Development of a stable Sf9 insect cell line to produce VSV-G pseudotyped baculoviruses.

Baculoviruses have shown great potential as gene delivery vectors in mammals, although their effectiveness in transferring genes varies across different cell lines. A widely employed strategy to improve transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus to pseudotype budded baculoviruses. It was obtained by inserting the VSV-G gene under the control of the very strong and infection-inducible pXXL promoter and was subsequently diluted to establish oligoclonal lines, which were selected by the fusogenic properties of VSV-G and its expression levels in infected cells and purified budded virions. Next, to enhance the performance of the cell line, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses were pseudotyped and the expression of the transgene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased across all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.

MVAΔ008 viral vector encoding the model protein OVA induces improved immune response against the heterologous antigen and equal levels of protection in a mice tumor model than the conventional MVA.

Modified vaccinia Ankara virus (MVA) is extensively used as a vaccine vector. We have previously observed that MVAΔ008, an MVA lacking the gene that codes for interleukin-18 binding protein, significantly increases CD8+ and CD4+ T-cell responses to vaccinia virus (VACV) epitopes and recombinant HIV antigens. However, the efficacy of this vector against pathogens or tumor cells remains unclear. Thus, the aim of this study was to evaluate the cellular immune response and the protection induced by recombinant MVAs encoding the model antigen ovalbumin (OVA). We used the MO5 melanoma tumor model (OVA-expressing tumor) as an approach for evaluating the vector-induced efficacy. Our results show that MVAΔ008-OVA (optimized vector) induced higher in vivo specific cytotoxicity and ex vivo T-cell IFN-γ responses against OVA than the conventional MVA vector. Importantly, the recombinant vectors were capable of controlling MO5 tumor growth. Indeed, the administration of MVAΔ008-OVA or MVA-OVA in prophylactic and therapeutic schemes provided total protection and longer survival of mice, respectively. Overall, our results demonstrate the improved immunogenicity and the protective capacity of MVAΔ008 against a heterologous model antigen. These findings suggest that MVAΔ008 constitutes an excellent candidate for vaccine development against pathogens or cancer therapy.

Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells.

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.

Bovine macrophages responses to the infection with virulent and attenuated Leptospira interrogans serovar Pomona.

Leptospirosis is a zoonosis, caused by pathogenic spirochetes of the genus Leptospira. Although cattle are usually the maintenance hosts of serovar Hardjo, Pomona is the most frequent serovar circulating in Argentina. The understanding of bovine innate immune response and the virulence of this serovar is important for future control measures. This work compares infection of bovine macrophages with the virulent L. interrogans sv Pomona strain AKRFB (P1) and its attenuated counterpart (P19). First, we confirmed attenuation in the hamster model. Mortality and lung hemorrhages occurred after P1 inoculation, while the survival rate was 100% in P19-infected animals. Cells infected with both strains showed statistically upregulated gene expression of pro-inflammatory cytokines, IL-1ß, IL-6 and TNFα. The level of expression of anti-inflammatory cytokine IL-10 was statistically different between strains. Increased expression of IL-10 was observed only in P1-infected cells. For the first time, we describe macrophages extracellular traps induced by infection of bovine macrophages (bMETs) with both, the virulent and attenuated Leptospira interrogans Pomona strains. P1 was found higher internalized when the phagocytosis was inhibited, suggesting a cell entrance of this strain also by an independent-phagocytosis pathway. Furthermore, P1 was higher colocalized with acidic and late endosomal compartments compared with P19. This data emphasizes the importance to deepen in Leptospira bovine macrophages particular invasion mechanisms and, furthermore, underline the value of studying the main hosts.

Systemic administration of imiquimod as an adjuvant improves immunogenicity of a tumor-lysate vaccine inducing the rejection of a highly aggressive T-cell lymphoma.

T-cell lymphomas include diverse malignancies. They are rare, some have low survival rates and they lack curative therapies. The aim of this work was to assess whether employing the TLR7 agonist imiquimod and the T-cell costimulatory molecule CD40 or the combination of both as adjuvants of a cell lysate vaccine could enhance the antitumor immune response using a murine T-cell lymphoma model. Immunization with LBC-lysate and imiquimod protected almost all vaccinated animals. A specific humoral and a Th1-type cellular immunity were induced in mice that rejected the lymphoma, characterized by an elevated number of CD4 + T-cells and secretion of IFN-γ, locally and systemically. In contrast, CD40 alone or in combination with imiquimod did not improve the protective response obtained with LBC-lysate and imiquimod. Systemic administration of imiquimod proved to have high potential to serve as a vaccine adjuvant for the treatment of T-cell lymphomas and was effective in this immunotherapy model.

Cyclophosphamide enhances the release of tumor exosomes that elicit a specific immune response in vivo in a murine T-cell lymphoma.

