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Background: The major structural protein of coronaviruses, the membrane (M) protein, can elicit the formation of protective antibodies, but little information is available about the M protein of porcine epidemic diarrhea virus (PEDV). Identification of epitopes on the PEDV M protein will be helpful in the elucidation of the antigenic properties of this protein., Results: One hybridoma cell line secreting anti-M protein monoclonal antibody (McAb) was generated and designated 4D4. To map the epitopes on the PEDV M protein, a total of 17 partially overlapping fragments covering the C-terminus of M protein were expressed as fusion proteins with a 6×His tag or a GST tag. A linear motif, 193TGWAFYVR200, was identified by enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis using McAb 4D4. The motif 195WAFYVR200 was the minimal requirement for reactivity, as demonstrated by removing amino acids individually from both ends of the motif 193TGWAFYVR200. The result of WB analysis showed that the 4D4-defined epitope could be recognized by PEDV-positive serum, but not transmissible gastroenteritis virus (TGEV)-positive serum. Furthermore, this epitope was highly conserved among different PEDV strains, as shown by alignment and comparison of sequences., Conclusion: A McAb, 4D4, directed against the M protein of PEDV, was obtained, and the 4D4-defined minimal epitope sequence was 195WAFYVR200. The McAb could serve as a candidate for development of a McAb-based antigen capture ELISA for detection of PEDV. The epitope identified provides a basis for the development of epitope-based differential diagnostic techniques and may be useful in the design of epitope-based vaccines.

Egypt, whose soil germinated the first civilization, monotheism, refined ethics and culture of sharing the abundance of extracted natural resources among its populace became the crucible proliferating de-novo genotypes of organic and moral maladies. The enigma is these mutations are synchronized by several factors, namely; failing medical health, if there is any, abundant filth, cultural bankruptcy, over population, dogmatic militarism, societal deprivation and characterization, etc. These domineering ingredients fossilized Egypt as of 1952 coup in an irrevocable national apoptosis, together with the crippled social justice and imbalanced distribution of wealth among Egyptians, rates of bacterial and viral evolution to second generation resistant to known medical interventions are expected to exponentially accelerate. Therefore, it deemed essential to elaborate on pollution and psychosis-induced inflammations and grievous crimes evoked by dogmatic cults at the breeding source, e.g., ghettos and sporadic locations of the homeless in Cairo, Alexandria and Upper Egyptian villages. While this second generation of viral and bacterial diseases could labor plagues threatening the precariously maintained so-called social fabric of Middle Eastern countries, that are uniquely segregating its populace according to their dogmatic affiliations and soaked into intolerance, it would definitely compromise the integrity of the expensively managed medical care system of developed countries.

Background: A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus., Results: By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture., Conclusions: These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.

A main complication of treatment of patients with hemophilia A is the development of anti-factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic. Finally, VWF, the carrier protein for fVIII in plasma, may play a role in immune recognition. We compared the immunogenicity of wild-type (wt) B domain-deleted fVIII and 2 inactive fVIII molecules, R372A/R1689A fVIII and V634M fVIII in fVIII(-/-) and fVIII(-/-)/VWF(-/-) mice. R372A/R1689A fVIII lacks proteolytic recognition sites and is not released from VWF. In contrast, V634M fVIII undergoes proteolytic cleavage and dissociation from VWF. No significant difference was observed in the immunogenicity of wt fVIII and V634M fVIII. R372A/R1689A fVIII was slightly less immunogenic in a subset of immunization regimens tested. High doses of wt fVIII were required to produce an immune response in fVIII(-/-)/VWF(-/-) mice. Our results indicate that a main component of the immune response to fVIII is independent of its procoagulant function, is both positively and negatively affected by its association with VWF, and may involve intrinsic elements of fVIII structure.

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.

