Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

A vaccine against S. pyogenes: Design and experimental immune response

Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund`s adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe. (c) 2009 Elsevier Inc. All rights reserved.

Photodynamic therapy for American cutaneous leishmaniasis: The efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance. (C) 2011 Elsevier Inc. All rights reserved.

Influência do vírus da hepatite G (GBV-C) na resposta imune frente à infecção por Leishmania chagasi

GB virus type C (GBV-C) appears to promote a Th1 response and is associated with prolonged survival in HIV-infected people. L. chagasi causes a spectrum of illness that varies from severe visceral leishmaniasis, a disease that in the majority of cases is fatal if not treated, to self resolution of infection and development of positive DTH response that is protective against symptomatic disease. To determine if GBV-C viremia might influence the outcome of Leishmania infection, we characterized GBV-C status in a cohort of subjects residing in a L. chagasi endemic area in Brazil. GBV-C viremia was more prevalent in blood donors from urban than in periurban regions of Natal, Brazil (16% and 7.5% respectively). Evidence of prior GBV-C (anti-E2 antibodies) was detected in 24% and 12%of these groups respectively. Anti-E2 increased with age (p= 0.0121). No difference in GBV-C viremia was found in the DTH+ and VL groups (p= 0.269); however, subjects with visceral leishmaniasis were more likely to have anti-E2 than DTH+ subjects (p=0.0012), and DTH induration was smaller in subjects with E2 antibodies (4.5 mm) compared those without (7.12 mm) (p= 0.002). Furthermore, the size of the Leishmania DTH response was greater in GBV-C viremica subjects (6.8 mm) compared to non-viremic subjects (3.3 mm; p= 0.0054). There findings suggest that GBV-C virus may promote a type 1 immune response that could influence the outcome of Leishmania infection

Antígenos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Associação entre fatores nutricionais e a resposta frente à infecção por Leishmania chagasi

Leishmania chagasi infection presents a wide spectrum of clinical outcomes, ranging from asymptomatic self resolving infection to disease, visceral leishmaniasis (VL). The exact mechanisms that lead the evolution of infection to disease are not understood. It is believed that malnutrition is a risk factor associated with VL development, although there are few human studies in the area. We aimed to assess the nutritional factors associated with the response to L. chagasi infection in Rio Grande do Norte. The study was conducted from December 2006 to January 2008. 149 children were assessed: 20 active VL cases, 33 children with VL history, 40 DTH+ asymptomatic children and 56 DTH-. Nutritional status was assessed using z scores for Weight/Age, Weight/Height, Height/Age, Body Mass Index (BMI), and mid-upper arm circumference/height (MUAC/height). Vitamin A status was determined by serum retinol concentrations and the modified-relative-dose-esponse test (MRDR). Breastfeeding time and birth weight were also evaluated. VL children presented compromised nutritional status when compared to the other groups using BMI and MUAC/age, with means -1,53 ± 1,10 and -1,48 ± 1,28 z scores, respectively (ANOVA, p < 0,05). VL children also showed lower vitamin A levels: 43% presented serum retinol < 20 µg/dL and 15% MRDR > 0,060. Birth weight was inverserly associated with the risk to belong the VL group (β = -0,00; OR = 0,84; 95% CI 0,73 - 0,99; p = 0,047), whereas more breastfeeding time was directly associated with the risk to belong to the DTH+ group (β = 0,02; OD = 1,16; 95% CI 1,01 - 1,33; p = 0,036). The nutritional variables evaluated were associated with the response to the L. chagasi infection, with malnutrition and compromised vitamin A status as markers of children who present with VL. Higher birth weight was associated with protection to disease, and higher breastfeeding time was associated with increased likelihood of an asymptomatic infection. The results show that modifiable nutritional aspects in the study population are associated with the response to the L. chagasi infection

Saccharomyces Cerevisiae cell wall dietary supplementation on the performance and intestinal mucosa development and integrity of broiler chickens vaccinated against coccidiosis

