Enhancing effects of immunoactive peptide FR48217 on immunological responses to vaccination by inactivated influenza virus

Enhancing effects of immunoactive peptide FR48217 on immunological responses to vaccination by inactivated influenza virus Taeko Kusumi*, Akira Yamada*, Meishi Cao t, Atsuo Tanaka t, Hiroshi Takenaka* and Jiro Imanishi* The adjuvant effect o f an immanoactive peptide, FR48217 ( FR ) , was investigated in mice vaccinated with inactivated influenza virus with and without FR (designated as V+ FR and V - F R , respectively). Mice were infected with influenza virus A/PR/8/34 28 days after vaccination. The survival rate indicated a strong adjuvant effect of FR on vaccination with inactivated influenza virus. No difference in interferon activity in the pulmonary lavage fluid was found between the V+ FR and V - FR groups. The titres of virus-specific IgA and IgG antibodies in the serum increased after vaccination in both V + FR and V - FR groups and were significantly higher in the V + FR group than in the V - F R group. On days 2 and 4 after viral infection, the serum antibody titres decreased sharply and then increased again rapidly. The free IgA and IgG antibody levels in the pulmonary lavage fluid of vaccinated mice started to increase after day 4. It is proposed that one of the mechanisms by which Fit has the adjuvant effect on vaccination is the increase of virus-specific IgA and IgG antibodies in the serum, which are diffused into the infection sites of the epithelial tissues in the respiratory tract to neutralize the virus at the early stages of viral infection.

Keywords:Adjuvant;inactivatedinfluenzavirus vaccine;immunoactivepeptide FR48217 Introduction Immunostimulants have been the subject of intensive studies in recent years because of their potential use as an adjuvant for vaccination as well as wide application in cancer therapy. Peptidoglycans, macromolecular structures from the bacterial cell wall, are one of the best known and the most effective immunostimulants1. For further understanding of the immunostimulation by peptidoglycans and for development of better immunostimulants, the portion of the peptidoglycan that is responsible for immunostimulation has to be identified. The muramyldipeptide (MDP), which is isolated from the peptidoglycan, has been extensively characterized and shown to enhance a variety of host immuno-responses2 including responses to vaccination 3. FK1564, which is isolated from another part of the peptidoglycan, and its derivative, FR48217 (FR) 5, have also been shown to have immunostimulating effects, including enhancement of macrophage activity6, defence against bacterial and viral infections7'8, and anti-tumour activities s. However, its adjuvant effect has not been investigated. The present study reports on the adjuvant effect of FR on the inactivated influenza virus vaccine. Simultaneous administration of immunostimulants with influenza virus vaccine is now under active investigation9 due to the unimpressive effect of vaccination 1°'11 using inactivated influenza virus or component vaccine. In the present study, attention was focussed on the *Department of Otorhinolaryngology, and tDepartment of Microbiology, Kyoto Prefectural University of Medicine, Kamikyo-ku, Kyoto, 602, Japan. (Received 10 December 1988; revised 27 February 1989) 0264-410X/89/040351-06$03.00 © 1989 8utterworth & Co. (Publishers) Ltd

adjuvant effects of FR on vaccination with the inactivated influenza virus in mice. We have found that FR increases the survival rate of mice against influenza virus infection by joint administration with inactivated influenza vaccine. We have investigated the mechanisms by which FR enhances immunological responses to vaccination by monitoring the titres of influenza virus-specific IgA and IgG antibodies in the serum and pulmonary lavage fluid, and interferon (IFN) activity in the pulmonary lavage fluid.

Materials and methods

....