Background: RH1 is one of the most clinically important blood group antigens in the field of transfusion and prevention of fetomaternal incompatibilities. New variant RHD alleles are regularly identified and their characterization is essential to ensuring patient safety., Study Design and Methods: Blood samples with uncertain RhD phenotypes not resolved by our first-line SNaPshot assay were sequenced for all 10 RHD exons. RHD zygosity was investigated. Flow cytometry was performed to determine RhD antigen density and epitope pattern., Results: Seven novel RHD alleles were identified. Six, that is, RHD(T55P), RHD(A85G), RHD(G132R), RHD(G132E), RHD(D403V), and DAR(T203A), resulted from nucleotide polymorphisms. The seventh, that is, RHD(S182WfsX46), resulted from a 4-bp deletion that led to a reading frame shift and the appearance of a premature stop codon. Study of RhD expression of the first five alleles at hemizygous state showed greatly reduced antigen densities ranging from 50 to 618 antigens per red blood cell (RBC). DAR(T203A) was classified as a partial D antigen with a weakened reactivity profile similar to that of DAR. As expected, no D antigen was detected on RBCs carrying the RHD(S182WfsX46) allele. In parallel, RhD expression of RHD(G336R)/weak D type 58, RHD(F410V), and suspected RHD(1-9)-CE was determined to be less than or equal to 50 antigens per RBC. RhAG/RhD(2) trimer model supports the observed phenotypes., Conclusion: Although the frequency of the new RHD alleles presented herein is low, their phenotypic and genotypic description adds to the repertoire of reported RHD alleles. These data can be useful for optimization of molecular screening tools., (© 2012 American Association of Blood Banks.)

The four serotypes of dengue virus (DENV) are the causative agents of the most prevalent mosquito-borne viral disease in human. No clinically approved antiviral therapy is currently available. Therapeutic antibodies represent a viable approach for potential treatment of DENV infection. We recently isolated a human monoclonal antibody (HM14c10) that selectively neutralizes DENV serotype 1 (DENV-1), but not serotypes 2, 3, and 4. Here we report the resistance profile of DENV-1 against HM14c10 in cell culture. Escape mutant viruses readily emerged by culturing wild-type DENV-1 in the presence of the HM14c10 antibody. Sequencing of resistant viruses revealed a single T51K substitution in the domain I/II hinge region of the viral envelope protein. Residue T51 is located within the HM14c10 epitope and is highly conserved among various DENV-1 isolates. Recombinant DENV-1 containing the T51K mutation could not be neutralized by HM14c10 in vitro or in vivo. Biochemical assay revealed that the T51K mutation completely abolished the antibody binding to the DENV-1 virion. Collectively, the results demonstrate that a single amino acid change in DENV envelope protein can confer resistance to a potent antibody through abolishing the antibody-virus interaction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The zinc transporter 8 (ZnT8) was recently identified as a common autoantigen in type 1 diabetes (T1D) and inclusion of ZnT8 autoantibodies (ZnT8Ab) was found to increase the diagnostic specificity of T1D., Objectives: The main aims were to determine whether ZnT8Ab vary during follow-up 1 year after diagnosis, and to relate the reactivity of three types of ZnT8Ab to the residual stimulated C-peptide levels during the first year after diagnosis., Subjects: A total of 129 newly diagnosed T1D patients <15 years was followed prospectively 1, 3, 6, and 12 months after diagnosis., Methods: Hemoglobin A1c, meal-stimulated C-peptide, ZnT8Ab, and other pancreatic autoantibodies were measured at each visit. Patients were genotyped for the rs13266634 variant at the SLC30A8 gene and HLA-DQ alleles., Results: The levels of all ZnT8Ab [ZnT8Arg (arginine), ZnT8Trp (tryptophan), ZnT8Gln (glutamine)] tended to decrease during disease progression. A twofold higher level of ZnT8Arg and ZnT8Gln was associated with 4.6%/5.2% (p = 0.02), 5.3%/8.2% (p = 0.02) and 8.9%/9.7% (p = 0.004) higher concentrations of stimulated C-peptide 3, 6, and 12 months after diagnosis. The TT genotype carriers of the SLC30A8 gene had 45.8% (p = 0.01) and 60.1% (p = 0.002) lower stimulated C-peptide 6 and 12 months after diagnosis compared to the CC and the CT genotype carriers in a recessive model., Conclusions: The levels of the Arg variant of the ZnT8 autoantibodies are associated with higher levels of stimulated C-peptide after diagnosis of T1D and during follow-up. Carriers of the TT genotype of the SLC30A8 gene predict lower stimulated C-peptide levels 12 months after diagnosis., (© 2012 John Wiley & Sons A/S.)

