OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of beta-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

Immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus

Objectives The aim of the present paper is to evaluate the immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus. Methods We performed a prospective and controlled study on a group of 54 SLE patients that were chosen at random to be or not to be vaccinated (28 were vaccinated and 26 were not). Twenty-eight healthy controls, of matching age and sex were also vaccinated. All were submitted to a questionnaire, physical evaluation and laboratory assays: lymphocyte immuno-phenotyping by flow cytometry, plasma varicella zoster virus (VZV) serology by ELISA and in vitro interferon gamma (IFN gamma) production by T-cells after stimulus with VZV antigen. They were evaluated before vaccination and at 30, 45, 180 and 360 days afterwards. Results We did not observe any differences in the frequency of adverse events in both vaccinated groups. At study entry, all individuals were seropositive for VZV antibodies. The serum VZV antibody titres similarly increased after vaccination. The frequency of flares and the SLEDAI score were also similar among the patients. Thirty days after vaccination the production of IFN gamma specific to VZV was lower in the SLE group compared to healthy, controls. In the follow-up we observed 4 cases of herpes zoster in the SLE unvaccinated group, but no zoster in the vaccinated group. Conclusion The varicella vaccine was well tolerated in SLE group, who had pre-existing immunity to varicella. The varicella vaccine immunogenicity measurement by serum antibody titres was appropriate. The incidence of HZ was lower in the vaccinated lupus group.

Cost-effectiveness of introducing the 10-valent pneumococcal conjugate vaccine into the universal immunisation of infants in Brazil

Background Cost-effectiveness studies have been increasingly part of decision processes for incorporating new vaccines into the Brazilian National Immunisation Program. This study aimed to evaluate the cost-effectiveness of 10-valent pneumococcal conjugate vaccine (PCV10) in the universal childhood immunisation programme in Brazil. Methods A decision-tree analytical model based on the ProVac Initiative pneumococcus model was used, following 25 successive cohorts from birth until 5 years of age. Two strategies were compared: (1) status quo and (2) universal childhood immunisation programme with PCV10. Epidemiological and cost estimates for pneumococcal disease were based on National Health Information Systems and literature. A 'top-down' costing approach was employed. Costs are reported in 2004 Brazilian reals. Costs and benefits were discounted at 3%. Results 25 years after implementing the PCV10 immunisation programme, 10 226 deaths, 360 657 disability-adjusted life years (DALYs), 433 808 hospitalisations and 5 117 109 outpatient visits would be avoided. The cost of the immunisation programme would be R$10 674 478 765, and the expected savings on direct medical costs and family costs would be R$1 036 958 639 and R$209 919 404, respectively. This resulted in an incremental cost-effectiveness ratio of R$778 145/death avoided and R$22 066/DALY avoided from the society perspective. Conclusion The PCV10 universal infant immunisation programme is a cost-effective intervention (1-3 GDP per capita/DALY avoided). Owing to the uncertain burden of disease data, as well as unclear long-term vaccine effects, surveillance systems to monitor the long-term effects of this programme will be essential.

CD8(+) T-cell-mediated immunity against malaria: a novel heterologous prime-boost strategy

