Productively infected murine Kaposi's sarcoma-like tumors define new animal models for studying and targeting KSHV oncogenesis and replication.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). KS tumors are composed of KSHV-infected spindle cells of vascular origin with aberrant neovascularization and erythrocyte extravasation. KSHV genes expressed during both latent and lytic replicative cycles play important roles in viral oncogenesis. Animal models able to recapitulate both viral and host biological characteristics of KS are needed to elucidate oncogenic mechanisms, for developing targeted therapies, and to trace cellular components of KS ontogeny. Herein, we describe two new murine models of Kaposi's sarcoma. We found that murine bone marrow-derived cells, whether established in culture or isolated from fresh murine bone marrow, were infectable with rKSHV.219, formed KS-like tumors in immunocompromised mice and produced mature herpesvirus-like virions in vivo. Further, we show in vivo that the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) enhanced viral lytic reactivation. We propose that these novel models are ideal for studying both viral and host contributions to KSHV-induced oncogenesis as well as for testing virally-targeted antitumor strategies for the treatment of Kaposi's sarcoma. Furthermore, our isolation of bone marrow-derived cell populations containing a cell type that, when infected with KSHV, renders a tumorigenic KS-like spindle cell, should facilitate systematic identification of KS progenitor cells.

Zidovudine-based lytic-inducing chemotherapy for Epstein-Barr virus-related lymphomas.

Treatment of Epstein-Barr virus (EBV)-related lymphomas with lytic-inducing agents is an attractive targeted approach for eliminating virus-infected tumor cells. Zidovudine (AZT) is an excellent substrate for EBV-thymidine kinase: it can induce EBV lytic gene expression and apoptosis in primary EBV+ lymphoma cell lines. We hypothesized that the combination of AZT with lytic-inducing chemotherapy agents would be effective in treating EBV+ lymphomas. We report a retrospective analysis of 19 patients with aggressive EBV+ non-Hodgkin lymphoma, including nine cases of acquired immune deficiency syndrome-associated primary central nervous system lymphoma (AIDS-PCNSL) treated with AZT-based chemotherapy. Our results demonstrate that high-dose AZT-methotrexate is efficacious in treating highly aggressive systemic EBV+ lymphomas in the upfront setting. In primary EBV+ lymphoma cell lines, the combination of AZT with hydroxyurea resulted in synergistic EBV lytic induction and cell death. Further, AZT-hydroxyurea treatment resulted in dramatic responses in patients with AIDS-PCNSL. The combination of AZT with chemotherapy, especially lytic-inducing agents, should be explored further in clinical trials for the treatment of EBV-related lymphomas.

Molecular studies and therapeutic targeting of Kaposi's sarcoma herpesvirus (KSHV/HHV-8) oncogenesis.

Kaposi's sarcoma herpesvirus or human herpesvirus-8 (KSHV/HHV-8) is the etiological agent of Kaposi's sarcoma (KS), an AIDS-defining angioproliferative neoplasm that continues to be a major global health problem and, of primary effusion lymphoma (PEL), a rare incurable B-cell lymphoma. This review describes the research from our laboratory and its collaborators to uncover molecular mechanisms of viral oncogenesis in order to develop new pathogenesis-based therapies to the KSHV-induced AIDS malignancies KS and PEL. They include the discovery of the viral angiogenic oncogene G protein-coupled receptor (vGPCR), the development of mouse models of KSHV and oxidative stress-induced KS, the identification of the role of Rac1-induced ROS in viral oncogenesis of KS and the development of novel therapeutic approaches able to target both latent and lytic oncogenic KSHV infection.

CD30 targeting with brentuximab vedotin: a novel therapeutic approach to primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent, monomethyl auristatin E. Brentuximab vedotin is an effective treatment of relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest, and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients.

Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare form of aggressive B cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Current chemotherapy approaches result in dismal outcomes, and there is an urgent need for new PEL therapies. Previously, we established, in a direct xenograft model of PEL-bearing immune-compromised mice, that treatment with the proteasome inhibitor, bortezomib (Btz), increased survival relative to that after treatment with doxorubicin. Herein, we demonstrate that the combination of Btz with the histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA, also known as vorinostat) potently reactivates KSHV lytic replication and induces PEL cell death, resulting in significantly prolonged survival of PEL-bearing mice. Importantly, Btz blocked KSHV late lytic gene expression, terminally inhibiting the full lytic cascade and production of infectious virus in vivo. Btz treatment led to caspase activation and induced DNA damage, as evidenced by the accumulation of phosphorylated γH2AX and p53. The addition of SAHA to Btz treatment was synergistic, as SAHA induced early acetylation of p53 and reduced interaction with its negative regulator MDM2, augmenting the effects of Btz. The eradication of KSHV-infected PEL cells without increased viremia in mice provides a strong rationale for using the proteasome/HDAC inhibitor combination therapy in PEL.

In vivo electroporation and non-protein based screening assays to identify antibodies against native protein conformations.

In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved.

HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration.

Plasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response. Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%) were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production by infected CD4+ cells, supporting an anti-HIV activity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-infected individuals.

