1.Project Description Section

1.1 Project Summary.

The COVID-19 pandemic has affected everyone; the spread of the virus has caused a steep increase in fatalities worldwide as well as hardships in all aspects of society. The development of vaccines capable of protecting against the virus has shown the capability of the scientific community; however, the differing production, storage, and distribution capacities have limited the reach of vaccines in certain regions. Adding these factors to the rapid transmission of the pathogen and the constant appearance of coronavirus variants makes controlling the spread a much more difficult task for these regions.

In developing countries especially, such conditions have been the bottleneck for vaccine accessibility. In Latin America, for example, access to vaccines is subject to additional problems caused by a lack of adequate infrastructure for storage and distribution of doses within the territories of each state. Therefore, alternatives capable of meeting the local needs of this region must be proposed. 

Our proposal addresses our national reality by considering all these elements, evaluating the vaccine design strategy, and adjusting to the prevailing conditions. Therefore, our research's objective consists of the design of a phagemid as a peptide display array for the development of neutralizing antibodies (Phagemid display as Peptide Array for neutralizing Antibodies, P(d)PANA). This proposal also considers efficiency parameters for the immune system response as well as biosafety parameters for the general public. One point to highlight is the Phage Display tool, which, although known within the field of molecular biology, its application as a means of vaccination against COVID-19 is just being evaluated, but with encouraging results (Kang et al, 2006; Staquicini et al, 2021)

P(d)PANA as a molecular biology technique is based on a bioinformatics approach in which the regions of the coronavirus spike protein with the highest antigenic potential are selected. This information allows the generation of recombinant proteins on the M13 phage envelope as a means of display. Given that the M13 phage presents an easy genetic edition with high thermostability (González-Cansino et al, 2019), the proposed vaccine design aims to solve the aforementioned problems. Thus, this method turns out to be one of the most ideal for mass production and storage at a low cost and with one of the best vaccination efficiencies for emerging economies in Latin America and the rest of the world.

1.2 Promotional Video.

1.3 Project Presentation Video. 

1.4 Education and Communication Infographic.

1.5 Team and Attributions.



2. Design and Results

2.1 Design Roadmap.

2.2 Excellence in Biological Engineering Design. 

Massive data filtering

   Given the need to generate a consensus spike protein sequence of the different SARCoV2 variants, GISAID.org used as a key database. It should note that the file downloaded from that website pertains only to sequences registered up to 28-07-2021. However, it should be noted that a large number of the sequences uploaded there present problems with the final availability of their data. In order to visualize this situation, free software Jalview has used as a reference, with which the following problems could detected:

• Duplicated sequences in amino acids
• Sequences with gaps between amino acids
• Incomplete sequences
• Sequences with errors and/or amino acid deletions
• Variable number of amino acids (excluding animal coronaviruses).

               In view of this situation, a script elaborated considering the parameters of:

• Year of registration: for the purposes of the project, only sequences from the present year 2021 up to the above-mentioned date taken.
• Protein length: Only for 1273 amino acids.
• Number of continuous gaps: These represented by null characters "X", and only those with less than 10 continuous gaps are accepted.
• Percentage of gaps: Only those sequences with a value of up to 10% of "X" taken into account.

               From such initial conditions, and from the subsequent application of the script on the downloaded data set, a high performance obtained for the visualization of functional sequences for the purposes of the project itself. From a 3.6 Gb fasta file with 2.6 million sequences it was possible to extract 57629 sequence in an 80.1 Mb fasta file. Latter file did read better by the Jalview software, and thus worked with it for the generation of the consensus sequence.

Immunoinformatic analysis:

               The consensus sequence generated with the Jalview program used in the immunoinformatic servers for the antigenic analysis. The sections used to divide the sequence represent the glycosylation protein sites mentioned by Watanabe et al. (2020). The reason for grouping immunoinformatic analyzes in these categories is due to the masking effect that the glycan shield has on the surface of the spike protein (Casalino et al., 2020).

               The key to the filtering and statistic representation of the data lies in the structure of the spike protein itself. Structure covered by sugars on its surface, also known as the glycan shield, which prevent adequate recognition by the immune system during the initial stages of infection. Therefore, the entire sialic protein is not functional for immunization; if the body were to establish a peptide that falls precisely on the amino acids covered within the glycan shield, it would be a deficient immunity. Thus, in order to identify the most useful peptides for the immunorecognition, the regions taken are those between glycosylation, since this favor the development of neutralizing antibodies in each individual.

