*Article* **Biomethane Community: A Research Agenda towards Sustainability**

**Idiano D'Adamo 1,\* and Claudio Sassanelli 2**

**Abstract:** The bioeconomy is an effective solution to align with the sustainability agenda and to meet the pressing calls for action from Cop26 on a global scale. The topic of the circular bioeconomy has gained a key role in the literature, while the theme of energy community is a basic form of social aggregation among stakeholders. This work focuses on biomethane and proposes a framework based on several criteria that are evaluated using a hybrid Analytic Hierarchy Process (AHP) and 10-point scale methodology. The results show that regulation and energy community are considered the two most relevant categories. The overall ranking of criteria sees the stakeholders' engagement as the most important, followed by more significant subsidies for small- and medium-sized plants and the principle of self-sufficiency applied at the inter-regional level. Subsequently, the Italian Adriatic corridor composed of four MMAP (Marche, Molise, Abruzzo, and Puglia) regions is considered as a case study in order to evaluate the possible environmental (854 thousand tons CO2eq year ) and economic (from 49 million EUR to 405 million EUR in function of plant size) benefits associated with potential biomethane production of 681.6 million m3. It is found that the biomethane community is an enabler of sustainability and this strategy can be used for sharing different natural resources.

**Keywords:** AHP; bioeconomy; biomethane; energy community; Italy; point scale; stakeholders' engagement; sustainability

#### **1. Introduction**

In a world constantly affected by wars, geopolitical risks, and a constantly surging need to modify production models to cope with resource depletion and scarcity, sustainability [1] is becoming the main challenge worldwide, involving different types of stakeholders in society. Sustainability is "development that meets the needs of the current generation without compromising the ability of future generations to meet their own needs" [2]. Sustainability is the balance point between economic prosperity, environmental improvement, and social equity, famously known as the three dimensions. The Triple Bottom Line captures the spectrum of values that organizations must embrace to be competitive given the increasing weight they are gaining [3].

Sustainable development [4] and the Circular Economy (CE) [5] are the dominant concepts suggesting new business models [6] that are able to reach the Sustainable Development Goals (SDGs) [7]. The CE proposes the criteria of narrowing, slowing, and closing resource loops [8] to promote sustainable development [9]. For this aim, the concept of waste is proposed as an added value [10,11].

Europe, with its strategy of the European Green Deal, aims to be the first climateneutral continent by 2050 [12]. Consequently, the European Commission explicitly seeks to achieve a just transition to a low-carbon energy system. The topic is very much felt in the literature and is becoming increasingly relevant. Some authors highlight three different perspectives: "energy justice occurring within community energy initiatives,

**Citation:** D'Adamo, I.; Sassanelli, C. Biomethane Community: A Research Agenda towards Sustainability. *Sustainability* **2022**, *14*, 4735. https:// doi.org/10.3390/su14084735

Academic Editor: Muhammad Abdul Qyyum

Received: 15 March 2022 Accepted: 13 April 2022 Published: 15 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

between initiatives and related actors, and beyond initiatives" [13]. Energy communities need government tools to develop, and a review of the topic highlighted four categories: (i) community planning and capacity; (ii) environmental protection; (iii) grid access; and (iv) payment-based [14]. For this scope, it is crucial to enhance the integration of the prosumer [15] but also to clarify the role of the stakeholders [16]. These concepts can be studied on individual projects [17] or can identify suitable indicators to measure performance [18].

In addition, the recent war in Ukraine has highlighted energy problems of national selfsufficiency for some countries, and society is no always able to reduce and limit the impact of the main industries on energy consumption (manufacturing [19], transportation [20], and both civil and industrial buildings [21]) through digital technologies adoption. In the current situation, to avoid a placebo effect, this is no longer sufficient, and a breakthrough is needed. The greater energy dependence on foreign countries and the greater dependence on speculative aspects related to the different actors involved in the chain of sale of the energy sources ask for urgent actions in the safe restoration and valorization of waste and biomass to produce valuable and competitive materials and energy [22]. The bioeconomy plays a key role in trying to answer this call, enabling the conversion of natural renewable resources, originating from the biological world, into energy sources [23,24]. The bioeconomy provides suitable answers to the requirements of sustainability in several areas [25,26].

Biomethane can be considered a reference model for the circular bioeconomy [27] through which sustainable best practices could be followed. Indeed, composed of different substrates (e.g., crop leftovers, organic components of municipal solid waste, etc.), it allows the displacement of non-renewable resources with biological ones, triggering the use of biomass and shrinking biowaste presence in landfills [28]. In addition, biomethane can be allocated for different uses (e.g., natural gas grids, energy source for vehicles) and/or converted into feed cogeneration units [29].

Even if different benefits deriving from biomethane adoption can be envisaged for different stakeholders of society levels [30–32], a quite heterogeneous and relevant set of issues can be registered that hinder its full diffusion (e.g., improvement in cleaning and upgrading technologies and processes [33], distrust towards related production plants, and regulation inefficiency [34]). To bridge all the issues related to the full adoption of biomethane towards a widespread solution in current society, pushing the criticalities analysis and leading policymakers to identify guidelines to be implemented to foster biomethane development, innovative frameworks are needed.

Indeed, there is a clear need for understanding how to distribute and deliver the latent value coming from biomethane among all the stakeholders in society through a sustainable hand [22]. The main issue is that to manage to achieve sustainability effectively, the huge quantity of different flows involved (of resources, energy, and wastes) needs a systematization of the related data and information [35]. The main means of bolstering this mechanism are not only digital technologies, which need to be implemented and exploited along the entire product lifecycle [36], but also innovation ecosystems, which are necessary to trigger cooperation and foster the birth of communities able to propose sustainable solutions addressing present and future necessities.

The sustainability bioeconomy aims to provide concrete answers to current problems but also to meet the needs of the literature [22]. For this reason, biomethane is proposed as a virtuous example of a circular bioeconomy, and its potential within an energy community is identified. A topic that needs attention and that finds little space is the use of biomass. Grounded on the concept of energy communities, the goal of this work is to build, propose, and assess a meso-level framework that is needed in CE models to analyze the large number of variables affecting rising biomethane communities. The framework is also engaged to explore the advantages deriving from cooperation dynamics characterizing the MMAP regions model.

The paper is structured as follows. Section 2 presents the research process, detailing the criteria chosen in the hybrid AHP and 10-point scale methodology conducted that are useful for building the biomethane framework development and for its application to the Italian context of the central and southern Adriatic regions. Section 3 shows the results of the survey related to the framework proposed, detailed with the category, local, and global priority (in Sections 3.1–3.3, respectively). It is also proposed in Section 3.4 a discussion to drive a future direction towards the sustainability of the biomethane community composed by the MMAP regions in Italy. Finally, Section 4 concludes the paper, raising the gaps of the research and unveiling that there is room for further studies to address the challenges to be addressed in the near future to bolster local communities in approaching sustainability.

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

A framework gains greater visibility when composed of a substantial number of criteria. The number of criteria can better explain the differences among conflicting criteria or to better delineate a topic. In this paper, we follow the Triple Bottom Line but choose not to propose the three dimensions as reference categories. We aim to identify how other categories influence these dimensions. The need to individuate categories arises from the substantial number of criteria chosen. The AHP is characterized by a small number of criteria and the aggregation of criteria within categories allows for comparison. The local priority and global priority method has precisely this objective.

This section is divided into six parts. In Section 2.1, the hybrid AHP and 10-point scale methodology are proposed with an identification of the experts in Section 2.2. A framework for assessing the impact of biomethane development is proposed in Section 2.3. Furthermore, the environmental and economic values associated with biomethane plants are shown in Sections 2.4 and 2.5. Finally, the case study is proposed in Section 2.6.

#### *2.1. Hybrid AHP and 10-Point Scale Methodology*

Decision models allow for the comparison of alternative solutions and decision making through quantitative data. These methods include AHP, which is used to evaluate energy projects [37,38]. The method proposed by Saaty [39] allows obtaining a priority list to be identified through pairwise comparisons based on expert judgments. The AHP, also known as the analytical hierarchy process, is a structured technique for organizing and analyzing complex decisions and is based on mathematics and psychology [40].

The dimension of the AHP comparison matrix ranges from one to ten factors but is typically set to seven ± two. When the number of criteria is very large, some authors have suggested the use of an integrative method applied to the biogas based on local–global priority [41]. The local priority measures the relevance of a criterion within the same category. The category priority evaluates the relevance of each category and, finally, the global priority is obtained as a product of the local priority and the category priority.

For each matrix, a nine-level scale can be used [42]: 1 → equally preferred; 2 → equally to moderately; 3 → moderately preferred; 4 → moderately to strongly; 5 → strongly preferred; 6 → strongly to very strongly; 7 → very strongly preferred; 8 → very strongly to extremely; and 9 → extremely preferred.