Evaluation of: Rodriguez D, Gonzalez-Aseguinolaza G, Rodriguez JR et al. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium. PLoS ONE 7(4), e34445 (2012). Recently, a vaccine against malaria was successfully tested in a human Phase III trial. The efficacy of this vaccine formulation, based on the Plasmodium falciparum circumsporozoite protein, was approximately 50% and correlated with the presence of antibodies specific to the infective stages of the malaria parasites. Different strategies are being pursued to improve vaccine efficacy levels. One such strategy is the induction of specific cytotoxic T cells that can destroy the intracellular hepatocyte stages of the malaria parasite. In this study, a novel vaccination protocol was developed to elicit strong immune responses mediated by CD8(+) cytotoxic cells specific to the circumsporozoite protein. As proof-of-concept, the authors used the rodent malaria Plasmodium yoelii parasite. The vaccination strategy consisted of a heterologous prime-boost vaccination regimen involving porcine parvovirus-like particles for priming and the modified vaccinia virus Ankara for the booster immunization, both of which expressed the immunodominant CD8 epitope of the P. yoelii circumsporozoite protein. Results from this experimental model were extremely meaningful. This vaccination strategy led to a significant T-cell immune response mediated by CD8(+) multifunctional T effector and effector-memory cells. However, most importantly for the malaria vaccine development was the fact that following a sporozoite challenge, immunized mice eliminated more than 97% of the malaria parasites during the hepatocyte stages. These results confirm and extend a vast body of knowledge showing that a heterologous prime-boost vaccination strategy can elicit strong CD8(+) T-cell-mediated protective immunity and may increase the efficacy of malaria vaccines.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Abstract Background Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection.

Improve protective efficacy of a TB DNA-HSP65 vaccine by BCG priming

Vaccines are considered by many to be one of the most successful medical interventions against infectious diseases. But many significant obstacles remain, such as optimizing DNA vaccines for use in humans or large animals. The amount of doses, route and easiness of administration are also important points to consider in the design of new DNA vaccines. Heterologous prime-boost regimens probably represent the best hope for an improved DNA vaccine strategy. In this study, we have shown that heterologous prime-boost vaccination against tuberculosis (TB) using intranasal BCG priming/DNA-HSP65 boosting (BCGin/DNA) provided significantly greater protection than that afforded by a single subcutaneous or intranasal dose of BCG. In addition, BCGin/DNA immunization was also more efficient in controlling bacterial loads than were the other prime-boost schedules evaluated or three doses of DNA-HSP65 as a naked DNA. The single dose of DNA-HSP65 booster enhanced the immunogenicity of a single subcutaneous BCG vaccination, as evidenced by the significantly higher serum levels of anti-Hsp65 IgG2a Th1-induced antibodies, as well as by the significantly greater production of IFN-γ by antigen-specific spleen cells. The BCG prime/DNA-HSP65 booster was also associated with better preservation of lung parenchyma.

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.

Prospective surveillance study of acute respiratory infections, influenza-like illness and seasonal influenza vaccine in a cohort of juvenile idiopathic arthritis patients

Background Acute respiratory infections (ARI) are frequent in children and complications can occur in patients with chronic diseases. We evaluated the frequency and impact of ARI and influenza-like illness (ILI) episodes on disease activity, and the immunogenicity and safety of influenza vaccine in a cohort of juvenile idiopathic arthritis (JIA) patients. Methods Surveillance of respiratory viruses was conducted in JIA patients during ARI season (March to August) in two consecutive years: 2007 (61 patients) and 2008 (63 patients). Patients with ARI or ILI had respiratory samples collected for virus detection by real time PCR. In 2008, 44 patients were immunized with influenza vaccine. JIA activity index (ACRPed30) was assessed during both surveillance periods. Influenza hemagglutination inhibition antibody titers were measured before and 30-40 days after vaccination. Results During the study period 105 ARI episodes were reported and 26.6% of them were ILI. Of 33 samples collected, 60% were positive for at least one virus. Influenza and rhinovirus were the most frequently detected, in 30% of the samples. Of the 50 JIA flares observed, 20% were temporally associated to ARI. Influenza seroprotection rates were higher than 70% (91-100%) for all strains, and seroconversion rates exceeded 40% (74-93%). In general, response to influenza vaccine was not influenced by therapy or disease activity, but patients using anti-TNF alpha drugs presented lower seroconversion to H1N1 strain. No significant differences were found in ACRPed30 after vaccination and no patient reported ILI for 6 months after vaccination. Conclusion ARI episodes are relatively frequent in JIA patients and may have a role triggering JIA flares. Trivalent split influenza vaccine seems to be immunogenic and safe in JIA patients.