Background: The mechanism by which HIV-1 induces AIDS is still unknown. Previously, synthetic peptides corresponding to the conserved immunosuppressive (isu) domain in gp41 of HIV-1 had been shown to inhibit proliferation and to modulate cytokine expression of immune cells. The question is, whether the viral gp41 can do the same., Results: We show for the first time that two trimeric forms of glycosylated gp41 released from transfected human cells modulated expression of cytokines and other genes in human PBMCs in the same manner, but at least seven hundred-fold stronger compared to that induced by the isu peptide. Single amino acid substitutions in the isu domain of gp41 introduced by site-directed mutagenesis abrogated this property. Furthermore, replication-competent HIV-1 with a mutation in the isu domain of gp41 did not modulate the cytokine expression, while wild-type virus did. Interestingly, most of the abrogating mutations were not reported in viral sequences derived from infected individuals, suggesting that mutated non-immunosuppressive viruses were eliminated by immune responses. Finally, immunisation of rats with gp41 mutated in the isu domain resulted in increased antibody responses compared with the non-mutated gp41. These results show that non-mutated gp41 is immunosuppressive in immunisation experiments, i.e. in vivo, and this has implications for the vaccine development., Conclusions: These findings indicate that the isu domain of gp41 modulates cytokine expression in vitro and suppresses antibody response in vivo and therefore may contribute to the virus induced immunodeficiency.

Mutations in the nucleophosmin gene (NPM1(mut)) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1(mut). In the present study, we were able to demonstrate both CD4(+) and CD8(+) T-cell responses against NPM1(mut). Ten peptides derived from wild-type NPM1 and NPM1(mut) were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr(51)-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4(+) T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1(mut) induced CD8(+) T-cell responses. A total of 33% of the NPM1(mut) AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4(+) T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8(+) and CD4(+) T cells. The results of the present study show that NPM1(mut) induces specific T-cell responses of CD4(+) and CD8(+) T cells and therefore is a promising target for specific immunotherapies in AML.

Background: Three influenza pandemics outbroke in the last century accompanied the viral antigen shift and drift, resulting in the change of antigenic property and the low cross protective ability of the existed antibody to the newly emerged pandemic virus, and eventually the death of millions of people. The antigenic characterizations of the viruses isolated in central China in 2004 and 2006-2007 were investigated in the present study., Results: Hemagglutinin inhibition assay and neutralization assay displayed differential antigenic characteristics of the viruses isolated in central China in two periods (2004 and 2006-2007). HA genes of the viruses mainly located in two branches in phylogeny analysis. 53 mutations of the deduced amino acids of the HA genes were divided into 4 patterns. Mutations in pattern 2 and 3 showed the main difference between viruses isolated in 2004 and 2006-2007. Meanwhile, most amino acids in pattern 2 and 3 located in the globular head of the HA protein, and some of the mutations evenly distributed at the epitope sites., Conclusions: The study demonstrated that a major antigenic drift had occurred in the viruses isolated in central China. And monitoring the antigenic property should be the priority in preventing the potential pandemic of H5N1 avian influenza virus.

Despite a central role in immunity, antibody neutralization of virus infection is poorly understood. Here we show how the neutralization and persistence of adenovirus type 5, a prevalent nonenveloped human virus, are dependent upon the intracellular antibody receptor TRIM21. Cells with insufficient amounts of TRIM21 are readily infected, even at saturating concentrations of neutralizing antibody. Conversely, high TRIM21 expression levels decrease the persistent fraction of the infecting virus and allows neutralization by as few as 1.6 antibody molecules per virus. The direct interaction between TRIM21 and neutralizing antibody is essential, as single-point mutations within the TRIM21-binding site in the Fc region of a potently neutralizing antibody impair neutralization. However, infection at high multiplicity can saturate TRIM21 and overcome neutralization. These results provide insight into the mechanism and importance of a newly discovered, effector-driven process of antibody neutralization of nonenveloped viruses.

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 recently described anti-human immunodeficiency virus type 1 (HIV-1) human mAb PG9 and PG16 are cross-clade broadly neutralizing. Therefore, it can be postulated that the targeted epitope(s) are highly conserved among variants of the entire group M. We analysed the sensitivity to PG9 and PG16 of pseudotyped viruses carrying envelope glycoproteins from the viral quasispecies of three HIV-1 clade CRF01&#95;AE-infected patients. The broad heterogeneity in sensitivity to PG9 and PG16, despite closely genetically related envelope glycoproteins issued from single individuals, allowed us to identify two gp120 cross-clade conserved residues, a lysine at position 168 in the V2 loop and an isoleucine at position 215 in the C2 region, whose substitutions were associated with resistance to PG9 and PG16. By site-directed mutagenesis, we confirmed both in clades B and CRF01&#95;AE that the substitutions K168E and I215M have a major impact on PG9 and PG16 neutralization sensitivity of pseudotyped viruses.

