PilVax - a novel peptide delivery platform for the development of mucosal vaccines

Abstract

Peptide vaccines are an attractive strategy to engineer the induction of highly targeted immune responses and avoid potentially allergenic and/or reactogenic protein regions. However, peptides by themselves are often unstable and poorly immunogenic, necessitating the need for an adjuvant and a specialised delivery system. We have developed a novel peptide delivery platform (PilVax) that allows the presentation of a stabilised and highly amplified peptide as part of the group A streptococcus serotype M1 pilus structure (PilM1) on the surface of the non-pathogenic bacterium Lactococcus lactis. To show proof of concept, we have successfully inserted the model peptide Ova_324-339 into 3 different loop regions of the backbone protein Spy0128, which resulted in the assembly of the pilus containing large numbers of peptide on the surface of L. lactis. Intranasal immunisation of mice with L. lactis PilM1-Ova generated measurable Ova-specific systemic and mucosal responses (IgA and IgG). Furthermore, we show that multiple peptides can be inserted into the PilVax platform and that peptides can also be incorporated into structurally similar, but antigenically different pilus structures. PilVax may be useful as a cost-effective platform for the development of peptide vaccines against a variety of important human pathogens.

Figure 1

Protein structure of the backbone pilin Spy0128 with selected peptide insertion sites. The protein structure was generated with the PDB Swiss Viewer version 4.1.0. using coordinates downloaded from the Brookhaven database (3B2M). The selected peptide insertion regions are shown in red. (A) Ribbon diagram structure. (B) Accessible surface structure.

Figure 2

Expression of the PilM1 structure on the surface of L. lactis with the model peptide Ova_324–339 inserted at selected sites within the Spy0128 backbone pilus protein. (A) Western blot analysis of L. lactis cell wall extracts (CWE) with antiserum specific for M1_Spy0128 (pilus backbone protein). The high molecular band patterns are indicative of pilus assembly. For L. lactis strains that showed pilus expression after peptide insertion, flow cytometry was used to compare the expression levels of M1_Spy0128 (B) and Ova_324–339 peptide (C). Error bars show the standard deviation from 3 independent experiments. **p<0.005; ***p<0.0005; one-way ANOVA followed by a Holm-Sidak test.

Figure 3

Intranasal immunisation of mice with L. lactis that expresses the M1 pilus with the model peptide Ova_324–339 inserted at the βE-βF loop region (PilM1-Ova) induces IgG responses. Groups of Balb/c mice (n = 5) were immunised intranasally with 1 × 10⁸ CFU live recombinant L. lactis PilM1-Ova. The data from two independent experiments were combined. L. lactis without inserted peptide (PilM1) or mixed with synthetic Ova_324–339 (PilM1 + Ova) were used as controls. Synthetic Ova_324–339 peptide alone (Ova) or mixed with Cholera Toxin B subunit adjuvant (Ova + CT-B) were used as additional controls. Total serum IgG titres against (A) immobilised recombinant Spy0128 or (B) commercial ovalbumin were measured by ELISA. **p < 0.005; ***p < 0.0005; ns = not significant; One-way ANOVA with Dunn’s multiple comparisons test. (C) Ova-specific responses by IgG subclasses. ***p < 0.0005; Mann-Whitney test.

Figure 4

Intranasal immunisation of mice with L. lactis PilM1-Ova induces systemic and mucosal IgA responses. Groups of Balb/c mice (n = 5) were immunised intranasally with 1 × 10⁸ CFU live recombinant L. lactis PilM1-Ova. The data from two independent experiments were combined. L. lactis without inserted peptide (PilM1) or mixed with synthetic Ova_324–339 (PilM1 + Ova) were used as controls. Synthetic Ova_324–339 peptide alone (Ova) or mixed with Cholera Toxin B subunit adjuvant (Ova + CT-B) were used as additional controls. (A) Specific IgA responses against recombinant Spy0128 were measured by ELISA. ELISAs were also performed to measure specific anti-ovalbumin IgA responses in serum (B), BAL fluid (C) and saliva (D). *p < 0.05; **p < 0.005; ***p < 0.0005; ns = not significant; One-way ANOVA with Dunn’s multiple comparisons test.

Figure 5

More than one peptide can be inserted in frame within a Spy0128 loop region. Cloning of a XhoI-SalI fragment into a XhoI site generates one non-cleavable hybrid site and one intact XhoI site downstream of the inserted DNA region. This allows the addition of consecutive DNA fragments (peptide sequences). A second peptide (J14) was added to the Ova_324–339 peptide within the PilM1 βE-βF loop region (PilM1-Ova-J14). (A) Western blot analysis of L. lactis cell wall extracts (CWE) with antiserum specific for M1_Spy0128. The high molecular band patterns are indicative of pilus assembly. (B) Flow cytometry analysis shows expression of M1_Spy0128 on the surface of L. lactis, but with notably reduced expression after insertion of peptides into the βE-βF loop region. Error bars show the standard deviation from 3 independent experiments. *p<0.05; ***p<0.0005; one-way ANOVA followed by a Holm-Sidak test.

Figure 6

Peptides can be inserted into loop regions from structurally related backbone pilus proteins. (A) The structures of the backbone pilus proteins found in serotype M18/T18 and serotype M28/T28 were modelled onto the Spy0128 (serotype M1/T1) crystal structure (3B2M) using the Swiss PDB modeling server. Despite low amino acid sequence identities, the models predicted conserved structures, which allowed the identification of loop regions equivalent to those analysed in Spy0128 (M1/T1). The model peptide Ova_324–339 was inserted into the βE-βF loop region of M18_Spy0128 using the same cloning strategy as for M1_Spy0128. (B) Western blot analysis of L. lactis cell wall extracts (CWE) with antiserum specific for M18_Spy0128. (C) Flow cytometry analysis with anti-M18_Spy0128 antiserum shows expression of M18_Spy0128 on the surface of L. lactis, but with notably reduced expression after insertion of peptides into the βE-βF loop region. Error bars show the standard deviation from 3 independent experiments. ***p<0.0005; one-way ANOVA followed by a Holm-Sidak test.