key: cord-0008824-0jarr7hj
authors: Lomonossoff, George P; Johnson, John E
title: Use of macromolecular assemblies as expression systems for peptides and synthetic vaccines
date: 2002-02-11
journal: Curr Opin Struct Biol
DOI: 10.1016/s0959-440x(96)80072-8
sha: 9e70c3e886b614f01913ba65af75a98223c85718
doc_id: 8824
cord_uid: 0jarr7hj

The past decade has witnessed the development of numerous systems for the presentation of antigens on the surface of self-assembling macromolecules. Although the sites for insertion were initially chosen empirically, the determination of the three-dimensional structures of a number of carrier macromolecules has enabled structure-based insertional mutagenesis to be used increasingly. Furthermore, it is now feasible to determine the structure of an inserted sequence as presented in a heterologous environment, making it possible to correlate the detailed structure of a peptide with its immunological properties.

Over the past 15 years, there has been much interest in the development of polypeptides derived from pathogens as novel vaccines. Though free peptides can confer protection, it is generally found that their immunogenicity can be enhanced by presenting them on the surface of a macromolecular assembly [1] . Initial studies involved chemically crosslinking synthetic peptides to a variety of moderately sized carrier proteins [2] . Subsequently, attention has turned to genetically fusing polypeptide sequences to self-assembling macromolecules, qb use such a method, it is necessary that the fused sequence does not interfere with the assembly process and that it is exposed on the assembled particle. Suitable sites have been identified either empirically or on the basis of a known three-dimensional (3D) structure. In this review; we examine the use of self-assembling systems as a method of presenting epitopes and the future prospects for such systems.

Since the pioneering work which demonstrated that filamentous phage could be used to express foreign antigens on the virion surface [3] , the uses to which such 'phage display' technology has been put have increased rapidly. In particular, the use of filamentous phage to display random libraries of amino acid sequences that bind to a variety of ligands has attracted considerable attention. Because this use of phage display has been extensively reviewed recently [4,5], it will not be discussed further here. We will, however, discuss the display of pre-defined polypeptides on the surface of filamentous phage.

The capsids of filamentous phage such as fd, fl and M13 consist of one major (pVIII) and four minor (pill, pVI, pVII and plX) proteins. There are approximately 2700 copies of the 50 amino acid long pVIII protein per virion, which form a helical structure around the genomic single-stranded DNA, and about five copies of each of the minor constituents [6] . To investigate the potential of filamentous phage as an epitope presentation system, de la Cruz et aL [7] inserted tetrapeptide repeats from the circumsporozoite protein of the malarial parasite Plasmodium falciparum between the functional domains of the minor capsid protein pill of phage fl. The recombinant phage were immunogenic in rabbits but gave variable responses in mice. To increase the number of copies of the insert on the modified phage, antigenic sequences were inserted near the N terminus of the major capsid protein, pVIII, of fd [8] and M13 [9] . However, it has proved impossible to recover assembled particles when the modified pVIII protein contains inserts longer than about six amino acids. This limit to the size of the insertion may reflect, constraints imposed during the initiation of assembly [10] . The insertion of a five amino acid sequence near the N terminus of pVIII does not appear to alter the architecture of the phage particles [9] and fibre diffraction studies [11 °°] indicate that such inserts are presented in an extended conformation between two ix helices on the surface of the virions, qb express longer sequences fused to pVIII, hybrid fd phage containing a mixture of modified and wild-type pVIII have been constructed. The resulting particles contained 10-30% of the modified protein. Such hybrids partic!es expressing cpitopes from Plasmodiumfa/ciparum [8, 12] or the V3 loop of tlIV-1 [131 have been shown to elicit an appropriate immune response when injected into experimental animals.