Exosomes are 60-150 nm small extracellular vesicles (EVs) released by most cells. Tumor-cell-derived exosomes, used as a vaccine, elicit a specific cytotoxic response against tumor cells, usually with a greater immunogenicity than tumor-cell lysates. However, the number of exosomes isolated from culture cells is limited. In recent studies, it was observed that cells respond to different stressor stimuli such as cytotoxic drugs, hypoxia, acidosis, or radiation by increasing the release of EVs. In this study, using the murine LBC T-cell lymphoma, we found that cyclophosphamide significantly increased EVs yield. These EVs express exosome marker proteins such as TSG-101, CD9, CD81, and CD63. Furthermore, similar humoral and cellular immune responses were induced in vivo by EVs isolated from LBC-tumor cells whether they were grown under normal culture conditions (EVs C) or in the presence of cyclophosphamide (EVs CTX). Mice vaccinated either with EVs C or EVs CTX were similarly protected against an intraperitoneal challenge with LBC tumor cells. CD4+ and CD8+ IFN-γ secreting cells were induced in immunized mice and a specific cytotoxic cellular immune response was elicited in vitro. These results demonstrate that a Th1 response was induced by immunization with the EVs. Our findings suggest that treatment of tumor cells with cyclophosphamide is a useful method to enhance the secretion of EVs in sensitive cell lines without altering their antitumor properties and thus may be used to produce antigens for future design of cancer vaccines.

Exosomes Isolated from Ascites of T-Cell Lymphoma-Bearing Mice Expressing Surface CD24 and HSP-90 Induce a Tumor-Specific Immune Response.

Extracellular vesicles (EVs), including endosome-derived nanovesicles (exosomes), are involved in cell-cell communication. Through transfer of their molecular contents, extracellular nanovesicles can alter the function of recipient cells. Due to these characteristics, EVs have shown potential as a new alternative for cancer immunotherapy. Tumor exosomes isolated from malignant ascites can activate dendritic cells, thereby priming the immune system to recognize and kill cancer cells. However, a suppressive role on tumor immune response has also been reported, suggesting that the neoplastic stage of carcinogenesis and the microenvironment where tumor cells grow may influence the amount of EVs released by the cell. This neoplastic stage and microenvironment may also impact EVs' components such as proteins and miRNA, determining their biological behavior. Most T-cell lymphomas have an aggressive clinical course and poor prognosis. Consequently, complementary alternative therapies are needed to improve the survival rates achieved with conventional treatments. In this work, we have characterized EVs isolated from ascites of mice bearing a very aggressive murine T-cell lymphoma and have studied their immunogenic properties. Small EVs were isolated by differential centrifugation, ultrafiltration, and ultracentrifugation at 100,000 × g on a sucrose cushion. The EVs were defined as exosomes by their morphology and size analyzed by electron microscopy, their floating density on a sucrose gradient, as well as their expression of endosome marker proteins ALIX, TSG-101; the tetraspanins CD63, CD9, and CD81. In addition, they contain tumor antigens, the marker for malignancy CD24, the heat shock protein HSP-70, and an unusual surface expression of HSP-90 was demonstrated. The administration of EVs isolated from ascites (EVs A) into naïve-syngeneic mice induced both humoral and cellular immune responses that allowed the rejection of subsequent tumor challenges. However, the immunization had no effect on a non-related mammary adenocarcinoma, demonstrating that the immune response elicited was specific and also it induced immune memory. In vitro analysis demonstrated that T-cells from EVs A-immunized mice secrete IFN-γ in response to tumor stimulation. Furthermore, tumor-specific CD4+ and CD8+ IFN-γ secreting cells could be efficiently expanded from mice immunized with EVs A, showing that a T helper 1 response is involved in tumor rejection. Our findings confirm exosomes as promising defined acellular tumor antigens for the development of an antitumor vaccine.

Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plant-derived VP2 as a strategy against IBDV.

