Search

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8 + T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS -derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (T reg ) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved T reg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced T reg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective T reg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall T reg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential T reg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of T reg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Vaccination and the use of neuraminidase inhibitors (NAIs) are currently the front lines of defense against seasonal influenza. The activity of influenza vaccines and antivirals drugs such as the NAIs can be affected by mutations in the influenza hemagglutinin (HA) protein. Numerous HA substitutions have been identified in nonclinical NAI resistance-selection experiments as well as in clinical specimens from NAI treatment or surveillance studies. These mutations are listed in the prescribing information (package inserts) for FDA-approved NAIs, including oseltamivir, zanamivir, and peramivir., Methods: NAI treatment-emergent H1 HA mutations were mapped onto the H1N1 HA1 trimeric crystal structure and most of them localized to the HA antigenic sites predicted to be important for anti-influenza immunity. Recombinant A/California/04/09 (H1N1)-like viruses carrying HA V152I, G155E, S162 N, S183P, and D222G mutations were generated. We then evaluated the impact of these mutations on the immune reactivity and replication potential of the recombinant viruses in a human respiratory epithelial cell line, Calu- 3., Results: We found that the G155E and D222G mutations significantly increased viral titers ~ 13-fold compared to the wild-type virus. The hemagglutination and microneutralization activity of goat and ferret antisera, monoclonal antibodies, and human serum samples raised against pandemic A(H1N1)pdm09 viruses was ~ 100-fold lower against mutants carrying G155E or D222G compared to the wild-type virus., Conclusions: Although the mechanism by which HA mutations emerge during NAI treatment is uncertain, some NAI treatment-emergent HA mutations correlate with decreased immunity to influenza virus.

C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We previously showed that single amino acid substitutions at seven positions in haemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in 11 representative H3 haemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at haemagglutinin position 145 was fully independent of the amino acid context of the representative haemagglutinins. Antigenic change caused by substitutions introduced at haemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the haemagglutinin can moderate the magnitude of antigenic change.

Background: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection., Objective: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively., Methods: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro., Results: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo., Conclusion: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48 A534V , a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48 A532V , SINV I538 and SINV T538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48 A534V was attenuated in vitro and in vivo Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48 A534V , differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48 A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease. IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates., (Copyright © 2018 Liu et al.)

In recent years, directed evolution has emerged as an efficient tool to develop and identify novel protein variants. Eimeria tenella microneme-2 (EtMIC2) is a promising vaccine candidate for use against E. tenella infection; however, it only yields partial protection. The present study aimed to improve the immunogenicity and protective efficacy of EtMIC2 through random mutagenesis. Mutagenesis gene libraries of EtMIC2 were generated using error-prone polymerase chain reaction (epPCR), and the corresponding variant proteins were displayed on the yeast cell surface. Variant EtMIC2 proteins with high immunogenicity were screened through fluorescence-activated cell sorting (FACS) based on the affinity between polyclonal antibodies and antigens. Seven effective variant proteins were screened out and heterogeneously expressed in Escherichia coli as subunit vaccines. The protective efficacy of the variant proteins against E. tenella infections was then evaluated in chicken. Two variant proteins (1130 and 2119) displayed higher immunogenicity and protective efficacy than the wild-type EtMIC2 protein against E. tenella infections, increasing body weight gains and significantly decreasing lesion scores and fecal oocyst shedding, and increasing sIgA antibody production and lymphocyte proliferation. These variants displayed potential for use in the development of subunit vaccines for coccidiosis in chickens. The present results also indicate that directed evolution technology is useful for improving the immunogenicity and protective efficacy of parasite antigens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.

Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hirudin III is an effective anti-coagulant; however, in 40% of treated patients, a high-titer of anti-Hirudin III IgG antibodies is observed. Development of antibody responses requires the activation of helper T lymphocyte (HTL), which is dependent on peptide epitopes binding to HLA class II molecules. Based on computational prediction softwares, four new mutants of Hirudin III, T4K, S9G, V21G, and V21K, had been designed with the aim of reducing the binding affinity of these HTL epitopes. The constructed mutants have been purified and assayed for bioactivity. Finally in vitro and in vivo cell-mediated responses were assessed and humoral immune assays were performed. All modified forms of Hirudin III were active, and showed significantly reduced human T-cell responses. All mutants indicated lower human IFN-γ level compared to native Hirudin, and V21K indicated lowest IFN-γ level. Mice immunized with T4K and V21K showed a significant reduction in total antibody responses and mouse IFN-γ levels. Mice immunized with V21K after 3rd immunization had lower T-cell proliferation compared to native Hirudin and other mutants. Based on these results, V21K is proposed as the best alternate Hirudin III candidate with lowest antigenicity. These findings validate our rational design strategy aimed at providing new active analogs of therapeutic proteins with reduced immunogenicity., (© 2017 APMIS. Published by John Wiley & Sons Ltd.)

An effective HIV vaccine remains elusive, and immunogens capable of eliciting protective host humoral immunity have not yet been identified. Although HIV/SIV infections result in the abundant production of CD4-induced (CD4i) antibodies (Abs), these Abs are not protective due to steric restrictions following gp120 binding to CD4 on target cells. Here we report that both DNA- and vaccinia-based vaccines encoding SIV mac239 gp160 readily elicited high levels of CD4i Abs in experimental animals. We identified a highly conserved N-linked glycosylation site N277 in the C2 region which strongly affected the immunogenicity of the CD4i Ab domain. Moreover, a single N277A substitution significantly enhanced the immunogenicity of the V2 domain yielding higher titers and frequency of anti-V2 Ab responses as determined by ELISA and yeast antigen display mapping, respectively. Importantly, immune sera elicited by the N277A-mutated gp160 exhibited elevated antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC activity correlated positively with the anti-V2 Ab titer yet, inversely with CD4i Ab titer. Thus, we identified a determinant of the CD4i domain that might affect vaccine-elicited anti-V2 Ab and ADCC responses to SIV mac239 . Our findings may have implications for design of immunogens to direct B cell recognition in the development of an Ab-based HIV vaccine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Context: - NRAS is a member of the RAS family oncoproteins implicated in cancer. Gain-of-function NRAS mutations were reported in a subset of colorectal cancers. These mutations occur at codons 12, 13, and 61 and are detected by molecular genetic testing. Recently, an antibody (clone SP174) became available to immunohistochemically pinpoint NRAS Q61R mutant protein. In malignant melanoma, NRAS Q61R mutant-specific immunohistochemistry was shown to be a valuable supplement to traditional genetic testing., Objective: - To evaluate the significance of NRAS Q61R mutant-specific immunohistochemistry in a cohort of colorectal carcinomas., Design: - A total of 1185 colorectal carcinomas were immunohistochemically evaluated with SP174 antibody. NRAS Q61R mutant-specific immunohistochemistry was validated by molecular genetic testing including Sanger sequencing, quantitative polymerase chain reaction (qPCR), and next-generation sequencing., Results: - Twelve tumors showed strong SP174 immunoreactivity. Sanger sequencing detected an identical c.182A>G substitution, causing NRAS Q61R mutation at the protein level, only in 8 SP174-positive cases. These results were confirmed by qPCR study. Subsequently, NRAS wild-type tumors with strong SP174 staining were evaluated by next-generation sequencing and revealed KRAS c.182A>G substitutions predicted to cause KRAS Q61R mutation. Review of colorectal carcinomas with known KRAS and NRAS genotype revealed that none of 62 wild-type tumors or 47 mutants other than Q61R were SP174 positive., Conclusion: - SP174 immunohistochemistry allows sensitive detection of NRAS and KRAS Q61R mutants. However, molecular genetic testing is necessary to determine specifically which RAS gene is mutated.