Influenza virus Influenza A/PR/8/34 (H1N1) virus (PR/8) was used throughout this study. The virus was purified by the method of Hosaka et al. 12 with a slight modification. PR/8 was grown in the allantoic sac of l 1-day-old embryonic chicken eggs. Cellular debris was removed from allantoic fluid by low-speed centrifugation (400g for 20 min, and 15 0009 for 3 h). The supernatant was used to infect mice. For further purification of the virus, the virus suspension was layered on a discontinuous sucrose gradient [30%(w/w) and 60%(w/w) sucrose] and centrifuged (100 0009 for 90 rain, and then 100 000g for 3 h). The virus band at the boundary of 30 and 60% sucrose solution was collected, resuspended in phosphate-buffered saline (PBS) and pelleted by centrifugation (1000009 for 90 rain). The pelleted virions were resuspended in PBS and used as purified virus. Preparation o f vaccine The vaccine was prepared by the method of Hosaka et al. 13. Briefly, the purified virions were solubilized with

Vaccine, Vol. 7, August 1989 351

Immunoactive peptide effect on responses to inactivated influenza virus vaccination: 7-. Kusumi et al.

CHs

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0.25% Nonidet P-40 and centrifuged at 100000g for 40 min. The supernatant was dialysed against PBS containing lmM MgCI2 and then centrifuged at 100 000g for 40 min. The pellet was resuspended in PBS and used as the inactivated influenza virus vaccine. CCA (chick cell agglutination) determination was carried out by Kanonji Institute, the Research Foundation for Microbial Disease of Osaka University (Kagawa, Japan). The influenza vaccine was administered intraperitoneally at a CCA value of 10, 1 or 0.1 CCA/mouse (0.2 ml).

Infection of mice Five-week-old female ICR mice (25 g body weight) were obtained from Shizuoka Agricultural Cooperative for Experimental Animals (Hamamatsu, Japan). Ten mice in a mesh cage made of stainless steel wire were put on a specially-constructed electric rotator in a desiccator, and intranasally infected with the influenza virus blown into the desiccator by a nebulizer. Tenfold excess of 50% lethal dose (10 LDso ) of virus was used.

Immuno stimulants FR (Figure 1) (lot. No. 402116S, a crystallized material), a gift from Fujisawa Pharmaceutical Co. Ltd (Osaka, Japan), was dissolved in 1%(w/v) NaHCO 3 and administered at a dose of 20, 2 or 0.2 mg kg- 1 (0.2 ml/mouse). FR and the vaccine were separately injected intraperitoneally into the mice. As control experiments, PBS was given to the mice.

Haemagglutination inhibition (HI) test On days 14 and 28 after the injection of vaccine and/or the immunostimulants, blood specimens were collected from the retro-orbital plexus. The HI test was performed essentially by the method of de St. Groth and Webster. 14.

Antibody determination by enzyme-linked immunosorbent assays ( ELISA ) Mice were anaesthetized with sodium pentobarbital (Pitman-Moore, Washington Crossing, N J, USA) and the whole blood was drawn from the heart. After tracheostomy, 1.5 ml of PBS was injected through the trachea by insertion of an 18-gauge needle and the lavage fluid was collected. Serum specimens were separated by centrifugation. Pulmonary lavage fluid specimens were centrifuged to remove cellular debris. Most lavage samples became bloody on days 6 and 8 after PR/8 infection because the mice were in the acute inflammatory period. All mice in the control group, which did not receive the advance injection of the immunostimulants and/or vaccine, were dead by day 8 after the infection. IgA and IgG antibodies in the serum and the pulmonary

