Phage display—A powerful technique for immunotherapy

Abstract

Phage display is a powerful technique in medical and health biotechnology. This technology has led to formation of antibody libraries and has provided techniques for fast and efficient search of these libraries.

The phage display technique has been used in studying the protein-protein or protein-ligand interactions, constructing of the antibody and antibody fragments and improving the affinity of proteins to receptors.

Recently phage display has been widely used to study immunization process, develop novel vaccines and investigate allergen-antibody interactions. This technology can provide new tools for protection against viral, fungal and bacterial infections. It may become a valuable tool in cancer therapies, abuse and allergies treatment. This review presents the recent advancements in diagnostic and therapeutic applications of phage display. In particular the applicability of this technology to study the immunization process, construction of new vaccines and development of safer and more efficient delivery strategies has been described.

Introduction

Phage display is a tool for molecular evolution, enzymology and study of antibodies. Moreover, it has been used in analyzing the protein-protein or protein-ligand interactions. This technique allows to identify and develop modulators of receptors and enzymes,^1-3 (to) determine the substrate specificity^4,5and (to) obtain stable proteins and enzymes for biotechnological and analytical applications.^6-8 Antibodies and antibody fragments originating from phage display are widely used in therapeutic development and diagnostics.^9-11 They are commonly used in methods such as ELISA,¹² immunoblotting,¹³ immunofluorescence microscopy,¹⁴ affinity chromatography,¹⁵ flow cytometry,¹⁶ microarrays assays,¹⁷ hemagglutination assays,¹⁸ bead based assays,¹⁹ proximity ligation assays,^20,21 molecular imaging,²² lateral flow strip assays,²³ immuno-PCR²⁴ and fluorescence resonance energy transfer (FRET).²⁵ Molecules displayed on phage surface were considered as direct diagnostic agents.²⁶ It was reported that by using the phage display technology, scFv fragment that recognizes glycated α7 subunit of the proteasome was obtained. This antibody fragment was defined as a tool for analyzing a marker of proteasomal malfunction, cellular viability, and a potential marker of aging.²⁷

Antibody phage display provides direct access to the genetic information of the binder, allowing a fast adaptation of the antibody format to the desired diagnostic assay.^12,13,17,22 The sensitivity of the diagnostic test could be increased by displaying on phage surface many copies of antibody fragments. Also the usage of the secondary antibodies with specificity for phage coat proteins could improve assay quality.

Phage display enables better understanding of the immunization process, therefore utilization of this technique in vaccine development has increased recently. This technology enables searching of immunogenic peptide sequences and constructing of novel vaccines. The phage display technology also provides new opportunities for vaccine delivery, which may lead to strengthening the effectiveness of immunization.

This review focuses on selected applications of phage display in immunization, vaccine development and investigation of allergen-antibody interactions, which can provide novel diagnostic and therapeutic agents.

Vaccine Delivery

Phage display is a powerful technique for designing novel vaccine delivery vehicles. With this method, phage display vaccine or phage DNA vaccine can be constructed. Also hybrid phage vaccine has been suggested. In the first model—the phage displayed vaccine, virions contain the antigen encoding gene which is expressed in fusion with coat protein. Antigen could be also attached to phage surface with artificial linker.^28,29 In phage DNA vaccine model, DNA vaccine that contains antigen gene cloned into eukaryotic cassette is packed within bacteriophage virion. Eukaryotic cassette is a part of vector DNA which contains three main sequences: promoter, ORF (open reading frame) and 3' untranslated region often with polyadenylation site. It was constructed to allow proper gene expression and correct folding of eukaryotic proteins in prokaryotic cells. The predominance of phage vaccines over standard DNA vaccines or using antigens per se is primarily the fact that virion protects the vaccine and makes it more stable for administration (especially in oral vaccines), storage and transport. Moreover, phages have been reported as the adjuvant-like particles, therefore this method allows to achieve better results at lower doses than standard vaccination.^30,31 Phage vaccines give higher and less variable immune response compared with standard vaccines. The hybrid phage vaccine is supposed to induce effectively humoral and cellular response. This model allows to display not only antigen but also targeting molecule, thus it appears to have a potentially significant advantage for goal directed therapy.^32,33

