Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

Protein expression of Myt272-3 recombinant clone and in silico prediction of a possible vaccine candidate against Mycobacterium tuberculosis

Purpose: To investigate the expression of Myt272-3 recombinant protein and also to predict a possible protein vaccine candidate against Mycobacterium tuberculosis . Methods: Myt272-3 protein was expressed in pET30a+-Myt272-3 clone. The purity of the protein was determined using Dynabeads® His-Tag Isolation & Pulldown. Protein sequence was analysed in silico using bioinformatics software for the prediction of allergenicity, antigenicity, MHC-I and MHC-II binding, and B-cell epitope binding. Results: The candidate protein was a non-allergen with 15.19 % positive predictive value. It was also predicted to be antigenic, with binding affinity to MHC-I and MHC-II, as well as B-cell epitope binding. Conclusion: The predicted results obtained in this study provide a guide for practical design of a new tuberculosis vaccine.

Assessment of an oral Mycobacterium bovis BCG vaccine and an inactivated M. bovis preparation for wild boar in terms of adverse reactions, vaccine strain survival, and uptake by nontarget species

Wildlife vaccination is increasingly being considered as an option for tuberculosis control. We combined data from laboratory trials and an ongoing field trial to assess the risk of an oral Mycobacterium bovis BCG vaccine and a prototype heat-inactivated Mycobacterium bovis preparation for Eurasian wild boar (Sus scrofa). We studied adverse reactions, BCG survival, BCG excretion, and bait uptake by nontarget species. No adverse reactions were observed after administration of BCG (n = 27) or inactivated M. bovis (n = 21). BCG was not found at necropsy (175 to 300 days postvaccination [n = 27]). No BCG excretion was detected in fecal samples (n = 162) or in urine or nasal, oral, or fecal swab samples at 258 days postvaccination (n = 29). In the field, we found no evidence of loss of BCG viability in baits collected after 36 h (temperature range, 11°C to 41°C). Camera trapping showed that wild boar (39%) and birds (56%) were the most frequent visitors to bait stations (selective feeders). Wild boar activity patterns were nocturnal, while diurnal activities were recorded for all bird species. We found large proportions of chewed capsules (29%) (likely ingestion of the vaccine) and lost baits (39%) (presumably consumed), and the proportion of chewed capsules showed a positive correlation with the presence of wild boar. Both results suggest proper bait consumption (68%). These results indicate that BCG vaccination in wild boar is safe and that, while bait consumption by other species is possible, this can be minimized by using selective cages and strict timing of bait deployment.

Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

BACKGROUND Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. METHODOLOGY/PRINCIPAL FINDINGS Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. CONCLUSIONS There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.

Characterization of the immune response induced by a commercially available inactivated bluetongue virus serotype 1 vaccine in sheep

The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1 (BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations, no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccination and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4(+), CD8(+), and WC1(+)), CD25(+) regulatory cells, or CD14(+) monocytes. After challenge with BTV1, the antibody response was much higher than during the boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14(+) monocytes, CD25(+) regulatory cells, and CD8(+) cytotoxic T lymphocytes.

Complications of pneumococcal bacteremia after 13-valent conjugate vaccine withdrawal

In the Region of Madrid, universal immunization with the 13-serotypes pneumococcal conjugate vaccine (PCV13) started in May 2010. In July 2012, public funding ceased. Vaccination coverage decreased from >95% to 82% in 2013 and to 67% in 2014. Our aim was to investigate the impact of PCV13 withdrawal from Madrid Region's universal immunization program on the incidence of complicated pneumococcal bacteremia. We performed a multi-center retrospective cohort study, from 2009 to 2014. Participants were children aged <14 years with Streptococcus pneumoniae bacteremia. Complications were defined as any condition requiring intensive care or surgery. Sequelae were conditions lasting ≥90 days. A total of 168 patients were recruited. One-fourth of both immunized and non-immunized patients had complications. Global complications increased after PCV13 withdrawal. A total of 28% of PCV13 serotypes presented complications. Complications due to PCV13 serotypes did not increase after July 2012. No-PCV13 serotypes increased progressively from 2009 on, and 23% presented complications. A significant risk of complications was found for patients with meningitis, empyema, C-reactive protein >100 mg/L, and serotype 1. A multivariate analysis indicated that complications were associated with meningitis and hospital admission after July 2012. Sequelae were significantly associated with children <2 years of age, meningitis and no-PCV13 serotypes. The incidence of complications due to PCV13 serotypes did not increase two years after PCV13 withdrawal. Nevertheless, all-serotypes complications increased. The likely cause was that no-PCV13 serotypes (associated with meningitis) are on the rise.