Figure 3: Schematic representation of the spike protein, its domains and glycosylation sites. Taken from Watanabe et al (2020)

               To understand rationale for immunoinformatics servers, each of the methods is best summarized in Table 1:

Table 1: Summary table of immunoinformatics tools used and parameters defined

     Likewise, given the objective of addressing the development of neutralizing antibodies, it has taken into account that the properties and peptides identified in the servers were from amino acids 1 to 583. This is because the S1 section of the protein is located in this region spike and is involved in the process of entry of the viral particle into human cells.

               Immunoinformatic analysis shows 182521 ligands for the MHC-I system, while 96748 ligands for the MHC-II, both systems associated with the presentation of antigens to T lymphocytes. I addition, most of these peptides are located in the last group between glycosylations of the sequence, ranging from 343 to 583 aa´s. On the other hand, the results obtained for antibodies evaluated different properties of the sequence, being the last group with the highest weight and abundance of available epitopes. Then the risk of allergenicity of the identified peptides evaluated. To do this, the Excel table files converted to Fasta-type files by means of a Python script, each with 100 peptides per file. With this new data set, they manually entered into the Vaxigen server. In this step, the antigenic potential of the peptide sequences ruled out or confirmed. Accordingly, a new library generated with the non-allergenic sections of the consensus sequence.

*Note: Representation of the total data obtained can appreciated in histograms from additional document on JOGL web page.

Consensus protein modeling

               The objective of this stage was to demonstrate the structural stability of the consensus protein. For consensus sequence, tructural modeling of the same carried out, using two strategies: By homology and De Novo. The first part of the modeling by comparison of sequences with structures already known in the Swiss-model servers. While for the second strategy used the AlphaFold2 server in Google Collab. From both methods, only one result obtained by homology, since, for the second tool, the resources assigned by the server were not sufficient for processing this protein. However, for the project standards, the results offered by Swiss-Model were sufficient.

               On the other hand, the sequence and structure of the consensus protein also compared with some of the spike proteins of the variants. From this result, it observed that there are no major changes in the consensus sequence or in its derived structure. This is because the same number of proline residues conserved with respect to the other variants. Therefore, it can be say that the 3D model obtained presents the necessary stable conformation.

Figure 4: protein swiss-model report for consensus protein

• 2.2.1 Standarization

       Regarding phagemid constriction, our work based on the genome of the bacteriophage M13. It characterized by being a non-lytic type filamentous virus whose only host is E.coli (Rakonjac et al, 2011). This phage is made up of eleven genes arranged in a semi-continuous manner with 2 non-coding intergenic regions (IR) with promoter and terminator sources respectively, and 5 of these genes (pIII, pVI, pVII, pVIII, pIX) give rise to capsid proteins (van Wezenbeek et al, 1980).

               Our modifications on the phage correspond to the insertion of two new genes and the change of an existing one. The incorporation of these new DNA sections proposed to be by the exponential cloning method by megapriming PCR (Ulrich et al, 2012). The reason for choosing this method over others is due to the limited insertion space, since it falls within a limited range within the IRs of the genome (Horton et al, 2013; Mathieu et al, 2014).

Figure 5: Schematic process for Exponential Megapriming PCR

               From M13 phage genome sequence proposed by van Wezenbeek et al (1980), each gene sequence tagged, including two of interest for the purposes of the project. These correspond to genes VII and IX, both whose proteins are part of two of the five phage capsid proteins, and which have a potential for peptide presentation (González -Mora et al, 2020).

               Additionally, it desired to preserve phage infectivity of the once purified. Reason why capsid protein III was not taken into account, despite being a popular system to express proteins of interest (POI) in its sequence, but whose main function is to anchor and enter E.coli (González -Mora et al, 2020).

               However, the limits of insertion of peptides in the key proteins mentioned above must considered. The first barrier lies in the insertion capacity of new amino acids in pVIII and pIX, with up to 10 amino acids and 1000 amino acids respectively. However, it also influences the amount of proteins available in the virus capsid, where pVIII has up to 2700 copies and its counterpart pIX only has five copies. Given this duality of aa's length and number of copies, the antigens that are exhibited on its surface should be prioritized (González-Mora et al, 2020). On the other hand, another influencing barrier is the genomic stability that these new recombinant genes and the sites where they inserted may have. Since, during the process of replication of the phage in its host, losses of the new genes can caused (Enshell-Seijffers et al, 2001; Horton et al, 2013; Mathieu et al, 2014).