Predominance of phage vaccines is based on fundamental properties of the virus itself such as high stability in wide range of pH, which facilitates the application and distribution in addition to a low cost of phage particle production. Moreover, phages are unable to proliferate in eukaryotic cells, thereby their application is safe. Also phage vaccine application does not cause side effects,³⁴ although there are some disadvantages of using prokaryotic viruses as vaccine delivery vehicles. The main problem is to display correctly folded, active epitope in a concentration sufficient to elicit an immune response. Also the administration route of the vaccine must be considered because there is a risk that after oral distribution phages will infect intestinal flora.³⁵ There are several strategies that could lead to enhance the oral administration of phage vaccines. First, the use of non-lytic phage may reduce the risk of infection and the destruction of the natural intestinal flora. Moreover, virions with damaged tail fibers are incapable of infection, but still can serve as a vaccine delivery vehicle. Phages that are stable in the gastro-intestinal tract could be successfully used in oral administration. although it is still more common to provide vaccines by subcutaneous or intramuscular injections.

Anti-cancer vaccines

To date, many anti-cancer vaccines based on phage display technology have been reported.^36-38 In phage DNA vaccines, cytokines, chemokines, costimulatory molecules and pathogen genes could prime the immune system. It was reported that the domain I fragment of pIII protein expressed as a fusion with an scFv can enhance T helper 1 humoral and cell-mediated antigen-specific immune response.³⁹ Moreover, CD8+ cytotoxic T-lymphocytes (CTL), that recognize tumor-associated antigenic epitopes expressed by human leukocyte antigen (HLA) class I molecules on the cancer cells, can be primed by vaccination.⁴⁰ The best target antigens are proteins such as telomerase, tyrosinase, gp100, MAGE, Melan-A/MART, MUC1, CEA, p53, Her-2/neu, survivin or Ras, which level is significantly different in cancer cells than in healthy tissue.⁴¹ The melanoma antigen MAGE-A1 is expressed in more than 50% of hepatocellular carcinoma, metastatic melanoma, non-small cell lung carcinoma, esophageal squamous cell carcinoma. Interestingly, phage vaccination with protein pVIII fused epitope of tumor-specific antigen (MAGE-A1_161–169) prevents and suppresses melanoma tumor growth and also enhances the NK cells activity.⁴² Moreover, vaccination with heat shock protein Hsp70-peptide complexes can induce protective immune responses. The method for identification of mimetics of this protein has been described. By the use of biopanning of a random phage display library, peptides that bind to Hsp70-peptide complexes from human breast carcinoma cells were identified. These peptides were used to identify Hsp70 mimetics, which could be used as modulators of the immune response against tumors.⁴³

Anti-viral vaccines

Phages have been also investigated as anti-viral vaccine delivery vehicles.^32,44 Cytotoxic T-lymphocyte (CTL) is an important factor not only in anti-cancer but also in anti-virus strategies^45,46 , including HIV infection control.⁴⁷ Thereby, ability of CTL phage displayed epitope to induce specific CTL response has been widely investigated.

In the recent study, phage display has been used to isolate and enhance a T-cell antigen receptor (TCR) from the infected person’s CTL. This TCR was specific for the SL9 peptide, an immunodominant HLA-A*02-restricted, HIVgag-specific SLYNTVATL sequence. It is noteworthy that the obtained high-affinity TCRs have targeted HIV-infected cells and have recognized all common escape variants of this epitope.⁴⁷

Interestingly, the DNA fragment encoding a hepatitis B virus epitope S_28–39 was packed into phagemid vector pC89. Hybrid phage containing a pVIII gene with the CTL epitope insert was constructed using the VASM13 helper phage and the BALB/c (H-2d) mice were immunized. After 8 d of immunization a MHC class I restricted hepatitis B specific CTL response occurred.⁴⁸ The phage lambda display system has been used for construction and characterization of cDNA expression library of human hepatitis C virus (HCV). . Several proteins displayed as fusion to the carboxy terminus of the phage lambda capsid protein D were accessible for interaction and affinity-selection with specific antibodies.⁴⁹

Furthermore, herpes simplex virus 1 (HSV-1) glycoprotein D is essential for the virus attachment and entry. It was shown that inoculation of BALB/c mice with phage DNA vaccine containing (HSV-1) glycoprotein D cassette results in enhanced both humoral and cellular immune responses.⁵⁰