All values are normalized to 1, and to verify the goodness of the results, the consistency ratio (CR) is calculated. The CR is calculated as the ratio between the consistency index and the random inconsistency. This value must be less than 0.10 [42]. This verification occurs automatically during the survey such that experts are not disturbed by an inconsistent assessment.

As a result, this work proposes a hybrid method in which AHP is used to assess local priority. Instead, the 10-point scale—which ranges from not at all (1) to extremely (10)—is chosen to evaluate category priority. This new approach emerged from the need to propose new methodological approaches that maintain a high quality of the result obtained but also from the time requirements related to experts (as highlighted in the pre-check phase of the survey).

#### *2.2. Identification of Experts*

The experts for this work were chosen, leading to 10 academic profiles. The invitation was made through an e-mail to those who have developed a Special Issue on scientific journals in the domains of biomethane, biogas, and, more generally, of bioenergy. The invitation was addressed to European colleagues and contained within it the objective of this work. Participation required a minimum number of years of experience equal to 10 and it was reported that the accessions would be chosen in chronological order. Once the adherence was received, the authors proceeded to organize an interview through Skype or Google Meet, lasting up to 1 h, in which they specifically reported what the study was aimed at, and feedback was also collected. It was important that the excel sheet includes only the necessary values and the self-check associated with the consistency ratio. Table A1 proposes the list of the experts with relative data on the role, country of work, and years of experience.

It should be noted that two of these ten experts were chosen in a phase called the pre-check phase of the survey in which the questionnaire was presented to receive feedback and the methodology was proposed in detail. In this phase, the necessity to replace the AHP methodology with the 10-point value emerged because it was considered more accessible. The motivation was not only due to the potential 9 × 9 matrix but also to the fact that, with a 10-point scale, the criteria were seen as non-conflicting. This information was re-proposed to the other experts as a modification of the first e-mail.

#### *2.3. Biomethane Framework Development*

The goal of this work was to assess a meso-level that is needed in CE models in order to provide a comprehensive framework for analysis [43]. To this end, the framework was built through an analysis of the literature [34,44–47] and the expertise acquired by the experts involved in this research, which led to considering a large number of variables and to ground the framework on the global/local priority approach [48].

Once the criteria were identified, it was decided to divide them into categories to allow for their comparison. To allow for a uniform comparison when evaluating global priority, an identical number of criteria per each category (in this case, chosen equal to three) was considered. As the criteria were chosen, the categories that could contain them were also identified. For the category priority, the number of nine was chosen.

Consequently, this survey consisted of nine 3 × 3 matrices related to the assessment of local priority conducted through the AHP method, while a 10-point scale was more preferred than an AHP-based 9 × 9 matrix to evaluate category priority.

It should be noted that the two pre-survey experts (see Section 2.2) validated the initial list of criteria but indicated changes to make the questions clearer. In addition, the two methodologies were compared (AHP and 10-point scale) to evaluate the category priority, from which emerged a preference for the second one. Furthermore, the values were then normalized to 1 to maintain consistency with what was obtained in the local priority evaluation phase and to make the calculation of global priority consistent.

The chosen categories addressed:

Among the categories, the three dimensions of sustainability were not considered to avoid triggering discussions about how the other categories might be viewed in contrast. However, all 27 criteria have an influence on the three dimensions of sustainability. In some categories, emphasis was placed on subsidies that are currently in place even in developed markets such as Italy. Table 1 proposes the list of all twenty-seven criteria, which is the first result of this work that was to be used as input for the survey.

**Table 1.** Biomethane framework.

#### *2.4. Environmental Analysis*

The environmental benefits associated with biomethane over the use of fossil sources are verified in several works: 23 g CO2eq MJ [49], 40 g CO2eq MJ [50], 53 g CO2eq MJ [51], and 62 g CO2eq MJ [52]. However, it is necessary to specify that several values are proposed for these analyses. The International Renewable Energy Agency (IRENA) estimates the greenhouse gas (GHG) emissions of biomethane according to the feedstock type: liquid manure 33 g CO2eq km , organic waste 48 g CO2eq km , and maize 66 g CO2eq km .  These data show that methane has a less negative impact on the environment than other sources and that biomethane has significant reductions. The highest value recorded for maize is caused by cultivation and harvesting processes.

In this research, three scenarios are considered, defined according to the literature [44]:

According to D'Adamo et al. [44], we defined a model to assess the potential reduction in terms of GHG emissions. It is necessary to make some assumptions to define the environmental savings associated with the use of a certain amount of biomethane. Considering that

a natural gas vehicle (NGV) has an annual mileage of 20,000 km and multiplying this value with that of the three scenarios examined, it is possible to calculate how much less impact an NGV has on the environment if it is fueled by biomethane compared to natural gas. The next step is to estimate how many NGVs can be used by dividing the potential biomethane calculated in the previous subsection and the consumption of one NGV (1333 m3). Finally, it is possible to estimate the overall emissions reduction by calculating the savings associated with a single NGV by their total number.

#### *2.5. Economic Analysis*

Economic analysis related to biomethane plants can be conducted on estimated production costs equal to 0.54–0.73 EUR m3 [29], 0.5–1.5 \$US m3 [53], and 90 EUR MWh [54], as well as on profitability values with a Net Present Value (NPV) that ranges from −585 thousand USD if subsidies were not provided to 5667 thousand USD [55], or from 0.49 million EUR to 132.7 million EUR based upon the mix of recovered waste [56]. However, in the analysis of the costs, the value associated with the externalities is not considered, and for this reason, it is suggested a minimal subsidy equal to 0.13 EUR m3 for biomethane production systems [57].

In March 2018, the Italian government adopted a policy decree (GU (Official Journal) no. 65 of 19 March 2018) to stimulate the development of biomethane [58]. This provides a value of incentive equal to 0.305 EUR m3 (single-counting) for the first ten years. Furthermore, this value is assumed equal to 0.61 EUR m3 (double-counting) if using some sustainable substrates (i.e., the Organic Fraction of Municipal Solid Waste (ofmsw) and by-products). The decree does not differentiate incentives by plant size.

In this research, two scenarios are considered, defined according to the literature [44]:

In the recent period, the conflict in Ukraine has led to a rise in costs, to which speculation has also been added, leading to the biomethane selling price (virtual trading point) being estimated at different values. The base value of 0.25 EUR m3 [44] is modified to 0.375 EUR m3 and 0.50 EUR m3 [22]. Table 2 proposes the economic profitability associated with the minimum and maximum scenarios for two distinct substrates as a function of biomethane selling price.

**Table 2.** NPV of biomethane plants [22,44]. Data are expressed in thousand EUR.

Another useful indicator to monitor the delay in the realization of the projects is the Discounted Do Nothing Cost (DDNC), which, when considered for 1 year, presents the following values [44]:

It should be noted that the decree provides a bonus for an alternative "biomethane producer and distributor" business model. This is an important aspect as the number of points of sale is currently not suitable to meet the needs of consumers, especially in some areas of the country.

#### *2.6. The Italian Context: Central and Southern Adriatic Regions*

The 2019 Integrated National Plan for Energy and Climate assigns a priority role to renewable gas in order to achieve the biofuel release targets set by the directive on the promotion of the use of energy from renewable sources—RED II—in the European Union. The total theoretical potential, which is calculated with data updated to 2016, is estimated to be approximately 6.2 billion m3 per year of advanced biomethane [59]. In particular, these authors have considered the following substrates: (i) straw; (ii) residues from the grape-wine chain; (iii) tomato peel residues; (iv) citrus juice residues; (v) residues from the olive oil industry; (vi) solid urban waste; (vii) zootechnical waste; (viii) sludge from wastewater purification; and (ix) milk whey. The aim of the Italian government is to inject 2.3 billion m3 of biomethane into the gas network by 2026, plus 1.1 billion m3 in transport within the Next Generation EU. The National Federation of Methane Distributors and Transporters (*Federmetano*) proposes the potential production of 8 billion m3 by 2030.

In this context, the regions of central–southern Italy on the Adriatic side (MMAP) have started forms of collaboration to strengthen the Adriatic ridge and constitute a point of overall connection with all of Italy and Europe [60]. This is a political gesture among regions of different political orientation but that are united in building the future of their communities. Italy's population exceeds 60 million, and MMAP regions account for about 12% (Figure 1).

