**Abstract**

Arbovirus of the *Flaviviridae* family represents an issue worldwide, particularly because it can lead to serious illness and death in some countries. There is still a great complexity in obtaining effective therapies and specific and sensitive diagnostic tests, due to the high antigenic similarity between them. This similarity may account for antibodies cross reactivity which has positive and negative consequences for the course of infectious diseases. Among dengue virus (DENV) serotype infections, the cross-reactivity can increase virus replication and the risk of a severe disease by a mechanism known as an antibody-dependent enhancement (ADE). The search for serological biomarkers through monoclonal antibodies (MAbs) that identify unique viral regions can assist in the differential detection, whereas the development of recombinant antibodies with a neutralizing potential can lead to the establishment of efficacious treatments. The Phage Display methodology emerged as one of the main alternatives for the selection of human MAbs with high affinity for a specific target. Therefore, this technology can be a faster alternative for the development of specific diagnostic platforms and efficient and safe treatments for flavivirus infections. In this context, we propose for this chapter a discussion about Phage Display as a strategy to obtain MAbs for DENV and other flaviviruses.

**Keywords:** antibody, Phage Display, dengue virus, flavivirus, therapy, diagnosis

### **1. Introduction**

When thinking about the development of virus detection and neutralization technologies whose bases of action are immunoglobulins, it is necessary to understand the structure of the viral particle of interest. In addition to the sequence of amino acid residues that make up the target epitopes, their position in the particle and their function in the process of infection and viral replication influence the design experiments aiming the obtantion of antibodies with a diagnostic and therapeutic potential.

The structures of flavivirus have been determined and studied, mainly, by combining cryo-electron microscopy with data from X-ray diffraction experiments using crystallography of viral proteins in the presence or not of antibody molecules. Results of this combination showed that the flavivirus is composed of a dense icosahedral electron nucleus and a lipid bilayer surrounding it. The genome comprises a sequence of ~10,700 nucleotides of a positive-sense RNA that encodes a polyprotein that is cleaved in 3 structural proteins, capsid protein (C), membrane protein (M), and envelope protein (E), and in 7 nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [1]. The NS1 protein plays a role in viral replication and is often shown to be a soluble antigen secreted into the bloodstream, interacting with components of the immune system. NS5 is the largest and most conserved nonstructural protein and acts in the transcription process of viral RNA [2, 3].

The icosahedral capsid is small and poorly organized, but it provides enough space for the genome and is surrounded by the envelope, so there are few connections between these structures, unlike what is found in other viruses. The viral envelope consists of two internal and concentric layers of phospholipids and an outer protein shell formed by regions of protein M and ectodomains of glycoprotein E organized in dimers which present protein determinants for the binding of the virus to the host cell (hemagglutination). Protein E contains three domains: domain I (DI), which is related to cell tropism and envelope organization; domain II (DII), which comprises the dimerization region and the fusion peptide; and domain III (DIII) with the function of binding to cell receptors, in the initial stage of viral infection [4, 5].

The fusion loop is a highly conserved region between dengue virus (DENV) serotypes and all flaviviruses, responsible for the late stage of infection, in which the virus interacts with the endosomal membrane, resulting in the release of the nucleocapsid in the cytoplasm. When the particle is in the lysosomal vesicle during the infection process, a structural reorganization of the viral envelope occurs; the fusion loop is exposed and inserted in the lysosomal membrane. After the formation of the fusion loop contact, protein E starts to organize itself into trimmers, resulting in the expansion of the viral particle and the approximation of the viral and lysosomal membranes, forming the fusion lipid pore that allows the release of the viral genome to the cytoplasm [3, 6, 7].

DENV serotypes show great heterogeneity in the structure of viral proteins. However, there is also antigenic similarity between DENV serotypes and, for some peptide sequences, between flaviviruses. Phylogeny studies of virus sequences, by estimating the antigenic distance between them, concluded that serotypes 1 and 3 are the most similar, serotype 2 was the second to diverge evolutionarily, and serotype 4 is the one that presents greater genetic difference. There is a 32% difference in the structure of protein E of the four DENV serotypes. Specific mutations in the genome result in the antigenic variability found in each serotype [7–9].