Other studies have described the platform for peptide display based on virus-like-particles (VLPs) of the RNA bacteriophage PP7. This platform was utilized in displaying the human papilloma virus type 16 (HPV16), minor capsid protein L2 epitope. After intramuscular injection of HPV16 L2 epitope displayed on PP7 VLPs the robust anti-HPV16 L2 serum antibodies were generated. Thus, vaccine based on this protein could provide more comprehensive protection against infection by diverse HPV types.^51-53

Although intensive research on anti-viral vaccines is conducted, much focused on a narrow range of diseases often associated with the appearance of tumors. It should be noted that the phage display technique has been used in the research on novel therapeutics that will specifically interfere with the transcription/replication complex of negative-strand RNA viruses (NSRV). Due to the high infectivity of these viruses and the possibility of calling epidemics and pandemics, this research is particularly important. NSRV can cause respiratory diseases (influenza virus, respiratory syncytial virus, measles and mumps viruses), encephalitis (rabies virus, Hendra and Nipah viruses; Rift Valley fever virus) or hemorrhagic fevers (Ebola and Marbug virus, Crimean-Congo hemorrhagic fever virus, Lassa virus). It is noteworthy that the transcription/replication complex is similar for all NSRV, thus broad-spectrum anti-viral therapeutics could be developed. Panning of large libraries by the phage display technique has led to the identification of five promising peptides, two of which have potentially broad-spectrum activity.⁵⁴ Furthermore, it was described that mice immunized with phage displayed vaccine that contain a protective B cell epitope of human respiratory syncytial virus gained a complete resistance to this virus.²⁸

Anti-parasite vaccines

Phage display strategies for anti-parasite therapeutics have been described.^55-58 One of them is based on an iterative subtraction strategy to identify potential vaccine antigens of Brugia malayi—the filarial parasites that cause lymphatic filariasis (a debilitating disease affecting over 120 million people in the tropical and sub-tropical countries). The cDNA library expressed on the surface of T7 phage was screened with human sera and five antigens (ALT-2, TPX-2, VAH, COX-2 and novel cuticular collagen Col-4) were identified. Vaccination of mice and jird with recombinant ALT-2 resulted in achieving high immunity level.⁵⁹ It is noteworthy that strategies for development of malaria vaccines have been reported. The abundant merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been successfully used in vivo as a protective immunogen.⁵⁵ Another vaccination approach to malaria is a phage displayed SM1 peptide that specifically binds to the same surfaces that are invaded by the malaria parasite and almost completely inhibits invasion of the midgut by ookinetes and invasion of the salivary glands by sporozoites.⁵⁶ Besides, host antibody response to the ring-infected erythrocyte surface antigen (RESA) from P. falciparum has been characterized by phage display library.⁶⁰

Anti-bacterial vaccines

Mimetics of polysaccharides were obtained by the phage display method. This strategy can be used for anti-bacterial vaccine development. Phage display libraries have been utilized in identification of Neisseria meningitidis serotypes A, B and C. Moreover, peptides selected by screening random phage library against Staphylococcus aureus RNAIII-activating protein were able to elicit immune responses in murine models.^61,62 The mouse monoclonal antibodies (mAbs) against Klebsiella pneumoniae MrkD adhesin were obtained by the hybridoma technique and screened against phage-displayed random library which resulted in (QKTLAKSTYMSA) mimotope selection that was able to mimic immunological properties of the native epitope of K. pneumoniae MrkD.⁶³ Furthermore, lambda display library of DNA fragments from Streptococcus pneumoniae and Mycoplasma pneumoniae genome was applied in identification of epitope-containing fragments. This study demonstrated that S. pneumoniae epitope is conserved in all Pneumococcus serotypes and S. mitis strains, while it is absent in other Streptococcus strains. It also allows to confirm the immunogenicity of M. pneumoniae adhesins P1 and P30 and to identify four novel immunogenic polypeptides.⁶⁴ Moreover, immunization of maternal mice with the phage particles displaying recombinant anti-idiotypic Ab fragments resulted in obtaining the streptococcal infection immunity by the neonatal mice.⁶⁵

Other studies focused on Pseudomonas aeruginosa have led to identification of outer membrane proteins encoding genes that are potential targets for anti-P. aeruginosa vaccine development.⁶⁶