This study was carried out to verify if Saccharomyces cerevisiae cell wall (SCCW) dietary supplementation (0.2%) was capable of protecting the intestinal mucosa of broiler chickens vaccinated against coccidiosis. Body weight gain, feed intake, feed conversion and intestinal mucosa morphometric parameters and epithelial loss were evaluated. In the experiment,400 day-old male chicks were distributed according to a completely randomized design in a 2x2 factorial arrangement. The following treatments were applied: T1 - no vaccination/ no SCCW supplementation; T2 - no vaccination/SCCW supplementation; T3 - vaccination/no SCCW supplementation; and T4 - vaccination/SCCW supplementation to four replicates of 25 birds each. Birds were vaccinated on the first day of age using a spray vaccine (Coccivac B®, Coopers), containing E. acervulina, E. maxima, E. mivati and E. tenella. S. cerevisiae cell wall was supplied from the first day of age. Live performance, intestinal morphometric parameters and epithelial loss were evaluated at 14, 21 and 28 days of age. Performance was affected by vaccination only at 21-days of age, when body weight gain was reduced in the vaccinated birds, but no body weight difference was observed on day 28. Vaccine also increased the crypt depth (p<0.05) in the duodenum and jejunum, suggesting a high cell activity in the crypt:villus transition area to maintain the epithelial cell turnover. Villi number/area (103,269 µm²) was not affected (p>0.05) by vaccine or cell wall supplementation, and epithelial loss was more pronounced in the duodenum and jejunum. In conclusion, the findings of this study suggest that S. cerevisiae cell wall supplementation may be an useful management tool to maintain the intestinal integrity of broilers vaccinated against coccidiosis.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

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

Humoral and cellular immune response generated by different vaccine programs before and after Salmonella Enteritidis challenge in chickens

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

Avaliação de dois clones de Escherichia coli recombinante quanto ao crescimento e expressão de antígenos de Leishmania chagasi (kmp11 e P36)

Visceral leishmaniosis caused by Leishmania chagasi, also known as calazar, presented, in the period from 1990 to 2005, tax of incidence in Brazil varying between 1 and 3 cases for 100 000 inhabitants. The Northeast region that up to the year of 2000 contributed with almost 90% of the registered cases is reducing his participation in the current decade, reaching 56% in 2005. Conventional leishmaniasis treatment is costly and it shows high toxicity, demanding more research for alternative treatments, with special interest in development of vaccines and diagnosis kits which include production of recombinant antigens by host cells. Escherichia coli has been the microorganism most studied and used as a host for recombinant protein production. Therefore, the aim of this work was to study the influence of induction on cellular growth and to verify the type of Leishmania chagasi antigens expression (intra or extracellular) during two recombinant E. coli clones (kmp11 and P36) cultivation in rotary incubator (shaker) using three different media (2xTY, TB, FASS+EL). For that, tests were carried out using conditions established in the literature for E. coli (37°C and 200 rpm) and media supplemented with antibiotics to guarantee that only competent cells grows. First, tests were carried out without induction in order to verify the two microorganisms kinetic behavior (growth and substrate consumption) in different media. Next, the induction was carried out through the addition of IPTG (1mM as final concentration), at the first hour of cultivation. It was observed that protein expression were intracellular for all clones and media tested, however the highest level of expression was clearly observed by the electrophoresis band density (intensity) for 2xTY medium and kmp11 protein. Although it contains the lowest substrate concentration, consequently, a reduced cellular concentration when compared to other media, it appeared that this medium and clone combination is the most indicated for recombinant protein production. Therefore, the objective of this work was achieved, since the interested proteins were produced. Consequently, this result motivates new studies for production optimization using different cultivation strategies

Influência das condições de cultivo na produção de antígenos recombinantes de Leishmania i. chagasi utilizando Escherichia coli M15 cultivada em incubador rotativo e biorreator

Escherichia coli has been one of the most widely used hosts in recombinant protein production, in both laboratory and industrial scale since the advent of recombinant DNA technology. Despite the substantial progress of studies on the molecular biology and immunology of infections, there is currently no medication-based prophylaxis capable of preventing leishmaniasis. As such, there is a great need to identify specific antigens for the development of vaccines and diagnostic kits against visceral leishmaniasis. Thus, the primary goal of the present study is to assess the influence of cultivation conditions on the production of Leishmania chagasi antigens, carried out in a rotating incubator and bioreactor. To that end, several assays were conducted to evaluate the kinetic behavior of antigens (648, 503) of Leishmania. i. chagasi in two different compositions of media (2xTY, TB), with and without an inducer. In order to improve expression, assays were performed in a benchtop bioreactor using the best conditions obtained in a rotating incubator, in addition to assessing the influence of stirring speed. Results show that high complexity of the cultivation medium favored kinetic growth of clones (648, 503). However, in assays submitted to induction by IPTG, this elevated complexity did not promote the expression of recombinant proteins. Expression of antigens 648 and 503 exhibited behavior associated with growth and, in terms of location, proteins 648 and 503 are intracellularly stored. Lactose may be the most adequate inducer in protein expression, when considering factors, cost, toxicity and stability. Elevated stirring may increase cell growth in clone 53, although it may not result in high concentrations for the protein of interest. On the other hand, positive results were obtained for all recombinant clones (648, 503) tested, confirmed by the electrophoretic profile

Photodynamic therapy for American cutaneous leishmaniasis: The efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The influence of natural rubber/Au nanoparticle membranes on the physiology of Leishmania brasiliensis

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

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

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