Victoria Cepeda

Biochemical Mechanisms of Cisplatin Cytotoxicity

Since the discovery by Rosenberg and collaborators of the antitumor activity of cisplatin 35 years ago, three platinum antitumor drugs (cisplatin, carboplatin and oxaliplatin) have enjoyed a huge clinical and commercial hit. Ever since the initial discovery of the anticancer activity of cisplatin, major efforts have been devoted to elucidate the biochemical mechanisms of antitumor activity of cisplatin in order to be able to rationally design novel platinum based drugs with superior pharmacological profiles. In this report we attempt to provide a current picture of the known facts pertaining to the mechanism of action of the drug, including those involved in drug uptake, DNA damage signals transduction, and cell death through apoptosis or necrosis. A deep knowledge of the biochemical mechanisms, which are triggered in the tumor cell in response to cisplatin injury not only may lead to the design of more efficient platinum antitumor drugs but also may provide new therapeutic strategies based on the biochemical modulation of cisplatin activity.

A Novel Poxvirus-based Vaccine (MVA-CHIKV) is Highly Immunogenic and Protects Mice against Chikungunya Infection

ABSTRACT There is a need to develop a single and highly effective vaccine against the emerging chikungunya virus (CHIKV), which causes a severe disease in humans. Here, we have generated and characterized the immunogenicity profile and the efficacy of a novel CHIKV vaccine candidate based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). MVA-CHIKV was stable in cell culture, expressed the CHIKV structural proteins, and triggered the cytoplasmic accumulation of Golgi apparatus-derived membranes in infected human cells. Furthermore, MVA-CHIKV elicited robust innate immune responses in human macrophages and monocyte-derived dendritic cells, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. After immunization of C57BL/6 mice with a homologous protocol (MVA-CHIKV/MVA-CHIKV), strong, broad, polyfunctional, and durable CHIKV-specific CD8+ T cell responses were elicited. The CHIKV-specific CD8+ T cells were preferentially directed against E1 and E2 proteins and, to a lesser extent, against C protein. CHIKV-specific CD8+ memory T cells of a mainly effector memory phenotype were also induced. The humoral arm of the immune system was significantly induced, as MVA-CHIKV elicited high titers of neutralizing antibodies against CHIKV. Remarkably, a single dose of MVA-CHIKV protected all mice after a high-dose challenge with CHIKV. In summary, MVA-CHIKV is an effective vaccine against chikungunya virus infection that induced strong, broad, highly polyfunctional, and long-lasting CHIKV-specific CD8+ T cell responses, together with neutralizing antibodies against CHIKV. These results support the consideration of MVA-CHIKV as a potential vaccine candidate against CHIKV. IMPORTANCE We have developed a novel vaccine candidate against chikungunya virus (CHIKV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). Our findings revealed that MVA-CHIKV is a highly effective vaccine against chikungunya virus, with a single dose of the vaccine protecting all mice after a high-dose challenge with CHIKV. Furthermore, MVA-CHIKV is highly immunogenic, inducing strong innate responses: high, broad, polyfunctional, and long-lasting CHIKV-specific CD8+ T cell responses, together with neutralizing antibodies against CHIKV. This work provides a potential vaccine candidate against CHIKV.

Human cytomegalovirus final envelopment on membranes containing both trans‐Golgi network and endosomal markers

The human cytomegalovirus (HCMV) has been shown to complete its final envelopment on cytoplasmic membranes prior to its secretion to the extracellular medium. However, the nature of these membranes has not been characterized. It is thought that HCMV acquires its final envelope from the trans‐Golgi network (TGN), though we and others have previously reported a role for endocytic membranes. Here we studied the localization of cellular markers in HCMV‐infected cells and in isolated viruses. Immunofluorescence staining indicated that HCMV induces the recruitment of TGN and endosomal markers to the virus factory. Immuno‐gold labelling of isolated viral particles and electron microscopy demonstrated the incorporation of TGN46, endosomal markers early endosomal antigen 1, annexin I, transferrin receptor and CD63, and the cation‐independent mannose 6‐phosphate receptor, which traffics between the TGN and endosomes into the viral envelope. Virus immunoprecipitation assays demonstrated that virions containing TGN46 and CD63 were infectious. This study reconciles the apparent controversy regarding the nature of the HCMV assembly site and suggests that HCMV has the ability to generate a novel membrane compartment containing markers for both TGN and endosomes, or that the membranes that HCMV uses for its envelope may be vesicles in transit between the TGN and endosomes.