Much of the genetic difference between flaviviruses is due to protein E, which can show up to 60% difference in its amino acid sequence. In the phylogenetic analysis of the viruses, the DENV serotypes are closer to the Zika virus (ZIKV), with approximately 45% difference, and have 50 and 60% dissimilarity with West Nile virus (WNV) and yellow fever virus (YFV), respectively. DENV and other flaviviruses vary dramatically in terms of the amino acid sequence of the glycosylation region and the content of glycans added to the surface of E and precursor membrane protein (prM). Many epitopes of protein E are unique to a DENV serotype [4, 9–11]. It can also be observed in ZIKV, which has the glycosylation site, in the DI of protein E, different in conformation and length of the loop that contains this glycosylation site. The carbohydrate associated with this residue can act as a virus binding site in host cells. Thus, differences in this region of glycosylation can influence cell tropism, infection, and pathogenesis of these viruses [12]. Another important characteristic of ZIKV is the insertion of an alanine residue in the carboxyl termination of DIII, which is associated with an increased stability of this virus [13].

**149**

basis of effective vaccines [19].

*Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies*

The hydrophobic sequence of the fusion loop appears to be the only epitope that is conserved among all flaviviruses; however, the degree of exposure in this region varies substantially among viruses [6]. Nonstructural proteins, NS1 and NS5, also present some epitopes conserved among the DENV serotypes and other members of the flavivirus genus, but their position also varies between viral strains. The relationships of antigenic similarity between flaviviruses generate immune responses that are configured as cross-reactions with protective or pathological

Immunoglobulins, or antibodies, are glycoproteins, expressed on the surface of B cells or secreted, that act in the neutralization and elimination of pathogens [16]. Antibodies are relatively flexible "Y"-shaped molecules made up of two heavy chains and two light chains, joined by an extensive network of non-covalent interactions, stabilized by disulfide bonds. Both types of chains are composed of constant and variable domains. The constant regions determine the functional properties of the antibody, and the variable regions determine the antigen-binding site. The light chain consists of a variable portion (VL) and a constant portion (CL) that can have two types of domains, kappa (ƙ) or lambda (λ). The heavy chain consists of a variable portion (VH) and three or four constant portions, depending on the class of the antibody, which are CH1, CH2, CH3, and CH4. The type of heavy chain determines the class, or isotype, of antibody, such as IgA, IgG, IgD, IgE,

The antibody molecule can be subdivided into portions of the crystallizable fragment (Fc) and antigen-binding fragment (Fab) region. The Fc portion has the constant domains (CH2, CH3, CH4), and the Fab portion consists of the VH-CH1 and VL-CL domains. The Fab portion retains the ability to bind to the antigen, and

Three segments containing variability can be identified in both the VH and VL domains. These segments are the hypervariable regions that determine antigen specificity and are more commonly called complementarity-determining regions (CDRs)—CDR1, CDR2, and CDR3. The combination of CDRs from a VH with CDRs from a VL forms the region of interaction with the epitope, called the paratope [17]. The variability of the antigen-binding regions is responsible for the ability of different antibodies to bind to many structurally diverse antigens [18]. **Figure 1**

In cognate antigen recognition, some naïve B cells can initiate somatic hypermutation, generating new variable domains, that can be selected based on their ability of antigen binding, usually with high affinity compared to germinal domains. After antigen recognition, naïve B cells differentiate into antibody-secreting plasma cells. These plasma cells secrete antibodies with high affinity and, can differentiate into memory B cells. Memory B cells are highly specialized cells for quickly recognizing the antigen in subsequent exposure, can persist for years, and provide long-term humoral protection for decades. These functional features of memory B cells are the

The knowledge about the mechanisms of antibody production and clonal selection of B cells led to the development of innovative hybridoma technology in 1975 [20]. The technique is based on the fusion of B lymphocytes with myeloma cells giving rise to hybrid cells that produce monoclonal antibodies (MAbs) continuously in vitro [21]. Therefore, MAbs are monovalent antibodies, which bind to the same

the Fc portion acts to mediate the effector functions of antibodies [17].

represents the structure of an IgG immunoglobulin and its domains.

epitope and are produced from a single B lymphocyte clone.

*DOI: http://dx.doi.org/10.5772/intechopen.93076*

characteristics [5, 14, 15].

**2. Immunoglobulins**

and IgM [17].

*Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies DOI: http://dx.doi.org/10.5772/intechopen.93076*

The hydrophobic sequence of the fusion loop appears to be the only epitope that is conserved among all flaviviruses; however, the degree of exposure in this region varies substantially among viruses [6]. Nonstructural proteins, NS1 and NS5, also present some epitopes conserved among the DENV serotypes and other members of the flavivirus genus, but their position also varies between viral strains. The relationships of antigenic similarity between flaviviruses generate immune responses that are configured as cross-reactions with protective or pathological characteristics [5, 14, 15].