Potential screening assay for undetectable viruses on the basis of their capacity to induce alpha interferon production.

High interferon production by plasmacytoid dendritic cells (PDC) was unexpectedly noted after their coculture with CD4(+) cells from a healthy donor whose cells subsequently showed human herpesvirus type 6 and 7 infections. This release of interferon was not observed with uninfected normal CD4(+) cells. Induction of PDC interferon production could help screen for covert virus infections.

Human immunodeficiency virus type 2 DNA vaccine provides partial protection from acute baboon infection.

We determined if the genetic adjuvants, granulocyte-macrophage colony stimulating factor (GM-CSF) and B7-2, could improve the immunogenicity and efficacy of an HIV-2 DNA vaccine. The vaccine consisted of the HIV-2 tat, nef, gag, and env genes synthesized using optimized codons and formulated with cationic liposomes. Baboons (Papio cynocephalus hamadryas) were immunized by the intramuscular, intradermal, and intranasal routes with these expression constructs and challenged with HIV-2(UC2) by the intravaginal route. In the first month after HIV-2 vaginal challenge, the baboons receiving the HIV-2 DNA vaccine with or without the genetic adjuvants had significant reductions in the viral loads in the peripheral blood mononuclear cells (PBMC) (P = 0.028) while the reductions in their plasma viremia were suggestive of a protective effect (P = 0.1). These data demonstrate that partial protection against HIV-2 vaginal challenge, as measured by reduced viral load, can be achieved using only a DNA vaccine formulation.

Evaluation of genetic immunization adjuvants to improve the effectiveness of a human immunodeficiency virus type 2 (HIV-2) envelope DNA vaccine.

In an effort to develop a more effective genetic immunization strategy for HIV, we developed an HIV-2 env DNA vaccine and evaluated three adjuvant formulations. The gp140 gene from HIV-2(UC2 )was synthesized using mammalian codons and cloned into a plasmid vector that expresses eukaryotic genes at high levels. We found that after three immunizations in mice, a novel cationic liposome formulation (Vaxfectin) was superior at inducing systemic and mucosal antibody responses compared to a naked DNA, a controlled release device (an Alzet minipump) and polysaccharide microparticles made from chitosan (P = 0.027). Vaxfectin also induced higher levels of systemic antibodies for each isotype and IgG subclass as well as levels of HIV-2-specific mucosal IgA (P = 0.034). When different routes of immunization were used with the Vaxfectin formulation, gp140-specific systemic antibody responses were highest by the intradermal route, mucosal antibody responses were highest by the intramuscular route, while the intranasal route was the least effective. These results suggest that this cationic liposome formulation is an important adjuvant to improve the effectiveness of genetic immunization strategies for AIDS, and that multiple routes of immunization should be employed for optimal efficacy for HIV vaccine candidates.

Enhancement of a human immunodeficiency virus env DNA vaccine using a novel polycationic nanoparticle formulation.

In an effort to develop a more effective DNA immunization strategy for HIV, we synthesized an HIV-2 env DNA vaccine and delivered it in a novel polycationic adjuvant formulation that forms nanoparticles in solution and enhances protein expression. The polycationic adjuvant contained imidazole moieties to facilitate endosomal escape. Nanoparticles containing the DNA vaccine plasmid were formed by electrostatic condensation with the polycationic adjuvant. We hypothesized that this formulation would improve immune responses to the gp140 env gene from HIV-2(UC2) by increasing the level of expressed antigen. We found that the nanoparticles were superior at inducing high levels of systemic antibody responses compared to naked DNA when delivered by the intradermal route in BALB/c mice. In addition, the nanoparticles induced higher levels of IgM, IgG, and IgA antibodies. These results suggest that nanoparticles may be an important adjuvant formulation to improve the effectiveness of genetic immunization and rationalize its use in the evaluation of vaccine candidates in non-human primate models for AIDS.

Enhancement of antibody responses to an HIV-2 DNA envelope vaccine using an expression vector containing a constitutive transport element.

Because immune responses to DNA vaccines in humans remains suboptimal, strategies need to be devised to facilitate expression of the vaccine in vivo. One method to improve response to a DNA vaccine is to construct plasmid vectors with leader sequences and post-transcriptional elements that facilitate export of transcribed RNA. In this study, we sought to determine if a mammalian expression vector (pND-14) containing a tissue plasminogen activator (TPA) leader sequence and a constitutive transport element (CTE) from simian retrovirus was superior to other mammalian expression vectors containing a post-transcriptional regulatory element (PRE) from hepatitis B virus (pCMV-link) or a minimal mammalian expression vector (pVAX1). Toward this objective, we evaluated protein expression of the HIV-2 envelope gene (gp140) in vitro and immune responses in immunized mice. We found that pVAX1 produced three- to fourfold lower levels of gp140 in vitro (5 ng/ml) in contrast to the pCMV-link and pND-14 vectors. When we immunized groups of mice intradermally with two of the HIV-2 gp140 DNA vaccine constructs, we found that pND-14 induced higher levels of envelope-specific systemic and mucosal antibodies than pCMV-link. We conclude that expression vectors for DNA vaccines should contain TPA and CTE sequences to facilitate immune responses.