Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189-201 and LLO91-99 and the GAPDH peptide, GAPDH1-22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1-22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91-99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1-22-specific CD4(+) and CD8(+) immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4(+) and CD8(+) epitopes

A Novel Therapy for Melanoma Developed in Mice: Transformation of Melanoma into Dendritic Cells with Listeria monocytogenes

Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4+ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8+ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal

Listeriosis cerebral en el modelo murino : patogénesis y prevención

RESUMEN: La microglía, células inmunes innatas del cerebro, juega un papel central en la listeriosis cerebral. En este trabajo se muestra como la microglía controla la infección por Listeria de manera diferente que los macrófagos. La infección de cultivos primarios microgliales y de líneas celulares murinas con Listeria dio lugar a dos patrones de expresión de genes implicados en la respuesta inmune en macrófagos. Mientras que el gen bacteriano hly parece ser responsable de ambos patrones transcripcionales en los macrófagos, Listeria induce en la microglía sólo la vía transcripcional regulada por el factor de necrosis tumoral (TNF). Listeria también reprime en microglía la respuesta inmune tardía recogida en dos grupos, la degradación microbiana y los genes inducibles por interferón (IFN). El gen bacteriano actA fue requerido en microglía para inducir respuestas reguladas por TNF y para reprimir la respuesta tardía. El aislamiento de fagosomas microgliales reveló un ambiente fagosomal incapaz de destruir Listeria. Esta estrategia transcripcional en microglía indujo altos niveles de TNF-α y MCP-1 y baja producción de otros compuestos neurotóxicos como el óxido nítrico, H2O2 e IFN tipo I. Esto disminuye la apoptosis neuronal. Esta estrategia bacteriana global sugiere que la microglía limita el patrón de inflamación de Listeria a través de respuestas mediadas por TNF para preservar la integridad del cerebro. Los casos clínicos de listeriosis neonatal se asocian con morbilidades cerebrales y la pérdida de fetos debido a complicaciones en el embarazo temprano o tardío, argumentando que la función de la microglía podría resultar alterada. Nosotros relacionamos la apoptosis microglial con listeriosis neonatal y morbilidad cerebral asociada a la listeriosis, proponiendo una nueva vacuna que revierte todos los efectos de la listeriosis y confiere inmunidad específica a LM.ABSTRACT: Microglia, the innate immune cells of the brain, plays a central role in cerebral listeriosis. In ths work, we present evidence that microglia control Listeria infection differently than macrophages. Infection of primary microglial cultures and murine cell lines with Listeria resulted in a dual function of the two gene expression programmes involved in immune responses in macrophages. Whereas the bacterial gene hly seems responsible for both transcriptional programmes in macrophages, Listeria induces in microglia only the tumor necrosis factor (TNF)-regulated transcriptional programme. Listeria also represses in microglia the late immune response gathered in two clusters, microbial degradation, and interferon (IFN)-inducible genes. The bacterial gene actA was required in microglia to induce TNF-regulated responses and to repress the late response. Isolation of microglial phagosomes revealed a phagosomal environment unable to destroy Listeria. This transcriptional strategy in microglia induced high levels of TNF-α and MCP-1 and low production of other neurotoxic compounds such as nitric oxide, hydrogen peroxide, and Type I IFNs. It´s display a low potential to trigger neuronal apoptosis. This overall bacterial strategy strongly suggests that microglia limit Listeria inflammation pattern through TNF-mediated responses to preserve brain integrity. Clinical cases of neonatal listeriosis are associated with brain morbidities and the loss of fetuses due to complications in early or late pregnancy, arguing microglia function might result altered. We link microglia apoptosis with neonatal listeriosis and listeriosis-associated brain morbidities, proposing a new vaccine that reverts all listeriosis effects and confer LM specific immunity.Este trabajo se ha financiado gracias a los proyectos: MINECO: SAF2009-08695 y SAF2012-34203; IDIVAL: INNVAL15/01; ISCIII: CIBER CIB16-NM009 y FIPSE 00- 00002755-1

A gold glyco-nanoparticle carrying a Listeriolysin O peptide and formulated with Advax™ delta inulin adjuvant induces robust T-cell protection against listeria infection

AbstractIn the search for an effective vaccine against the human pathogen, Listeria monocytogenes (Listeria), gold glyconanoparticles (GNP) loaded with a listeriolysin O peptide LLO91–99 (GNP-LLO) were used to immunise mice, initially using a dendritic cell (DC) vaccine approach, but subsequently using a standard parenteral immunisation approach. To enhance vaccine immunogenicity a novel polysaccharide adjuvant based on delta inulin (Advax™) was also co-formulated with the GNP vaccine. Confirming previous results, DC loaded in vitro with GNP-LLO provided better protection against listeriosis than DC loaded in vitro using free LLO peptide. The immunogenicity of GNP-LLO loaded DC vaccines was further increased by addition of Advax™ adjuvant. However, as DC vaccines are expensive and impracticable for prophylactic use, we next asked whether the same GNP-LLO antigen could be used to directly target DC in vivo. Immunisation of mice with GNP-LLO plus Advax™ adjuvant induced LLO-specific T-cell immunity and protection against Listeria challenge. Protection correlated with an increased frequency of splenic CD4+ and CD8+ T cells, NK cells and CD8α+ DC, and Th1 cytokine production (IL-12, IFN-γ, TNF-α, and MCP-1), post-challenge. Enhanced T-cell epitope recruitment post-challenge was seen in the groups that received Advax™ adjuvant. Immunisation with GNP-LLO91–99 plus Advax™ adjuvant provided equally robust Listeria protection as the best DC vaccine strategy but without the complexity and cost, making this a highly promising strategy for development of a prophylactic vaccine against listeriosis

