**2. Literature review and hypotheses development**

#### **2.1. Accounting and sustainable development**

Traditional accounting tools do not provide information tailored to the specific needs of environmental issues. Environmental information is often embedded in aggregates of costs and revenues, which do not allow the benefits and losses inherent in this area to be identified. This inefficient allocation of costs complicates the decision-making process.

The occurrence of environmental accounting is consistent with the improvement of environmental awareness that started in the 1970s. The unawareness of different leaders about environmental concerns has given way to rebuff. Increasingly, the focus of firms progressed toward mindfulness, and the appearance of a commitment vis-à-vis the protection of the environment emerged [1].

The need to integrate environmental considerations into decision-making is one of the basic strategies for sustainable development. Sustainability requires taking greater responsibility for decisions, which calls for changes in the legal and institutional frameworks to emphasize the public interest. The law alone cannot impose the public interest; this requires broad public participation in decision-making that affects the environment. When the environmental impacts of the proposed project are high, it requires that there be mandatory public scrutiny of such projects [2].

Income smoothing is one of the most common forms of earnings management, and managers try to maintain the stability of net income by influencing the timing of certain financial events or by selecting specific accounting methods or both. Companies generally prefer to show a stable trend in income growth and do not want to show the volatility of profits, the rise in some periods, and decline in others. To achieve this balance, companies try to maintain income stability.

this area. Then, in 2002, the United Nations Conference in Johannesburg stressed the importance of adopting adequate environmental controls and information systems at different levels in countries that can be used as a basis for political decisions, mainly through the Global

The World Bank is not far behind; it has published recommendations and offers courses to raise corporate social responsibility (CSR) awareness among companies. The Organization for Economic Co-operation and Development (OECD) has made simple recommendations but is a forerunner with papers dating back to 1976. Environmental accounting provides more information and promotes transparency and accountability for political action for the envi-

On another hand, green or universal accounting is slowly entering the world of finance. Global warming, biodiversity, pollution, water consumption, noise pollution, employability, and the fight against discrimination are beginning to be integrated into accounting plans. The objective, in addition to financial performance, is the company's ability to live in harmony with its physical and social environment. An important change in the economic model was evident, and the subject is taken very seriously by a handful of auditors, experts, and researchers.

Traditional accounting tools do not provide information tailored to the specific needs of environmental issues. Environmental information is often embedded in aggregates of costs and revenues, which do not allow the benefits and losses inherent in this area to be identified. This

The occurrence of environmental accounting is consistent with the improvement of environmental awareness that started in the 1970s. The unawareness of different leaders about environmental concerns has given way to rebuff. Increasingly, the focus of firms progressed toward mindfulness, and the appearance of a commitment vis-à-vis the protection of the envi-

The need to integrate environmental considerations into decision-making is one of the basic strategies for sustainable development. Sustainability requires taking greater responsibility for decisions, which calls for changes in the legal and institutional frameworks to emphasize the public interest. The law alone cannot impose the public interest; this requires broad public participation in decision-making that affects the environment. When the environmental impacts of the proposed project are high, it requires that there be mandatory public scrutiny

Income smoothing is one of the most common forms of earnings management, and managers try to maintain the stability of net income by influencing the timing of certain financial events or by selecting specific accounting methods or both. Companies generally prefer to show

ronment by bringing the economy and the environment closer together.

**2. Literature review and hypotheses development**

inefficient allocation of costs complicates the decision-making process.

**2.1. Accounting and sustainable development**

ronment emerged [1].

of such projects [2].

Reporting Initiative (GRI) and the Global Compact.

18 Sustainability Assessment and Reporting

Management is based on the relative performance of the company now and in the future. When current profits are low and expected future profits are strong, the manager borrows profits from the future period for use in the current period. When current profits are good, management tends to save them for potential use in the future.

Several studies have dealt with the concept of preparing income for study and analysis. Fudenberg and Tirole [3] referred to the concept of income as all the methods and processes are used by management in business organizations to reduce income to reduce the degree of risk in the company's investments. Ashari et al. [4] referred to the introduction of income as a set of mechanisms whereby profits are reduced in periods of a significant increase in income and they increase in periods where income falls significantly.

## **2.2. Relation between sustainability reporting and income smoothing: hypothesis development**

A growing number of organizations are making sustainable development a major focus of communication. If sustainable development is designated as the ideal new structure or "skeleton" of a society going through a global crisis, communication is the blood that feeds it. I know the magnitude of the operational challenges that companies face when trying to understand and use this new approach. However, developing an integrated approach to sustainability based communication is an equally difficult challenge. There are perceptions of issues of concern: understanding stakeholder expectations varies, and traditional structures are firmly established.

Indeed, accounting allows the aggregation of all the financial information of the firm and communicating it to stakeholders with interest in the financial management of the company. Accounting can also be used to measure other types of financial information about the company's environmental and social performance, helping managers to make strategic decisions [5]. Several concepts have appeared in parallel with this concern for sustainability, for example, eco-accounting, the "green economy," the carbon footprint among others.

Eco-accounting is a reworking of the traditional accounting system, integrating the internal and external environmental and social and economic costs inherent in the total life cycle of the product. This practice allows managers to review the profitability of their products by considering the environmental and social impacts. For example, an executive could recognize the cost of managing and disposing of waste by property or by department and thus review its profitability. The attribution of a specific cost to an environmental or social effect makes it possible to measure the inefficiency of current methods and to identify new sources of potential savings [6]. However, some costs are difficult to quantify. In this case, the company may allocate an approximate cost to them.

A majority of the sustainability literature relies on institutional theory, which states that firms establish actions for external legitimacy. Conferring to the legitimacy theory, firms attempt to justify their activities as reliable with the norms and values "required" by society because they are permitted to continue their actions through a social treaty. If a firm does not act according to society's rules, a legitimacy gap appears, affecting the firm's very durability [7]. Legitimacy is insight or postulation that the actions of an organization are needed or suitable within some focus on the norms, values, and beliefs in the social area. It increases when society and pertinent stakeholders enhance a company's behavior as correct and convenient; consequently, firms communicate to the relevant public that they work in tandem with norms and values of society. A firm may also report its envisioned objective in social matters along with the use of reporting to adjust the discernment of its actions or hide unethical comportment or the weak quality of its financial information to protect or enhance its legitimacy [8].

A final sample of 94 of the 146 nonfinancial listed companies was obtained (see Appendix 1).

Sustainability Reporting and Income Smoothing: Evidence from Saudi-Listed Companies

http://dx.doi.org/10.5772/intechopen.79219

21

In this section, I describe the variables of the study beginning with the principle variables

Sustainability reporting represents the dependent variable, which is measured by an index. The index includes many items related to CSR, economic, and environmental governance, updated to the new guidelines of the International Organization of Standardization (ISO) 26000. These items are measured via content analysis, grouped in dimensions that describe

In the field of management sciences, the most commonly used method in the analysis of qualitative data, especially interviews, is content analysis [5, 11, 12] as pointed out by [13], p.202: "The place of content analysis is becoming increasingly important in social research, particularly because it offers the possibility of methodically dealing with information and testimonies that have a certain degree of depth and complexity, such as semi-directive interviews." The sustainability reporting score is obtained after an analysis of the quality and quantity of published sustainability information. The assessment of the quality of the information content delivered by the company consists of coding the content delivered according to two modalities: quantitative information and general information. Quantitative information is of better quality and reliability than general information because it is considered to have better

To measure the sustainability reporting index, I evaluate the degree of firm communication by coding a grid of items relating to sustainability, assigning values from zero to four dependent on the quantity or quality of reported information in the firm's website. The rating of the grid of items related to sustainability reporting is based on a score from zero to three; three points are given for an item described in monetarily or quantitatively, two when an item is described precisely, one for an item discussed in general, and zero for no information about the item [15, 16]. Two persons made the valuation of items, and then I made a rapprochement

Regarding the explanatory variable, income smoothing, the firm is supposed to smooth its results if it presents low variability results as referenced to a level considered normal. From a methodological point of view, it is necessary to define the object of smoothing, the duration of study of the smoothing, and the statistical tool allowing evaluation of the variability of the

I use the ratio of cash flow volatility to earnings volatility to measure income smoothing. This measure presents the extent to which accrual accounting has smoothed out the underlying volatility of the firm's operations, which is reliable with previous research on income smoothing [8, 18]. Cash flow (earnings) volatility is the standard deviation of cash flows from

I discarded companies created after 2008 and those that do not have a website.

**3.2. Variables and measurement**

social and environmental areas.

informational influence [14].

between the two results [17].

result.

*3.2.1. Independent and explanatory variables*

(dependent and explanatory) and then the control variables.

According to the institutional theory, sustainability reporting is observed as one of the central concepts that organizations rely on to prove that they work with society's rules [9]. A company's legitimacy is influenced by its sustainability reputation, as well as by its financial situation; sustainability reporting and financial reporting are instruments that outline the stakeholders' insights of these two reputational features [10].

Firms use signaling of their qualities and then act dependably with ethical values. As stated by signaling theory, a pertinent signal should be noticeable to the community. Sustainability reporting is a visible signal to socially responsible behavior. Likewise, it is a costly behavior and cost rise with the volume of the given information.

Regarding the impact of CSR on financial performance, some studies in stakeholder theory suggest a positive link between the two concepts (social impact hypothesis) since it is supposed to improve the satisfaction of the whole. The company's stakeholders, and consequently, the reputation of the company, favor better economic and financial performance. Others, belonging to a liberal trend, establish a negative link (trade-off hypothesis), a socially responsible commitment of the company increasing costs and leading to misuse of capital, causing competitive disadvantages.