Introduction: Epidermal growth factor receptor (EGFR) gene mutations are usually detected by direct sequencing to identify patients with advanced pulmonary adenocarcinomas as candidates for tyrosine kinase inhibitor therapy. The aim of the study was to evaluate the efficacy of EGFR mutation-specific antibodies in identifying EGFR-mutated adenocarcinomas., Materials and Methods: Thirty-three consecutive cases of pulmonary adenocarcinomas sequenced for EGFR mutations were retrieved from our files. Immunohistochemistry was performed with the rabbit monoclonal antibodies E746-A750del (6B6) and L858R (43B2). The results obtained using the 2 procedures were statistically compared by Coehn κ and by calculation of sensitivity and specificity., Results: There were 21 women and 12 men, ranging in age from 48 to 78 years. All cases were lung adenocarcinomas, 23 primaries and 10 metastatic. The mutational spectrum was as follows: 12 cases mutated in exon 19 (9 with E746-A750del, 1 with homozygote L747-T751del, 1 with L747-P753del, 1 with E747-S752del), 6 in exon 21 (5 with L858R, 1 with L861Q+L862L), and 15 EGFR wild type. Immunohistochemistry detected 6/9 cases with an E746-A750del mutation (κ=0.744, sensitivity: 66.7%, specificity: 100%) and 5/5 cases with an L858R mutation (κ=1, sensitivity: 100%, specificity: 100%). Four cases showed faint and focal immunostaining and were interpreted as negative. All other cases were negative. Overall, the 2 antibodies had 61.1% sensitivity and 100% specificity for EGFR mutations., Conclusions: EGFR mutation-specific antibodies may represent a first-line screening tool to identify patients as candidates for tyrosine kinase inhibitor therapy.

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.

Bovine herpesvirus type 1 (BHV-1) envelope protein U(L)49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. Earlier, we have constructed a BHV-1U(L)49.5Δ30-32 CT-null virus and determined that in the infected cells, TAP inhibition and MHC-I down regulation properties of the virus are abolished. In this study, we compared the pathogenicity and immune responses in calves infected with BHV-1U(L)49.5Δ30-32 CT-null and BHV-1 wt viruses. Following primary infection, both BHV-1 wt and BHV-1U(L)49.5Δ30-32 CT-null virus replicated in the nasal epithelium with very similar yields. BHV-1 antigen-specific CD8+ T cell proliferation as well as CD8+ T cell cytotoxicity in calves infected with the BHV-1U(L)49.5Δ30-32 CT-null virus peaked by 7 dpi (P<0.05) which is 7 days earlier than that of BHV-1 wt-infected calves. Further, virus neutralizing antibody (VN Ab) titers and IFN-γ producing peripheral blood mononuclear cells (PBMCs) in the U(L)49.5 mutant virus-infected calves, also peaked 7 days (IFN-γ; P<0.05) and 14 days (VN Ab; P<0.05) earlier, respectively. Therefore, relative to wt in the BHV-1U(L)49.5 mutant virus-infected calves, primary neutralizing antibody and cellular immune responses were induced significantly more rapidly., (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.

We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.

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.

We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.

Background: The human epidermal growth factor receptor (EGFR) is an important therapeutic target in oncology, and three different types of EGFR inhibitors have been approved for the treatment of cancer patients. However, there has been no clear association between the expression levels of EGFR protein in the tumours determined by the FDA-approved EGFR PharmDx kit (Dako) or other standard anti-EGFR antibodies and the response to the EGFR inhibitors., Method: In this study, we investigated the potential of our anti-EGFR monoclonal antibodies (mAbs; ICR9, ICR10, ICR16) for immunohistochemical diagnosis of wild-type EGFR and/or the type-III deletion mutant form of EGFR (EGFRvIII) in formalin-fixed, paraffin-embedded human tumour specimens., Results: We found that the anti-EGFR mAb in the EGFR PharmDx kit stained both wild-type and EGFRvIII-expressing cells in formalin-fixed, paraffin-embedded sections. This pattern of EGFR immunostaining was also found with our anti-EGFR mAb ICR16. In contrast, mAbs ICR10 and ICR9 were specific for the wild-type EGFR., Conclusion: We conclude that mAbs ICR9 and ICR10 are ideal tools for investigating the expression patterns of wild-type EGFR protein in tumour specimens using immunohistochemistry, and to determine their prognostic significance, as well as predictive value for response to therapy with EGFR antibodies.

