Eur. J. Immunol. 1991. 21: 1337-1344

Passive transfer of respiratory tract IgA against influenza A virus hemagglutinin


Shin-ichi Tamura., Hirono Funato., Yoshihiro Himbayashi., Yujiro Suzuki*, Takashi Nagamhe*, Chikara Aizawa* and Takeshi Kurata.

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules

Department of Pathology, National Institute of Health. and Department of Technology, The Kitasato Institute*, Tokyo

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (HlNl), ArYamagata (HlN1) or AFukuoka (H3N2) virus, together with cholera toxinB subunit as an adjuvant, were examined for protection against PR8 infection; PR8 H A and Amamagata HA immunization conferred complete protection, while A/Fukuoka H A immunization failed to confer protection. In parallel with protection, PR8 HA-, ANamagata HA-, and AFukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount deteced in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from ArYamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A F u kuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza Avirus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.

1 Introduction

Among the Ab to influenzaA viruses, secretory IgA, whose presence in the upper respiratory tract correlated The hemagglutinin (HA) of influenza A virus is a major with resistance to homologous virus infection [7,8], is external glycoprotein, involved in attachment of the virus thought to play an essential role in defence against to cells and in penetration of the virus into the cell during influenza virus. In vitro studies on the ability of IgA to the initial stages of infection [ l].The antigenic properties of prevent adherence of the viruses to various cells also HA in humans have changed radically in recent history suggest that respiratory mucosal IgA serves to inhibit the (antigenic shift), resulting in the current three subtypes H1, initial stages of infection in v i m [9]. Moreover, the facts H2 and H3 [2, 31. These have also changed slightly (anti- that secretory IgA in the nasal wash has a wider spectrum of genic drift), producing variant viruses with minor differ- activity against some A-type influenza viruses than circuences within a subtype. Therefore, the extent of protection lating antibodies, would render the IgA an effective against challenge virus infection by prior antiviral immunity cross-protective agent against some of drift viruses [ 10-121. depends on the closeness of the relation of the antigenic The fact that the cross-reactive secretory IgA can provide properties of HA between the prior virus and the challenge the resistance to drift viruses of the same subtype of virus. Ab directed against HA are associated with resis- influenza A virus [13] also supports that secretory IgA tance to the homologous virus infection in humans and in would play a much more critical role in protecting the host laboratory animals [2-61. Moreover, the anti-HA Ab may against the drift virus infection than serum IgG. These prevent other drift virus infection by cross-reacting with studies suggest that the respiratory tract IgA, directed another virus HA, whereas the Ab may not confer cross- against influenza A virus HA, by itself can provide not only protection against the homologous virus, but also crossprotection against different subtype virus infection. protection against some of heterologous drift viruses, although this possibility has not yet been verified directly. [I 92041 This could be approached by passive transfer experiments, employing IgA Ab purified from respiratory secretions of Correspondence: Shin-ichi Brnura, Department of Pathology, mice immunized with different influenza A virus HA National Institute of Health, 2-10-35 Kamiosaki, Shinagawa-ku, molecules. Tokyo 141,Japan Abbreviations: HA: Hemagglutinin CTB: Cholera toxin B subunit EIDSo: 50% Egg-infecting dose LDm: 50% Lethal dose 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

In previous reports, we demonstrated an intranasal inoculation of inactivated influenza virus vaccines together with the B subunit of cholera toxin (CTB) into BALBk mice Oo14-2980/91/0606-1337$3S O + .25/0


S. Tamura, H. Funato,Y. Hirabayashi et al.

resulted in high levels of nasal IgA Ab in parallel with protection against viral infection [14, 151. The use of this mucosal immunization procedure has enabled us to obtain high levels of respiratory tract IgA Ab against HA molecules and to conduct preliminary passive transfer experiments [16]. The transfer experiment suggested that HAspecific IgA can provide protection against homotypic virus infection. Under these circumstances, the present study was designed to extend the observations quantitatively and to evaluate further the role of respiratory tract IgA anti-HA Ab in the cross-protection against influenza A virus infection. Correlation of the presence of HA-reactive IgA in the upper respiratory tract with protection against viral infection was examined first in different HA-immunized mice. Then, the respiratory tract IgA Ab from the immunized mice were transferred intranasally to naive mice and their ability to protect the host against the challenge infection was examined. Moreover, the duration of the ability was studied.The results demonstrate that protection against homologous virus challenge, and heterologous virus challenge within the same subtype, can be provided in different HA-immunized mice, in parallel with the induction of strain-specific and cross-reacting IgA anti-HA Ab on the upper respiratory tracts. Furthermore, they demonstrate that passive transfer of the strain-specific and the cross-reacting IgA Ab can protect naive mice from challenge with homologous virus and heterologous virus within the same subtype. The level of protection conferred is quantitatively related to the amount of transferred IgA, which persist in the respiratory tract in an active form for at least 3 days.