In addition to the filamentous phage, the coat proteins of other types of bacteriophage have also been modified to express foreign sequences. For example, the coat protein of the small spherical RNA-containing bacteriophage MS2 has been modified to express epitopes up to 24 amino acids long [14] . Particles of MS2 contain 180 copies of a single coat protein arranged in a T =3 lattice (Fig. la) and the foreign sequences were inserted in the N-terminal 13 hairpin [15] . When expressed in Escherichia co~i, the modified coat proteins can self assemble into empty virus-like particles which are immunogenic. In view of the fact that the structures of a number of MS2 mutants have been solved recently [16], this system may provide a means of determining the 3D structure of inserted sequences. Two systems involving bacteriophage X have also been developed for displaying foreign protein sequences. These involve making fusions either to the C terminus of V tail protein [17] or to the N terminus of the D capsid protein [18"]. In the former case, only a fraction of the V proteins are modified and the system allows the expression of proteins as large as tetrameric 13-galactosidase; in the latter case, all copies of the D protein could be modified to express peptides of significant length.

These systems arc based on proteins which can self assemble when expressed in either a homologous or heterologous system. Although many of the examples described below are based on macromolecular assemblies produced by viral proteins, the particles themselves are not infectious.

The Ty element from Saccharomyces cerevisiae encodes a 50kDa protein (TYA) which can self assemble into particles which range in size between 15 and 39nm [19] . Particles of a more homogeneous size (ll-16nm) can be produced when a truncated form of TYA containing amino acids 1-381 is expressed in yeast [19, 20] . It has proved possible to fuse substantial lengths of heterologous sequences (>20 kDa) after amino acid 381 without abolishing the ability of the particles to assemble; particles expressing portions of HIV-1 proteins at this site were weakly immunogenic [20] . Immunological analysis of the 1-381 particles indicates that although the N terminus of the TYA protein is on the exterior of the assembled particles, the C terminus lies on the interior [21]. This probably explains the relatively poor immunogenicity of sequences fused to the C terminus. Analysis of regions of the TYA proteins essential for particle formation indicates that there are many non-essential sequences, including the N-terminal 40 amino acids [22" ]. This information should greatly assist the development of new TYA-based systems.

Two self-assembling proteins from hepatitis B virus have been developed as epitope-presentation systems. The first to be developed was based on the 22nm particles formed by the surface antigen (HBsAg). These particles contain lipids and approximately 100 molecules of 226 amino acid long S protein. Expression of this protein in a variety of systems leads to production of lipoprotein particles similar to those found during infection. Initial The self-assembly properties of the coat proteins of two plant viruses have been used to design potential antigen-presentation systems. In the first instance, an eight amino acid epitope from VP1 of poliovirus type 3 was fused to the C terminus of the tobacco mosaic virus (TMV) coat protein and the fusion protein expressed in E. coil [44] . As with wild-type coat protein, the modified protein could assemble into helical virus-like rods when extracts were acidified, greatly aiding purification. The purified rods were shown to be able to elicit the production of anti-poliovirus neutralizing antibodies in rats. More recently, it has been shown that co-expression in E. coil of both the TMV coat protein and single-stranded RNAs containing the TMV origin of assembly can give virus-like particles without the need to acidify the medium [45] . Because RNA-containing particles are more stable than protein-only helices, especially at higher pHs, the latter system could be adapted as an improved way of expressing foreign sequences on TMV virus-like particles. The second plant virus coat protein to be used was from the potyvirus Johnsongrass mosaic virus. Epitopes can be fused to either the N or C terminus of the protein without abolishing the ability of the protein to polymerize into virus-like particles when expressed in E. coil [46] . In one case, the assembled particles were shown to be capable of raising an appropriate immune response in mice.

The group of animal viruses most extensively investigated as potential presentation systems are the Picornaviridcee.

The capsids of these viruses comprise three major structural proteins: VP1, VP2 and VP3, arranged in a pseudo T=3 (P=3) lattice (Fig. lc) . Atomic resolution structures of a number of members of the group have been available since the 1980s and one member of the group, poliovirus, has been used extensively as a live attenuated vaccine for many years. These features have made picornaviruses particularly attractive candidates for structure-based insertion of foreign epitopes. In the case of poliovirus, foreign inserts have been expressed as replacements in the 13B-13C loop (Fig. 2) of VP1 and in a number of cases, the particles have been shown to be immunogenic [47, 48] . A high resolution structure has been obtained of a poliovirus type 1 chimaera expressing an antigenic site from poliovirus type 2 [49] . Examination of the structure revealed that there were changes not only in the conformation of the inserted loop but also in the neighbouring loops that were required to accommodate the new sequence. Thus, even when chimaeras are made between two closely related viruses, structural changes can occur when a sequence is placed in a heterologous context.