Different immunogens such as subunit, DNA or live viral-vectored vaccines against Infectious Bursal Disease virus (IBDV) have been evaluated in the last years. However, the heterologous prime-boost approach using recombinant modified vaccinia Ankara virus (rMVA), which has shown promising results in both mammals and chickens, has not been tried against this pathogen yet. IBD is a highly contagious and immunosuppressive disease of poultry that affects mainly young chicks. It is caused by IBDV, a double-stranded RNA virus carrying its main antigenic epitopes on the capsid protein VP2. Our objective was to evaluate the immune response elicited by two heterologous prime-boost schemes combining an rMVA carrying the VP2 mature gene (rVP2) and a recombinant VP2 protein produced in Nicotiana benthamiana (pVP2), and to compare them with the performance of the homologous pVP2-pVP2 scheme usually used in our laboratory. The SPF chickens immunized with the three evaluated schemes elicited significantly higher anti-VP2 antibody titers (p<0.001) and seroneutralizing titers (p<0.05) and had less T-cell infiltration (p<0.001), histological damage (p<0.001) and IBDV particles (p<0.001) in their bursae of Fabricius when compared with control groups. No significant differences were found between both heterologous schemes and the homologous one. However, the rVP2-pVP2 scheme showed significantly higher anti-VP2 antibody titers than pVP2-rVP2 and a similar tendency was found in the seroneutralization assay. Conversely, pVP2-rVP2 had the best performance when evaluated through bursal parameters despite having a less potent humoral immune response. These findings suggest that the order in which rVP2 and pVP2 are combined can influence the immune response obtained. Besides, the lack of a strong humoral immune response did not lessen the ability to protect from IBDV challenge. Therefore, further research is needed to evaluate the mechanisms by which these immunogens are working in order to define the combination that performs better against IBDV.

Evaluation of different heterologous prime-boost immunization strategies against Babesia bovis using viral vectored and protein-adjuvant vaccines based on a chimeric multi-antigen.

Protection against the intraerythrocytic bovine parasite Babesia bovis requires both humoral and cellular immune responses. Therefore, tailored combinations of immunogens targeted at both arms of the immune system are strategies of choice to pursue sterilizing immunity. In this study, different heterologous prime-boost vaccination schemes were evaluated in mice to compare the immunogenicity induced by a recombinant adenovirus, a modified vaccinia Ankara vector or a subunit vaccine all expressing a chimeric multi-antigen. This multi-antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: Merozoite Surface Antigen - 2c (MSA-2c), Rhoptry Associated Protein - 1 (RAP-1) and Heat Shock Protein 20 (HSP20). Both priming with the adenovirus or recombinant multi-antigen and boosting with the modified vaccinia Ankara vector achieved a high degree of activation of TNFα and IFNγ-secreting CD4(+) and CD8(+) specific T cells 60days after the first immunization. High titers of specific IgG antibodies were also detected at the same time point and lasted up to day 120 of the first immunization. Only the adenovirus - MVA combination triggered a marked isotype skew for the IgG2a antibody subclass meanwhile for the other immune traits analyzed here, both vaccination schemes showed similar performances. The immunological characterization in the murine model of these rationally designed immunogens led us to propose that adenoviruses as well as the bacterially expressed multi-antigen are highly reliable primer candidates to be considered in future experiments in cattle to test protection against bovine babesiosis.

Immunomodulatory effect of baculovirus in chickens: How it modifies the immune response against infectious bursal disease virus.

Several reports have shown that baculoviruses (BVs) have strong adjuvant properties on the mammalian immune system. Recent studies of our group demonstrated the ability of BV to stimulate the innate immunity in chickens. In this investigation, we aimed to assess the potential antiviral effect of BV given both, before and after infectious bursal disease virus (IBDV). In the first case, specific pathogen free chickens were intravenously inoculated with 5 × 10(7) pfu of Autographa californica nuclear polyhedrosis virus and 3 h later were orally administered 2.5 × 10(5) egg infectious doses 50 of IBDV. In the second case, chickens received IBDV 3 h before BV inoculation. Five days later, chickens were bled and euthanized. RNA from the bursa was analyzed for cytokine production. Also, bursae were used for virus recovery, and processed for lymphocyte isolation. The results showed that the administration of BV 3 h after the inoculation with IBDV produced important changes in the effect that IBDV causes in the bursa. BV reduced the infiltration of T lymphocytes, decreased the expression pattern of IL-6 and IFN-γ and inhibited IBDV replication. The results herein presented demonstrate that this Lepidopteran virus shows antiviral activity in chickens under experimental conditions. Investigations under field conditions have to be done to probe this strategy as a valuable sanitary tool for the treatment and prevention of chicken diseases.

An Oral Salmonella-Based Vaccine Inhibits Liver Metastases by Promoting Tumor-Specific T-Cell-Mediated Immunity in Celiac and Portal Lymph Nodes: A Preclinical Study.

Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination.

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

Modulation of innate immunity in chickens induced by in vivo administration of baculovirus.

Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3(+)CD4(+) T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3(+)CD4(+) T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved.

Activation of the immune response against Infectious Bursal Disease Virus after intramuscular inoculation of an intermediate strain.