According to the signal theory, the incentives emanating from sustainability reporting and unethical comportment, such as income smoothing, have been well documented. Based on the following theories, propositions, and arguments, I can present the following hypothesis:

*Hypothesis:* The more companies disclose about sustainability, the more their managers are motivated to smooth income.


**Abstract**

Vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) is an exciting new procedure that combines the best of laparoscopic and transvaginal surgery. The skills of a laparoscopic surgeon are applied to this approach which offers several advantages over traditional laparoscopy. First, the recovery of a vaginal procedure is shorter and less painful. Second, there is no abdominal incision which avoids potential for wound infection, herniation, pain and unsightly scarring. Third, the surgeon is seated with more comfortable ergonomics than traditional laparoscopy. Fourth, the blood supply is controlled very early in the procedure reducing overall blood loss. Fifth, the specimen for removal is quite close to the operator which enables less crossing of instruments and allows larger scopes with better illumination to be used. Finally, where traditional laparoscopy progresses to a smaller and smaller surgical area as the operation proceeds deeper into the pelvis, vNOTES is continually moving out of the pelvis with greater room for specimen manipulation and visualization. Advantages over traditional transvaginal surgery include the ability to examine the entire abdomen, the safety of direct visualization of the pedicles for adnexal removal, and the ability to perform abdominal procedures including lymph node removal, omentectomy, appendectomy, and biopsies not previously available to the vaginal approach.

**Keywords:** vNOTES, minimally invasive surgery


**8. Intention for formation HABs**

There is no conclusive report available for the causes of HABs, unfortunately, the causes of HABs are uncertain to date. Though, some of the factor which are thought

accountable for causing HABs are briefly described here. An attempt made in **Figure 2** to show interface between some environmental events and microorganism with HABs.

Coastal contamination from a variety of source including household and industrialized effluents is most imperative factors in the growth of HABs. The majority times eutrophication by nutrient enrichment outcomes as blooms of algal growth, from which several are toxic to humans and as well as marine organisms. Contemporary research suggests that eutrophication and climate change are two most important processes that help for proliferation and expansion of Cyanobacterial blooms [51]. It is also important to know that nutrient enrichment can modify the species framework of ecological system [52]. Also inhabitant biota gets displace as the surroundings becomes enriched with nitrates and phosphates [53]. As the coastal eutrophication and improved offshore nutrient concentration taking place offshore due to vertical integration have been linked with the expansion of large biomass, eventually foremost to harmful impacts on ecosystems, human health and fisheries resources. At the same time if eutrophication increases nitrogen and phosphorus inputs, the ratio of these nutrients to silicates becomes very high. This favors non-diatom species including several harmful species. Additionally, it is believed that high concentration of phosphorus, and a low total nitrogen to total phosphorus (TN:TP) TN: TP ratio, are favorable for the production of Cyanobacteria blooms. Recent studies, represents that Cyanobacteria usually dominates in lakes with low TN/TP ratio and are rare in lakes with high TN:TP ratios [54, 55]. Cyanobacteria dominate in lakes where TN:TP mass ratio is below 29:1.

Nutrients consequent from anthropogenic activities have resulted in the increase in HAB account also at some places, unusual heavy rains have resulted in blooms of *L. polyedrum* owing to nutrient rich runoff into the coastal waters. Blooms of Dinoflagellate *Pfiesteria* are found in estuaries of middle and southern Atlantic coasts. The main factors controlling cycles of Dinoflagellates, includes water salinity, pH, nutrients and temperature to one side from these, studies in North California have illustrated that they thrive well near sources of organic phosphates released from sewage treatment plants [56, 57], talk about some Coastal and Continental Shelf Zone (CCSZ) and also Open Oceanic Zone (OOZ) of the Indian segment, were algal blooms can develop only when the calculated rate of biomass increase exceeds the rate of loss generally the grazing and sedimentation rates. As once a bloom develops, it persist for a long epoch under low growth if the rate of loss is small. Still the interactive effects of future eutrophication and changed climate on harmful algal blooms are versatile, and according to current knowledge such processes are likely to enhance the magnitude and frequency of these events. Temperature rise and precipitation associated with climate change falls into broad ranges, also qualms exist in their upshot stratification as North Sea flushing species like *Dinoflagellates* and *Raphidophytes* increase considerably. Some species of *D. acuminata, P. minimum, F. japonica and C. antique* are observed in this region frequently, representing an increase in HAB [58]. Discrepancy in temperature affects circulation patterns, and causes variation in the physical structure of water column that supports occurrence of HABs [59].

#### **9. HAB and impacts**

It is very important to consider harmful Cyanobacterial blooms (cHABs) as they are have noteworthy socioeconomic and environmental outlay, by having impact on water quality, drinking water, agriculture, fisheries, tourism, food web pliability, habitats, along with anoxia and fish kills [60]. Also high biomass accumulation

**43**

*Considering Harmful Algal Blooms*

frequent coughing.

**10. Oceans upwelling**

the southwest coast of India.

**11. Unhealthy coral reefs**

*DOI: http://dx.doi.org/10.5772/intechopen.94771*

and degradation of algal blooms possibly leads to depletion of dissolved oxygen, light attenuation and clogging of fish gills, resulting in fish kills and thousands of other marine life, as direct degradation of the ecosystem [61]. The most critical impacts of algal blooms are on human being health, toxins, produce HABs cause acute and chronic health effects in mammals including humans. For example toxins produced during harmful algal blooms are some of the natural toxic substances directly killing fish/shellfish and other marine life also accumulating in fish and sea food leading to human poisoning after ingestion of contaminated sea food [62]. Sometimes toxins produced during algal blooms may not found toxic to fish and other marine existence. On the other hand, they accumulate in fish and mollusks and move up the food chain and showing shocking impact on humans. Therefore aquatic toxin diseases are categorized into two types as. Shellfish carry toxins that facilitate to paralytic, neurotoxic, diarrheic and amnesic shellfish poisoning. Next type of poisoning is through mollusks tend that occurs during algal blooms. Fish takes toxin that escort to ciguatera and tetrodotoxin poisoning. Poisoning of fish is found more localized and also associated with parts of specific reefs and fishes. Sometimes bloom occurrence of species of Dinoflagellate *Pfiesteria* in estuaries of some middle and Southern Atlantic coast hint that anthropogenic stress on marine environment has caused fish kills and related health hazards in humans also [56]. Species of *Pfiesteria* are also known to cause lesions in fishes. Additionally, we humans can be exposed to toxins that are directly released into water and air. This occur as expected, cell disruption caused through human activities including water treatment. As known such phenomenon frequently occurs in the Gulf of Mexico where residents and beach goers are exposed to toxins through seas spray. Toxins can then be inhaled and lodged in the nose and throat and can down into the lungs. General symptoms associated with this are irritation in respiratory system and

Ascending motions caused due to oceanic circulation is well-known as 'Upwelling', which bring into being some affects to the environmental conditions, beside increases the nutrient content in euphotic zone thus increasing the productivity of the province. Noteworthy findings [63] suggested various factors inducing upwelling off the south west coast of India [64], also have worked to come across the occurrence of the upwelling route along the Dakshina Kannada Coast of India and description shows that upwelling was found to occur from month of March to October along the coast this could be one of the factor for occurrence HABs along

Unhealthy coral reefs play a very imperative role in the formation of blooms, as healthy coral reefs are free of external algal growth [65]. Unhealthy conditions/ death of corals is generally for the reason that pollution of oil or depositions of sediments leading to encrustations of corals by calcareous materials and algae, plus may in turn lead to the death of zooplanktons and some higher fishes in the food web. Also endolithic algal bloom can cause disease named White Syndrome (WS), entailing of distinct lines between healthy and strong corals and dead ones. Such endolithic algae, including *Ostreobium* Spp. penetrate the coral tissues of tabular *Acropora* Spp., in turn affecting the corals with micro-lessions, which

#### *Considering Harmful Algal Blooms DOI: http://dx.doi.org/10.5772/intechopen.94771*

*Environmental Issues and Sustainable Development*

accountable for causing HABs are briefly described here. An attempt made in **Figure 2** to show interface between some environmental events and microorganism with HABs. Coastal contamination from a variety of source including household and industrialized effluents is most imperative factors in the growth of HABs. The majority times eutrophication by nutrient enrichment outcomes as blooms of algal growth, from which several are toxic to humans and as well as marine organisms. Contemporary research suggests that eutrophication and climate change are two most important processes that help for proliferation and expansion of Cyanobacterial blooms [51]. It is also important to know that nutrient enrichment can modify the species framework of ecological system [52]. Also inhabitant biota gets displace as the surroundings becomes enriched with nitrates and phosphates [53]. As the coastal eutrophication and improved offshore nutrient concentration taking place offshore due to vertical integration have been linked with the expansion of large biomass, eventually foremost to harmful impacts on ecosystems, human health and fisheries resources. At the same time if eutrophication increases nitrogen and phosphorus inputs, the ratio of these nutrients to silicates becomes very high. This favors non-diatom species including several harmful species. Additionally, it is believed that high concentration of phosphorus, and a low total nitrogen to total phosphorus (TN:TP) TN: TP ratio, are favorable for the production of Cyanobacteria blooms. Recent studies, represents that Cyanobacteria usually dominates in lakes with low TN/TP ratio and are rare in lakes with high TN:TP ratios [54, 55]. Cyanobacteria dominate in lakes where TN:TP mass ratio is