When human rhinovirus 14 (HRVI4) was investigated, it was found that the 13B-13C loop of.VP1 was not a suitable site of insertion; however, it was possible to isolate some viable chimaeras with inserts in the [3E-13F loop (Fig. 2) of VP2 [50] . To generate viable chimaeras displaying sequences from the V3 loop of HIV-1, a system of random systematic mutagenesis of the sequences flanking the conserved hexapeptide was employed [51"°] . Fifteen out of 25 chimaeras produced in this way were neutralized by one or more of a panel of four anti HIV-1 V3 loop antibodies. The differing responses observed indicate that the flanking residues have a major effect on the antigenicity of the presented sequence. Using a technique related to that used in phage display technology, it has proved possible to immunoselect particularly interesting rhinovirus-based chimaeras from a library. [52, 53] .

An enveloped RNA animal virus, Sindbis virus, has been developed as a potential source of live attenuated vaccine. Random insertional mutagenesis has been used to construct infectious Sindbis virus chimaeras expressing epitopes from Rift Valley fever virus [541. Suitable sites for insertion were found in the E2 and E3 glycoproteins.

In the case of insertions into E2, the antigens were expressed on the virion surface and could stimulate a partially protective immune response against Rift Valley fever virus.

Plant viruses have a number of potential advantages as polypeptide presentations systems: they, are often easy to grow in large (gram) quantities, purification is usually straightforward and many are extremely, stable. The Comoviridae are bipartite positive-strand icosahedral RNA viruses which are related to picornaviruses [55] (Fig. 1) .

Comparison of the structures of three members of the group showed that the [BB-]3C loop (Fig. 2 ) of the S protein was particularly variable and, like the corresponding loop in VP 1 of picornaviruses, was exposed on the virion surface loop has been shown to tolerate the insertion of foreign sequences [57,58"°]. Modified CPMV particles expressing a sequence from gp41 of HIV-1 have been shown to be able to elicit the production of anti-HIV neutralizing antibodies in three strains of mice [59,60], raising the prospect that such particles could form the basis of novel vaccines. In addition it has proved possible to obtain crystals of a chimaera expressing a 14 amino acid insert derived from VP1 of HRV14 which diffract to near atomic resolution [61] . Analysis of the diffraction data has allowed the structure of the insert to be determined (Fig. 3) .

Comparison of the structure of the loop expressed on CPMV with that which it adopts in its native environment confirms that presentation of sequence on the surface of a heterologous particle can have a profound effect on its structure.

Initial attempts to modify the coat protein of TMV concentrated on making extensions to the exposed C terminus. The results indicated that particle formation is inhibited if every copy of the protein is modified 

Recent work has highlighted the importance of structural constraints on the immunogenicity of peptides expressed on heterologous proteins [66°•]. Clearly the optimization of the sites of insertion to achieve the highest possible levels of antigenicity/immunogenicity of an expressed peptides is an important stage in the development of novel vaccines. The increasing availability of data regarding the structures of self-assembling proteins should assist the identification of such sites. Of possibly even greater significance is the prospect of being able to determine the structure of peptide when expressed on the surface of a heterologous particle and to correlate changes in immunogenicity with variations in the 3D structure of the insert. This could lead to 'fine tuning' of expression systems so that they fully realize their potential.

