Takashi Nagamine

Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit

Secretory IgA antibodies in mucosa are known to play an essential role in protection against various infectious agents. To enhance the induction of protective mucosal antibodies, influenza HA vaccine was inoculated intranasally into mice with the B subunit of cholera toxin (CTB), which is known to be an excellent mucosal self-adjuvanting molecule. This combination resulted in high levels of antiviral IgA antibodies in nasal secretions and enhanced serum haemagglutinin-inhibiting (HI) antibodies 4 weeks after inoculation, compared with the inoculation of vaccine alone which induced only a low level of HI serum antibodies and no local IgA antibodies. (Subcutaneous or intraperitoneal inoculation of the vaccine with CTB failed to induce detectable nasal antiviral IgA antibodies). Levels of nasal IgA and serum HI antibodies increased in a dose-dependent fashion with increasing nasal doses of both vaccine and CTB, and correlated with the degree of protection against viral challenge. A greater protective effect was seen with cholera toxin than with its B subunit. Moreover, a second administration of vaccine alone, 4 weeks after the inoculation of the vaccine with CTB, elevated the level of the antiviral IgA nasal antibodies to 10-100 times higher than that of the primary response. These results suggest that either CT or CTB could be used as a potent adjuvant to induce protective secretory antibodies by nasal vaccination against pathogens impinging on respiratory mucosa.

Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine

Cholera toxin B subunit (CTB) and Escherichia coli heat-labile toxin (LTB) (2 micrograms), each supplemented with a trace amount of cholera toxin (CT) (0.02-20 ng), were examined for the adjuvant effect on antibody (Ab) response against influenza inactivated HA (haemagglutinin) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation of the vaccine (1.5 micrograms) and the CT-containing CTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The primary inoculation of the vaccine with CTB alone did not induce either anti-HA IgA or IgG Ab response, or haemagglutination-inhibition Ab responses in the serum. The vaccine with less than 2 ng of CT also failed to induce Ab response. On the other hand, the vaccine with CT-containing CTB induced a high Ab response, which increased depending on the CT dose. Moreover, the second vaccine induced a response more than ten times higher than the primary one and the response increased depending on the CT dose. Similar enhancement was found in the local anti-HA IgA Ab response in the nasal wash. Such synergistic effects were observed also between LTB and CT. The amount of Ab produced by the synergism was considered to be enough to protect against virus infection. These results suggest that CTB (or LTB) containing a trace amount of CT (about 0.1%) can be used practically as a potent adjuvant for nasal vaccination of humans against influenza.

Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination.

The antibody responses to influenza virus A/PR/8/34 HA vaccine and protection against virus challenge in mice given the vaccine together with the B subunit of cholera toxin (CTB) intranasally were compared with those in mice given the vaccine with CTB perorally, intraperitoneally or subcutaneously. Intranasal vaccination induced remarkably higher levels of antiviral IgA antibodies in both respiratory washings and serum than did other routes of vaccination. The titres of antiviral IgG antibodies in respiratory washings and serum, and haemagglutination-inhibiting (HI) antibodies in serum, were similar after intranasal and parenteral vaccination. Oral vaccination, however, induced low levels of antiviral IgG antibodies but no detectable HI antibodies. Moreover, intranasal immunization elicited significantly higher titres of antiviral IgA antibodies in intestinal secretions in comparison with oral immunization. In contrast, parenteral immunization failed to induce these IgA antibodies. In virus challenge studies, a greater protective effect was seen after intranasal and intraperitoneal vaccination than after other routes of vaccination. These results suggest that intranasal inoculation of combined HA vaccine and CTB is superior to oral or parenteral inoculation in protecting mice. Furthermore, the intestinal antiviral IgA responses suggest that intranasal administration of CTB-combined vaccines could be effective not only against respiratory pathogens but also against enteropathogens.