Poly(ADP-Ribose) Polymerase-1 (PARP-1) Inhibitors in Cancer Chemotherapy

Poly(ADP-ribose) polymerases (PARPs) are defined as a family of cell signaling enzymes present in eukaryotes, which are involved in poly(ADP-ribosylation) of DNA-binding proteins. The best studied of these enzymes (PARP-1) is involved in the cellular response to DNA damage so that in the event of irreparable DNA damage overactivation of PARP-1 leads to necrotic cell death. Inhibitors of PARP-1 activity in combination with DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. When DNA is moderately damaged, PARP-1 participates in the DNA repair process and the cell survives. However, in the case of extensive DNA damage PARP-1 overactivation induces a decrease of NAD+ and ATP levels leading to cell dysfunction or even to necrotic cell death. So, due to PARP-1 involvement in cell death, pharmacological inhibition of PARP-1 activity by PARP-1 inhibitors may constitute a suitable target to enhance the activity of antitumor drugs through inhibition of necrosis and activation of apoptosis. PARP-1 inhibitors such as 3-aminobenzamide, 1,5-dihydroxyisoquinolinone and the recently patented tryciclic benzimidazoles have shown potent inhibitory effects of PARP-1 activity in tumor cells. The present review gives an update of the state-of-the-art of inhibition of PARP-1 activity as adjuvant therapy in cancer treatment.

Poly(ADP-ribose) Polymerase-1 Inhibitor 3-Aminobenzamide Enhances Apoptosis Induction by Platinum Complexes in Cisplatin-Resistant Tumor Cells

Cisplatin is one of the most widely used antitumor drugs. However, as all the anticancer drugs currently used in clinic, cisplatin shows the phenomenon of drug resistance (intrinsic or acquired) against a wide variety of tumors. Poly (ADP-ribose) polymerase-1 is an enzyme involved in DNA repair and apoptotic cell death, which may be inhibited to increase cisplatin chemosensitivity of tumor cells so that cisplatin resistance may be circumvented. In the present study we report that PARP-1 inhibitor 3-aminobenzamide (3-AB) increases the cytotoxic activity of the platinum compounds cisplatin, trans-[PtCl(2)(4-picoline)(piperazine)] and transplatin against CH1cisR cisplatin-resistant ovarian tumor cells. In fact, a concentration of 3-AB of 1 mM not only increases the cytotoxic activity of these platinum complexes but also switches the mode of cell death from necrosis to apoptosis. Altogether, these data suggest that pharmacological modulation of PARP-1 by inhibitors may be a suitable strategy to fight against tumor resistance to platinum drugs.

Rab27a Is Required for Human Cytomegalovirus Assembly

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.

A role for the SNARE protein syntaxin 3 in human cytomegalovirus morphogenesis

As an enveloped virus, replication of human cytomegalovirus (HCMV) is dependent on interaction with cellular membrane systems. Its final envelopment occurs into intracellular membranes prior to its secretion. However the mechanisms underlying these processes are poorly understood. Here, we show that HCMV infection induces expression of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3), a component of the cellular machinery for membrane fusion. STX3 was located at the plasma membrane and at the assembly site where it was found associated with virus wrapping membranes by immunogold labelling. Depletion of STX3 using RNA interference reduced HCMV production, while expression of a STX3 construct resistant to RNAi inhibition enhanced virus production. Ultrastructural examination of the assembly site in HCMV‐infected STX3‐depleted cells showed fewer mature virions and more viruses undergoing final envelopment. In contrast, silencing of STX3 did not affect herpes simplex virus type 1 production. The mechanism through which STX3 affected HCMV morphogenesis likely involved late endosomes/lysosomes since STX3 depletion reduced the expression of lysosomal membrane glycoproteins. Our results demonstrate a function for STX3 in HCMV morphogenesis, and unravel a new role for this SNARE protein in late endosomes/lysosomes compartments.