Papers of particular interest, published within the annual period of review, have been highlighted as: :208-213. The structure of a mutant of M13 harbouring a five amino acid insertion near the N terminus of the major coat protein (pVlll) was determined to 7 resolution by fibre diffraction. Examination of the structure reveals that the inserted sequence is presented in an extended conformation between two helices on the surface of the virions, mimicking the presentation of peptides in an MHC complex. This configuration may contribute to the high level of immunogenicity of peptides presented at the N terminus of gp8. This paper represents the first example of the determination of the structure of a peptide as presented on the surface of a heterologous particle. . Bacteriophage X is used as a presentation system, with peptides being added to the N terminus of the 11 kDa D protein present in 420 copies on the T=7 lattice on the virus head. To produce large protein D fusion display libraries without having to clone many fragments of DNA directly into X DNA, the authors use a site-specific recombination system in vivo to incorporate plasmids encoding D fusions into the phage genome. Assembly of the bacteriophage occurs normally and the peptide is accessible on the virus surface. The system will probably be most useful for the display of sequences obtained from cDNA libraries.

Burns show that regions from both the N and C terminus as well as certain internal regions of the protein can be deleted without abolishing particle assembly. The findings will be useful in the further development of Ty particles as antigen-presentation systems.

Valenzuela Chimaeric human parvovirus VP2 subunits containing epitopes from herpes simplex virus and mouse hepatitis virus were constructed and expressed in insect cells. The subunits spontaneously assembled into particles when the peptides were fused either to the N terminus of VP2 or in a loop predicted to be exposed on the basis of the 3D structure of canine parvovirus. Peptides inserted into the loop appeared to be more exposed than those at the N terminus, demonstrating the value of knowledge of the 3D structure of the carrier.

Sedlik C, Sarraseca J, Rueda P, Leclerc C, 

Design, construction and characterisation of poliovirus antigen chimaeras

The construction and characterization of poliovirus antigen chimaeras presenting defined regions of the human T lymphocyte marker CD4

Three-dimensional structure of a mouseadapted type 2/type 1 poliovirus chimera

Design and construction of rhinovirus chimeras incorporating immunogens from polio, influenza and human immunodeficiency viruses

Use of random systematic mutagenesis to generate viable human rhinovirus 14 chimaeras displaying human immunodeficiency virus type 1 V3 loop sequences

Using random mutagenesis of two residues on either side of the insert, a selection of linkers was produced that optimized replication of the chimaeras. The random systematic mutagenesis methodology described in this paper should be generally useful for the rapid insertion of foreign sequences

Libraries of human rhinovirus-based HIV vaccines generated using random systematic mutagenests

Chimeras from a human rhinovirus 14-human immunodeficiency virus type 1 (HIV-1) V3 loop seroprevalence library Induce neutrallsing responses against HIV-I

Picornaviruses and their relaUves in the plant kingdom

detailed discussion of structure-based insertional mutagenesis is presented with particular reference to the plant virus CPMV. The discussion should be helpful in the selection of potential sites of insertion for a variety of different systems

Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides

\~ arc indebted to all our colleagues who supplied preprints of work in press.

The icosahedral plant virus CPMV was used to express epitopes from several animal viruses as insertions into the capsid S protein. In two out of three cases, the modified particles grow as well as wild-type CPMV. Particles expressing an epitope from HRV14 are shown to be able to elicit the production of anti-HRV14 antibodies when injected into rabbits. This is the first example of the use of an infectious plant virus as an epitope-presentation system.

McLain L, Porta Epitopes from the malarial parasite were expressed on the surface of the helical plant virus TMV. Two sites for insertion were identified: the C terminus of the viral coat protein and an exposed loop. The resulting chimaeras grew well and the particles had the antigenic properties of the insert. In view of the ease of propagation and stability of TMV, this approach could prove an effective way of producing novel vaccines.

Fitchen J, Beachy RN, Hein MB: Plant virus expressing hybrid coat protein with added murlne epitope elicits autoantibody response. Vaccine 1995, 13:1051-1057.

Benito A, Mateu MG, Villaverde A: Improved mimicry of a foot • -and mouth disease virus antigenic site by a viral peptide displayed on 13-galactosldase surface. Biotechnology 1995, 13:801-804. The antigenicity of several recombinant E. coil ~-galactosidases displaying the immunodominant epitope from FMDV in different sites on the enzyme surface was studied. For each site, the inserted peptide showed a different specificity with a set of anti-FMDV monoclonal antibodies directed against the epitope. The results indicate that the precise way a sequence is presented on the surface of a macromolecule can dramatically influence its immunological properties.