Nutrients consequent from anthropogenic activities have resulted in the increase in HAB account also at some places, unusual heavy rains have resulted in blooms of *L. polyedrum* owing to nutrient rich runoff into the coastal waters. Blooms of Dinoflagellate *Pfiesteria* are found in estuaries of middle and southern Atlantic coasts. The main factors controlling cycles of Dinoflagellates, includes water salinity, pH, nutrients and temperature to one side from these, studies in North California have illustrated that they thrive well near sources of organic phosphates released from sewage treatment plants [56, 57], talk about some Coastal and Continental Shelf Zone (CCSZ) and also Open Oceanic Zone (OOZ) of the Indian segment, were algal blooms can develop only when the calculated rate of biomass increase exceeds the rate of loss generally the grazing and sedimentation rates. As once a bloom develops, it persist for a long epoch under low growth if the rate of loss is small. Still the interactive effects of future eutrophication and changed climate on harmful algal blooms are versatile, and according to current knowledge such processes are likely to enhance the magnitude and frequency of these events. Temperature rise and precipitation associated with climate change falls into broad ranges, also qualms exist in their upshot stratification as North Sea flushing species like *Dinoflagellates* and *Raphidophytes* increase considerably. Some species of *D. acuminata, P. minimum, F. japonica and C. antique* are observed in this region frequently, representing an increase in HAB [58]. Discrepancy in temperature affects circulation patterns, and causes variation in the physical structure of water

It is very important to consider harmful Cyanobacterial blooms (cHABs) as they are have noteworthy socioeconomic and environmental outlay, by having impact on water quality, drinking water, agriculture, fisheries, tourism, food web pliability, habitats, along with anoxia and fish kills [60]. Also high biomass accumulation

**42**

column that supports occurrence of HABs [59].

**9. HAB and impacts**

below 29:1.

and degradation of algal blooms possibly leads to depletion of dissolved oxygen, light attenuation and clogging of fish gills, resulting in fish kills and thousands of other marine life, as direct degradation of the ecosystem [61]. The most critical impacts of algal blooms are on human being health, toxins, produce HABs cause acute and chronic health effects in mammals including humans. For example toxins produced during harmful algal blooms are some of the natural toxic substances directly killing fish/shellfish and other marine life also accumulating in fish and sea food leading to human poisoning after ingestion of contaminated sea food [62]. Sometimes toxins produced during algal blooms may not found toxic to fish and other marine existence. On the other hand, they accumulate in fish and mollusks and move up the food chain and showing shocking impact on humans. Therefore aquatic toxin diseases are categorized into two types as. Shellfish carry toxins that facilitate to paralytic, neurotoxic, diarrheic and amnesic shellfish poisoning. Next type of poisoning is through mollusks tend that occurs during algal blooms. Fish takes toxin that escort to ciguatera and tetrodotoxin poisoning. Poisoning of fish is found more localized and also associated with parts of specific reefs and fishes. Sometimes bloom occurrence of species of Dinoflagellate *Pfiesteria* in estuaries of some middle and Southern Atlantic coast hint that anthropogenic stress on marine environment has caused fish kills and related health hazards in humans also [56]. Species of *Pfiesteria* are also known to cause lesions in fishes. Additionally, we humans can be exposed to toxins that are directly released into water and air. This occur as expected, cell disruption caused through human activities including water treatment. As known such phenomenon frequently occurs in the Gulf of Mexico where residents and beach goers are exposed to toxins through seas spray. Toxins can then be inhaled and lodged in the nose and throat and can down into the lungs. General symptoms associated with this are irritation in respiratory system and frequent coughing.


**4. Discussion**

In the SPR-POF sensing apparatus here considered, the operative RI ranges were from about 1.33 to 1.41 (see Figure 2). In aqueous matrices with low RI, an RI in this range is obtained when a thin MIP layer is deposited on the gold surface so that the plasmonic wave partially penetrates in the overlying aqueous medium, as shown in Figure 1b. The preparation of the MIP layer reported in the experimental part allowed this goal to be achieved, i.e., having an RI of the layer (MIP plus water solution) in the suitable range. The resonance wavelength shift of the sensor with MIP in water, with respect to that of the bare platform in water, was around 50–150 nm. Similar shifts were previously observed in sensors with MIP's receptor. For example, a value as high as 150 nm in sensors for TNT [13] and nicotine [19], and about 80 nm in a 2-FAL sensor [27] were obtained. Lower Δ*λ* was observed in the case of a monolayer of molecular receptor as in the case of an aptamer [17] or a metal ligand [16,19], confirming that the shift is higher when a higher amount of receptor is present at the resonant surface. As shown in the present investigation (Figure 2), the resonance wavelength should not exceed about 800 nm, so that an MIP layer that is not too thick must be used.

On the other hand, this makes the sensor sensitive to the RI of the overlying liquid, which thus must be carefully controlled.

For this reason, when measuring a real life sample, for example wine, using the sensing method proposed, any RI variation due to the matrix composition must be avoided. This aspect is relevant when thin MIP layers are considered, i.e., thinner than the plasmonic wave penetration in the dielectric. In that case, the Δ*λ* must always be measured in solution with the same RI. The matrix effects are avoided by equilibrating the sample with the sensor and then changing the sample with a solvent with a proper RI (for example, an aqueous buffer), for recording the spectrum and measuring Δ*λ*. In the present work, to demonstrate the method's effectiveness, pure water was selected as the liquid for registering the spectrum for the analysis of 2-FAL in a white wine sample. An alternative

approach could be applied, using a much thicker MIP layer; however, in this case, the RI of the dielectric over gold could not be suitable for the measurements since the resonance wavelength could be outside the operative range.


**5. Conclusions**

This chapter discussed the most important Bayesian frameworks and their applications. These frameworks play a crucial role in statistical and causal inferences. Learning from observations is one of the most fundamental concepts. Learning should be unbiased, systematic, replicable, generalizable, sample and other resources efficient and also provide a sufficient information gain. Since learning problems are often mathematically intractable, Bayesian reasoning can guide development of corresponding numerical algorithms. The algorithms can be considered to be concise representations of data, which can be generated by these algorithms. Bayesian frameworks allow building much more sophisticated models and carry out more complex computer simulations.

*Bayesian Methods and Monte Carlo Simulations DOI: http://dx.doi.org/10.5772/intechopen.108699*

Bayesian experiment design is especially important due to a widespread adoption of computer simulations in designing and analyzing various real-world systems. Research in machine learning is slowly moving from statistical correlations-based models to models exploiting causal relationships. As discussed in this chapter, the causal relationships in SCM (or SEM) can be analyzed by statistical inference methods, which were developed for Bayesian probabilistic models. Bayesian machine learning assigns a distribution to data labels. It improves the performance of semisupervised learning and enables automated data labeling and data label corrections. Bayesian optimization is particularly useful for optimizing real-world complex systems, which are very expensive to evaluate. Both Bayesian optimization and Bayesian machine learning are very active areas of research with many exciting open research problems. Bayesian Monte Carlo simulations aim at providing a systematic, semiautomated, explainable simulation framework. They augment the set of observations and leverage SEM or SCM in order to move from traditional descriptive simulations to predictive and even prescriptive simulations. Such research is still little explored in the existing literature.


**4. Use of a combination of particulate allograft (DFDBA) and platelet concentrates (PRF) in oral and maxillo-facial surgery**

In our department, we routinely use a combination of particulate allograft (DFDBA 300–500 μm) and platelet concentrates (PRF) for the preservation of alveolar bone volume after dental extractions, for sinus grafts (sinus lifts), and for horizontal and vertical bone grafts.

The DFDBA comes from the cortical part of the long bones of the lower limbs of deceased tissue donors in the Erasmus Hospital Bone and Tissue Bank. It is collected by the Erasmus Bone and Tissue Bank team. The samples are then demineralized in HCl to obtain a residual calcium value <10%. Sterilization is then performed using two chemical baths of NaOH and H202 validated by the Institut Pasteur for the destruction of bacteria and viruses and finally undergoes Gamma 25 kGy irradiation at the end of the process. The different particle sizes are obtained by sieving.

To assess whether this DFDBA and PRF mixture was favorable for post-extraction bone filling, we conducted clinical and histological/histomorphometric research.

First, a retrospective radiological clinical study was conducted on 56 patients for whom 95 extractions had been done, to evaluate vertical alveolar bone loss 3 months after tooth extraction, to find out if a combination of particulate allograft (300–500 μm) and PRF could be beneficial for the patient, with predictable results, *The Combined Use of Particulate Allografts (DFDBA) and Platelet Concentrates in Oral… DOI: http://dx.doi.org/10.5772/intechopen.111848*

to preserve alveolar bone volume. Three months after tooth extraction, the mean of vertical loss of the midbuccal bone wall was 0.72 (SD 0.71) mm (5.53%), indicating a good potential for the technique using DFDBA 300–500 μm and platelet concentrates in alveolar bone preservation [25].

In a second study [26], a retrospective clinical study was performed. A total of 84 patients were included with 247 dental implants, to compare peri-implant bone loss at implants placed in alveolar sockets filled with a particulate allogenous bone graft (DFDBA 300–500 μm) and platelet concentrates *versus* at implants placed in the native bone. At 6 and 12 months, the peri-implant bone loss in sockets preserved with DFDBA and platelet concentrates was similar to the peri-implant bone loss in native bone.

