Recombinant baculovirus expressing the FrC-OVA protein induces protective antitumor immunity in an EG7-OVA mouse model.

BACKGROUND: The baculovirus (BV) Autographa californica multiple nuclear polyhedrosis virus has been used in numerous protein expression systems because of its ability to infect insect cells and serves as a useful vaccination vector with several benefits, such as its low clinical risks and posttranslational modification ability. We recently reported that dendritic cells (DCs) infected with BV stimulated antitumor immunity. The recombinant BV (rBV) also strongly stimulated peptide-specific T-cells and antitumor immunity. In this study, the stimulation of an immune response against EG7-OVA tumors in mice by a recombinant baculovirus-based combination vaccine expressing fragment C-ovalbumin (FrC-OVA-BV; rBV) was evaluated. RESULTS: We constructed an rBV expressing fragment C (FrC) of tetanus toxin containing a promiscuous MHC II-binding sequence and a p30-ovalbumin (OVA) peptide that functions in the MHC I pathway. The results showed that rBV activated the CD8+ T-cell-mediated response much more efficiently than the wild-type BV (wtBV). Experiments with EG7-OVA tumor mouse models showed that rBV significantly decreased tumor volume and increased survival compared with those in the wild-type BV or FrC-OVA DNA vaccine groups. In addition, a significant antitumor effect of classic prophylactic or therapeutic vaccinations was observed for rBV against EG7-OVA-induced tumors compared with that in the controls. CONCLUSION: Our findings showed that FrC-OVA-BV (rBV) induced antitumor immunity, paving the way for its use in BV immunotherapy against malignancies.

Baculovirus Infection of Human Monocyte-Derived Dendritic Cells Restricts HIV-1 Replication.

Acquired immune deficiency syndrome (AIDS) is mainly caused by infection with human immunodeficiency virus-1 (HIV-1) and still poses a global threat for which we lack a protective or therapeutic vaccine. Dendritic cells (DCs) play a major role in the onset of HIV infection, providing one of the primary sites of HIV replication, and also act as viral reservoirs in vivo. Previous studies have shown that baculovirus (BV) induces strong host immune responses against infections and malignancies. In this study, we infected human monocyte-derived DCs with recombinant BV (AcCAG-gag) and showed that AcCAG-gag-infected human DCs underwent maturation and produced interferon alpha and other proinflammatory cytokines accompanied by increases in the mRNA and protein expression levels of APOBEC3 (A3A, A3F, and A3G), proteins associated with the inhibition of HIV-1 replication. Surprisingly, HIV-1 inhibition is also observed in human DCs infected with a wild-type BV, as determined by the production of inflammatory cytokines, the expression of A3, and a reduction in the p24 level. Our findings outline the mechanism underlying the inhibition of HIV-1 in BV-infected human DCs and pave the way for the use of BV as an effective tool for immunotherapy against HIV-1.

HIV-1 Gag-virus-like particles inhibit HIV-1 replication in dendritic cells and T cells through IFN-α-dependent upregulation of APOBEC3G and 3F.

Human immunodeficiency virus-1 (HIV-1) infection and the acquired immune deficiency syndrome (AIDS) pandemic remain global threats in the absence of a protective or a therapeutic vaccine. HIV-1 replication is reportedly inhibited by some cellular factors, including APOBEC3G (A3G) and APOBEC3F (A3F), which are well known inhibitors of HIV-1. Recently, HIV-1 Gag-virus-like particles (Gag-VLPs) have been shown to be safe and potent HIV-1 vaccine candidates that can elicit strong cellular and humoral immunity without need of any adjuvant. In this report, we stimulated human monocyte-derived dendritic cells (DCs) with Gag-VLPs and we demonstrated that Gag-VLP-treated DCs (VLP-DCs) produced interferon alpha (IFN-α), along with an increase in mRNA and protein expression of A3G and A3F. Gag-VLPs inhibited HIV-1 replication not only in DCs themselves, but also in cocultured T cells in an IFN-α-dependent manner. In addition, A3G/3F content in HIV virions released from VLP-DCs increased. Both the increase in A3G/3F expression and the inhibition of HIV-1 replication were reversed by anti-IFN-α or anti-IFNAR antibodies. Our findings in this study provide insight into the mechanism of Gag-VLP-induced inhibition of HIV-1 replication in DCs and T cells.

HIV-1 Gag-virus-like particles induce natural killer cell immune responses via activation and maturation of dendritic cells.