Listeriolysin O (LLO) has been proposed as a potential carrier or adjuvant molecule in the vaccination field. However, the cytotoxic and pro-apoptotic effects of LLO are the major limitations for this purpose. Here, we have performed a preclinical safety evaluation and characterized a new potential adjuvant application for a non-cytolytic LLO mutant (dtLLO) to enhance and modulate the immune response against the envelope (E) protein from dengue virus. In addition, we have studied the adjuvant effects of dtLLO on human immune cells and the role of membrane cholesterol for the binding and proinflammatory property of the toxoid. Our in-vivo results in the murine model confirmed that dtLLO is a safer molecule than wild-type LLO (wtLLO), with a significantly increased survival rate for mice challenged with dtLLO compared with mice challenged with wtLLO (P < 0·001). Histopathological analysis showed non-toxic effects in key target organs such as brain, heart, liver, spleen, kidney and lung after challenge with dtLLO. In vitro, dtLLO retained the capacity of binding to plasma membrane cholesterol on the surface of murine and human immune cells. Immunization of 6-8-week-old female BALB/c mice with a combination of dtLLO mixed with E protein elicited a robust specific humoral response with isotype diversification of immunoglobulin (Ig)G antibodies (IgG1 and IgG2a). Finally, we demonstrated that cholesterol and lipid raft integrity are required to induce a proinflammatory response by human cells. Taken together, these findings support a potential use of the dtLLO mutant as a safe and effective adjuvant molecule in vaccination., (© 2016 British Society for Immunology.)

HIV-1 passage in cell culture in the presence of chemokine receptor antagonists can result in selection of viruses with env mutations that confer resistance to these inhibitors. In the present study, we examined the effect of HIV-1env mutations that confer resistance to CXCR4 antagonists on envelope (Env) sensitivity to neutralizing antibodies (NAbs). Serial passage of CXCR4-tropic HIV-1 NL4-3 in PM1/CCR5 cells under CXCR4 antagonists KRH-3955, AMD3100 and AMD070 yielded two KRH-3955-resistant, one AMD3100-resistant and one AMD070-resistant viruses. These viruses had multiple env mutations including the Env gp120 V3 region. The majority of viruses having these CXCR4 antagonist-resistant Envs showed higher sensitivity to NAbs 447-52D, b12 and 2F5 targeting the V3 region, the gp120 CD4-binding site and the gp41 membrane proximal region, respectively, compared to NL4-3 WT virus. Recombinant NL4-3 viruses with the V3-coding region replaced with those derived from the CXCR4 antagonist-resistant viruses showed increased sensitivity to NAbs b12, 2F5 and 447-52D. Molecular dynamics simulations of Env gp120 outer domains predicted that the V3 mutations increased levels of fluctuations at the tip and stem of the V3 loop. These results indicate that mutations in the V3-coding region that result in loss of viral sensitivity to CXCR4 antagonists increase viral sensitivity to NAbs, providing insights into our understanding of the interplay of viral Env accessibility to chemokine receptors and sensitivity to NAbs.

Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.

Longitudinal analysis of chronic hepatitis C virus (HCV) infection has shown that the virus has several adaptive strategies that maintain persistence and infectivity over time. We examined four serum samples from the same chronically infected HCV genotype 4a patient for the presence of IgG antibody-associated virus. RNA was isolated from antibody-associated and antibody-free virions. Subsequent to sequence analysis, 27 aa hypervariable region 1 (HVR1) peptides were used to test the humoral immune escape. We demonstrated that differential peptide binding of Fab was associated with a single amino acid change. We provide direct evidence of natural humoral immune escape by HCV within HVR1.

Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines?

