Introduction Of The Brand New Bacteriophage T4 Dual Sites Display System

Bacteriophage T4 display system is a SOC- and HOC- dual sites expression and a display system for exogenous poly-peptides / proteins at high copy numbers with biological activity.

Background:

In the phage expression and display field, the bacteriophage T4 capsid is composed of three essential structural proteins: major scaffold capsid protein gp23 (930~960 copies per capsid), and two minor capsid proteins: gp24 (vertex protein, 55 copies) and gp20 (portal vertex protein 12 copies) (1, 2, 3, 4).

In addition, the outer surface of T4 virion is coated with two dispensable capsid proteins SOC (810 ~ 960 copies per capsid, molecular mass 9.4 kDa) and HOC (155 ~160 copies, 40.5 kDa). These two proteins are regularly displayed on the T4 icosahedral lattice (1, 2). SOC and HOC are nonessential capsid accessory proteins and bind strongly to the capsid following capsid assembly.

The capsid expansion triggered by DNA packaging creates HOC and SOC capsid binding sites (2, 3, 4). SOC binds in trimeric arrays on the hexagonal gp23 surface lattice and HOC binds in the center of the lattice. Following refolding- processing- maturation of the major protein gp23, SOC binds in equal molar quantity to cleaved gp23.

The foreign peptide / protein can be displayed by fusion to SOC and HOC on T4 capsid either on a mature viral particle or poly head for display purpose. Our system consists of diverse SOC-, HOC- delivery plasmids, and variant mutated T4 phage expression and display vectors.

In our T4 phage vectors DT4-Z1, DT4-Zh--1, DT4-B-1, and DT4-ZBM-1, both SOC and HOC genes are perfectly deleted from natural T4 phage genome, unlike the other researcher that used T4 display vector only with these two genes carrying artificial mutated stop codon.

The SOC and HOC delivery plasmids of our system contain special homologous recombination elements and function- reporter gene. At first, the desired exotic gene will be cloned to fuse with the SOC or HOC gene on the delivery plasmid, then infect host E. coli with coupled T4 display vector phage.

The desired exogenous gene will be homogenously recombined into T4 phage genome and the target protein will be expressed under the control of T4 mature late promoter and encapsidated / integrated onto T4 capsid surface SOC and HOC sites.

The poly- peptide / protein will be regularly displayed on the T4 icosahedral surface lattice in relative spaced and in natural intact structural conformation. To obtain 110 nm × 84 nm (length × width) recombinant T4 phage strain, scientist could propagate the desired T4 recombinant phage strain to high number of progeny (107 phage /cell), according to different usage purposes by different purification methods. Then the recombinant T4 phage particle can be used as the candidate for vaccine, diagnosis, and protein display.

The phage genome is inserted with exotic DNA gene with lysogenic life cycle as temperate T4 phage. T4 phage particles will multiply themselves in E. coli by carrying the cloned gene and expressed its protein on capsid surface of T4 phage through generations.

Unlike the method of intact genome T4 phage display, it only manipulates on plasmid expressed protein level binding, the displayed target protein is not able to multiply with T4 phage virion together through the generation of T4 phage (5).

On the delivery plasmid a T4-IPIII- CTS (capsid targeting sequence) scaffold proteinase P21 site (12) was added and positioned between the SOC or HOC and fused exotic protein to cleave out the expressed target protein from SOC or HOC fusion partner with proteinase P12.(12).

Merit and character of phage display:

Phage display is a powerful technique for identifying peptide or proteins that have desirable biological and pharmaceutical properties. T4 phage display makes it possible to construct macromolecular complexes that display hundreds of identical copies of designated protein as antigens, antibody / scFv or enzymatic activities on a single macromolecular particle.

Such complexes of display will find prevalent applications especially in the vaccine development. The powerful R&D values of T4 phage display included in protein / peptide library construction for protein / peptide optimization, identification of structurally constrained peptide, leader target discovery, cytokine receptor agonists and drug receptor agonists / inhibitors ligand binding site / active domain discovery.

Assembling massively multimeric copies of rare, expensive, or unstable enzymatic activities onto T4 macromolecular platform could generate the candidate of the biopharmaceutical reagents.