Virological and Immunological Characterization of Novel NYVAC-Based HIV/AIDS Vaccine Candidates Expressing Clade C Trimeric Soluble gp140(ZM96) and Gag(ZM96)-Pol-Nef(CN54) as Virus-Like Particles

ABSTRACT The generation of vaccines against HIV/AIDS able to induce long-lasting protective immunity remains a major goal in the HIV field. The modest efficacy (31.2%) against HIV infection observed in the RV144 phase III clinical trial highlighted the need for further improvement of HIV vaccine candidates, formulation, and vaccine regimen. In this study, we have generated two novel NYVAC vectors, expressing HIV-1 clade C gp140(ZM96) (NYVAC-gp140) or Gag(ZM96)-Pol-Nef(CN54) (NYVAC-Gag-Pol-Nef), and defined their virological and immunological characteristics in cultured cells and in mice. The insertion of HIV genes does not affect the replication capacity of NYVAC recombinants in primary chicken embryo fibroblast cells, HIV sequences remain stable after multiple passages, and HIV antigens are correctly expressed and released from cells, with Env as a trimer (NYVAC-gp140), while in NYVAC-Gag-Pol-Nef-infected cells Gag-induced virus-like particles (VLPs) are abundant. Electron microscopy revealed that VLPs accumulated with time at the cell surface, with no interference with NYVAC morphogenesis. Both vectors trigger specific innate responses in human cells and show an attenuation profile in immunocompromised adult BALB/c and newborn CD1 mice after intracranial inoculation. Analysis of the immune responses elicited in mice after homologous NYVAC prime/NYVAC boost immunization shows that recombinant viruses induced polyfunctional Env-specific CD4 or Gag-specific CD8 T cell responses. Antibody responses against gp140 and p17/p24 were elicited. Our findings showed important insights into virus-host cell interactions of NYVAC vectors expressing HIV antigens, with the activation of specific immune parameters which will help to unravel potential correlates of protection against HIV in human clinical trials with these vectors. IMPORTANCE We have generated two novel NYVAC-based HIV vaccine candidates expressing HIV-1 clade C trimeric soluble gp140 (ZM96) and Gag(ZM96)-Pol-Nef(CN54) as VLPs. These vectors are stable and express high levels of both HIV-1 antigens. Gag-induced VLPs do not interfere with NYVAC morphogenesis, are highly attenuated in immunocompromised and newborn mice after intracranial inoculation, trigger specific innate immune responses in human cells, and activate T (Env-specific CD4 and Gag-specific CD8) and B cell immune responses to the HIV antigens, leading to high antibody titers against gp140. For these reasons, these vectors can be considered vaccine candidates against HIV/AIDS and currently are being tested in macaques and humans.

A Phase I Randomized Therapeutic MVA-B Vaccination Improves the Magnitude and Quality of the T Cell Immune Responses in HIV-1-Infected Subjects on HAART

Trial Design Previous studies suggested that poxvirus-based vaccines might be instrumental in the therapeutic HIV field. A phase I clinical trial was conducted in HIV-1-infected patients on highly active antiretroviral therapy (HAART), with CD4 T cell counts above 450 cells/mm3 and undetectable viremia. Thirty participants were randomized (2:1) to receive either 3 intramuscular injections of MVA-B vaccine (coding for clade B HIV-1 Env, Gag, Pol and Nef antigens) or placebo, followed by interruption of HAART. Methods The magnitude, breadth, quality and phenotype of the HIV-1-specific T cell response were assayed by intracellular cytokine staining (ICS) in 22 volunteers pre- and post-vaccination. Results MVA-B vaccine induced newly detected HIV-1-specific CD4 T cell responses and expanded pre-existing responses (mostly against Gag, Pol and Nef antigens) that were high in magnitude, broadly directed and showed an enhanced polyfunctionality with a T effector memory (TEM) phenotype, while maintaining the magnitude and quality of the pre-existing HIV-1-specific CD8 T cell responses. In addition, vaccination also triggered preferential CD8+ T cell polyfunctional responses to the MVA vector antigens that increase in magnitude after two and three booster doses. Conclusion MVA-B vaccination represents a feasible strategy to improve T cell responses in individuals with pre-existing HIV-1-specific immunity. Trial Registration ClinicalTrials.gov NCT01571466

Novel insights on the progression of intermediate viral forms in the morphogenesis of vaccinia virus

Morphogenesis of vaccinia virus (VACV) is a complex structural process in which the capture of all cytoplasmic stages is difficult due to the rapid transition between the different viral forms. Taking advantage of two VACV mutants (M65 and M101) with defined genetic alterations, we described by transmission electron microscopy (TEM) of ultrathin sections novel potential transition viral forms (Ts) with reorganization of the immature virus (IV) membrane and construction of the internal core, and illustrated the envelopment steps from the mature virus (MV) to the wrapped virus (WV) stages. Our observations allowed us to propose a sequence of structural events for VACV assembly that provides key clues about VACV morphogenesis.