Mucosal immunity and vaccine development

Mucosae constitute the major entry for most microbial pathogens but also innocuous antigens derived from ingested food, airborne matter or commensal bacteria. A large and highly specialized innate and adaptative mucosal immune system protects the mucosal surfaces and the body interior from potential injuries from the environment. The mucosal immune system has developed a variety of immune mechanisms to discriminate between non-pathogenic and pathogenic invaders. It is able to maintain tolerance against the plethora of environmental antigens and to induce potent protective immunity to avoid mucosal colonisation and organism invasion by dangerous microbial pathogens. Mucosal immunisation with appropriate antigens and immunostimulatory molecules may induce potent protective immunity against harmful pathogens. Alternatively, mucosally-induced tolerance against auto-antigens or allergens may be generated by mucosal administration of these antigens alone or with immunomodulators potentiating regulatory responses. Here, we review the properties of the mucosal immune system and briefly discuss the advances in the development of mucosal vaccines for protection against infections and for the treatment of inflammatory disorders such as autoimmune diseases or type I allergies.     

Ultrafiltration improves ELISA and Endopep MS analysis of botulinum neurotoxin type A in drinking water

The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260 pg/mL and 21 pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5 μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7 pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Binding of Clostridium botulinum neurotoxin to gangliosides

The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides. These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes.     

Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

We recently reported the primary structures, antimicrobial activities and cDNA precursors of nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorranaishikawae. Their cDNA clones revealed a highly conserved approximately 60 bp region upstream of the start codon. This conserved region was used in the “shotgun” cDNA cloning method to reveal additional cDNAs encoding novel antimicrobial peptides of O.ishikawae. After sequencing 344 clones, we identified novel 13 cDNAs encoding dermal peptides in addition to the previously identified nine antimicrobial peptides. These 13 unique cDNAs encoded precursor proteins each containing a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg/Lys processing site and a dermal peptide at the C-terminus. The dermal peptides were members of the palustrin-2 (two peptides; termed palustrin-2ISc and palustrin-2ISd), nigrocin-2 (one peptide; nigrocin-2ISc), brevinin-1 (one peptide; brevinin-1ISa), odorranain-M (one peptide; odorranain-MISa) and entirely novel peptides (eight peptides; ishikawain-1-8). Although palustrin-2ISd and odorranain-MISa had few antimicrobial activities, palustrin-2ISc and nigrocin-2ISc possessed a broad-spectrum of growth inhibition against bacteria. Brevinin-1ISa had the most potent antimicrobial activities against the Gram-positive bacteria and the fungus but not the Gram-negative bacterium, Escherichiacoli. However, eight novel peptides showed no growth inhibition against these microorganisms.     

DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.     

Sublingual mucosa: A new vaccination route for systemic and mucosal immunity

Needle-free vaccine delivery has become a global priority, both to eliminate the risk of improper and unsafe needle use and to simplify vaccination procedures. In pursuit of greater ease of vaccination, a number of needle-free delivery routes have been explored, with mucosal routes being perhaps the most prominent. Since the vaccine administration route significantly affects immune responses, numerous researchers are attempting to develop alternative vaccine delivery methods including a mucosal route. My group's recent studies demonstrate the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Sublingual administration conferred effective protection against a lethal challenge with influenza virus (H1N1) or genital papillomavirus. Moreover, CCR7-CCL19/CCL21-regulated dendritic cells are responsible for activation of T and B cells following s.l. administration. This review highlights current knowledge about the safety and effectiveness of s.l. vaccination and describes how s.l. vaccination can induce both systemic and mucosal immunity.     

Enzymatic autocatalysis of botulinum A neurotoxin light chain

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.     

Nature of intracellular type A botulinum neurotoxin.

The neurotoxin in cells of young Clostridium botulinum type A culture was extracted with lysozyme. Highly purified neurotoxin preparation, obtained by processing the extract in two chromatographic steps had only unnicked (single-chain) molecules of molecular weight comparable to that of the dichains isolated from type A crystals. Trypsinization converted the unnicked molecules into dichains whose component subunits were of sizes indistinguishable from those of the neurotoxin from crystals. The enzymatic treatment increased toxicity of crude extract 30-fold but did not activate the purified intracellular neurotoxin preparation. The results indicated that intracellular type A botulinum neurotoxin is unnicked, is not fully activated, and is activated in the time between its extraction and purification. Since trypsinization nicked all of the single chains without increasing toxicity, nicking was not causally related to toxicity activation.     

Benzoquinones as inhibitors of botulinum neurotoxin serotype A

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC₅₀ values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC₅₀ competitive inhibitors that depend on the BQ moiety for activity.     

Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin

Cholera toxin (CT) has been found to be an extremely potent immunogen for mucosal IgA responses when administered via the intestine. This study has examined both mucosal and systemic immune responses after feeding CT and compared these responses with those obtained after feeding keyhole limpet hemocyanin (KLH), another protein that is strongly immunogenic in mice. Feeding CT to mice resulted not only in IgA antibody in intestinal secretions but also resulted in substantial plasma IgG and IgA antibody levels. Feeding KLH in much larger quantity resulted in little or no antibody response in intestinal secretions or plasma. Lymphoid cells from various tissues of mice fed CT were cultured in vitro for 10 days and the supernatant was tested for antibody to CT. Spontaneous antibody synthesis (no antigen added to cultures) was present in cultures of each cell type, but IgG anti-CT was found mainly in cultures of spleen and mesenteric lymph node cells and IgA anti-CT mainly in cultures of Peyer's patch and lamina propria cells. Peyer's patch cells cultured with CT as antigen synthesized both IgG and IgA anti-CT, suggesting that the antibody response to both isotypes originated in this site. Helper T cell activity for both IgA and IgG anti-CT was detected in spleens, mesenteric lymph nodes, and Peyer's patches. Lastly, when KLH and CT were fed to mice at the same time, an intestinal IgA anti-KLH and plasma IgG anti-KLH response was stimulated, a response pattern similar to that occurring to CT after CT was fed alone. We conclude that mucosal stimulation by CT generates both a systemic IgG and mucosal IgA response to this antigen, and that CT can cause a similar pattern of response to an unrelated protein antigen when both are administered into the intestine at the same time. The data favor the idea that both the IgG and IgA responses originate in GALT and then disseminate to other tissues. We propose that CT accomplishes these effects by altering the regulatory environment within GALT.     

Characterization of the subunits of botulinum neurotoxin type A

A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out. The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold. In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified. Alanine was found to be the N-terminus of the heavy chain. The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains. The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.     

A型肉毒神经毒素理化及生物学特性研究

目的探究A型肉毒神经毒素的理化及生物学特性,为A型肉毒神经毒素制品的研制提供依据。方法采用SDS-PAGE、HPLC测定A型肉毒神经毒素的纯度;采用毛细管凝胶电泳分析其在非还原条件及还原条件下各组分的相对分子质量;采用等电点聚焦电泳测定其等电点;对其N-末端氨基酸序列进行测序,并将测序结果与NCBI blast数据库中A型肉毒梭菌Hall株的氨基酸序列作比对,确证其各组分的成分;对A型肉毒毒素复合物及A型肉毒神经毒素进行口服毒性分析,并对3批A型肉毒神经毒素原液和其半成品进行生物学稳定性研究。结果 A型肉毒神经毒素纯度99.00%;非还原条件下其相对分子质量约为152 000,N-末端氨基酸序列ALNDLQINVN,为完整的A型肉毒神经毒素;还原条件下,由相对分子质量约为101 000、N-末端氨基酸序列为ALNDLQINVN的重链和相对分子质量约为101 000、N-末端氨基酸序列为PFVNKQFNYK的轻链组成;等电点为4.91;口服A型肉毒神经毒素的小鼠LD50为1.50×107U/kg,是其复合物的7.9倍;3批A型肉毒神经毒素原液在2~8℃放置28 d生物学活性无下降,平均比活性为2.13×108U/mg,是其复合物的7.1倍;3批半成品在18~26℃放置28 d生物学活性无下降。结论 A型肉毒神经毒素纯度较高,非还原条件下为单链,还原条件下裂解为轻链和重链,其原液及其半成品的生物学稳定性较好。     

Structure and biological activity of botulinum neurotoxin

Botulinum neurotoxin appears to undergo structural alterations after synthesis and also before it inhibits neurotransmitter release. Discussions and conjectures are presented in this context: 1. At what sites on the 150 kDa neurotoxin does posttranslational proteolytic processing occur? 2. Does neurotransmitter inhibition depend on separation of a segment of the neurotoxin from the rest of the molecule? 3. At what step in the intoxication pathway does activation of neurotoxin (enhanced lethality following limited proteolysis) manifest? 4. Can the receptor binding parameters (based on bovine brain synaptosome and lipid membrane), channel forming property (lipid bilayer membrane) and intracellular inhibitory activity (based on permeabilized chromaffin and PC 12 cells) provide clues to differences in the lethal potency between the neurotoxin serotypes? In addition, the following issues are considered: 5. The spontaneous fragmentation of isolated 50 kDa light chain, after its separation from 100 kDa heavy chain, 6. Effect of specific chemical modification of Arg, His, Lys, Trp, Tyr and Asp/Glu residues of types A, B and E neurotoxins on lethality and antigenicity, and 7. Development of second generation toxoids.     

Proteolysis of synthetic peptides by type A botulinum neurotoxin

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.     

Mechanism of substrate recognition by botulinum neurotoxin serotype A

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting neurotransmitter-carrying vesicle fusion to the plasma membrane of peripheral neurons. Unlike other zinc proteases, BoNTs recognize extended regions of SNAP25 for cleavage; however, the molecular basis for this extended substrate recognition is unclear. Here, we define a multistep mechanism for recognition and cleavage of SNAP25 by BoNT/A. SNAP25 initially binds along the belt region of BoNT/A, which aligns the P5 residue to the S5 pocket at the periphery of the active site. Although the exact order of each step of recognition of SNAP25 by BoNT/A at the active site is not clear, the initial binding could subsequently orient the P4'-residue of SNAP25 to form a salt bridge with the S4'-residue, which opens the active site allowing the P1'-residue access to the S1'-pocket. Subsequent hydrophobic interactions between the P3 residue of SNAP25 and the S3 pocket optimize alignment of the scissile bond for cleavage. This explains how the BoNTs recognize and cleave specific coiled SNARE substrates and provides insight into the development of inhibitors to prevent botulism.     

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

AIMS: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). METHODS AND RESULTS: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. CONCLUSION: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.     

A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.