Non-immunogenic recombinant staphylokinase versus alteplase for patients with acute ischaemic stroke 4·5 h after symptom onset in Russia (FRIDA): a randomised, open label, multicentre, parallel-group, non-inferiority trial.

BACKGROUND: Non-immunogenic staphylokinase is modified recombinant staphylokinase with low immunogenicity, high thrombolytic activity, and selectivity to fibrin. We aimed to assess the safety and efficacy of a single intravenous bolus of non-immunogenic staphylokinase compared with alteplase in patients with acute ischaemic stroke within 4·5 h after symptom onset. METHODS: We did a randomised, open-label, multicentre, parallel-group, non-inferiority trial in 18 clinical sites in Russia. We included patients aged 18 years and older with a diagnosis of acute ischaemic stroke (up to 25 points on the National Institutes of Health Stroke Scale). The study drug had to be administered within 4·5 h after the onset of symptoms. Patients were randomly assigned to receive either non-immunogenic staphylokinase (10 mg) or alteplase (0·9 mg/kg, maximum 90 mg), both administered intravenously. The randomisation sequence was created by an independent biostatistician using computer-generated random numbers. 84 blocks (block size of four) of opaque sealed envelopes were numbered sequentially from 1 to 336 and were opened in numerical order. Patients were unaware of their assigned treatment and were assessed by the study investigators who were also unaware of the treatment assignment on all trial days. Emergency department staff, who administered the assigned drug and opened the envelopes, were not masked to treatment. The primary efficacy endpoint was a favourable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. The margin of non-inferiority was established as 16% for the difference in mRS score of 0-1 on day 90. Non-inferiority was tested using Welch's t-test for the primary outcome only. Endpoints were analysed in the per-protocol population, which comprised all randomly assigned patients who completed treatment without any protocol violations; this population was identical to the intention-to-treat population. This trial is completed and registered at ClinicalTrials.gov, NCT03151993. FINDINGS: Of 385 patients recruited from March 18, 2017, to March 23, 2019, 336 (87%) were included in the trial. 168 (50%) patients were randomly assigned to receive non-immunogenic staphylokinase and 168 (50%) to receive alteplase. The median duration of follow-up was 89 days (IQR 89-89). 84 (50%) of 168 patients in the non-immunogenic staphylokinase group had a favourable outcome at day 90 compared with 68 (40%) of 168 patients in the alteplase group (odds ratio [OR] 1·47, 95% CI 0·93 to 2·32; p=0·10). The difference in the rate of favourable outcome at day 90 was 9·5% (95% CI -1·7 to 20·7) and the lower limit did not cross the margin of non-inferiority (pnon-inferiority <0·0001). Symptomatic intracranial haemorrhage occurred in five (3%) patients in the non-immunogenic staphylokinase group and in 13 (8%) patients in the alteplase group (p=0·087). On day 90, 17 (10%) patients in the non-immunogenic staphylokinase group and 24 (14%) patients in the alteplase group had died (p=0·32). 22 (13%) patients in the non-immunogenic staphylokinase group had serious adverse events, compared with 37 (22%) patients in the alteplase group (p=0·044). INTERPRETATION: Non-immunogenic staphylokinase was non-inferior to alteplase for patients with acute ischaemic stroke. Mortality, symptomatic intracranial haemorrhage, and serious adverse events did not differ significantly between groups. Future studies are needed to continue to assess the safety and efficacy of non-immunogenic staphylokinase in patients with acute ischaemic stroke within the 4·5 h time window, and to assess the drug in patients with acute ischaemic stroke outside this time window with reperfusion CT or magnetic resonance angiography followed by thrombectomy if necessary. FUNDING: The Russian Academy of Sciences.

Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice.

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/- or C1q-/- mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors. In-vitro studies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/- than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first "hit" is recipient inflammation and the second "hit" is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Novel neutralizing human monoclonal antibodies against tetanus neurotoxin.

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)-reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix-reactive antibodies provided enhanced protection compared to the use of each antibody alone.

