbidity and mortality associated with respiratory viral infections pose a major public health problem. Respiratory viral infections compound the seriousness of many other medical conditions and in some instances result in chronic disabling sequelae (Sherter and Polnitsky, 1981; Stempel and Boucher, 1981; Welliver, 1983). Thus prevention and/or limitation of respiratory tract viral

NEAT MONOCLONAL

ANTIBODY

1:10 DILUTION

Fig. 5. Passive immunization with IgA monomer vs oligomer. Gel filtration was used to separate MAb IgA 380 into monomeric and oligomeric fractions. Mice (7-X/group) were intranasally immunized with either monomer or oligomer 1 h before and 4 and 24 h after intranasal inoculation with 2.5 X 10” pfu Sendai virus.

infections would significantly reduce viral associated hospitalizations and/or absences from school and work. To this end, extensive research into vaccines for respiratory viruses has been conducted over the last several decades but truly effective vaccines have yet to be developed. Most of this research has concentrated on parenterally delivered vaccines despite repeated demonstration that viral specific antibodies in mucosal secretions often have a higher correlation with resistance to infection of the upper airways than do serum antibodies (Clements et al., 1983; Liew et al., 1984; Mills et al., 1971; Smith et al., 1966). Secretory IgA is the major immunoglobulin in these secretions and as such is the primary immunologic barrier encountered by viruses at the mucosal surface (Bienenstock, 1984; Kilian et al., 1988; Mestecky, 1987). Containment of viruses to the upper airway by secretory IgA should prevent spread of infection to the lower respiratory tract and hence many of the complications resulting from pulmonary infections. Similar to active immunization studies, most previous passive immunization protocols have employed parenteral administration of IgG antibody prior to virus infection. In general, in animal models, passive systemic immunization with IgG can provide significant protection from infection (Orvell and Norrby, 1977; Orvell and Grandien, 1982; Prince et al., 1985a). In a Sendai virus system, intramuscular immunization of mice with rabbit antisera or anti-Sendai IgG MAb attenuated subsequent viral infection as measured by lung viral titers four days after viral challenge (Orvell and Norrby, 1977; Orvell and Grandien, 1982). In another animal model, using respiratory syncytial virus (RSV) in the cotton rat, Prince et al. demonstrated that passive intraperitoneal immunization with convalescent RSV antisera could provide resistance to RSV infection (Prince et al., 1985a). Moreover, resistance was greater in the lungs than in the nose, again suggesting that serum IgG plays a more prominent role in protecting the lower respiratory tract than the upper airway. Further studies by Prince et al. demonstrated that administration of pooled normal human IgG with high anti-RSV titers to cotton rats with established RSV infections significantly hastened viral clearance (Prince et al.,

1985b). Likewise, owl monkeys treated with the same preparations of IgG on the fifth day following intratracheal infection with RSV had reduced virus titers 2-3 days later, in both the lung and nose (Hemming et al., 1985). The importance of local antibody concentration was suggested by the demonstration that the therapeutic effect of topically administered neutralizing human immunoglobulin was 160 times greater than parenteral inoculation as measured by reduction in lung RSV titers in cotton rats (Prince et al., 1987). Although in these studies the immunoglobulin was primarily IgG, the results imply that local anti-viral antibody is particularly important in abrogating viral infection. Recently, Renegar and Small documented that intravenous administration of anti-influenza oligomeric IgA antibody to mice could lead to transport of significant levels of antibody into the airway (Renegar and Small, 1991a,b). In addition, animals passively immunized with anti-influenza IgA in this way were protected against subsequent virus challenge. Unlike oligomeric IgA, intravenous injection of similar virus neutralizing doses of anti-influenza IgG MAb did not protect mice. In addition, IgA-mediated protection could be abrogated by the intranasal administration of antiserum against the LYchain of IgA. As suggested by Renegar and Small’s work, and since a selective, secretory component dependent transport mechanism enables IgA, but not IgG, to reach mucosal secretions, both active and passive immunization protocols for the upper airway should be designed to generate a local IgA response rather than a systemic IgG response. This approach should be more effective in preempting initial viral invasion, at the same time minimizing inflammation and hence disabling sequelae. In the current work, passive topical immunization with IgA was generally as efficacious as with IgG in providing resistance to viral infection. There did not appear to be an inherent viral neutralizing advantage of any isotype of antibody at extremes (low or high) of concentration (Figs. 2-4). Although the various subclasses of IgG differ in their inflammatory and functional properties, IgA achieved equivalent anti-viral prophylaxis regardless of the subclass of IgG with which it was compared. Thus, IgG’s ability to activate complement and promote phagocytosis may not enhance its viral neutralizing capacity compared to that of IgA, at least when pre-existing antibody is present. We previously demonstrated that IgA antibody by itself can prevent respiratory viral infection when administered intranasally to mice (Mazanec et al., 1987). Our current work suggests that IgA is as efficacious as IgG in neutralizing virus in vivo in the murine airway. This result, along with the fact that IgA but not IgG is selectively transported into the airway, provides strong impetus for developing mucosal immunization protocols that generate high IgA titers in respiratory secretions. When IgA’s low phlogistic potential is also considered, vaccines which optimize an IgA response seem highly desirable.

Acknowledgements We thank Steven Emancipator Janet Robinson, David Fletcher,

for help in statistical analysis, Norma Sigmund, Candice Roskoph, and Janet Peterra for technical

11

assistance, and Trudy Sansbury and Cheryl Diane Gilliam for preparation of the manuscript. This work was supported by National Institutes of Health Grants HL-37117, HL-02002, AI-11949, and AI-26449, American Lung Association Research Grant, and a Parker B. Francis Fellowship Award to Mary B. Mazanec.

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