Yukari Hagiwara

A dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants

In the development of mucosal vaccines, cholera toxin (CT) has been shown to be an effective adjuvant and to induce both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its innate toxicity. Recent vaccine development efforts have emphasized nasal application of antigen and CT for the induction of mucosal IgA responses. When we examined potential toxicity of CT for the central nervous system (CNS), both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development.

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.

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.

Enterotoxin-Based Mucosal Adjuvants Alter Antigen Trafficking and Induce Inflammatory Responses in the Nasal Tract

ABSTRACT The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1β or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

Protective Mucosal Immunity in Aging Is Associated with Functional CD4+ T Cells in Nasopharyngeal-Associated Lymphoreticular Tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4+ T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4+, CD45RB+ T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer’s patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.

Genetically manipulated bacterial toxin as a new generation mucosal adjuvant.

Cholera toxin (CT) and heat‐labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co‐administered protein antigen (Ag). Both LT and CT induce B7‐2 expression on antigen‐presenting cells (APCs) for subsequent co‐stimulatory signalling to CD4+ T cells. CT directly affects CD4+ T cells activated via the TCR–CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)‐4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL‐4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4+ Th2 cells, which provided effective help for the Ag‐specific mucosal immunoglobulin (Ig)A, as well as serum IgG1, IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7‐2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4+ T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.

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.

Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin).

Inactivated influenza vaccine was administered intranasally to BALB/c mice together with an adjuvant (cholera toxin B subunit [CTB] supplemented with a trace amount of the whole toxin, CTB*) and its ability to induce innate immunity and confer protection against influenza was examined. Nasal wash virus titres 3 days after inoculation of homologous viruses were measured as an index of the ability of the vaccine to confer protection in mice immunized with either CTB*-combined vaccine or CTB* alone 1-21 days previously. The results were as follows. (1) Partial but significant reduction of the nasal-wash virus titres (prevention) was detected beginning 3 days after the vaccination, that is, 2 days earlier than the appearance of both virus-specific antibody-forming cells (AFCs) in the nasal-associated lymphoid tissue (NALT) and virus-specific IgA antibody responses in the nasal washes of mice immunized with the CTB*-combined vaccine. (2) The protection, detected on day 3 and peaking on day 5 but lost by day 21, was also conferred in mice immunized with CTB* alone. (3) The non-specific prevention was detected at doses of more than 0.3 microg of CTB*/mouse. (4) The nonspecific protection beginning 3 days after the immunization involved the enhanced expression of cytokine mRNAs (IL-15 and IL-18), considered responsible for natural killer (NK) cell activation, by the non-T cell populations in the NALT. (5) Normal NALT cells, when cultured in vitro with CTB*, secreted IL-1beta within a few hours in culture. These results demonstrate that the CTB*-combined vaccine, when given intranasally into mice, can confer nonspecific protection against influenza beginning 3 days after the vaccination and that CTB* also possessed this ability to confer protection non-specifically and temporarily by inducing the secretion of IL-1beta, one of the most important cytokines that initiates both innate and adaptive immunity, and also NK cell activity.

Effects of intranasal administration of cholera toxin (or Escherichia coli heat-labile enterotoxin) B subunits supplemented with a trace amount of the holotoxin on the brain.

Effects of intranasal administration of cholera toxin (CT) [or Escherichia coli heat-labile enterotoxin (LT)] B subunits supplemented with a trace amount of the holotoxin, CTB* or LTB*, on the brain were examined in BALB/c mice by comparing with those of the intracerebral injection. Intracerebral injection of CTB* at doses more than 10 microg/mouse caused significant body weight loss and dose-dependent death within 7 days, with localization of conjugates of horseradish peroxidase with CTB (HRP-CTB) in the ventricular system and in the perineural space of olfactory nerves of the nasal mucosa 3 h after injection. Intracerebral injection of CTB* at doses less than 3 microg/mouse (or LTB* at doses less than 22.7 microg/mouse) did not cause any significant body weight loss for 7 days, with localization of HRP-CTB in the brain but not in the nasal mucosa. On the other hand, intranasal administration of 10 microg of CTB* caused localization of HRP-CTB in the nasal mucosa but not in the brain 3 h after administration and caused body weight loss even after 30 administrations. Neither any histological changes of brain tissues nor marked changes in serum biochemical parameters were found in mice after the 30 administrations of CTB* or LTB*. These results suggest that 0.1 microg of CTB* or LTB*, which is known to be close to the minimal effective dose as an adjuvant for nasal influenza vaccine in mice and corresponds to 100 microg per person, can be used as a safe nasal adjuvant without adversely affecting the brain.

Mutants of cholera toxin as an effective and safe adjuvant for nasal influenza vaccine

The effectiveness and safety of mutants of cholera toxin (CT) as an adjuvant for nasal influenza vaccine was examined. Four CT mutants, called CT7 K (Arg to Lys), CT61F (Arg to Phe), CT112 K (Glu to Lys), and CT118E (Glu to Gln), were produced by the replacement of one amino acid at the A1-subunit using site-directed mutagenesis. All these mutants were confirmed to be less toxic than native CT when the toxicity was analysed by using Y1 adrenal cells in vitro. When high (1 microg) and low (0.1 microg) doses of these CT mutants, together with high (1 microg) and low (0.1 microg) doses of influenza A/PR/8/34 virus (H1N1) vaccine, respectively, were administered intranasally into BALB/c mice in a two dose regimen (twice, 4 weeks apart), they produced both anti-PR8 hemagglutinin (HA) IgA and IgG antibody (Ab) responses roughly in a dose-dependent manner. The relatively low level of anti-HA Ab responses, induced by the low dose CT mutants, were enough to provide complete protection against the homologous virus infection. Under these vaccination conditions, no anti-CTB IgE Ab responses were induced. The mutant CT112 K, which showed a relatively high adjuvant activity, the lowest toxicity and relatively high yields in a bacterial culture, seems to be the most effective and safest adjuvant for nasal influenza vaccine among those examined. The low dose of CT derivatives or vaccine used in the mouse model (0.1 microg/20 g mouse) corresponded to 100 microg/20 kg, the estimated dose per person. A tentative plan for safety standards for human use of CT (or LT) derivatives as an adjuvant of nasal influenza vaccine is discussed.