Fetweh H. Al-Saleem

Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route

ABSTRACT Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.

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.

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.

Localization of the sites and characterization of the mechanisms by which anti-light chain antibodies neutralize the actions of the botulinum holotoxin

The recombinant, catalytically active light chain of botulinum toxin type A was evaluated as a potential vaccine candidate. Previous studies have shown that the light chain can elicit protective immunity in vivo. [Kiyatkin N, Maksymowych AB, Simpson LL. Induction of immune response by oral administration of recombinant botulinum toxin. Infect Immun 1997;65(11):4586-91], but the underlying basis for this observation was not determined. In the present study, antibodies directed against the light chain were shown to act at three different sites in the body to produce neutralization. Firstly, these antibodies acted to block toxin absorption into the body. This was demonstrated in vitro, in studies on binding and transport of toxin across epithelial monolayers, and in vivo, in studies on inhalation poisoning. Secondly, anti-light chain antibodies acted to promote clearance of toxin from the general circulation. This was demonstrated in vivo in studies on toxin levels in blood and in parallel studies on toxin accumulation in liver and spleen. Finally, anti-light chain antibodies acted to protect cholinergic nerves from botulinum toxin action. This was demonstrated in two types of in vitro assays: rate of paralysis of murine phrenic nerve-hemidiaphragm preparations and extent of binding to Neuro-2a cells. When taken together, these data show that anti-light chain antibodies can evoke three layers of protection against botulinum toxin.

The role of systemic handling in the pathophysiologic actions of botulinum toxin.

The ability of botulinum toxin to poison cholinergic nerve transmission is a dynamic phenomenon that involves not only the actions of the toxin on the body but also the actions of the body on the toxin. The former has been the subject of intense research, whereas the latter has received almost no attention. Therefore, a series of studies were performed to characterize systemic handling of botulinum toxin. The results indicated that the toxin reaches the general circulation (transcytosis across epithelial cells) without obvious changes in structure or biological activity. The general circulation acts as a holding compartment until there is adequate fractional distribution to neuromuscular junctions to produce blockade of transmission. During its transit through this compartment, the toxin 1) undergoes little biotransformation, 2) does not accumulate significantly in circulating cells, and 3) remains largely in the free state. In naive animals, the t1/2 for toxin in the general circulation is approximately 10 h, and at any given point in time, there is little uptake in nontarget organs (liver, kidney, heart, and lung). In immunized animals, toxin clearance from the general circulation is rapid, and there is substantial accumulation of antibody-antigen complexes in liver. Thus, enhanced clearance from the circulation is a major mechanism by which active immunization can protect against poisoning.

Analysis of the Mechanisms That Underlie Absorption of Botulinum Toxin by the Inhalation Route

ABSTRACT Botulinum toxin is a highly potent oral and inhalation poison, which means that the toxin must have an efficient mechanism for penetration of epithelial barriers. To date, three models for toxin passage across epithelial barriers have been proposed: (i) the toxin itself undergoes binding and transcytosis; (ii) an auxiliary protein, HA35, transports toxin from the apical to the basal side of epithelial cells; and (iii) an auxiliary protein, HA35, acts on the basal side of epithelial cells to disrupt tight junctions, and this permits paracellular flux of toxin. These models were evaluated by studying toxin absorption following inhalation exposure in mice. Three types of experiments were conducted. In the first, the potency of pure neurotoxin was compared with that of progenitor toxin complex, which contains HA35. The results showed that the rate and extent of toxin absorption, as well as the potency of absorbed toxin, did not depend upon, nor were they enhanced by, the presence of HA35. In the second type of experiment, the potencies of pure neurotoxin and progenitor toxin complex were compared in the absence or presence of antibodies on the apical side of epithelial cells. Antibodies directed against the neurotoxin protected against challenge, but antibodies against HA35 did not. In the final type of experiment, the potency of pure neurotoxin and toxin complex was compared in animals pretreated to deliver antibodies to the basal side of epithelial cells. Once again, antibodies directed against the neurotoxin provided resistance to challenge, but antibodies directed against HA35 did not. Taken collectively, the data indicate that the toxin by itself is capable of crossing epithelial barriers. The data do not support any hypothesis in which HA35 is essential for toxin penetration of epithelial barriers.

Immunization of mice with the non-toxic HC50 domain of botulinum neurotoxin presented by rabies virus particles induces a strong immune response affording protection against high-dose botulinum neurotoxin challenge.

We previously showed that rabies virus (RABV) virions are excellent vehicles for antigen presentation. Here, a reverse genetic approach was applied to generate recombinant RABV that express a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin type A (BoNT/A) and RABV glycoprotein (G). To promote surface expression and incorporation of HC50/A into RABV virions, the RABV glycoprotein (G) ER translocation sequence, various fragments of RABV ectodomain (ED) and cytoplasmic domain were fused to HC50/A. The HC50/A chimeric proteins were expressed on the surface of cells infected with all of the recombinant RABVs, however, the highest level of surface expression was detected by utilizing 30 amino acids of the RABV G ED (HV50/A-E30). Our results also indicated that this chimeric protein was effectively incorporated into RABV virions. Immunization of mice with inactivated RABV-HC50/A-E30 virions induced a robust anti-HC50/A IgG antibody response that efficiently neutralized circulating BoNT/A in vivo, and protected mice against 1000 fold the lethal dose of BoNT/A.

A Human Monoclonal Antibody that Binds Serotype A Botulinum Neurotoxin

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

Evidence that botulinum toxin receptors on epithelial cells and neuronal cells are not identical: implications for development of a non-neurotropic vaccine.

Botulinum toxin typically interacts with two types of cells to cause the disease botulism. The toxin initially interacts with epithelial cells in the gut or airway to undergo binding, transcytosis, and delivery to the general circulation. The toxin then interacts with peripheral cholinergic nerve endings to undergo binding, endocytosis, and delivery to the cytosol. The receptors for botulinum toxin on nerve cells have been identified, but receptors on epithelial cells remain unknown. The initial toxin binding site on nerve cells is a polysialoganglioside, so experiments were performed to determine whether polysialogangliosides are also receptors on epithelial cells. A series of single mutant and dimutant forms of the botulinum toxin type A binding domain (HC50) were cloned and expressed. One of these (dimutant HC50 AW1266L,Y1267S) was shown to have lost its ability to bind nerve cells (phrenic nerve-hemidiaphragm preparation), yet it retained its ability to bind and cross human epithelial monolayers (T-84 cells). In addition, the wild-type HC50 and the dimutant HC50 displayed the same ability to undergo binding and transcytosis (absorption) in a mouse model. The fact that the dimutant retained the ability to cross epithelial barriers but did not possess the ability to bind to nerve cells was exploited to create a mucosal vaccine that was non-neurotropic. The wild-type HC50 and non-neurotropic HC50 proved to be comparable in their abilities to: 1) evoke a circulating IgA and IgG response and 2) evoke protection against a substantial challenge dose of botulinum toxin.

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.