### **2. Immunoglobulins**

*Dengue Fever in a One Health Perspective*

using crystallography of viral proteins in the presence or not of antibody molecules. Results of this combination showed that the flavivirus is composed of a dense icosahedral electron nucleus and a lipid bilayer surrounding it. The genome comprises a sequence of ~10,700 nucleotides of a positive-sense RNA that encodes a polyprotein that is cleaved in 3 structural proteins, capsid protein (C), membrane protein (M), and envelope protein (E), and in 7 nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [1]. The NS1 protein plays a role in viral replication and is often shown to be a soluble antigen secreted into the bloodstream, interacting with components of the immune system. NS5 is the largest and most conserved nonstructural protein and acts in the transcription process of viral RNA [2, 3].

The icosahedral capsid is small and poorly organized, but it provides enough space for the genome and is surrounded by the envelope, so there are few connections between these structures, unlike what is found in other viruses. The viral envelope consists of two internal and concentric layers of phospholipids and an outer protein shell formed by regions of protein M and ectodomains of glycoprotein E organized in dimers which present protein determinants for the binding of the virus to the host cell (hemagglutination). Protein E contains three domains: domain I (DI), which is related to cell tropism and envelope organization; domain II (DII), which comprises the dimerization region and the fusion peptide; and domain III (DIII) with the func-

tion of binding to cell receptors, in the initial stage of viral infection [4, 5].

the viral genome to the cytoplasm [3, 6, 7].

The fusion loop is a highly conserved region between dengue virus (DENV) serotypes and all flaviviruses, responsible for the late stage of infection, in which the virus interacts with the endosomal membrane, resulting in the release of the nucleocapsid in the cytoplasm. When the particle is in the lysosomal vesicle during the infection process, a structural reorganization of the viral envelope occurs; the fusion loop is exposed and inserted in the lysosomal membrane. After the formation of the fusion loop contact, protein E starts to organize itself into trimmers, resulting in the expansion of the viral particle and the approximation of the viral and lysosomal membranes, forming the fusion lipid pore that allows the release of

DENV serotypes show great heterogeneity in the structure of viral proteins. However, there is also antigenic similarity between DENV serotypes and, for some peptide sequences, between flaviviruses. Phylogeny studies of virus sequences, by estimating the antigenic distance between them, concluded that serotypes 1 and 3 are the most similar, serotype 2 was the second to diverge evolutionarily, and serotype 4 is the one that presents greater genetic difference. There is a 32% difference in the structure of protein E of the four DENV serotypes. Specific mutations in

Much of the genetic difference between flaviviruses is due to protein E, which can show up to 60% difference in its amino acid sequence. In the phylogenetic analysis of the viruses, the DENV serotypes are closer to the Zika virus (ZIKV), with approximately 45% difference, and have 50 and 60% dissimilarity with West Nile virus (WNV) and yellow fever virus (YFV), respectively. DENV and other flaviviruses vary dramatically in terms of the amino acid sequence of the glycosylation region and the content of glycans added to the surface of E and precursor membrane protein (prM). Many epitopes of protein E are unique to a DENV serotype [4, 9–11]. It can also be observed in ZIKV, which has the glycosylation site, in the DI of protein E, different in conformation and length of the loop that contains this glycosylation site. The carbohydrate associated with this residue can act as a virus binding site in host cells. Thus, differences in this region of glycosylation can influence cell tropism, infection, and pathogenesis of these viruses [12]. Another important characteristic of ZIKV is the insertion of an alanine residue in the carboxyl termina-

the genome result in the antigenic variability found in each serotype [7–9].

tion of DIII, which is associated with an increased stability of this virus [13].

**148**

Immunoglobulins, or antibodies, are glycoproteins, expressed on the surface of B cells or secreted, that act in the neutralization and elimination of pathogens [16]. Antibodies are relatively flexible "Y"-shaped molecules made up of two heavy chains and two light chains, joined by an extensive network of non-covalent interactions, stabilized by disulfide bonds. Both types of chains are composed of constant and variable domains. The constant regions determine the functional properties of the antibody, and the variable regions determine the antigen-binding site. The light chain consists of a variable portion (VL) and a constant portion (CL) that can have two types of domains, kappa (ƙ) or lambda (λ). The heavy chain consists of a variable portion (VH) and three or four constant portions, depending on the class of the antibody, which are CH1, CH2, CH3, and CH4. The type of heavy chain determines the class, or isotype, of antibody, such as IgA, IgG, IgD, IgE, and IgM [17].

