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Mucosal Vaccine Targeting Improves Onset of Mucosal and Systemic Immunity to Botulinum Neurotoxin A¹

Massimo Maddaloni^*, Herman F. Staats, Dagmara Mierzejewska, Teri Hoyt^*, Amy Robinson^*, Gayle Callis^*, Shunji Kozaki, Hiroshi Kiyono^¶, Jerry R. McGhee^||, Kohtaro Fujihashi^|| and David W. Pascual^2,*

^* Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717; Department of Pathology, Duke University Medical Center, Durham, NC 27710; Department of Food Chemistry, Institute of Food Research, Polish Academy of Science, Olsztyn, Poland; Laboratory of Veterinary Epidemiology, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai-shi, Osaka, Japan; ^¶ Department of Microbiology and Immunology, Division of Mucosal Immunology, Institute for Medical Sciences, University of Tokyo, Tokyo, Japan; and ^|| Department of Microbiology and Department of Pediatric Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a -trefoil (Hctre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hctre or Hctre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hctre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hctre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hctre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hctre-dosed mice. Mice immunized with adjuvanted Hctre-Ad2F or Hctre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hctre-Ad2F or Hctre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hctre Ab titers even in the absence of adjuvant. This study shows that the Hctre structure can confer protective immunity and that use of Hctre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Botulism occurs from the infection by Clostridium botulinum or by ingestion of its neurotoxin complex, resulting in flaccid paralysis (1, 2). Due to its potency, the illicit dissemination of botulinum neurotoxins (BoNT)³ poses a major threat to the unprotected populace. Of the seven serotypes, serotypes A, B, and, to a minor extent, E are typically associated with botulism in humans, whereas serotype C mostly affects domestic animals (1, 2). The toxins are each naturally synthesized as a single 150-kDa polypeptide, requiring posttranslational proteolysis to yield a 50-kDa fragment or L chain, containing the catalytic activity, and a 100-kDa component or H chain (100 kDa), containing the translocation domain in the N terminus and the cell-binding domain in the C terminus (Hc). Upon binding to the target cell, the translocation domain in the N terminus translocates the L chain to the cytosol, and the L chain endopeptidase activity inactivates a group of proteins (termed SNARE proteins) required for release of acetylcholine at the neuromuscular junction resulting in flaccid paralysis (3, 4, 5, 6).

The current vaccine is a pentavalent one with as little at 10% of the toxoid preparation representing neurotoxoid (7), suggesting that much of the immune response induced by the vaccine is directed against vaccine components that will not contribute to protection against BoNT lethal challenge. Because formalin is used to inactivate BoNT to produce the toxoids for vaccine formulation, such chemical modification of the toxins results in a considerably altered tertiary structure (8) needed to elicit neutralizing Abs (9, 10, 11). Thus, better formulations are needed. To this end, structural analysis of the catalytic and binding sites of C. botulinum BoNT serotype B reveals that the Hc is structurally subdivided into a N-terminal subdomain, forming a jelly-roll motif, and a C-terminal subdomain, forming -trefoil structure (5). We hypothesize that this -trefoil structure contains the important neutralizing epitopes. In fact, in a previous study, neutralizing Abs against Hc BoNT serotype A (BoNT/A) exhibited two major protective epitopes, BoNT/A_455–661 and BoNT/A_1150–1289, which conferred 75% protection, and one minor protective epitope, BoNT/A_780–939, which conferred 28% protection (9). The neutralizing epitope BoNT/A_1150–1289 conferred 75% protection and spanned about two-thirds of the -trefoil region. Taken together, these results hint that the intact -trefoil structure would be a potential candidate for an optimized vaccine.

Replication-deficient adenovirus vectors (12) and other viruses (13) have been experimentally used to correct genetic deficiencies, but these viral vectors have remained, in large part, immunogenic. Despite this condition, adenovirus vectors retain the advantage of transfecting the airway epithelium (14, 15). However, examination of the adenovirus components shows that adenovirus adhesion is mediated by the interaction between its attachment or fiber protein and receptors on the cell surface (15). Study of adenovirus (16) and reovirus (17) attachment proteins reveals that they share a common feature in that they are homotrimeric to form a high-energy structure that springs open upon binding its cell receptor, thus triggering a series of events ultimately leading to the entry of the virus into the cell. Therefore, it may be possible that adenovirus 2 fiber protein (Ad2F) could potentially be used in lieu of the intact virus for mucosal targeting.

In an effort to improve the current BoNT vaccine, we adopted a two-prong approach to create a mucosal vaccine against BoNT/A. First, we questioned whether the -trefoil structure, which is responsible for the cell-binding activity, would be sufficient to elicit neutralizing Abs because this structure is maintained in all serotypes, as well as in tetanus toxoid (18). Accordingly, our vaccine includes this -trefoil structure. In addition, we hypothesize that a fusion protein between this -trefoil structure from BoNT/A genetically fused to adhesin Ad2F attachment protein may enhance its immunogenicity and uptake by the common mucosal immune system.

In this study, we showed that the addition of the mucosal-targeting molecule, Ad2F, enhanced onset of plasma and mucosal Ab responses. Both vaccines (with and without Ad2F) conferred complete protection from systemic challenge with as much as 20,000 LD₅₀ of the native BoNT/A. Upon evaluation of mucosal Abs to the immunodominant Hc as a -trefoil (Hctre) structure, mice dosed with Hctre-Ad2F showed the best protection against the BoNT/A complex.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
DNA manipulations

As shown by others (19), initial attempts to express the C. botulinum gene encoding the Hc BoNT/A gene in yeast failed to yield any protein. Consequently, a synthetic gene encoding for Hc BoNT/A amino acids R864 to L1295 (GenBank accession no. X52066) was designed for optimized expression in the yeast Pichia pastoris. Factors taken into account for the synthetic gene were yeast codon bias, the reduction of C. botulinum A/T content, and the necessity for not depleting any particular tRNA pools. The Hctre spans from position S1096 to L1295, as predicted by others (18). To clone the Hctre domain, we included 15 more amino acids upstream from the predicted Hctre beginning with K1076 to L1295 to facilitate proper folding of the relevant domain.

