Single low-dose un-adjuvanted HBsAg nanoparticle vaccine elicits robust, durable immunity.

Chitosan nanoparticles were evaluated as a vaccine delivery system for hepatitis B surface antigen (HBsAg) in the absence of adjuvant. Nano-encapsulated HBsAg (HBsAg chitosan-NP) was endocytosed more rapidly and efficiently by dendritic cells compared to soluble HBsAg. FRET analysis demonstrated that intact nanoparticles were taken up by DCs. To determine the immunogenicity of adjuvant-free nano-encapsulated HBsAg, mice were immunized with a single dose of non-encapsulated HBsAg, HBsAg chitosan-NP, or HBsAg alum. Mice immunized with adjuvant-free nanoparticle elicited anti-HBs antibodies at significantly higher titers compared to mice immunized with HBsAg alum. Elevated numbers of BAFF-R(+) B cells and CD138+ plasma cells account for the heightened anti-HBs response in nanoparticle immunized mice. Increases in Tfh cells provide a mechanism for the accumulation of anti-HBs secreting cells. Thus, chitosan nanoparticle vaccines represent a promising un-adjuvanted platform to generate robust and durable immunity to HBsAg and other subunit antigens following a single low-dose administration. FROM THE CLINICAL EDITOR: In this study, chitosan nanoparticle vaccines are demonstrated as a promising un-adjuvanted platform to generate robust and durable immunity to HBsAg and other subunit antigens following a single low-dose administration in a murine model. The authors also demonstrated superior antibody response induction compared with non-encapsulated HBs antigen and HBsAg aluminum.

Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling.

The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI) to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/-) DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.

Novel nanoparticles for the delivery of recombinant hepatitis B vaccine.

We describe the use of methoxypolyethylene glycol-poly(lactide-co-glycolide) nanoparticles as a delivery system for recombinant hepatitis B surface antigen (HBsAg). Evaluation of the stability and release kinetics of nanoencapsulated HBsAg in vitro in serum revealed an initial burst effect and a subsequent slower release of the antigen. Importantly the antigenicity was not destroyed by the encapsulation process, because upon release it was able to react with an anti-HBs antibody. Bone marrow-derived dendritic cells showed efficient uptake of the nanoparticle vaccine as visualized by confocal imaging. To determine whether nano-encapsulated HBsAg was capable of eliciting an immune response in the absence of an adjuvant, mice were immunized with the nanoparticle vaccine or with nonencapsulated recombinant HBsAg. In mice immunized with the nanoparticle vaccine, anti-HBs antibodies were detected at significantly earlier time points than in mice immunized with the nonencapsulated recombinant HBsAg.

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.