**Figure 1.** MMAP regions.

Table 3 proposes data on the potential of biomethane from substrates relative to the regions examined [59]. However, for the ofmsw, the values proposed by *Istituto superiore per la protezione e la ricerca ambientale* (ISPRA) were considered [61]—Table 4. The conversion factor (*tons o f msw m*3 biomethane) proposed in Table 3 was considered, 75% of separate collection was assumed for all regions (a value currently achieved by the Veneto region), and the weight of organic waste on the total calculated in Table 3 was considered.

**Table 3.** Potential biomethane in MMAP regions (data in thousand m3) [59].

**Table 4.** Data on waste in MMAP regions [61].

Thus, our estimates of an increase in separate collection to 75% for all regions predict an increase in the value of 60,530 thousand m3 of biomethane. The final value is 77,523 thousand m3 of biomethane (an increase of about 17 million m3). Consequently, the potential biomethane in MMAP regions is assumed equal to 681,574 million m3.

#### **3. Results**

This section presents results regarding the relevance of the framework criteria associated with biomethane. Sections 3.1 and 3.2 show the results for category and local priority, respectively, while Section 3.3 shows the results for global priority. Section 3.4 shows the sustainable benefits associated with MMAP regions.

#### *3.1. The Assessment of Category Priority*

The aggregation phase of the judgments provided by the experts consists of receiving the weighted data for each category (Table A2) and each criterion (Table A3). Their product determines the global priority, the result of which obviously depends on the incidence of the two specific components. In particular, the weight of each expert is the same during the aggregation phase. The experts evaluated the incidence of the categories and, in six cases, the maximum value of 10 was assigned to both regulation and the energy community. It should be pointed out that, in four cases, this value was also assigned to substrates and plant size. For the other five categories, the maximum value has never been assigned. It should be pointed out that normalization for this weight does not take place in such a way that the sum of all is equal to 1 but is conducted by comparing the absolute value obtained by the experts divided by the maximum value (equal to 10).

The results show that the four categories proposed above have the highest value that is also close to 10 (Figure 2): regulation (0.96), energy community (0.95), and substrates and plant size (both 0.93). However, all nine categories were considered to be of some relevance

as the lowest value is given to communication with a weight of 0.74. Other categories have the following weight: technology (0.88), final use (0.84), Nimby (0.83), and Nimto (0.82).

#### **Figure 2.** Category priority.

Regulation is seen as a key element as it dominates the sector, defining the guidelines to be followed. Similarly, the theme of the energy community has recently been introduced by the European Union in the Clean Energy for all Europeans package of 2019. It is worth highlighting how the energy community theme has been characterized by the presence of subsidies that play a key role in the development of the sector [48,62]. The same subsidies are used to assess the theme of plant size and for substrates (although not implicitly for the latter category).

#### *3.2. The Assessment of Local Priority*

The analysis conducted within each category made it possible to highlight the role of the specific criteria. In this subsection, the specific result is assessed accordingly, while in the next subsection, the interconnections are identified.

As far as regulation is concerned, all the experts assigned the greatest importance to R2—self-sufficiency principle (inter-regional level and not national)—which assumed a weight of 0.54, followed by R1—self-sufficiency principle (regional level)—with 0.30 (Figure 3). Therefore, the meso-approach is preferred to a macro or micro model as it is considered that the theme of sustainability cannot be circumscribed to a restricted geographical area, which is, in any case, called upon to meet objectives with respect to national performance. In particular, the relationship between local and regional authorities is seen as a strategic lever for moving towards ambitious goals. Some authors have highlighted the relevance of quantifying the potential of biomethane for strategic choices by defining the relevance of the territorial context of reference [63].

**Figure 3.** Local priority (regulation).

The analysis of substrates shows that unsustainable choices cannot be made and, therefore, each area must deal with residues, waste, and raw materials that do not, however, lead to a worsening of eco-systems. This weight of 0.93 is divided between S2—only

sustainable substrates (regional or inter-regional)—with 0.51, and S3—only sustainable substrates (national)—with 0.42 (Figure 4). In particular, the sustainability of the resources used also suggests the use of resources coming from outside the area in question as the environmental balance is, in any case, compensated. In fact, the recovery of resources is linked to a longer transport than expected. The idea prevails that the inability of some areas to grasp sustainable opportunities can be a source of competitive advantage for others. Criterion S2 was considered most important to eight experts, while criterion S3 was considered most important to the other two. The relevance of substrates for energy and sustainable purposes is also evident in the different contributions that can be provided by resources that are classified as first, second, and third generation substrates [64].

**Figure 4.** Local priority (substrates).

Regarding the size of the plants, it is clear that larger plants present significant economic results due to the economies of scale. In addition, this can also depend on incentive policies that do not distinguish between subsidies according to size. Experts did not consider this a particularly critical aspect (P3—subsidies not differentiated by plant size—with 0.30). However, they assigned greater importance to P1—more significant subsidies for small–medium size—with 0.62 (Figure 5). In particular, it emerges that small-scale plants can support sustainable development in areas that do not have a large quantity of raw materials. The risk run is that the non-profitable nature of such plants would rightly not allow their realization. All experts agreed on the greater relevance of criterion P1. The role of size should be properly identified in order to capture both the exploitation of available resources and public acceptance. This is an essential aspect that could be reflected in causing significant delays to the implementation of the work. However, such delays can also be determined by aspects related to bureaucracy [44].

**Figure 5.** Local priority (plant size).

The issue of final use is one of the great advantages of biomethane that takes advantage of its flexibility. However, the perception is that electrical use can be reduced given the presence of other renewable sources and it is hoped that greater use will be made of transport—F2 with 0.60 (Figure 6). The contribution of renewables in the transport sector is still marginal in most European countries. However, the experts did not underestimate the flexible role of biomethane and, therefore, the mix is still given relevance, albeit less significant—F3 with 0.33. All experts agreed on the greater relevance of criterion F2. The purposes of biomethane use are all relevant as they contribute to the reduction in fossil fuels in different applications. However, a comparison of them should be verified in the function of the energy portfolio of the country under analysis [65].

**Figure 6.** Local priority (final use).

The technology category shows that the biogas–biomethane chain is considered mature, but there is space for improvement. In particular, during the interviews with experts, it emerged that particular studies should be placed on how to increase the yield of raw materials. Therefore, the maturity of the technology takes a low weight not because it is considered a weak factor, but because other aspects must be valued. The academic world clearly highlights the importance of collaboration and, therefore, factor T1—enterprise– university relationships—takes on a weight of 0.42. However, the factor considered most important was T3—internally produced plant components—with 0.50 (Figure 7). This figure should also be understood in the period in which the survey was conducted, in which the importance for companies of internally producing the components they use most emerged. Experts were split almost down the middle on the most relevant criterion (six of them identified T3 while the others identified T1). Studies of biomethane in Europe provide experiences that can be examined in other countries as well. The relevance of the connection between technology, subsidies, and domestic production of components plays a favorable role in supporting the development of the sector [66].

**Figure 7.** Local priority (technology).

As far as Nimby is concerned, the experts unanimously penalized this phenomenon if it develops for residues produced in their own area of competence, while they assigned a slight relevance to criterion N3—Nimby with residues not produced in your area—with 0.25. On the other hand, all agreed to consider criterion N1—stakeholders' engagement—the most relevant in this category and the weight assigned was decidedly very significant compared to the others, with an average weight of 0.70 (Figure 8). In particular, sustainable development is also associated with inclusive development and provides for decision-making models that involve all stakeholders. It was highlighted by experts as often being underestimated, but the achievement of which allows implementing good actions and to be a model of best practice. The correct balance between personal interest and the interest of an organization theorized by the school of human relations is the basis of this concept. The topic of stakeholder engagement is not well discussed in the

literature and is one of the topics that will need to be explored [67]. In fact, this aspect turns out to be a decisive factor towards the implementation of energy community models [22].

**Figure 8.** Local priority (Nimby).

The topic of the energy community is analyzed by considering whether its aggregation should be encouraged. The answer provided by the experts was clear. However, the way in which this happened sees seven of them favoring the subsidy while three favored the tax deduction. These aspects are reflected in the final weight, where, however, the difference is not so great: criterion E2—bonus for installations in an energy community (subsidies)—has a weight of 0.51, which is greater than the 0.44 associated with criterion E1—bonus for installations in an energy community (tax deduction)—Figure 9. The subsidy is seen with interest because it stimulates greater production of output. Instead, tax deduction typically affects investment costs. However, the need emerges to introduce a form of incentive to stimulate this ecological transition and to encourage such models of aggregation. A crucial aspect is therefore the contribution of local community energy initiatives to support a decentralized sustainable energy system. The key elements highlighted are the type of organization, the level of activities, and the development of a shared vision. All this translates into a very communicative slogan "power to the people" [68].