A phase I clinical trial of a new 5-valent rotavirus vaccine

We conducted a phase I, double-blind, placebo-controlled trial to evaluate a new 5-valent oral rotavirus vaccine’s safety and immunogenicity profiles. Subjects were randomly assigned to receive 3 orally administered doses of a live-attenuated human-bovine (UK) reassortant rotavirus vaccine, containing five viral antigens (G1, G2, G3, G4 and G9), or a placebo. The frequency and severity of adverse events were assessed. Immunogenicity was evaluated by the titers of anti-rotavirus IgA and the presence of neutralizing antibodies anti-rotavirus. No severe adverse events were observed. There was no difference in the frequency of mild adverse events between experimental and control groups. The proportion of seroconversion was consistently higher in the vaccine group, for all serotypes, after each one of the doses. The 5-valent vaccine has shown a good profile of safety and immunogenicity in this small sample of adult volunteers.

Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006–2007

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

Imunogenicidade da vacina brasileira contra hepatite B em adultos.

OBJETIVO: Avaliar a imunogenicidade e segurança da vacina contra hepatite B, após o aumento na concentração do antígeno HBsAg para 25 μg, em comparação à vacina de referência. MÉTODOS: Ensaio com alocação aleatória e mascaramento simples, comparando a VrHB-IB (Instituto Butantan) com a vacina de referência (Engerix B®, Glaxo Smith Kline). Os voluntários, entre 31 e 40 anos de idade (n=419), foram alocados aleatoriamente ao grupo experimental (n=216) ou ao grupo controle (n=203), e receberam três doses de vacina. A primeira dose foi administrada no momento do recrutamento, a segunda e terceira 30 e 180 dias depois respectivamente, entre 2004 e 2005. Amostras de sangue foram colhidas para análise sorológica antes da randomização, e após a segunda e terceira doses. Foi realizada a vigilância ativa de eventos adversos durante os cinco primeiros dias após a vacinação. As diferenças foram avaliadas pelos testes do qui-quadrado e exato de Fisher, com nível de significância de 5%. RESULTADOS: Não se observaram eventos adversos graves. A soroporteção foi confirmada em 98,6% (213/216) dos voluntários do grupo experimental, em comparação a 95,6% (194/203) do grupo controle. Os títulos geométricos médios foram de 12.557 e 11.673, respectivamente. CONCLUSÕES: A vacina brasileira foi considerada equivalente à vacina de referência e seu uso recomendado para adultos.

A phase I randomized, double-blind, controlled trial of 2009 influenza A (H1N1) inactivated monovalent vaccines with different adjuvant systems

Methods We conducted a phase I, multicenter, randomized, double-blind, placebo-controlled, multi-arm (10) parallel study involving healthy adults to evaluate the safety and immunogenicity of influenza A (H1N1) 2009 non-adjuvanted and adjuvanted candidate vaccines. Subjects received two intramuscular injections of one of the candidate vaccines administered 21 days apart. Antibody responses were measured by means of hemagglutination-inhibition assay before and 21 days after each vaccination. The three co-primary immunogenicity end points were the proportion of seroprotection >70%, seroconversion >40%, and the factor increase in the geometric mean titer >2.5. Results A total of 266 participants were enrolled into the study. No deaths or serious adverse events were reported. The most commonly solicited local and systemic adverse events were injection-site pain and headache, respectively. Only three subjects (1.1%) reported severe injection-site pain. Four 2009 influenza A (H1N1) inactivated monovalent candidate vaccines that met the three requirements to evaluate influenza protection, after a single dose, were identified: 15 μg of hemagglutinin antigen without adjuvant; 7.5 μg of hemagglutinin antigen with aluminum hydroxide, MPL and squalene; 3.75 μg of hemagglutinin antigen with aluminum hydroxide and MPL; and 3.75 μg of hemagglutinin antigen with aluminum hydroxide and squalene. Conclusions Adjuvant systems can be safely used in influenza vaccines, including the adjuvant monophosphoryl lipid A (MPL) derived from Bordetella pertussis with squalene and aluminum hydroxide, MPL with aluminum hydroxide, and squalene and aluminum hydroxide.