In a recent study [27] to evaluate the bone quality of sinus and alveolar grafts following filling with particulate allogenous bone (DFDBA 300–500 μm) and platelet concentrate (PRF), we realized a prospective interventional clinical and histomorphometric study. A total of 40 bone cores, 2 mm in diameter, were taken from 21 patients: 22 from grafted alveoli, 7 from grafted sinus sites, and 11 from native bone used as a control. Fixed, paraffin-embedded samples were subjected to histological staining with hematoxylin–eosin and Masson's trichrome. Bone maturity of the samples was evaluated by two independent operators using histomorphometric analysis. There existed a greater proportion of lamellar neoformed bone than woven neoformed bone as the healing time increased. Moreover, there was also an increasing proportion of newly formed bone in the grafted sockets as a function of healing time (average: 41.22% ≤ 5 months, 55.89% ˃ 5 months). Resorption of DFDBA particles also appears to be correlated with healing time in the grafted socket (average: 15.43% ≤ 5 months, 13.72% ˃ 5 months). We were able to conclude that performing sinus lift and alveolar socket preservation techniques using DFDBA and PRF results in high-quality, mature bone tissue according to histological criteria.

Our studies show that the use of particulate DFDBA mixed with platelet concentrate (PRF) leads to good clinical results with regard to post-extraction bone preservation and success rate of implants placed in the grafted bone. Moreover, a histomorphometric study shows that this technique results in a high-quality mature bone according to histological criteria.

#### **4.1 Horizontal and vertical bone grafting**

For bone grafting of the maxillas, autologous bone grafting has long been considered the gold standard owing to its osteogenic, osteoconductive, and osteoinductive properties [28]. However, this graft, which is taken from intra- or extraoral donor sites, limits the amount of bone available and requires a second invasive surgical procedure, thus increasing the risk of post-operative morbidity [29]. Moreover, it features a mean resorption rate of 50% [28].

Demineralized freeze-dried bone allograft (DFDBA) in the particulate form has been used successfully for many years in the Stomatology and Maxillofacial Surgery Department of the Erasmus Hospital for bone grafting procedures in maxillo-facial and implant surgery.

Its particulate character (particles whose diameter is between 300 and 500 μm and 500–1000 μm) allows its easy adaptation to the recipient bone bed. Nevertheless, the forces applied by the soft tissues can compromise its stability and the maintenance of

the volume of the graft, and it is necessary to add a mechanical means of maintenance of volume during the integration period of the bone graft.

The principle of guided bone regeneration (GBR) consists in placing the graft material under a support acting as a space maintainer, to prevent the non-osteogenic tissues from interfering with bone regeneration [30].

To this end, the use of titanium osteosynthesis screws arranged as a "tent peg" is currently considered an effective therapeutic procedure in the treatment of significant vertical bone deficits (**Figure 1**) [31].

We recently performed a 2-year clinical prospective study [submitted] to quantify the study of horizontal oral bone ridge augmentation using GBR with an association of particulate allografts mixed with platelet-rich fibrin, collagen membrane, and tenting screws. This study aimed to radiologically evaluate the horizontal bone gain. A total of 42 patients with an insufficient alveolar bone width for dental implant placement were treated with a GBR technique using a mixture of particulate allograft (demineralized freeze-dried bone allograft 300–500 and 500–1000 μm), advanced platelet-rich fibrin (A-PRF), resorbable collagen membranes, and screw tents (1.2 mm in diameter). Bone gains were measured by cone-beam computed tomography (CBCT) at 9.1 ± 2.0 months post-operatively. A significant mean increase (P < 0.001) of 3.2 ± 0.9 mm was observed regardless of bone defect location and without complications during the entire post-operative follow-up. All patients benefited from implant placement following the bone augmentation protocol.

We were able to conclude that maxillomandibular bone augmentation in the horizontal plane by the guided bone regeneration technique using a mixture of particulate allograft, platelet concentrates (A-PRF), and screw-tent in combination with a flap closure technique tension-free mucoperiosteal was a procedure that provides reliable, predictable, and reproducible clinical results. This guided bone regeneration technique differs from previous studies on the subject by its qualitative aspects. Indeed, it is easy to use, considerably reducing operating time, with a low complication rate and an absence of morbidity associated with the donor site in the context of autologous bone harvesting. In addition, all the materials and methods used in the context of this technique are reimbursed for the patient.

#### **Figure 1.**

*The placement of osteosynthesis screws arranged in a "tent peg" to counter the forces exerted by the soft tissues. After placement of the demineralized freeze-dried bone allograft (DFDBA) and platelet-rich fibrin (PRF) mixture against the recipient bone bed, a collagenic membrane is positioned to cover the graft. The mucosal flap can be put back in place with sutures.*

*The Combined Use of Particulate Allografts (DFDBA) and Platelet Concentrates in Oral… DOI: http://dx.doi.org/10.5772/intechopen.111848*

#### *4.1.1 Clinical case: Horizontal bone grafting*

This 51-year-old woman, totally edentulous at the maxilla, presents with significant horizontal bone resorption of the upper jaw (Class IV by Cawood et al. [32]). She wants an implant-supported rehabilitation solution, and it is necessary to perform bone reconstruction before placing implants (**Figure 2**).

Computed tomography (CT) scan in left (B) and right (C) premolar areas shows important horizontal loss of the maxillary bone (**Figures 3**–**6**).

#### **Figure 2.**

*Intrabuccal clinical view (A) showing horizontal bone resorption of the maxilla.*

#### **Figure 3.**

*Surgical procedure. A mucoperiosteal incision is made (A) and a mucoperiosteal flap is released. Screws (1.2 mm diameter) are placed (B), to allow guided bone regeneration and four temporary implants are placed avoid prosthesis pressure on the grafted site. A mix of particulate allograft (demineralized freeze-dried bone allograft (DFDBA) 300–500 μm) and platelet concentrate (platelet-rich fibrin (PRF)) is applied between the screws, in tied contact with bone, and covered with a collagen membrane then by PRF membranes (C). Flap is sutured in place without any tension (D).*

#### **Figure 4.**

*Radiological assessment 9 months later. The panoramic X-ray (A) and the computed tomography (CT) scan in right premolar (B), left premolar (C), and anterior (D) areas show a bone gain of between 4 and 5 mm over the entire area that has been grafted.*

**Figure 5.** *The placement of six implants 9 months after the grafting procedure, together with removal of tenting screws.*

#### *4.1.2 Clinical case: Vertical bone grafting*

This 31-year-old woman underwent exeresis of a myxoma of the anterior part of the maxilla 9 years ago. Consecutively, she had lost a part of anterior maxilla, as well as teeth 11, 12, and 13. Clinically, there is a vertical bone loss, together with retracted scar gingiva (**Figures 7**–**11**).

*The Combined Use of Particulate Allografts (DFDBA) and Platelet Concentrates in Oral… DOI: http://dx.doi.org/10.5772/intechopen.111848*

#### **Figure 6.**

*The final prosthetic result. Six months after the placement of implants, the patient can be provided with a fixed upper bridge screwed on implants.*

#### **Figure 7.**

*Clinical view showing vertical bone loss in the area where a myxoma of the anterior part of the maxilla had been removed 9 years ago.*

#### **Figure 8.**

*Panoramic X-ray (A) and computed tomography (CT)-scan axial (B) and sagittal (C) views show the voluminous loss of bone in the anterior region of the maxilla.*

#### **Figure 9.**

*The day of bone reconstruction, a mucoperiosteal flap is raised, then 1.2 mm screws are placed (A), then a mixing of particulate demineralized freeze-dried bone allograft (DFDBA) 300–500 and 500–1000 (2/1)/platelet-rich fibrin (PRF) are compacted against the recipient bone bed (B), the PRF membranes are placed to cover the graft (C), and the site is sutured together with anterior shift of gingival papillas (D).*

#### **Figure 10.**

*Nine months later, X-ray assessment: Panoramic X-ray (A), cephalometric R-ray (B), and computed tomography (CT) scan in axial (C) and sagittal (D) views show a good bone reconstruction in the grafted area.*

*The Combined Use of Particulate Allografts (DFDBA) and Platelet Concentrates in Oral… DOI: http://dx.doi.org/10.5772/intechopen.111848*

#### **Figure 11.**

*Two implants could be placed into the bone graft, and the patient was provided with a fixed bridge screwed on implants.*

### **5. Conclusion**

Particulate allografts are commonly used in oral and maxillo-facial surgery. Particulate Demineralized Freeze Dried Bone Allograft (DFDBA) is mainly used in a granulometry 300–500 μm. Mixed with platelet concentrates, it is a material that is easy to handle and to mold in place on bone beds.

Clinical and histomorphometric studies carried out by our team show that the use of this DFDBA and PRF combination gives good results for post-extraction bone volume conservation, maxillary sinus grafts, horizontal and vertical maxillary augmentations.

### **Conflict of interest**

The author declares no conflict of interest.


**Figure 11.**

*The measured drop in local ionization rate as a function of distance from the cathode.*

20 m of actual travel). At 120 V the attenuation was 20% at a distance of around 1.5 m from the cathode. It should be pointed out that because of the cycloidal electron motion, the true mean free path is many times greater than this. But as a practical matter, with 100–120 V anode voltage, the non-uniformity would be about 20% for a 1500 mm broad beam, and if a cathode were placed at each end (which is easily done), the intrinsic non-uniformity would fall to well under 1% (**Figure 11**).

The spacing and profiles of the electrodes, and the suppression voltage, are modeled with OPERA/ELECTRA, and can be optimized for voltages from 2 keV to 60 keV with ease. **Figure 12** shows a 3 keV case. The beam is self space-charge neutralized, without the addition of neutralizing electrons from a gun. Great care was taken to avoid any unshielded connections to electrodes, etc., which should disrupt this neutralization. For the present we do not use a dynamically adjustable electrode gap, because the high precision required to optimize tuning along the full arc chamber length would be very mechanically challenging.