Protein secretion related toxicity can be avoided in lytic T4 phage and protein folding takes place in a fundamental different intracellular environment to prevent the secretion of fusion protein. One of the most valuable and different feature of our system is the ability to express the large protein (>500 amino acids) in an ordered array on T4 phage surface.

The major advantages of T4 system:

1. The ability of displaying multi-copy of two different peptide / proteins to create the “high concentration/density hot spot” of expressed protein to achieve its Kd and simulate a relevant protein-protein interaction. Unlike the proteins used in the phage M13 and other display system, phage T4 HOC and SOC are nonessential for phage infectivity and productivity. They are added onto the capsid surface during the phage assembly process. Therefore, it is capable of simultaneously displaying two different proteins / peptides, spacing-separately outside T4 envelope surface.(10 )
2. The exogenous protein / peptide could be fused to both N- and C- terminal of T4 SOC and HOC proteins at higher copy number than other filamentous phage systems. For example, M13 can only display the short peptides in high copy number from the N-terminal of major coat protein gpVIII, whereas only one or a few copies of polypeptide can be displayed from a minor coat protein gpIII.(13).
3. Secretion of the fusion protein does not occur. The secretory apparatus of E. coli will not eliminate T4 phage displayed proteins/peptides, as it may happen with M13. Unlike the M13 and yeast system, the fusion protein to be displayed with T4 is secreted through the host membrane, posing toxicity and folding problems for some cloned genes. The T4 SOC and HOC system overcome these limitations and secretion- related toxicity can be avoided in the lytic T4 phage (1, 2, 3, 4). Moreover, in icosahedral phage T4 assembly, protein folding takes place in the cytoplasmic milieu and has distinct advantage over the filamentous phages for the displayed proteins.
4. Large protein insertion capability with T4. T4 is able to display proteins with various sizes up to 800 amino acid (aa) length on SOC, more than 1000 aa on HOC, and with various sequences in a much less restricted stringency ways (6-11). One of the most valuable features of the SOC and HOC display system is the possibility of binding large protein domains cluster in an ordered array on the phage surface.
5. The foreign proteins could be displayed both on T4 capsids and polyheads (long planar tubules of the major capsid protein). It is displayed not only on mature phage but also on intermediate empty coat which allows potential for structure determination of displayed proteins. Polyheads have an even higher carrying capacity for display, the copy number is at least twice as high as the SOC display system and it is capable of presenting up to ~103 copies per virus particle and more than 104 per polyhead on giant T4 phage particle of V3-sized, and perhaps much larger protein domains (3,6.).
6. T4 phage particles possess high antigenicity without adjuvant. Arise from T4 phage coat major scaffold protein gp23 and surface accessory HOC’s higher antigenicity enhanced immune response in animals. Animal immunization with the recombinant T4 phage product does no need conventional immune-adjuvant to achieve the same effects (10, 11).

Experimental Achievement:
T4 phage based vaccine R&D:

The T4 phage SOC or HOC display system we had successfully displayed peptides and proteins with immunological activities antigenic activity: Human immunodeficiency virus 1 (HIV) gp120-V3 loop (43 aa) (6); Poliovirus –VP1(312 aa) (6); Foot-and-mouth disease virus (FMDV) VP1 (213 aa), GH-loop extension (70 aa; 72 aa; 34 aa) and P1 (720 aa) (11); CSFV- mE2 (123 aa) and E2 (371 aa) (10); Very Virulent Infectious Bursal Disease Virus (vvIBDV) VPII (415 aa)(14); Avian Influenza viurs (AIV) HA (450 aa); Newcastle Disease Virus (NDV) antigen cluster (550 aa)(15).

All of the above antigens were displayed on the T4-SOC site as SOC fused protective antigen and elicited out neutralizing antibody in animal model. We have conducted the protective test against challenge with strong pathogenic virus of FMDV, vvIBDV and NDV in China.

The result of the FMDV vaccine is briefly shown here: on mice immunized with T4-FMDV full-length coat immunogen P1, immunized via oral given and injection, both oral given group and injection group got 100% protection from challenge with field strong FMDV (11).

On chicken immunized with T4-vvIBDV-VPII immunogen, got 100% protect from Very Virulent Infectious Bursal Disease Virus challenge (14). On chicken immunized with T4-NDV immunogen, got 80% protect from strong pathogenic Newcastle Disease Virus challenge (15).