Towards in silico IBV vaccine design: defining the role of polymorphism in viral attenuation

The gammacoronavirus, Infectious Bronchitis Virus (IBV), is a respiratory pathogen of chickens. IBV is a constant threat to poultry production as established vaccines are often ineffective against emerging strains. This requires constant and rapid vaccine production by a process of viral attenuation by egg passage, but the essential forces leading to attenuation in the virus have not yet been characterised. Knowledge of these factors will lead to the development of more effective, rationally attenuated, live vaccines and reduction of the mortality and morbidity caused by this pathogen. M41 CK strain was egg passaged four times many years ago at Houghton Poultry Research Station and stored as M41-CK EP4 (stock virus at The Pirbright Institute since 1992). It was the first egg passage to have its genome pyrosequenced and was therefore used as the baseline reference. The overall aim of this project was to analyse deep sequence data obtained from four IBV isolates (called A, A1, C and D) each originating from the common M41-CK EP4 (ep4) and independently passaged multiple times in embryonated chicken eggs (figure 1.1). Highly polymorphic encoding regions of the IBV genome were then identified which are likely involved in the attenuation process through the formation of independent SNPs and/or SNP clusters. This was then used to direct targeted investigation of SNPs during the attenuation process of the four IBV passages. A previously generated deep sequence dataset was used as a preliminary map of attenuation for one virulent strain of IBV. This investigation showed the nucleocapsid and spike as two highly polymorphic encoding regions within the IBV genome with the highest proportion of SNPs compared to encoding region size. This analysis then led to more focussed studies of the nucleocapsid and spike encoding region with the ultimate aim of mapping key attenuating regions and nucleotide positions. The 454 pyrosequencing data and further investigation of nucleocapsid and spike encoding regions have identified the SNPs present at the same nucleotide positions within analysed A, A1, C and D isolates. These SNPs probably play a crucial role in viral attenuation and universal vaccine production but it is not clear if independent SNPs are also involved in loss of virulence. The majority of SNPs accumulated at different nucleotide positions without further continuation in Sanger sequenced egg passages presenting S2 subunit (spike) and nucleocapsid as polymorphic encoding regions which in nature remain highly conserved.

Design of the ATAQ peptide and its evaluation as an immunogen to develop a Rhipicephalus vaccine

2016

Serum concentrations of acute phase proteins and immunoglobulins of calves with rotavirus diarrhea.

2015

Serum concentrations of acute phase proteins and immunoglobulins of calves with rotavirus diarrhea.

2016

Dissection of the protective response elicited by the DOMV component of the 4CMenB vaccine

Neisseria meningitidis serogroup B is the major etiological agent of meningitis and life-threatening sepsis, against which two vaccines are licensed. The 4CMenB vaccine is composed of three major protein antigens (fHbp, NHBA and NadA) and detergent-extracted outer membrane vesicles (DOMV) from the NZ98/254 strain. DOMV are safe, immunogenic and able to raise bactericidal antibodies, mainly attributed to the immunodominant PorA protein. Nevertheless, DOMV offer a complex reservoir of potentially immunogenic proteins, whose relative contribution in protection is still poorly characterized. By testing antisera from vaccinated infants in serum bactericidal assay, we observed that the addition of DOMV in the vaccine formulation enhanced breadth of coverage compared to recombinant proteins alone against a panel of 11 meningococcal strains mismatched for the vaccine antigens. To unravel the DOMV components involved in such protection, 30 DOMV antigens were cloned and expressed in Escherichia coli as recombinant proteins and/or in vesicles to maintain their native conformation. Samples obtained were both included in tailor-made protein-microarrays to immunoprofile the antibody repertoire raised by DOMV-containing formulations and were individually used for mouse immunization studies to assess their ability to induce bactericidal antibodies. The protein-array immunosignature of mouse DOMV/4CMenB antisera unveiled a subset of 8 DOMV-reactive proteins potentially responsible for the additional protective responses. The antisera derived from mouse immunizations showed high levels of antibodies and recognized the corresponding antigen across different meningococcal strains. Among the protein-array reactive antigens, OpcA, NspA and PorB induced antibodies able to kill 10 of the 11 genetically diverse meningococcal strains and the specificity of the protective role of OpcA and PorB was also confirmed in 4CMenB infant vaccinee sera. In conclusion, we identified additional PorA-independent antigens within DOMV involved in broadening the coverage of 4CMenB, thus supporting the key role played by vesicles in this multivalent formulation.