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lavage fluid were determined by the ELISA method of van Wyke et al.15 with some modification. The purified PR/8 virus was incubated with the lysis buffer (0.05 M Tris-HCl buffer, pH 7.5, 0.5%(v/v) Triton X-100, and 0.6 u KC1)at room temperature for 5 min at a concentration of 2000 HAU/50 #1, and then diluted to a final concentration of 200 HAU/50/tl with PBS, and used as the antigen for ELISA. This antigen was placed in a 96-weU plate (50 /A/well) (Nunc, Denmark) and incubated at room temperature for 2 h. After washing twice with PBS containing 0.05%(v/v) Tween 20 (PBS-T), 100/A of PBS containing l%(w/v) BSA was added to each well and incubated at room temperature for 1 h. After washing twice with PBS-T, 50 /A of the serum sample or the pulmonary lavage fluid sample was added to each well and incubated at 4°C for 16 h. These samples were serially diluted. After the reaction, each well was washed four times with PBS-T. Then 50/A of rabbit anti-mouse IgA antibody conjugated to horseradish peroxidase (HRP) (Miles Laboratories, Naperville, IL) or goat anti-mouse IgG antibody conjugated to HRP (ICN Immunobiologicals, Lisle, IL) was added and incubated at room temperature for 1 h. Labelled antibodies were diluted 200-fold with PBS containing 1% BSA. Labelled antibodies were tested for their specificity using mouse IgA, IgG 1, IgG2a , IgG2b , IgM and IgE. After the addition of labelled antibodies, each well was washed four times with PBS-T and 100 #l of the substrate solution [50 mM citrate buffer pH 7.4, 10 ml; 1% H 2 0 2 , 40 /,tl; 40 mM 2,2'-azino-di(3-ethylbenzothiazoline-6-sulphonate),50 /A] was added. After incubation at room temperature for 30 min, absorbance of each well at 405 nm was measured by an ELISA plate reader (Multiskan, Titertek, Finland). The control wells were incubated with the serum or pulmonary lavage fluid of normal mice, or were those without PR/8 antigens. The geometric mean (rh) and the standard error (s.e.) of the absorbance were calculated and the well that showed an absorbance larger than + 3 s.e. of the control well was determined as a positive well. Antibody titres were determined as the lowest dilution in positive samples.

Assay of IFN activity IFN activity in the pulmonary lavage fluid samples was determined by the cytopathogenic effect (CPE) reduction method, using L929 cells and vesicular stomatitis virus (VSV). L929 cells (5 x 104 cells) were added to a microplate well containing serial twofold dilutions of samples and incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 18 h. Then, 10-50 TCIDso of VSV was added to each well. After incubation at 37°C for 18 h, CPE was observed under the microscope. The reciprocal of the sample concentration that reduced CPE was expressed in laboratory IFN units. The values were calibrated against those for an international reference preparation (code G002-902-026) and expressed in terms of international units (IU). Results

Survival rate Figure 2 shows the time course of the survival rate after PR/8 infection. The survival rate of the group administered both FR (2 mg kg- 1) and 1 CCA of vaccine was higher than that of the group that received the vaccine

Immunoactive peptide effect on responses to inactivated influenza virus vaccination: T. Kusumi et al.

H I titres in the serum

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Adjuvant effects of immunostimulants were investigated in terms of PR/8-specific HI titre in the serum. Figure 3a shows that the HI titre of the group administered 1 CCA of vaccine with 20 or 0.2 mg kg-1 of FR increased significantly (p<0.05, t test) on days 14 and 28 after inoculation compared with the group given 1 CCA of vaccine without FR. Among the animals administered 10 CCA of vaccine (Figure 3b), only those vaccinated with 20 mg kg- 1 of FR showed a significant increase in HI titre compared with the animals given the vaccine alone (p<0.05, t test) on day 14. On day 28, with 10 CCA of vaccine, no significant difference in the HI titre was detected between the groups with and without FR, probably because a high level of HI titre could be induced by the vaccine alone.

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The immunostimulating effect of FR (0.2 mg kg- 1) on vaccination was investigated in terms of the titre of PR/8-specific IgA and IgG antibodies in the serum and in the lavage fluid. In this study, 1 CCA of vaccine/mouse was used. Figure 4 shows the PR/8-specific IgA (a) and IgG (b) antibody titres in the pulmonary lavage fluid of the lung, which is the major site of invasion, infection

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only after day 8. Table 1 lists the effect of vaccination with various amounts of vaccine and FR on the survival rate on day 21 after PR/8 infection. The survival rate of the group that received both 1 CCA of vaccine and FR was significantly higher than that of the group given only 1 CCA of vaccine (p < 0.05, Fisher's exact probability test). The survival rate of the mice given only FR was the same as that of the control group (p = 1), which indicates that FR by itself has no anti-influenza effect. These results indicate that FR has an adjuvant effect on vaccination with the inactivated influenza virus. Joint administration of 1 CCA of vaccine and FR is roughly as effective as administration of 10 CCA of vaccine, which induces a high level of immunological responses by the antigen alone.