To clone the Hc BoNT/A gene into the expression vector, the synthetic gene encoding for Hc BoNT/A was amplified with primers containing EcoRI and SalI restriction sites. Likewise, to clone the Hctre, this gene was amplified from the synthetic Hc BoNT/A using primers containing EcoRI and SalI restriction sites. In both cases, the 5' primers containing the EcoRI site also provided an ATG initiation codon embedded into an optimal Kozak’s sequence. The PCR products were cloned into a pCRII TOPO cloning vector (Invitrogen Life Technologies), excised with EcoRI and SalI, and cloned into the P. pastoris expression vector pPICZ B cut with EcoRI and XhoI. Such a vector is designed to provide a C-terminal histidine tag for subsequent protein purification. Adenovirus 2 fiber was amplified from genomic adenovirus 2 DNA (a gift from Dr. J. Chroboczek, European Molecular Biology Laboratory, Grenoble, France). Attempts to express the entire Ad2F were unsuccessful; thus, only the C-terminal region, starting from G378 to E582, was cloned. This region encompasses 1) a short stretch of amino acids rich in Gly (4 of 15), 2) the region containing the trimerizing domain, and 3) the region containing the globular domain, commonly referred to as the "knob," which is important for interacting with the coxsackievirus/adenovirus receptor on the cell surface (15). This region proved to be easily expressed in P. pastoris, even when fused to a variety of Ags or fluorescent proteins.

To make Hctre-Ad2F, Hctre was amplified with primers generating EcoRI and SalI ends cloned and excised with EcoRI and SalI. The mentioned region of Ad2F was amplified with primer generating SalI and KpnI ends, cloned, and excised with the corresponding enzymes. The vector pPICZ B was cut with EcoRI and KpnI. Finally, Hctre-Ad2F was assembled via a tripartite ligation. Primers were designed to allow in-frame junction between the different components and the formation of a flexible joint between Hctre and the Ad2F. To make Red2-Ad2F, a similar strategy was adopted. Red2 gene was amplified with primers generating EcoRI and SalI ends using pDsRed2 (BD Biosciences) as template, and fused to Ad2F as described. These constructs were transformed into P. pastoris by electroporation.

Protein expression, purification, and FACS analysis

Recombinant proteins were produced by shifting the carbon source from glycerol to methanol, as required with P. pastoris. Briefly, yeast cells were cultured in yeast nitrogen base-glycerol until OD 0.5–1.0. Cells were then harvested by centrifugation and resuspended in yeast nitrogen base-methanol for 36–48 h. Pelleted biomass was either used right away or stored at –80°C until needed. Cells were disrupted with a bead-beater in ice, and debris were cleared by centrifugation, followed by filtration through a 1.2-µm prefilter, then by filtration through a 0.45-µm filter under vacuum. The cleared supernatant was then applied to a Talon column (BD Biosciences), as per the manufacturer’s instruction. Purified proteins were eluted, titrated, and loaded onto a 12% polyacrylamide gel. Recombinant Hctre proteins were analyzed by Coomassie-stained SDS-PAGE to assess the quality of the protein. The Red2-Ad2F was blotted and probed with a commercial rabbit anti-Red2 Abs (BD Biosciences).

Recombinant Ad2F-binding assay

The mouse fibroblast L929 cells, human HeLa cells, and human 293 cells (American Type Culture Collection) were grown according to the complete medium (CM): RPMI 1640 (Invitrogen Life Technologies), 10% FBS (Atlanta Biologicals), 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin. Confluent cells were detached by trypsinization, washed, and resuspended in FACS buffer (10 g/L of Dulbecco’s PBS (Sigma-Aldrich) plus 2% FBS). Cells were incubated with 25 µg of Red2-Ad2F or Red2 (BD Clontech), and incubated at room temperature for 45 min. Cells were washed once with 3 ml of FACS buffer and resuspended in 200 µl of FACS buffer. Bound fluorescence was analyzed with a FACSCalibur (BD Biosciences).

Histological preparation

Mice were nasally dosed with 50 µg Red2-Ad2F, and after 90 min, mice were euthanized to harvest heads. The skin, tongue, and lower jaw were removed, and the remaining portion was embedded into OCT (Sakura Finetek) with the top of turbinates oriented in the bottom of plastic cryomold to avoid cutting teeth. Turbinates were then snap frozen at –80°C. Turbinate frozen sections were cut at –30°C in a Leica Cryocut 1850 (Leica Microsystems) using Instrumedics CryoJane Tape Transfer System and a D profile tungsten carbide cryostat knife (Delaware Diamond Knives) to retain the undecalcified bone with epithelial lining on a slide. Frozen sections were air dried 30 min, then cover-slipped unfixed using Prolong Gold ready-to-use Anti-Fade Reagent with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes).

Immunizations

Specific pathogen-free BALB/c mice were obtained from the National Cancer Institute and maintained in the Animal Resources Center at Montana State University (Bozeman, MT). All mice were kept under pathogen-free conditions in individually ventilated cages under HEPA-filtered, barrier conditions and fed sterile food and water ad libitum. The mice were free of bacterial and viral pathogens, as determined by Ab screening and by histopathologic analysis of major organs and tissues. Mice between age 5 and 8 wk were immunized nasally with 50 µg of Hctre or Hctre-Ad2F plus 5 µg of cholera toxin (CT; List Biological Laboratories), and boosted with their respective vaccines on days 7 and 14 postprimary immunization with 2 µg of CT. For i.m. immunizations, BALB/c mice were immunized with equimolar amounts of Hctre Ag, which represented 50 µg of Hctre-Ad2F and 25 µg of Hctre on days 0, 7, and 14 with or without 1.0 µg of CT given at the anterior tibial tuberosity to ensure delivery into the muscle (20).

