Augmentation of Anti-Influenza Virus Hemagglutinin Bifidobacterium ...

CLINICAL

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DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1994, P. 244-246

1071-412X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Vol. 1, No.

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Augmentation of Anti-Influenza Virus Hemagglutinin Antibody Production by Peyer's Patch Cells with Bifidobacterium breve YIT4064 HISAKO YASUI,* NORIKO NAGAOKA, AND KAZUHITO HAYAKAWA Yakult Central Institute for Microbiological Research, Yaho, Kunitachi, Tokyo 186, Japan Received 20 August 1993/Returned for modification 11 October 1993/Accepted 24 November 1993

It has been shown that protection against influenza virus is more closely correlated with antibodies at the mucosal surfaces of the respiratory tract than with serum antibodies (7, 15, 16, 20). Antibody production in the respiratory mucosal tissue, as well as in the intestinal and genital mucosal tissues and mammary, salivary, and lacrymal glands, occurs as a result of dissemination of antigen-sensitized and immunoglobulin A (IgA) isotype-committed cells from gut-associated lymphoid tissues (1, 9). Chen et al. (2-4) have actually demonstrated that commercial influenza vaccine administered by gavage to mice pretreated with cimetidine can produce significant levels of IgA, demonstrable in bronchial washes, and can induce protection against a lethal challenge to the lung that is equal to protection induced by systemically administered vaccine. The precursor cells of the IgA-producing cells are present in Peyer's patches (PP), the intestinal lymphoid tissues in which antigen processing and intestinal cellular events of the immune response in the gut occur (13). It has been shown that a large amount of bifidobacteria inhabit the intestines of healthy breast-fed infants, in contrast to those of formula-fed infants (11); they are thought to play a role in resistance to infection in humans and animals (6, 14, 22); and the number of the organisms decreases in the intestines of young and old adults (12). Hence, wide implantation of bifidobacteria in the intestines is done with yogurt and cultures of bifidobacteria. We have found one strain (Bifidobacterium breve YIT4064) capable of inducing large quantities of IgA among many strains of bifidobacteria isolated from human feces by the murine PP cell culture method. When the organism was administered orally, along with cholera toxin (CT), to mice, the amount of anti-CT IgA antibody in feces and anti-CT IgA antibody production and proliferation in PP cells were significantly greater than after administration of only CT or CT and B. breve Ka-6 (YIT4079), which did not induce IgA in the in vitro PP cell culture. Therefore, in vitro IgA induction with a PP cell culture correlated with in vivo IgA induction in the intestinal lumen after oral administration (24). In the present study, by using the murine PP cell culture method, we investigated whether B. breve YIT4064 can augment anti-influenza virus hemagglutinin (HA) antibody production by PP cells. A licensed influenza HA vaccine (Kaketsuken, Kumamoto, Japan) was used as the antigen and

