Correction: In Vitro Evolution and Affinity-Maturation with Coliphage Qß Display.

[This corrects the article DOI: 10.1371/journal.pone.0113069.].

In vitro evolution and affinity-maturation with Coliphage qß display.

The Escherichia coli bacteriophage, Qß (Coliphage Qß), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qß RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qß display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qß resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qß icosahedral shell by electron microscopy. Evolution of Qß-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qß selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qß-G-H loop, validating the requirement of the tandem pair epitope. Qß-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

SplitCore: an exceptionally versatile viral nanoparticle for native whole protein display regardless of 3D structure.

Nanoparticles displaying native proteins are attractive for many applications, including vaccinology. Virus-based nanoparticles are easily tailored by genetic means, commonly by inserting heterologous sequences into surface-exposed loops. The strategy works well with short peptides but is incompatible with the structures of most native proteins, except those with closely juxtaposed termini. Here we overcome this constraint by splitting the capsid protein of hepatitis B virus, one of the most advanced and most immunogenic display platforms, inside the insertion loop (SplitCore). The split parts, coreN and coreC, efficiently form capsid-like particles (CLPs) in E. coli and so do numerous fusions to coreN and/or coreC of differently structured proteins, including human disease related antigens of >300 amino acids in length. These CLPs induced high-titer antibodies, including neutralizing ones, in mice. The concept was easily expanded to triple-layer CLPs carrying reporter plus targeting domains, and should be applicable to protein-based nanoparticle design in general.

Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins.

Virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. Presentation on the icosahedral hepatitis B virus capsid (HBcAg) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 B cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. Even some complete proteins were successfully inserted but others, e.g. the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi, impaired formation of capsid-like particles (CLPs). This difference can be rationalized by the requirement for the termini of the insert to fit into the predetermined geometry of the two acceptor sites in the carrier. We reasoned that cleavage of one of the two bonds connecting insert and carrier should relieve these constraints, provided the cleaved protein fragments remain competent to support the particle structure. Indeed, HBcAg CLPs containing a recognition site for tobacco etch virus (TEV) protease in the c/e1 loop remained intact after cleavage, as did CLPs carrying a 65-residue peptide insertion. Most importantly, in situ cleavage of a core-OspA fusion protein by coexpressed TEV protease strongly enhanced CLP formation compared with the uncleaved protein. These data attest to the high structural stability of the HBcAg CLP and they significantly widen its applicability as a carrier for heterologous proteins. This approach should be adaptable to any protein-based particle with surface-exposed yet sequence-internal loops.

Development of hepatitis B virus capsids into a whole-chain protein antigen display platform: new particulate Lyme disease vaccines.

The immunogenicity of peptides and small protein fragments can be considerably enhanced by their presentation on particulate carriers such as capsid-like particles (CLPs) from hepatitis B virus (HBV). HBV CLPs are icosahedral nanoparticles formed by 90 or 120 core protein dimers. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop on the HBV capsid protein, are especially immunogenic. Here we investigated whether the HBV core protein can be exploited as a vaccine carrier for whole-chain protein antigens, using two clinically relevant proteins derived from a bacterial human pathogen, the Lyme disease agent Borrelia burgdorferi. For this purpose we analyzed CLP formation by core fusions with the entire 255-amino-acid ectodomain of outer surface lipoprotein A (OspA), and with two distinct, 189 amino acid long variants of the dimeric OspC (OspC(a), OspC(b)) of B. burgdorferi. OspA appropriately inserted into the HBV core protein yielded a multimerization-competent fusion protein, termed coreOspA. Although only partially assembling into regular CLPs, coreOspA induced antibodies to OspA, including the Ig isotype profile and specificity for the protective epitope "LA-2", with an efficiency similar to that of recombinant lipidated OspA, the first generation vaccine against Lyme disease. Moreover, coreOspA actively and passively protected mice against subsequent challenge with B. burgdorferi. Fusions with the two OspC variants were found to efficiently form regular CLPs, most probably by OspC dimerization across different core protein dimers. In mice, both coreOspC preparations induced high-titered antibody responses to the homologous but also to the heterologous OspC variant, which conferred protection against challenge with B. burgdorferi. The data demonstrate the principal applicability of HBV CLPs to act as potent immunomodulator even for structurally complex full-length polypeptide chains, and thus open new avenues for novel vaccine designs.

Hepatitis B virus capsid-like particles can display the complete, dimeric outer surface protein C and stimulate production of protective antibody responses against Borrelia burgdorferi infection.

Hepatitis B virus capsid-like particles (CLPs), icosahedral assemblies formed by 90 or 120 core protein dimers, hold promise as immune-enhancing vaccine carriers for heterologous antigens. Insertions into the immunodominant c/e1 B cell epitope, a surface-exposed loop, are especially immunogenic. However, display of whole proteins, desirable to induce multispecific and possibly neutralizing antibody responses, can be restrained by an unsuitable structure of the foreign protein and by its propensity to undergo homomeric interactions. Here we analyzed CLP formation by core fusions with two distinct variants of the dimeric outer surface lipoprotein C (OspC) of the Lyme disease agent Borrelia burgdorferi. Although the topology of the termini in the OspC dimer does not match that of the insertion sites in the carrier dimer, both fusions, coreOspCa and coreOspCb, efficiently formed stable CLPs. Electron cryomicroscopy clearly revealed the surface disposition of the OspC domains, possibly with OspC dimerization occurring across different core protein dimers. In mice, both CLP preparations induced high-titered antibody responses against the homologous OspC variant, but with substantial cross-reactivity against the other variant. Importantly, both conferred protection to mice challenged with B. burgdorferi. These data show the principal applicability of hepatitis B virus CLPs for the display of dimeric proteins, demonstrate the presence in OspC of hitherto uncharacterized epitopes, and suggest that OspC, despite its genetic variability, may be a valid vaccine candidate.

A fusion product of the complete Borrelia burgdorferi outer surface protein A (OspA) and the hepatitis B virus capsid protein is highly immunogenic and induces protective immunity similar to that seen with an effective lipidated OspA vaccine formula.

The immunogenicity of peptides and protein fragments can be considerably enhanced by their presentation on particulate carriers such as capsid-like particles (CLP) from hepatitis B virus (HBV). Here we tested the suitability of the HBV capsid protein as a carrier for a relevant full-length pathogen-derived protein antigen. The entire 255-amino acid ectodomain of the outer surface protein A (OspA) from Borrelia burgdorferi, the causative agent of Lyme disease, was inserted into the major B cell epitope of the HBV capsid, yielding a multimerization-competent fusion protein, termed coreOspA. CoreOspA, consisting only in part of regular CLP, induced antibodies to OspA, including the Ig isotype profile and specificity for the protective epitope LA-2, with an efficiency similar to that of recombinant lipidated OspA, the first generation vaccine against Lyme disease. Moreover, coreOspA actively and passively protected mice against subsequent challenge with B. burgdorferi. The data demonstrate the capacity of the HBV capsid protein to act as a potent immunomodulator even for full-length and structurally complex polypeptide chains and thus opens new avenues for novel vaccine designs.