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

Avaliação da eficácia vacinal e imunogenicidade de antígenos de Ascaris suum na ascaridíase experimental

Exportado OPUSMade available in DSpace on 2019-08-13T22:27:09Z (GMT). No. of bitstreams: 1 disserta__o_ana_clara_gazzinelli___vers_o_final_arquivo.pdf: 3215597 bytes, checksum: c2ec7504e6b55a14aea20d6baa25b472 (MD5) Previous issue date: 17A ascaridíase humana possui uma distribuição cosmopolita sendo caracterizada também como a doença tropical mais prevalente e negligenciada do mundo. Estudos recentes estimam que aproximadamente 800 milhões de pessoas estejam infectadas em todo o mundo. Estratégias para intervenções profiláticas contra a ascaridíase humana são limitadas, uma vez que o sistema de educação em saúde e saneamento básico nas regiões endêmicas para a doença são extremamente precários, o que resulta em elevadas taxas de reinfecção mesmo após tratamento específico. Em busca da melhoria da qualidade de vida, de uma menor morbidade principalmente nos indivíduos de áreas endêmicas, buscamos o desenvolvimento de uma vacina que fosse capaz de garantir a proteção a longo prazo contra uma das principais geohelmintoses em questão. Dessa forma, por meio da imunização com diferentes antígenos de Ascaris suum, sendo: 1extrato bruto de verme adulto; 2extrato bruto de tegumento de verme adulto; e 3extrato bruto de larva infectante, o presente estudo objetivou a identificação de novos candidatos vacinais visando o desenvolvimento de uma nova estratégia profilática contra a ascaridíase. Neste estudo avaliou-se a eficácia vacinal por meio da quantificação da carga parasitária no pulmão e no lavado bronco alveolar dos animais previamente imunizados e posteriormente desafiados com ovos infectantes do parasito. Além disso, foram realizados a avaliação da imunogenicidade dos candidatos vacinais por meio do perfil de citocinas sistêmicas e teciduais, além do funcionamento pulmonar e o perfil de leucócitos presentes no lavado bronco alveolar. Por fim, foram também avaliados a participação humoral na proteção vista da carga parasitária e o perfil de produção de anticorpos após as imunizações e após infecção. Nossos resultados demonstraram uma redução significativa da carga parasitária nos animais imunizados com os antígenos de A. suum sendo o antígeno bruto de larva chegando a aproximadamente 61% de proteção, seguido pelo antígeno bruto de tegumento com 59% e por fim o antígeno bruto de verme adulto com 51% de redução da carga. Quanto ao perfil de citocinas sistêmicas foi observado uma relevante regulação da citocina IL-5 nos animais imunizados com os diferentes antígenos de A. suum, e uma significativa produção de IL-10 induzida pelo antígeno TEG em relação aos animais não imunizados. Quanto aos níveis de anticorpos IgG antígeno-específico, foi observado um aumento gradual e progressivo conforme as imunizações com um aumento principalmente das subclasses IgG1 e IgG3. Posteriormente, diante da investigação da participação humoral na redução da carga, observamos que animais submetidos ao protocolo de transferência passiva com anticorpos anti-antígenos de A. suum, apresentaram cerca de 65% de proteção frente a infecção. A partir destes resultados, podemos inferir que os antígenos de A. suum são potenciais candidatos à investigação e nos provam que é viável a utilização e desenvolvimento de uma vacina, visando principalmente o controle e diminuição da carga parasitária nos indivíduos infectados das regiões endêmicas.Human ascariasis has a cosmopolitan distribution and is also characterized as the most prevalent and neglected tropical disease in the world. Recent studies estimate that about 800 million people are infected worldwide. Strategies for prophylactic interventions against human ascariasis are limited, since the education system in health and basic sanitation in regions endemic for the disease is extremely precarious, resulting in high reinfection rates even after specific treatment. In order to improve the quality of life and lower morbidity, especially in individuals from endemic areas, we are lookinf for the development of a vaccine that would guarantee the long-term protection against the main soil transmitted helminths in general. Thus, through immunization with different Ascaris suum antigens, being: crude extract of adult worm; crude extract of adult worm tegument; and crude extract of infective larvae, the present study aimed at the identification of new vaccine candidates aiming the development of a new prophylactic strategy against ascariasis. In this study we evaluated the vaccine efficacy by quantifying the parasite load in the lung and alveolar bronchoalveolar lavage of animals previously immunized and then challenged with infecting eggs of the parasite. In addition, the immunogenicity of the vaccine candidates was evaluated through the systemic and tissue cytokine profile, as well as the pulmonary function and the leukocyte profile present in the bronchoalveolar lavage. Finally, the humoral participation in the protection of parasite load and the profile of antibody production after immunizations and after infection were also evaluated. Our results demonstrated a significant reduction of parasite load in the animals immunized with A. suum antigens with the crude larval antigen reaching approximately 61% protection, followed by the crude tegument antigen with 59% and finally the crude adult worm antigen with 51% load reduction. Regarding the systemic cytokine profile, a significant regulation of the IL-5 cytokine in the animals immunized with the different A. suum antigens and a significant IL-10 production induced by the TEG antigen in relation to the non-immunized animals was observed. As for antigen-specific IgG antibody levels, a gradual and progressive increase was observed according to the immunizations with an increase mainly of IgG1 and IgG3 subclasses. Subsequently, in view of the investigation of the humoral participation in the reduction of the load, we observed that animals submitted to the passive transfer protocol with anti-antigen antibodies of A. suum presented approximately 65% protection against infection. From these results, we can infer that A. suum antigens are potential candidates for research and prove that it is feasible to use and develop a vaccine, mainly aimed at controlling and reducing the parasite burden in infected individuals in endemic regions