The antibody molecule can be subdivided into portions of the crystallizable fragment (Fc) and antigen-binding fragment (Fab) region. The Fc portion has the constant domains (CH2, CH3, CH4), and the Fab portion consists of the VH-CH1 and VL-CL domains. The Fab portion retains the ability to bind to the antigen, and the Fc portion acts to mediate the effector functions of antibodies [17].

Three segments containing variability can be identified in both the VH and VL domains. These segments are the hypervariable regions that determine antigen specificity and are more commonly called complementarity-determining regions (CDRs)—CDR1, CDR2, and CDR3. The combination of CDRs from a VH with CDRs from a VL forms the region of interaction with the epitope, called the paratope [17]. The variability of the antigen-binding regions is responsible for the ability of different antibodies to bind to many structurally diverse antigens [18]. **Figure 1** represents the structure of an IgG immunoglobulin and its domains.

In cognate antigen recognition, some naïve B cells can initiate somatic hypermutation, generating new variable domains, that can be selected based on their ability of antigen binding, usually with high affinity compared to germinal domains. After antigen recognition, naïve B cells differentiate into antibody-secreting plasma cells. These plasma cells secrete antibodies with high affinity and, can differentiate into memory B cells. Memory B cells are highly specialized cells for quickly recognizing the antigen in subsequent exposure, can persist for years, and provide long-term humoral protection for decades. These functional features of memory B cells are the basis of effective vaccines [19].

The knowledge about the mechanisms of antibody production and clonal selection of B cells led to the development of innovative hybridoma technology in 1975 [20]. The technique is based on the fusion of B lymphocytes with myeloma cells giving rise to hybrid cells that produce monoclonal antibodies (MAbs) continuously in vitro [21]. Therefore, MAbs are monovalent antibodies, which bind to the same epitope and are produced from a single B lymphocyte clone.

#### **Figure 1.**

*Classical structure of an antibody. Structure of a class G immunoglobulin, representing the two portions of the molecule: two Fabs that correspond to the antigen-binding fragment and an Fc that corresponds to the crystallizable fragment. A type G antibody consists of two heavy polypeptide chains, each containing a VH and three constant domains (CH1, CH2, and CH3), and two light chains, each containing a VL and a CL. CDRs are three regions of hypervariability present in each of the variable domains. In addition to the natural format of the antibody, it is possible to generate recombinant antibodies such as the single-chain variable fragment (scFvs).*

MAbs interact with a single epitope allowing a specific reactivity and affinity for target antigens. This feature is a great advantage over polyclonal antibodies, which have different epitope specificities and affinities [22, 23]. For this reason, MAbs have a broad clinical applicability in therapy for various illness, including cancer, transplant rejection, and autoimmune, infectious, hematologic, and cardiovascular diseases. Moreover, MAbs can play a significant role in the diagnosis and as antibody-drug conjugate for drug delivery. Thus, MAbs are considered a powerful tool for a wide range of medical applications.

#### **2.1 Cross-reactivity of antibodies**

Antibodies that bind to different flaviviruses are able to promote both the neutralization of the infection and the increase of the virus capture, such as by the interaction of immune complexes with Fc receptors expressed in certain cell types. The creation of alternative routes of entry of the viral particle into cells by low-neutralizing antibodies, during secondary infections of flavivirus, results in increased levels of viral replication and pathogenicity. This mechanism constitutes a phenomenon called antibody-dependent enhancement (ADE) [24, 25].