     According to the immunoinformatic and structural analyzes of the consensus sequence, antigens presence correspond to those identified within the region 343 to 583 from it sequence, and that correspond to the receptor binding domain (RBD). Based on this, it was decided that for the pVIII protein a non-allergenic and 12 aa antigenic peptide from the aforementioned region would be used, while for pIX the entire domain itself would be used, since it has a globular-like structure in its structure.

               An important point to highlight is that the insertion of new sequences within those that encode pVIII and pIX obey a series of parameters for their correct incorporation, and of which following will mention:

A.     pVIII:

• Modification at the C-terminal: The insertion of any motif other than the base sequence be made at the carboxyl terminal end of the polypeptide, due to the way of assembly in the virus capsid (Enshell-Seijffers et al, 2001)
• Two classes of pVIII: Being the protein with the highest abundance in the capsid, its modification can affect the stable form in which they are incorporated into the virus capsid, which is why the support of the helperphage with a wild type copy is needed, or the same phage with a wild type sequence and a recombinant one (Wang & Yu2004).
• A synonymous sequence: In the case of using the strategy of a phage with two pVIII genes, the recombinant region must have a synonymous sequence for the base of pVIII. This is because spontaneous deletions can be generated during the replication of the virus if it has two exact copies of the same gene (Enshell-Seijffers et al, 2001)

B.     pIX:

• Modification at the N-terminal: insertion of any motif of the base sequence be made at the initial aminoacyl end of the polypeptide, since it corresponds to the surface exposed towards exterior of the capsid (Mohammadi et al, 2016; Tornetta et al. al, 2010). Likewise, the insertion must be after the Methionine and Serine codons respectively (Nilssen, 2011)
• Flexible linker: it corresponds to a peptide sequence of no more than 20 amino acids that acts as a bridge between POI and pIX (Mohammadi et al, 2016 ; Nilssen, 2011).

               Finally, the last gene of interest is the tetracycline resistance gene. A sequence already standardized within the iGEM community and that has its own promoter and terminator domains (Balley, 2018). This genetic piece corresponds to the metabolic marker for the conservation of the M13 phage genome within E.coli

               Based on all of the above, a phagemid with the two recombinant proteins (pVIII and pIX) and the tetracycline resistance gene (TetR) chosen as strategy. In the case of pVIII, it decided to leave Wild type DNA sequence and add a synonymous sequence of nucleotides + antigenic peptide nucleotides. Now, for pIX, its approach was different, because it involves only the modification of the N-terminal end of its amino acid sequence, therefore, only the linker sequence from Mohammadi et al (2016) + RBD inserted.

               In the next step we explain more how to be each gene to Biopart.

• 2.2.2 Optimization.

        For the construction of the pVIII codon synonymous sequence, the original sequence provided by van Wezenbeek et al (1980) and the GenScript (SF) codon frequency table taken as reference. Subsequently, the nucleotides organized by amino acids of pVIII with the synonymous codons for them (Table 3). Based on this, the codons were alternated, for the aa's with a single codon we proceeded to alternate with the next one, while for the aa's with 3 or more codons we proceeded to randomly select any of the options available for the construction of the synonymous sequence

Table 3: Condos and alternative codons for synonymous pVIII sequence construction

               To verify the similarity between the original and the new sequence, it was compared using the Pairwise Sequence Alignment tool from EMBOSS Needle (2021). The result provided by the server presented a 65.18% similarity regarding the alignment of the codons (see figure bellow)

Figure 6: Alignment sequence for pVIII and Synonym pVIII

               In another vein, the peptide sequence for pVIII and the linker + polypeptide for pIX converted to E.coli nucleotides using the JCat tool (Groote et al, 2005). After obtaining the nucleotides for both antigens, they incorporated into the corresponding sections within the genes. In the pVIII gene it was incorporated before the TGA (Stop) codon, and for pIX it was inserted after the Methionine and Serine codons respectively.

       By the way, for each of the aforementioned parameters, the new genetic constructs are available in below table and figure.

