Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains.

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

RBC Adherence of Immune Complexes Containing Botulinum Toxin Improves Neutralization and Macrophage Uptake.

In the paralytic disease botulism, the botulinum neurotoxin (BoNT) passes through the bloodstream to reach and inactivate neuromuscular junctions. Monoclonal antibodies (mAbs) may be useful BoNT countermeasures, as mAb combinations can rapidly clear BoNT from the blood circulation. We have previously shown that the BoNT-neutralizing potency of mAbs can be improved through red blood cell (RBC) immunoadherence. For example, a fusion protein (FP) that adheres biotinylated mAbs to the RBC surface enabled a pair of mAbs to neutralize 5000 LD50 BoNT/A in the mouse protection assay. Here, we added two mAbs to that combination, creating a 4-mAb:FP complex that neutralized 40,000 LD50 BoNT/A in vivo, and analyzed functional correlates of neutralization. The FP enhanced potency of BoNT/A immune complexes, providing the greatest magnitude of benefit to the 4-mAb combination. RBC binding of a BoNT/A complexed with 4-mAb:FP exhibited a bi-phasic clearance process in vivo. Most of the complexes were cleared within five minutes; the rest were cleared gradually over many hours. Peritoneal macrophages showed better uptake of the 4-mAb complex than the 3-mAb complex, and this was not affected by the presence of the FP. However, the addition of RBCs to the 4-mAb:FP BoNT/A doubled macrophage uptake of the complexes. Lastly, the 4-mAb:FP BoNT/A complex synergistically induced M2 macrophage polarization, as indicated by IL-10 expression, whether or not RBCs were present. RBC-targeted immunoadherence through the FP is a potent enhancer of mAb-mediated BoNT/A neutralization in vivo, and can have positive effects on BoNT/A sequestration, immune complex uptake, and macrophage activation.

Mechanisms of enhanced neutralization of botulinum neurotoxin by monoclonal antibodies conjugated to antibodies specific for the erythrocyte complement receptor.

Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP+mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2h, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb+HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3h after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics.

Oral vaccination with an adenovirus-vectored vaccine protects against botulism.

We have previously shown that an adenovirus vectored vaccine delivered intramuscularly or intranasally was effective in protection against botulism in a mouse model. The adenoviral vector encodes a human codon-optimized heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). Here, we evaluate the same vaccine candidate as an oral vaccine against BoNT/C in a mouse model. To elicit protective immunity, the mice were orally vaccinated with a single dose of 1×10(4) to 1×10(7)plaque forming units (pfu) of the adenoviral vector. The immune sera, collected six weeks after oral vaccination with 2×10(7)pfu adenovirus, have shown an ability to neutralize the biological activity of BoNT/C in vitro. Additionally, animals receiving a single dose of 2×10(6)pfu adenovirus or greater were completely protected against challenge with 100×MLD(50) of BoNT/C. The data demonstrated the feasibility to develop an adenovirus-based oral vaccine against botulism.

Progress in cell based assays for botulinum neurotoxin detection.

Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the 'gold standard' assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies.

Expression, purification and development of neutralizing antibodies from synthetic BoNT/B LC and its application in detection of botulinum toxin serotype B.

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.

Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.

Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes.

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LH(N), comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH(N) fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LH(N)/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10(3) 50% lethal dose (LD(50)) of BoNT/A(1) with an 50% effective dose (ED(50)) of 50 ng of the vaccine. In similar experiments, the LH(N)/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A(1), A(2), and A(3), which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH(N)/B vaccine was also highly efficacious, giving an ED(50) of approximately 140 ng to a challenge of 10(3) LD(50) of BoNT/B(1). In addition, LH(N)/B provided single-dose protection in mice against BoNT/B(4) (nonproteolytic toxin subtype).

An adenoviral vector-based mucosal vaccine is effective in protection against botulism.

A replication-incompetent adenoviral vector encoding the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C) was evaluated as a mucosal vaccine against botulism in a mouse model. Single intranasal inoculation of the adenoviral vector elicited a high level of H(C)50-specific IgG, IgG1 and IgG2a in sera and IgA in mucosal secretions as early as 2 weeks after vaccination. The antigen-specific serum antibodies were maintained at a high level at least until the 27th week. Immune sera showed high potency in neutralizing BoNT/C as indicated by in vitro toxin neutralization assay. The mice receiving single dose of 2 x 10(7) p.f.u. (plaque-forming unit) of adenoviral vector were completely protected against challenge with up to 10(4) x MLD(50) of BoNT/C. The protective immunity showed vaccine dose dependence from 10(5) to 2 x 10(7) p.f.u. of adenoviral vector. In addition, animals receiving single intranasal dose of 2 x 10(7) p.f.u. adenoviral vector could be protected against 100 x MLD(50) 27 weeks after vaccination. Animals with preexisting immunity to adenovirus could also be vaccinated intranasally and protected against lethal challenge with BoNT/C. These results suggest that the adenoviral vector is a highly effective gene-based mucosal vaccine against botulism.

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

BACKGROUND: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

Molecular recognition of botulinum neurotoxin B heavy chain by human antibodies from cervical dystonia patients that develop immunoresistance to toxin treatment.

