Humoral immunity against bacterial toxins has been studied for over a century. This research journey started when Behring and Kitasato established the existence of humoral immunity by showing that passive transfer of antibodies from the blood of immunized animals could protect non-immune animals against diphtheria [1]. Serum therapy was commonly used to treat infectious diseases until the discovery of sulfonamide in the 1930s [2]. Today serum therapy remains the only prophylactic and therapeutic option against many toxin-mediated and viral diseases. Toxins are excellent target antigens for antibodies, as they are usually structurally distinct from the self-antigens expressed by the host cells. In contrast, anti-infective drugs are usually not selective enough to differentiate pathogenic strains from non-pathogenic flora and thereby have the potential to cause a long-term disruption on the flora dynamics [3]. To date specific antibody is the only compound that can neutralize toxin and they are attractive for therapeutic development because antimicrobial drugs can kill the microbes but does not eradicate pre-formed toxins.

The development of hybridoma technology in 1975 allowed, for the first time, the functional and structural analysis of individual immunoglobulin molecules [4]. Individual antibody molecules can now be isolated in monoclonal preparations and produced in an unlimited supply. The availability of monoclonal antibodies (mAbs) with defined specificity and expressing a single isotype significantly reduces its toxicity relative to immune serum [5], despite the fact that non-human mAbs used directly as therapeutics against human diseases are still immunogenic [6]. The high purity of the preparation also enhances the biological activity per mass of protein [7], since all immunoglobulins are specific for the target toxin. This is particularly important for the development of prophylactic and therapeutic antibodies against infectious and toxin-mediated diseases [8]. In addition to the neutralizing activity that is mediated by the binding of the variable region to toxin, the Fc region of mAb mediates various effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC), complement activation, and opsonization [9]. The combined potential of an immunoglobulin molecule for microbe eradication, toxin neutralization, and the enhancement of host immune system make mAbs attractive candidates for the next generation of magic bullet against infectious diseases.

The toxin neutralizing efficacy of an antibody molecule is usually defined by the activity of its monoclonal preparation. In general, a mAb can be classified as protective, indifferent, or disease-enhancing depending on how it modifies the course of infection or toxemia measured by various types of protection assays. Protective mAb benefits the host primarily by neutralizing the toxin, while disease-enhancing mAb intensifies its toxicity. Indifferent mAb binds to the toxin with no apparent effect on toxicity and host damage. This paper reviews the literature on mAbs to six toxins listed on Pubmed and analyzes the prevalence of the various functional types and isotypes. All the mAbs described result from immunization with whole toxin, toxin component/subunit, or clinical vaccine. Uncharacterized mAbs were not included in our analysis. Only those studies that described the first appearance of a particular mAb are listed—the follow up studies about the chimerization or humanization of a previously reported mAb will not be discussed in this review. In addition, we acknowledge that a significant number of toxin-binding fragment antigen binding (Fab) and single-chain variable fragment (scFv) have been generated as alternative forms of antibody-derived reagents that possess great potential in prophylaxis and therapeutics [10,11,12], but this paper will focus only on mAbs that originate from hybridoma cells.

The mAb studies listed in Table 1 are divided into two groups, with the first one (Group A) describing two or more categories of mAbs (among protective, indifferent, and disease-enhancing mAbs), and the second (Group B) describing a single category of mAbs (Figure 1A). In Group A 14% of the mAbs are protective, 2% are disease-enhancing, and the remainders are indifferent with no measurable effect on the host (Figure 1B). In contrast, for the studies that described only one category of mAbs, 84% of which are protective against toxins (Figure 1C). Humoral responses produce complex polyclonal mixtures with different specificities, isotypes, and affinities, and it is possible that the antibodies reported in Group A are more representative of natural antibody responses from immunization. The fact that a high percentage of the mAbs described in Group B is protective suggests that these studies are skewed to report primarily mAbs with prophylactic or therapeutic potential. A positive result publication bias could have led to significantly fewer publications for those studies that generated non-productive or indifferent mAbs. Consequently, the indifferent mAbs with no apparent benefits to the host are usually suggested as potential diagnostic or research tools. Not surprisingly, given the lack of therapeutic potential, disease-enhancing mAbs are never described on their own in any toxin-mAb studies. Disease-enhancing mAbs have been historically labeled as deleterious by-products of immunizations, while some of them are shown to enhance toxicity by increasing the activation rate of toxin components and through the interaction with the Fc region [13,14,15,16,17,18,19,20,21].

The field of antibody studies revolves around the discovery of novel and useful immunoglobulins against diseases, so it is not surprising that most effort is selectively given to characterize and report those mAbs with therapeutic potential (Figure 1D). While more than 20 mAbs have been developed and marketed as therapeutics against multiple types of cancers and inflammatory diseases, to date only one mAb is licensed for an infectious disease [22]. Palivizumab was approved by FDA in 1998 for the prevention of respiratory syncytial virus (RSV) infection in high-risk pediatric patients [23,24]. Despite of the very low approval rate of mAb against infectious diseases, research institutes and companies still devote large amount of time and resources in that area given that they remain the most reliable means to neutralize toxins. However, the effort in mAb discovery and development has always been spent on the candidates with the greatest potential given limited resources and keen competition [25], and this is evident in the fact that the majority of studies in Group B is focused on protective antibodies (Figure 1C).