Development of dominant epitope-based vaccines encoding Gp63, Kmp-11 and Amastin against visceral leishmaniasis.

The most dangerous form of leishmaniasis is Visceral leishmaniasis (VL). The elimination of VL depends not only on agent treatments but also on effective vaccines against Leishmania parasites. Epitope-based vaccines composed of alternative short antigenic epitopes have the advantages of MHC epitope easy designing, which has broad application prospects. In a previous study, we analyzed Leishmania Gp63, Kmp-11 and Amastin protein sequence in silico, and found that the amino acid fragments of Gp63 (138-360aa), Kmp-11 (1-91aa) and Amastin (1-72aa) were rich in dominant epitopes. In this study, we used the three amino acid fragments as multi-epitope vaccine candidates to construct DNA and protein vaccines. BALB/c mice were vaccinated with the DNA and protein vaccines by DNA prime-protein boost strategy and challenged with Leishmania promastigotes. To evaluate vaccine immunogenicity and immunoprotection, serum specific antibody titers and cytokines were detected using ELISA, splenic CD3+, CD4+ and CD8+ cells were analyzed by flow cytometry, livers were made into pathological sections to observe pathological changes, and splenic parasitic loads were quantified using qPCR. The results showed that the increased specific IgG titers from vaccinated mice supported the vaccine immunogenicity. The increased cytokines (IFN-γ, IL-12 and TNF-α), splenic CD3+, CD4+ and CD8+ T cells and hepatic granulomas, and the decreased splenic parasitic loads (parasite reduction rates of Gp63, Kmp-11 and Amatin groups were 89%, 86% and 79%, respectively) from immunized mice post-infection were suggested the good immunoprotection of the vaccines. Our study demonstrated that vaccines based on the dominant epitopes of Gp63, Kmp-11 and Amastin with DNA prime-protein boost vaccination strategy showed significant immune effects against Leishmania, especially the Gp63 group showed a nearly 90% parasites reduction rate. This study will provide references for visceral leishmaniasis epitope vaccine design and immune strategy selection.

Association of Pre-diagnostic Antibody Responses to Escherichia coli and Bacteroides fragilis Toxin Proteins with Colorectal Cancer in a European Cohort.

Experimental evidence has implicated genotoxic Escherichia coli (E. coli) and enterotoxigenic Bacteroides fragilis (ETBF) in the development of colorectal cancer (CRC). However, evidence from epidemiological studies is sparse. We therefore assessed the association of serological markers of E. coli and ETBF exposure with odds of developing CRC in the European Prospective Investigation into Nutrition and Cancer (EPIC) study.Serum samples of incident CRC cases and matched controls (n = 442 pairs) were analyzed for immunoglobulin (Ig) A and G antibody responses to seven E. coli proteins and two isoforms of the ETBF toxin via multiplex serology. Multivariable-adjusted conditional logistic regression analyses were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association of sero-positivity to E. coli and ETBF with CRC.The IgA-positivity of any of the tested E. coli antigens was associated with higher odds of developing CRC (OR: 1.42; 95% CI: 1.05-1.91). Dual-positivity for both IgA and IgG to E. coli and ETBF was associated with >1.7-fold higher odds of developing CRC, with a significant association only for IgG (OR: 1.75; 95% CI: 1.04, 2.94). This association was more pronounced when restricted to the proximal colon cancers (OR: 2.62; 95% CI: 1.09, 6.29) compared to those of the distal colon (OR: 1.24; 95% CI: 0.51, 3.00) (pheterogeneity = 0.095). Sero-positivity to E. coli and ETBF was associated with CRC development, suggesting that co-infection of these bacterial species may contribute to colorectal carcinogenesis. These findings warrant further exploration in larger prospective studies and within different population groups.

HLA-DRB1 molecules and the presentation of anchor peptides from RhD, RhCE, and KEL proteins.