Despite the extensive efforts that have been made to combat acquired immune deficiency syndrome (AIDS), the number of people infected each year with human immunodeficiency virus type 1 (HIV-1) is still increasing worldwide, and a safe and effective vaccine to control HIV infection is urgently needed. Recently, the natural killer (NK) cell-mediated innate immune response, which represents the first line of defense against infections, has attracted attention for its role in combating HIV infection and disease progression. In the present study, we investigated the immunogenic ability of HIV-1 Gag-virus-like particles (Gag-VLPs) to induce NK cell immune responses in vitro and in vivo. Gag-VLPs efficiently activated human monocyte-derived dendritic cells (MDDCs), eliciting MDDC maturation with an associated increase in the surface expression of CD80, CD86 and MHC classes I and II, MDDC proliferation and proinflammatory cytokine production. Gag-VLP-treated MDDCs subsequently activated autologous NK cells, leading to their proliferation and production of interferon-γ and to the upregulation of NK cell cytotoxicity against YAC-1 cells and HIV-1-infected CD4(+) T cells. In addition, we introduced a 2-phase immunization strategy in BALB/c mice to assess the role of DCs in the induction of NK cell immune responses by Gag-VLPs in vivo. Our findings reveal that Gag-VLPs efficiently activate DCs, which in turn induce innate and Gag-specific immune responses in NK cells.

Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses.

We previously reported that the baculovirus induced a strong host immune response against infections and malignancies. Among the immune cells, the dendritic cells were most strongly infected and activated by the baculovirus, although the exact mechanism remained unclear. Here, we evaluated the non-specific immune responses of bone marrow-derived dendritic cells (BMDCs) after infection by a wild-type baculovirus. MHC class I and II molecules and co-stimulation molecules (CD40, CD80, and CD86) on BMDCs were up-regulated by baculovirus infection. At the same time, the BMDCs produced pre-inflammatory cytokines (IL-6, IL12p70, and TNF-alpha) and IFN-alpha. NK cells showed IFN-gamma production, CD69 up-regulation, and enhanced cytotoxicity when they were co-cultured with baculovirus-infected BMDCs. T cells showed IFN-gamma production, CD69 up-regulation, and cell proliferation. Ex vivo analysis performed in vitro produced similar results. These findings suggested that baculovirus-infected dendritic cells induce non-specific immune responses and can be used as an immunotherapeutic agent against viral infections and malignancies, together with present therapeutic drug regimens.

Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy.

BACKGROUND: Hepatitis C virus (HCV) is one of the main causes of liver-related morbidity and mortality. Although combined interferon-alpha-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs) inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA) expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1) and the origin of latent viral DNA replication (OriP) sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV) replicon cells. RESULTS: We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. CONCLUSION: These findings indicate that we successfully constructed a long-term transgene (shRNA) expression vector (Ac-EP-shRNA452) using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA) expression vector was evaluated with the view of establishing highly effective therapeutic agents that can be further developed for HCV gene therapy applications.

Pycnogenol, a procyanidin-rich extract from French maritime pine, inhibits intracellular replication of HIV-1 as well as its binding to host cells.

A procyanidin-rich extract from French maritime pine, Pycnogenol(R) (PYC), is known as an antioxidant that exerts a variety of physiological activities and is widely used in human beings. We report here that PYC inhibits not only human immunodeficiency virus type-1 (HIV-1) binding to host cells, but also its replication after entry in susceptible cells in vitro. Prominent biochemical alterations induced by PYC were the elevated expression of an intracellular antioxidant protein, manganese superoxide dismutase (Mn-SOD), and the inhibition of phosphorylation of the ribosomal S6 protein. Interestingly, ectopic expression of Mn-SOD inhibited HIV-1 replication as well. Inhibition of HIV-1 replication associated with induced expression of Mn-SOD in cells treated with PYC suggests the potential of this natural antioxidant inducer as a new anti-HIV-1 agent.

Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction.

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA synthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.

Production and characterization of a monoclonal antibody specific to Nef-associated factor 1 (Naf1)/A20-binding inhibitor of NF-kappaB activation (ABIN-1).

Cellular protein Naf1 (Nef-associated factor 1) or ABIN-1 (A20-binding inhibitor of NF-kappaB activation) is an important cellular protein, expressed in various human tissues and T-cell lines. Naf1 protein has two isoforms (Naf1alpha and Naf1beta) with different C-termini, produced by alternative splicing. Naf1alpha and Naf1beta have approximately 2800 and 2600 nucleotides, with an open reading frame of 1941 and 1781 nucleotides, encoding the 72-kDa Naf1alpha and 68-kDa Naf1beta proteins, respectively. In the present study, we generated a monoclonal antibody (MAb) against human Naf1, which recognizes full-length, endogenous Naf1 of both isotypes. For this purpose, recombinant 6xHis and myc-tagged N-terminal Naf1(38135), Naf1(N) protein was produced by using the baculovirus expression system. Recombinant Naf1(N) protein was used to immunize Balb/c mice, and a hybridoma cell line producing stable and highly specific MAb with strong affinity to Naf1 was established. We further characterized this antibody by immunofluorescent assay and Western blot analysis to confirm effectiveness in detecting recombinant and endogenous Naf1. By Western blot analysis of recombinant Naf1-N fusion proteins with overlapping N-terminal sequences, the epitope targeted by anti-Naf1 MAb was determined as the 81-88-amino acid region of human Naf1.