Functional role of respiratory tract haemagglutinin-specific IgA antibodies in protection against influenza

Intranasal inoculation of haemagglutinin (HA) purified from influenza virus A/PR/8/34 (PR8, H1N1) together with cholera toxin B subunit, into Balb/c mice resulted in complete protection against PR8 infection in parallel with the induction of high levels of HA-specific IgA and IgG antibodies on the respiratory tract. The respiratory tract IgA and IgG were purified from nasal and lung washings of the immunized mice using affinity columns, and their HA-specific activities were measured by enzyme-linked immunosolvent, plaque neutralization and haemagglutination inhibition assays. The purified IgA and IgG had the following properties: (1) They were able to neutralize virus in vitro. (2) The purified IgA included major antibodies directed against PR8 virus and minor antibodies cross-reactive with A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) virus, while the purified IgG included major antibodies to the homotypic virus, minor antibodies to the H1N1 virus and only a trace amount of antibodies to the H3N2 virus. (3) When separated on a Sephacryl column, most of the IgA anti-HA activities occurred in the polymeric fractions of purified IgA, whereas the IgG anti-HA activities occurred in the monomeric fractions. (4) When passively administered to normal mouse respiratory tract before infection, the purified IgA protected against PR8 infection. These results suggest that HA-specific, polymeric IgA antibodies on the respiratory tract by themselves provide not only protection against the homotypic virus but also higher levels of heterotypic immunity than IgG.

Antibody responses in volunteers induced by nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin

Evaluation of the efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit (LTB) containing a trace amount of the holotoxin (LT) in inducing antibody responses among volunteers, which was conducted during the winter season of 1993-1994, is reported. A trivalent inactivated vaccine, composed of A/Yamagata/32/89 (H1N1), A/Kitakyusyu/159/93 (H3N2) and B/Bangkok/163/90 influenza virus strains, was used alone or together with the adjuvant, recombinant LTB supplemented with 0.5% recombinant LT (LTB*). The volunteers were divided into two groups: 73 volunteers (mean age 35.0 +/- 12.0 years) inoculated intranasally (i.n.) with LTB*-combined vaccine and 49 volunteers (37.9 +/- 11.3) inoculated i.n. with the vaccine alone. Vaccination was done twice 4 weeks apart. Salivary secretory IgA and serum hemagglutination-inhibiting (HI) antibodies were measured before and 8 weeks after the primary vaccination. For the sake of convenience, more than a 1.4-fold rise in IgA antibody response (units of specific IgA antibody per microgram of total IgA) and a fourfold or greater rise in HI antibody titer after vaccination were regarded as a positive antibody response. Thirty-seven (50.3%) and 36 (49.3%) of the 73 vaccinees, respectively, given the nasal LTB*-combined vaccine showed positive IgA and HI antibody responses to one or more of the three vaccine strains. In comparison, positive antibody responses in the group given vaccine alone were 32.7% for IgA and 30.6% for HI antibody. There was a significant difference between these two groups. These results suggest that the nasal LTB*-combined vaccine could enhance the production of higher levels not only of serum HI antibody but IgA antibodies in the respiratory tract than do the nasal vaccine alone.

Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine

Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response. The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT. Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype. These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza.

Enhancement of protective antibody responses by cholera toxin B subunit inoculated intranasally with influenza vaccine