Isocitrate dehydrogenase 1 (IDH1) mutations have been detected in gliomas and other tumors. Although IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in cytosol, mutated IDH1 proteins possess the ability to change α-KG into the oncometabolite D-2-hydroxyglutarate (D-2HG). Several monoclonal antibodies (mAbs) specific for IDH1 mutations have been established, such as H09, IMab-1, and HMab-1 against IDH1-R132H, which is the most frequent IDH1 mutation in gliomas. In this study, we established a novel high-sensitive mAb HMab-2, which reacts with IDH1-R132H but not with wild type IDH1 in ELISA. HMab-2 reacted only with IDH1-R132H, not with wild type IDH1/2 and other IDH1/2 mutants in Western-blot analysis. Furthermore, HMab-2 recognized IDH1-R132H more sensitively compared with our previously established HMab-1. HMab-2 detected endogenous IDH1-R132H protein expressed in glioblastoma in immunohistochemical analysis. HMab-2 is expected to be useful for the diagnosis of IDH1-R132H-bearing tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Current commercial PCV2 vaccines are almost based on PCV2a and have been shown to be effective in reducing PCV2a and PCV2b viremia and PCV2-associated lesions and diseases. The recent emergence of novel mutant PCV2 (mPCV2) strains and linkage of mPCV2 with cases of porcine circovirus associated disease (PCVAD) in pig herds have raised concerns over emergence of vaccine-escape mutants and reduced efficacy of PCV2a-based vaccines. The aim of this study was to determine the ability of a commercial PCV2a-based vaccine developed by our laboratory to protect conventional pigs against experimental challenge with mPCV2 at 9 weeks of age. Twenty 4-week-old pigs free of PCV2 infection were randomly divided into four treatment groups with 5 pigs each. Two groups were unvaccinated as positive and negative controls. Another two groups were vaccinated with the commercial PCV2a-based vaccine (PCV2-LG strain, China) at 4 weeks of age and identical booster immunization was conducted 3 weeks post primary immunization. At 9 weeks of age, all pigs except the negative control were challenged with a mutant PCV2b/YJ (mPCV2b/YJ) with two amino acids elongation in capsid protein. The experiment was terminated 28 days after challenge. Under the conditions of this study, vaccinated pigs were protected against PCV2 viremia and lesions whereas unvaccinated pigs were not. Moreover, mPCV2b/YJ infection was demonstrated in positive control and almost all had macroscopic or microscopic lesions consistent with PCVAD while negative control did not develop PCVAD. This study indicates that mPCV2b/YJ infection alone can trigger PCVAD development and that the commercial vaccine (PCV2-LG) is still effective in protecting conventional pigs against the emerging mPCV2b/YJ strain in China., (Copyright © 2015. Published by Elsevier Ltd.)

We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein Sci., 22, 213-221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin., (Copyright © 2014. Published by Elsevier B.V.)

The molecular structure of protein and epitope mapping strategies are required to engineer the epitopes of a protein. In the present study, IgE binding regions of osmotin were identified and mutated to obtain hypoallergenic variant. The three dimensional (3-D) model of osmotin obtained by homology modeling comprised of a characteristic thaumatin-like fold. This model was used to predict IgE binding regions of osmotin. These regions were mutated and three mutant proteins with four mutations (Ma, Mb and Mc) and one with six mutations (Mabc) were expressed and purified to homogeneity. IgE binding of the mutant proteins was evaluated by in vitro studies using patients' sera. Ma, Mb and Mc demonstrated reduction in IgE binding of 73%, 83% and 77%, respectively, whereas Mabc showed complete abrogation of IgE binding. Ma, Mb and Mc showed inhibition of 48%, 44% and 38%, respectively to osmotin, while Mabc showed 24% inhibition at 10 μg with pooled patients' sera. Osmotin reached effective concentration at 50% inhibition (EC50) at 3 ng and none of the mutant proteins reach the EC50 value. The immunological response to mutant proteins was examined in mice. Blood, bronchoalveolar lavage fluid spleen and lung tissue were excised from mice for analysis. The mice treated with mutant proteins showed significant reduction in IgE and IgG1 levels as compared to mice given osmotin (p<0.001). Th2 cytokines level in splenocyte supernatant and BALF of mice given mutant proteins were significantly lower (p<0.001), accompanied with significant reduction in cellular infiltration in lungs (p<0.001). In conclusion, osmotin structure was predicted by homology modeling and IgE binding regions predicted were mutated to obtain a hypoallergenic protein., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Myasthenia gravis (MG) is usually caused by antibodies against the muscle acetylcholine receptor (AChR). Plasmapheresis and immunoadsorption are often used to treat non-responsive patients. Antigen-specific immunoadsorption would remove only the pathogenic autoantibodies reducing side-effects. We expressed AChR extracellular domain mutants for use as specific adsorbents, and characterized them. Antigenicity and capacity for autoantibody binding were improved compared to the wild-type proteins. Adsorption appeared to be fast, as high plasma flow-rates could be applied. The bound autoantibodies were eluted repeatedly, allowing column reuse, without compromise in efficiency. Overall, the adsorbents were specific, efficient and suitable for use in therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A neoantigen derived from mutant IDH1 can promote antitumor CD4(+) T-cell and antibody responses., (©2014 American Association for Cancer Research.)

