General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector. (C) 2012 Elsevier Ltd. All rights reserved.

Mutations in the H, F, or M proteins can facilitate resistance of measles virus to neutralizing human anti-MV sera

Although there is currently no evidence of emerging strains of measles virus (MV) that can resist neutralization by the anti-MV antibodies present in vaccinees, certain mutations in circulating wt MV strains appear to reduce the efficacy of these antibodies. Moreover, it has been hypothesized that resistance to neutralization by such antibodies could allow MV to persist. In this study, we use a novel in vitro system to determine the molecular basis of MV's resistance to neutralization. We find that both wild-type and laboratory strain MV variants that escape neutralization by anti-MV polyclonal sera possess multiple mutations in their H, F, and M proteins. Cytometric analysis of cells expressing viral escape mutants possessing minimal mutations and their plasmid-expressed H, F, and M proteins indicates that immune resistance is due to particular mutations that can occur in any of these three proteins that affect at distance, rather than directly, the native conformation of the MV-H globular head and hence its epitopes. A high percentage of the escape mutants contain mutations found in cases of Subacute Sclerosing Panencephalitis (SSPE) and our results could potentially shed light on the pathogenesis of this rare fatal disease.

Biological and protective properties of immune sera directed to the influenza virus neuraminidase

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCE The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Prevalence of hepatitis B and C serological markers among first-time blood donors in Brazil: A multi-center serosurvey

Little data are available on the seroprevalence of, and risk factors for hepatitis B and C viruses (HBV and HCV) infection in Latin American countries. A multi-center serosurvey was conducted among 3,598 first-time blood donors (65% men) from Sao Paulo, Salvador and Manaus in Brazil. The gender-specific seroprevalences of antibodies against hepatitis B core antigen (anti-HBc) and of the hepatitis B surface antigen (HBsAg) in anti-HBc-positive sera were measured, and risk factors analyzed by gender. The gender-specific seroprevalences of antibodies against HCV (anti-HCV) were measured, but risk factors for HCV were not determined. Anti-HBc and HBsAg seroprevalences were not significantly different in men [101/2,341 (4.31%) and 4/2,229 (0.18%), respectively] and women [65/1,237 (5.25%) and 8/ 1,169 (0.68%), respectively], whereas the seroprevalence of anti-HCV was higher in women (12/1,238 [0.97%] vs. 9/2,353 [0.38%]; odds ratio [OR] = 2.49; 95% confidence interval [Cl]: 1.0-6.0). No significant difference for HBV infection was found across the three study sites or by ethnic group. The seroprevalence of anti-HBc increased with age, but decreased with education level in both genders. Lifetime number of sexual partners was associated with anti-HBc prevalence among men (OR = 1.95; 95% Cl: 1.2-3.1), but not women. The seroprevalence of HBV and HCV was low among Brazilian blood donors, and exposure increased with age in both genders.

Rat nephrotoxic nephritis. The relation between the type and intensity of induced histological lesions and the content of antiglomerular basement membrane antibodies of the inoculated serum

Six groups of 6 rats received equal doses (0.8 ml/100 g of body weight) of different rabbit anti rat kidney sera. The titer of anti GBM antibodies in the sera was evaluated by indirect immunofluorescent test in isolated GBM (IIT GBM). Rats of groups 1, 2, 3, 5, 6 received anti rat GBM sera with titers of 1/320, 1/240, 1/160, 1/60, 1/30 respectively. Group 4 received anti rat kidney serum with a titer of 1/80. The rats of group 1 died from 1 to 5 minutes after inoculation and their kidney were congested, with hialine trombi occluding arterioles and glomerular capillaries. The rats of group 2 and one of group 3 died from 2 to 15 days after inoculation and diffuse cortical necrosis was found. The remaining rats were sacrificed 2 months after inoculation. The kidneys were normal in control group; chronic membranoproliferative glomerulonephritis was observed in group 3 and 4, membranoproliferative glomerulonephritis in group 5 and minimal changes in group 6. By immunofluorescence rabbit gammaglobulin was seen in GBM of group 3, 4, 5 and 6. The IIT GBM performed in the eluates of the kidneys revealed the presence of heterologous antibody in groups 1, 2, 3, 4, 5 and 6 and autologous antibody in groups 3, 4 and 5. One concludes that the IIT GBM identifies and quantifies antibodies which have the property of damaging the kidney.

Effects of tunicamycin on glycoprotein antigens of Aspergillus fumigatus

Tunicamycin, which inhibits N-glycosylation of proteins, was used as a tool to determine the type of linkage which occurs in glycoprotein antigens of Aspergillus fumigatus. When A. fumigatus extracts were electrophoretically separated and blotted then probed with anti-Aspergillus patients' sera, differences in antigenic profiles were noted when tunicamycin-treated samples were compared with controls. Tunicamycin had no detectable effect on the cellular proteinases of A. fumigatus, most of which are glycosylated. Some enzymatic components were lacking when extracellular proteinases were compared with those of control samples. The major catalase component of A. fumigatus is a concanavalin A (Con A)-binding glycoprotein. In cultures grown in the presence of tunicamycin, partiallydeglycosylated catalase components were obtained which could be distinguished from the native catalase by their altered mobilities in polyacrylamide gels. The effect of deglycosylation on catalase antigens was monitored using an antiserum raised to a ConA-binding fraction of A fumigatus mycelium. These antibodies bound both to the native glycoprotein and the partially deglycosylated material. These latter two were largely unaffected when incubated with an antiserum raised to a non-ConA-binding fraction of A. fumigatus which is essentially carbohydrate free. The ability to produce partially-glycosylated antigens of A. fumigatus offers a model to study the effect of basic structural modifications on both the enzymatic and antigenic activities of these molecules.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera and techniques

This thesis reports on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60°C.

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.