HLA-B*4006 is the most common allele amongst Indians. It belongs to the 'HLA-B44 supertype' family of alleles that constitute an important component of the peptide binding repertoire in populations world over. Its peptide binding characteristics remain poorly examined. The amino acid sequence and structural considerations suggest a small, poorly hydrophobic 'F' pocket for this allele that may adversely affect the interaction with the C terminal residue of the antigenic peptide. Contribution of auxiliary anchor residues (P3) of the peptide has also been indicated. To examine these aspects by in silico analysis, HLA-B*4001, 4002, and 4006 alleles were modeled using HLA-B*4402 as a template. Eleven peptides, known to bind alleles of this family, were used for docking and molecular dynamics studies. Interaction between the amino group (main-chain) of P3 residue and Tyr99 of the alleles was seen in majority of peptide-complexes. Hydrophobic interactions between Tyr7 and Tyr159 with N terminal residues of the peptide were also seen in all the complexes. Replacement of Trp95 by leucine in HLA-B*4006 resulted in reduction of binding free energy in 8 out of 9 complexes. In summary, the analysis of the modeled structures and HLA-peptide complexes strongly supports the adverse effect of Trp95 at pocket F and the possible role of the third residue of the antigenic peptide as an auxiliary anchor in HLA-B*4006 peptide complexes. In the light of suggested promiscuous peptide binding pattern and association with risk for tuberculosis/HIV for this allele, the ascertainment of the predicted effects of Trp95 and role of P3 residue as an auxiliary anchor by this preliminary in silico analysis thus helps define direction of the further studies.

Background: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions., Methods: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays., Results: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen., Conclusions: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens., (Copyright © 2012 S. Karger AG, Basel.)

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays., Methodology/principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation., Conclusions/significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties.

Background: There is a strong need for a recombinant subunit vaccine against fowl cholera. We used a reverse vaccinology approach to identify putative secreted or cell surface associated P. multocida proteins that may represent potential vaccine candidate antigens., Principal Findings: A high-throughput cloning and expression protocol was used to express and purify 71 recombinant proteins for vaccine trials. Of the 71 proteins tested, only one, PlpE in denatured insoluble form, protected chickens against fowl cholera challenge. PlpE also elicited comparable levels of protection in mice. PlpE was localized by immunofluorescence to the bacterial cell surface, consistent with its ability to elicit a protective immune response. To explore the role of PlpE during infection and immunity, a plpE mutant was generated. The plpE mutant strain retained full virulence for mice., Conclusion: These studies show that PlpE is a surface exposed protein and was the only protein of 71 tested that was able to elicit a protective immune response. However, PlpE is not an essential virulence factor. This is the first report of a denatured recombinant protein stimulating protection against fowl cholera.

Respiratory Syncytial Virus (RSV) is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F) surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

Background: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored., Methodology/principal Findings: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer., Conclusions/significance: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level., (© 2012 Lin et al.)

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.

Antibodies against the CD4 binding site (CD4bs) on the HIV-1 spike protein gp120 can show exceptional potency and breadth. We determined structures of NIH45-46, a more potent clonal variant of VRC01, alone and bound to gp120. Comparisons with VRC01-gp120 revealed that a four-residue insertion in heavy chain complementarity-determining region 3 (CDRH3) contributed to increased interaction between NIH45-46 and the gp120 inner domain, which correlated with enhanced neutralization. We used structure-based design to create NIH45-46(G54W), a single substitution in CDRH2 that increases contact with the gp120 bridging sheet and improves breadth and potency, critical properties for potential clinical use, by an order of magnitude. Together with the NIH45-46-gp120 structure, these results indicate that gp120 inner domain and bridging sheet residues should be included in immunogens to elicit CD4bs antibodies.

Human herpesvirus 6 (HHV-6) is a T cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6A and HHV-6B, based on differences in their genetic, antigenic, and growth characteristics and cell tropisms. The function of HHV-6B should be analyzed more in its life cycle, as more than 90% of people have the antibodies for HHV-6B but not HHV-6A. It has been shown that the cellular receptor for HHV-6A is human CD46 and that the viral ligand for CD46 is the envelope glycoprotein complex gH/gL/gQ1/gQ2; however, the receptor-ligand pair used by HHV-6B is still unknown. In this study, to identify the glycoprotein(s) important for HHV-6B entry, we generated monoclonal antibodies (MAbs) that inhibit infection by HHV-6B. Most of these MAbs were found to recognize gQ1, indicating that HHV-6B gQ1 is critical for virus entry. Interestingly, the recognition of gQ1 by the neutralizing MAb was enhanced by coexpression with gQ2. Moreover, gQ1 deletion or point mutants that are not recognized by the MAb could nonetheless associate with gQ2, indicating that although the MAb recognized the conformational epitope of gQ1 exposed by the gQ2 interaction, this epitope was not related to the gQ2 binding domain. Our study shows that HHV-6B gQ1 is likely a ligand for the HHV-6B receptor, and the recognition site for this MAb will be a promising target for antiviral agents.

Context: Some tumor markers, including carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21-1), are used for the detection of lung cancer; however, their use is limited because of low sensitivities and high false-positive rates., Objectives: To investigate the usefulness of an anti-p53 assay in detecting lung cancer and to compare the anti-p53 to CEA and CYFRA 21-1 tumor markers., Design: Serum samples were collected from 82 patients with lung cancer. Sera were also collected from 79 patients with or without benign pulmonary disease for the control group. All 161 specimens were assayed for CEA, CYFRA 21-1, and anti-p53. The diagnostic performances of these markers were compared using receiver operating characteristic analysis., Results: The receiver operating characteristic area under the curve values of CYFRA 21-1, CEA, and anti-p53 for discriminating lung cancers from benign or healthy conditions were 0.79, 0.81, and 0.79, respectively. Area under the curve for the 3 markers in combination was 0.90. The sensitivities of those markers for lung cancer detection were respectively 39.0%, 53.7%, and 34.1% at 94.9% specificity, and the cutoff levels at those sensitivities and specificities were 4.5 ng/mL for CYFRA 21-1, 5.4 ng/mL for CEA, and 2.7 U/mL for anti-p53. We found 79.3% positive results for patients with lung cancer by any of the 3 markers, and 12.2% were positive only for anti-p53. All patients without cancer had negative results for 2 or all 3 markers., Conclusions: Anti-p53 combined with other conventional markers is helpful in increasing the sensitivity and specificity for detecting lung cancer.

Lassa virus (LASV; Arenaviridae) is responsible for severe hemorrhagic fevers in Africa. LASV nucleoprotein (NP) plays important roles in regulating viral transcription and replication and in inhibiting type I interferon (IFN) production. The NP C-terminal domain contains a 3'-to-5' exonuclease activity involved in suppressing IFN induction. We have established a murine polymerase (Pol) I reverse genetics system for LASV, showing that residues D389 and G392 of NP were critical for LASV viability, while the D389A/G392A and D389T/392A double mutants were severely altered in the ability to suppress IFN in macrophages and dendritic cells. Assessing their attenuation in vivo may open new perspectives in vaccinology.

Background: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co-administration of t-PA and a TAFIa inhibitor enhances t-PA treatment., Objective: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t-PA induced clot lysis., Results: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A(147) -C(305) -I(325) -I(329) -Y(333) -Q(335) ), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16-fold molar excess of nanobody over TAFIa, IC(50) 's range between 0.38- and > 16-fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t-PA concentration. The nanobodies were found to bind in the active-site region of TAFIa and their time-dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition., Conclusion: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa., (© 2011 International Society on Thrombosis and Haemostasis.)

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.

Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0-D1-D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an 'innate HLA sensor' domain. Second, whereas the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species., (© 2011 Macmillan Publishers Limited. All rights reserved)

We constructed functional recombinant single chain Fv (scFv) against bovine herpes virus type 1 (BoHV-1), aetiological agent of respiratory and genital diseases in cattle for which available vaccines do not provide adequate protection. The scFv against BoHV-1 with 18 amino acid long flexible linker (scFv3-18L; monomeric form) recognized target antigen and, also, neutralized BoHV-1 in vitro. A comparison with recombinant scFv with 7 amino acid linker against BoHV-1 (scFv1-7L), capable of forming diabodies, indicated that a relatively higher concentration (two-fold) of monomer scFv3-18L is needed for virus neutralization as compared to scFv1-7L. A single point replacement mutation (Asp98 to Gly98) in the framework-3 (FR3) variable-region of scFv with 18 amino acid linker (scFv4m-18L), however, affected the viral neutralization in a dose-dependent manner where 2.7 fold higher mutant scFv concentration was required to achieve virus neutralization. Despite differences in dose-dependent viral neutralization of the mutant scFv-18L, it detected viral antigen in an immunofluorescent assay. The outlined experiments demonstrate that recombinant scFv against BoHV-1, whether expressed as scFv or diabody, provide an effective antibody based therapeutic and immunodiagnostic protein. Further, single point substitution mutation in the FR3 can affect viral neutralization dynamics without affecting qualitative viral antigen recognition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Leishmania eukaryotic initiation factor (LeIF) antigen, a Leishmania protein, was shown to induce IL-12, IL-10 and tumour necrosis factor-α (TNF-α) production by human monocytes-derived macrophages and dendritic cells from healthy individuals. This cytokine-inducing activity was previously found to be located in the amino-terminal region of LeIF protein. This study aimed at characterizing the cytokine-inducing activity of Leishmania infantum LeIF [Leishmania (L.) infantum (LieIF)] and at defining the fragments necessary for inducing cytokine secretion. Eleven rationally designed recombinant polypeptides, corresponding to the entire LeIF protein or parts of it, were expressed and used to stimulate monocytes from healthy individuals. Leishmania (L.) infantum was able to induce IL-12p70, IL-10 and TNF-α secretion in human monocytes. In addition, both amino- (1-226) and carboxyl-terminal (196-403) parts of the protein were shown to induce significant levels of the three cytokines analysed. However, IL-12p70-inducing activity was not significant when monocytes were stimulated with the fragments 129-226 and 129-261, inferring that IL-12p70-inducing activity was primarily located within amino acids 1-129 and 261-403. Although the full-length LieIF protein was a more potent inducer than the tested fragments, a significant cytokine-inducing activity was maintained in smaller amino acid regions. This work suggests that cytokine-inducing activity of LieIF or its parts could be exploited in vaccination or immunotherapy protocols., (© 2011 Blackwell Publishing Ltd.)

The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike.

APOBEC3G (A3G) is packaged into human immunodeficiency virus type 1 (HIV-1) virions unless HIV-1 virion infectivity factor (Vif) counteracts it. Virion A3G restricts HIV-1 reverse transcription and integration in target cells. Some A3G in producer cells colocalizes with specific cytoplasmic structures, in what are called "A3G complexes" here. Functional effects of producer cell A3G complexes on HIV-1 replication were studied. HeLa cells were cotransfected with HIV-1 constructs producing pseudoviruses, as well as either wild-type (WT) A3G or a mutant A3G (C97A, Y124A, W127A, or D128K A3G). Pseudovirus particle production was decreased from cells expressing any of the A3Gs that formed complexes by 24 h after transfection, relative to cells with C97A A3G that did not form detectable A3G complexes by 24 h or A3G-negative cells. The intracellular HIV-1 Gag half-life was shorter in cells containing A3G complexes than in those lacking complexes. HIV-1 virion output was decreased in a single round of replication from a T cell line containing A3G complexes (CEM cells) after infection with Vif-negative HIV-1, compared to Vif-positive HIV-1 that depleted A3G. Levels of production of Vif-negative and Vif-positive virus were similar from cells not containing A3G (CEM-SS cells). Knockdown of the mRNA processing body (P-body) component RCK/p54, eliminated A3G complex formation, and increased HIV-1 production. We conclude that endogenous A3G complexes in producer cells decrease HIV-1 production if not degraded by Vif.