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Immunoactive peptide effect on responses to inactivated influenza virus vaccination: T. Kusumi et al.

group on days 14 and 28 after administration of vaccine (on days - 14 and 0 in Figure 5a and 5b, p<0.05, t test). After infection, no statistically significant differences were observed between the V + FR and V - F R groups in the IgA and IgG antibody titres in the serum. It is of particular interest to notice that both IgA and IgG antibody titres in the serum were markedly decreased on days 2 and 4 after infection and then they increased again after day 6.

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pulmonary lavage fluid of mice after vaccination (1 CCA) with (O) or without (O) FR (0.2 mg kg-1), and after infection with PR/8 (10 LDso) 28 days after vaccination. Each point represents the mean, and the bar indicates the standard error of the mean for three mice. Standard error of the mean is not shown when this value fell within the symbol used to denote the mean. Anti-PR/8 IgA and IgG antibodies were not detected in control mice and values for them are not presented

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Figure 4 Titres of PR/8-specific IgA (a) and IgG (b) antibodies in the

and proliferation of influenza virus. Before viral infection (on days - 14 and 0 in Figure 4a), the IgA titre did not increase in either the animals vaccinated with FR (designated as V + FR) or those not given FR ( V - F R ) . After infection, the IgA titre increased in both V + FR and V - F R groups from day 6, but no difference was observed between these groups on days 6 and 8. On day 14, the IgA antibody titre in the V + F R group was lower than that in the V - F R group (p < 0.05, t test). As is shown in Figure 4b, the IgG antibody titre in the V + FR group was somewhat lower than in the V - FR group on day 14 (p =0.058, t test). Figure 4b shows the tendency of the IgG antibody titre in the pulmonary fluid to increase in the V + FR group 28 days after vaccination (day 0 in Figure 4b, p = 0.068). Figure 5 shows PR/8-specific IgA (a) and IgG (b) antibody titres in the serum. Both IgA and IgG titres were higher in the V + F R group than in the V - F R

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Immunoactive peptide effect on responses to inactivated influenza virus vaccination: T. Kusumi et al.

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than in the vaccinated groups on days 2, 4, and 6 after infection (p<0.05). No difference in IFN activity was found between the V + FR and V - F R groups.

Discussion FR raised the survival rate against influenza virus infection by joint administration with 1 CCA of vaccine in mice. The titres of HI antibody and PR/8-specific IgA and IgG antibodies in the serum before viral infection were increased in the V+ FR group compared with those of the V - F R group. From these results, it is concluded that FR possesses adjuvant effects on vaccination with inactivated influenza virus. However, when an amount of vaccine 10 times larger (10 CCA) was used, no difference in the survival rate was observed between the V + F R and V - F R groups (Table 1), because a high level of immunological response was induced by the antigen alone. The dose of influenza vaccine currently used in Japan is 350 CCA (700 CCA/ml x 0.5 ml). Although it is very difficult to compare doses given to different species, several CCA to 25 g mice may be comparable to 350 CCA to a 50 kg human adult. Therefore, the results reported here suggest that, with the use of FR, the antigen dose could be reduced to a lower level, which would increase the safety of influenza virus vaccination. (Concerning the toxic side-effects of FR, Izumi et al. reported that FR caused loss of body weight in healthy mice on day 2 after subcutaneous injection of 6 mg kg-1 of FR (Ref. 5), and Oku et al. showed that the 50% lethal dose for mice was > 500 mg kg- 1 (Ref. 8). The dose of FR used most often in this study was 0,2 mg kg- 1, which is much less than the doses above. We have not determined the minimal effective dose.) The cell wall of Mycobacterium tuberculosis is known to enhance humoral and cellular immunity 16, and one of