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4(+) T-helper-1 (TH1) responses. CD4(+) TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4(+) T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Background: During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei., Methodology/principal Findings: (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination., Conclusion/significance: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis.

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

Aims: The presence of a BRAF mutation is a strong marker for poor prognosis of colorectal carcinoma (CRC), and can be used as evidence of a sporadic mechanism of mismatch repair deficiency. BRAF mutation may also predict resistance to EGFR-targeted therapy. A BRAF V600E-specific antibody has recently become commercially available. The aim of this study was to determine whether immunohistochemistry can predict BRAF mutations in CRC., Methods and Results: Immunohistochemistry was performed on 52 genotyped CRC cases (17 BRAF mutant, 18 KRAS mutant, 17 BRAF/KRAS wild-type) with monoclonal antibody VE1. Cytoplasmic staining was observed in 71% of BRAF V600E mutant tumours (moderate or strong staining in 50% of these cases). Weak cytoplasmic staining was observed in 17% of KRAS mutant tumours and 35% of wild-type tumours. Non-specific nuclear staining was common. The sensitivity and specificity of immunohistochemistry with VE1 for BRAF mutation were 71% and 74%, respectively; when only moderate or strong staining was considered to be positive, the specificity was 100%, but the sensitivity only 35%., Conclusions: Immunohistochemistry with VE1 is not a useful surrogate for genotyping in CRC. Although moderate or strong cytoplasmic staining is specific for BRAF V600E mutations, this antibody is insufficiently sensitive to serve as an effective screening tool., (© 2013 John Wiley & Sons Ltd.)

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.

HBV surface antigen (HBsAg) variants may impair diagnosis or allow the virus to escape vaccine-induced immunity and their circulation in the population can represent a Public Health threat. Their prevalence, however, is not yet completely established. Evidence indicates that amino acid substitutions within HBsAg can lead to conformational changes which allow mutated HBV to escape the vaccine-induced antibodies used in the screening tests. In such scenario, the aim of this study was to investigate the prevalence of HBV S-Gene escape mutants by sequencing the gene in a cohort of Ligurian patients monitored for viral load, genotype and drug resistance and to evaluate the risk of false negative HBsAg detection by routine screening tests. From 2007 to 2011, in 256 consecutive samples from Ligurian HBV positive patients sequencing assay for detection of RT/S-Gene mutations using Trugene HBV Genotyping kit (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) was performed. Serological HBV tests and viral load were also performed. Analyzed sequences revealed G145R mutation in 8/256 (3.1%) examined sequences, it was alone in 5 patients and accompanied by other HBsAg mutations in 3 samples. HBsAg resulted undetectable by 3 of the 8 samples, derived from patients with multiple mutations: T126I-T131A-C139Y-E/D144G, T126I-M133L, and P120Q-T126I. The emergence of these mutants, at least the G145R, has already been addressed as a public health concern because of its capability of escaping the immune system. In the present study we point out a second aspect connected with their existence and with similar potential negative impact on public health, that is their capability of escape punctual detection.

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

Background: CD72 is an inhibitory co-receptor expressed on B cells. We previously demonstrated significant association of the polymorphism of the CD72 gene with susceptibility to human systemic lupus erythematosus (SLE) in individuals carrying a SLE-susceptible FCGR2B genotype (FCGR2B-232Thr/Thr). The human CD72 locus generates a splicing isoform that lacks exon 8 (CD72Δex8) as well as full-length CD72 (CD72fl), and the CD72 polymorphism regulates exon 8 skipping., Results: Here we demonstrated that individuals carrying the disease-protective CD72 genotype exhibit significantly lower serum immunoglobulin levels than do individuals carrying other CD72 genotypes (P < 0.05). Although expression level of CD72fl in the peripheral blood B cells was similar regardless of CD72 genotype, the protein level of CD72Δex8 was increased in individuals carrying the disease-protective CD72 genotype, suggesting a crucial role of CD72Δex8 in regulation of antibody production. By expressing these human CD72 isoforms in mouse cell lines, we further demonstrated that CD72Δex8 is accumulated in endoplasmic reticulum (ER) and fails to regulate BCR signaling whereas human CD72fl is efficiently transported to the cell surface and inhibits signaling through the B cell antigen receptor (BCR), as is the case for mouse CD72., Conclusion: Human CD72 polymorphism appears to regulate antibody production as well as susceptibility to SLE by regulating expression of ER-localizing CD72Δex8.