BACKGROUND: Antigens from the Rh and Kell systems are recognized as the most immunogenic in clinical practice. This study evaluated the possible molecular mechanisms involved in the interaction of antigenic peptides with the DRB1 molecules, which help to explain the high frequency of anti-K and association of D + C antibodies in transfusion and incompatible pregnancy. STUDY DESIGN AND METHODS: We included 201 patients with antibodies against antigens from the Rh and Kell systems and compare them with 174,015 controls. HLA-DRB1 genotyping and in silico analysis were performed. The NetMHCIIpan software was used to identify RhD-, RhCE-, and KEL-derived anchor peptides that bind to DRB1 molecules. RESULTS: HLA-DRB1*15 is associated with an increased risk of D, C, E, and K alloimmunization, while the HLA-DRB1*01 and *12 alleles are overrepresented in patients with anti-C and anti-D, respectively. In silico analysis showed that three polymorphic points (60I, 68S, and 103S) common to C and D antigens can be presented by several DRB1 molecules, including DRB1*15:01. The DRB1*09:01 molecule, although not showing statistical significance, was able to interact strongly with almost all five anchor peptides from the sequence containing the polymorphic determinants of E antigen, except 217-WMFWPSVNS-225. CONCLUSION: The DRB1*15 molecule has specific physicochemical characteristics in residues 11P and 13R in the P4 pocket that can favor the response to various antigenic peptides. Anti-K alloimmunization is unrestricted for interaction with specific DRB1 molecules, which suggests that almost all individuals in our population have DRB1 molecules capable of binding to KEL-derived anchor peptides and produce anti-K when stimulated.

Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

Engineered antigen containing epitopes from Loxosceles spp. spider toxins induces a monoclonal antibody (Lox-mAb3) against astacin-like metalloproteases.

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 (184ENNTRTIGPFDYDSIMLYGAY205) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism.

Poly(I:C) causes failure of immunoprophylaxis to red blood cells expressing the KEL glycoprotein in mice.

Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/ß). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.

Do red cell alloantibodies continue to challenge breast fed babies?

BACKGROUND: Newborns have limited specific immune capability at birth, owing to delayed and constrained development of adaptive immunity. To supplement this period the mother passively transfers antibodies to the child either transplacentally or through breast milk. When maternal alloimmunisation occurs through foreign or fetal red cell surface antigens, stimulating the production of immunoglobulin G (IgG) antibodies, these IgG antibodies can cross the placenta and cause haemolytic disease of the fetus and the newborn. OBJECTIVE: We present two case reports of a neonate and an infant in whom IgG red cell alloantibodies were transferred through maternal breast milk. METHODS: Maternal serum, baby's serum and expressed breast milk samples were tested for the presence of red cell alloantibodies using gel card. Antibody screening, antibody identifications and titres alongside monospecific direct antiglobulin test, IgG subtypes were performed using the standard methods. RESULTS: In the first case, a 6-month-old child was incidentally found to have positive antibody screen. Anti-KELL1 was identified, which was also present in maternal serum and breast milk. The second neonate was evaluated for haemolysis and was found to have anti-D. Anti-D was also detected in the maternal serum and breast milk. Both babies did not have any sensitising events. The first baby was asymptomatic, but the second baby had ongoing haemolysis until 1 month. CONCLUSION: We report that maternal anti-KELL1 and anti-D antibodies were present in breast milk and were capable of being transferred to a feeding child. Our case report also raises interesting and unanswered immunologic fundamentals that should be considered in neonates with unexplained anaemia or delayed and persistent haemolysis.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-, mitogen-activated protein kinase-, and Nrf2-dependent pathway.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) causes colitis and diarrhea, and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers. These diseases are dependent on ETBF-secreted toxin (BFT). Dendritic cells (DCs) play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1 (HO-1) is involved in the regulation of DC function. AIM: To investigate the role of BFT in HO-1 expression in DCs. METHODS: Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2-/- knockout mice. DCs were exposed to BFT, after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR, EMSA, fluorescent microscopy, immunoblot, and ELISA. RESULTS: HO-1 expression was upregulated in DCs stimulated with BFT. Although BFT activated transcription factors such as NF-κB, AP-1, and Nrf2, activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs. Instead, upregulation of HO-1 expression was dependent on Nrf2 activation in DCs. Moreover, HO-1 expression via Nrf2 in DCs was regulated by mitogen-activated protein kinases such as ERK and p38. Furthermore, BFT enhanced the production of reactive oxygen species (ROS) and inhibition of ROS production resulted in a significant decrease of phospho-ERK, phospho-p38, Nrf2, and HO-1 expression. CONCLUSION: These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Construction of a novel Staphylokinase (SAK) mutant with low immunogenicity and its evaluation in rhesus monkey.