the effective elements in the cell wall is the peptidoglycanl that contains repeated sequences of GlcNAc-MurNAcL-Ala-D-isoGln-meso-Azpm-D-Ala (where A2pm = 2,6diaminopimeric acid), which sequences are commonly found in bacterial cell walls. FR, isolated from Streptomyces, also includes a part of this repeated subunit of the peptidoglycan (docosanoyl-L-Ala-o-Glu-meso-A2pm-DAla). Ellouz et al. 2 concluded that MDP (MurNAc-L-AlaD-isoGln) was the smallest effective structural unit of peptidoglycan and di- and tripeptides without muramic acid had no adjuvant effects since these peptides failed to increase antibodies in the serum and to induce delayed hypersensitivity (cellular immunity) to ovalbumin used as an antigen. On the other hand, Fleck et al)7 found that a peptide without muramic acid, L-Ala-D-Glumeso-A2pm-D-Ala, enhanced delayed hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine (antigen). Our findings with FR are consistent with the data obtained by Fleck et al. and indicate that the tripeptide without muramic acid is capable of inducing the adjuvant effect. FR by itself has no antiviral effect against influenza infection as shown in Figure 2 and Table 1: the group of mice administered 20, 2 or 0.2 mg kg-1 of FR without vaccine died at the same rate as the group of control mice. By contrast, Oku et al. s reported a survival rate of 100% against herpes simplex virus infection by administration of FR (6 mg kg- 1 of FR was administered subcutaneously to mice once a week for 3 weeks after infection). They suggested that FR enhanced non-specific defence mechanisms of the host, such as macrophage activation, induction of IFN in peritoneal exudate cells, enhancement of IFN production, and natural killer cell activity. Since influenza virus infection takes place locally in the epithelial cells on the surface of the respiratory tract, it is concluded that the non-specific immunological responses in the respiratory tract epithelial tissues were not enhanced by the intraperitoneal administration of FR without vaccine. The concentration of locally secreted antibodies in the epithelial tissues is more directly related to protection against influenza infection than the antibody concentration in the serum because the influenza virus infects the epithelial cells and spreads directly from cell to cell rather than diffusing through the blood flow is. Balkovic and Six 19 measured the immunoglobulin titres and quantity of albumin in the serum and in the pulmonary lavage fluid after vaccination with inactivated virus and after influenza infection, and concluded that most of the IgA and IgG antibodies detected in the pulmonary lavage fluid were derived from the serum. Yoshida et al. 2° found enhancement of leukotriene release from alveolar macrophages upon influenza infection and ascribed it to the increased permeability of lung vessels after viral infection. These findings suggest that the antibodies are transferred from the serum to the lung tissues for local protection against the virus. Figure 5 shows that vaccinated mice produce both PR/8-specific IgA and IgG antibodies in the serum, and that there is a temporary decrease of titre in the serum just after the viral infection. This, together with the results by Balkovic and Six and Yoshida et al., suggests that the serum antibodies are transported to the lung epithelial tissues and consumed to neutralize the virus upon infection. These findings and the survival rates shown in Figure 2 indicate that the increased serum antibody levels

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Immunoactive peptide effect on responses to inactivated influenza virus vaccination: T. Kusurni et al.

greatly contribute to raising the survival rate. However, since we have not examined cellular immunity, the possibility cannot be denied that vaccination with FR also enhances cellular immunological responses.

Acknowledgement The authors are grateful to Professor Osamu Mizukoshi at Kyoto Prefectural University of Medicine for his support and encouragement, and also to Fujisawa Pharmaceutical Co. Ltd, for supplying FR48217.