ELISA to detect BoNT/A -trefoil Abs

Standard ELISA protocols were followed to quantitate the Abs induced to BoNT/A Hctre. Recombinant BoNT/A Hctre (5 µg/ml) was used to coat Maxisorp microtiter plates (Nunc) overnight at 4°C. Immune plasma, fecal extracts, or nasal washes from individual mice were evaluated for their activity against the Hctre. Fecal samples were extracted from the supernatants of 10% (w/v) suspension of sample and PBS with 50 µg/ml soybean trypsin inhibitor (Sigma-Aldrich). Nasal washes were collected by intubation of the trachea to access the nasopharyngeal cavity. An 1-inch long tygon tubing (internal diameter 0.010 inches, outside diameter 0.030 inches; Cole-Parmer) was attached to a 1.0-ml syringe, an approach used to avoid any blood contamination of the nasal washes. A total of 200 µl of PBS was inserted via the trachea, and the exudate from the nares was collected into microfuge tubes. Cells and debris were removed by centrifugation for 10 min at 10,000 x g. HRP-goat anti-mouse IgA, IgG, IgG1, IgG2a, and IgG2b (Southern Biotechnology Associates) or biotinylated rat anti-mouse IgE mAb (BD Pharmingen) plus HRP-goat anti-biotin Abs (Vector Laboratories) were used for detection. Following 90 min of incubation at 37°C and a washing step, the specific reactivity was determined by the addition of an enzyme substrate, ABTS (Moss) at 100 µl/well. Absorbance was measured at 415 nm on a Kinetics Reader model EL312 (Bio-Tek Instruments). Endpoint titers were defined as the highest reciprocal of dilution of sample giving an absorbance at OD₄₁₅ above 0.100 OD units above negative controls after 1 h of incubation at 25°C. For total IgE ELISA, a similar protocol was followed as for Ag-specific ELISA already described, but wells were coated with rat anti-mouse IgE mAb (2.0 µg/ml; BD Pharmingen). After blocking, serial dilutions (beginning at 2⁵) of individual serum were done and added to coated wells in duplicate and incubated overnight at 4°C. After washing wells, 2.0 µg/ml biotinylated rat anti-mouse IgE was incubated for 2 h at 37°C, washed, then incubated with 1/500 dilution of HRP-goat anti-biotin for 1 h at 37°C. Wells were developed, as described, and a specific amount of IgE was extrapolated from a standard curve derived from wells incubated with an IgE anti-trinitrophenyl mAb (BD Pharmingen).

Lymphocyte isolation

Lymphocytes were isolated from nasal passages, submaxillary glands, small intestinal lamina propria, Peyer’s patches, mesenteric lymph nodes (LN), spleens, nasal-associated lymphoreticular tissue, and head and neck LN. Splenic, Peyer’s patch, mesenteric LN, nasal-associated lymphoreticular tissue, and head and neck LN mononuclear cell suspensions were obtained by conventional methods using dounce homogenization (21, 22) and yielding >95% viability by trypan blue exclusion. To isolate mononuclear cells from nasal passages and submaxillary glands, the tissues were minced and suspended in medium containing 300 U/ml Clostridium histolyticum type IV collagenase (Worthington Biochemical), followed by two 30-min digestions at 37°C or a single digestion for 45 min, respectively, in spinner flasks. The small intestinal lamina propria samples were isolated, as previously described (21, 22). After incubation, the digestion mixtures were passed through Nitex mesh (Fairview Fabrics) to remove undigested tissues. Mononuclear cells were subjected to Percoll (Amersham Biosciences) density gradient centrifugation, and the cells were interfaced between 40 and 75% Percoll (21, 22). Viability of >95% was noted for lymphocytes isolated from each tissue, as determined by trypan blue exclusion.

B cell Ab ELISPOT

Ab-forming cells (AFC) were enumerated using IgA and IgG Ag-specific ELISPOT assays similar to those previously described (21, 22, 23). Mixed cellulose ester membrane-bottomed microtiter plates (MultiScreen-HA; Millipore) were coated with 5.0 µg/ml recombinant Hc BoNT/A in sterile PBS. For total IgA or IgG AFC determinations, wells were coated with 5 µg/ml goat anti-mouse IgA or IgG (Hc-specific) Abs (Southern Biotechnology Associates) in sterile PBS overnight at 25°C. The plates were blocked at 37°C for 2 h in CM. A total of 100 µl of cells from each tissue at varying concentrations was added to the wells, and the plates were incubated at 37°C overnight. Cells were removed, and the plates were washed, as previously described (21, 22, 23). For detection of AFC responses, 100 µl of 1.0 µg/ml goat anti-mouse IgG and IgA-HRP conjugates (Southern Biotechnology Associates) were added to the wells, and the plates were incubated overnight at 4°C. After washing, the wells were developed with 100 µl of AEC (Moss), and the reaction was allowed to continue until spots developed (30 min). The reaction was stopped with H₂O, the plates were allowed to dry overnight, and AFC were enumerated by counting under a low-power dissecting microscope (Leica).

Cytokine ELISPOT

Groups of BALB/c mice (5–10/group) were euthanized 3 wk after the last immunization to collect spleens. Total splenic, head and neck LN, and mesenteric LN mononuclear cells (5 x 10⁶/ml) were resuspended in CM, and restimulated with 10 µg/ml recombinant Hc BoNT/A, OVA (tissue-culture grade; Sigma-Aldrich), or media in the presence of 10 U/ml human IL-2 (PeproTech) for 2 days at 37°C. Cells were washed and resuspended in CM. Stimulated lymphocytes were then evaluated by IFN--, IL-4-, IL-5-, IL-6-, and IL-10-specific ELISPOT assays, precisely as previously described (22, 24).

