Shin-ichi Tamura

Protection Against Influenza Virus Infection in Polymeric Ig Receptor Knockout Mice Immunized Intranasally with Adjuvant-Combined Vaccines

The role of secretory IgA in conferring cross-protective immunity was examined in polymeric (p)IgR knockout (KO) mice immunized intranasally with different inactivated vaccines prepared from A/PR/8/34 (H1N1), A/Yamagata/120/86 (H1N1), A/Beijing/262/95 (H1N1), and B/Ibaraki/2/85 viruses and infected with the A/PR/8/34 virus in the upper respiratory tract (RT)-restricting volume. In wild-type mice, immunization with A/PR/8/34 or its variant (A/Yamagata/120/86 and A/Beijing/262/95) vaccines conferred complete protection or partial cross-protection against infection, while the B-type virus vaccine failed to provide protection. The protection or cross-protection was accompanied by an increase in the nasal A/PR/8/34 hemagglutinin-reactive IgA concentration, which was estimated to be >30 times the serum IgA concentration and much higher than the nasal IgG concentration. In contrast, the blockade of transepithelial transport of dimeric IgA in pIgR-KO mice reduced the degree of protection or cross-protection, in parallel with the marked increase in serum IgA concentration and the decrease in nasal IgA concentration (∼20 and 0.3 times those in wild-type mice, respectively). The degree of the reduction of protection or cross-protection was moderately reversed by the low but non-negligible level of nasal IgA, transudates from the accumulated serum IgA. These results, together with the absence of the IgA-dependent cross-protection in the lower RT and the unaltered level of nasal or serum IgG in wild-type and pIgR-KO mice, confirm that the actively secreted IgA plays an important role in cross-protection against variant virus infection in the upper RT, which cannot be substituted by serum IgG.

Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin- and neuraminidase-expressing DNAs

The ability of plasmid DNA encoding hemagglutinin (HA), neuraminidase (NA) or matrix protein (M1) from influenza virus A/PR/8/34 (PR8) (H1N1), and mixtures of these plasmid DNAs (HA + NA and HA + NA + M1) to protect against homologous or heterologous virus infection was examined in BALB/c mice. Each DNA was inoculated twice, 3 weeks apart, or four times, 2 weeks apart, at a dose of 1 microg of each component per mouse by particle-mediated DNA transfer to the epidermis (gene gun). Seven days after the last immunization, mice were challenged with a lethal homologous or heterologous virus and the ability of each DNA to protect the mice from influenza was evaluated by observing lung virus titers and survival rates. The administration of a plasmid DNA mixture of either (HA + NA) or (HA + NA + M1) provided almost complete protection against the PR8 virus challenge, and this protection was accompanied by high levels of specific antibody responses to the respective components. The degree of protection afforded in these groups is significantly higher than that in mice given either HA- or NA-expressing DNA alone, which provided only a partial protection against PR8 challenge or that in mice given M1-expressing DNA, which failed to provide any protection. In addition, both of the plasmid DNA mixtures (HA + NA) and (HA + NA + M1) showed a slight tendency to provide cross-protection against an A/Yamagata/120/86 (H1N1) virus challenge, and this was accompanied by a relatively high level of cross-reacting antibodies. Thus, there was no clear difference between the ability of the HA + NA and HA + NA + M1 plasmid DNA mixtures in providing protection against either a PR8 or heterologous virus challenge. These results suggest that in mice immunized by gene gun, a mixture of plasmid DNAs encoding HA and NA can provide the most effective protection against the virus challenge. The addition of the M -expressing plasmid DNA to this mixture does not enhance the degree of protection afforded.

Cross-Protection against H5N1 Influenza Virus Infection Is Afforded by Intranasal Inoculation with Seasonal Trivalent Inactivated Influenza Vaccine

Abstract Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005–2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C12U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor—3 agonist, poly(I): poly(C12U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

Cross-protection against a lethal influenza virus infection by DNA vaccine to neuraminidase.

Cross-protection against a lethal influenza virus infection was examined in BALB/c mice immunized with plasmid DNAs encoding the neuraminidase (NA) from different subtype A viruses. Each NA-DNA was administered twice, 3 weeks apart, at the dose of 1 microg per mouse by particle-mediated DNA transfer to the epidermis (gene gun) or at a dose of 30 microg per mouse by electroporation into the muscle. Three weeks after the second vaccination, the mice were challenged with lethal doses of homologous or heterologous viruses and the ability of each NA-DNA to protect the mice from influenza was evaluated by determining the lung virus titers, body weight and survival rates. The H3N2 virus NA-DNA conferred cross-protection against lethal challenge with antigenic variants within the same subtype, but failed to provide protection against infection by a different subtype virus (H1N1). The degree of cross-protection against infection was related to titers of the cross-reacting antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against infection not only with homologous virus but also with drift viruses.

