IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity

Comorbidity associated to Ascaris suum infection during pulmonary fibrosis exacerbates chronic lung and liver inflammation and dysfunction but not affect the parasite cycle in mice.

Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice

Submitted by Nuzia Santos ([email protected]) on 2017-01-20T16:39:47Z No. of bitstreams: 1 ve_Nogueira_Denise_Multiple Exposures_CPqRR_2016.pdf: 7283772 bytes, checksum: 9287d516a1c1fa65a4453682a7bd231f (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-01-20T17:32:40Z (GMT) No. of bitstreams: 1 ve_Nogueira_Denise_Multiple Exposures_CPqRR_2016.pdf: 7283772 bytes, checksum: 9287d516a1c1fa65a4453682a7bd231f (MD5)Made available in DSpace on 2017-01-20T17:32:40Z (GMT). No. of bitstreams: 1 ve_Nogueira_Denise_Multiple Exposures_CPqRR_2016.pdf: 7283772 bytes, checksum: 9287d516a1c1fa65a4453682a7bd231f (MD5) Previous issue date: 2016Universidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, Brasil/Universidade Federal do Mato Grosso. Instituto de Biologia e Ciencia da Saude. Cuiabá, MT, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Departamento de Patologia Geral. Instituto de Ciencias Biologicas. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Fisiologia e Biofisica Laboratorio de Imunologia e Mecânica Pulmonar. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Fisiologia e Biofisica Laboratorio de Imunologia e Mecânica Pulmonar. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Departamento de Patologia Geral. Instituto de Ciencias Biologicas. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Laboratorio de Imunologia Celular e Molecular. Belo Horizonte, Brasil/University of Nottingham. United KingdomUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Fisiologia e Biofisica Laboratorio de Imunologia e Mecânica Pulmonar. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciencias Biologicas. Departamento de Parasitologia. Laboratorio de Imunologia e Genomica de Parasitos. Belo Horizonte, MG, Brasil/Universidade Federal do Mato Grosso. Instituto de Biologia e Ciencia da Saude. Cuiabá, MT, BrasilAscaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host's homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris

Mononuclear and granulocyte cell counting in the blood at different time points after <i>A</i>. <i>suum</i> experimental infection.

<p>(A) Lymphocyte cell counts. (B) Monocyte cell count. (C) Neutrophil cell counts. (D) Eosinophil cell counts. Filled circles–non-infected group; Open circles–single-infected group; and divided circles–reinfected group. Two-way ANOVA test followed by multiple comparison test were used to compare the variances between the groups. Results are shown as the mean ± SEM and were represented ‘*’ and ‘#’. * <i>p</i>< 0.05; ** <i>p</i>< 0.01 *** <i>p</i>< 0.001 and **** <i>p</i>< 0.0001 represent the differences between all groups in the respective time; and # <i>p</i><0.05 and ## <i>p</i><0.01 represent the differences to the non-infected group.</p

Histopathological visualization of lesions caused by larval migration in the liver on the 4^th day post-infection and area of the lesions caused by larval migration.

<p>(A and B) Non-infected mouse; (C and D) Single-infected mouse with the presence of necrosis (arrowheads) and mild inflammatory infiltrate (arrows); (E and F) Reinfected mouse with presence of intense inflammatory infiltrate (arrows) and necrosis (*). Lower magnification Bar scale = 50 μm. Higher magnification Bar scale = 200 μm. (G) Area of lesion caused by larval migration in liver on the 4^th day post-infection. Mann-Whitney test was used to evaluate differences between groups.</p

Experimental design of <i>Ascaris suum</i> single- and reinfection.

<p>Experimental design of <i>Ascaris suum</i> single- and reinfection.</p

Assessment of lung mechanics after single or multiple <i>Ascaris</i> infection in mice.

<p>Forced spirometry was performed to investigate the injury by modifications in lung functions. The parameters assessed were Functional Vital Capacity (A), Inspiratory Capacity (B), Dynamic Compliance Forced (C), Chord Compliance (D), Expiratory Volume at 100 msec (E) and Lung Resistance (F). Kruskal-Wallis test followed by Dunn´s multiple comparisons test was used to evaluate differences among groups. Results are shown as the mean ± SEM. * <i>p</i> < 0.05; ** <i>p</i> < 0.01.</p

Number of larvae recovered from host organs.

<p>(A) Liver on the 4^th day post-infection; (B) lung on the 8^th day post-infection; (C) BAL on the 8^th day post-infection; and (D) gut on the 12^th day post-infection. Filled circles–single infection (SI) group; Open circles–reinfection (RE) group. Mann-Whitney test was used to assess differences between groups and are depicted in the graphs by the p values.</p

Histopathological visualization of the lesion caused by larval migration in the lungs on the 8^th day post-infection and area of the lesion caused by larval migration.

<p>(A and B) Non-infected mouse; (C and D) Single-infected mouse with slight thickening of the septum at the expense of inflammatory infiltrates, hyperaemia and haemorrhage (*); (E and F) Reinfected mouse with inflammatory infiltrate, intense hyperaemia and haemorrhage, causing extensive thickening of the septum (*). Lower magnification Bar scale = 50 μm. Higher magnification Bar scale = 100 μm. (G) Area of the lesion caused by larval migration and inflammation in the lung on the 8^th day post-infection; Mann-Whitney test was used to assess differences between the groups. (H-I) Optical density representing the MPO and EPO activity in the lung at 8 days post-infection. (H) EPO production in the lung on the 8^th day post-infection. (I) MPO production in the lung on the 8^th day post-infection. Kruskal-Wallis test followed by Dunn´s multiple comparisons test was used to evaluate differences between groups. The p values in the graphs represent the significant differences.</p

Levels of haemoglobin, total protein, mononuclear and granulocyte cell counts in the BAL on the 8^th day post-infection.

<p>(A) Haemoglobin levels in BAL on the 8^th day post-infection. (B) Larvae from <i>A</i>. <i>suum</i> surrounded by leukocytes in the BAL 8 days post-infection. (C) Total protein levels in the BAL on the 8^th day post-infection. (D) Total leukocytes counts in the BAL on the 8^th day post-infection. (E) Macrophage counts in the BAL. (F) Lymphocyte cell counts in the BAL. (G) Neutrophil cell counts in the BAL. (H) Eosinophil cell counts in the BAL. Kruskal-Wallis test followed by Dunn´s multiple comparisons test was used to evaluate differences among the groups. Results are shown as the mean ± SEM and were represented * and # for was used where * <i>p</i>< 0.05; ** <i>p</i>< 0.01 and *** <i>p</i>< 0.001 for the differences among all groups in the respective time; and # <i>p</i>< 0.05 and ## <i>p</i><0.01 for differences to the control group at the same time.</p