Table 4: Gene insertion for phagemid

Figure 7:Schematic representation for key proteins

        van Wezenbeek et al (1980) have given confusing numbering for the M13 phage genome, thus a new numbering system was developed according to the sequence provided by the same author. From this new order, each of the genes and regions of the virus identified according to the sequences of the genes identified by Suggs & Ray (1979), van Wezenbeek et al (1980), Kim et al (1981), Russel & Model (1989), Schaller, H. (1979) and Smeal et al (2017). All work made with Benchling digital tool, from which the following figure obtained:

Figure 8: M13 Phage genome

        Once each of sequences pVIII, pIX and TetR were defined, we proceeded to design their constructs to make the Bioparts. As mentioned in the previous point, the insertion of foreign genes into the M13 phage genome will carried out by means of the TAL technique. According to this protocol, it requires the incorporation of flanking primers of the exogenous gene, and that they recognize specific domains of the genome for incorporation (Horton etal, 2013; Mathieu et al, 2014).

               According to Mathieu et al(2014) recommendations, the design of these primers follows those of a conventional PCR, but with the difference that they must contain an affinity section to the gene of interest and another alignment section for genome recognition. Therefore, for each of the aforementioned genes of interest, we proceeded to design the respective primers with dual functionality. All this was done by selecting 5'→3' regions of the aforementioned intergenic regions, for subsequent incorporation as ends of each of the external genes.

               Once the sequences obtained, we proceeded to generate their respective primers by means of the NCBI Primer-BLAST tool, which presented the following results:

Table 5: Primers for ovelaping insertion for each gene

Finally, we have a register for each biopart showed:

·        IDLBB_001851-iDLBB_001900: recombinant pIX +linker +RBD

·        IDLBB_001851-iDLBB_001901: synonymous pVIII+ antigenic peptide

·        IDLBB_001851-iDLBB_001902: Forward primer to pIX

·        IDLBB_001851-iDLBB_001903: Reverse primer to pIX

·        IDLBB_001851-iDLBB_001904: Forward primer to pVIII

·        IDLBB_001851-iDLBB_001905: Reverse primer to pVIII

·        IDLBB_001851-iDLBB_001906: Forward primer to SpVIII-R

·        IDLBB_001851-iDLBB_001907: Reverse primer to SpVIII-R

·        IDLBB_001851-iDLBB_001908: Forward primer to TetR

·        IDLBB_001851-iDLBB_001909: Reverse primer to TetR

Figure 9: Phagemid

• 2.2.3 Build and Test

Replication of the genetic material of Sars-CoV-2 on the strain DH5⍺F’ of Escherichia coli

               To insert the genetic material of Sars-CoV-2 inside Escherichia coli bacteria, the virus needs to become a bacteriophage. To achieve this, it is necessary to realize a recombinant homology process between Sars-CoV-2 and the bacteriophage M13. The recombinant homology allows the exchange of the genotypic and phenotypic characteristics from both virulent structures to obtain the organism expected (Chen et al., 2019). Which would be the phagemid based on bacteriophage M13 modified, so it has spike protein in its capsid (affecting in the least the proteins that allow any metabolic process). Thus, the genetic material found on the capsid’s interior would be a hybrid between both virulent organisms. Moreover, the molecular markers that are being transferred are: the resistance genes to the tetracycline (tet) and the modified genes that correspond to the phage, protein VIII and IX.

               Nevertheless, in proof no producing a bacteriophage that could attack the strains of E. coli found in the digestive microbiota, which could have a negative effect on the activation of the T regulatory cells (Maldonado, 2021). E experimentation will be with the strain DH5⍺F’ because is a modified version of DH5⍺ (Kostylev et al., 2015), one of the most common strains of this bacteria that is utilized in laboratory work. So, it does not represent any health security problem at first sight. The difference between both strains is that DH5⍺F’ have a pili and its antecessor does not. The selection of a strain with pili is important because it allows an easier interaction between the bacteriophage and the bacteria (Chen et al., 2019).

Design of the bioreactor

               To design a reactor is necessary to understand the productive process that will be made and how the parameters that affect the process development will be measured (Rojas y González, 2011). Knowing this, the fermenter will be a stirred tank of 14 liters. At the bottom of the tank will be a magnetic surface that with a magnet will mix the tanks' contain. Additionally, to guarantee the use of the correct operation conditions (temperature, pression, pH, and others) there will be a constant revision in the tank with the use of measurement instruments for each variable. There will be a thermometer, a pH meter, a barometer, and a probe of O2 and CO2. There will only be one measurement instrument for each variable because the mixing in the tank will guarantee a unique measure of every parameter at the time of revision.

