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Targeting the unwilling antigen

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By:  John P. O’Rourke and Pamela R. Hall
Department of Pharmaceutical Sciences
University of New Mexico

Peptides play many crucial roles in biology, including infectious disease, but are difficult to target by vaccines because they are too small to be immunogenic. Vaccine-mediated induction of a neutralizing antibody response to such peptides generally requires ligation to a much larger, highly immunogenic protein. Anti-peptide vaccine design is made more difficult when the peptide is susceptible to conformational changes which result in loss of its native function, which would negatively impact the induction of neutralizing antibodies.  In a recent report in PLoS One (1), we described a vaccine discovery platform based on virus-like particles (VLPs) which we used to overcome these challenges and provide protection against skin infection caused by the bacterial pathogen Staphylococcus aureus.

Virus-like particles (VLPs) are derived from viruses whose coats consist of one or more proteins arranged geometrically into dense, repetitive arrays. Because these structures are largely unique to microbial antigens, they are highly immunogenic in mammals and actually form the basis of FDA approved vaccines for the prevention of human papilloma virus (HPV) infection (2). Due to their innate immunogenicity, VLPs can dramatically enhance the adaptive immune response to peptides presented on the VLP surface.

In the work discussed in our paper we used a VLP technology first described by Peabody and Chackerian (3, 4). Like other VLPs, ours confers high immunogenicity to any epitope it displays, but it has the added advantage of allowing affinity-selection of epitope mimics in a process akin to phage display. Based on the RNA bacteriophage MS2, our technology depends on a version of MS2 coat protein engineered to tolerate the insertion of diverse peptides. This means that high-complexity random-sequence peptide libraries can be presented on VLPs, and subjected to biopanning on any arbitrary receptor molecule (e.g. a neutralizing antibody). Then, because the VLP also encapsidates its own mRNA, the affinity-selected sequences can be recovered by reverse transcription and PCR (Figure 1). Like conventional phage display, MS2 VLP display allows for identification of mimics of native epitopes, but because it reliably displays foreign peptides at high density (and therefore at high immunogenicity) it can be used directly as a potent vaccine immunogen. This permits preservation in the vaccine immunogen of the structural context present during affinity optimization of the mimotope for its target. The ultimate aim is to select accurate immunogenic epitope mimics that can elicit antibodies with neutralizing activities similar to that of the selecting antibody. This is the approach we used to identify mimotopes of a cyclized autoinducing peptide (AIP) involved in S. aureus pathogenesis.

Hall 1

S. aureus uses a bacterial communication system called quorum-sensing (QS) via the accessory gene regulator (agr) virulence operon to coordinate changes in gene expression required for invasive skin infection. This communication is mediated by agr-encoded AIPs, ranging from seven to nine amino acids in length, which are secreted by S. aureus and signal for agr activation and tissue invasion by binding to cell surface receptors on the same or neighboring S. aureus bacteria. Therefore, blocking AIP-mediated agr-activation can limit the pathogenesis of invasive skin infection.  However, AIPs are innately non-immunogenic because of their size and, due to their cyclic nature, are subject to inactivating conformational changes, making them extremely challenging vaccine targets. In a previous effort to overcome this challenge, Park et al. (5) identified an anti-AIP antibody induced in mice in response to a carrier protein conjugated, synthetic AIP derivative.  Furthermore, passive transfer of the resulting monoclonal antibody, AP4-24H11, protected mice against S. aureus skin infection. This suggested that AP4-24H11 could be used to screen for a stable, peptide mimotope of AIP4 for utilization as part of a VLP-based active vaccine against agr-mediated S. aureus infection.

To demonstrate the feasibility of using our VLP-based random sequence peptide library to produce active, mimotope vaccines, we panned the library against AP4-24H11 (5) and assessed the protection afforded vaccinated mice against agr-mediated S. aureus skin infection (1). In early screening, we identified two vaccine candidates (identified as peptide 2- and peptide 4-VLPs) which showed promise in limiting agr-mediated ulceration and dermonecrosis during S. aureus skin infection. Furthermore, mice vaccinated with a mixture of these VLPs (peptide 2/4-VLPs), showed significantly reduced dermonecrosis and pro-inflammatory cytokine production at the site of infection compared with mice vaccinated with saline or VLP-control (Figure 2). Not surprisingly, compared to controls, mice vaccinated with peptide 2/4-VLPs also showed reduced production of an important agr-regulated virulence factor, alpha-hemolysin, at the site of infection, as would be expected with peptide 2/4-VLP vaccination-induced production of an AIP neutralizing antibody response. Importantly, the amino acid sequences of peptides 2 and 4 (IGMSQSK and SGIMPH, respectively) differed from that of the target AIP (YSTCYFIM), providing additional support for the role of these VLP-presented peptides as conformational/antigenic mimics of the S. aureus QS peptides.

Hall 2

To date, there is no approved vaccine to prevent S. aureus infection or to limit its pathogenesis. As S. aureus continues to acquire antibiotic resistance, multiple approaches to combat this pathogen will likely be required, including the use of therapeutic monoclonal antibodies as well as prophylactic vaccines. To our knowledge, ours is the first report of an active vaccine targeting the secreted AIPs of the S. aureus QS system. Additionally, this work provides in vivo evidence of the broader potential for the use of VLP-based random sequence peptide libraries for identification of efficacious antigenic mimotopes for preventing infectious disease.

The studies described above were conducted by the following investigators and first reported in (1):

John P. O’Rourke
Seth M. Daly
Kathleen D. Triplett
David Peabody
Bryce Chackerian
Pamela R. Hall

 

1.              O’Rourke JP, Daly SM, Triplett KD, Peabody D, Chackerian B, Hall PR. 2014. Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway. PloS One. 9:e111198.

2.              Schiller JT, Lowy DR. 2014. Raising Expectations For Subunit Vaccine. J Infect Dis.

3.              Chackerian B, Jdo CC, Peabody J, Peabody DS. 2011. Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles. Journal of Molecular Biology. 409:225-237.

4.              Peabody DS, Manifold-Wheeler B, Medford A, Jordan SK, Caldeira JdC, Chackerian B. 2008. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2. Journal of Molecular Biology. 380:252-263.

5.              Park J, Jagasia R, Kaufmann GF, Mathison JC, Ruiz DI, Moss JA, Meijler MM, Ulevitch RJ, Janda KD. 2007. Infection control by antibody disruption of bacterial quorum sensing signaling. Chem Biol. 14:1119-1127.

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