BoNT intoxication challenge and mouse neutralization assay

To assess the protective value of the Hctre vaccine, BALB/c mice were immunized nasally with equimolar doses of Hctre or Hctre-Ad2F with or without CT as adjuvant, as previously described. For the first experiment, one group of mice was immunized nasally with 2 µg of BoNToxoid/A (List Biological Laboratories) plus 2 µg of CT on days 0, 7, 14, and 21. Mice were monitored for Ab titers to Hctre, Hc BoNT/A, and BoNT/A. On day 63 or day 96, mice were challenged i.p. with 20,000 or 2000 LD₅₀ BoNT/A (2 x 10⁸ mouse LD₅₀/mg, Lot no. A121004-01; Metabiologics), respectively, in 200 µl of PBS containing 0.2% gelatin, as previously described (23). Mice were monitored hourly for the first 6 h and then daily for signs of morbidity, including difficulty breathing and lack of mobility. Mice were euthanized when signs of morbidity were observed.

To determine whether nasal immunization with Hctre immunogens was able to induce protective Abs in mucosal secretions against BoNT/A, we picked a mouse neutralization assay using BoNT/A complex (3.8 x 10⁷ mouse LD₅₀/mg, batch no. MA0305, lot no. A121005-01; Metabiologics). In this assay, filter-sterile fecal extracts from naive or day 21 samples were combined with BoNT/A complex (1/2 final dilution of fecal extract) and incubated at room temperature for 30 min before i.p. injection into naive BALB/c mice. As a positive control, some of the naive fecal extracts were spiked with immune sera from mice immunized nasally with Hc BoNT/A plus CT at a final dilution of 1/110. If the anti-Hctre Abs in the fecal extracts were able to neutralize BoNT/A in the complex, the mice would not exhibit BoNT/A intoxication. If the fecal extracts did not contain BoNT/A-neutralizing Abs, the mice would exhibit signs of BoNT/A toxicity, including shortness of breath and immobility. Mice were monitored twice daily for up to 5 days. Morbid mice were euthanized in accordance with the Institutional Animal Care and Use Committee of both institutions.

Statistical analysis

An ANOVA, followed by Tukey’s method, was used to evaluate differences between variations in Ab titers, Ab AFC, and cytokine-forming cell (CFC) levels and discerned to the 95% confidence interval. The Kaplan-Meier method (GraphPad Prism; GraphPad) was applied to obtain the survival fractions following intoxication with BoNT/A or the BoNT/A complex. Using the Mantel-Haenszel log rank test, the p value for statistical differences between surviving BoNT/A challenge and Hctre plus CT, Hctre-Ad2F plus CT, Hctre alone, Hctre-Ad2F alone, or BoNToxoid plus CT vaccinated mice or fecal extracts derived from vaccinated and naive mice were discerned at the 95% confidence interval.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Recombinant Ad2F exhibits cell-binding activity

We hypothesized that the addition of a mucosal-targeting ligand, i.e., Ad2F, would possibly enhance vaccine uptake. To test for this response, several recombinant proteins were produced for this study (Fig. 1A). The Hc fragment of the C. botulinum neurotoxin A (Hc BoNT/A) is a small molecule subdomain corresponding to the binding domain for Hc referred to as the -trefoil (Hctre) structure. A fusion protein between the Hctre and the Ad2F referred to as the Hctre-Ad2F was also assessed. Finally, a fusion between the Red2 fluorescent protein and Ad2F referred to as Red2-Ad2F was used. The recombinant Hc, Hctre, and Hctre-Ad2F migrated with the expected molecular mass of 58, 27.8, and 50 kDa, respectively, as determined by SDS-PAGE (Fig. 1B) or 60 kDa for Red2-Ad2F, as assessed by Western blot (Fig. 1C). To assess whether the Ad2F could bind to the mucosal epithelium, binding studies to human HeLa and 293A cells were performed using the Red2-Ad2F (Fig. 1D). The Red2-Ad2F was able to bind to both HeLa and 293A cells, but not the recombinant Red2. Neither Red2-Ad2F nor Red2 were able to bind to mouse L cells, suggesting that the Ad2F was indeed functional. To test whether this protein could bind to mouse epithelium, BALB/c mice were nasally dosed with Red2-Ad2F and after 90 min, nasal passages were evaluated for protein binding. As depicted in Fig. 1E, the Red2 fluorescence was associated with the nasal epithelium on its luminal surface.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. Construction of novel BoNT/A vaccines using the -trefoil (tre) structure of the BoNT/A Hc adapted with and without the adhesin Ad2F. A, Schematic depictions of BoNT/A Hc, Hctre/A (Hctre), Hctre genetically fused to Ad2F (Hctre-Ad2F), and the Red2 fluorescent protein genetically fused to Ad2F (Red2-Ad2F). B, Coomassie blue stained 12% SDS-PAGE of the Hctre vaccines: Hc (58.8 kDa), Hctre-Ad2F (50 kDa), and Hctre (27.8 kDa). C, The Red2-Ad2F migrates with the expected molecular mass of 60 kDa as detected by Western blot analysis using a polyclonal rabbit anti-Red2 Ab. D, FACS analysis of Red2-AdF binding to human epithelial HeLa and 293A cells (open histograms), but not to L929 fibroblasts. E, Red2-Ad2F binds to mouse nasal epithelium. BALB/c mice were given 50 µg of Red-Ad2F nasally, and 90 min later, nasal passages were harvested for frozen sections and evaluated for Red2 fluorescence (on apex of turbinate cells) together with DAPI nuclear (blue) stain. F, An adjacent section was cut for H&E staining.