**Figure 9.** Local priority (energy community).

With regard to Nimto, it is highlighted how this aspect also influences the results. The judgment of the experts on this aspect was unanimous. However, the judgment changed as to who should be attributed greater responsibility in the non-realization of strategic work. In fact, criterion I1—Nimto determined by local politicians—has a weight of 0.51 and was considered the most relevant by seven experts. The others assigned it to criterion I2—Nimto determined by national politicians—with a weight of 0.44 (Figure 10). The rationale that emerges is that we do not always see a sharp distinction between these two categories based on the actual responsibilities that are laid out in the laws. What does emerge, however, is how political non-decision making can delay change towards ecosystem defenses. Support schemes and innovation are among the main forces that drive investment in renewable energy technologies, and both involve considerable uncertainty. These aspects identify the ability to pursue the fight against climate change. However, one element that can destabilize these choices is uncertainty [69].

**Figure 10.** Local priority (Nimto).

Finally, the communication aspects show that, additionally in this case, the experts agreed on the need to implement changes compared to the current situation. Criterion C2—transparent site in which to report the results—achieved by the sorting and recovery of waste has a weight of 0.42, while criterion C1—organization of webinars/public meetings—is considered more relevant with 0.54 (Figure 11). These data highlight, on the one hand, that autonomy is given to people, to their digital skills. Similarly, interaction, both in human and digital form, is important as it is considered fundamental for dispelling doubts and perplexities. Almost all the experts (nine) identified C1 as the most relevant. However, key aspects of the development of biomethane plants are represented by appropriate communication models [22,70]. It should be emphasized that the literature shows that the topic of the circular economy needs to be more appropriately addressed [71].

**Figure 11.** Local priority (communication).

#### *3.3. The Assessment of Local Priority*

The results show that global priority does not follow the order defined by category priority. In fact, in first place in the ranking, we find criterion N1 (with a value of 0.581), despite the fact that the Nimby category occupies only the seventh position in the relative ranking associated with the categories. This figure can be explained by what was determined within the local priority for this category: N1 has a weight of 70%, which is the most significant among all the cases analyzed.

In numerical terms, in second place in the global ranking is placed criterion P1 (with a value of 0.577). Additionally in this case, with 62%, this occupies the second most significant weight in terms of local priority when considering all the numerical values, even if the plant size category is the third most significant. In third place in terms of local priority, criterion F2 with 60% occupies the fourth place in the global ranking with 0.504, with the category to which it belongs positioned in sixth place. The most relevant category, regulation, sees its most significant criterion (R2) in third place in the global ranking with 0.518.

Among the top ten criteria in the global ranking (which have a value of at least 0.400), we find all the criteria positioned in first place in all categories. The only category that has two criteria is the energy community with criterion E2 positioned in fifth place in the global ranking with 0.485. This result can be explained by the minimal difference between the first two criteria that have a distance of seven percentage points (the same happens for the Nimto category).

In addition, the nine least relevant criteria for each category occupy the last positions in the overall ranking. It is worth noting that eight of the nine criteria have a weight as local priority less than 0.10, and all these criteria have a global priority value of less than 0.100. The exception is criterion R3 (regulation—self-sufficiency principle (national level)), which has a local weight of 0.16 and a global weight of 0.154—Table 5.

**Table 5.** Global priority.

These results highlight that the theme of the biomethane community can be defined as an enabling factor towards sustainability. The integration between the different criteria allows for highlighting how the aggregation between regions is a winning element because it allows for pursuing the spirit of a European community and can be suitable for intercepting public funds available. In addition, collaboration would allow the aggregation of skills and resources and could have greater weight as a leading player in a market.

The essential elements for an energy community foresee the collaboration among all the actors involved in which one could think of a bottom-up decision-making model that is not, however, confirmed by the relevance given to Nimto (the joint weight given both to the political and national class should be underlined), which could push to a top-down model. Clearly, the solution lies in stakeholder engagement where choices are made with everyone's input, but if a final choice is not reached (and it is necessary to make such a choice in order to not reduce future opportunities), a leader who can synthesize is needed. Incentives should privilege small plants to allow a greater spectrum of choices by providing some territories lacking raw materials with this option as well. The choice of biomethane towards transport appears to be the desired end use, and substrates from one's own geographical area can be preferred but others can be accepted if they follow sustainability criteria. The formation of an energy community should be subsidized and, in particular, there is a push for a choice that would increase the production of this energy carrier. Another aspect considered critical is to strengthen the industrial fabric to increase the competitive advantage of a territory.

#### *3.4. Biomethane Community Composed of MMAP Regions: A Future Direction towards Sustainability*

The results obtained from Section 3.3 have highlighted how the theme of the energy community, and specifically that represented by biomethane, is capable of providing various points for reflection. Sustainability requires the contribution of everyone, and during the green transition, it is clear that global scenarios based on competitiveness will change. The recent war in Ukraine, which has led to the death of many children, has highlighted the need for a change in mentality. A sustainable approach as reported in the editorial in which this new section within the *Sustainability* journal was introduced [22] has the ambition of being a meeting point among different stakeholders. A metaphor could indicate a port where ships from different parts of the world can dock, where ideas, projects, and ambitions of everyone can be gathered, but with a special eye on new generations. The Adriatic corridor and its four regions, MMAP, are fortunate to have uncontaminated territories where the relationship between man and nature is not disfigured. There are ideas for improvement that can be pursued and circular bioeconomy models can support the achievement of the Sustainable Development Goals [3].

The strategy can proceed in this direction by expanding the fields of collaboration and creating what could be called the sustainable innovation hub, the idea of an innovation ecosystem based on the involvement of different stakeholders, a result that emerges unequivocally from this research. Sustainable innovation hubs are grounded on the concept of Digital Innovation Hubs (DIHs), i.e., ecosystems that assist companies to improve their competitiveness through innovations and fostering the implementation of up-to-date digital technologies [72,73]. Involving different stakeholders belonging to a heterogeneous ecosystem, DIHs provide a set of supportive services that help companies to become more competitive by improving their business by means of digital technology [74].

The four regions have already signed important agreements in terms of infrastructure, transport, and communications, leaving aside political affiliation, which is certainly an element to be stressed. The need for teamwork is emphasized. This work, in a simple way, aims to unite this great strategic project with the results obtained previously, highlighting how the theme of biomethane development can be an element of sustainable success. This is confirmed not only by the environmental and economic results associated with them but also by a substantial number of social opportunities and related economic opportunities induced that could be connected. In addition, it would allow resources to be acquired at lower values and would allow all interested parties to present valid projects by choosing appropriate technologies, adopting models of public participation and reasoning on how this meso-approach reaches the macro-level of sustainability and is then architected at the micro-level on where to install these plants.

Economic estimates are that ofmsw is treated separately while the other substrates are all incorporated at the by-products level. Environmental estimates consider one cubic meter of biomethane regardless of its source of origin. Figures 12 and 13 provide environmental and economic results.

The obtained results are based on an estimate of potential biomethane production of 681.6 million m3 for the four regions examined (an increase of 17 million compared to values proposed by Pierro et al. [59]). It is evident that this value also includes raw materials currently used for other purposes (e.g., for electricity production through biogas) and that all raw materials are recovered at the technical factors assumed in the reference study. It is also true that an increase in the percentage of separate waste collection has been considered. These data clearly call for the virtuous contribution of all stakeholders. It is clear that ofmsw plants are more profitable than by-products, but a reduction in the profitability of these plants should be envisaged with a simultaneous reduction in the bill paid by citizens, rewarded for good separate collection. In fact, nowadays, it is not only necessary to differentiate but also to do it correctly. This obviously translates into lower costs for the companies that treat such waste in a better energy yield from a technical point of view. As for by-products, it is necessary to inform all stakeholders of the advantages

they could have from the recovery of these residues and their sending first to an anaerobic digestion plant and then to an upgrading plant.

**Figure 12.** Potential environmental results (thousand tons CO2eq).

The economic results also show that as the sale price of biomethane increases, the plants would become more profitable for investors. However, a higher sales price would have a negative impact on consumers. Thus, it is necessary to be vigilant of these aspects. Sustainability is the meeting point of all stakeholders, thus it cannot only support renewable plant owners but also consumers. Taxes that are paid on resources that are more environmentally damaging are considered appropriate. In addition, attention should be paid to the less affluent income groups who do not have the opportunity to take advantage of these sources. It could happen that these groups would have to pay higher prices (taxes on fossil fuels), which would worsen their difficult economic situation.

In this period, great attention has been given to the development of electric vehicles, which, if powered by green sources and with proper battery disposal, are able to provide important environmental results. However, no less important is the role that can be played by NGVs, which are known to pollute less than diesel and gasoline, are a widely known technology, and are typically chosen by consumers for their savings. Evidently, these aspects can be confirmed if natural gas is gradually replaced by green gas. However, we would like to point out that, currently, the energy policy of countries should be projected towards a strong expansion of renewables but the transition should not disregard the use of some fossil sources. Among these, gas has the lowest impact, and the European Commission has indicated this source as necessary for this transition.

