CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W, Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525-532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, VI., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66,331-341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1` in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high beta sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae. (c) 2007 Elsevier Inc. All rights reserved.

Protection against high-dose homologous infection in calves immunized with intestine or membrane extracts from Haemonchus placei

Control of Haemonchus placei, one of the most important cattle nematodes in Brazil, relies on the use of anthelmintics. However, there is a need for integrated control, which includes active immunization. The aim of this work was to assess the protection afforded to calves by immunization with adult H. placei extracts against a high-dose challenge infection, a condition frequently found in the tropics. Holstein calves aged 8-10 months were immunized four times with intestinal extracts (Group D) or with a Triton X-100-soluble fraction of adult H. placei (Group A), challenge-infected with 120,000 infective larvae and sacrificed 40 days later. Immunized animals had higher IgG titers than the controls against tested fractions after the 2nd immunization, peaking after the 4th. Sera from both immunized groups recognized bands of similar apparent mass in both antigenic preparations, some of which were similar in molecular weight to Haemonchus contortus antigens with known protective effect to sheep. Egg counts were 49% and 57% lower in Groups A and D than in controls, respectively. High levels of protection were observed in two of the four calves in Group D, as evidenced by very low worm numbers recovered at necropsy, absence of eggs in the uteri of the recovered females and reduced worm length. Group D animals also showed milder signs of anemia than the other infected animals. Results demonstrate that protection against homologous high-dose challenge can be achieved by immunizing calves with H. placei gut antigens. (C) 2007 Elsevier B.V. All rights reserved.

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Lower serum IgA levels in horses kept under intensive sanitary management and physical training

Quantity and variety of environmental antigens, age, diet, vaccine protocols, exercising practice and mucosal cytokine microenvironment are factors that influence serum immunoglobulin (Ig) levels. IgA, IgG, IgG(T) and IgM were quantified in 60 horses, which were classified into two groups, `intensive` or `relaxed`, according to sanitary standards of the facilities and physical exercise to which animals were subjected to. The `intensive` group presented lower means for all isotypes, but only IgA presented a significant (P < 0.0064) difference when compared to the `relaxed` group. This suggests that mucosal immunity found in the `intensive` group is lower when compared to the `relaxed` group. Our data suggest that athlete horses may be less poised to mount an effective mucosal immunity response to environmental challenges and should not be considered by the same perspectives as a free-ranging horse.

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.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

Susceptible-infected-recovered and susceptible-exposed-infected models

Two stochastic epidemic lattice models, the susceptible-infected-recovered and the susceptible-exposed-infected models, are studied on a Cayley tree of coordination number k. The spreading of the disease in the former is found to occur when the infection probability b is larger than b(c) = k/2(k - 1). In the latter, which is equivalent to a dynamic site percolation model, the spreading occurs when the infection probability p is greater than p(c) = 1/(k - 1). We set up and solve the time evolution equations for both models and determine the final and time-dependent properties, including the epidemic curve. We show that the two models are closely related by revealing that their relevant properties are exactly mapped into each other when p = b/[k - (k - 1) b]. These include the cluster size distribution and the density of individuals of each type, quantities that have been determined in closed forms.

Cyclooxygenase-2 and vascular endothelial growth factor expression in 5-fluorouracil-induced oral mucositis in hamsters: evaluation of two low-intensity laser protocols

The aim of this study was to investigate the mechanisms whereby low-intensity laser therapy may affect the severity of oral mucositis. A hamster cheek pouch model of oral mucositis was used with all animals receiving intraperitoneal 5-fluorouracil followed by surface irritation. Animals were randomly allocated into three groups and treated with a 35 mW laser, 100 mW laser, or no laser. Clinical severity of mucositis was assessed at four time-points by a blinded examiner. Buccal pouch tissue was harvested from a subgroup of animals in each group at four time-points. This tissue was used for immunohistochemistry for cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and factor VIII (marker of microvessel density) and the resulting staining was quantified. Peak severity of mucositis was reduced in the 35 mW laser group as compared to the 100 mW laser and control groups. This reduced peak clinical severity of mucositis in the 35 mW laser group was accompanied by a significantly lower level of COX-2 staining. The 100 mW laser did not have an effect on the severity of clinical mucositis, but was associated with a decrease in VEGF levels at the later time-points, as compared to the other groups. There was no clear relationship of VEGF levels or microvessel density to clinical mucositis severity. The tissue response to laser therapy appears to vary by dose. Low-intensity laser therapy appears to reduce the severity of mucositis, at least in part, by reducing COX-2 levels and associated inhibition of the inflammatory response.

