Humoral and cell-mediated immune responses to different doses of attenuated vaccine against avian infectious bronchitis virus

The antibody and cellular immune responses against infectious bronchitis virus (IBV) were evaluated at mucosal sites of chickens after immunization with various doses of an attenuated vaccine at 1 day of age. The correlation of these immune responses with protection of tracheal tissues was evaluated after experimental infection of these birds. Significantly reduced tracheal pathologic effects, measured according to ciliostasis and histology lesions, and reduced viral load were observed only in the full-dose vaccinated group at 5 days post-infection (dpi), while incomplete protection was observed for the subdose vaccinated groups. Moreover, birds of vaccinated groups, especially with full dose, developed higher levels of lachrymal IBV-specific IgG and IgA and increased the expression of cell-mediated immunity (CMI) genes, such as gamma interferon (IFNγ), CD8+ T cell marker, and granzyme homolog A more rapidly. In addition, these humoral and cellular immune responses evaluated at mucosal sites correlated significantly with tracheal protection against homologous IBV challenge in a vaccine dose-dependent manner. The results indicate that IgG, IgA and CD8+ T cell responses developed at mucosal sites after IBV vaccination of day-old chicks, could be taken as good correlates of protection against this virus. © 2013, Mary Ann Liebert, Inc.

Inflammatory and Cell-Mediated Immune Responses in the Respiratory Tract of Chickens to Infection with Avian Infectious Bronchitis Virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Energy deficit does not affect immune responses of experimentally infected pacu (Piaractus mesopotamicus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Humoral Immune Responses of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n = 5), orally in liquid form (n = 5), and subcutaneously (n = 6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n = 6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

CD25(+) T cell depletion impairs murine squamous cell carcinoma development via modulation of antitumor immune responses

Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1 beta, IL-10, IL-12, IL-13, interferon-gamma, transforming growth factor-beta and tumor necrosis factor-alpha, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.

5-Lipoxygenase Deficiency Impairs Innate and Adaptive Immune Responses during Fungal Infection

5-lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO-/- mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO-/- mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Infliximab partially impairs the anti-Mycobacterium tuberculosis immune responses of severe psoriasis patients with positive tuberculin skin-test

Background Infliximab and etarnecept are now widely used for treating severe psoriasis. However, these drugs, especially infliximab, increased the risk of tuberculosis reactivation. Surprisingly, epidemiological data suggest that the tuberculosis rate in patients taking infliximab in Sao Paulo State, Brazil, is similar to that of some developed, non-endemic countries. Objective The aim of this study was to better understand the effect of infliximab on Mycobacterium tuberculosis (Mtb) immune responses of psoriasis patients in an endemic setting (Brazil). Methods We evaluated the tuberculosis-specific immune responses of severe psoriasis patients and healthy individuals, both tuberculin skin test (TST) positive, in the presence/absence of infliximab. Patients had untreated severe psoriasis, no co-morbidities affecting the immune responses and a TST >10 mm. Healthy TST+ (>10 mm) individuals were evaluated in parallel. PBMC cultures from both groups were stimulated with different Mycobacterium tuberculosis (Mtb) antigens (ESAT-6, 85B and Mtb lysate) and phytohemagglutinin, with or without infliximab (5 mu g/mL). Parameters evaluated were TNF-alpha, IFN-gamma and IL-10 secretion by ELISA, overnight IFN-gamma ELISpot and lymphocyte proliferative response (LPR). Results Infliximab almost abolished TNF-alpha detection in PBMC supernatants of both groups. It also significantly reduced the LPR to phytohemagglutinin and the Mtb antigens as well as the IFN-gamma levels secreted into day 5 supernatants in both groups. There was no concomitant exaggerated IL-10 secretion that could account for the decreases in these responses. ELISpot showed that, contrasting with the central-memory responses above, infliximab did not affect effector-memory INF-gamma-releasing T-cell numbers. Conclusions Infliximab affected some, but not all aspects of the in vitro antituberculosis immune responses tested. The preserved effector-memory responses, putatively related to exposure to environmental mycobacteria, may help to explain the lower than expected susceptibility to tuberculosis reactivation in our setting. Received: 29 December 2010; Accepted: 9 March 2011

The spider acylpolyamine Mygalin is a potent modulator of innate immune responses

Mygalin is an antibacterial molecule isolated froth the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-gamma synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-gamma activation. In addition, Mygalin-activated macrophages produced TNF-alpha but not IL-1 beta, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-gamma and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection. (C) 2012 Elsevier Inc. All rights reserved.

Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection

Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Evaluation of the acquired immune responses to Plasmodium vivax VIR variant antigens in individuals living in malaria-endemic areas of Brazil

Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Maturation of humoral immune responses : Studies on the effects of antigen type, apoptosis and age

The humoral immune response is dependent on the formation of antibodies. Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the germinal center (GC) reaction. The GC is the main site for class switch, somatic hypermutation and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent antigens (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the T cell compartment is more affected by age than the B cell compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or antibody mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a plasma cell biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

Anti-parasitic and anti-viral immune responses in insects

Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.