2 Materials and methods 2.1 Purifkation of HA Influenza A/PR/8/34 (PR8, HlNl), A/Yamagata/120/86 (Amamagata, HlN1) viruses were grown in embryonated eggs, then purified and concentrated. HA was purified according to the procedure of Phelan et al. [17]. Briefly, the purified influenza virions were suspended in 1% NP40 and incubated with agitation for 1 h at room temperature.This preparation was layered onto a continuous 5%-25% sucrose gradient and centrifuged. The appropriate gradient fractions containing HA and neuraminidase were collected and then separated into their constituent glycoproteins using the neuraminidase inhibitor N-(p-aminopheny1)oxamic acid-coupled agarose (Sigma, St. Louis, MO). The biochemical purity of the HA preparations was confirmed by analysis using polyacrylamide gel electrophoresis. The absence of neuraminidase was verified by assay for the enzyme with fetuin [18]. 2.2 Immunization of mice BALB/c female mice, 6 weeks old, were obtained from Japan SLC, Inc. (Hamamatsu-shi, Japan) and used in all experiments. Mice were anesthetized by an i.p. injection of sodium amobarbital(O.25 ml of a solution of 1 pg/ml), then immunized by dropping intranasally 10 pl of PBS containing the required dose of purified H A alone or in combination with CTB (Sigma; [14]).

Eur. J. Immunol. 1991. 21: 1337-1344

2.3 Serum, nasal wash and bronchoalveolar wash specimens Serum specimens were collected from mice by drawing whole blood from the heart. Nasal wash specimens were collected from mice after drawing whole blood by washing the nasal cavity of the head, which was separated from the body with the submaxilla, three times with a total of 1 ml PBS containing 0.1% BSA and 0.1% NaN3 (washing solution). Bronchoalveolar wash specimens were obtained by injecting a total of 2 ml washing solution, twice, into the trachea and lungs, which were separated from the body [19]. Nasal wash and bronchoalveolar wash specimens were sometimes pooled as respiratory tract wash specimens and used as a source of respiratory tract IgA. Blood contamination of respiratory secretions was estimated by counting the red blood cells and comparing them with the counts in serially diluted blood, for five groups of specimens pooled from five mice. The contaminations of nasal wash and bronchoalveolar wash specimens were 0.03 f 0.02% (mean f SD) and 0.59 f 0.17%, respectively. The nasal wash and bronchoalveolar wash specimens were centrifuged to remove cellular debris and stored at -30°C until tested. 2.4 Purification of IgA Ab Respiratory tract wash specimens, collected from immunized mice, were concentrated to one-tenth of their original volume on a Centriprep-30 (Amicon Corp., Lexington, MA). To purify the secretory IgA in the concentrated washings, IgG was first removed by passing the concentrated washings through a protein G-Sepharose column (Mab Trap G ; Pharmacia LKB, Piscataway, NJ) to which most of the secretory IgA did not bind. The effluent was then passed through an affinity column constructed by coupling goat anti-mouse a chain Ab (Zymed Laboratories, San Francisco, CA) to tresyl-activated Sepharose 4B (Pharmacia). Secretory IgA was eluted from the column with 0.1 M ammonium hydroxide and 0.05 M diethylamine, pH 11.5 [9].The eluate was neutralized with HCI, dialyzed against PBS and then concentrated.The IgG contamination of the purified IgA preparation, determined by ELISA (see Sect. 2.5), was < 3 % , while IgA contamination of the purified IgG preparation was < 2%. The purified IgA contained monomeric (about 20%) and polymeric IgA (about SO%)),when fractionated on a Sephacryl S-300 HR column (Pharmacia LKB) in PBS containing 0.01% BSA [161.

2.5 ELISA The concentration of total IgA or IgG was determined by ELISA as described previousy [ 14,201. Briefly, ELISA was performed with EIA plates (Costar, Cambridge, MA) and the following reagents: first, goat anti-mouse IgA (a chain specific) or goat anti-mouse IgG (y chain specific), second, serum or respiratory tract washings, third, goat anti-mouse IgA or IgG conjugated with alkaline phosphatase, and finally p-nitrophenylphosphate. The chromogen produced was measured by absorbance at 410nm in an SJeia Autoreader (model er-8000, Sanko Junyaku Co., Ltd., Tokyo, Japan).The purified mouse myeloma IgA (Miles

Eur. J. Imrnunol. 1991. 21: 1337-1344

Passive transfer of respiratory tract IgA against influenza A virus hemagglutinin

Laboratories, Naperville, 1L) or purified mouse IgG (Cappel Products,West Chester, PA) was used as a standard for 1000 ng/ml of total IgA or IgG. The amount of IgA or IgG Ab against HA molecules purified from PR8, ANamagata or AEukuoka viruses was also measured by ELISA. The procedure was the same as described above, except that the plate was coated with purified HA, and that 100 ng/ml of purified HA-specific IgA or IgG was used as a standard. The standards were prepared by passing the respiratory tract IgA or IgG from the HA-immunized mice through an affinity column constructed by coupling H A molecules to tresyl-activated Sepharose 4B (Pharmacia). The Ab concentration of unknown specimens was determined from the standard regression curve constructed for each assay on the SJeia Autoreader using a programme. 2.6 Virus titrations Mice were infected with a lethal dose of mouse-adapted influenza PR8 virus, containing lo4.’ EIDsdmouse (40x LDso), under the light anesthesia using sodium amobarbital by dropping intranasally 20 pl of virus suspension in the PBScontainingO.l% BSA [14].Three dayslater, lungs were removed and homogenized to yield a 10% suspension in PBS, after which they were centrifuged. Serial dilutions (1 : 10) of the individual lung homogenates were prepared and each dilution was injected into five embryonated eggs. The lung virus titer of each mouse was expressed as EIDSdml [21]. Under these conditions, the significant reduction in the lung virus of the immunized mice (less than 104.9EIDSdml) correlated with long-term survival. 2.7 Statistics Comparisons between experimental groups were evaluated by Student’s t-test. Probability (p) values

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H...
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