To test whether Ad2F could efficiently deliver Hctre to the mucosa, mice were immunized nasally on days 0, 7, and 14. A kinetic analysis was performed and showed that the Ad2F could effectively stimulate a rapid secretory IgA (sIgA) Ab response to the Hctre, whereas nasal immunization with Hctre alone could not (Fig. 2A). Only weak plasma IgG anti-Hctre Ab titers were elicited with Hctre-Ad2F and none with Hctre alone. Thus, in subsequent studies, all immunizations were conducted with CT as mucosal adjuvant. Adjuvanted doses of Hctre-Ad2F resulted in a rapid onset of sIgA Ab titers when compared with onset for adjuvanted dosages of Hctre, which was delayed by 2 wk (Fig. 2A). In a similar fashion, adjuvanted doses of Hctre-Ad2F also resulted in a rapid onset of plasma IgG titers when compared with onset for dosages of Hctre plus CT, which was delayed by 1 wk (Fig. 2B). Differences in plasma IgG1 and IgG2a, but not IgG2b responses, were observed between the two immunization groups (Fig. 3B). Hctre-specific IgE and total IgE levels were also measured during peak Ab responses on day 28. Mice nasally immunized with Hctre, Hctre-Ad2F, or Hctre-Ad2F plus CT failed to show Hctre-specific IgE (Fig. 3C); in the group of mice nasally dosed with Hctre plus CT, only one in 10 mice showed an IgE titer of 2⁶.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. The addition of the mucosal-targeting ligand, Ad2F, enhances time for mucosal Ab production. BALB/c mice nasally immunized on days 0, 7, and 14 with Hctre or Hctre-Ad2F, either alone or in combination with CT, and fecal IgA (A) and plasma IgG (B) Ab titers against the Hctre were determined by standard ELISA methods. In the absence of CT, nasal immunization with Hctre-Ad2F elicited a robust and rapid onset of secretory IgA (sIgA) Abs, whereas mice nasally dosed with Hctre only produced a weak sIgA Ab response. Both groups produced weak plasma IgG anti-Hctre titers. Upon CT coadministration, a rapid onset and sustained sIgA and plasma IgG Ab titers were produced when nasally vaccinated with Hctre-Ad2F, but this rapid onset was not evident with the Hctre-dosed mice. Thus, for the remaining studies, only responses in adjuvanted mice were evaluated. Data represent the mean ± SEM (n = 10 mice). *, p = 0.001; **, p = 0.015; and ***, p = 0.02 represent the statistical differences in sIgA and IgG anti-Hctre levels between mice given the same vaccine with CT as adjuvant compared with mice dosed with their respective vaccine without CT.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Nasal immunization with Hctre-Ad2F and CT shows increased secretory IgA (sIgA) (A) anti-Hctre titers with significant differences in plasma IgG1 and IgG2a titers (B) but no changes in total IgE levels (C). Data represent mean ± SEM (n = 10) sampled at day 35 postprimary immunization or sampled at day 28 for total IgE levels. Only one of 10 mice from the Hctre plus CT-dosed group showed a plasma IgE anti-Hctre titer of 26; none of the other groups showed a Hctre-specific IgE titer. *, p < 0.001 and **, p = 0.003 represent the differences in sIgA, IgG1, and IgG2a Ab titers between mice nasally dosed with Hctre-Ad2F plus CT and mice dosed with Hctre plus CT, respectively.

Nasal immunization with Hctre-Ad2F enhances IgA AFC in nasal passages and submaxillary glands

To determine the distribution of Ag-specific B cells following nasal vaccination, an Ab ELISPOT was performed on lymphoid tissues for Hctre-Ad2F plus CT or Hctre plus CT-treated mice (Fig. 4). Nasal immunization with Hctre-Ad2F plus CT stimulated a pronounced number of Ag-specific IgA AFC in nasal passages and submaxillary glands when compared with mice dosed with the adjuvanted Hctre (p < 0.001) (Fig. 4A). Ag-specific IgA AFC were also significantly increased in Peyer’s patches (p < 0.05). The remaining Ag-specific IgA AFC response for the small intestinal lamina propria, spleen, and nasal-associated lymphoreticular tissue was the same for both groups. Evaluation of Ag-specific IgG AFC showed only subtle changes in small intestinal lamina propria, Peyer’s patches, spleen, nasal passages, and nasal-associated lymphoreticular tissue (Fig. 4C).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Nasal immunization with Hctre-Ad2F plus CT as adjuvant enhances the number of IgA AFC in nasal passages (NP), submaxillary glands (SMG), and Peyer’s patches (PP) (A) when compared with Hctre plus CT, but showed slightly less IgG AFC response in nasal passages, Peyer’s patches, and small intestinal lamina propria (iLP) (C). B cell ELISPOT was conducted 4 wk postprimary immunization and Hc BoNT/A-specific (A and C) and total AFC were measured (B and D). Data are expressed as mean ± SEM of three separate experiments of AFC/1 x 106 lymphocytes. *, p 0.001; **, p 0.016; ***, p < 0.05.

Nasal immunization with Hctre-Ad2F plus CT as adjuvant elicits a mixed Th cell response