Pregnancy Vaccination with Gold Glyco-Nanoparticles Carrying Listeria monocytogenes Peptides Protects against Listeriosis and Brain- and Cutaneous-Associated Morbidities

Listeriosis is a fatal infection for fetuses and newborns with two clinical main morbidities in the neonatal period, meningitis and diffused cutaneous lesions. In this study, we vaccinated pregnant females with two gold glyconanoparticles (GNP) loaded with two peptides, listeriolysin peptide 91–99 (LLO91–99) or glyceraldehyde-3-phosphate dehydrogenase 1–22 peptide (GAPDH1–22). Neonates born to vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice presented clear brain affections and cutaneous diminishment of melanocytes. Therefore, these nanoparticle vaccines are effective measures to offer pregnant mothers at high risk of listeriosis interesting therapies that cross the placenta

Frequencies of LLO_296–304 specific CD8⁺ T cells in spleens of B16F10 treated mice or non-treated after LM^WT therapy.

<p>^aC57BL/6 mice non-treated with murine melanoma were inoculated with saline <i>i</i>.<i>p</i> for 7 days and next injected <i>i</i>.<i>p</i> with LM^WT for 3 days as described in <i>Materials and Methods</i>. Splenocytes from mice treated with LM^WT were incubated with recombinant dimeric H-2K^b: Ig fusion protein (BD Biosciences) loaded with LLO_296–304 peptide. The staining cocktail contained the dimeric fusion protein loaded with the peptides, CD8 and anti-IFN-gamma antibodies. CD8⁺ cells were gated for anti-IFN-gamma staining (% Gated dimer-CD8) to calculate the frequencies of CD8⁺-LLO_296–304. Results are expressed as percentages of triplicate samples ± SD. <i>P</i><0.05.</p><p>^bB16F10 murine melanoma were inoculated <i>i</i>.<i>p</i> into C57BL/6 mice for 7 days and next injected <i>i</i>.<i>p</i> with LM^WT for 3 additional days as described in <i>Materials and Methods</i>. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. <i>P</i><0.05.</p><p>^cB16F10 murine melanoma pre-infected with LM^WT was inoculated into C57BL/6 mice for 7 days as described in Materials and Methods. Splenocytes from mice were incubated with recombinant dimeric H-2Kb: Ig fusion protein as in a. <i>P</i><0.05.</p><p>Frequencies of LLO_296–304 specific CD8⁺ T cells in spleens of B16F10 treated mice or non-treated after LM^WT therapy.</p

<i>Listeria</i> infection of melanoma induces nitric oxide and iNOS expression.

<p>^aB16F10 murine melanoma or BMDC were infected with LM^WT (B16F10-LM^WT, BMDC-LM^WT) or non-infected (B16F10-NI, BMDC-NI) for 24 hours.</p><p>^bNO produced was measured in cell supernatants. Results are expressed as nmol of NO produced by 10⁵ cells (mean ± SD, <i>P</i><0.005) obtained with triplicate samples.</p><p>^cCell surfaces markers of B16F10 and BMDC infected or not with LM^WT were analysed by FACS using the following antibodies: CD11c-PE, iNOS-FITC, CD86-V450 and MHC-II-APC. Samples were acquired using FACSCanto flow cytometer. Results are expressed as the percentages of positive cell (mean ± SD, <i>P</i><0.005).</p><p><i>Listeria</i> infection of melanoma induces nitric oxide and iNOS expression.</p

<i>Listeria</i> vaccination of melanoma shows a dual action, tumour regression by apoptosis and bacterial clearance.

<p><b><i>A</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) for 7 (7-D) or 15 days (15-D). Mice were bled, sacrificed and treated for histological analysis as described in <i>Material and Methods</i>. Images correspond to sections of peritoneum infiltrates or liver metastases. <b><i>B</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) as in <b><i>A</i></b> for none (NT), 7 (7-D) or 15 days (15-D). Spleens from sacrificed mice (n = 5) were stained for histological analysis using different antibodies as described in <i>Material and Methods</i> and images correspond to sections. Results are expressed as percentages of positive cells (mean ± SD) (<i>P</i> < 0.05). <b><i>C</i></b>, C57BL/6 female were inoculated <i>i</i>.<i>p</i>. with 5 x 10⁵ B16F10/mice (n = 5) for 7 days and next injected <i>i</i>.<i>p</i>. or not (NT) with 5 x 10³ bc/mice of different LM strains (LM^WT or LM^ΔLLO) for 5 additional days. Mice were sacrificed, bled to collect sera and photographed before collecting melanoma and lungs. Images correspond to the peritoneum of mice and the recovered melanoma. Plots correspond to measurements of diameters of collected melanoma. Results are expressed as the mean ± SD (<i>P</i> < 0,05). <b><i>D</i></b>, Melanoma recovered from LM^WT or LM^ΔLLO vaccinated mice or from non-vaccinated mice (NV) as in <b><i>C</i></b> were analysed for early and late apoptosis by FACS according to <i>Materials and Methods</i> after double staining with 7-AAD (IP labelled) and annexin V (anexina labelled). Results are expressed as the percentages of late apoptotic cells, necrotic death, (Q2 area corresponding to double positive for 7-AAD and annexin V cells) and the percentages of early apoptotic cells, programmed cell death (Q4 area corresponding to annexin V positive while 7-AAD negative cells) (mean ± SD) (<i>p</i> < 0.05).</p