Regulatory monocytes in helminth infections: insights from the modulation during human hookworm infection

Abstract Background While the macrophage polarization is well characterized in helminth infections, the natural heterogeneity of monocytes with multiple cell phenotypes might influence the outcome of neglected diseases, such hookworm infection. Here, we report the profile of monocytes in human hookworm infections as a model to study the regulatory subpopulation of monocytes in helminth infections. Methods Blood samples were collected from 19 Necator americanus-infected individuals and 13 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated, and immunophenotyping was conducted by flow cytometry. The expressions of genes encoding human nitric oxide synthase (iNOS), interleukin 4 (IL-4), arginase-1 (Arg-1) and glyceraldehyde 3-phosphate dehydrogenase were quantified by qPCR. Plasma levels of IL-4 were determined by sandwich ELISA. Unpaired t-tests or Mann-Whitney tests were used depending on the data distribution. Results Hookworm infected individuals (HWI) showed a significant increase in the number of monocytes/mm3 (555.2 ± 191.0) compared to that of the non-infected (NI) individuals (120.4 ± 44.7) (p < 0.0001). While the frequencies of CD14+IL-10+ and CD14+IL-12+ cells were significantly reduced in the HWI compared to NI group (p = 0.0289 and p < 0.0001, respectively), the ratio between IL-10/IL-12 producing monocytes was significantly elevated in HWI (p = 0.0004), indicating the potential regulatory activity of these cells. Measurement of IL-4 levels and gene expression of IL-4 and Arg-1 (highly expressed in alternatively activated macrophages) revealed no significant differences between the NI and HWI groups. Interestingly, individuals from the HWI group had higher expression of the iNOS gene (associated with a regulatory profile) (20.27 ± 2.97) compared to the NI group (11.28 ± 1.18, p = 0.0409). Finally, individuals from the HWI group had a significantly higher frequency of CD206+CD23+IL-10+ (7.57 ± 1.96) cells compared to individuals from the NI group (0.35 ± 0.09) (p < 0.001), suggesting that activated monocytes are a potential source of regulatory cytokines during hookworm infection. Conclusions Natural hookworm infection induces a high frequency of circulating monocytes that present a regulatory profile and promote the downmodulation of the proinflammatory response, which may contribute to prolonged survival of the parasite in the host