Ionizing collisions are not the only inelastic collisions that can occur. Neutral excitation and resulting subsequent light emission also occur. For this reason, viewing the glow of the ion beam can be useful but misleading, for the glow is generated by electron transitions resulting from charge-exchange and other collisions with residual gas, so the emitted light is mainly from excited neutral atoms, so some of the halo around the beam may come from excited neutrals which travel outside the beam before decaying.

Discharge uniformity can be improved by two methods. The first is simple to implement. The gas flow to the different gas ports can be adjusted in real time to adjust the uniformity of the extracted beam. Crude adjustment is particularly easy: the current on the suppression electrode is proportional to ions which strike the electrode ideally zero. The suppression current therefore measures beam de-tuning. Initially the


Author details

Robert Stephen Mulholland SpaMedica Plastic Surgery Center (Private Plastic Surgery Practice), Toronto, Canada

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapteris distributed underthe terms oftheCreative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

BodyTite®: The Science and Art of Radiofrequency Assisted Lipocoagulation (RFAL)… DOI: http://dx.doi.org/10.5772/intechopen.83446


**5. Conclusions**

The goal of this chapter was to present the historical, functional, and processual perspectives on working on dreams in CBT. Issues related to dreams are rarely raised during therapeutic sessions in this therapeutic approach. Although research on dreams had an important place when cognitive therapy was established by Beck, ideological, financial, and pragmatic reasons led to its abandonment. Only a few methods related to dreams have been elaborated within CBT, for instance the DMR method of dream interpretation created by Montangero [2] and the guidelines for using dreams defined by Freeman and White [8]. There are very few studies on them; the only exception is the cognitive-experimental model of dream interpretation established by Hill [4]. This model has great empirical support; however, it is not dedicated to CBT. Because of the lack of research on the use of dreams in CBT, cognitive-behavioral therapists are not trained in this area and therefore do not work with their patients' dreams. This vicious circle should be broken by conducting empirical studies about the use of dream analysis in the course of CBT, creating manuals for work with dreams, as well as training programs for therapists about such work. As has been presented, working with dreams in CBT can be useful and beneficial when elaborating on the functional perspective. Dreams can not only facilitate the therapeutic process, but also broaden the self-knowledge of the patient, provide clinical information for the therapist, and be a measure of therapeutic change [26]. Dream analysis can be incorporated into all of the stages of CBT. It is possible to refer to it during clinical diagnosis and case formulation, realization of therapeutic goals, and evaluation and preservation of therapeutic achievements. For dream content, the same methods as for working with automatic thoughts can be used. There is potential for dream analysis within CBT; however, this area is still waiting to be explored by cognitive-behavioral therapists, their patients, and researchers interested in CBT and dreams.

#### **Author details**

Dagna Skrzypińska\* and Barbara Szmigielska \*Address all correspondence to<EMAIL_ADDRESS>Institute of Psychology, Unit of Sleep Psychology, Jagiellonian University, Krakow, Poland


*Article* **Stratified Particle Filter Monocular SLAM**

**Pawel Slowak \* and Piotr Kaniewski**

> Faculty of Electronics, Military University of Technology, ul. gen. S. Kaliskiego 2, 00-908 Warsaw, Poland<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** This paper presents a solution to the problem of simultaneous localization and mapping (SLAM), developed from a particle filter, utilizing a monocular camera as its main sensor. It implements a novel sample-weighting idea, based on the of sorting of particles into sets and separating those sets with an importance-factor offset. The grouping criteria for samples is the number of landmarks correctly matched by a given particle. This results in the stratification of samples and amplifies weighted differences. The proposed system is designed for a UAV, navigating outdoors, with a downward-pointed camera. To evaluate the proposed method, it is compared with different samples-weighting approaches, using simulated and real-world data. The conducted experiments show that the developed SLAM solution is more accurate and robust than other particle-filter methods, as it allows the employment of a smaller number of particles, lowering the overall computational complexity.

**Keywords:** SLAM; autonomous navigation; particle filter; monocular camera; IMU; UAV


*Article* **A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site**

**Siva Krishna Angalakurthi 1,†, David J. Vance 2,†, Yinghui Rong 2, Chi My Thi Nguyen 3, Michael J. Rudolph 3, David Volkin 1, C. Russell Middaugh 1, David D. Weis 4 and Nicholas J. Mantis 2,\***

Received: 26 November 2018; Accepted: 11 December 2018; Published: 17 December 2018

**Abstract:** In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot ("cluster 3") located in close proximity to RTA's active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1–3.3 physically occlude RTA's active site cleft, while the single antibody in subcluster 3.4 associates on the active site's upper rim.

**Keywords:** toxin; antibody; camelid; vaccine; biodefense; hydrogen exchange-mass spectrometry

#### **1. Introduction**

Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins and classified as a biothreat agent due to its high potential to induce morbidity and mortality after inhalation [1–3]. The toxin is a ~65 kDa heterodimeric glycoprotein from the castor bean plant (*Ricinus communis*) consisting of a binding subunit (RTB) and an enzymatic subunit (RTA). RTB is a galactose/N-acetyl galactosamine (Gal/GalNAc)-specific lectin that promotes toxin attachment and entry into mammalian cells [4]. RTA is an RNA N-glycosidase (EC <IP_ADDRESS>) that depurinates a conserved adenosine within the sarcin-ricin loop (SRL) of 28S rRNA, thereby stalling ribosome translocation [5,6]. At the structural level, RTA is a globular protein with a total of 10 β-strands (A–J) and seven α-helices (A–G). RTA folds into three distinct domains: domain 1 (residues 1–117) is dominated by a six-stranded β-sheet, domain 2 (residues 118–210), by five α-helices, and domain 3 (residues 211–267), which interfaces with RTB

through hydrophobic interactions and a single disulfide bond [7,8]. RTA's active site constitutes a shallow pocket formed at the interface of the three domains [8,9]. Active site residues include Tyr80, Tyr123, Glu177, Arg180, and Trp211 (Figure 1A) [10].

**Figure 1.** Structure of enzymatic subunit (RTA) with active site residues and IB2's epitope highlighted. (**A**) The residues that constitute the RTA's active site are in α-helices C, G and E, and a loop between β-strands e and f. The following residues are colored: Tyr80 (green); Tyr123 (red); Glu 177 (blue); Arg 180 (cyan) Trp211 (orange). (**B**) IB2's epitope on RiVax adapted from previous publication [11]. The color shading corresponds to strong (deep blue) and intermediate (light blue) protection. No significant interaction is colored gray.

Inhalation of ricin results in severe lung inflammation characterized by an influx of neutrophils, alveolar edema, and hemorrhage, presumably initiated by the intoxication of alveolar macrophages and lung epithelial cells [1,12,13]. Non-human primates (NHPs) exposed to ricin by aerosol succumb to the effects of the toxin within 24–52 h [12,14]. At the present time, medical intervention following ricin exposure is strictly supportive [15]. However, vaccination strategies have shown great promise in affording complete or near complete protection against ricin intoxicosis in mice and NHPs [16]. For example, intramuscular administration of RiVax, a non-toxic thermostabilized recombinant RTA-based subunit vaccine adjuvanted with aluminum salts, to Rhesus macaques was sufficient to confer immunity to a lethal dose (LD) ricin challenge delivered by aerosol [14]. In vivo neutralization

of ricin toxin following vaccination is associated with onset of anti-RTA IgG antibodies in serum and bronchoalveolar lavage (BAL) fluid [13,14,17].

Monoclonal (mAb) and polyclonal (pAb) antibody responses in mice, rabbits, and NHPs elicited by RiVax vaccination are directed against four spatially distinct immunodominant regions on RTA, which we refer to as epitope clusters 1–4 [11,18–23]. A combination of competition ELISAs, X-ray crystallography, and hydrogen exchange-mass spectrometry (HX-MS) has revealed key secondary elements associated with each cluster. Cluster 1 encompasses RTA's β-strand h (residues 113–117) and α-helix B (94–107), a protruding immunodominant secondary structure element previously known to be a target of potent toxin-neutralizing antibodies [24]. Cluster 2 consists of two subclusters: one involving α-helix A (14–24) and α-helices F–G (184–207) and the other encompassing β-strands d-e (62–69) and parts of α-helices D–E (154–164). Cluster 3 involves α-helices C (121–135) and G (207–217) near RTA's active site, while Cluster 4 is proposed to form a diagonal sash from the front to back of RTA spanning β-strands b, c, and d (35–59). Our long-term goal is to generate a comprehensive molecular B-cell epitope map of each of these clusters and define the specific antibody-contact points on RTA that render the toxin inactive. Such information will be invaluable in efforts to deconvolute the complex human antibody response profile to ricin toxin and RiVax [25].

While much has been learned about clusters 1 and 2 over recent years, comparatively little is known about cluster 3, as it is defined by only a single mAb called IB2 [11]. IB2 was first identified as a toxin-neutralizing mouse mAb that, in competition ELISAs, proved to be distinct from other mAbs in our collection at the time [18,26]. IB2 can passively protect mice against a 5 × LD50 ricin challenge by injection, indicating it has neutralizing activity in vivo, and must, by definition, interact with an important element on ricin toxin. As noted above, we recently demonstrated by HX-MS analysis that IB2 recognizes an epitope involving RTA's α-helix C (residues 121–135) and α-helix G (residues 207–217), which is not only in close proximity to RTA's active site but includes two active site residues, Tyr123 and Trp211 (Figure 1B). However, efforts to interrogate cluster 3 in more detail have been hindered by the absence other cluster 3-specific mAbs. Indeed, recent screens of B-cell hybridomas derived from RiVax and ricin toxoid immunized mice failed to identify additional cluster 3 antibodies [27].

