Baculovirus-driven protein expression in insect cells: A benchmarking study.

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.

Novel bifunctional single-chain variable antibody fragments to enhance virolysis by complement: generation and proof-of-concept.

When bound to the envelope of viruses, factor H (FH), a soluble regulator of complement activation, contributes to the protection against a potent immune defense mechanism, the complement-mediated lysis (CML). Thus, removing FH from the surface renders viruses, such as HIV, susceptible to CML. For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment (scFv) based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains (short consensus repeats 19-20; SCR1920) of FH. We used Pichia pastoris as expression system in common shake flasks and optimized expression in high density bench top fermentation. Specific binding of recombinant scFv was proven by flow cytometry. The recombinant scFv-SCR significantly enhanced CML of F-MuLV in vitro implying that FH binding to the viral surface was impaired by the scFv-SCR. This novel concept to enhance virolysis may provide a new approach for antiviral treatment.

Complement factor H-derived short consensus repeat 18-20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells.

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.

Targeting viral antigens to CD11c on dendritic cells induces retrovirus-specific T cell responses.

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.

Emerging role for complement in HIV infection.

PURPOSE OF REVIEW: New evidence is provided that the complement system is not only an effective component of the innate immunity, but is also involved in bridging innate and adaptive immune response to control retroviral infections. RECENT FINDINGS: The complement contributes to the control of retroviral replication by various strategies, such as complement-mediated lysis, triggering of B-cell responses by trapping the virus on follicular dendritic cells in the germinal center or enhancing of antigen presentation and thus the induction of virus-specific cytotoxic T lymphocytes. HIV has evolved mechanisms to escape from complement-meditated neutralization and counteracts these immune responses by escaping from lysis, using follicular dendritic cells as anchor to generate a latent viral reservoir and enhancing the infection of antigen-presenting cells. SUMMARY: This review will discuss the complex interactions of complement and complement receptors with retroviruses and review the escape mechanisms, which protect this virus family from complement-mediated destruction.

Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.

Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential.

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response.

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.