The reduction in geopolitical risk and the production of domestically produced energy are necessary steps for the energy development of a country and for energy communities to move towards these directions. The previous analysis has underlined the role of subsidies, which are justified if it is considered at present that several fossil sources are agreed. However, their value cannot be fixed forever in the long term but should be reduced gradually while assuming, however, a stable regulatory framework.

The results of this work assume that at the usual selling price of biomethane and not subject to increases, there would be 49 million EUR in the minimum scenario and 405 million EUR in the maximum scenario (see Figure 13). The choice of plant size must be referred to individual territories. However, some considerations emerge:

The associated DDNC 1-year amounts to 2263 million EUR in the minimum scenario and 19,333 million EUR in the maximum scenario. The delay in the non-construction of the plants has led to greater energy dependence on foreign countries, greater dependence on speculative aspects related to the different actors involved in the chain of sale of the final product, and, above all, a non-contradiction of environmental aspects. The results of this work show a reduction of 854 thousand tons CO2eq year in the baseline green scenario and 777 tons CO2eq year and 931 thousand tons CO2eq year in the alternative ones (see Figure 12).

#### **4. Conclusions**

This work contributes to clarifying how the theme of biomethane, a virtuous model of the circular bioeconomy, is relevant to the reduction in geopolitical risks. The transport sector is called to reduce its environmental impact. The biogas–biomethane supply chain moves towards this direction as it can be used as a vehicle fuel, but it can also be used for other purposes (e.g., it can be distributed into the natural gas grid or converted into cogeneration units).

At present, those who have economic interests in fossil fuels are speculating because they are aware that, today, many countries depend on this raw material. The future foresees the presence on the market of an alternative resource, and this will inevitably lead to a reduction in economic opportunities associated with those who use fossil fuels. It is necessary to identify the transition to advance renewable sources and, in the same way, allow businesses and citizens to have controlled prices.

From a methodological point of view, this work proposes a hybrid approach based on AHP and 10-point value to determine the incidence of the criteria, and this model is suitable when it comes to consider weights based on the local–global priority in which the category priority is calculated considering a consistent number of criteria.

From a conceptual point of view, the model of a united Europe is based on the breaking down of barriers among several countries. The idea of a collaboration of the four regions of the Adriatic corridor (Marche, Molise, Abruzzo, and Puglia) has proved to be a winning strategic idea. The strategy can proceed in this direction by expanding the fields of collaboration and creating what could be called the sustainable innovation hub.

From an operational point of view, the energy communities are able to create new forms of market and alternatives to centralized structures to combine the interests of multiple subjects. However, their realization is by no means simple as it requires an approach based on the concept of shared value. Biomethane is an example of a shared resource that can affect these territories.

The work has some limitations:

However, this work reports economic and environmental results that are verified with an implementation plan for the development of biomethane, whose values may vary as a result of changes in critical variables. Nevertheless, findings underline that the biomethane community is seen as an enabling factor towards sustainable development.

The ecological transition is a great challenge and the increase in energy costs cannot be described as being caused by the development of sustainable sources. Obviously, this change will affect the interests of some companies and investors whose portfolios were based on fossil sources. However, the transition cannot be to the disadvantage of companies and citizens. For this reason, it should be monitored, and cooperation between the private and public sectors is required. Renewables can take advantage of the funds that are made available, but these projects should be implemented quickly in order to avoid generating costs of doing nothing. In addition, the intervention of a third party is required where abnormal market phenomena are created. This outcome would allow for proper movement towards the goals of the Next Generation EU.

The MMAP project may be able to combine tourism and industry to attract young people from all over the world and it may represent a strategic crossroads within the corridors of integrated logistics. The element that can make the difference is the ability to be a team creating a strong, cohesive, resilient, inclusive, and sustainable community.

**Author Contributions:** Conceptualization, I.D. and C.S.; methodology, I.D.; formal analysis, C.S.; data curation, C.S.; writing—original draft preparation, I.D. and C.S.; writing—review and editing, I.D. and C.S.; supervision, I.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable because the survey is not involved with any data of human subjects (1) through intervention or interaction with the individual, or (2) identifiable private information.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to experts used in the AHP methodology.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Table A1.** Survey participants.

**Table A1.** *Cont.*

#### **Table A2.** Category weights provided by ten experts.

**Table A3.** Local weights provided by ten experts.

#### **References**


14. Conclusion

Thrombocytopenia may be a symptom of APS. Paradoxically, it refers to an elevated thrombotic tendency, not bleeding risk. The causes of decreased platelet number are complex: antiphospholipid antibodies that bind to platelets activate them and induce thrombosis. Thrombocytes are consumed during this process. On the other hand, thrombocytopenia is the consequence of destruction by immune mechanisms, like in the case of ITP. Sometimes, it might be the only sign of APS.

Diagnosis is not easy; other causes of thrombocytopenia must be excluded by careful examinations.

Thrombocytopenia in APS is usually mild and per se does not require any treatment. Patients with or without thrombocytopenia require anticoagulant medication in case of venous thrombotic event while low-dose aspirin or clopidogrel is recommended after arterial thrombosis. For OAPS, the combination of anticoagulant and thrombocyte aggregation inhibitor therapy is advised. CAPS is a life-threatening disease with a rapid progression of multiorgan failure. Even application of high-dose corticosteroids, plasma exchange, intravenous immunoglobulin and complement inhibitor agent mortality is very high.


Currently, we have very limited knowledge about anti-NA immunity. To develop a broadly protective vaccine based on NA, there are several critical questions that need to be answered. Firstly, although the NAI antibodies have been determined as an independent correlate of protection in humans (Couch et al., 2013; Monto et al., 2015), this needs to be further validated by the NA-only vaccine constructs in animal and human models. Secondly, very little is known about the breadth of NA immunity. The literature discussed earlier has consistently demonstrated a subtype-specific protection (e.g., within N1 or N2) of NA-based vaccines in animal models. Considering the repertoire of influenza viruses infecting humans and animals (including N1, N2, N3, N7, and N9 encompassing both NA group 1 and 2) (**Figure 1**), a successful NA-based vaccine should be designed to elicit broad protection covering both NA groups. Hence, the determination of conserved regions or epitopes hidden in NA is urgently needed. Thirdly, the mechanisms by which NA antibodies contribute to protection are not completely understood. Many NAI antibodies inhibit its enzymatic activity and thus prevent the release of newly formed viral particles. However, the extent to which NAI antibody titers may be considered protective has not been determined yet. Evaluation of cross-protection against mismatched or heterologous strains may be even more complicated. While ADCC was shown to be involved in protection by nonneutralizing NA antibodies (Jegaskanda et al., 2014, 2017a; Wohlbold et al., 2017), other protective mechanisms are yet to be further elucidated. Further isolation and characterization of broadly protective NA antibodies is required for better design of NA-based vaccines. Comprehensive reviews on NA-based immunity and the perspectives on current knowledge gaps to be addressed may be found in specialized reviews (Wohlbold and Krammer, 2014; Krammer et al., 2018a).

#### UIV AGAINST INFLUENZA B VIRUSES

Besides IAVs, ∼25% of all human influenza virus infections in each epidemic season is caused by IBVs that are classified into two distinct lineages, Victoria-like and Yamagata-like lineages (Ambrose and Levin, 2012). The current seasonal influenza vaccine is prepared in a trivalent or quadrivalent formulation, depending on the inclusion of one or two lineages of IBV antigens. Although priority is given to IAVs owing to a greater impact, IBVs may be more vulnerable targets against which to develop a UIV (**Table 1**) because of their low antigenic diversity and lack of animal reservoir (**Figure 1**) (Tan et al., 2018). Indirect evidence is being accumulated by the isolation of cross-protective antibodies against IBVs. Several broadly protective antibodies binding to head or stalk domain of influenza B HA have been isolated in humans. Overall, these monoclonal antibodies show lineage-specific neutralizing activity in vitro. Further, in vivo protection against both lineages was also demonstrated in mice by passive transfer, through non-neutralizing antibodydependent effector functions (Shen et al., 2017; Hirano et al., 2018; Vigil et al., 2018; Asthagiri Arunkumar et al., 2019; Liu et al., 2019b). Notably, some of B HA stalk antibodies demonstrated extremely broad binding ability (Hirano et al., 2018) or protection against both IAVs and IBVs (Dreyfus et al., 2012). Influenza B NA-reactive broadly neutralizing antibodies were also isolated in animals and humans. Seasonal QIV induced NA antibodies with broad and potent antiviral activity against both lineages in humans (Piepenbrink et al., 2019). Additionally, murine NA antibodies also showed broad protection against both lineages of IBV (Wohlbold et al., 2017).