Whereas isolation of additional IB2-like mouse mAbs has not been fruitful, we did recently identify 21 unique heavy chain-only single-domain camelid antibodies (VHHs) that are competed by IB2 for binding to ricin toxin (D. Vance, C. Shoemaker, N. Mantis, manuscript in preparation) [23]. We wished to characterize these VHHs in detail with respect to their binding affinities, epitopes, and capacities to neutralize ricin. In this report, we localized by HX-MS the epitopes of all 21 of these VHHs. We found that the 21 VHHs fall within one of four distinct but overlapping subclusters (3.1–3.4) that share at least one secondary element contacted by IB2. Only two of the 21 VHHs, V6D4 and V1D3, have appreciable toxin-neutralizing activity (TNA), which we speculate is due to their epitope specificity along with strong binding affinity to toxin. This work furthers our overall goal of constructing a complete B-cell epitope map of ricin toxin.

#### **2. Materials and Methods**

#### *2.1. RiVax and VHH Production*

RiVax was expressed and purified from *E. coli*, as described [28]. Please note that RiVax differs from native RTA at two positions, which render the enzyme inactive: there is an Ala at position 80 substituted for Tyr, and a Met at position 76 in place of Val [29]. RiVax also lacks high mannose residues normally found on RTA, due to the fact that RiVax is expressed in *E. coli*. In addition, the RiVax used here has the addition of an Ala at the N-terminus, which we denoted as residue 0 for simplicity. VHHs were expressed in *E. coli* as either thioredoxin- and E-tagged constructs or tag-free variants [22].

#### *2.2. Competition ELISA*

NUNC microtiter plates (Fisher Scientific, Hampton, NH) were coated with competitor mAbs (1 μg/mL in Phosphate Buffered Saline (PBS)) overnight at 4 ◦C and then blocked for 2 h with 2% goat serum (Gibco, Gaithersburg, MD, USA) in 0.1% PBST. Ricin (1 μg/mL) (Vector Labs, Burlingame, CA, USA) was then captured by the mAbs and probed with VHH analytes at 330 nM. Bound VHHs were detected with an anti-E-tag-HRP secondary antibody (Bethyl Labs, Montgomery, TX, USA) and developed with SureBlue 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate (SeraCare, Milford, MA, USA). After quenching with 1 M phosphoric acid (Sigma Aldrich, Carlsbad, CA, USA), absorbance was read at 450 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). % inhibition was calculated by comparing absorbance of captured VHHs on each mAb-ricin complex with that of the absorbance of each VHH captured onto SylH3-ricin, where SylH3 is an anti-RTB mAb that does not interfere with the binding of any VHHs to RTA's cluster 3.

#### *2.3. Vero Cell Cytotoxicity Assay*

Vero cells were detached from culture dishes with trypsin (Gibco), seeded into white 96-well cell culture treated plates (Fisher Scientific) (100 uL per well, 5 × 104 cells/mL) and allowed to adhere overnight. The cells were then treated with Dulbecco's Modified Eagle Medium (DMEM) alone, ricin alone (10 ng/mL), or a mixture of ricin with VHHs at five-fold dilutions. After 2 h at 37 ◦C, the culture medium was changed, and the cells were incubated at 37 ◦C for ~48 h. Viability was assessed using CellTiter-GLO (Promega, Madison, WI, USA). All treatments were performed in triplicate and repeated at least three times.

#### *2.4. Affinity Determinations*

VHH association and dissociation rates were determined by SPR using a ProteOn XPR36 system (Bio-Rad Inc., Hercules, CA, USA). Ricin was immobilized on a general layer compact (GLC) chip (Bio-Rad Inc.) equilibrated in PBS-0.005% Tween running buffer at a flow rate of 30 μL/min. Following EDAC [N-ethyl-N=-(3-dimethylaminopropyl) carbodiimide hydrochloride] (200 mM)–sulfo-NHS (N-hydroxysulfosuccinimide) (50 mM) activation (3 min), ricin was diluted in 10 mM sodium acetate (pH 5.0) at either 4 μg/mL or 2 μg/mL and coupled for 2 min. A third vertical channel received only acetate buffer and served as a reference channel. The surfaces were deactivated using 1 M ethanolamine for 5 min. A ProteOn array system multichannel module (MCM) was rotated to the horizontal orientation for affinity determination experiments. Each VHH was serially diluted in running buffer and then injected at 50 μL/min for 180 s, followed by 1 to 3 h of dissociation. After each experiment, the chip was regenerated with 10 mM glycine (pH 1.5) at 100 μL/min for 18 s, until the response unit (RU) values had returned to baseline. All kinetic experiments were performed at 25 ◦C. Kinetic constants for the antibody/ricin interactions were obtained with ProteOn Manager software 3.1.0 (Bio-Rad Inc.) using the Langmuir fit model.

#### *2.5. HX-MS*

HX-MS experiments for epitope mapping were conducted essentially as described previously [11]. Briefly, a H/DX PAL™ robotic system (LEAP Technologies, Morrisville, NC, USA) was used for sample preparation, mixing and injection. For the free RiVax, 4 μL of 20 μM RiVax stock solution was incubated with 36 μL of deuterated buffer (10 mM sodium phosphate, 150 mM sodium chloride, pD 7.4). For the bound states, the stock solution had a final concentration of 20 μM RiVax and 40 μM VHH resulting in 1:2 molar ratio of RiVax:VHH. Four μL of the stock was incubated with 36 μL of deuterated buffer. Samples were incubated at 25 ◦C for five HX times between 13 s and 24 h and subsequently quenched using 200 mM phosphate-4 M guanidine hydrochloride solution (pH 2.5) held at 0 ◦C. The quenched samples were then injected onto an immobilized pepsin column where proteolysis occurs overlapping peptides from RiVax. The peptides were desalted using a C18 trap and separated using a segmented

gradient with water/acetonitrile/0.1% formic acid on a C18 column (Zorbax 300SB-C18 2.1 × 50 mm, 1.8 μm particle diameter, Agilent, Santa Clara, CA, USA). The entire liquid chromatography system (immobilized pepsin column, C18 trap and a C18 RP-UHPLC column) was kept in a refrigerated cabinet that is maintained at 0 ◦C to minimize back exchange. Nevertheless, the first two residues in a peptide generally undergo rapid back exchange [30]. RiVax peptides were analyzed by an QTOF mass analyzer (model 6530, Agilent Technologies, Santa Clara, CA, USA) for their increase in mass i.e., for deuterium uptake. All HX-MS measurements were based on triplicate independent HX reactions of each labeling time.

#### *2.6. Data Analysis*

The HX-MS data processing was carried out using HDExaminer (version 2.3, Sierra Analytics, Modesto, CA, USA). A total of 138 peptides (Table S1) that cover the entire sequence of RiVax were analyzed. For each peptide, the magnitude of protection from each HX time was averaged and normalized to its peptide length to obtain a ΔHX value, ΔHX = HXbound − HXbound, as described previously [11]. The propagated standard error in delta HX was estimated as described in [31]. The magnitudes of delta HX values of overlapping peptides that span the entire RiVax are then classified using K-means clustering into three categories and were colored as follows: strong protection, intermediate protection, no significant protection. For visualization, the HX-MS results were mapped onto the crystal structure of RiVax (PDB: 3SRP) [32] using PyMoL (The PyMOL Molecular Graphics System; Schrodinger LLC, San Diego, CA, USA). For better visualization purpose, only overlapping peptides that fall in strong and intermediate protection category are colored.

#### **3. Results**

#### *3.1. Identification and Characterization of Cluster 3 VHHs*

Using a variety of screening strategies that are described in detail in separate manuscripts (D. Vance, J. Tremblay, C. Shoemaker, N. Mantis, manuscript in preparation) [23], we identified from different phage-displayed alpaca single chain libraries a total of 21 VHHs whose binding to ricin toxin was partially or completely inhibited by IB2 in a capture ELISA (Figure 2). The competitive ELISA was designed such that IB2 was immobilized on microtiter plates and then allowed to capture ricin in solution. The plates were washed to remove unbound ricin and then probed with query VHHs, as described in the figure legend and Materials and Methods. The DNA sequences and mAb competition profiles of ten of the VHHs were reported in a recent study, although only two (JNM-D1 and V1B11) HX-MS epitopes were described [23].

To further differentiate among the 21 VHHs, they were subjected to a more comprehensive competition array using a panel of nine additional RTA-specific mAbs representing cluster 1 (PB10, WECB2), cluster 1–2 (SWB1), cluster 2 (PH12, TB12, PA1, SyH7), and cluster IV (JD4, GD12) [11]. The competition ELISA revealed a wide range of profiles (Figure 2), indicating the 21 VHHs, as a whole, represent a diversity of epitopes on RTA. Indeed, the predicted CDR3 amino acid sequences of the 21 VHHs suggest they represent at least 11 different B-cell lineages: five unique VHHs and 16 others that fell into one of six sequence families (Table 1; Figure S1).

**Figure 2.** VHH binning by competition ELISA. (**A**) Ricin was captured on microtiter plates by anti-RTA mAbs (indicated along the top panel) or an anti-RTB mAb (SylH3) as a control. The plates were then probed with individual VHHs as indicated on the left most column and detected with a secondary E-tag antibody. Binding inhibition was calculated as 100 − (100 × (AmAb-Ricin/ASylH3-Ricin)) where interference by SylH3 was assumed to be negligible. The colored scale bar on far right indicates % inhibition. VHHs JIV-F5 and JIY-E1 were used as controls, since they are known to bind epitopes on RTA outside of IB2's footprint. (**B**) The IB2 values from panel A are re-plotted for clarity to compare relative IB2 inhibition values and color coded based on subcluster designations described later in the manuscript. The two VHHs with toxin-neutralizing activity are denoted with an \*.