Comparison of the ability of viral protein-expressing plasmid DNAs to protect against influenza

The ability of plasmid DNA encoding various influenza viral proteins from the A/PR/8/34 (H1N1) virus to protect against influenza was compared in BALB/c mice. The plasmid DNA encoded hemagglutinin (HA), neuraminidase (NA), matrix protein (M1), nucleoprotein (NP) or nonstructural protein (NS1) in a chicken beta-actin-based expression vector (pCAGGS). Each DNA was inoculated twice 3 weeks apart at a dose of 1 microgram per mouse by particle-mediated DNA transfer to the epidermis (gene gun). Seven days after a second immunization, mice were challenged with the homologous virus and the ability of each DNA to protect mice from influenza was evaluated by decreased lung virus titers and increased survival. Mice, given HA- or NA-expressing DNA, induced a high level of specific antibody response and protected well against the challenge virus. On the other hand, mice given M1-, NP-, or NS1-DNA failed to provide protection, although M1- and NP-DNAs did induce detectable antibody responses. These results indicate that both HA- and NA-expressing DNAs for the surface glycoproteins are most protective against influenza from among the various viral protein-expressing DNAs used here.

Protection and antibody responses in different strains of mouse immunized with plasmid DNAs encoding influenza virus haemagglutinin, neuraminidase and nucleoprotein.

Protection against influenza virus infection and antibody responses in mice vaccinated with plasmid DNAs encoding haemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) were compared among BALB/c (H-2d), B10 (H-2b) and C3H (H-2k) mice. Mice were inoculated with each DNA construct twice, 3 weeks apart, at a dose of 1 microg per mouse by particle-mediated DNA transfer (gene gun) to the epidermis. They were challenged with a lethal dose of the homologous virus 7 days after the second vaccination. NA-DNA provided significant protection in all strains of mouse, whereas HA-DNA afforded significant protection only in BALB/c mice. The serum antibody titres against NA or HA molecules in BALB/c, C3H and B10 mice were high, intermediate and low, respectively. NP-DNA failed to provide protection in any strain of mouse, and elicited low titres of anti-NP antibodies. These results suggest that NA-DNA can be used as a vaccine component to provide effective protection against influenza virus infection in various strains of mouse.

Roles of anti-hemagglutinin IgA and IgG antibodies in different sites of the respiratory tract of vaccinated mice in preventing lethal influenza pneumonia.

The roles of IgA and IgG antibodies (Abs) against hemagglutinin (HA) in the prevention of lethal influenza pneumonia in vaccinated mice were examined in terms of distribution and concentration of the Abs in the mucus or the serous fluid in different sites of the respiratory tract (RT), mucosa of the nose, trachea, bronchi and bronchioli and the alveolar epithelia of pulmonary acinus. First, the surface areas of the tracheal, bronchial and bronchiolar mucosa and alveolar epithelia were measured to be 20, 260 and 217, 433 mm(2), respectively, using serial tissue sections of the trachea and lungs. Then, the volumes of the tracheal mucus, the bronchial and bronchiolar mucus and the serous fluid of alveolar epithelia were estimated to be 0.2, 2.6 and 21.7 mm(3), respectively, by calculating each from the surface area and an assumed thickness of the mucus layer (0.01 mm) or that of the serous fluid (0.0001 mm). Next, anti-HA IgA and IgG Ab responses in the nasal wash, the trachea-lung wash and the trachea wash were measured in BALB/c mice immunized intranasally with an adjuvant-combined A/PR/8/34 (H1N1) virus vaccine and challenged with a lethal dose of the virus. Then the values of Ab responses were converted to the mucus and serous fluid Ab concentration based on two premises that the serum Abs diffuse at a constant rate to the surface of the tracheal, bronchial and bronchiolar mucosa, and that the active transepithelial transport of IgA Abs does not work in the alveolar epithelia. Results showed that 21.4 microg/ml IgA Abs and 3.6 microg/ml IgG Abs in the tracheal mucus (19.1 and 0.3% of the trachea-lung wash IgA and IgG Ab amounts, respectively), 5.9 microg/ml IgA Abs and 3.6 microg/ml IgG Abs in the bronchial and bronchiolar mucus (66.0 and 3.4% of the trachea-lung wash IgA and IgG Ab amounts, respectively) and about 0.1 microg/ml IgA Abs and 12.3 microg/ml IgG Abs in the serous fluid of alveolar epithelia (14.9 and 96.3% of the trachea-lung wash IgA and IgG Ab amounts, respectively) were present in the vaccinated mice, at which concentrations influenza pneumonia was prevented. Thus, 96.3% of anti-HA IgG Abs in the trachea-lung wash work on the alveolar epithelia, whose surface area is about 800 times larger than that of tracheal, bronchial and bronchiolar mucosa and seem to play a more important role than the mucosal IgA Abs in the prevention of lethal influenza pneumonia.