To learn what are the supportive Th cells for the observed Ag-specific B cell responses, adjuvanted Hctre-Ad2F-dosed or Hctre-dosed mice were evaluated for CFC. Lymphocytes from spleen, head and neck LN, and mesenteric LN were cultured and assayed for the production of IFN-, IL-4, IL-5, IL-10, and IL-13. Following 48 h of stimulation, lymphocytes were analyzed by cytokine ELISPOT method. Although the responses varied among the tissues examined, for the most part, a mixed Th cell phenotype was observed when triggered with Hc BoNT/A. In the spleen, IFN-, IL-4, and IL-5 CFC were induced to equivalent levels for both dosing groups, but increased numbers of IL-10- and IL-13-producing cells were detected in the Hctre-Ad2F-dosed mice (Fig. 5A). In the head and neck LN, IL-4, IL-5, and IL-10 were not significantly different between either dosing group; however, IFN- (p < 0.001) and IL-13 (p < 0.05) were particularly enhanced in Hctre-Ad2F-dosed mice (Fig. 5B). In the mesenteric LN, IL-5 and IL-10 were similar for both groups; however, Hctre-Ad2F-dosed mice exhibited significantly less IFN- (p < 0.011), IL-4 (p < 0.05), and IL-13 (p < 0.001) CFC (Fig. 5C).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Mucosal targeting enhances head and neck LN IFN- and IL-13 production following nasal immunization with Hctre-Ad2F. Three weeks after their final immunization, Hctre-Ad2F plus CT-dosed and Hctre plus CT-dosed mice were evaluated for CFC responses in the spleen (A), head and neck LN (HNLN) (B), and mesenteric LN (MLN) (C) by a cytokine ELISPOT method. Immune lymphocytes were isolated and cultured with Hc BoNT/A (Hc), OVA, or media only for 2 days, and then evaluated for IFN-, IL-4, IL-5, IL-10, and IL-13 CFC. Data shown are mean ± SEM of CFC/1 x 106 lymphocytes from a total of three experiments. *, p < 0.001; **, p 0.011; ***, p < 0.05.

Two challenge studies with BoNT doses that would cause intoxication in humans were conducted using immunized mice. In the first experiment, groups of BALB/c mice were nasally dosed with equimolar amounts of Hctre (25 µg) and Hctre-Ad2F (50 µg) with CT (2 µg). One control group was given BoNToxoid/A (2 µg) with CT, and another control group was left unimmunized. Mice were immunized four times at weekly intervals and assessed for immune titers on day 57 postprimary immunization. ELISA were performed using Hctre, Hc BoNT/A, or native BoNT/A as coating Ags (Fig. 6A). Both groups of mice dosed with either Hctre or Hctre-Ad2F showed elevated Ab endpoint titers against all three Ags; however, the Hctre-Ad2F-dosed group showed a slightly reduced titer against Hc BoNT/A. Mice immunized with BoNToxoid showed poor Ab reactivity against the immunizing immunogen, as well as to Hctre and Hc BoNT/A (Fig. 6A). On day 63, mice were challenged i.p. with 20,000 mouse LD₅₀, which is equivalent to 1.0 human LD₁₀₀ (3), and both the Hctre-dosed or Hctre-Ad2F-dosed groups showed complete protection (Fig. 6B). In addition, neither group showed signs of BoNT intoxication. Surprisingly, the mice immunized with the BoNT/A toxoid failed to show any protective efficacy and succumbed to intoxication as quickly as naive mice (all within 3 h).

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Nasal immunization with adjuvanted Hctre-Ad2F or Hctre, but not with BoNToxoid/A, confers complete protection in BALB/c mice subsequently challenged i.p. with 20,000 LD50 BoNT/A on day 63 postprimary immunization. Immunizations were conducted on days 0, 7, 14, and 21, and the groups were subdivided as follows: Group 1 (n = 3 mice) given 25 µg of Hctre plus CT (2 µg); Group 2 (n = 4 mice) given 50 µg of Hctre-Ad2F (to ensure that equivalent amount of Hctre was given); Group 3 (n = 3 mice) given 2 µg of BoNToxoid/A; and Group 4 (n = 4 mice) was left as naive. A, On day 57, plasma IgG titers from each group were measured against Hctre, BoNT/A Hc, or native BoNT/A. The mice dosed with adjuvanted Hctre showed a greater endpoint titer against Hc BoNT/A than mice dosed with Hctre-Ad2F; however, both dosing groups showed similar endpoint titers against native BoNT/A. *, p = 0.002. B, On day 63, mice were challenged i.p. with 20,000 mouse LD50 and monitored for survival for 5 days. Both the adjuvanted Hctre-dosed and Hctre-Ad2F-dosed mice showed a 100% survival rate. **, p < 0.05.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Nasal immunization with adjuvanted Hctre-Ad2F or Hctre confers complete protection in BALB/c mice subsequently challenged i.p. with 2000 LD50 BoNT/A on day 96 postprimary immunization. Immunizations were conducted on days 0, 7, and 14, and the groups were subdivided as follows: Group 1 (n = 5 mice) given 25 µg of Hctre plus CT (2 µg); Group 2 (n = 5 mice) given 50 µg of Hctre-Ad2F (to ensure equivalent amount of Hctre was given) plus CT (2 µg); Group 3 (n = 8 mice) given 2 µg of CT only; Group 4 (n = 5 mice) given 25 µg of Hctre; and Group 5 (n = 5 mice) given 50 µg of Hctre-Ad2F. A, On day 84, plasma IgG titers from each group were measured against Hctre and BoNT/A Hc, and equivalent endpoint titers were obtained between groups 2 and 3 and between groups 4 and 5. Plasma IgG titers from each group were significantly greater than titers in the control CT-dosed group. *, p < 0.001. B, On day 96, mice were challenged i.p. with 2000 mouse LD50 and monitored for survival for 5 days. Both the adjuvanted Hctre-dosed and Hctre-Ad2F-dosed mice showed a 100% survival rate, unlike the mice dosed with Hctre or Hctre-Ad2F alone, which did not. *, p 0.002 and **, p = 0.015.

To determine whether mucosal secretions contain neutralizing Abs can passively protect against BoNT/A, an in vivo mouse neutralization assay was used with the BoNT/A complex. The BoNT/A complex was used in these studies to mimic natural exposure to BoNT. In this assay, sterile-filtered fecal extracts were combined with 6.5 LD₅₀ of the BoNT/A complex, and incubated at room temperature for 30 min before i.p. injection into naive BALB/c mice. Sterile fecal extracts were prepared from mice immunized with Hctre-Ad2F plus CT or Hctre plus CT, from naive mice, or from naive samples spiked with immune anti-Hctre plasma. All of the mice given the naive fecal extracts succumbed within 24 h (Fig. 8). Mice given the naive fecal extract containing the added anti-Hctre Abs were protected against challenge with the BoNT/A complex (p = 0.005). Likewise, all mice given the fecal extracts from Hctre-Ad2F plus CT-dosed mice survived, and 75% of the mice given fecal extracts from mice dosed with Hctre and CT survived the challenge with the BoNT/A complex (p = 0.005). These collective results showed the feasibility of nasal immunization with our novel BoNT/A immunogens to induce neutralizing Abs in mucosal secretions that protect against intoxication with BoNT or BoNT complex.