Intravesical Bacillus Calmette-guérin Efficiently Reduces P70s6k1 But Not 4e-bp1 Phosphorylation In Nonmuscle Invasive Bladder Cancer.

We characterized the functional consequences of intravesical bacillus Calmette-Guérin on the molecular mechanism of the AKT/mTOR signaling pathway in nonmuscle invasive bladder cancer. To our knowledge this has not been reported previously. At age 7 weeks female Fischer 344 rats received 1.5 mg/kg MNU intravesically every other week for 6 weeks. They were randomized at 10 per group to MNU (0.2 ml vehicle), bacillus Calmette-Guérin (10(6) cfu Connaught strain), rapamycin (15 μg/ml) and bacillus Calmette-Guérin plus simultaneous rapamycin, each intravesically for 6 weeks. At week 15 the bladders were collected for histopathology, immunohistochemistry and immunoblot to determine p-AKT, Rictor, Raptor, p-4E-BP1, p-p70S6K1, p-AMPK-α, p-mTOR and p-p53. Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (pTis) predominated in the MNU group while normal urothelium, papillary and flat hyperplasia were more common in treated groups. Nonmuscle invasive bladder cancer treated with bacillus Calmette-Guérin showed suppression of p70S6K1 but not 4E-BP1 phosphorylation. This suggests that 4E-BP1 is regulated differently than p70S6K1, escaping the bacillus Calmette-Guérin action that occurs in a mTOR independent manner. The association of bacillus Calmette-Guérin with rapamycin but not rapamycin monotherapy affected p70S6K1 and 4E-BP1 phosphorylation with no features of in situ carcinoma (pTis). The activation status of p70S6K1 and 4E-BP1 might be used to stratify patients who could benefit from targeting such molecular elements with multitarget/multidrug intravesical therapy. In the future 4E-BP1 might be a worthwhile new target for bacillus Calmette-Guérin refractory nonmuscle invasive bladder cancer.

Produção de micropartículas de alginato contendo Flavobacterium columnare inativada pelo método de emulsão para vacinação de peixes por via oral

Alginate microparticles were prepared by an emulsion method aiming oral controlled release of antigens to fish. The effects of emulsification temperature and impeller type on particle morphology, average diameter, and size distribution were evaluated. Microparticles contaning formalin-killed Flavobacterium columnare cells (a model antigen) were prepared and characterized regarding bacterial release and particle stability when exposed to Nile tilapia (Oreochromis niloticus) typical gastrointestinal conditions. This methodology allowed the production of microparticles containing up to 14.3 g/L of bacterin, stable at a pH range from 2.0 to 9.0 for 12 h and smaller than 35 μm.

Perspectives of vaccination in Chagas disease revisited

The perspectives for a Chagas Disease vaccine 30 years ago and today are compared. Antigens and adjuvants have improved, but logistic problems remain the same. Sterilizing vaccines have not been produced and animal models for chronic Chagas have not been developed. Vector control has been successful and Chagas incidence has come to a halt. We do not have a population candidate to vaccination now in Brazil. And if we had, we would not know how to evaluate the success of vaccination in a short time period. A vaccine may not seem important at the moment. However, scientific reasons and incertitudes about the future recommend that a search for a vaccine be continued.

Perfil sociodemográfico e padrão de utilização dos serviços de saúde do Sistema Único de Saúde (SUS), 2003- 2008

O objetivo foi analisar a evolução do perfil de utilização de serviços de saúde, entre 2003 e 2008, no Brasil e nas suas macrorregiões. Foram utilizados dados da PNAD. A utilização de serviços de saúde foi medida pela proporção de pessoas que procuraram e foram atendidas nas 2 semanas anteriores e pelos que relataram internação nos últimos 12 meses, segundo SUS e não SUS. Foram analisadas as características socioeconômicas dos usuários, o tipo de atendimento e de serviço e os motivos da procura. A proporção de indivíduos que procuraram serviços de saúde não se alterou, assim como a parcela dos que conseguiram atendimento (96%), entre 2003 e 2008. O SUS respondeu por 56,7% dos atendimentos, realizando a maior parte das internações, vacinação e consultas e somente 1/3 das consultas odontológicas. Em 2008, manteve-se o gradiente de redução de utilização de serviços de saúde SUS conforme o aumento de renda e escolaridade. Houve decréscimo da proporção dos que procuraram serviços de saúde para ações de prevenção e aumento de procura para problemas odontológicos, acidentes e lesões e reabilitação. O padrão de utilização do SUS por região esteve inversamente relacionado à proporção de indivíduos com posse de planos privados de saúde.