A Nontoxic Chimeric Enterotoxin Adjuvant Induces Protective Immunity in Both Mucosal and Systemic Compartments with Reduced IgE Antibodies

A novel nontoxic form of chimeric mucosal adjuvant that combines the A subunit of mutant cholera toxin E112K with the pentameric B subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli was constructed by use of the Brevibacillus choshinensis expression system (mCTA/LTB). Nasal immunization of mice with tetanus toxoid (TT) plus mCTA/LTB elicited significant TT-specific immunoglobulin A responses in mucosal compartments and induced high serum immunoglobulin G and immunoglobulin A anti-TT antibody responses. Although TT plus native CT induced high total and TT-specific immunoglobulin E responses, use of the chimera molecule as mucosal adjuvant did not. Furthermore, all mice immunized with TT plus mCTA/LTB were protected from lethal systemic challenge with tetanus toxin. Importantly, the mice were completely protected from influenza virus infection after nasal immunization with inactivated influenza vaccine together with mCTA/LTB. These results show that B. choshinensis-derived mCTA/LTB is an effective and safe mucosal adjuvant for the induction of protective immunity against potent bacterial exotoxin and influenza virus infection.

Protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing DNAs with electroporation

Electroporation for the transfer of plasmid DNA encoding influenza virus hemagglutinin (HA) into muscle or nasal mucosa was tried in BALB/c mice to examine the efficacy of this method for inducing anti-HA immune responses and providing protection against homologous A/PR/8/34 (PR8) virus infection. Mice were immunized by two injections, 3 weeks apart, of HA-DNA with electroporation into the muscle wherein a pair of electrode needles was inserted to deliver the electric pulses. One or 3 weeks after the immunization, the mice were infected with a lethal dose of the PR8 virus. Ten micrograms or more of HA-DNA/dose induced strong serum anti-HA IgG antibody (Ab) responses, in which both IgG1 and IgG2a were predominant, and weak cytotoxic T lymphocyte responses. These immune responses were sufficient to provide efficient protection against the lethal infection. In addition, mice were immunized by dropping HA-DNA (12 microg) three times, 2 weeks between each dose into nostrils where each of two electrode needles was placed on the right nostril or the palate. One week after the immunization, the mice were infected with a sublethal dose of the PR8 virus. The DNA immunization by electroporation provided reduced nasal virus titers, in parallel with a relatively high levels of serum anti-HA IgG Ab and a slight nasal anti-HA IgA Ab production. The intranasal administration of cholera toxin before HA-DNA immunization by electroporation enhanced the nasal IgA Ab production together with enhancement of the efficiency of protection. These results suggest that electroporation can be used as one of the efficient gene delivery systems for the transfer of influenza DNA-vaccine into muscle or nasal mucosa to provide protection against influenza virus infection.

Enhanced resistancy of thioredoxin-transgenic mice against influenza virus-induced pneumonia

Thioredoxin (TRX) is a small redox-active protein with anti-oxidant effect and redox-regulating functions. Using TRX transgenic (Tg) mice in which human TRX is overexpressed systemically under the control of beta-actin promoter, the effects of influenza virus infection were examined in TRX Tg mice and wild type C57BL/6 mice. (1) Median lethal dose (LD50) against influenza virus infection in wild-type C57BL/6 mice was 10(-5.3) dilution, while that of TRX Tg mice was 10(-4.2) dilution. Thus, TRX Tg mice were more resistant against the virus infection than wild-type mice. (2) The body weights of wild-type mice 7 days after infection with a sublethal dose of the virus (10(-6) dilution) decreased significantly, whereas those of TRX Tg mice increased slightly. (3) Histopathology of the lung at 3 weeks after sublethal infection of influenza virus showed that severe alveolar or bronchiolar destruction was observed in wild-type mice, while mild viral pneumonia was seen in the TRX Tg mice. (4) Local (IgA) and systemic (IgG) antibody productions against influenza virus hemagglutinin in mice surviving 3 weeks after infection were similar between wild-type and TRX Tg mice. These results indicate that overexpression of TRX in Tg mice suppresses the inflammatory overshoot of viral pneumonia caused by influenza virus infection, resulting in the reduction of mortality without affecting the hosts systemic immune responses to the infection. TRX may play some important roles in regulating the inflammatory process in the primary host defense against infection.