We also used both SOC and HOC dual sites displayed on one T4 virion surface for Classic Swine Fever Virus (CSFV) major antigenic determinants clustered mE2 (123 aa) on SOC site and full-length CSFV primary antigen E2 (371 aa) on HOC site, the humoral immunity and cellular immunity were measured in 100 mice (10).

The immune effects of single site display, bipartite display, and conventional E. coli plasmid expression were observed. The animal immune response data showed that the elicited antibody titers with phage T4 display are higher than plasmid based method, and double site display the antibody titers are three times higher than single site display in animal model.(10).

Dr. Rao used both SOC and HOC single site display of Neisseria meningitidis PorA antigen (36 aa) has been investigated with different approaches of T4 system. (5) As toT4 vaccine administration, several routes have been explored through traditional injection, oral administration and intestine mucosal immunization as well.

For T4 phage recombinant particle safety investigation:

T4 phage may occur naturally in the animal and human intestine. Scientists have already performed T4 phage safety test in human by orally given and it is considered safe with non-pathogenic side effects since it infects enteric E. coli but not human cells (16).

For T4 phage based antibody activity:

We displayed Egg White Lysozyme (EWL) - IgG VH-linker-VL, (271aa) on T4-SOC site (8).

For T4 phage based receptor function:

We displayed HIV-1 CD4 receptor domain 1 and 2, on T4-HOC site (8).

For T4 phage based enzyme activity:

We displayed FMDV envelope structure processing proteinase 3C, on T4-SOC site (11);

For T4 phage based peptide libraries construction:

Randomized display peptide libraries (9mer), displayed on both T4-SOC and HOC sites

For trying T4 phage based carcinoma immune-therapy:

We constructed a series of T4 -EGF,EGFR, VEGFR recombinant phage strains.

For T4 phage based scFv libraries construction:

Using bird flu human substrain virus immunized mouse spleen, we are able to construct a single chain Fv antibody library. This work co-operates with research institute in China.

For T4 phage based HIV diagnosis & vaccine construction:

SBIR grant submitted.

Prospect of commercial application:
Potential on vaccine development:

In vaccine development, antigens displayed with T4 phage platform proved to be more stable and effective as immunogens than synthetic peptides or recombinant proteins.

By presenting them on large macromolecular platform, we proved T4 recombinant phage can elicit protective immune response for antigens; we also investigated to dispense T4 with or without adjuvants, or greatly reduce the amount of antigen required for immunization (11).

Phage T4 prohead and virion particles can be propagated to a large number in E. coli host. The desired protein /peptide expressed T4 recombinant particles are easily being purified by CsCl, glycerol, or sugar gradient centrifuge procedure and directly used as immunogens in animals to stimulate neutralizing antibody production.

T4 vaccine platform is highly attractive for developing multivalent vaccines because it can accommodate large protein antigens and present high copies on both T4 capsid surface Soc- and Hoc- sites and is cost effective.

Therefore phage T4 dual sites display emerges as a potentially powerful method for the preparation of immunogens and for phage based vaccine production with no potential biohazard risk for human as traditional inactivated pathogen vaccine.

• Oral given T4 phage based safe vaccine for animal and human.
  
• Diagnosis kit production for a series of human and animal infectious disease, employing T4-displayed antigen detect antibody.
  
• Production of T4-phage surface displayed scFv antibody library, employing T4-displayed antibody detect pathogens /antigens in animal and human bodies, and research usages.
  
• Biopanning tool, just like mast the art of finding a needle in haystack, for scientific research kit development. Our experiment has established a biopanning procedure by T4 display system (6).
  
• In biotechnology market, generating novel biotechnological reagents, such as the rare, expensive, or unstable enzymatic activities onto T4 macromolecular platforms.
  
• Scientific research kit development with T4- phage macromolecular complex as desirable protein-protein interactions platform, also involved in cell receptors and enzymes. ( ref. Cortese,R.,Monaci,P.,Nicosia,A.,Luzzago,A.,Felici,F.,Galfre,G.,Pessi,A.,Tramontano,A.,and Sollazzo,M., 1995, Curr. Opin. Biotechnol. 6:73-80.
  