Infectious bursal disesase is a highly contagious, wide spread immunosuppressive chicken disease caused by the Infectious Bursal Disease Virus (IBDV). IBDV is a two segmented double-strand RNA virus, member of the Birnaviridae family. In order to study the interaction between IBDV and the immune system, chickens were exposed to an intermediate IBDV strain by intramuscular route, and using Real Time PCR the expression of a panel of avian cytokines and chemokines in duodenum, spleen and bursa of Fabricius was analyzed. Also, splenic nitrite (NO2) production and the frequencies of different mononuclear cell populations were evaluated by Griess reaction and flow cytometry, respectively. Intramuscular (i.m.) IBDV inoculation promoted an over expression of proinflammatory cytokines IL-6, IL-15 and gIFN in spleen, which correlated with an increase of gIFN plasma concentration measured by ELISA, together with an increment of NO2 concentration in splenocyte supernatants at 1dpi. Results obtained in the present work showed that IBDV of intermediate virulence, given i.m., induced similar effects to those previously described for highly virulent IBDV in early innate immune responses. Considering that the i.m. route is the route of choice for the delivery of new generation vaccines, and that the use of recombinant antigens also requires the addition of adjuvants for proper immune stimulation, results presented here could contribute to identify suitable cytokines to be used or to be stimulated when utilizing subunit vaccines, for the improvement of prevention tools for avian health.

Complete rejection of a T-cell lymphoma due to synergism of T-cell receptor costimulatory molecules, CD80, CD40L, and CD40.

The equal importance of the qualitative and quantitative characteristics of antigen presentation as well as the set of costimulatory signals provided by antigen presenting cells to T-cells in determining the outcome of T-cell responses at the time of antigen recognition is now clear. Moreover, an important function in innate mechanisms has been recently attributed to costimulatory molecules demonstrating their relevant role in different stages of immune response. In this paper, we demonstrated the ability of CD40L (CD154) and CD80 costimulatory molecules expression in a T-cell lymphoma to induce both T-cell dependent and independent immune responses leading to an important anti-tumor effect. CD40 expression by LBC cells enhanced only T-cell dependent anti-tumor immune response resulting in tumor rejection. Furthermore, this work represents the first report to describe complete tumor rejection after co-inoculation of lymphoma cells transfected with CD40L and CD80 in either presence or absence of CD40 expressing lymphoma cells. In addition, this synergistic effect resulted in long lasting immunity to parental tumor cells. Co-inoculation of tumor cells each genetically modified to express a different costimulatory molecule circumvents the need to co-transfect genetically unstable tumor cells and represents an option for those weakly or non-immunogenic tumors where either treatment alone proved to be inefficient. This strategy represents a promising approach for inducing anti-tumor immunity and provides a new rational design of cancer therapies.

Transgene expression enhancement in T-lymphoma cell lines.

In transfection protocols, the expression levels of the transgene is important to define, still is difficult to obtain in certain cell lines such as those derived from T-lymphoma cells. In this study we evaluate transgene expression kinetics in the presence and absence of two well known transcription activators such as phorbol-12-myristate13-acetate (PMA) and Ionomicin (IO). Three murine T lymphoma cell lines (LBC, EL4 and BW5147) were transfected by electroporation using green fluorescent protein (GFP) as a reporter gene and analyzed by flow cytometry. Addition of PMA/IO resulted in a significant increase of the Mean Fluorescence Intensity but not in GFP-positive cell percentages, either in transient or stable transfected LBC and EL4 cells. Remarkable, BW5147 cells showed low GFP induction with a significant increment only in stable transfected cells. Our results demonstrated that CMV promoter activity can be enhanced in transfected lymphoma cells by PMA/IO suggesting that transgene expression levels can be optimized by means of the use of transcription activators.

IL-2, IL-10, IL-15 and TNF are key regulators of murine T-cell lymphoma growth.

We studied the role of IL-2, IL-15, IL-10, TNF and IL-2 receptor complexes (IL-2R) produced constitutively by a T-cell lymphoma line (LBC) on their own proliferation. The constitutive expression of surface alpha, beta and gamma chains IL-2R was detected in tumor cells by flow cytometry. Using reverse-transcription PCR, mRNA for IL-2, IL-15, IL-10 and TNF were found to be present in LBC. In addition, tumor cells were found to constitutively express intracellular IL-2, IL-15, IL-10 and TNF. Despite the production of these cytokines by tumor cells, specific neutralising antibodies did not inhibit LBC proliferation; surprisingly, anti-IL-15 increased LBC cell growth. We also demonstrated that recombinant IL-2 or IL-15 enhanced LBC cell proliferation. Our data suggest that endogenous IL-2 and IL-15 may trigger the proliferation of lymphoma LBC cells, and so their growth could be regulated, at least partly, by IL-2/IL-15/IL-2R system. In addition, IL-10 and TNF, immunosuppressor and pro-metastatic cytokines, respectively, may promote the in vivo growth of the tumor. The fact that leukaemia-lymphoma cells produce simultaneously both IL-2 and IL-15 should be taken into consideration in the design of immunotherapy protocols directed to IL-2R.