Non-structural protein 1 (NS1) counteracts the production of host type I interferons (IFN-α/β) for the efficient replication and pathogenicity of influenza A viruses. Here, we reveal another dimension of the NS1 protein of avian influenza A viruses in suppressing IFN-β production in cultured cell lines. We found that allele A NS1 proteins of H6N8 and H4N6 have a strong capacity to inhibit the activation of IFN-β production, compared with allele B from corresponding subtypes, as measured by IFN stimulatory response element (ISRE) promoter activation, IFN-β mRNA transcription and IFN-β protein expression. Furthermore, the ability to suppress IFN-β promoter activation was mapped to the C-terminal effector domain (ED), while the RNA-binding domain (RBD) alone was unable to suppress IFN-β promoter activation. Chimeric studies indicated that when the RBD of allele A was fused to the ED of allele B, it was a strong inhibitor of IFN-β promoter activity. This shows that well-matched ED and RBD are crucial for the function of the NS1 protein and that the RBD could be one possible cause for this differential IFN-β inhibition. Notably, mutagenesis studies indicated that the F103Y and Y103F substitutions in alleles A and B, respectively, do not influence the ISRE promoter activation. Apart from dsRNA signalling, differences were observed in the expression pattern of NS1 in transfected human and mink lung cells. This study therefore expands the versatile nature of the NS1 protein in inhibiting IFN responses at multiple levels, by demonstrating for the first time that it occurs in a manner dependent on allele type.

The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -β target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein.

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.

Soluble forms of the HIV-1 receptor CD4 (sCD4) have been extensively characterized for more than 2 decades as promising inhibitors and components of vaccine immunogens. However, they were mostly based on the first two CD4 domains (D1D2), and numerous attempts to develop functional, high-affinity, stable soluble one-domain sCD4 (D1) have not been successful because of the strong interactions between the two domains. We have hypothesized that combining the power of structure-based design with sequential panning of large D1 mutant libraries against different HIV-1 envelope glycoproteins (Envs) and screening for soluble mutants could not only help solve the fundamental stability problem of isolated D1, but may also allow improvement of D1 affinity while preserving its cross-reactivity. By using this strategy, we identified two stable monomeric D1 mutants, mD1.1 and mD1.2, which were significantly more soluble and bound Env gp120s more strongly (50-fold) than D1D2, neutralized a panel of HIV-1 primary isolates from different clades more potently than D1D2, induced conformational changes in gp120, and sensitized HIV-1 for neutralization by CD4-induced antibodies. mD1.1 and mD1.2 exhibited much lower binding to human blood cell lines than D1D2; moreover, they preserved a β-strand secondary structure and stability against thermally induced unfolding, trypsin digestion, and degradation by human serum. Because of their superior properties, mD1.1 and mD1.2 could be potentially useful as candidate therapeutics, components of vaccine immunogens, and research reagents for exploration of HIV-1 entry and immune responses. Our approach could be applied to other cases where soluble isolated protein domains are needed.

Prostate-specific membrane antigen (PSMA) is a membrane protein that is overexpressed manifold in prostate cancer and provides an attractive target for therapy. PSMA ADC is an antibody-drug conjugate (ADC) that consists of a fully human anti-PSMA monoclonal antibody conjugated to monomethylauristatin E through a valine-citrulline linker. In this study, the antitumor activity of PSMA ADC was evaluated against a panel of prostate cancer cell lines in vitro and in a novel in vivo model of taxane-refractory human prostate cancer. In vitro cell killing was efficient for cells with abundant PSMA expression (>10(5) molecules/cell; IC(50) ≤ 0.022 nmol/L) and 1,000-fold less efficient for cells with undetectable PSMA (IC(50) > 30 nmol/L). Intermediate potency (IC(50) = 0.80 nmol/L) was observed for cells with approximately 10(4) molecules of PSMA per cell, indicating a threshold PSMA level for selective cell killing. Similar in vitro activity was observed against androgen-dependent and -independent cells that had abundant PSMA expression. In vitro activity of PSMA ADC was also dependent on internalization and proper N-glycosylation/folding of PSMA. In contrast, less potent and nonselective cytotoxic activity was observed for a control ADC, free monomethylauristatin E, and other microtubule inhibitors. PSMA ADC showed high in vivo activity in treating xenograft tumors that had progressed following an initial course of docetaxel therapy, including tumors that were large (>700 mm(3)) before treatment with PSMA ADC. This study defines determinants of antitumor activity of a novel ADC. The findings here support the clinical evaluation of this agent in advanced prostate cancer.