Influenza virus vaccination in kidney transplant recipients: serum antibody response to different immunosuppressive drugs

Introduction: This study prospectively accessed the immune response to the inactivated influenza vaccine in renal transplant recipients receiving either azathioprine or mycophenolate mofetil (MMF). Side effects were investigated. Methods: Sixty-nine patients received one dose of inactivated trivalent influenza vaccine. Antihemagglutinin (HI) antibody response against each strain was measured before and one to six months after vaccination. Results: Geometric mean HI antibody titers for H1N1 and H3N2 strains increased from 2.57 and 2.44 to 13.45 (p = 0.001) and 7.20 (p < 0.001), respectively. Pre- and post-vaccination protection rates for H1N1 and H3N2 increased from 8.7% to 49.3% (p < 0.001); and 36.3% (p < 0.001) and seroconversion rates were 36% and 25.3%, respectively. There was no response to influenza B. The use of MMF reduced the H1N1 and H3N2 protection rates and the seroconversion rate for the H1N1 strain when compared with the use of azathioprine, and subjects transplanted less than 87 months also had inferior antibody response. Adverse events were mild and there were no change on renal function post-vaccination. Conclusion: Renal transplant patients vaccinated against influenza responded with antibody production for in. uenza A virus strains, but not for in. uenza B. Use of MMF and shorter time from transplantation decreased the immune response to the vaccine.

Yellow fever vaccination coverage among children in Brazilian capitals

Brazil recommends universal yellow fever (YF) vaccination for children who reside in or travel to endemic areas. We conducted a household survey to calculate YF vaccine coverage among children 18-30 months of age in 27 capital cities. A total of 9285 children were surveyed in the 15 cities with YF fever universal vaccination; 7290(79%) had documented evidence of YF vaccination by 12 months of age, 7996 (86%) by 18 months of age, and 8479 (91%) prior to the survey. In 12 cities with selective YF vaccination coverage was only 1% by 18 months of age. YF fever vaccination can be improved to reach all children where vaccine is recommended. (C) 2010 Elsevier Ltd. All rights reserved.

Household survey of hepatitis B vaccine coverage among Brazilian children

We conducted a multi-stage household cluster survey to calculate hepatitis B vaccine coverage among children 18-30 months of age in 27 Brazilian cities. Hepatitis B vaccine is administered at birth, 1 month and 6 months of age by Brazil`s national immunization program. Among 17,749 children surveyed, 40.2% received a birth dose within one day of birth, 94.8% received at least one dose of hepatitis B vaccine, and 86.7% completed the three-dose series by 12 months of age. Increased coverage with the birth dose and administration of hepatitis B in combination with diphtheria-tetanus-pertussis-Haemophilus influenzae type b antigens could improve protection against hepatitis B. (C) 2009 Elsevier Ltd. All rights reserved.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

The facial affective scale as a predictor for pain unpleasantness when children undergo immunizations.

Needle fear is a common problem in children undergoing immunization. To ensure that the individual child's needs are met during a painful procedure it would be beneficial to be able to predict whether there is a need for extra support. The self-reporting instrument facial affective scale (FAS) could have potential for this purpose. The aim of this study was to evaluate whether the FAS can predict pain unpleasantness in girls undergoing immunization. Girls, aged 11-12 years, reported their expected pain unpleasantness on the FAS at least two weeks before and then experienced pain unpleasantness immediately before each vaccination. The experienced pain unpleasantness during the vaccination was also reported immediately after each immunization. The level of anxiety was similarly assessed during each vaccination and supplemented with stress measures in relation to the procedure in order to assess and evaluate concurrent validity. The results show that the FAS is valid to predict pain unpleasantness in 11-12-year-old girls who undergo immunizations and that it has the potential to be a feasible instrument to identify children who are in need of extra support to cope with immunization. In conclusion, the FAS measurement can facilitate caring interventions.

Methods and background characteristics of the TOHNN study : a population-based study of oral health conditions in northern Norway

OBJECTIVES: The aim of the Tromstannen - Oral Health in Northern Norway (TOHNN) study was to investigate oral health and dental-related diseases in an adult population. This article provides an overview of the background of the study and a description of the sample characteristics and methods employed in data collection. STUDY DESIGN: Cross-sectional population-based study including a questionnaire and clinical dental examination. METHODS: A randomly selected sample of 2,909 individuals (20-79 years old) drawn from the population register was invited to participate in the study. The data were collected between October 2013 and November 2014 in Troms County in northern Norway. The questionnaire focused on oral health-related behaviours and attitudes, oral health-related quality of life, sense of coherence, dental anxiety and symptoms from the temporomandibular joint. The dental examinations, including radiographs, were conducted by 11 dental teams in 5 dental offices. The examination comprised of registration of dental caries, full mouth periodontal status, temporomandibular disorders, mucosal lesions and height and weight. The participants were grouped by age (20-34, 35-49, 50-64 and 65-79) and ethnicity (Norwegian, Sámi, other European and other world). RESULTS: From the original sample of 2,909 individuals, 1,986 (68.3%) people participated, of whom 1,019 (51.3%) were women. The highest attendance rate was among women 20-34 years old (80.3%) and the lowest in the oldest age group of women (55.4%). There was no difference in response rate between rural and urban areas. There was a positive correlation between population size and household gross income (p < 0.001) and education level (p < 0.001). The majority of Sámi resided in smaller municipalities. In larger cities, most participants used private dental health care services, whereas, in rural areas, most participants used the public dental health care service. CONCLUSION: The TOHNN study has the potential to generate new knowledge on a wide range of oral health conditions beneficial to the population in Troms County. Due to the high participation rate, generalization both nationally and to the circumpolar area ought to be possible.