In line with these observations, chimeric HA strategy has also been tested for a UIV against IBVs. Chimeric HAs consisting of HA head domain from IAV and stalk domain from IBV, delivered as a DNA vaccine (prime), followed by two boosting immunizations with protein vaccines into mice, provided broad protection against both the lineages as well as an ancestral strain of IBV (Ermler et al., 2017). Mosaic HA is an advanced version, in which major antigenic sites of type B HA head domain were replaced by those of type A HA head so that antibodies directed to conserved regions both in the head and stalk domains of type B HA could be induced (Sun et al., 2019). The mosaic B HA provided broad protection against both lineages of IBV, through non-neutralizing ADCC-mediating antibody responses. There are only a few studies reporting B NA-based vaccine offering cross-lineage protection. A study showed that mice immunized with recombinant B NA protein of B/Yamagata/16/88 were protected from homologous Yamagatalike and Victoria-like lineages (Wohlbold et al., 2015). Another study demonstrated that a B NA-based vaccine inhibited the transmission of both homologous and heterologous IBVs in Guinea pig model (McMahon et al., 2019). As compared to IAVs, very little effort has been made so far to develop a UIV against IBVs. However, considering the limited diversity and variability (**Figure 1**) compared to IAVs, further identifications of broadly protective B cell and T cell epitopes would make it possible to develop a pan-influenza B vaccine in the near future (Tan et al., 2018).

## T CELL IMMUNITY AS AN ESSENTIAL FACTOR FOR TRULY UNIVERSAL INFLUENZA PROTECTION

A vast majority of current efforts to develop a UIV are focused on inducing antibody response toward surface glycoproteins, M2e, HA, and NA. However, T cell immunity has been acknowledged as a potential immune correlate of broad protection against influenza infections (Sridhar, 2016; Clemens et al., 2018). T cell immunity may not provide sterilizing or neutralizing immunity against influenza viruses but improves the standard of care by reducing the disease severity and duration of infection, facilitating recovery from illness (Sridhar, 2016). It, therefore, seems that multiple immune arms including both antibodies and T cell immunity are critical to provide a truly universal protection against highly variable influenza viruses. The influenza-specific T cell immunity is known to be highly cross-reactive by recognition of conserved peptides between different subtypes of IAV (Assarsson et al., 2008; Kreijtz et al., 2008; Lee et al., 2008; van de Sandt et al., 2014). Extensive studies have proven the protective role of vaccination or infection-induced cross-reactive CD8+ T cells in various animal models (Kreijtz et al., 2007, 2009; Bodewes et al., 2011; Hillaire et al., 2011). Additionally, in humans, CD8+ T cells offered cross-protection across seasonal, pandemic, avian IAVs, and both lineages of IBVs (Gras et al., 2010; Hayward et al., 2015; van de Sandt et al., 2015; Wang et al., 2018b). The majority of cross-reactive T cell epitopes of IAVs are derived from internal proteins; among >70 T cell epitopes identified between H5N1 and H3N2 viruses, only one derived from HA and other from internal proteins (Lee et al., 2008). This is not surprising given that the conservation rate of internal proteins is >90%, whereas that of surface HA and NA is only 40– 70% among IAVs (Lee et al., 2008), which shows that inducing T cell immunity directed to internal proteins of influenza virus may provide a basis of developing T cell-based UIVs.

Despite poor sequence homology between the HAs of IAVs and IBVs, HA stalk harbors not only extremely broad B cell epitopes but also T cell epitopes encompassing both types of influenza viruses. The fusion peptide in HA stalk contains a cross-reactive CD4+ T cell epitope conserved in both IAVs and IBVs, although its protective role has not been examined in vivo (Babon et al., 2012). A number of CD4+ and CD8+ T cell epitopes are highly conserved in internal proteins (Terajima et al., 2013). A recent study has discovered a universal human CD8+ T cell epitope in PB1 (NMLSTVLGV PB1413−412) that is identical across IAVs, IBVs, and ICVs (Koutsakos et al., 2019). The preexisting memory PB1413−412+CD8+ T cells were detected in the blood and lung tissues of healthy donors and clonally expanded upon infection with IAV or IBV. This report not only showed the existence of heterotypic memory CD8+ T cells in humans that could be induced by exposure to influenza viruses, but also presents the prospect of designing a T cell-based UIV. However, these cross-reactive T cells were not induced in HHD-A2 mouse model despite multiple influenza infections or vaccinations and the protective role of the T cells could not be assessed in the study. Nonetheless, the existence of a number of cross-reactive T cell epitopes between IAVs and IBVs provides an avenue to address to a UIV.

Several T cell-based vaccine candidates are in different stages of clinical development, the major underlying strategy of which is to deliver multiple T cell epitopes derived from different viral antigens including internal as well as surface antigens (Sridhar, 2016). Delivery platforms include replicating or nonreplicating viral vectors derived from vaccinia or adenovirus, recombinant VLPs, recombinant protein or peptide vaccines, and DNA vaccines (Sridhar, 2016; Clemens et al., 2018). Modified vaccinia Ankara (MVA) vector encoding NP and M1 was shown to induce robust T cell responses and provide cross-protection against multiple subtypes in animals and humans (Antrobus et al., 2012; Powell et al., 2013; Hessel et al., 2014; Folegatti et al., 2019). The baculovirus VLPs carrying influenza HA/NA and M1 offered cross-protection where T cells played a significant role in protection in mice (Hemann et al., 2013; Keshavarz et al., 2019). Synthetic peptides or fusion proteins harboring multiple conserved T cell epitopes have also been evaluated for immunogenicity and protective efficacy in animal models (Adar et al., 2009; Rosendahl Huber et al., 2015). While the vaccine approaches described above deliver exogenous antigens and induce CD4+ T cells as well as CD8+ T cells by crosspresentation, DNA vaccines are designed to predominantly activate cytotoxic CD8+ T cells to recognize endogenously expressed antigens presented on MHC class I molecules. In fact, the first report on DNA vaccines was targeted to influenza virus (Cohen, 1993; Ulmer et al., 1993), but initial success in a mouse model did not well-translate into higher animal models due to poor performance (Porter and Raviprakash, 2017). To date, much progress has been made to improve the efficacy of DNA vaccine against influenza virus, encompassing rational design of antigens and expression vectors and the development of novel adjuvants and delivery methods (Lee et al., 2018a). Candidate universal DNA vaccines encoding NP, matrix proteins, or PB1 were shown to decrease viral load and cross-protect against heterologous challenges in diverse animal models including mice, pigs, ferrets, and macaques (Ulmer et al., 1993; Tompkins et al., 2007; Price et al., 2009; Bragstad et al., 2013; Wang et al., 2015; Koday et al., 2017). Further studies are required for to refine DNA vaccine approaches as a stand-alone UIV. Recent studies have indicated that DNA vaccines may serve an attractive component of prime-boost strategy, considering it as a very effective means to priming immune system when preexisting immunity is low (Ledgerwood et al., 2013; DeZure et al., 2017). Despite the potential for broad protection, the safety issues need to be monitored closely, especially because of the documented rise in pathological consequences associated with CTL responses (Peiris et al., 2010; Duan and Thomas, 2016).

#### LIVE ATTENUATED INFLUENZA VACCINE AS AN ALTERNATIVE STRATEGY

## Cross-Protective Immunogenicity of LAIV

LAIV has been approved for clinical use in humans since 2003 and is proven effective in preventing influenza infections (Mohn et al., 2018). The protection of LAIV is superior to IIVs due to multifaceted immune arms including antibody responses and cell-mediated responses that operate systemically and locally (Jang and Seong, 2013a,b; Sridhar et al., 2015). Further, displaying a whole set of viral antigens in a native conformation into the host immune system presents a definite advantage of LAIV to generate better protective immunity than the other strategies relying on a limited set of antigens. As discussed above, T cell immunity directed to the conserved viral epitopes constitutes the cornerstone of cross-protection. A large number of researches have shown that T cell responses induced by LAIV are critical for broad protection against heterologous and heterosubtypic influenza infections in animal models (Cheng et al., 2013; Jang and Seong, 2013a; Rekstin et al., 2017). In young children, only LAIVs were shown to induce durable and potent T cell responses, while developing similar levels of antibody response as compared to IIVs (Belshe et al., 2000; Hoft et al., 2011; Mohn et al., 2015, 2017). Despite well-documented cross-protection, LAIV has received little attention to develop a UIV. This may be attributed to the general belief that LAIV is not effective at inducing broadly neutralizing antibodies against conserved domains in surface antigens (e.g., M2e or HA stalk). However, close attention is recently being given to LAIV as an alternative platform as a potent and cross-protective vehicle than previously thought, through inducing multifaceted immune correlates including T cell response and antibody-mediated effector functions (Jang and Seong, 2013a, 2014).