CD4+ T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways

Submitted by Repositório Arca ([email protected]) on 2019-04-24T17:44:00Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-08-22T12:31:14Z (GMT) No. of bitstreams: 2 ve_Hojo-Souza_Natália_etal_INI_2015.pdf: 673291 bytes, checksum: 5de19dfdb1440f05f52dfb3676dbc81a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-22T12:31:14Z (GMT). No. of bitstreams: 2 ve_Hojo-Souza_Natália_etal_INI_2015.pdf: 673291 bytes, checksum: 5de19dfdb1440f05f52dfb3676dbc81a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Research Centre in Tropical Medicine. Porto Velho, RO, Brazil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Research Centre in Tropical Medicine. Porto Velho, RO, Brazil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro, RJ, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Federal University of Minas Gerais. Institute of Biological Science. Department of Parasitology. Belo Horizonte, MG, Brasil.Background: Reduction in the number of circulating blood lymphocytes (lymphocytopaenia) has been reported during clinical episodes of malaria and is normalized after treatment with anti-malaria drugs. While this phenomenon is well established in malaria infection, the underlying mechanisms are still not fully elucidated. In the present study, the occurrence of apoptosis and its pathways in CD4+ T cells was investigated in naturally Plasmodium vivax-infected individuals from a Brazilian endemic area (Porto Velho – RO). Methods: Blood samples were collected from P. vivax-infected individuals and healthy donors. The apoptosis was characterized by cell staining with Annexin V/FITC and propidium iodide and the apoptosis-associated gene expression profile was carried out using RT2 Profiler PCR Array–Human Apoptosis. The plasma TNF level was determined by ELISA. The unpaired t-test or Mann–Whitney test was applied according to the data distribution. Results: Plasmodium vivax-infected individuals present low number of leukocytes and lymphocytes with a higher percentage of CD4+ T cells in early and/or late apoptosis. Increased gene expression was observed for TNFRSF1B and Bid, associated with a reduction of Bcl-2, in individuals with P. vivax malaria. Furthermore, these individuals showed increased plasma levels of TNF compared to malaria-naive donors. Conclusions: The results of the present study suggest that P. vivax infection induces apoptosis of CD4+ T cells mediated by two types of signaling: by activation of the TNFR1 death receptor (extrinsic pathway), which is amplified by Bid, and by decreased expression of the anti-apoptotic protein Bcl-2 (intrinsic pathway). The T lymphocytes apoptosis could reflect a strategy of immune evasion triggered by the parasite, enabling their persistence but also limiting the occurrence of immunopathology

New insights into the immunopathology of early Toxocara canis infection in mice

BACKGROUND: Nematodes of the genus Toxocara are cosmopolitan roundworms frequently found in dogs and cats. Toxocara spp. can accidentally infect humans and cause a zoonosis called human toxocariasis, which is characterized by visceral, ocular or cerebral migration of larval stages of the parasite, without completing its life cycle. In general, chronic nematode infections induce a polarized T(H)2 immune response. However, during the initial phase of infection, a strong pro-inflammatory response is part of the immunological profile and might determine the outcome and/or pathology of the infection. METHODS: Parasitological aspects and histopathology during larval migration were evaluated after early T. canis experimental infection of BALB/c mice, which were inoculated via the intra-gastric route with a single dose of 1000 fully embryonated eggs. Innate immune responses and systemic cytokine patterns (T(H)1, T(H)2, T(H)17 and regulatory cytokines) were determined at different times after experimental challenge by sandwich ELISA. RESULTS: We found that experimental infection with T. canis induced a mix of innate inflammatory/T(H)17/T(H)2 responses during early infection, with a predominance of the latter. The T(H)2 response was evidenced by significant increases in cytokines such as IL-4, IL-5, IL-13 and IL-33, in addition to increasing levels of IL-6 and IL-17. No significant increases were observed for IL-10, TNF-α or IFN-γ levels. In parallel, parasitological analysis clearly revealed the pattern of larval migration through the mouse organs, starting from the liver in the first 24 h of infection, reaching the peak in the lungs on the 3rd day of infection and finally being found numerously in the brain after 5 days of infection. Peripheral leukocytosis, characterized by early neutrophilia and subsequent eosinophilia, was remarkable during early infection. The tissue damage induced by larvae was evidenced by histopathological analysis of the organs at different time points of infection. In all of the affected organs, larval migration induced intense inflammatory infiltrate and hemorrhage. CONCLUSION: In conclusion, these new insights into early T. canis infection in mice presented here enabled a better understanding of the immunopathological events that might also occur during human toxocariasis, thus contributing to future strategies of diagnosis and control