Die Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen

„Synthese von Glycopeptiden und Glycopeptid-Protein-Konjugaten mit einer Partialstruktur des tumorassoziierten Mucins MUC1 zur Entwicklung von Tumorvakzinen“ Das Glycoprotein MUC1 ist in Tumorepithelzellen sonderlich stark überexprimiert und wegen der vorzeitig einsetzenden Sialylierung sind die Saccharid-Epitope der O-Glycanketten stark verkürzt (sog. tumorassoziierte Antigene). Dadurch werden auch bisher verborgene Peptidepitope des Glycoprotein-Rückgrates auf der Zelloberfläche der Epithelzellen zugänglich, die als fremd von den Zellen des Immunsystems erkannt werden können. Dies macht das MUC1-Zelloberfächenmolekül zu einem Zielmolekül in der Entwicklung von Tumorvakzinen. Diese beiden strukturellen Besonderheiten wurden in der Synthese von Glycohexadecapeptiden verbunden, indem die veränderten tumorassoziierten Saccharidstrukturen TN-, STN- und T-Antigen als Glycosylaminosäure-Festphasenbausteine synthetisiert wurden und in das Peptidepitop der Wiederholungseinheit des MUC1 durch Glycopeptid-Festphasensynthese eingebaut wurden. Wegen der inhärenten schwachen Immunogenität der kurzen Glycopeptide müssen die synthetisierten Glycopeptidstrukturen an ein Trägerprotein, welches das Immunsystem stimuliert, gebunden werden. Zur Anbindung der Glycopeptide ist ein selektives Kupplungsverfahren nötig, um definierte und strukturell einheitliche Glycopeptid-Protein-Konjugate zu erhalten. Es konnte eine neue Methode entwickelt werden, bei der die Konjugation durch eine radikalische Additionsreaktion von als Allylamide funktionalisierten Glycopeptiden an ein Thiol-modifiziertes Trägerprotein erfolgte. Dazu wurde anhand von synthetisierten, als Allylamide modifizierten Modellaminosäuren untersucht, ob diese Reaktion generell für eine Biokonjugation geeignet ist und etwaige Nebenreaktionen auftreten können. Mit dieser Methode konnten verschiedene MUC1-Glycopeptid-Trägerprotein-Konjugate hergestellt werden, deren immunologische Untersuchung noch bevorsteht. Das tumorassoziierte MUC1 nimmt in der immundominanten Region seiner Wiederholungseinheit eine knaufartige Struktur ein. Für die Entwicklung von selektiven Tumorvakzinen ist es von großer Bedeutung möglichst genau die Struktur der veränderten Zelloberflächenmoleküle nachzubilden. Durch die Synthese von cyclischen (Glyco)Peptiden wurde dieses Strukturelement fixiert. Dazu wurden olefinische Aminosäure Festphasenbausteine hergestellt, die zusammen mit den oben genannten Glycosylaminosäuren mittels einer Glycopeptid-Festphasensynthese in acyclische Glycopeptide eingebaut wurden. Diese wurden dann durch Ringschlussmetathese zyklisiert und im Anschluss reduziert und vollständig deblockiert. In einem dritten Projekt wurde der Syntheseweg zur Herstellung einer C-Glycosylaminosäure mit einer N-Acetylgalactosamin-Einheit entwickelt. Wichtige Schritte bei der von Glucosamin ausgehenden Synthese sind die Keck-Allylierung, eine Epimerisierung, die Herstellung eines Brom-Dehydroalanin-Derivates und eine B-Alkyl-Suzuki-Miyaura-Kreuzkupplung sowie Schutzgruppenoperationen. Der racemische Baustein konnte dann in der Peptid-Festphasensynthese eines komplexen MUC1-Tetanustoxin-Konjugates eingesetzt werden.