Anti-fungal vaccines

Strengthening the immune response against fungal infection has been intensively investigated. Specific epitope LKVIRK of Candida albicans heat shock protein 90 was genetically inserted into the phage major coat protein pVIII using a phagemid vector system and investigated in vivo in C57BL/6 mice as anti-fungal vaccine. Immunization led to achieve higher titers of epitope LKVIRK-specific serum IgG as compared with those immunized with heat-killed C. albicans (HK-CA).⁶⁷ Protective immune responses mediated by hybrid-phage expressing Candida albicans heat shock protein 90 epitope (DEPAGE) in C57BL/6J mice were also evaluated. This epitope, expressed as fusion with pVIII, has been reported to induce the specific antibody response, enhance delayed-type hypersensitivity (DTH) response, natural killer (NK) cell activity and concanavalin A (ConA)-induced splenocyte proliferation.⁶⁸ Phage display based method of identification of short peptide sequences that can distinguish C. albicans from other closely related species has been also reported.^69,70

Anti-drug vaccines and abuse treatment

Addictions are a major societal and health problem and the addiction syndrome is similar between different drugs of abuse and could be described as a chronic relapsing brain disorder with neurobiological changes.⁷¹ Recently, immunopharmacotherapy treatment of drug abuse has been widely discussed and anti-drug vaccines against methamphetamine,⁷² phencyclidine, opiates, nicotine⁷³ and cocaine,⁷⁴ have become the object of intensive research.^75,76 Moreover, phage display peptide libraries and biopanning are considered a tool for early diagnosis and prognosis of chronic alcohol consumption.⁷⁷ Antibody fragments have been applied toanti-idiotypic cocaine vaccines, cocaine-specific scFv fragments, nicotine-specific IgG and methamphetamine-specific scFv.^72-74

Phages ability to penetrate the central nervous system (CNS) was applied to generate phage displayed cocaine-binding proteins. This anti-cocaine antibodies block the psychostymulatory effects of cocaine. One of them is GNC92H2 phage displayed protein, which delivered intranassally might serve as anti-drug therapeutic.⁷⁵ The potential of this approach has been demonstrated, however the kinetic constant of this protein is insufficient for clinical treatment. Nevertheless, phage displayed enzymes such as butyrylcholinesterase (BChE), the major cocaine-metabolizing enzyme and bacterial cocaine esterase (CocE), the most efficient cocaine degradation biocatalyst might be displayed on phage surface to enable them to access the CNS and protect them from proteolysis.⁷⁸

Anti-sperm Contraceptive Vaccines

To date, several human scFv antibody fragments that recognize sperm antigens were described as potential contraceptives.⁷⁹ The phage display technique was used to obtain antibodies that react in a concentration-dependant manner with fertility-related sperm antigens.

The contraceptive effect of cFA-1,⁸⁰ YLP₁₂,⁸¹ P10G,⁸² A9D,⁸³ and SP56⁸⁴ peptides has been investigated in vivo in murine model. All vaccines show maximum 75% reduction in mice fertility, however, this result cannot be directly related to the effectiveness of the vaccine in humans.⁸⁵ Furthermore, an immunocontraceptive vaccine for men was considered.⁸⁶ Phage display libraries were used to select several peptides of 7 and 12 amino acids with binding specificity to canine zona pellucida (ZP) glycoprotein, one of the key proteins in the sperm-oocyte binding process. The NNQSPILKLSIH peptide was found to induce an immune response and to stimulate anti-sperm antibodies production.⁸⁷ Studies on the contraceptive effect of sperm antigens include also LDH-C4, 80 kDa HSA, Eppin and Izumo, however they have not led to clinical trials.⁸⁸

Investigation of Allergen-Antibody Interactions

Cloning and characterization of diverse types of allergens is essential for investigating the allergen-IgE interactions. Phage display is an advanced technology that can lead to development of novel diagnostic and therapeutic agents.. Fab fragments with high affinity for the IgE-binding epitope of the allergen could be applied to neutralize allergens in vivo and to prevent binding of IgE to FcεRI receptor and thereby block histamine release. The obtained mimotopes could serve as anti-allergic vaccines and might be safer than the currently used natural allergens, which evoke IgE responses.⁸⁹

