Frederik W. van Ginkel

Cutting Edge: The Mucosal Adjuvant Cholera Toxin Redirects Vaccine Proteins into Olfactory Tissues

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT (125I-CT) or CT-B subunit (125I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with 125I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas 125I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

Immunizations with pneumococcal surface protein A and pneumolysin are protective against pneumonia in a murine model of pulmonary infection with Streptococcus pneumoniae

Intranasal infection of mice with certain strains of capsular group 19 Streptococcus pneumoniae can result in focal pneumonia in the absence of bacteremia. Using this model of murine pneumonia, we demonstrated that immunization with recombinant forms of either pneumococcal surface protein A (PspA) or PdB (a genetically detoxified derivative of pneumolysin) elicited significant protection against focal pulmonary infection. This may be the first demonstration that a proposed vaccine antigen can protect against pneumococcal pneumonia. The best protection was obtained by immunizing mice with a mixture of PspA and PdB, indicating that the protection elicited by these antigens can complement each other. This result is in agreement with previous studies that used pneumococcal sepsis and nasal colonization models and demonstrate that the best protein vaccines for prevention of infection may be those that include more than one protection-eliciting pneumococcal protein.

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.

Heterosubtypic Immunity to Influenza A Virus Infection Requires B Cells but Not CD8+ Cytotoxic T Lymphocytes

Heterosubtypic immunity (HSI), defined as protective cross-reactivity to lethal infection with influenza A virus of a serotype different from the virus initially encountered, is thought to be mediated by cross-reactive cytotoxic T lymphocytes (CTL). This study provides direct evidence for the role of effector CTL versus B cells in HSI in mice with a targeted disruption in the alpha chain of CD8 molecule (CD8(+) T cell deficient) or the immunoglobulin mu heavy chain (B cell deficient), respectively. CD8(+) T cell-deficient mice developed complete HSI. These mice displayed normal humoral immune responses, as determined by titers of subtype cross-reactive antibodies and virus-neutralizing antibodies specific for the immunizing influenza strain. In contrast, HSI was not observed in B cell-deficient mice, although these mice could mount cross-reactive CTL responses. These results show that B cells are required for HSI and provide new insight into the mechanisms of HSI, with significant implications in vaccine development.

Pneumococcal carriage results in ganglioside-mediated olfactory tissue infection

Streptococcus pneumoniae cause considerable morbidity and mortality, with persistent neurological sequelae, particularly in young children and the elderly. It is widely assumed that carriage occurs through direct mucosal colonization from the environment whereas meningitis results from invasion from the blood. However, the results of published studies can be interpreted that pneumococci may enter the brain directly from the nasal cavity by axonal transport through olfactory nerves. This hypothesis is based on findings that (i) teichoic acid of the pneumococcal cell wall interact with gangliosides (GLS), (ii) the interaction of GLS with cholera toxin leads to axonal transport through the olfactory nerves into the brain, and (iii) viruses enter the brain through axonal transport into olfactory nerves. After nasal inoculation, we observe high numbers of pneumococci in nasal washes and the olfactory nerves and epithelium. Significant numbers of pneumococci also infected the olfactory bulbs, brain, and the trigeminal ganglia. The absence of bacteremia in this model makes it unlikely that the bacteria entered the brain from the blood stream. Recovery of colony-forming units from the brain, lungs, olfactory nerves, and epithelium and nasal washes was inhibited by incubating pneumococci with GLS before nasal inoculation. These findings, confirmed by PCR and immunohistochemistry, support a GLS-mediated process of infection and are consistent with pneumococci reaching the brain through retrograde axonal transport.

Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci

ABSTRACT We demonstrated that during colonization with Streptococcus pneumoniae the nasal mucosal tissues of mice support two populations of pneumococci. Transparent-phase pneumococci can be readily washed from the outer surface, while a second population composed of primarily opaque-phase pneumococci is released only by homogenization of the nasal tissue. The fact that the opaque phase has previously been associated with invasion and the fact that opaque-phase pneumococci were released by homogenization of previously washed nasal tissue suggest that the opaque-phase pneumococci may have invaded the nasal tissue. Consistent with this hypothesis was our observation that there was inflammation in portions of the nasal mucosa of the colonized mice but not in the mucosa of noncolonized mice, but this observation did not prove the hypothesis. If the opaque-phase pneumococci released from the nasal tissue were from within the tissue and/or if resistance of the opaque-phase subpopulation to antibody, complement, and phagocytes is essential for long-term carriage, it seems likely that the virulence factors of S. pneumoniae that are necessary for killing humans exist to facilitate carriage. Although this speculation is unproven, the observation that there are separate populations of pneumococci during colonization may help guide future attempts to understand the biology of nasal colonization by this pathogen.

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.

Complement-Dependent Acute-Phase Expression of C-Reactive Protein and Serum Amyloid P-Component

The acute-phase response (APR) is regulated by TNF-α, IL-1β, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1β and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1β gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1β to promote up-regulation of the CRP and SAP genes.

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.

Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice

ABSTRACT Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/− mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/− mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.