We determined the entire profile of the continuous antigenic regions recognized by blocking antibodies (Abs) in sera from 30BoNT/B-treated cervical dystonia (CD) patients who developed unresponsiveness to treatment. The sera protected mice against a lethal dose of BoNT/B. We analyzed Ab binding to a panel of 60 synthetic 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and encompassed the entire BoNT/B heavy (H) chain (residues 442-1291). Most Abs recognized a limited set of peptides but the pattern and Ab levels bound varied with the patient, consistent with genetic control of immune responses and with responses to each epitope being separately controlled. Abs were bound by peptides (in decreasing order): C1 (residues 848-866), C10 (974-992), C16 (1058-1076), C14 (1030-1048), N15 (638-656), N21/N22 (722-740/736-754), N24/N25 (764-782/778-796) and N29 (834-852). Peptides N3/N4 (470-488/484-502), N27 (806-824), C2 (862-880), C4 (890-908), C6/C7 (918-936/932-950), C17 (1072-1090), C24 (1170-1188), C29 (1240-1258) and C31 (1268-1291) exhibited low Ab binding. The remaining peptides bound little or no Abs. Of the 30 antisera, 28 (93.3%) had Abs that bound to peptides C1, C10, C14 or C16, and 27 (90.0%) bound to peptide N22. No peptide was recognized by all the antisera, but peptide combinations N24+C1, N22+N24+C1, N24+C1+C10, C10+C14+C16, N22+N24+C1+C10, C1+C10+C14+C16 or N22+N24+C1+C10+C14 bound blocking Abs in 30 (100%) antisera. BoNT/B-treated CD patients had higher Ab levels and bound to more epitopes (at least 11) than did BoNT/A-treated patients (5 regions). The regions recognized by anti-BoNT/B Abs occupied surface areas that displayed no correlation to surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. These regions afford candidates for epitope-specific manipulation of anti-toxin immune responses.

Dominant antigenic peptides located at the heavy chain terminal of botulinum neurotoxin B contain receptor-binding sites for synaptotagmin II.

Botulism toxicity is caused by botulinum neurotoxins (BoNTs) that bind to the surface of the nerve terminals through the C-terminal portion of the heavy chain (Hc) and gain access to the neuron cells, thereby blocking the release of neurotransmitters into the synapse by the light chain. It has been recently found that synaptotagmin II (syt II) is the specific protein receptor of BoNTs; however, the molecular basis underlying the interaction between BoNTs and the receptor has been poorly understood. In this study, the recombinant fragment from the luminal domain of the human protein receptor syt II was prepared. Further, the Hc antigenic peptides of BoNTs type B (BoNTb) containing the possible epitopes were synthesized and analyzed for checking their ability for specific recognition and binding by using completely protective polyclonal antibody and were then evaluated for checking their ability to elicit protective immunity in mice against lethal toxin challenge. Finally, the interaction between the Hc-dominant antigenic peptides and syt II was analyzed. We found that two peptides located at the Hc terminal of the BoNTb showed the general character of the dominant antigenic epitopes in terms of protection against BoNTb intoxication. Further, they were found to contain the receptor-binding sites through the specific recognition of the receptor syt II. Both were derived from the uninterrupted segments of the amino acid residues H(1241-1277) of BoNTb. Kinetic data of the antigen-receptor interaction were depicted in real-time and calculated as affinity constant of 2.99x10(-7)M.

A natural human IgM antibody that neutralizes botulinum neurotoxin in vivo.

Affinity-matured human antibodies have demonstrated efficacy as countermeasures for exposure to botulinum neurotoxin (BoNT), which is the cause of the disease botulism category A select bioterror agent. Little is known, however, about the potential role of natural (un-mutated) antibodies in the protective immune response to BoNT. Here we describe the cloning of two human IgM antibodies that bind serotype A BoNT. Both are un-mutated IgM antibodies, consistent with an origin in naive B cells. One of the antibodies is able to fully neutralize a lethal dose of serotype A BoNT in vivo. These results suggest that the natural human antibody repertoire may play a role in protection from exposure to biological toxins.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins.

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display.

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo.

Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine.

Botulinum neurotoxins cause botulism, a neuroparalytic disease in humans and animals. We constructed a replication-incompetent adenovirus encoding a synthesized codon-optimized gene for expression of the heavy chain C-fragment (H(C)50) of botulinum neurotoxin type C (BoNT/C). This recombinant human serotype 5 adenoviral vector (Ad5) was evaluated as a genetic vaccine candidate against botulism caused by BoNT/C in a mouse model. A one-time intramuscular injection with 10(5) to 2 x 10(7)pfu of adenoviral vectors elicited robust serum antibody responses against H(C)50 of BoNT/C as assessed by ELISA. Immune sera showed high potency in neutralizing the active BoNT/C in vitro. After a single dose of 2 x 10(7)pfu adenoviral vectors, the animals were completely protected against intraperitoneal challenge with 100 x MLD(50) of active BoNT/C. The protective immunity appeared to be vaccine dose-dependent. The anti-toxin protective immunity could last for at least 7 months without a booster injection. In addition, we observed that pre-existing immunity to the wild-type Ad5 in the host had no significant influence on the protective efficacy of vaccination. The data suggest that an adenovirus-vectored genetic vaccine is a highly efficient prophylaxis candidate against botulism.

Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants.

Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products.