**Table 1.** VHH Families based on CDR3 similarity.

\*, indicates VHHs with toxin-neutralizing activity; The following VHHs were not assigned to a family: JNM-A11, JNM-D1, V1B11, V5A2, V7H7.

The binding kinetics of each VHH for ricin holotoxin was determined by surface plasmon resonance (SPR). Twelve of the 21 VHHs had dissociation constants (*Kd*) of greater than 1 nM, while the remaining nine had dissociation constants ranging from 0.2–1 nM (Table 2; Figure S2). The VHHs were also tested for ricin TNA in a Vero cell assay. Only two VHHs, V6D4 (IC50, 200 nM) and V1D3 (IC50, 80 nM), had demonstrable TNA (Figure S3). Neutralizing activity was not solely a function of binding affinity, as several VHHs with KDs comparable to V6D4 and V1D3 lacked detectable neutralizing activity. For that reason, we sought to localize the epitopes on RTA recognized by each of the 21 VHHs with the expectation that such information would offer insight into the basis of toxin-neutralizing activity.

**Table 2.** Cluster 3 VHH TNA and Binding Affinities.

a, determined by SPR with Langmuir fit model; b, 1/Ms; c, 1/s.

#### *3.2. VHH Epitope Mapping by HX-MS*

We have previously used HX-MS to localize more than two dozen VHH and mAb epitopes on RTA or on RiVax, an attenuated recombinant RTA subunit vaccine antigen with point mutations at positions V76 and Y80 [11,23,27,31,33]. We used RiVax in place of RTA because it is non-toxic to humans and therefore poses no hazard to research staff. RiVax also assumes a tertiary structure essentially identical to RTA [32]. Therefore, HX-MS was performed with RiVax in the presence of two-fold molar excess

of each of the cluster 3 VHHs at five exchange times between 13 s and 24 h. Epitope assignment was based on reduced (slower) HX exchange for peptides in the presence of a VHH, as compared to RiVax alone. For example, in the presence of V6B4, the HX rate in the peptide corresponding to RiVax residues 57–60 was unaltered, whereas there was much slower exchange observed for the peptide corresponding to residues 206–218 (Figure 3). While reduced hydrogen exchange is generally attributed to direct antibody-protein interaction, we cannot necessarily exclude possible allosteric effects that may occur upon antibody engagement, especially when reduced exchange is observed at a distance not consistent with being part of a core epitope [11].

**Figure 3.** Hydrogen exchange (HX) kinetics of two representative RiVax peptides in presence of V6B4. Hydrogen deuterium exchange kinetics of two representative RiVax peptides in presence of V6B4. (**A**) Peptide 14 (56–59), where the HX rate was not affected by association with V6B4. (**B**) Peptide 94 (205–217) where the rate of HX was substantially slowed by V6B4. Significance limit for HX differences was defined as described in the Experimental section.

#### *3.3. Identification of Epitope Subclusters*

The results of epitope mapping studies revealed that the Cluster 3 VHHs grouped within four subclusters, referred to as 3.1–3.4 (Table 3; Table S2;). Subcluster 3.1 involves contact with RiVax α-helices C and G, a profile very similar to mAb IB2. Subcluster 3.2 encompasses α-helices B, C and G, while subcluster 3.3 covers α-helices B and G, but not α-helix C. Finally, subcluster 3.4 encompasses α-helices C and E, but not G. Each of these subclusters is now described in more detail.

**Table 3.** Localization of epitopes on RiVax recognized by representative Cluster 3 VHHs.

a, Peptides on RiVax are indicated in supplementary Table S1. \*, indicates VHHs with toxin-neutralizing activity. Underline indicates intermediate protection determined by HX-MS.

Subcluster 3.1: Sixteen of the 21 VHHs shared an HX-MS profile involving contact with α-helices C and G, which we refer to as subcluster 3.1 (Table 3; Table S2; Figures S4 and S5). While the HX-MS profiles of the VHHs within 3.1 were qualitatively similar, there were quantitative differences that may be significant in terms of neutralizing activity. For example, V1D3, one of the two VHHs with toxin-neutralizing activity, had a binding pattern virtually identical to IB2 in that it strongly protected α-helix C (peptides 54–55, residues 127–135) and the C-terminus region (peptides 132–134, residues 249–255) (Figures 4 and 5). Moreover, V1D3 demonstrated intermediate protection of α-helix G (peptide 91, residues 205–210), as well as strands i and j (peptides 112–116, residues 226–243). In contrast, V6B4, an antibody without toxin-neutralizing activity, strongly protected RiVax residues 119 to 133 (peptides 48–51), corresponding to α-helix C, and residues 205–217 (peptides 91 to 102), corresponding to α-helix G (Figures 4 and 5). However, V6B4 differed from V1D3 in three respects. First, V6B4 had stronger protection of α-helix G than C, as compared to V1D3. Second, V1D3 interacted with β-strands i and j, while V6B4 did not, possibility indicating that V1D3 overall contact interface with RiVax is larger than V6B4's. Finally, the patterns of protection in α-helix C are distinct. In case of V6B4, the entirety of α-helix C is strongly protected, while in the case of V1D3 it is only the C-terminal end that is strongly protected (Figures 4 and 5). V1D3 also caused intermediate protection in the N-terminal end of helix G, while V6B4 protected all of helix G. It is unclear if these differences in α-helix C and α-helix G protection explain V1D3's TNA.

**Figure 4.** HX-MS analysis of RiVax bound to two VHHs in subcluster 3.1. The ΔHX values for each RiVax peptide are shown for VHHs (**A**) V6B4 and (**B**) V1D3. The ΔHX values are clustered using k-means clustering into three categories: strong (deep blue), intermediate (light blue) or no significant protection (gray). The dotted lines represent "3σ" confidence intervals for statistically significant changes in hydrogen exchange.

**Figure 5.** Epitope localization for two subcluster 3.1 VHHs. HX protection categories shown in Figure 4 were mapped onto the crystal structure of RiVax for (**A**) V6B4 and (**B**) V1D3. The most relevant secondary structure elements, α-helices C and G and β-strands i and j, are labeled. The color shading corresponds to strong (deep blue), intermediate (light blue) or no significant protection (gray), as represented in Figure 4.

The competition ELISA with the panel of RTA-specific mAbs revealed additional degrees of difference among the 16 VHHs in subcluster 3.1 (Figure 2). Not only was there a clear gradation of competition with IB2 (range 25–90%), but there were marked disparities with other mAbs. For example, V1B11 is a potent inhibitor of WECB2, V1D3 stood out because of competition with SWB1, while JNM-D1 competes strongly with SyH7. Because the footprints of all 10 anti-RTA mAbs have been defined, we can infer from the various inhibition profiles how different VHHs engage RTA. Thus, looking directly at the RTA active site, with RTB oriented on the bottom, we predict that V1B11 likely approaches RTA from the top down, V1D3 likely from top left, and JNM-D1 likely from bottom left.

Subcluster 3.2: Three VHHs, V6D8, V6F12 and V7H7, were grouped within subcluster 3.2 based on a common HX-MS profile encompassing α-helices B, C and G (Figures 6 and 7). For example, V6D8 strongly protected α-helices B (residues 92–107; peptides 35–39), C (residues 123–135; peptides 49–54), G (residues 211–217; peptides 102) and a short region near the C-terminus of α-helix G (residues 247–255, peptides 129–134). V6F12 shared a binding profile with V6D8, which was not surprising since the two VHHs are likely from the same B-cell lineage (Table 1; Figure S1). Although the protection profiles of V6D8, V6F12, and V7H7 were qualitatively similar, and all three VHHs were competed by IB2 to a similar degree, the magnitudes of protection in the three secondary structural features were distinct. V6D8 and V6F12 interacted primarily with α-helices B and C, and secondarily with α-helix G. V7H7, by contrast, primarily protected several overlapping peptides in α-helix G, and secondarily protected α-helices B and C (except for one peptide in α-helix C). Finally, HX-MS indicated

that V6D8, V6F12, and V7H7 each contact α-helix B, which has been postulated as being a neutralizing hotspot on RTA [22]. However, none of the VHHs within this subcluster had any detectable TNA, possibly because their binding affinities do not achieve a minimum threshold required to inactivate ricin. Other previously described VHHs that engage α-helix B and have potent toxin-neutralizing activities each have binding affinities of less than 200 pM, including JIV-F5 (19 pM), JIY-E5 (191 pM), and JPF-A9 (102 pM) [21,22,33]. This contrasts with V7H7, the strongest binder in subcluster 3.2, which has a binding affinity of ~500 pM.

**Figure 6.** HX-MS analysis of RiVax bound to two VHHs in subcluster 3.2. The ΔHX values for each RiVax peptide are shown for VHHs (**A**) V6D8 (**B**) V6F12 and (**C**) V7H7. The ΔHX values are clustered using k-means clustering into three categories: strong (deep blue), intermediate (light blue) or no significant protection (gray). The dotted lines represent "3σ" confidence intervals for statistically significant changes in hydrogen exchange.