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

Lung larva reduction (A) and stunted development (B) in mice immunized with rAs16 formulated with ISA720 on Day 8 after being challenged with 2,500 <i>A</i>. <i>suum</i> eggs.

<p>Lung larvae are presented as the mean ± S.D (N = 15). The asterisks indicate statistically significant differences in larval reduction compared to the PBS or adjuvant control groups (*<i>p</i>< 0.05, ***<i>p</i>< 0.001). The larvae collected from lung Baermann culture were observed under 4x objective lens.</p

Properties of recombinant As14 and As16 proteins expressed in <i>P</i>. <i>pastoris</i> X-33.

<p>(<b>A</b>) SDS-PAGE of purified rAs14 and rAs16 proteins (1 μg each). (<b>B</b>) Western blot probed with sera from mice infected with <i>A</i>. <i>suum</i> eggs (diluted 1:3,000) shows that only rAs16 was recognized, but not rAs14 (all antigens 50 ng). (<b>C</b>) Western blot with anti-rAs16 mouse sera (1:3,000). (<b>D</b>) Western blot with anti-rAs14 mouse sera (1:3,000) (all antigens 50 ng). (<b>E</b>) Sequence comparison between As16 and As14 proteins shows 47% sequence identity and 66% similarity.</p

Mouse immune responses to the immunization of rAs16 and rAs14.

<p><b>(A)</b> Anti-As16 and anti-As14 IgG1and IgG2a titers (Log10) in sera of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720, as measured by ELISA. The IgG1/IgG2a titer ratio for rAs16 immunization is higher than for the rAs14 immunization (2662:1 and 206:1, respectively). <b>(B)</b> Cytokine profiles (IFN-γ, IL-2, IL-4, IL-5 and IL-10) of BALB/c mice immunized with rAs16 and rAs14 formulated with ISA720. Cytokine levels were determined in supernatants of splenocytes after being re-stimulated with rAs16 or rAs14 (25 μg/ml) for 48 hours. Results are shown as means ± standard deviation (SD) and individual data points for each group (n = 5), **<i>p</i><0.01, NS, non-significant.</p

Protective immunity induced by immunization of mice with rAs16 formulated with different adjuvants (Alhydrogel, MPLA and Addavax).

<p>(<b>A</b>) Lung larval count on Day 8 after challenge with 2,500 <i>A</i>. <i>suum</i> eggs. Values are presented as the mean ± S.D. The asterisks indicate statistically significant differences (*<i>p</i> < 0.05) in lung larval reduction compared to the PBS and adjuvant control groups (n = 15). (<b>B</b>) Anti-As16 IgG1 and IgG2a titers (Log₁₀) in sera from BALB/c mice immunized with rAs16 formulated with different adjuvants as measured by ELISA. Values are shown as means ± S.D and individual data points (n = 15). (<b>C</b>) Cytokine profiles (IL-2, IL-4, IL-5 and IL-10, IL-12, GM-CSF, IFN-γ and TNF-α) of BALB/c mice immunized with rAs16 formulated with different adjuvants. Cytokines detected in supernatants of splenocytes after stimulation with rAs16 (25 μg/ml) for 48 hours. Data are presented as means ± S.D and individual values for each group (n = 5). *p<0.05, **p<0.01 ***p<0.001, ****p<0.0001.</p