View larger version (11K): [in this window] [in a new window]   FIGURE 8. Abs in mucosal samples are protective against challenge with the BoNT/A complex. Day 21 mucosal samples were evaluated for their BoNT-neutralizing capacity using a mouse neutralization assay. Filter-sterilized soluble fecal extracts (diluted 1/2) from Hctre-Ad2F plus CT dosed (n = 4 mice) () and Hctre plus CT dosed (n = 4 mice) (), from naive mice (n = 8) (), or from naive mice, but spiked with immune mouse plasma (n = 7) () were reacted with 6.5 LD50 BoNT/A complex for 30 min, then were injected i.p. into naive BALB/c mice. All mice treated with naive fecal extract and the BoNT/A complex became moribound within 1 day, and all of the mice given the naive fecal extract spiked with immune plasma survived. Complete protection was obtained in all mice treated with the BoNT/A complex plus fecal extracts from mice nasally immunized with Hctre-Ad2F plus CT. This survival fraction was not statistically different from mice given fecal extracts from Hctre plus CT only. *, p = 0.005 for survival fractions from treatment groups compared with mice given naive fecal samples.

Intramuscular immunization with Hctre or Hctre-Ad2F elicits elevated Ab titers

To assess how the Ad2F-targeting molecule may enhance the immunogenicity of the Hctre, i.m. immunization studies with Hctre or Hctre-Ad2F with and without CT adjuvant were performed. BALB/c mice were dosed with equimolar amounts of Hctre vaccine given alone or together with 1.0 µg of CT adjuvant on days 0, 7, and 14. Ab titers were assessed weekly and showed that two doses of Hctre-Ad2F were sufficient to produce elevated IgG Ab titers when compared with mice similarly dosed with Hctre by day 14 (Fig. 9A). In fact, the Ab titers induced by Hctre-Ad2F remained significantly greater through day 28. The addition of adjuvant modestly improved the IgG Ab response induced by Hctre-Ad2F. In addition, the Hctre-Ad2F plus CT group showed enhanced IgG Ab titers when compared with the Hctre plus CT group (Fig. 9A). Secretory IgA Ab titers were induced by all groups, but clearly mice dosed with Hctre-Ad2F with or without CT showed the greatest sIgA response peaking on day 21 (Fig. 9B), but these Ab responses subsequently deteriorated to sIgA levels obtained in mice immunized with Hctre or Hctre plus CT. Thus, these data show that the inclusion of the Ad2F molecule improves the immunogenicity of Hctre when given parenterally even in the absence of adjuvant.

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Parenteral immunization with Hctre-Ad2F enhances serum IgG and mucosal IgA Ab responses to Hctre. BALB/c mice i.m. immunized on days 0, 7, and 14 with Hctre or Hctre-Ad2F, either alone or in combination with CT. Plasma IgG (A) and fecal IgA (B) Ab titers against the Hctre were determined by standard ELISA methods. In the absence of CT, i.m. immunization with Hctre-Ad2F elicited a robust and rapid onset of IgG and sIgA Abs, whereas mice i.m. immunized with Hctre produced a delayed IgG and a weak sIgA Ab response. By day 35, both groups had equivalent IgG and sIgA anti-Hctre titers. Upon CT coadministration with Hctre-Ad2F or Hctre, a rapid onset of plasma IgG Ab titers were produced, but mice vaccinated with Hctre-Ad2F showed greater Ab responses. SIgA Ab titers were greater in the Hctre-Ad2F plus CT-dosed group than titers in mice similarly dosed with Hctre. Thus, these studies show that targeting with Ad2F enhances Ab responses to Hctre. Data represent mean ± SD (n = 5 mice). *, p 0.001; **, p = 0.005; and ***, p = 0.024 represent the statistical differences in sIgA and IgG anti-Hctre levels between mice given the same vaccine with or without CT. , p 0.001 and , p 0.009 represent differences between mice given Hctre-Ad2F or Hctre or given Hctre-Ad2F plus CT or Hctre plus CT.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In an effort to develop an alternative BoNT vaccine with improved efficacy, we adopted a two-prong strategy using BoNT/A as a vaccine prototype. We and others have recently identified the Hc containing the cell-binding domain in the C terminus as a potential vaccine candidate (11, 31) that contains a -trefoil structure (18). The Hctre is a structure common to all serotypes (18), and this motif is even repeated in the progenitor toxin complex (32). This Hctre domain has been implicated in previous studies using a peptide analysis approach to determine the protective epitopes for Hc BoNT (9, 10). In the first study (9), three Hc regions in BoNT/A were identified for eliciting protection: BoNT/A_455–661, BoNT/A_780–939, and BoNT/A_1150–1289, conferring 78, 29, and 75% protection, respectively, after challenge with two LD₉₉ dosed. In the second study, the synthetic peptide corresponding to Hc BoNT/A_1230–1253 was 40% protective in mice against a lethal BoNT/A challenge (10). Thus, it appeared from these studies that, at a minimum, peptides encompassing a portion of the Hctre were protective. In addition, mAbs to Hc BoNT/A, some of which contain epitopes within the -trefoil structure, have also been shown to be protective (10, 30).

