Saccharomyces cerevisiae-derived HBsAg preparations differ in their attachment to monocytes, immune-suppressive potential, and T-cell immunogenicity

Expression of the hepatitis B virus S protein results in the formation of a lipoprotein particle, the hepatitis B surface antigen (HBsAg). Such particles, produced in Saccharomyces cerevisiae, bind to the cell surface of monocytes through interaction with the lipopolysaccharide binding protein and the lipopolysaccharide receptor, CD14. This attachment is suggested to depend on the presence of charged phospholipids in the particles. In addition, such particles interfere with the lipopolysaccharide and interleukin-2-induced activation of monocytes. In the present study, it is reported that of three Saccharomyces cerevisiae-derived HBsAg preparations, two have a reduced capacity to bind to monocytes. A correlation with a reduced potential to inhibit the lipopolysaccharide-induced activation of monocytes and an increased potential to stimulate HBsAg-specific T-cell proliferation is observed. Surprisingly, differences in phospholipid content that might explain these observations, were not detected. (C) 2003 Wiley-Liss, In

Production, characterization and in vitro testing of HBcAg-specific VHH intrabodies

Hepatitis B virus (HBV) infections represent a global health problem, since these account for 350 million chronic infections worldwide that result in 500 000-700 000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes

Enhancement of Polymeric Immunoglobulin Receptor Transcytosis by Biparatopic VHH

The polymeric immunoglobulin receptor (pIgR) ensures the transport of dimeric immunoglobulin A (dIgA) and pentameric immunoglobulin M (pIgM) across epithelia to the mucosal layer of for example the intestines and the lungs via transcytosis. Per day the human pIgR mediates the excretion of 2 to 5 grams of dIgA into the mucosa of luminal organs. This system could prove useful for therapies aiming at excretion of compounds into the mucosa. Here we investigated the use of the variable domain of camelid derived heavy chain only antibodies, also known as VHHs or Nanobodies®, targeting the human pIgR, as a transport system across epithelial cells. We show that VHHs directed against the human pIgR are able to bind the receptor with high affinity (∼1 nM) and that they compete with the natural ligand, dIgA. In a transcytosis assay both native and phage-bound VHH were only able to get across polarized MDCK cells that express the human pIgR gene in a basolateral to apical fashion. Indicating that the VHHs are able to translocate across epithelia and to take along large particles of cargo. Furthermore, by making multivalent VHHs we were able to enhance the transport of the compounds both in a MDCK-hpIgR and Caco-2 cell system, probably by inducing receptor clustering. These results show that VHHs can be used as a carrier system to exploit the human pIgR transcytotic system and that multivalent compounds are able to significantly enhance the transport across epithelial monolayers

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity

Hepatitis B Virus Lacks Immune Activating Capacity, but Actively Inhibits Plasmacytoid Dendritic Cell Function

Chronic hepatitis B virus (HBV) infection is caused by inadequate anti-viral immunity. Activation of plasmacytoid dendritic cells (pDC) leading to IFNα production is important for effective anti-viral immunity. Hepatitis B virus (HBV) infection lacks IFNα induction in animal models and patients and chronic HBV patients display impaired IFNα production by pDC. Therefore, HBV and HBV-derived proteins were examined for their effect on human pDC in vitro. In addition, the in vitro findings were compared to the function of pDC derived from chronic HBV patients ex vivo. In contrast to other viruses, HBV did not activate pDC. Moreover, HBV and HBsAg abrogated CpG-A/TLR9-induced, but not Loxoribine/TLR7-induced, mTOR-mediated S6 phosphorylation, subsequent IRF7 phosphorylation and IFNα gene transcription. HBV/HBsAg also diminished upregulation of co-stimulatory molecules, production of TNFα, IP-10 and IL-6 and pDC-induced NK cell function, whereas TLR7-induced pDC function was hardly affected. In line, HBsAg preferentially bound to TLR9-triggered pDC demonstrating that once pDC are able to bind HBV/HBsAg, the virus exerts its immune regulatory effect. HBV not only directly interfered with pDC function, but also indirectly by interfering with monocyte-pDC interaction. Also HBeAg diminished pDC function to a certain extent, but via another unknown mechanism. Interestingly, patients with HBeAg-positive chronic hepatitis B displayed impaired CpG-induced IFNα production by pDC without significant alterations in Loxoribine-induced pDC function compared to HBeAg-negative patients and healthy controls. The lack of activation and the active inhibition of pDC by HBV may both contribute to HBV persistence. The finding that the interaction between pDC and HBV may change upon activation may aid in the identification of a scavenging receptor supporting immunosuppressive effects of HBV and also in the design of novel treatment strategies for chronic HBV