References 1 Holton, J.B. and Schwab, J.H. Adjuvant properties of bacterial cell wall mucopeptides. J. Immunol. 1966, 96, 134 2 EIIouz, F.A., Adam, A., Ciorbaru, R. and Lederer, E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 1974, 59, 1317 3 Webster, R.G., Glezen, W.P., Hannoun, C. and Laver, W.G. Potentiation of the immune responses to influenza virus subunit vaccines. J. Immunol. 1977, 119, 2073 4 Gotoh, T., Nakamura, K., Iwai, M., Aoki, H. and Imanaka, H. Studies on a new immunoactive peptide, FK156. 1. Taxonomy of the producing strains. J. Antibiot. 1982, 35, 1280 5 Izumi, S., Nakahara, K., Gotoh, T., Hashimoto, S., Kino, T., Okuhara, M., Aoki, H. and Imanaka, H. Antitumor effect of novel immunoactive peptides, FK156 and its synthetic derivatives. J. Antibiot. 1983, 35, 566 6 Mine, Y., Watanabe, Y., Tawara, S., Yokota, Y. and Nishida, M. Immunoactive peptides, FK156 and FK565. 3. Enhancement of host defense mechanisms against infection. J. Antibiot. 1983, 35, 1059 7 Mine, Y., Yokota, Y., Wakai, Y., Fukada, S. and Nishida, M.

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Immunoactive peptides, FK156 and FK565. 1. Enhancement of host resistance of microbial infection. J. Antibiot. 1983, 36, 1010 8 0 k u , T., Imanishi, J. and Kishida, T. Antiviral effect of two aryloligopeptides, FR41565 and FR46217. Antiviral Research 1986, 6, 233 9 Nerome, K. Influenza vaccine no atarashii kaihatsu kenkyu. (in Japanese) C/in. Virol. 1985, 13, 289 10 Clements, M.L., Betts, R.F. and Murphy, B.R. Advantage of live attenuated cold-adapted influenza A virus over inactivated vaccine for A,fiNashington/80 (H3N2) wild-type virus infection. Lancet 1984, 8, 705 11 Barry, D.W., Staton, E. and Mayner, R.E. Inactivated influenza vaccine efficacy: diminished antigenicity of split-product vaccines in mice. Infect. Immunol. 1974, 10, 1329 12 Hosaka, Y. and Hosokawa, Y. Virions with glutaraldehyde-treated red blood cells. Intervirology 1977, 8, 1 13 Hosaka, Y., Yasuda, Y. and Fukai, K. Hemolysis by liposomes containing influenza virus hemagglutinins. J. Virol. 1983, 46, 1014 14 de St. Groth, F. and Webster, R.G. Disquisitions on original antigenic sin. 1. Evidence in man. J. Exp. Med. 1966, 124, 331 15 van Wyke, K.L., Hinshaw, V.S., Bean, W.J. and Webster, R.G, Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies. J. Gen. Virol. 1980, 35, 24 16 Freund, J. and McDermott, K. Sensitization to horse serum by means of adjuvants. Proc. Exp. Biol. Med. 1942, 46, 548 17 Fleck, J., Mock, M., Tytgat, F., Nauciei, C. and Minck, R. Adjuvant activity in delayed hypersensitivity of peptidic part of bacterial peptidoglycans. Nature 1974, 250, 517 18 Sonoguchi, T., Sakoh, M., Kunita, N., Satsuta, K., Noriki, H. and Fukumi, H. Reinfection with influenza A (H2N2, H3N3, and H1N1) viruses in Japan. J. Infect. Dis. 1986, 153, 33 19 Balkovic, E.S. and Six, H.R. Pulmonary and serum isotypic antibody responses of mice to live and inactivated influenza virus. Am. Rev. Respir. Dis. 1986, 134, 6 20 Yoshida, S., Saitoh, H., Takenaka, H. and Mizukoshi, O. Leukotriene C release by mouse alveolar macrophages in viral infection. Jpn. J. Immunoallergol. Otorhinolaryngol. 1986, 4, 14