               When the sensors perceive an out-of-range lecture will give the following answers:

·        When the temperature is not the expected one, a heat exchange blanket will be turned on. If the temperature is below the range, the heat exchange blanket will have hot water in its interior, in contrast, it will have cold water when the interior’s temperature tank is higher.

·        When the pH is not the expected one, a buffer solution will be released at the necessary volume to establish the correct pH. If the pH is lower, a based buffer solution will be released. If the pH is higher, an acid buffer solution will be released in the interior of the reactor.

·        The pressure tends to increase in a reactor because the organisms are producing some gases like O2 or CO2 (the production depends on the organism utilized). When the sensors perceive a high concentration of products that could harm the bioprocess or a higher pressure than the expected one a valve will open, and it will release the production gases.

Moreover, the bioreactor will have an extraction system that goes from the fermenter to the filtrate system to allow the extraction of phages in a secure way.

Fermentation conditions

               The experimentation will be in anaerobic conditions to avoid lysis on the cell walls of Escherichia coli, which provide a better and correct interaction between the cell walls of the bacteria and the bacteriophage. Also, this bacterium needs a culture medium that has a carbon rich fount and an antibiotic to avoid the growth of an unwanted microorganism (Hernández, 1974). The process will be at atmospheric pressure, under a pH that varies between 6 and 7.5, and at two different temperatures in the different phases of the productive process.

               The culture will have two phases. The first phase is when the bacterium is being inoculated in the culture medium, the inoculated concentration needs to be defined in an experimental way. The culture medium is conformed with glucose, tryptophan and tetracycline. According to the bibliography, 15 µl of Escherichia coli DH5⍺F’ submerged in a broth rich in tetracycline should form colonies after an incubation of 16 hours under a 37ºC temperature (Biolabs inc, 2016).

               After an optimum bacteria production is reached, the temperature will drop to 29ºC because at this condition exists a metabolic balance between the bacterial and viral growth (Warner et al. 2014). Next, the bacteriophage M13 modified will add to the fermentation and it will inoculate for a determinate time that needs to be measured experimentally. Nevertheless, it is probable that by adding the viral organism the needs for the bacteria’s food increase and the bacteria’s production diminished. So, the optimum bacteria production needs to be calculated based on the volume of bacteriophages added and the concentration of these viral organisms wanted. Also, the rate and concentrations of the culture medium that is added to the reactor after the infection of the bacteriophage needs to be measured experimentally.

Filtration

               After the production of phages comes the separation process that consists in ultracentrifugation. Which generates a centrifuge force that separates the macromolecules of the mixing according to its different sizes. The velocity that the molecules acquire during the centrifugation called sedimentation velocity and it determines the final location of the molecule after this process (Fields, 2019). This methodology allows the separation between the phages from the bacterium and unwanted matter. Additionally, it is a common process; its machinery can found at different prices and models, which allows a greater economic accessibility. 

2.3. Human Practices

      The covid19 pandemic in Venezuela is an issue that is already part of Venezuelan society, and at present, it accepted as part of the bitter reality that the country is experiencing. For most Venezuelans within the country, the same concern is present: When will they be able to vaccinate? In view of this problem, the local option for our territory is the design of a vaccine whose cost of production, storage and distribution is the most optimal possible, but which maintains the safety and efficiency for the development of immunity against the coronavirus. Consequently, the use of the phage-display technique for the presentation of antigenic peptides derived from SARS-CoV-2 is the most efficient and viable option to address these needs, not only for our country, but also for the rest of the emerging economies of Latin America.

2.4. Integrated Human Practices.

        Due to this indifference, several experts in the area of health and biology have spoken out about it. One of them is Dr. Flor Pujol, IVIC virologist, who has been in charge of clarifying the myths and realities about the coronavirus in the media. In view of this, the team decided to contact her to get her recommendations on how we should carry out our project and how best to approach it. We also consulted with our PI about the biological risks to considered for the administration of our vaccine design. He recommended reading Baldo et al (2021) as a key reference on the environmental risks that have studied on the commercial vaccines distributed this year.

2.5. Impact on the Sustainable Development Goals (SDGs)

Figure 8: Team sustainable development objectives 

        SDG 3 is the most important for our work, since we seek to contribute to the improvement of health conditions and quality of life in our country by means of a vaccine design that is capable of offering the necessary safety and protection against COVID19. Preventing and reducing the death and health consequences of covid19 infection is the rationale for the design of our vaccine candidate, since the pandemic is not yet over and the speed of vaccination and accessibility of vaccines in Venezuela remains limited.

               With regard to SDG 9, we consider it complementary to our research approach. The reason for this is that the design and implementation of this technology would expand the productive capacity of the region for the accessibility of vaccines, not only against COVID19 but also against new emerging diseases in the world. The main factor for the delay of vaccination in the world lies in the speed at which vaccines produced, stored and distributed. Additionally, in our region there is a poor infrastructure for vaccine storage and transportation. One way to correct this is to change the basis of vaccine design, making it more stable at room temperature and producing it at a higher speed at a lower cost. In other words, an optimized industrial infrastructure must create to meet these needs.

               Finally, SDG 17 implies that in order to achieve our two previous objectives, partnerships and cooperation systems must established to make this a reality. These partnerships would have to integrate different sectors of society such as citizens, scientists and politicians. 

       On the other hand, we consider that the most convenient SDGs for some iGEM design equipment summarized in the following table:

Table 6: Most related SDG for some igem design teams

3.Multidisciplinary Excellence

3.1 Improvement of an existing iGEM design.

  The only previous design used for our work corresponds to the Tetracycillin resistance gene (TetR) mentioned above. This biopart developed by the iGEM Austin-Texas team. Because for our production system (E.coli), it is a perfectly tested and standardized gene.

Figure 10: Screen Capture TetR biopart

3.2 Collaboration.

        Throughout the competition we participated in different events and meetings with different researchers and teams, each of them contributed to our project an approach on how to work and improve our proposal.

               The events where we had a space to present ourselves and share with the academic community and general public were: Interview by BiotecLatina, Forum of Synthetic Biology in Latin America, iGEM LATAM FEST and the Venezuela-Ecuador bioinformatics symposium. In the case of the first one, we decided to participate in an open interview via Instagram Live, where we presented part of our objectives and work approaches.

Figure 11: Screen capture for Bioteclatina interview

    In the second activity, we had the opportunity to present together with the teams of Lecchain-UTEC, iGEM Yucatan, iGEM UNALM-UNMSN the problems, challenges and opportunities of synthetic biology in our region and the approaches of our projects to meet such needs.

Figure 12: Record Synthetic Biology in Latam forum

        For the next activity, we had the opportunity to know not only the presentations of some igem design teams and to expose ours as well, but also the culture of the countries of origin of each of them.

Figure 13: iGEM LATAM Fest

        In the fourth event, in the space of the symposium we elaborated and presented posters with the preliminary advances of our project to the bioinformatics community of Ecuador and Venezuela. In this last event, we received advice and questions on how to improve our design.

Figure 14: Poster for Bioinformatics Symposium

        In addition, we held meetings with the IXORA, Lecchain-UTEC, Mikuytec, Biotec-EC teams. With each of them we discussed the different aspects of our designs and how to improve them. Thanks to the IXORA and Lecchain teams we were able to test some bioinformatics tools for protein structural modeling. For Mikuytec and Biotec-Ec we were able to share our knowledge to advice on how to apply or approach some aspects of their respective designs.

Figure 15: Call meetings Collage with Ixora, Biotect-EC and Lecchain-UTEC teams

Special thanks to:

·        Felix Moronta: For being our PI, believing in our project and guiding us on Biosafety regulations.

·        Rafael Puche and Deliana Infante: For motivating us to participate in the Bioinformatics Symposium and helping us with crowdfunding project promotion.

·        Solmar Varela: For advising us on how to design our scientific poster for the bioinformatics symposium.

·        To all the Venezuelan community in the country and abroad that have supported us with the fundraising campaign for our project.

·        We also acknowledge the donations from individuals and foreigners for our project.