#### UIV Approaches Using LAIV

In the UIV approaches reported so far, LAIV was used either as a component in prime-boost regimens with other different vaccine formats such as IIV, DNA vaccine, or recombinant protein vaccine. Alternatively, LAIVs were also studies as a standalone vaccine given in single or multiple doses. A reassortant LAIV expressing a chimeric HA was constructed under the genetic background of Russian strain (A/Leningrad/134/17/57 cold-adapted virus) and used as a boosting vaccine in a ferret model (Nachbagauer et al., 2017). Notably, the LAIV-IIV regimen showed greater protective efficacy against the pandemic H1N1 challenge than the IIV-IIV regimen in terms of viral loads in the respiratory tissues, despite 32-fold lower stalk antibody titers in serum. Several factors were presumed to account for this disconnect between stalk antibody titers and protection efficacy, including anti-NA immunity, mucosal IgA antibodies, cellmediated immunity, and non-specific innate immune responses offered by the LAIV. Similar results were obtained when using a different LAIV strain (A/Ann Arbor/6/60 cold-adapted strain) (Nachbagauer et al., 2018). In a subsequent study performed by the same group, chimeric HA-containing the LAIV-LAIV (A/Leningrad/134/17/57 cold-adapted strain) vaccine regimen was compared with the LAIV-IIV combination in terms of protection efficacy in a ferret model, in which the LAIV-LAIV vaccine regimen conferred superior protection against pandemic H1N1 and H6N1 challenges than the LAIV-IIV (Liu et al., 2019a). Another group tested a vaccination regimen comprising only LAIV (A/Leningrad/134/17/57 cold-adapted strain) as primeboost vaccination in a mouse model (Isakova-Sivak et al., 2018). To enhance the breadth of protection, an internal gene of cold-adapted virus was replaced with the wild type. This study compared the immunogenicity and cross-protection between chimeric HA-containing LAIVs and natural HA-containing LAIVs. The chimeric HA-containing LAIVs induced higher HA stalk antibody titers and showed better cross-protection against heterologous infection with various group 1 IAVs. Thus, a cooperative role of cell-mediated immunity and HA stalk antibodies was suggested, although their individual contribution to protection were not assessed directly. It would be interesting to investigate if the cross-protection could be extended to group 2 influenza viruses such as H3 or H7 strains.

Besides the Leningrad and Ann Arbor strains, an independent cold-adapted vaccine strain (X-31ca) that was previously used for trivalent seasonal vaccine (Jang et al., 2014), H5N1 pre-pandemic vaccine (Jang et al., 2013c), and 2009 pdmH1N1 vaccine (Jang et al., 2012, 2013a), was recently tested as a UIV in a mouse model (Jang et al., 2018). Mice vaccinated with single or two doses of X-31 ca LAIVs were completely protected against lethal challenge of heterosubtypic strains encompassing both HA group 1 and 2 IAVs. Interestingly, boosting with heterosubtypic LAIVs carrying different HA and NA surface antigens showed more potent cross-protection than homologous boosting. T cell immunity and NK cell-mediated ADCC activity was demonstrated to contribute significantly to the observed cross-protection. As the first report of pan-influenza A protection covering both HA groups, these results merit further studies in a ferret model for clinical relevance. Hence, the LAIVs appear to be a powerful tool to develop a UIV that confers broad and potent cross-protection as a stand-alone vaccine or in combination with other strategies.

## Future Prospects of LAIV-Based UIVs

While the LAIV presents a remarkable prospect for a broadly protective influenza vaccine, several important issues need to be addressed for it to serve as a reliable vaccine modality. The protection efficacy of a LAIV substantially differs with age. The estimated protection efficacy of a LAIV is 80% in young children and only 40% in adults, to the matched strains (Jefferson et al., 2008, 2010). As for T cell immune responses, clinical studies indicate that LAIVs induce better T cell response than IIVs in both children and adults (He et al., 2006; Subbramanian et al., 2010; Hoft et al., 2011). However, clinical studies have reported that LAIVs are not effective at inducing T cell responses in adults and the elderly, perhaps due to preexisting "vector" immunity, which limits the replication of LAIVs (He et al., 2006; Forrest et al., 2008; Hoft et al., 2011). Considering that both humoral and cell-mediated immunity contribute to broad protection, it will be important to elucidate how preexisting immunity or immunologic imprinting affects B cell and T cell immune response induced by LAIVs in humans (Gostic et al., 2016; Henry et al., 2018). For this purpose, animal models mimicking preexisting immunity and controlled human challenge studies will be needed. Considering that LAIVs mimic natural infection, a fundamental question remains to be answered: if infection (or vaccination with LAIV) is effective for conferring protection, why are humans vulnerable to repeated infections with homologous or heterologous strains? At the population level, currently used cold-adapted LAIVs provide a relatively low protection rates (∼40% in adults) even against matched strains (Jefferson et al., 2010). However, little is known whether individuals who successfully acquired protective immunity by a cold-adapted LAIV were protected against other heterologous strain(s) in the next epidemic. This may be directly addressed by a wellcontrolled longitudinal cohort study using human challenge models. Some studies showed that LAIVs were able to generate cross-reactive T cell responses in children for up to 1 year after vaccination, as a basis of long-term cross-protection in humans (Mohn et al., 2015, 2017).

Most of the LAIV-based approaches are based on coldadapted attenuated strains (Nachbagauer et al., 2017, 2018; Jang et al., 2018; Liu et al., 2019a). These strains acquire multiple attenuation mutations in the internal genes during the coldadaptation process, which contribute to genetic stability to overall attenuation (Jang et al., 2016). A common concern for live vaccines is safety issues, especially those associated with potential reversion of attenuating mutation(s) into virulence during vaccination. LAIVs acquired multiple attenuation mutations among various internal genes and proven safe as seasonal influenza vaccines (especially for A/Ann Arbor strain as the master strain for FluMist). However, genetically engineered LAIVs with a limited set of attenuation mutations, e.g., NS1 deletion or elastase-dependent HA cleavage (Talon et al., 2000; Stech et al., 2005), may require additional monitoring on safety.

Defining the precise correlates of protection represents the most challenging step in the development of a UIV (Erbelding et al., 2018). Significant efforts were made to identify the protection mechanisms of HA stalk-based vaccines, suggesting that direct neutralization in combination with Fc-dependent indirect effector mechanisms mediated by stalk antibodies were the primary correlates of protection. In contrast to HA stalkbased vaccines, LAIVs elicit multiple immunological factors including serum IgG antibodies and mucosal IgA antibodies to surface antigens (HA, NA, and M2) and cell-mediated immunity to internal antigens, synergistically contributing to protection. However, their quantitative relationship to protection has not been determined, not even for homologous protection (Sridhar et al., 2015; Mohn et al., 2018), let alone for cross-protection against heterologous infection. The development of standardized assays to quantitatively measure T cell-mediated protection is particularly challenging, as the magnitude and the subset of T cells critical for protection is likely to differ according to strains of LAIV and challenge viruses. Further, mucosal IgA antibodies are believed to correlate with cross-protection, but it is still challenging to measure the neutralizing activity or effector functions of mucosal IgA antibodies. The complicated nature of LAIV-induced immunity, including non-neutralizing antibodies and diverse subsets of T cells, present a bottleneck to identifying precise correlates of protection.

Another important aspect of LAIV-based strategies lies on the LAIV strains. During the past century, H1N1 and H3N2 subtypes of influenza A viruses were the most prevalent strains in humans, causing annual epidemics as well as occasional pandemics, except for the temporal circulation of H2N2 during 1957–1968 (Kilbourne, 2006) (**Figure 6**). Accordingly, seasonal influenza vaccines are recommended to include H1N1 and H3N2 vaccine antigens for more than 40 years since 1977. Therefore, it is likely that most contemporary population has preexisting T cell immunity to H1N1 and H3N2 strains through natural infections or vaccinations. It should be remembered that currently licensed LAIVs (A/Ann Arbor/6/60 ca and A/Leningrad/134/17/57 ca) are of H2N2 subtypes. Probably, the nature of strain itself does not really matter for seasonal influenza vaccine, for which strain-specific immunity is focused on the surface HA antigen. However, for eliciting cross-protection, the role of conserved region become important (see section HA stalk-based UIV approaches, Conserved targets in HA other than stalk, NA as a novel target for a UIV for HA/NA and section T cell immunity as an essential factor for truly universal influenza protection for internal proteins). It is likely that human populations under 50 years of age (born after 1968 when H2N2 became extinct) has little preexisting immunity against H2N2, but predominantly against H1N1 and H3N2 viruses. It will therefore be worthwhile to examine whether cold-adapted LAIVs of H1N1 (Jang et al., 2018) or H3N2 origin (non-existent, to our knowledge) offer a beneficial effects on boosting the preexisting cross-reactive T cell immunity and antibody effector functions (section Mode of protection by a UIV; **Figure 3**).

