Pathogenesis of domestic pigs submitted to mycobacterial sensitizations previous to experimental infection with Mycobacterium bovis

Aim of study: To demonstrate the virulence of a Mycobacterium bovis local pig isolate in order to contribute to a better understanding of the pathological and immunological consequences of M. bovis infection in previous sensitized animals. Area of study: Buenos Aires, Argentina Material and methods: One group of ten pigs received two oral doses of killed M. bovis suspension and a comparative intradermal tubercu-lin test (CIT) (multiple sensitized) and then was infected with the M. bovis strain. Another group only received the CIT (single sensitized) and the infective dose. Humoral immune response was followed monthly, and gross pathology, histopathological and bacteriological analysis were performed at necropsy 100 days after infection. Main results: M. bovis oral infection induced lesions and allowed bacterial growth in most of the animals. Previous sensitization with killed M. bovis suspension slightly raised the intensity of the response, as the multiple sensitized group showed higher lesion scores and humoral response. Research highlights: Although the differences in lesion scores were not statistically significant, oral route infection after sensitization can modify the course of infections towards a fast development of lesions with a higher fibrotic component suggestive of increased resistance to infection in the right conditions.Fil: Cuerda, Maria Ximena. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Colombatti Olivieri, María Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Gravisaco, María J.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Marfil, Maria Jimena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barandiaran, Soledad. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sevilla, Iker A.. Centro de Investigación. Neiker - Tecnalia; EspañaFil: Garrido, Joseba M.. Centro de Investigación. Neiker - Tecnalia; EspañaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Zumárraga, Martín José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Juste, Ramón A.. Centro de Investigación. Neiker - Tecnalia; EspañaFil: Santangelo, María de la Paz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Baculovirus Capsid Display Potentiates OVA Cytotoxic and Innate Immune Responses

Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination

Innate immune response in mice injected with BV.

<p>(A) Induction of inflammatory cytokines by BV. C57BL/6 mice were i.v. injected with 5×10⁷ PFU BV (•) or with supernatants of uninfected insect cells (SN, □), and at the times shown sera were collected and assayed for IL-6, IFN-γ and IL12p40 by ELISA. Sera collected before the injection were employed as control. Results are representative of two independent experiments. The line depicted in each group corresponds to the mean of each group of dots. (B, C) Activation of NK, NKT and T cells by BV. C57BL/6 mice were i.v. injected with 5×10⁷ PFU BV, and 3 hours post injection splenic populations were incubated in the presence of BFA for an additional 6 hours. Then, cells were labeled for CD49b and CD3 markers and intracellular IFN-γ. (B) A representative dot plot of the proportion of NK (CD49b+ CD3-), NKT (CD49b+ CD3+) and T (CD49b- CD3+) cells in the spleen of a control mouse and (C) the percentage of NK, NKT and T cells (left) and proportion of IFN-γ+ NK, NKT and T cells after BV injection (right) are shown. Results are representative of two independent experiments and are expressed as mean +/− SEM (n = 4). (D) <i>In vivo</i> maturation of DCs by BV. C57BL/6 mice were injected i.v. with saline, 5×10⁷ PFU BV-WT and 30 ng or 30 µg CpG-containing oligodeoxinucleotide (CpG-ODN) 1826. Eighteen hours later, their CD11c+ spleen cells were labeled with anti-CD86 and analyzed on a FACSCanto II flow cytometer. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) and as the frequency of CD86+ cells in total DCs. Results are representative of at least two independent experiments.</p

BV induce BMDCs maturation.

<p>(A) Maturation of BMDCs by BV. BMDCs were incubated for 18 hours with BV-WT or mock. Then, cells were stained with anti-CD11c and one of the following antibodies: anti-CD40, CD86 or I-A^b, and analyzed on a FACSCanto II. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) for each indicated molecule in total DCs. (B) Production of inflammatory cytokines. BMDCs from C57BL/10 ScCr (TLR4 deficients) and C57BL/6 (TLR4 competents) were incubated for 18 hours with BV-WT, or a supernatant from mock infected Sf9 cells (SN), and then levels of IL-6 and IL-12 p40 were determined in supernatants of BMDCs by ELISA. Results are representative of two independent experiments.</p

Anti-OVA CTL response in mice injected with BV-OVA is long-lasting and not affected by repeated immunization with the same vector.

<p>(A, B) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. One hundred and ten days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered to determine (A) the percentage of specific <i>in vivo</i> killing by flow cytometry and (B) IFN-γ content by ELISA in supernatants of spleen cells from immunized mice, cultured for 48 hours in the presence of OVA protein. (C) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT or PBS. Seven and fourteen days later, spleen cells were recovered and cultured with BV-WT (MOI 5) for 48 hours. The IFN-γ content in supernatants was determined by ELISA (D) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. Fifteen days later, mice received a second i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS, combined as shown in the figure. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered and the percentage of specific <i>in vivo</i> killing was determined by flow cytometry. *, p<0.05; ***, p<0.001. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 5).</p

Estimation of the number of CpG motifs in AcNMPV genome.