3.3 Entrepreneurship and Innovation.

        The design of vaccines against COVID-19 is one of the main goals of global scientific research. From numerous studies on this disease, different approaches to immunization strategy for humans have emerged. Each takes advantage of the knowledge of molecular and cellular biology in their respective approaches, thus offering efficiency and safety in the respective administration to the public. However, all WHO-approved vaccines have faced a bottleneck due to technical limitations in their production, storage and distribution, from the simplest vaccines using inactivated coronavirus (Sinopharm, Sinovac) to the most complex vaccines such as those based on mRNA (BioNTech and Moderna) or viral vectors (Johnson & Johnson and Astrazeneca). Because of this, there is a priority to develop other processes for the manufacture, testing and distribution of new vaccines to ensure a constant supply of new doses for the population. It is for this reason that our team proposes a vaccine candidate that is capable of both meeting such needs and offering sufficient safety and efficiency for robust immunization against coronavirus and its variants. Therefore, the aim of our research consists in the design of a phagemid-displary as peptide-array for neutralizing antibodies (PdPANA) against the SARS-CoV-2 spike protein. This concept starts from a mainly bioinformatics approach to select from the protein of interest the regions with the highest antigenic potential, and based on this, to generate recombinant proteins on the M13 phage coat as a means of displaying them.

               The reasons for choosing a peptide presentation system based on the M13 phage are due to the very advantages that this system offers over the other strategies mentioned above:

·        Easy gene editing, the insertion of exogenous genes can be performed via PCR.

·        Rapid production: Since it is a non-lytic phage, and its host is an E.coli, it facilitates rapid and inexpensive production (de Vries et al, 2021; González-Mora et al, 2020).

·        High thermal stability: It does not require a cooling chain for storage, withstanding up to 70°C (Gonzáles-cansino et al, 2019)

·        It does not require the use of adjuvants: The phage capsid proteins are capable of triggering an innate immune response (de Vries et al, 2021; González-Mora et al, 2020)

               These advantages over other vaccine designs are what make phage-based vaccines the most cost-effective option for production, storage, and distributi. Motivated by this, our project focuses on optimizing such a system to be functional for the fight against SARS-CoV-2, and that in terms of manufacturing it results in the most efficient to be easily reproducible on an industrial scale and at a lower cost.

               Therefore, our business model would focused on designing and producing the vaccination system against COVID19 for emerging economies not only in our region, but also in rest of the world. Also, for further studies, the concept could be improved so that it can be adapted to new zoonotic or local clinical diseases in developing countries.

3.4 Education and science communication.

        Our communication and scientific dissemination efforts were subject to the aforementioned symposium and forum. Additionally, we were through social networks (Twitter, Instagram and LinkedIn) updating the content inherent to the topics of our project as well as its progress.

3.5 Policy, biosafety and/or biosecurity.

        According to t WHO for the manufacture and distribution of vaccines in the world (WHO, 2013), the research phases (WHO/PAHO, 2020) and the research by Baldo et al (2021) on the environmental risks involved in the manufacture and administration of vaccines in the world, any initiative to immunize the human population must go through the respective controls. For this reason, once we have the necessary conditions to approach laboratory trials, we will consider the latent risks and the necessary protocols will respected.

               For each of the steps in the design and testing of our vaccine approach, it must performed under the international biosafety standards specified by the WHO (2005). Thus, a biosafety level 3 laboratory is required, due to the handling of E.coli DH5αF', which despite not being virulent, when infected by phagemid can become a system of concern due to the presence of the tetracycline resistance gene.

               On the other hand, it should note that there are no proven risks in mammals regarding adverse effects of phage-based vaccines (Staquicini et al, 2021). This is because the phagemid only infects its host, i.e., it only affects E.coli, therefore, there are no dangers regarding changes in the human genome.

3.6 Arts and creativity activities and results.

        Regarding the design and artwork of our project, most of it focused on the use of free tools such as Canva and digital libraries of icons, backgrounds and vectors such as freepik. However, it is worth mentioning the external collaboration of two people outside the team.

·        Mariana Saavedra: Biologist and digital illustrator, who gave us her direct support for the design fanart design of twice of project Instagram post.

Figure 14: B-lymphocyte Fanart by @Unanchoa

·        Danelys Sanchez: Biology student and scientific illustrator, who gave us her talent for the design of the team's logotype and logo.

Figure 15: PdPANA Logo

• Gabriel Sojo: A young ilustrator for this colaboration for one post on our instagram account

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