consisted of formulated influenza virus AlYamagata/32/89 (HIN1), A/Peking/352/89 (H3N2), and B/Bankok/163/90 HAs. B. breve YIT4064, which induces large quantities of IgA, and B. breve YIT4079, which does not induce IgA, were used as adjuvants. These organisms were incubated in reduced GAM broth medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 37°C for 48 h, harvested by centrifugation at 7,000 x g for 10 min at 4°C, washed three times with phosphate-buffered saline, and heated at 100°C for 30 min. PP cells were prepared by treating intestines of BALB/c mice with dispase (23). They were then cultured by the method of Mishell and Dutton (10), with the following modifications. PP cells (5 x 105 or 1 x 106) in 0.2 ml of Eagle's minimum essential medium (Nissui) were cultured in 96-well trays (Nunc, Roskilde, Denmark) with various concentrations of licensed influenza virus HA antigen and B. breve YIT4064 or YIT4079 in a 37°C, 5% CO2 incubator and were fed 0.02 ml of a nutrient mixture per well daily. Supplementation of Eagle's minimum essential medium and the composition of the nutrient mixture were described previously (23). One day after culture initiation, HA antigen was removed by centrifugation. After 6 more days, anti-influenza virus HA antibody levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). HAs (3 ,ug/ml) were coated on the wells of a 96-well ELISA plate (Nunc) by using carbonate buffer (pH 9.6). The culture supernatant (various dilutions) or antiserum to HA was added to each well. Then horseradish peroxidase-labeled goat antimouse IgA (1:1,000; Zymed Laboratories, South San Francisco, Calif.), IgG (1:2,000; Cappel Laboratories, Cochranville, Pa.), or IgM (1:5,000; Cappel Laboratories) was added to the wells. The wells were extensively washed between incubations. O-Phenylendiamine solution (0.4 mg of citrate buffer [pH 5.01 per ml) containing 0.02% H202 was added to each well. After the reaction was stopped by addition of 2.5 M H2SO4, the absorbance of the contents of the wells was measured at 492 nm with a Titertek Multiscan apparatus (Flow Laboratories Inc., McLean, Va.). The levels of anti-HA IgA, IgM, and IgG antibodies were expressed as units of which one was the A492 equivalent to a 1:200, 1:2,000, or 1:200,000 dilution of the respective anti-HA serum. Anti-HA serum was prepared after injection of 100 ,ug of HA antigen with complete Freund adjuvant and a booster of 50 ,ug of HA antigen intraperitoneally into BALB/c mice. When HA antigen (0.34 or 3.4 ,ug/ml) was added to PP cell cultures, anti-HA IgA and IgM antibodies, but not anti-HA IgG antibodies, were produced (Table 1). Anti-HA IgA and

* Corresponding author. Mailing address: Yakult Central Institute for Microbiological Research, 1796 Yaho Kunitachi, Tokyo 186, Japan. Phone: (0425) 77-8960. Fax: (0425) 77-3020.

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Bifidobacterium breve YIT4064 was tested as an adjuvant for oral influenza vaccine by the murine Peyer's patch cell culture method. The organism augmented production of anti-influenza virus hemagglutinin immunoglobulin A antibody by Peyer's patch cells in response to addition of hemagglutinin. These antibodies may be disseminated to the respiratory mucosal tissue and prevent influenza virus infection.

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VOL. 1, 1994

TABLE 1. Augmentation of anti-influenza virus HA antibody Expt no. and antibody class

Concn of influenza virus HA (p.g of protein/ml)

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production by mouse PP cells with B. breve YIT4064' Antibody titer

No adjuvant

(U/ml)"

YIT4064

YIT4079

0.181 ± 0.069

ND"

0.606 ± 0.009d 0.830 ± 0.023d

ND ND

1

0 IgA

IgG

IgM

0.34

3.4 0 0.34 3.4 0 0.34 3.4

0.030 ± 0.044 0.119 ± 0.016 0.050 ± 0.009 0 ± 0.003 0 ± 0.011 0 ± 0.009 0 ± 0.075 2.774 ± 0.434

0 ± 0.009 0 ± 0.015

ND ND

2.660 ± 0.226

0 ± 0.009 13.934 ± 0.368d 18.009 ± 0.406d 27.849 ± 0.981d

ND

0.040 ± 0.009 0.136 ± 0.007 0.213 ± 0.007 0±0 0 0 0 0 2.830 ± 0.660 4.151 ± 0.566 21.604 ± 0.775

0.306 ± 0.036e 0.420 ± 0.021d 0.406 ± 0.009" 0±0 0 0 0 0 39.057 2.736d 37.547 ± 1.981" 56.698 ± 0.660"

0.081 ± 0.011 0.047 ± 0.010 0.216 ± 0.010 0±0 0 0 0 0 7.830 ± 0.750" 1.792 ± 0.283 25.283 ± 0.849

ND ND

ND

2

IgG IgM

0 0.34 3.4 0 0.34 3.4 0 0.34 3.4

"In experiment 1, B. breve (100 ,ug/ml) and influenza virus HA antigen were added to PP cell (2.5 x 10"/ml) cultures. In experiment 2, B. breve (250 ,ug/ml) and influenza virus HA antigen were added to PP cell (5 x 106/ml) cultures. One day after culture initiation, the influenza virus HA antigen was removed by centrifugation. After 6 more days, antibody levels in the culture supernatants were measured by ELISA. " The statistical significance of the difference between the experimental and control groups was determined by Student's t test. 'ND, not done. d"p < 0.001. eP < 0.01.

