[Maturation and Antigen Loading Protocols Influence Activity of Anticancer Dendritic Cells].

The practical use of dendritic cell-based vaccines in anticancer therapy is limited by a lack of standards for dendritic cell (DC) generation, as well as standard procedures for controlling their activation and the technique of DC loading with nucleic acids encoding tumor antigens. Analyzing the currently available data, the most promising cocktails for DC maturation were selected and a comparative study of the cocktails and time of maturation on the capacity of DC to activate T-cell immune response has been performed. A study of the expression of surface markers and the production of IL-12, IL-6, and IL-10 cytokines, as well as the efficacy of T-cell activation showed that the use of the standard 7-day maturation protocol is preferable to the 4-day maturation protocol. Cocktails composed of TNF-α, IL-lß, IFN-α, IFN-γ, and poly(I:C), as well as TNF-α, IL-lß, IFN-γ, R848, and PGE2 were shown to be the most efficient activators of DCs. A comparison of the efficacy of different methods of DNA transfection into DCs and RNA delivery using alphavirus vectors demonstrated the superiority of magnet-assisted transfection (MATra) to other protocols.

A Semliki Forest virus expression system as a model for investigating the nuclear import and export of hepatitis B virus nucleocapsid protein.

HBV core protein (HBc), the major component of nucleocapsid is known to have an essential role in the virus life cycle as the transporter of virus genome to the host nucleus; however, molecular details of the intracellular transport of HBc remain unknown. In this study, we investigated the intracellular distribution of the HBc protein resulting from Semliki Forest virus (SFV)-driven HBc gene and HBV RNA pregenome (pgRNA) expression in BHK-21 cells. Fluorescent confocal microscopy revealed extensive HBc protein synthesis in the cytoplasm 12 hr post infection (p.i.) with recombinant pSFV1/HBc or pSFV1/HBVpgRNA viruses. Twenty-four hr p.i., the HBc protein showed asymmetric, predominantly nuclear localization in most cells. However, 42 hr p.i., the number of cells containing intranuclear HBc protein, dramatically decreased to ~ 5%, while cytoplasmic HBc protein was detected in all cells. Remarkably, 24 hr p.i. with pSFV1/HBVpgRNA virus, cytoplasmic HBc protein colocalized with HBs protein. We conclude that a HBs-HBc interaction partially prevents the transport of HBc from the cytoplasm to nucleus. The SFV-driven system can be used for identification of new factors involved in the HBV nuclear import and export.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

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.

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon.

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.