Plant virus particles carrying tumour antigen activate TLR7 and induce high levels of protective antibody

Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-? by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant

Porphyromonas gingivalis FimA Type II - PVXCP Fusion DNA Vaccine Expression in Mammalian Cells

Abstract Objective: It has been shown that Porphyromonas gingivalis (P. gingivalis) FimA is able to induce antibody production and protection against bone loss in animal model. P. gingivalis FimA is therefore the candidate antigen in periodontitis vaccine production. In this study, DNA vaccine was produced by fusion sequences of FimA type II, the most prevalent type associated with periodontitis, and potato virus X coat protein (PVXCP), the immune-enhancing molecule. The ability of this DNA vaccine to be expressed in mammalian cells was investigated.Methods: The DNA vaccine was constructed in a fusion form of FimA and PVXCP (FimA -PVXCP) DNA vaccine, in which the mammalian expression plasmid pcDNA3 was used as a backbone plasmid. FimA type II gene was amplified from genomic DNA of P.gingivalis FimA type II by PCR. The FimA PCR product was inserted into predigested pcDNA3 containing PVXCP sequence; and consequently FimA was fused to the N-terminal side of PVXCP. The resulting plasmid was transfected into human embryonic kidney (HEK293) cells. FimA-PVXCP RNA expression in transfected cells was detected by RT-PCR. The fusion protein inside the cells and the protein secreted into medium were analyzed by Western Blot analysis using anti-PVXCP antibody. Results: RT-PCR of RNA extracted from pcDNA3. FimA-PVXCP transfected HEK293 cells showed the expected band size of 1.9 kb of FimA-PVXCP sequence. FimA-PVXCP protein was detected by Western blot analysis both in cell lysate of the transfected cells and in the medium. However, the secreted protein appeared to be larger than the protein remained inside the cells. Conclusion: FimA-PVXCP DNA vaccine was able to be expressed in HEK293 cells as confirmed by RT-PCR and Western blot analysis. Keywords : P.gingivalis vaccine, Periodontitis vaccine, DNA vaccine, Protein expression in mammalian cell

Plant viral vaccine delivery as a platform to induce cellular immunity against cancer antigens

Immunotherapy with immunostimulatory antibodies has become a breakthrough therapeutic modality for solid tumours. However, only up to 50% of patients benefit and hence there is a renewed interest to vaccination to direct the immune attack against tumour antigens to widen the range of patients who benefit. Modern vaccine platforms such as viral-like particles have already demonstrated success in cancer prevention and the challenge is now to harness these approaches in patients with established malignancies. We have focused on a novel vaccine platform utilising a plant viral particle (PVP), which has been previously demonstrated that this single strand RNA (ssRNA) containing vaccine activated the immune mechanisms closely resembling the natural antiviral defence. Based on our previous mouse work, with sensing of viral RNA by plasmacytoid dendritic cells (pDCs), IFN-α was generated to activate conventional DCs (cDCs), and then prime Th1 immunity. We also confirmed that PVP with ssRNA packaged inside was capable to activate human DCs. The induction of anti-tumour antibody was another outcome. Subsequently, we explored this vaccine platform for induction of cellular responses against defined cancer targets. Initially using PVP loaded with a single model peptide, the induction of a rapid CD8 response of high magnitude was represented. In order to extend the application of this platform, the clinically relevant cancer testis antigens MAGEC1 and PASD1 were set as vaccine targets. In HLA-A2 transgenic (HHD) mouse model, PVP simultaneously targeting MAGEC1 and PASD1 induced CTLs, which were able to lyse human cancer cells expressing these antigens. We were accordingly keen to expend applicability to patient cohorts beyond HLA-A2+ patients, PVP loaded with whole protein antigens was subsequently tested. The vaccine induced CD8 and CD4 T-cell responses against epitopes delivered through the whole antigen were both detected. Of these, this flexible and economical platform can be adapted easily to deliver any antigenic peptides including multiple epitopes as well as whole proteins and may provide a universal platform for vaccination against cancer

Linked CD4 T cell help: broadening immune attack against cancer by vaccination

In the last decade, immunotherapy with monoclonal antibodies targeting immunological check points has become a breakthrough therapeutic modality for solid cancers. However, only up to 50 % of patients benefit from this powerful approach. For others vaccination might provide a plausible addition or alternative. For induction of effective anticancer immunity CD4+ T cell help is required, which is often difficult to induce to self cancer targets because of tolerogenic mechanisms. Our approach for cancer vaccines has been to incorporate into the vaccine design sequences able to activate foreign T cell help, through genetically linking cancer targets to microbial sequences (King et al. in Nat Med 4(11):1281–1286, 1998; Savelyeva et al. in Nat Biotechnol 19(8):760–764, 2001). This harnesses the non-tolerized CD4 T cell repertoire available in patients to help induction of effective immunity against fused cancer antigens. Multiple immune effector mechanisms including antibody, CD8+ T cells as well as CD4 effector T cells can be activated using this strategy. Delivery via DNA vaccines has already indicated clinical efficacy. The same principle of linked T cell help has now been transferred to other novel vaccine modalities to further potentiate immunity against cancer targets

Induction of protective anti-Id antibody by Id conjugate vaccines.

<p><b>(A)</b> and <b>(B)</b>. Mice were vaccinated with Id-SA, Id-PVP, PVP alone or Id-CP DNA at day 0 (priming) and again at day 21 (boosting). <b>(A)</b>. Total anti-Id IgG antibody was measured three weeks after priming (primary response) and two weeks after boosting (secondary response). U/ml represents antibody levels compared to an internal standard. Each symbol represents an individual mouse, with horizontal lines representing medians. The data has been combined from two representative, independent experiments, in which all groups were included. Three stars indicates significance of p<0.001, using the Mann-Whitney statistical test. <b>(B)</b>. Protection from lymphoma challenge. Two weeks after boosting mice were challenged i.v. with 5x10⁴ BCL1 lymphoma cells. Mice were culled upon reaching a predetermined level of splenomegaly. One star indicates significance of p = 0.02, using the log rank (Mantel-Cox) statistical test. A representative graph from three independent experiments is shown. <b>(C)</b>. The Id-PVP vaccine was compared with Id-KLH plus or minus the adjuvant alum, following the same time course as in <b>(A)</b>. Total anti-Id IgG antibody levels are shown from two representative, independent experiments. Each symbol represents an individual mouse with bars representing median values. Two stars indicate 0.001(A-C). 4–10 mice were used per group per experiment.</p

Antibody isotype analysis.

<p><b>(A)</b>. The levels of anti-Id IgG antibody isotypes induced by Id-PVP and Id-KLH vaccines with or without alum were compared two weeks after booster vaccinations. U/ml represents the level of serum antibody compared to an internal standard. The data are combined from two independent experiments. Each symbol represents the response from an individual mouse, bars are medians. Mann-Whitney statistics were used for analysis, with one star indicating 0.01(B). Anti-Id antibody isotype titres were measured two weeks after booster injection. The vaccines included Id-PVP and Id-KLH both with and without alum, the Id-CP DNA vaccine and Id-SA. Mean titres from three mice per group from one representative experiment are shown, with error bars showing standard deviation.</p