IgM antibody production by PP cells after addition of HA and B. breve YIT4079 was almost the same as that achieved by addition of only HA. However, production of anti-HA IgA and IgM antibodies by PP cells after addition of HA and B. breve YIT4064 was significantly greater than that achieved by addition of HA and B. breve YIT4079 or only HA (Table 1). B. breve YIT4064 augmented the production of IgA and IgM antibodies against HA antigen by PP cells. Augmentation of IgA and IgM anti-HA antibody production by PP cells after addition of B. breve YIT4064 occurred without addition of HA antigen, too. It was thought that PP cells might have been already primed with cross-reactive antigen. We have found that production of interleukin 1 (IL-1), IL-4, and IL-S and the numbers of surface IgA-positive cells and IgA-producing cells in PP cells after addition of B. breve YIT4064 are greater than those found after no addition or addition of B. breve YIT4079 (25). Therefore, it is speculated that B. breve YIT4064 activates macrophages and T helper type II cells and induces IL-1, IL-4, and IL-5; these cytokines accelerate the differentiation of B cells into IgM or IgA-producing cells; and anti-HA IgM and IgA antibody production by PP cells after addition of HA antigen and B. breve YIT4064 is significantly greater than after addition of only HA antigen. Hence, it is assumed that B. breve YIT4064 administered orally along with HA augments the differentiation of IgA precursor cells in PP and the appearance of anti-influenza virus IgA antibody in the respiratory mucosal tissue and enhances the efficacy of oral vaccination with influenza virus. Attractive attributes of oral vaccines include the simplicity of administration, which does not require sterile syringes and needles and trained personnel; less stringent requirements for preparation of orally delivered antigens than for injectable ones; and fewer problems with storage of dry, lyophilized oral vaccines than with liquid injectable vaccines. However, as the

antigens administered orally are inactivated by gastric acid and digestive enzymes, it is difficult for them to be taken up and processed by PP. Therefore, immune responses after oral administration of antigen are weaker than after systemic immunization. The immunogenicity of vaccines is augmented by administration of potent adjuvants. A number of promising new adjuvants, immunostimulating complexes, cholera toxin subunit B, proteoliposomes, 6-O-acylmuramyldipeptide, a lipid A analog, and others, have been reported to enhance the immune responses of animals and humans to influenza vaccines (5, 8, 17-19, 21). However, adjuvants which stimulate mucosal immunity and do not cause undesirable side effects after oral administration have not been discovered. B. breve YIT4064 administered orally shows weak antigenicity and has significant adjuvant activity against CT antigen administered orally (24). Further, the strain is present in the intestines of healthy breast-fed infants. Therefore, it is safe as an adjuvant for oral vaccination. Our results suggest that B. breve YIT4064 may act as an adjuvant for oral influenza vaccine. We are now studying whether oral administration of B. breve YIT4064 enhances the immune response to oral influenza vaccine and leads to effective protection against influenza virus infection. In the preliminary experiments, anti-HA antibody levels in nasal wash specimens of mice given HA antigen and B. breve YIT4064 orally were significantly higher than those in mice given only HA antigen orally, and the antibodies inhibited HA activity. B. breve YIT4064 may become an available adjuvant for oral vaccination. REFERENCES 1. Bergmann, K.-C., and R. H. Waldman. 1989. Oral immunization with influenza virus: experimental and clinical studies. Curr. Top.

Microbiol. Immunol. 146:83-89. 2. Chen, K. S., D. B. Burlington, and G. V. Guinnan. 1987. Active


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