The heterologous nature of SAK, a thrombolytic drug, elicits high titers of neutralizing antibodies, which limits its clinical use. Here, we aim to establish a SAK mutant with equivalent activity to the wild type but reduced antigenicity, which may allow for multiple injections. Biosun software was used to predict SAK antigenic epitopes, and several main epitopes were modified by gene deletion and mutation. Ten SAK mutants were constructed, and their thrombolytic activity and immunogenicity were analyzed in vitro. SAK6, with a high expression level (45%), similar thrombolytic activity, and lower antibody reaction, was chosen for in vivo analysis in rhesus monkey. In the nearly 8-month experimental period, the antibody level of the SAK6 group was significantly lower than that of the SAK group. Moreover, only 5% of SAK activity was retained, whereas 75.6% of SAK6 activity was retained after incubating with respective antiserum. Overall, these results demonstrated that SAK6, established through comprehensive site-directed mutagenesis program, had identical thrombolytic activity to SAK, low immunogenicity, and less side effects, demonstrating its efficient clinical potential for thrombus disease.

Novel multi-epitope protein containing conserved epitopes from different Leishmania species as potential vaccine candidate: Integrated immunoinformatics and molecular dynamics approach.

Leishmaniosis, caused by intracellular parasites of the genus Leishmania, has become a serious public health problem around the world, and for which there are currently extensive limitations. In this work, a theoretical model was proposed for the development of a multi-epitope vaccine. The protein GP63 of the parasite was selected for epitopes prediction, due to its important biological role for the infection process and abundance. IEDB tools were used to determine epitopes B and T in Leishmania braziliensis; besides, other conserved epitopes in three species were selected. To improve immunogenicity, 50S ribosomal protein L7 / L12 (ID: P9WHE3) was used as a domain of adjuvant in the assembly process. The folding arrangement of the vaccine was obtained through homologous modeling multi-template with MODELLER v9.21, and a Ramachandran plot analysis was done. Furthermore, physicochemical properties were described with the ProtParam tool and secondary structure prediction combining GOR-IV and SOPMA tools. Finally, a molecular dynamics simulation (50 ns) was performed to establish flexibility and conformational changes. The analysis of the results indicates high conservancy in the epitopes predicted among the four species. Moreover, Ramachandran plot, physicochemical parameters, and secondary structure prediction suggest a stable conformation of the vaccine, after a minimum conformational change that was evaluated with the free energy landscape. The conformational change does not drive any substantial change for epitope exposition on the surface. The vaccine proposed could be tested experimentally to guide new approaches in the development of pan-vaccines; vaccines with regions conserved in multiple species.

A hemorrhagic metalloprotease of Egyptian Cerastes vipera venom: Biochemical and immunological properties.

A hemorrhagic metalloprotease (CVHT1) was isolated from Cerastes vipera (CV) viper venom and characterized in a set of biochemical and immunological assays. A simple two-step purification procedure included gel filtration and ion-exchange increase the purity of enzyme 39-fold with specific activity of 20,200 Umg-1 compared to 520 Umg-1 for crude venom. CVHT1 is a dimer enzyme with two subunits of ~60 kDa. The LC-MS/MS analysis of CVHT1 revealed that the identified peptides show high homology to other P-III snake venom zinc-metalloproteases. The activity of CVHT1 showed stability at pH (6.5-8.5) and temperature (30-60 °C) with optima at pH 8.5 and 60 °C. Activators for CVHT1 included Mg+2, Zn+2, Ca+2, K+, Ba+2 and Na+, while the full inhibition was given by other tested ions, SH-group reagents and metalloproteinase inhibitors. The CVHT1 potentially digested gelatin, fibrinogen, fibronectin and inhibited the platelet aggregation. The hemorrhagic and proteolytic activities of medically important Egyptian viper venoms were highly cross-neutralized by anti-CVHT1. The anti-CVHT1 increased the survival time of mice injected with lethal dose of CV venom to 23 ±â€¯2.5 h compared to the mice injected with venom alone 0.52 ±â€¯0. 05 h. This study could be useful for production of safer and more efficient therapeutic anti-venom.

