Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Adipose-derived stem cells cultured in autologous serum maintain the characteristics of mesenchymal stem cells Human adipose tissue is known to be an attractive and readily available source of mesenchymal stem cells (MSC), which are becoming increasingly popular for application in regenerative medicine. Most of the protocols currently used for in vitro expansion of MSC include fetal bovine serum (FBS) supplementation. When MSC are cultured in such a way for clinical applications this rises concerns about immunogenicity of FBS proteins. A possible solution to this problem is the use of autologous serum (AS) instead of FBS. In this study we investigated whether adipose-derived stem cells (ADSC), cultivated in medium containing AS, maintain characteristics of MSC. The results show that the obtained ADSC were plastic adherent, rapidly dividing (doubling time 40 ± 4 hours), spindle-shaped cells with fibroblastoid morphology and exhibited normal karyotype. No less than 95% of the obtained cells displayed MSC surface markers, including CD73, CD90 and CD105, but showed no expression of the hematopoietic markers CD34 and CD45. ADSC cultured in the presence of AS underwent in vitro differentiation into adipocytes, osteoblasts and chondroblasts, confirmed by Oil Red O, Alizarin Red S and Alcian Blue stains, respectively. These findings suggest that ADSC may be expanded in the AS without the loss of characteristics of MSC.

BackgroundSubviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution.FindingsThe findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization.ConclusionsThe large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.