The first phase of mAb development against most toxins began in the 80s and slowed down in the early 90s, a result from the wide application of the hybridoma technology and the successful isolation of toxins and their components (Figure 2). The second phase began after the increase of research spending on biological warfare toxins and the approval of the Project BioShield Act in 2004 that specifically aimed “to provide protections and countermeasures against chemical, radiological, or nuclear agents that may be in a terrorist attack against the United States by giving the National Institutes of Health (NIH) contracting flexibility, infrastructure improvements, and expediting the scientific peer review process, and streamlining the Food and Drug Administration (FDA) approval process of countermeasures” [118]. Consequently, the number of mAb studies on National Institute of Allergy and Infectious Diseases (NIAID) biodefense category A and B priority pathogens and their toxins, such as anthrax toxin, ricin toxin and Staphylococcus enterotoxin B (SEB) increased significantly (Figure 2) [118]. Many of these mAbs were further chimerized and humanized (data not shown). In 2009, Human Genome Sciences delivered 20,000 doses of Raxibacumab, a human IgG1 mAb as a treatment of inhalation anthrax, to the US Strategic National Stockpile, and an additional 45,000 doses were ordered later in the same year [119,120]. In contrast, for diseases with a less obvious threat profile and better vaccine/treatment such as those mediated by Shiga toxin, Shiga-like toxin, and pertussis toxin, the mAb development has slowed down since the late 90s (Figure 2). Progress in the generation of prophylactic and therapeutics mAbs against biodefense pathogens has recently been reviewed [121].

Isotype function is important in designing antibody therapeutics and the alteration of isotype subclass can increase protective efficacy [122,123]. The reported percentage of IgG for protective and indifferent mAbs listed in Table 1 is 93% and 81%, respectively (Figure 3). There are about 5 fold more IgM for indifferent mAbs than protective mAbs. Interestingly, 44 of the 45 listed indifferent IgM are raised against either anthrax toxin or Shiga/Shiga-like toxin. A possibility for the relative scarcity of IgM comparing to IgG raised against toxins is that protein antigens trigger T-cell dependent responses resulting in B cell memory, and consequently, IgG becomes the primary isotype of an immunoglobulin to protein antigens. IgG1 is the predominant IgG subclass (Figure 3) [124], but the choice of immunization adjuvants can skew the immune system to produce more IgG of other subclasses [125,126]. Alternatively, the scarcity of IgM may have represented a bias for IgG in preserving hybridomas or lack of screening for IgM in the hybridoma development. IgM as a pentamer with large molecular weight is more difficult to purify, which makes it less attractive as therapeutics candidate. IgA plays important roles in mucosal immunity including the respiratory and gastrointestinal tracts, with primary functions of intracellular neutralization and immune exclusion [9]. However, protective IgA mAbs against toxins are rare that they are only reported in a ricin toxin study [82]. It should be noted that the nomenclature of IgG subclasses between human and mouse is different [127]. Although there is no exact correspondence in function between murine and human isotypes, human IgG1 is generally thought to be analogous to murine IgG2a or IgG2c depending on the mouse strains while human IgG2, IgG3, and IgG4 are considered analogous to murine IgG3, IgG2b, and IgG1, respectively. Although many murine mAbs have been used therapeutically in humans, advances in antibody engineering technologies allow the construction of chimeric and humanized antibodies which have the advantage of being less immunogenic and confer human constant region function [128].

Several caveats are observed from the mAb studies listed in Table 1, which may render the results difficult to interpret and compare. Some of them are discussed below, and the aims are to highlight the diversity of methods and experimental parameters in mAb works, and help guiding the design of future protection studies.

The properties of mAb that include high specificity, purity, and ability to enhance host immune system make immunoglobulins attractive candidates for the next generation of therapeutics. While mAb development is primarily focused on the candidates with the greatest medicinal potential, one should be cautious that the mAbs categorized as non-protective on single antibody screen, which comprise the majority of naturally-produced antibodies (Figure 1B), could be false-negative results. In other words, it is possible that those antibodies are biologically active when they function in combination with other antibodies. Combination testing could reveal new properties of these so called non-protective or indifferent antibodies with the caveat that evaluating antibodies in combinations would increase considerable cost and complexity to any screen for useful mAbs. However, the promising outcomes from mAbs cocktail against viral diseases and synergy between mAb and anti-infective drugs [9] suggest that this approach may unveil the potential of previously neglected mAbs. Perhaps the next generation technology will shift the focus of antibody development from single antibody screening to combination testing. Finally, continuous advancement of mAb engineering in the areas of mAb bispecificity, glycosylation, and Fc region modification will lay a solid foundation for the future mAb therapeutic era against toxin-mediated diseases.