Background: The quantification of hepatitis-B surface antigen (HBsAg) is useful to identify inactive carriers in chronically HBV infected patients and to predict interferon treatment outcome., Objective: To compare two quantitative HBsAg assays for the detection of HBsAg mutants expressed in vitro., Study Design: HBsAg mutants (n=35) were expressed in HuH7 cells and the supernatants were tested in two different quantitative HBsAg assays (Architect, Abbott and Elecsys, Roche)., Results: All HBsAg mutants were detected by both assays, but in general the results of the Architect system were higher than those of the Elecsys system. The detection of HBsAg mutants in comparison to wild type was similar using both assays. However, HBsAg mutation T123A was under quantified by the Architect, whereas HBsAg mutations P142L, P142S and G145K yielded lower results in the Elecsys system., Conclusions: Even though HBsAg assays are optimised for the detection of HBsAg mutants, discrepant results were obtained for some HBsAg mutants in two quantitative HBsAg assays. These findings have to be considered when testing samples from one patient with two different quantitative HBsAg assays., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The development and testing of detection methodologies for biothreat agents are by their very nature complicated by the necessity to handle hazardous materials. Toxoids prepared by thermal or chemical inactivation are often used in place of the native toxin; however, the process of detoxification can decrease the agent's ability to be detected at similar concentrations. One method to overcome this limitation is the use of toxin mutants which have altered amino acid sequences sufficient to abrogate or greatly reduce their toxic activity. While this method of toxoid preparation is much more controlled, there is still no guarantee that the resulting product will be equal in detectability to the native toxin. In this work, we have evaluated the utility of two recombinantly expressed Staphylococcal Enterotoxin B (SEB) mutants, a single point mutant (Y89A), and a mutant with three amino acids changed (L45R, Y89A, Y94A), to act as surrogates for SEB in immunoassays. We evaluated the affinity of a number of anti-SEB monoclonal antibodies (mAb) and an anti-SEB single domain antibody (sdAb) for SEB and its surrogates. One of the mAb's affinity was decreased by a factor of 3000 for the triple mutant, and another mAb's affinity for the triple mutant was decreased by 11-fold while the others bound the mutants nearly as well as they did the native toxin. MAGPIX sandwich immunoassays were used to evaluate the ability of all combinations of the recognition reagents to detect the SEB mutants in comparison to SEB and a chemically inactivated SEB. These results show that recombinant mutants of SEB can serve as much more useful surrogates for this hazardous material relative to the chemically inactivated toxin; however, even the point mutant impacted limits of detection, illustrating the need to evaluate the utility of toxin mutants on a case-by-case basis depending on the immunoreagents being employed.

Our previous research showed that immulectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and β-1,3-glucan, and IML-2 plays an important role in cellular encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2Δ220-244 and IML-2Δ220-257), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2Δ220-244 and IML-2Δ220-257, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO activation, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions.

A panel of 6 neutralizing monoclonal antibodies (MAbs) raised against A/Moscow/IIV01/2009 (H1N1) virus isolated during the 2009 pandemic was used for the selection of 26 escape mutants. The mutants were characterized in immune cross-reactions with the panel of MAbs. The sequencing of the mutant HA genes revealed 5 amino acid positions recognized by monoclonal antibodies: 129, 156, 158, 159, and 190 (H3 numbering). The amino acid positions were distributed in two epitopes belonging to antigenic sites Sa and Sb. The mutant HAs exhibited variations in the affinity to synthetic high molecular mass sialic acid-containing receptor analogues. Results are discussed in connection with the antigenic drift potential of the "swine-like" pandemic 2009 influenza virus., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells. Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary 'high affinity' receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ and IL-2Rγ. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 'superkine' (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.