As for IBVs, two distinct lineages diverged and circulated after 1985, which necessitates the incorporation of Victoria-like and Yamagata-like lineages in seasonal influenza vaccines. This leads us to raise a possibility of using an ancestral influenza B strain (such as B/Lee/40) before divergence into two different lineages as a UIV candidate for IBVs. Establishing new cold-adapted LAIV strains is a time-consuming and laborious. However, recent advances in reverse genetics and rational approaches to attenuate viral virulence have enabled the rapid conversion of a wild type virus into a novel LAIV strain.

The redirection of host immune responses from surface proteins toward internal proteins may be achieved by rational vaccination strategies with LAIVs. For example, the down regulation of expression levels of HA or NA in a LAIV (Yang et al., 2013) is likely to result in preferable induction of T cell immunity to internal proteins. Alternatively, vaccination with LAIV carrying HA from non-human influenza viruses such as H5 or H9 may be effective at boosting preexisting T cell immunity to internal proteins in humans. Given the cross-reactivity of T cell immunity between IAVs and IBVs, A type LAIVs and B type LAIVs may be administered as a bivalent formulation or by sequential vaccination to induce improved protection.

#### OTHER STRATEGIES FOR UIVs

A number of alternative strategies are being investigated for their potential to serve as a UIV platform. First approach is to use infection-competent but replication-defective viruses. For instance, the M2 knock-out influenza virus (M2SR) rescued from M2-producing cells is able to infect cells but does not produce progeny virus due to the lack of functional M2 protein. The M2SR was shown to induce strong cell-mediated and humoral immunity and to provide broad protection against both homologous and heterologous influenza virus challenge in mice and ferrets (Sarawar et al., 2016; Hatta et al., 2017, 2018). A conceptually similar to M2SR, PB2 knock-out influenza virus could be produced in PB2-expressing cells as a vaccine.

The protection efficacy of PB2-KO vaccine was tested against diverse influenza strains in mice (Victor et al., 2012; Uraki et al., 2013; Ui et al., 2017). Pseudotyped influenza A virus also presents a similar replication-incompetent virus approach. The pseudotyped influenza virus lacking HA produced in HAexpressing cells can infect cells and express all the viral proteins except for HA. Studies have shown that the vaccination of mice and ferrets with the pseudotyped influenza virus generated a vigorous T cell response and reduced lung viral loads and weight loss after challenge with homologous and heterologous influenza viruses (Powell et al., 2012; Baz et al., 2015). These approaches based on replication-defective viruses should be rigorously evaluated for safety, considering potential reversion into virulence by acquiring the wild type gene by reassortment with circulating viruses (Lowen, 2017).

Secondly, mRNA vaccines have emerged as a promising alternative to conventional vaccine platforms against various infectious diseases including the influenza virus (Pardi et al., 2018a). Initially, the mRNA vaccines successfully induced protective B cell and T cell immune response in mice, ferrets, and pigs (Petsch et al., 2012). In subsequent studies, mRNA vaccines encoding HA induced neutralizing antibodies and T cell immunity essential for heterologous protection in mice and ferrets (Brazzoli et al., 2016). Notably, mRNA vaccines encoding HA elicited HA stalk antibodies in mice, rabbits, and ferrets, and the stalk antibody responses were associated with protection against homologous and heterologous influenza virus infection in mice (Pardi et al., 2018b), depicting promising potential as a UIV platform.

## CONCLUSIONS AND PROSPECTS

During the past few years, several critically important issues have emerged in developing a UIV. While identification of a number of broadly protective antibodies presents an optimistic prospect for pan-influenza therapeutics, induction of such antibodies at a sufficiently protective level by vaccination has not been accomplished yet. A variety of viral targets have been identified to induce broad protection, including M2e and the conserved regions in the HA, such as stalk, RBS, and VE, and the longneglected NA has emerged as an essential target for durable and broad protection. In virtually all cases, the rationale for broad protection is to redirect the immune responses from the variable immunodominant regions to conserved immunosubdominant regions. While effective in eliciting cross-protection, concerns were raised to non-neutralizing antibodies that potentially trigger the enhancement of disease. T-cell immunity has also been considered as an important correlate of cross-protection. LAIVs are able to induce multifaceted immune response and thus have increasingly received close attention as a promising vaccine platform, either alone, or in boosting format. Preexisting immunity or immunologic imprinting, established by prior exposure to influenza by infection or vaccination, may influence or thwart the desired breadth of immune response by UIVs. Therefore, disparities may exist between pre-clinical evaluation of a UIV without due recognition of immune imprinting and human challenge studies. Lastly, the correlates of protection and their precise molecular mechanisms have not been determined yet, which remains a significant gap between development and licensing of a UIV. Given the limited antigenic diversity of IBVs among humans and the lack of animal reservoir, paninfluenza B virus vaccine may be attainable in the near future. On the other hand, IAVs continually change their antigenicity via antigenic drift and shift and zoonotic spillover from animal reservoirs, requiring multi-faceted immune arms to increase the breadth of protection. Judicious choice of antigens and their efficient designing to confer a broad protection for a prolonged period are required to counterfeit the immune evasion and

#### REFERENCES

provide a truly universal protection against the ever-changing influenza viruses.

#### AUTHOR CONTRIBUTIONS

BS designed and conceived the review. YJ and BS investigated the literature and wrote the manuscript.

#### FUNDING

This work was supported by grants from National Research Foundation of Korea (NRF-2018R1D1A1B07048881; NRF-2018M3A9H4079358) and also supported by grant from the Ministry of Food and Drug Safety of the Republic of Korea (19172MFDS255).

MVA-NP+M1 mnufactured on the AGE1.CR.pIX avian cell line. Vaccines 7:33. doi: 10.3390/vaccines7010033

attenuated pre-pandemic H5N1 influenza vaccines. Vaccine 31, 3339–3346. doi: 10.1016/j.vaccine.2013.05.080

of influenza virus-like particles in a mouse model. J. Cell Physiol. 226, 1110–1117. doi: 10.1002/jcp.28339

that contains ectodomains of matrix protein 2. Vaccine 21, 2616–2626. doi: 10.1016/S0264-410X(03)00040-9

by Tobacco mosaic virus-based vectors. Curr. Pharm. Des. 19, 5587–5600. doi: 10.2174/13816128113199990337

cross-reactive human monoclonal antibodies. J. Virol. 85, 13463–13467. doi: 10.1128/JVI.05193-11

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Jang and Seong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

digital media

of impactful research

article's readership


**6. Conclusions**

The data in the literature suggest that OAEs represent a valid noninvasive instrument, which can monitor the cochlear damage entity in patients affected by MD, and could also monitor its progress. OAEs are not just reliable tools, but they are a low-cost methodology in comparison to standardized MD monitoring methods, such as electrocochleography.

The data in the literature shows that alterations in the inner ear caused by the presence of a hydrops can be monitored accurately with OAEs. Glycerol results can also be monitored successfully. Some reports suggest that a DPOAE protocol can be more suitable for MD monitoring tasks, but considering the mechanisms of OAE generation this might not be true. TEOAEs and DPOAEs can similarly detect inner ear alterations as long as these affect the basilar membrane mobility and the functioning of outer hair cells.

Although promising, this field of research still needs to be expanded, where experimental studies are lacking. It is likely that in the future, either implementing our knowledge among MD, or implementing the OAE technology, the application of the OAE for patients affected by MD could be further expanded and offered with more information to the clinical practice.


**10.  However, information about breast cancer screening is still limited and affects our ability to diagnose breast cancer at an early stage. Knowledge and strong evidence are essential to provide the rationale for the early detection of breast cancer, and this Special Issue aims to highlight ways to further improve survival and quality of life in women diagnosed with breast cancer. As a physician and breast radiologist who sees the harm of advanced breast cancers at diagnosis, I dream of the ways that we can reduce the rate of advanced breast cancer. Marie Curie said, "I was taught that the way of progress was neither swift nor easy." As illustrated in this Special Issue, with improved screening practices that incorporate breast tissue density, we are making progress. Let us keep this in the realm of the possible and minimize the harm of breast cancer to our patients.

**Conflicts of Interest:** The authors declare no conflict of interest.