Effects of the B subunit of cholera toxin (CTB) on the primary antibody responses to influenza virus A/PR/8/34 (PR-8) (H1N1) HA vaccine and on protection against viral challenge were investigated in Balb/c mice which were immunized intranasally with both the vaccine and CTB. The dose of CTB (greater than or equal to 1 microgram) inoculated with the vaccine (greater than or equal to 0.15 microgram) induced high responses of both antiviral IgA antibodies in the nasal wash and haemagglutinin-inhibiting (HI) antibody in the serum, enough to provide complete protection against viral challenge four weeks after immunization. High levels of antibody were maintained for more than 16 weeks after inoculation, affording complete protection during this interval. The inoculation of HA vaccine prepared from influenza viruses A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with CTB provided partial protection against PR-8 infection, with production of antiviral IgA antibodies which were cross-reactive to PR-8 antigens whereas immunization with CTB and HA vaccine prepared from a different type of influenza virus (B/Ibaraki/2/85) failed to protect against PR-8 infection. These results indicate that CTB can produce an augmented and persistent antibody response to PR-8 HA vaccine, which is cross-protective to other A-type virus infections. The mechanisms by which CTB enhances the protective antibody responses to the nasally inoculated vaccine were investigated. The ability of CTB to augment antibody responses was lost, either when CTB was inoculated via the intravenous or subcutaneous route, or when CTB was introduced into nasal site one day before or after the vaccine inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria–pertussis–tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 microg) was intranasally (i.n.) inoculated with influenza vaccine (10 microg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3-14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 microg) was i.n. inoculated with the vaccine (10 microg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27-50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 microg) and influenza HA vaccine (1 microg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 microg) each and diphtheria-pertussis-tetanus (DPT) vaccine (10 microg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 microg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.

Effectiveness of cholera toxin B subunit as an adjuvant for nasal influenza vaccination despite pre-existing immunity to CTB

In a previous paper, it was shown that cholera toxin B subunit (CTB) augments the production of protective antibodies to influenza virus when CTB is inoculated intranasally into Balb/c mice together with influenza HA vaccine. The present study was designed to determine whether the effectiveness of CTB as a potent adjuvant for nasal vaccination could be limited by pre-existing immunity to CTB. Mice were sensitized by intranasal inoculation of either 1 or 0.05 microgram of CTB 2, 4 and/or 6 weeks before nasal vaccination. They were then vaccinated by either a single inoculation of vaccine together with 1 microgram of CTB or a two-dose regimen, composed of a primary inoculation of vaccine together with 0.05 microgram of CTB and a subsequent inoculation of vaccine alone. Levels of nasal IgA antibodies to CTB increased with the increase of the dose of CTB and the frequency of CTB-inoculation. Pre-existing immunity to CTB, however, did not significantly reduce the levels of both nasal IgA and serum haemagglutination-inhibiting (HI) antibodies to influenza virus and did not change the ability of the vaccinated mice to resist viral challenge. These results suggest that a relatively low dose of CTB could be inoculated repeatedly into animals as an adjuvant for nasal vaccination.

Protection against influenza virus infection by a two-dose regimen of nasal vaccination using vaccines combined with cholera toxin B subunit

The effectiveness of the two-dose regimen, composed of a primary intranasal inoculation of influenza A-type virus HA vaccine together with B subunit of cholera toxin (CTB) and the subsequent intranasal inoculation of vaccine alone 4 weeks later, was examined. In mice given a relatively high dose of virus A/PR/8/34 (PR-8, H1N1) HA vaccine (1.5 micrograms) both as a primary antigen with CTB (1 microgram) and as the second antigen, the secondary responses of both antiviral IgA antibodies in nasal wash and haemagglutination-inhibiting (HI) antibody in serum were much higher than those of primary responses and persisted for greater than 12 weeks after the second inoculation. Even in mice that received reduced amounts of a primary vaccine (0.03 microgram) [prepared from virus PR-8, A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2)] together with reduced amounts of CTB (0.05 microgram), the subsequent inoculation of PR-8 vaccine produced both nasal IgA and serum HI antibodies and provided complete protection against homologous A-type virus (PR-8) infection. Moreover, the combination of the reduced amounts of heterologous A-type virus vaccine (A/Yamagata or A/Fukuoka) with CTB for primary inoculation and the secondary heterologous A-type virus vaccine [A/Yamagata, A/Kumamoto/37/79 (H1N1), or A/Fukuoka] resulted in high levels of cross-reactive IgA antibodies and partial cross-protection against PR-8 infection. On the other hand, a second inoculation of B/Ibaraki/2/85 vaccine failed to produce cross-reactive antibodies and to protect against PR-8 infection.(ABSTRACT TRUNCATED AT 250 WORDS)