• Construction of T4- phage displated peptide library showing up to several tens of amino acid length.
  
• Try on phage therapy. Created ERE evade T4 phage strain by “Serial Passage” method. (ref : Merril,C.R.Biswas,B.,Calton,R.,Jensen,N.C., Greed,G.J.,Zullo,S., and Adhya,S: 1996 Proc.Natl.Acad.Sci.USA, 93:3188-3192 ).

Reference:

1. Black, L. w., Showe, and A.C.Steven.1994. Morphogenesis of the T4 head, p.218-253. In J.D.Karam (ed) ,Bacteriophage T4.ASM Press, Washington, D .C
2. Yanagide, M. 1977. Molecular organization of the shell of theT even bacteriophage head. II Arrangement of subunits in the head shells of giant phages. J. Mol. Biol. 109:515-537.
3. Ishii,T.,and M.Yanagida.1977,The two dispensable structural proteins (Soc and Hoc) of the T4 phage capcid; their purification and properties, isolation and characterization of the defective mutants, and their binding with the defective heads in vitro. J.Mol. Biol. 109:487-514.
4. Fokine, A., Chipman, P.R., Leiman, P.G., Mesyanzhinov, V.V., Rao, V.B., and Rossmann, M.G. (2004) Molecular architecture of the prolate head of bacteriophage T4. Proc. Natl. Acad. Sci. (USA) 101, 6003-6008.
5. Jiang J, L.Abu-Shilbayeh, and V.B. Rao. 1997. Display of a porA Peptide from Neisseria meningitidis on the Bacteriophage T4 Capsid Surface, Infect Immu. 65: 4770~4777
6. Ren, Z.J., Lewis, G.K., Wingfield, P.T., Locke, E.G., Steven, A.C., and Black, L.W. (1996). Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4. Protein Science 5: 1833-1843.
7. Ren, Z.J., Baumann, R.G., and Black, L.W. (1997). Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector. Gene 195, 303-311.
8. Ren, Z.J. and Black, L.W. (1998) Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid. Gene 215, 439-444.
9. Ren, Z.J and Lai, D. (2005) : T4 Phage Antibody Display to Develop Antibodies for New, Emerging or Engineered Threats. Executive Summary, Pharmaceutical International, published by Copybook Solutions, web: www.pharmaceutical-int.com, Vincogen.
10. Wu J.M., Tu C.C., Yu X.R., Zhang M.L., Zhang N.Z., Nie W.X., Zhao M.Y., Ren Z.J (2005): Bacteriophage T4 nanoparticle Capsid Surface SOC and HOC Bipartite Display with Enhanced Classical Swine Fever Virus Immunogenicity: A Powerful Immunological Approach, ( submitted to J. Virological Method )
11. Ren Z.J, Tian C.J., Zhao M.Y., Ling S. R., Zhu M.W., Wu J.Y., Lan H.Y., and Bi Y.Z (2005):Oral Foot-and-mouth disease virus whole envelop vaccine on recombinant bacteriophage T4 nanaparticle surface: 100% protection from potency challenge in mice, ( submitted to Vaccine)
12. Mullaney, M. M.and Black,L.W.: Capsid Targeting Sequence Targets Foreign Proteins into Bacteriophage T4 and Permits Proteolytic Processing, J.Mol.Biol.(1996) 261:372-385
13. Lannolo,G.Minenkova,O.,Petruzelli,R., and Cesareni,G: J. Mol.Biol. (1995)248: 835-844
14. 14. Cao Y.C., Shi Q.C, Ma J.Y., Xie Q.M., Bi Y.Z.,2005, Vaccination against Very Virulent Infectious Bursal Disease Virus using recombinant T4 bacteriophage displaying viral protein VP2, Acta Biochimica et Biophysica Sinica, 37: 657–664
15. 15. Liuyou, Bi Y.Z., Cao Y.C.2005,Construction and immunogenecity of recombinant phage T4 expresing the F gene of Newcastle Disease Virus, Scientia Agricultura Sinica, 38: 1270-1274.
16. Anne Bruttin and Harald Brussow, 2005,Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy, Antimicrobial Agents and Chemotherapy, 49: 2874-2878

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