**Figure 7.** Epitope locations of V6D8, V6F12 and V7H7 on RiVax. HX protection categories shown in Figure 6 were mapped onto the structure of RiVax for (**A**) V6D8 (**B**) V6F12 and (**C**) V7H7. Secondary structure elements including α-helices B, C and G and β-strand h are labeled. Intermediate protection by V6D8 is spread over much of RiVax's surface and the magnitudes of protection are low. Therefore, only strongly protected elements are mapped onto the crystal structure of RiVax. The color shading corresponds to strong (deep blue) or no significant protection (gray).

Subcluster 3.3: The third subcluster is populated by V6D4, which had weak toxin-neutralizing activity (IC50 ~200 nM) in the Vero cell cytotoxicity assay. HX-MS analysis demonstrated strong protection of α-helix G (peptide 102, residues 211–217) and intermediate protection of α-helix B (peptides 35–39, residues 92–107). V6D4 also protected a short region in the C-terminus of RiVax, but not α-helix C itself (Figure 8). Whether V6D4's neutralizing activity is a result of contact with

α-helix B is unclear, though its high affinity for ricin (*Kd* = 222 pM) may put it above any relevant affinity threshold.

**Figure 8.** Epitope mapping of V6D4 from subcluster 3.3. (**A**) Relative levels of protection of RiVax peptides by V6D4 as defined by HX-MS. The color shading corresponds to strong (deep blue), intermediate (light blue) or no significant protection (gray), as represented in Figure 4. (**B**) The HX protection categories, as shown in panel A, were mapped onto the crystal structure of RiVax. Secondary structures α-helices B and G are labeled.

Subcluster 3.4: The fourth subcluster is also populated by a single antibody, JNM-A11. JNM-A11 showed strong protection of residues in RTA's α-helix C (peptides 49–51; residues 124–133) and intermediate protection of the N-terminal region of α-helix E (peptides 70 and 71; residues 162–168) and β-strand h (peptides 45 and 46; residues 108–122) (Figure 9). JNM-A11 did not protect α-helix G, which differentiates it from the 20 other VHHs in cluster 3. JNM-A11's competition profile against a panel of RTA-specific mAbs is consistent with results obtained by HX-MS. Namely, JNM-A11 competed with both Cluster 1 (PB10, WECB2) and cluster 1–2 (SWB1) mAbs (Figure 2A). Finally, JNM-A11 did not neutralize ricin, despite a strong binding affinity (*Kd* = 212 pM). Since JNM-A11 appears to target α-helix C almost exclusively, we infer that contact with α-helix C alone is not sufficient to affect ricin function.

**Figure 9.** Epitope mapping of JNM-A11 from cluster 3.4. (**A**) Relative levels of protection of RiVax peptides by JNM-A11, as defined by HX-MS. The color shading corresponds to strong (deep blue), intermediate (light blue) or no significant protection (gray), as represented in Figure 4. (**B**) The HX protection categories, as shown in panel a, are mapped onto the crystal structure of RiVax. Secondary structures α-helices C and E and β-strand h are labeled.

#### **4. Discussion**

As part of our long-standing effort to generate a comprehensive B-cell epitope map of ricin toxin, we have characterized 21 unique VHHs that share the common property of being within the shadow of IB2 based on competition ELISAs. IB2 is a toxin-neutralizing mAb that engages with α-helices C and G on RiVax and defines so-called epitope cluster 3 [11,18]. Cluster 3 is of interest because it encompasses the residues on RTA involved in ribosome inactivation [10,34,35]. The 21 VHHs originated from different phage-displayed libraries, each generated from alpacas immunized with ricin toxin antigens, including RiVax (D. Vance, C. Shoemaker, N. Mantis, manuscript in preparation) [23]. As a result, of epitope mapping studies by HX-MS, the 21 VHHs were further grouped into four distinct subclusters (3.1–3.4) based on their interactions with RiVax α-helix C and α-helix G, as well as other local secondary structures including α-helix B, α-helix E, and β-strand h (Figure 10). The fact that all 21 VHHs engage RiVax via α-helix C and/or α-helix G explains the observed competition with IB2 by ELISA (Figure 2). However, we are unable to explain exactly why V1D3 and V6D4 are the only VHHs within cluster 3 that have toxin-neutralizing activity, since other VHHs have similar footprints on ricin and nearly identical binding affinities as V1D3 and V6D4 but are devoid of neutralizing activity. We can only speculate that neutralizing activity is due to specific residue contacts or combinations of contact that are not apparent by HX-MS epitope mapping methodologies (see below).

**Figure 10.** Visual representation subcluster 3 binding sites on ricin toxin. (**A**) Linear depiction of RTA with arrows denoting β-strand secondary structure and coils indicating α-helices, as per Protein Data Bank (PDB) format. Below, the colored bars denote epitope coverage for each of the VHH subclusters 3.1–3.4. The colors correspond to secondary structures highlighted in panel B. Horizontal line below refers to RTA amino acid residue number. (**B**) Surface representations of ricin (PDB 2AAI) using PyMol showing the regions of protection for each of the four subclusters (3.1–3.4). Colors are as follows: RTA, light gray; RTB, dark gray; active site, yellow; α-helix B, blue; α-helix C, red; α-helix G, green; α-helix E, orange; β-strand h, purple.

RTA's active site consists of a large solvent-exposed cleft on one face of the molecule [10,34–37]. Active site residues include Tyr80, Tyr123, Arg180, and Glu177, which are involved in stacking the purine ring of target adenosine moiety (Tyr80, Tyr123) and transition state stabilization (Glu177, Arg180). Viewing the active site pocket head on, α-helices C (121–135) and G (207–217) would be located at 10 o'clock and 7 o'clock, respectively (Figure 10). Thus, antibodies in subclusters 3.1, 3.2 and 3.3 would be expected to physically occlude (straddle) or even occupy the active site pocket, whereas the single antibody (JNM-A11) in subcluster 3.4 is probably associated with upper rim (11 o'clock) of the active site (Figure 10). To examine these possibilities, efforts are ongoing to solve the X-ray crystal structures of all 21 of these VHHs in complex with RTA.

The current study also highlights both the advantages and shortcomings of HX-MS for use in B-cell epitope mapping. On the upside, the HX-MS pipeline proved to be robust and relatively high throughput due to the fact that we had already established a RiVax peptide map and baseline HX kinetics [11]. HX-MS was able to assess RiVax-VHH binding in solution and parse cluster 3 epitopes into four subclusters that we are currently compared to interaction sites observed by X-ray crystallography. On the other hand, HX-MS provides only peptide level resolution in terms of defining actual antibody contacts on the target antigen and cannot reveal subtle interactions that may ultimately be of consequence to toxin-neutralizing activity. As a case in point, we recently described two VHHs (JPF-A9 and V8A7) with essentially identical HX-MS profiles but that differ in both binding affinity and toxin-neutralizing activity as a result of a single residue difference in CDR2 [33]. Coupling HX-MS with high density competition ELISAs and/or site-directed mutagenesis can significantly improve epitope definition [23,38–40]. The magnitude of HX protection will depend on the affinity and kinetics of binding. Lower affinity generally leads to weaker protection against HX, thereby making it more difficult to resolve the epitope from allosteric effects. However, in practice we have found that introduction of point mutations in VHHs that led to ~10-fold differences in binding affinity (e.g., 0.4 to 4 nM) did not notably alter their HX profiles [31]. Since each epitope mapping data set is treated independently, our analysis still finds the most strongly protected regions.

At this point in time, more than 30 alpaca B-cell epitopes and more than a dozen murine B-cell epitope on RTA have been reported [18,19,21–23,41–44]. The availability of this dense epitope map and a collection well characterized antibodies has already proven to have utility in terms of pre-clinical evaluation of RiVax and other candidate RTA-based vaccine antigens. In one instance the mAbs were used as tools in competition ELISAs to demonstrate epitope use within humans and non-human primates vaccinated with RiVax [14]. More recently, the mAbs were used to evaluate the integrity of key neutralizing epitopes on RiVax during long-term storage [25]. The 21 VHHs described here focused around RTA's active site now add to that growing list of critical reagents.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4468/7/4/45/s1, Table S1: RiVax peptic peptides, Table S2: HX-MS analysis of VHHs in subcluster 3.1, Figure S1: Alignment of cluster 3 VHH families, Figure S2: Representative sensorgrams of Cluster 3 VHHs, Figure S3: Representative toxin-neutralizing activities of cluster 3 VHHs, Figure S4: HX-MS analysis of RiVax bound to VHHs in subcluster 3.1, Figure S5: Epitope localization of subcluster 3.1 VHHs on the surface of RiVax.

**Author Contributions:** Study conceptualization, N.J.M., C.R.M. and D.D.W.; methodology, D.J.V., C.M.T.N., M.J.R., D.D.W. and N.J.M.; formal analysis, S.K.A., D.J.V., D.V., D.D.W. and N.J.M.; investigation, S.K.A., D.J.V., Y.R.; writing—original draft preparation, S.K.A., D.J.V.; writing—review and editing, D.J.V., D.V., D.D.W., N.J.M.; supervision, D.V., C.R.M., D.D.W., N.J.M.; project administration, N.J.M.; funding acquisition, N.J.M.

**Funding:** This work was supported by Contract No. HHSN272201400021C to N.J.M. from the National Institutes of Allergy and Infectious Diseases, National Institutes of Health.

**Acknowledgments:** We thank Chuck Shoemaker and Jacqueline Tremblay (Tufts University) for generating alpaca VHH libraries used in this study. We gratefully acknowledge Beth Cavosie for administrative support. We thank the Wadsworth Center's Biochemistry and Immunology Core facility for assistance with SPR. DDW gratefully acknowledges an equipment loan from Agilent Technologies.

**Conflicts of Interest:** The authors declare no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

#### **References**

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