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Paragraph: The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.) , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system)

No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , 

In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) .

Question: How may the display be achieved?
by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.)