Screening of random phage libraries allows mapping of allergen epitopes and leads to obtain a variety of allergen mimetics. Phage display has been used to generate IgE Fab library, which allows to select four Fab clones that were specific for the Phleum pratense grass (timothy-grass) allergen Phl p 5.⁹⁰ The CDR3 region was rich in aromatic amino acids which increase the allergen cross-reactivity of specific IgE.⁹¹ The rPhl p 5a domain contains several IgE epitopes in a configuration optimal for efficient effector cell activation, interacts with serum IgE from 76% of grass pollen-allergic patients and reveals an extremely high allergenic activity in basophil histamine release as well as skin test experiments.⁹² The selection of anti-idiotypic antibodies against Phl p 5a-specific IgE directly from the B-cell repertoire of a grass pollen allergic individual had led to identification of five different Fab clones with anti-idiotypic specificity for anti-Phl p 5a-IgE. It was reported that phagemid DNA was used to produce two soluble recombinant anti-idiotypic Fab clones in E. coli, both of which induced anti-Phl p 5a-specific antibodies in immunized BALB/c mice.⁹³

Recently, phage display has been used to characterize the epitopes and identify the mimotopes for antibodies to the Lolium (ryegrass) pollen allergen Lol p 2,⁹⁴ Betula (birch) pollen allergen Bet v 1,⁹⁵ Prunus dulcis (almond) allergen,⁹⁶ Charybdis feriatus allergen (crab)⁹⁷ and to the Dermatophagoides pteronyssinus (home dust) allergen Der p 1.⁹⁸ Moreover, the cDNA display libraries based on pJuFo phagemid were utilized to define fungal (Aspergillus fumigatus),⁹⁹ peanut (Arachis hypogaea) and wheat (Triticum aestivum L.) protein allergens.¹⁰⁰ For instance, a panel of six Arachis hypogaea allergens was isolated by the phage display technology. Two of them were previously described as major peanut allergens (Ara h 1¹⁰¹ and Ara h 2¹⁰²), allergens Ara h 4, Ara h 6 and Ara h 7 have been shown to contain similar sequence to seed storage proteins and the last one, Ara h 5 was similar to a well-known plant allergen profilin. Phage display can offer solutions in allergen studies that will certainly affect the development of novel diagnostic tools and therapeutic agents.

Development of New Vaccine and Diagnostic Candidates

Several novel vaccines and diagnostic candidates, developed by the use of phage display were described earlier.^51,72-74 Recently, the usage of the phage display technique to identify new potential vaccines and diagnostic candidates has been described. This technique was utilized to identify three antigens: a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029), that could serve as diagnostic agents in Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides. Interestingly, the prototype of the diagnostic test resulted in 100% sensitivity and specificity.¹⁰³ Also, phage display allowed to identify novel immunogenic proteins and generate antibodies against Salmonella typhimurium. These proteins could be used to establish a diagnostic kit with very high specificity in comparison to commonly used methods.¹⁰⁴ It was suggested that MAP-31, a four-branch multiple antigen peptide, may be a novel vaccine candidate against Staphylococcus aureus.¹⁰⁵ A novel mechanism of marking morbillivirus vaccines, using rinderpest virus (RPV) has been recently described. Moreover, this method was considered to be utilized in development of marked vaccines for peste des petits ruminants virus (PPRV).¹⁰⁶ It was shown that immunogens carrying large combinatorial libraries of mutated epitope variants (VELs) induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. This VELs with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids, could serve as vaccines against antigenically variable pathogens including HIV-1 virus.¹⁰⁷ Also the immunogenicity of different conjugates of epitope EC26–2A4 localizing to the membrane proximal external region (MPER) of the HIV-1 transmembrane protein gp41 has been studied. This EC26–2A4 MPER epitope was reported as a promising vaccine candidate for HIV-1 virus.¹⁰⁸

Conclusion

Since its creation in 1985, phage display has become a technology of great importance in medicine and biotechnology. With this inspiring technology, mechanisms of many diseases can be investigated, which may lead to development and improvement of diagnostic methods and therapies. Moreover, through phage display new drugs and vaccines may be available on the market, allowing treatment and protection against many diseases such as autoimmune and cancer diseases or life-threatening infections. Further research should focus on studying the immune response, which may lead to improvement of the method for rapid antibody selection and development of new therapies. It can be concluded that the role of phage display in diagnostic and medical applications within the next decades will continue to increase.