Different studies have been conducted to understand the effects of crossreactive memory antibodies on subsequent flavivirus exposures. Many of them reported that the opsonization of the virus with weakly neutralizing antibodies led to the increase of the viral production and of pro-inflammatory mediators. This could lead to the suppression of the antiviral immune response, worsening the clinical condition of the disease [5, 7]. ADE has already been reported in in vitro experiments of infection of cells that express Fc receptors and in vivo experiments

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*Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies*

the low protection induced by vaccines targeting DENV serotypes [27].

virus through the formation of interspecific antibody memory [28, 29].

used for the selection of bacteriophages that recognize antigens.

of vaccination with flavivirus [11, 26]. From the results of these experiments, ADE is pointed out as one of the main causes of severe forms of DENV infection and of

An opposite effect of cross-reacting antibodies has also been demonstrated, an increase in protection against secondary infections by flavivirus, which has resulted in potent neutralization and rapid induction of affinity maturing immune responses against heterologous flaviviruses. Studies in endemic areas of flavivirus showed protection against Zika virus infection in patients with a previous experience with DENV. A humoral response capable of potentially neutralizing both species of flavivirus was assembled from the expansion of cross-reaction memory B cell clones, even in the absence of DENV circulation. Thus, previous flavivirus infections can lead to both cross-neutralization and increased pathogenicity of the

The potential of cross-reaction immunity to trigger protection or pathology depends on the profile, quality, and magnitude of the immune responses induced by antibodies. The ADE reaction is a factor that should be considered in the development of therapeutic antibodies and vaccines for infections by flavivirus. Different approaches have been tested to shift the ADE profile to a cross-protection profile in

The principle of the Phage Display is the presentation of libraries of molecules on the surface of a bacteriophage (phage), allowing the identification of a wide range of biomolecules, including peptides, antibodies, and other proteins. The Phage Display methodology was first described in 1985, by George Smith and colleagues. Through the expression (display) of polypeptides on the phage surface (phage) M13, it was possible to perform the mapping of antibody epitopes by screening them using random peptide libraries [30]. In 1990, McCafferty and colleagues [31] demonstrated that it was also possible to fuse genes that encode an entire antibody domain, in the form of a scFv to the sequence of one of the bacteriophage's coat proteins. This approach allowed that this methodology could also be

Later, in 1994, Winter refined the Phage Display technology through a guided selection strategy of human antibody fragments from Phage Display repertoires for a single-antigen epitope, using rodent MAbs as a model [32]. The first all-human antibody produced, using Winter's Phage Display technique, to be marketed for use in humans was adalimumab (Humira), approved by the United States Food and Drug Administration (FDA) in 2002 for the treatment of rheumatoid arthritis [33]. It is noteworthy that George P. Smith and Gregory P. Winter received the Nobel Prize in chemistry in 2018 for the Phage Display of peptides and antibodies (Nobel Prize, 2018), a true tool for molecular evolution *in vitro* emphasizing the importance of this technique in obtaining biomolecules for various applications. Phages are single-stranded viruses that infect Gram-negative bacteria and are used mainly for the purpose of gene cloning and expression of recombinant proteins, in addition to basic molecular biology studies. The particle coating is composed of five different proteins, pIII, pVI, pVII, pVIII, and pIX; proteins responsible for DNA replication include pII, pV, and pX; and the assembly proteins are pI, pIV, and pXI. All of the five proteins contribute to the stability of the phage particle; however, pIII is also necessary for the recognition and infection of the host cell [33]. Through genetic manipulation, sequences of billions of peptides, protein variants, and antibody fragments can be cloned into a vector associated with the

*DOI: http://dx.doi.org/10.5772/intechopen.93076*

heterogeneous infections of these viruses.

**3. Phage Display**

#### *Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies DOI: http://dx.doi.org/10.5772/intechopen.93076*

of vaccination with flavivirus [11, 26]. From the results of these experiments, ADE is pointed out as one of the main causes of severe forms of DENV infection and of the low protection induced by vaccines targeting DENV serotypes [27].

An opposite effect of cross-reacting antibodies has also been demonstrated, an increase in protection against secondary infections by flavivirus, which has resulted in potent neutralization and rapid induction of affinity maturing immune responses against heterologous flaviviruses. Studies in endemic areas of flavivirus showed protection against Zika virus infection in patients with a previous experience with DENV. A humoral response capable of potentially neutralizing both species of flavivirus was assembled from the expansion of cross-reaction memory B cell clones, even in the absence of DENV circulation. Thus, previous flavivirus infections can lead to both cross-neutralization and increased pathogenicity of the virus through the formation of interspecific antibody memory [28, 29].

The potential of cross-reaction immunity to trigger protection or pathology depends on the profile, quality, and magnitude of the immune responses induced by antibodies. The ADE reaction is a factor that should be considered in the development of therapeutic antibodies and vaccines for infections by flavivirus. Different approaches have been tested to shift the ADE profile to a cross-protection profile in heterogeneous infections of these viruses.
