Versatile virus-like particle carrier for epitope based vaccines

Abstract

Background: Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.

Methodology/principal findings: Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.

Conclusions/significance: AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

Figure 1. Schematic representation of the four AP205 coat protein fusion constructs.

Genes encoding an epitope to be introduced at the N-terminus of AP205 using a short spacer (in plasmid pAP378) or a long spacer (in plasmid pAP382) are cloned as NcoI-Kpn2I cassettes, while for introduction of epitopes to the C-terminus of AP205 coat protein using a short spacer (in plasmid pAP409) or a long spacer (in plasmid pAP405) a Kpn2I- Mph1103I cassette is used. AP205 cp stands for AP205 coat protein sequence.

Figure 2. Electron micrographs of negatively stained AP205-Ang II VLPs.

AP205 VLPs displaying the Ang II peptide fused to the C-terminus (AP441 – short linker, AP442 – long linker), or the N-terminus (AP446 – short linker, AP447 – long linker) of AP205 coat protein.

Figure 3. AP205-Ang II VLPs analyzed by dodecyl sulphate PAGE and Western blot.

After purification by gel filtration, AP205 VLPs carrying Ang II peptide fused to the C-terminus (AP441 and AP442) or N-terminus (AP446 and AP447) of AP205 coat protein were heated at 95°C in sample buffer for 5 minutes prior to loading. (a) Coomassie-stained LDS-PAGE of corresponding chimeric proteins; (b) Western blot with an AP205 coat protein-specific rabbit serum; (c) Western blot with an Ang II-specific mouse serum. The migration of all four AP205 coat proteins carrying Ang II is retarded in comparison to wt AP205 coat protein (130 aa). Accordingly, migration of coat protein fused to AP205 coat protein using a long spacer (AP442 and AP447) is slower than the migration of coat proteins containing a short spacer (AP441 and AP446).

Figure 4. Display of Ang II on AP205 VLP assessed by inhibition ELISA.

Ang II was conjugated to RNAse, and coated on ELISA plates. The anti-Ang II serum was pre-incubated with serial dilutions of the 4 VLPs displaying Ang II or wt AP205, and subsequently added to the wells coated with RNAse-Ang II. Signals shown on the Figure are normalized and are given as % binding. The VLP concentration [µg/ml] was log transformed. (a) VLPs displaying Ang II at the C-terminus of AP205 coat protein using a short (AP441) or long spacer (AP442) or wt AP205 VLP (AP205). (b) VLPs displaying Ang II at the N-terminus of AP205 coat protein using a short (AP446) or long spacer (AP447) or wt AP205 VLP (AP205).

Figure 5. Immunogenicity of D2 peptide displayed on AP205 VLPs.

AP205 VLPs with the D2 peptide fused to the C-terminus (AP418, short linker, and AP420, long linker) and to the N-terminus (AP421, short linker, and AP422, long linker) were injected s.c. at a dose of 25 µg twice, on day 0 and 14. Sera were collected on day 21, and IgG titre measured by ELISA. Pre-immune titre was too low to be determined, and was arbitrarily attributed the value of 1∶100, the lowest dilution of serum used in the assay.

Figure 6. Inhibition of GnRH function in mice immunized with AP205-GnRH.

(a) Testis weight in male C57BL/6 mice immunized with 50 µg AP205 GnRH, or untreated, on day 0 and 21. Mice were sacrificed on day 70. There was a 38% decrease in testis weight in the AP205-GnRH group compared to the untreated control group (p = 0.003, t-test). (b) Anti-GnRH IgG antibody titre measured by ELISA. The mice were immunized on day 0, and 21 with 50 µg AP205-GnRH, and anti-GnRH ELISA titres measured on day 21, 28, 45, and 70. (c) Testosterone level measurements. Testosterone levels were measured by ELISA in sera collected on day 0, 21, 28, 45 and 70. Error bars are the standard error of the mean. The median of the testosterone level, averaged between day 28 and 70, was 81% lower in the AP205-GnRH treated animals when compared to untreated animals (p = 0.008, Mann-Whitney test).

Figure 7. Protection of mice by immunization with M2-AP205 against a lethal influenza challenge.

Mice were immunized on day 0 and 18 with M2-AP205 or AP205, and challenged with 4× the lethal dose of influenza Pr8 (H1N1) on day 31. (a) Anti-M2 and anti-AP205 IgG titre on day 18 and 25 in mice immunized with M2-AP205. (b) Anti-AP205 IgG titer on day 18 and 25 in mice immunized with AP205 only. (c) Survival curve. 4 control animals had to be sacrificed on day 10 due to excessive weight loss and temperature drop. Morbidity was assessed by measurement of the animal weights (d) and temperature drop (e).