In the study, a gene encoding Tat protein N terminal 1- 21 amino acid residues-deleted mutant (Tat22-101) was amplified by PCR from a full length Tat gene of human immunodeficiency virus type 1, and the prokaryotic expression plasmid pET32a-Tat22-101 was constructed. After identification by digestion with endonucleases and sequencing, the recombinant plasmid pET32a-Tat22-101 was transformed into E. coli BL21(DE3) and expressed with IPTG induction. The mutant fusion protein with deleted Tat N terminal was purified by an affinity chromatography column Ni(2+)-NTA and subsequently identified by SDS-PAGE and Western blotting. The results showed that the molecular weight of the mutant protein was approximately 26.9kD. Furthermore, BALB/c mice were immunized with the mutant protein and the anti-sera were collected. ELISA results showed that the mutant protein preserved its immunogenicity, particularly it could improve the production of antibodies to other epitopes in addition to the N terminal epitope of Tat protein, which might provide some valuable information for the study of Tat functions as well as for development of potential novel HIV Tat vaccine.

The transcription factor GATA3 plays an essential role during T cell development and T helper 2 (Th2) cell differentiation. To understand GATA3-mediated gene regulation, we identified genome-wide GATA3 binding sites in ten well-defined developmental and effector T lymphocyte lineages. In the thymus, GATA3 directly regulated many critical factors, including Th-POK, Notch1, and T cell receptor subunits. In the periphery, GATA3 induced a large number of Th2 cell-specific as well as Th2 cell-nonspecific genes, including several transcription factors. Our data also indicate that GATA3 regulates both active and repressive histone modifications of many target genes at their regulatory elements near GATA3 binding sites. Overall, although GATA3 binding exhibited both shared and cell-specific patterns among various T cell lineages, many genes were either positively or negatively regulated by GATA3 in a cell type-specific manner, suggesting that GATA3-mediated gene regulation depends strongly on cofactors existing in different T cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Clinical application of staphylococcal enterotoxin C2 (SEC2) was restricted during the cure of malignant tumor due to its side-effects. The aim of this study was to obtain SEC2 mutant, preserving the important functional sites responsible for the T-cell stimulatory activities but removing the sites responsible for emetic activity, through truncation of SEC2. It would efficiently solve the question of SEC2 side-effect. According to the results of methyl thiazol tetrazolium (MTT) assay in vitro, novel truncated SEC2 mutant (NSM) efficiently stimulated T-cell proliferation and inhibited the growth of such tumor cells as human colorectal cancer cells (Cx-1) and human breast cancer cells (MCF-7) in vitro. Activities of T cell stimulating and anti-tumor of NSM were similar to those of SEC2. According to results of animal experiments, the mutant no longer induced emetic response even if the dose was a 10-fold excess of the amount of SEC2 required. And also, NSM obviously inhibited the tumor growth in tumor-bearing mice. Therefore, we obtained novel truncated staphylococcal enterotoxin C2 mutant, which could efficiently inhibit the growth of tumor cells. It will become novel anti-tumor agents with the lowest side-effects and best treatment effects in clinic.

Spleen tyrosine kinase Syk provides critical transducer functions for a number of immune cell receptors and has been implicated in the generation of several forms of leukemias. Catalytic activity and the ability of Syk to interact with other signaling elements depend on the phosphorylation status of Syk. We have now identified and quantified the full spectrum of phosphoacceptor sites in human Syk as well as the interactome of Syk in resting and activated B cells by high-resolution mass spectrometry. While the majority of inducible phosphorylations occurred on tyrosine residues, one of the most frequently detected phosphosites encompassed serine 297 located within the linker insert distinguishing the long and short isoforms of Syk. Full-length Syk can associate with more than 25 distinct ligands including the 14-3-3γ adaptor protein, which binds directly to phosphoserine 297. The latter complex attenuates inducible plasma membrane recruitment of Syk, thereby limiting antigen receptor-proximal signaling pathways. Collectively, the established ligand library provides a basis to understand the complexity of the Syk signaling network., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)