**6. Development of a phage display panning strategy**

The phage display technology is based on the integration of a gene encoding a peptide or a protein fused with the phage coat proteins was first described by George Smith in 1985 [38]. The most broadly used coat proteins for display are the PVIII and PIII proteins; however, other coat proteins likewise been utilized for display. As a result of its high copy number (~2700 copies), the PVIII protein has been just utilized for the display of small peptides due to conformational issues hampering capsid formation. The PIII system, on the other hand, with its low copy number (5 copies), allows the display of larger molecules such as recombinant antibodies [12]. The first phage display system as shown in **Figure 4** displaying antibodies was explained by *Mc Cafferty et al*. in 1990. They effectively showed variable regions of antibody on phages by using immunoglobulin variable genes of hybridomas and B cells [39]. After its innovation, phage display technology has been extensively used for the research and discovery of antibodies or peptides against a large variety of antigens in many fields of application such as toxicology, drug discovery, immunization, epitope mapping and virus or toxin neutralization by using phage peptide and antibody libraries. Phage display technology has been intensively used for the production of neutralizing antibodies as shown in **Table 1** [40].

Various antibody libraries of different methodologies and strategies have been screened against SARS-CoV-2 spike protein and its receptor binding domain (RBD). Few studies have focused on screening previously developed libraries against SARS-CoV and MERS-CoV and finding cross-reactive antibodies. Others have performed screenings against semisynthetic or synthetic antibody libraries [41]. Phage displayed single-domain antibody was previously developed from llama, which simultaneously neutralizes the S antigen of SARS-CoV and also help in the neutralization of S antigen of the pseudotyped virus SARS-CoV-2 as a bivalent human IgG Fc-fusion protein. A selected antibody has high affinity to RBD, for this a library

**Figure 4.** *Schematic presentation of phage display systems (Bazan et al. [12]).*


*Role of Phage Therapy in COVID-19 Infection: Future Prospects DOI: http://dx.doi.org/10.5772/intechopen.96788*

#### **Table 1.**

*Phage display strategies for neutralizing antibody development (*BalcioĞlu et al*. [15]).*

constructed and screened against the RBD domain of the SARS-CoV-2 spike antigen known as phage displayed synthetic human Fab library [42].

ELISA and pseudo typed virus neutralization assay. A phage-displayed singledomain antibody library has been developed by grafting naive CDR regions into the framework region of an allele in the human antibody heavy chain variable region. They made affinity selection against the RBD domain and the S1 subunit of SARS-CoV-2 and chose several neutralizing antibodies, including a "cryptic" epitope located in the spike's trimeric interface.A site directed screening was performed in a naive human scFv antibody library and domain antibody library by phage display against SARS-CoV-2 RBD. After several rounds of screening, they obtained 9 enriched clones from the domain antibody library and a single clone from the scFv antibody library. The scFv clone was reformatted into a human IgG1 antibody, while the domain antibody clones were fused with human Fc tag. A potential neutralizing effect of these recombinant antibody structures revealed with pseudotyped virus neutralization assay [15].

The future of phage therapy is not necessarily to replace current therapies, rather there is potential for clinical applications to enhance and provide another treatment for infections. Research in this area is likely to grow at an exponential rate. However, the full potential of phage therapy can only be accomplished when there is transparency and an eagerness to share knowledge as well as resources. Preferably, phage libraries should be freely accessible through a network of collaboration, information on preparation and delivery methods for phages implied for clinical usage should be well documented. Phage articulation and delivery are also critical considerations in order to direct activity to targeted areas and maximize efficacy. In fact, use of phage therapy already appears to be as of now gives off an impression of being composed in different nations, and by major public health institutes such as Therapeutic Goods Administration (TGA) (Australia), Food and Drug Authority (FDA) (United States of America) and the European Medicines Agency (EMA) (Europe). Importantly, a universal code of ethics should

### *Bacteriophages in Therapeutics*

be established and regulatory bodies reach a consensus on the exchange of information, usage of phages as treatment and reporting of treatment outcomes. Due to the critical nature of the rise of multiple drug resistance (MDR), expanding the urgency for phage therapy to be implemented as standard consideration, alternative therapies to be translated into clinical applications need to be expedited. A concerted effort with both national and international partners could see phage therapy being translated into standard care in the next 5 years [33].