The second attribute of our approach is the addition of a mucosal-targeting molecule Ad2F. Replication-deficient adenovirus vectors have widely been used in vaccination regimens (33, 34), and these vectors have been proven efficacious in their ability to stimulate protective immune responses (35, 36). However, as with any vaccine platform, concerns arise as to whether repetitive application of these vectors can limit their use (33, 34). Similar concerns are applicable with our methods, as well; however, again their application may be beneficial in a prime-boost strategy involving vaccination with the Hctre, both with or without Ad2F. It was evident from this study that the recombinant fusion protein with Ad2F could bind to the mouse nasal epithelium enabling uptake of the Hctre-Ad2F vaccine. Its use rapidly stimulated both mucosal IgA and systemic IgG Abs when Ad2F was present. Because nasal immunization with Hctre ultimately developed to similar anti-Hctre Ab titers as with Hctre-Ad2F, subsequent immunizations may only require Hctre. These studies will be pursued. When tested parenterally, our results showed that the inclusion of Ad2F-binding moiety enhanced plasma IgG and fecal IgA responses. This finding is of interest because i.m. immunization in the absence of CT adjuvant showed elevated IgG Ab titers as early as after two doses of Hctre-Ad2F and remained elevated at least until day 35 postprimary immunization. Although the coadministration of CT adjuvant clearly enhanced the IgG Ab titer, this increase was, at best, 9.2-fold. Additional studies will be taken to discern whether more vaccine can enhance this Ab response in the absence of adjuvant or whether the amount of adjuvant can be reduced. Such study will require evaluation of more suitable adjuvants approved for human use. How Ad2F is improving the systemic IgG response after i.m. immunization, at this point, can be speculated. Ad2F binds the coxsackie-adenovirus receptor (reviewed in Refs. 37, 38) and is expressed in a variety of tissues (39, 40). Thus, the parenteral delivery of Hctre-Ad2F may be taken up more efficiently because of coxsackie-adenovirus receptor expression. Alternatively, the Ad2F may enhance the immunogenicity of Hctre. Moreover, the Hctre also induced elevated Abs alone, albeit, lesser than Hctre-Ad2F. However, mucosal IgA Abs were induced to greater levels when using the Hctre-Ad2F than when using Hctre, although both showed only low mucosal Ab levels after day 35, suggesting long term mucosal memory was not induced. Nonetheless, these results show the feasibility of using either Hctre-Ad2F or Hctre in parenteral immunization paradigm.

Our study showed that the Hctre is strongly immunogenic when delivered nasally. As was evident, the Hctre stimulated 100% protection to mice when challenged i.p. with either 20,000 or 2,000 LD₅₀. This immunity was found to be long lasting as noted by the day 96 challenge. In addition, we found that the Abs within mucosal secretions were protective. Using a mouse neutralization assay, sterile fecal extracts protected against 6.5 LD₅₀ of the BoNT/A complex. Intoxication with the BoNT complex is 100-fold more toxic than purified BoNT (41, 42), when using the gastric route of exposure. This BoNT complex would most likely represent the form of toxin dissemination in bioterrorism. Our results suggest that the protective value of these mucosal Abs, at a minimum, approximates the lower end protection (2,000 LD₅₀) found with systemic Abs. It also shows that our vaccine formulation is better than using BoNToxoid as a nasal vaccine immunogen for the induction of protective humoral immunity in mucosal secretions. For example, fecal extracts from 75 to 100% of mice nasally immunized with Hctre plus CT or Hctre-Ad2F plus CT, respectively, protected mice against a lethal BoNT/A complex challenge. However, when a similar assay was performed with fecal extracts from mice nasally immunized with BoNToxoid, only one-third of the mice were protected against one LD₅₀ of BoNT/A complex (23). Clearly, this evidence showed that immunization with the Hctre is protective and is a better immunogen than the toxoid.

Although no differences in protection were noted between mice dosed with the Hctre or the Hctre-Ad2F, it is important to note that the addition of the Ad2F acted as a mucosal accelerant in which anti-Hctre sIgA, and plasma IgG Ab titers were more quickly induced. Although Hctre-Ad2F given without adjuvant was able to induce some Ab titers to the Hctre, elevated plasma IgG Ab titers did not become elevated as when in the presence of an adjuvant. It may be more advantageous to give Hctre-Ad2F without adjuvant as a booster, rather than to initiate the Ab response, and thus lessen the need for mucosal adjuvants. Studies are currently addressing this possibility. The use of the Hctre immunogen may also be beneficial to elicit a polyclonal antiserum to neutralize against BoNT intoxication. Because a portion of the neutralizing epitopes are retained in the Hctre (9, 10, 11), Abs directed against this cell-binding domain may even be comparable to the use of mAbs.

	Acknowledgments

 
We are grateful to Dr. J. Chroboczek (European Molecular Biology Laboratory, Grenoble, France) for the gift of adenovirus 2 genomic DNA. We thank Carol Riccardi for assistance with the FACS analysis, Shaun M. Kirwan for technical assistance and Nancy Kommers for assistance in preparing this manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Public Health Service Grants DE-13812, AI-56286, AI-18958, DE-12242, AI-43197, and DC-04976, and in part by Montana Agricultural Station and the U.S. Department of Agriculture Formula Funds. The Veterinary Molecular Biology flow cytometry facility was supported in part by the Center of Biomedical Research Excellence P20 RR-020185 from the National Institutes of Health/National Center for Research Resources.

² Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, P.O. Box 173610, Montana State University, Bozeman, MT 59717-3610. E-mail address: dpascual{at}montana.edu

³ Abbreviations used in this paper: BoNT, botulinum neurotoxin; Hctre, -trefoil stucture of Hc for BoNT; Ad2F, adenovirus 2 fiber protein; AFC, Ab-forming cell; CFC, cytokine-forming cell; CM, complete medium; DAPI, 4',6-diamidino-2-phenylindole; CT, cholera toxin; LN, lymph node. sIgA, secretory IgA.

Received for publication April 5, 2006. Accepted for publication July 21, 2006.

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