Conjugation of staphylokinase with the arabinogalactan-PEG conjugate: Study on the immunogenicity, in vitro bioactivity and pharmacokinetics.

Staphylokinase (SAK) is a bacterial protein with profibrinolytic activity. However, SAK suffers from short serum half-life and high immunogenicity. PEGylation with high Mw (20 kDa or 40 kDa) could decrease the immunogenicity and prolong the serum half-life of the proteins. However, the PEGylated protein could induce the anti-PEG antibodies and its bioactivity was significantly decreased. Arabinogalactan (AG) is a health-promoting substance with numerous biological activities. Conjugation of AG is an alternative strategy to solve the above-mentioned problems. However, conjugation with AG significantly decreased the bioactivity of a protein by shielding the bioactive domain. Here, AG conjugation and PEGylation were combined to improve the therapeutic efficacy of SAK. PEG with low Mw (2 kDa or 5 kDa) acted as a linker to conjugate AG from Larix. As compared with SAK-AG (22.3%), the conjugates (SAK-P2K-AG and SAK-P5K-AG) largely maintained the bioactivity of SAK (73.8% and 62.9%). The two conjugates both showed an 8-fold decrease in the SAK-specific IgG titers and a prolonged serum half-life. Moreover, the conjugates did not render any apparent toxicity to the heart, liver and renal functions of mice. Thus, our conjugation strategy is promising for the development of an effective long-acting therapeutic protein.

Transfused platelets enhance alloimmune responses to transfused KEL-expressing red blood cells in a murine model.

BACKGROUND: Factors influencing the development of alloantibodies against blood group antigens on transfused red blood cells are poorly defined. We hypothesised that transfused platelets may act as a danger signal to recipients and affect humoral immune responses to transfused red blood cells. MATERIALS AND METHODS: Platelet-rich plasma prepared from wild-type C57BL/6 or CD40L knock-out donors was transfused into wild-type or CD40L knock-out recipients. Leucoreduced red blood cells from transgenic donors expressing high levels of the human KEL glycoprotein in an erythrocyte-specific manner (KELhi donors) were transfused after the platelets, and anti-KEL responses were measured longitudinally. In some experiments, recipients were treated with poly (I:C), monoclonal CD40L-blocking antibody, or CD4-depleting antibody prior to transfusion. RESULTS: Transfusion of wild-type C57BL/6 platelets or treatment with poly (I:C) prior to KELhi red blood cell transfusion led to an anti-KEL alloimmune response in wild-type recipients. Transfusion of platelets from wild-type but not CD40L knock-out donors prior to KELhi red blood cell transfusion led to an IgG anti-KEL alloimmune response in CD40L knock-out recipients; unexpectedly, transfusion of platelets from CD40L knock-out donors prior to KELhi red blood cell transfusion led to a robust anti-KEL alloimmune response in wild-type recipients. Recipient treatment with MR1 CD40L-blocking antibody or CD4-depleting antibody prevented KEL alloimmunisation altogether. DISCUSSION: Transfused platelets serve as an adjuvant in this T-dependent murine model of anti-KEL red blood cell alloimmunisation, with CD40/CD40L interactions being involved to some degree but with additional mechanisms also playing a role. These findings raise questions about the role that transfused or endogenous platelets may play in other innate/adaptive immune responses.

Identification of a linear B-cell epitope in the catalytic domain of bothropasin, a metalloproteinase from Bothrops jararaca snake venom.

Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B-cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti-venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti-BotEp1 serum cross-reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti-BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non-immunized animal). Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.