<p>The frequency at which each CpG hexamer appeared in the BV genome was determined by using the GenBank accession number for the complete genome of AcNPV NC 001623.</p

BV-OVA induces CD8 T cell activation.

<p>Splenic CD11c+ cells (2×10⁵) purified from C57BL/6 mice were incubated with BV-WT, BV-OVA or BV-OVAsur at the indicated multiplicity of infection (MOI) for 120 minutes and washed twice. Then, DCs were cultured for 3 days with CFSE-labeled CD8 T cells from OT-I mice (having a transgenic T cell receptor which recognizes OVA_257-264 in the context of H-2K^b). T cell proliferation and CD25 expression were analyzed by flow cytometry, after labeling cells with an anti-CD3 antibody and exclusion of dead cells with 7AAD. (A) A representative dot plot overlay of cell proliferation vs CD25 expression of CD8 T cells cultured with DCs, which were preincubated with BV-OVA (black dots) or BV-WT (gray dots) at a MOI of 30. (B) Percentage of CD8 T cells undergoing one or more rounds of proliferation and (C) IFN-γ content, assessed by ELISA, in supernatants of culture of CD8 T cells with DCs preincubated with BV-OVA or BV-WT. (D) T cell proliferation and (E) CD25 expression of CD8 T cells co-cultured with DCs preincubated with BV-OVA (▪), BV-OVAsur (▴) or BV-WT (○). Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 4).</p

Anti-OVA CTL response in mice injected with BV-OVA.

<p>C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA, BV-OVAsur, BV-WT + OVA (30 ng or 1 mg), OVA alone (1 mg) or PBS. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes as target cells. (A) A representative histogram of remaining CFSE^high and CFSE^low cells in control and BV-OVA immunized mice 20 hours after injection of target cells is shown. (B) Percentage of specific <i>in vivo</i> killing of one representative experiment. (C) IFN-γ content in supernatants of spleen cells from immunized mice determined by ELISA. Spleen cells were recovered and cultured for 48 hours in the presence of OVA protein or BV-WT. As control, spleen cells without stimulus were also cultured. *, p<0.05; **, p<0.01. (D, E) IFN-γ intracellular staining on splenocytes from immunized mice with 30 ng OVA+BV-WT, BV-WT, BV-OVA, OVA alone or PBS, as indicated above. Spleen cells were recovered and cultured for 12 hours in the presence of OVA protein or OVA_256-264, and incubated in the presence of brefeldin A for 6 additional hours. Then, cells were labeled for CD4 or CD8 markers and intracellular IFN-γ. (D) A representative dot plot is shown. (E) Percentage of IFN-γ+ CD4 or CD8 T cells. **, p<0.01; ***, p<0.001. (F) Comparison of specific <i>in vivo</i> killing between mice immunized with BV-OVA vs BV-OVAsur. ***, p<0.001. (G) Comparison of the frequency of IFN-γ-producing CD8 T cells in splenocytes from BV-OVA vs BV-OVAsur immunized mice. **, p<0.01. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 4).</p

Therapeutic effects of Salmonella Typhi in a mouse model of T-cell lymphoma

In this study, we assessed the effectiveness of a live, attenuated Salmonella enterica serovar Typhi (S. Typhi) vaccine strain as a cancer immunotherapy in a mouse model of metastatic T-cell lymphoma. EL4 tumor-bearing C57BL/6J mice immunized with S. Typhi strain CVD 915, by injection into the tumor and the draining lymph node areas, displayed a significant decrease in tumor growth, a reduction in the mitotic index (MI) of tumors, a delayed development of palpable lymph node metastases and most importantly improved survival, compared to untreated mice. Besides, complete tumor regression was achieved in a small number of bacteria-treated mice. A successful therapeutic response associated with a significant reduction of tumor mass was evident as early as 5 days after treatment. The administration of Salmonella to tumor-bearing mice promoted early cellular infiltration (mainly neutrophils) within the tumor, and was accompanied by a decreased intratumoral interleukin 10 production as well as by leukocyte expansion in tumor draining lymph nodes. A tumor-specific memory immune response was induced in most of cured animals, as evidenced by the lack of tumor growth after a rechallenge with the same tumor. EL4 cells cultured with live Salmonella failed to proliferate and underwent apoptosis in a dose-dependent, timedependent, and contact-dependent manner. To our knowledge, these results demonstrate for the first time the efficacy of a S. Typhi vaccine strain as an oncolytic and immunotherapeutic agent against a highly malignant tumor and support the use of S. Typhi-based vaccine strains in cancer therapy.Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Gravisaco, María J.. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias; ArgentinaFil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Pasetti, Marcela F.. University of Maryland; Estados UnidosFil: Herschllik, Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: de Toro, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Rodriguez, Carla Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Comision Nacional de Energia Atomica; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Larotonda, Gerardo. Lavet Lab Argentina; ArgentinaFil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin