Agonistic Bivalent Human scFvs-Fcγ Fusion Antibodies to OX40 Ectodomain Enhance T Cell Activities against Cancer.

(1) Background: Understanding how advanced cancers evade host innate and adaptive immune opponents has led to cancer immunotherapy. Among several immunotherapeutic strategies, the reversal of immunosuppression mediated by regulatory T cells in the tumor microenvironment (TME) using blockers of immune-checkpoint signaling in effector T cells is the most successful treatment measure. Furthermore, agonists of T cell costimulatory molecules (CD40, 4-1BB, OX40) play an additional anti-cancer role to that of checkpoint blocking in combined therapy and serve also as adjuvant/neoadjuvant/induction therapy to conventional cancer treatments, such as tumor resection and radio- and chemo- therapies. (2) Methods and Results: In this study, novel agonistic antibodies to the OX40/CD134 ectodomain (EcOX40), i.e., fully human bivalent single-chain variable fragments (HuscFvs) linked to IgG Fc (bivalent HuscFv-Fcγ fusion antibodies) were generated by using phage-display technology and genetic engineering. The HuscFvs in the fusion antibodies bound to the cysteine-rich domain-2 of the EcOX40, which is known to be involved in OX40-OX40L signaling for NF-κB activation in T cells. The fusion antibodies caused proliferation, and increased the survival and cytokine production of CD3-CD28-activated human T cells. They showed enhancement trends for other effector T cell activities like granzyme B production and lysis of ovarian cancer cells when added to the activated T cells. (3) Conclusions: The novel OX40 agonistic fusion antibodies should be further tested step-by-step toward their safe use as an adjunctive non-immunogenic cancer immunotherapeutic agent.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

Genome assembly and annotation of Periplaneta americana reveal a comprehensive cockroach allergen profile.

BACKGROUND: One of the most common cockroach types in urban areas, the American cockroach (Periplaneta americana), has been reported to impose an increased risk of allergies and asthma. Limited groups of allergens (Per a 1-13) have been identified in this species due to the lack of genome-related information. METHODS: To expand the allergen profile of P. americana, genomic, transcriptomic, and proteomic approaches were applied. With the support of a high-quality genome assembled using nanopore, Illumina, and Hi-C sequencing techniques, potential allergens were identified based on protein homology. Then, using enzyme-linked immunosorbent assay, selected allergens were tested in Thai patients allergic to P. americana. RESULTS: A chromosomal-level genome of P. americana (3.06 Gb) has been assembled with 94.6% BUSCO completeness, and its contiguity has been significantly improved (N50 = 151 Mb). A comprehensive allergen profile has been characterized, with seven novel groups of allergens, including enolase (Per a 14), cytochrome C (Per a 15), cofilin (Per a 16), alpha-tubulin (Per a 17), cyclophilin (Per a 18), porin3 (Per a 19), and peroxiredoxin-6 (Per a 20), showing IgE sensitivity in enzyme-linked immunosorbent assay. A new isoallergen of tropomyosin (Per a 7.02) and multiple potential isoallergens of Per a 5 were revealed using bioinformatics and proteomic approaches. Additionally, comparative analysis of P. americana with the closely related Blattodea species revealed the possibility of cross-reaction. CONCLUSION: The high-quality genome and proteome of P. americana are beneficial in studying cockroach allergens at the molecular level. Seven novel allergen groups and one isoallergen in Per a 7 were identified.

Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase.

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.

Human Transbodies to Reverse Transcriptase Connection Subdomain of HIV-1 Gag-Pol Polyprotein Reduce Infectiousness of the Virus Progeny.

HIV-1 progeny are released from infected cells as immature particles that are unable to infect new cells. Gag-Pol polyprotein dimerization via the reverse transcriptase connection domain (RTCDs) is pivotal for proper activation of the virus protease (PR protein) in an early event of the progeny virus maturation process. Thus, the RTCD is a potential therapeutic target for a broadly effective anti-HIV agent through impediment of virus maturation. In this study, human single-chain antibodies (HuscFvs) that bound to HIV-1 RTCD were generated using phage display technology. Computerized simulation guided the selection of the transformed Escherichia coli-derived HuscFvs that bound to the RTCD dimer interface. The selected HuscFvs were linked molecularly to human-derived-cell-penetrating peptide (CPP) to make them cell-penetrable (i.e., become transbodies). The CPP-HuscFvs/transbodies produced by a selected transformed E. coli clone were tested for anti-HIV-1 activity. CPP-HuscFvs of transformed E. coli clone 11 (CPP-HuscFv11) that presumptively bound at the RTCD dimer interface effectively reduced reverse transcriptase activity in the newly released virus progeny. Infectiousness of the progeny viruses obtained from CPP-HuscFv11-treated cells were reduced by a similar magnitude to those obtained from protease/reverse transcriptase inhibitor-treated cells, indicating anti-HIV-1 activity of the transbodies. The CPP-HuscFv11/transbodies to HIV-1 RTCD could be an alternative, anti-retroviral agent for long-term HIV-1 treatment.

A Component-Resolved Therapeutic Vaccine for Cockroach Allergy Made of Per a 9 and Transforming Growth Factor-ß Homologue, an Immunosuppressive Protein of Brugia malayi.

Allergen-specific-immunotherapy (ASIT) can cause long-term resolution of allergic diseases, reduces drug use and chances of new allergen sensitization. Nevertheless, therapeutic vaccine and data on ASIT efficacy for cockroach (CR) allergy are relatively scarce. In this study, efficacy and mechanism of a novel intranasal vaccine consisting of liposome (L)-entrapped mixture of American CR (Periplaneta americana) major allergen (Per a 9) and immunosuppressive protein of Brugia malayi nematode named transforming growth factor-beta homologue (TGH) in treatment of CR allergy were investigated along with two other vaccines (L-Per a 9 alone and L-TGH alone). All three vaccines could reduce pathogenic type 2 response and lung immunopathology in the vaccines-treated CR-allergic mice, but by different mechanisms. L-Per a 9 caused a deviation of the pathogenic type 2 to type 1 response (IFN-γ-upregulation), whereas the L-(TGH + Per a 9) and L-TGH generated regulatory immune responses including up-expression of immunosuppressive cytokine genes and increment of serum adenosine and lung indoleamine-2,3-dioxygenase-1 which are signatures of regulatory T cells (Tregs) and tolerogenic dendritic cells, respectively. The L-(TGH + Per a 9) should be further evaluated towards clinical application, as this vaccine has a propensity to induce broadly effective therapeutic effects for inhalant allergies.

Human Antibodies to VP4 Inhibit Replication of Enteroviruses Across Subgenotypes and Serotypes, and Enhance Host Innate Immunity.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease that usually affects infants and young children (<5 years). HFMD outbreaks occur frequently in the Asia-Pacific region, and these outbreaks are associated with enormous healthcare and socioeconomic burden. There is currently no specific antiviral agent to treat HFMD and/or the severe complications that are frequently associated with the enterovirus of serotype EV71. Therefore, the development of a broadly effective and safe anti-enterovirus agent is an existential necessity. In this study, human single-chain antibodies (HuscFvs) specific to the EV71-internal capsid protein (VP4) were generated using phage display technology. VP4 specific-HuscFvs were linked to cell penetrating peptides to make them cell penetrable HuscFvs (transbodies), and readily accessible to the intracellular target. The transbodies, as well as the original HuscFvs that were tested, entered the enterovirus-infected cells, bound to intracellular VP4, and inhibited replication of EV71 across subgenotypes A, B, and C, and coxsackieviruses CVA16 and CVA6. The antibodies also enhanced the antiviral response of the virus-infected cells. Computerized simulation, indirect and competitive ELISAs, and experiments on cells infected with EV71 particles to which the VP4 and VP1-N-terminus were surface-exposed (i.e., A-particles that don't require receptor binding for infection) indicated that the VP4 specific-antibodies inhibit virus replication by interfering with the VP4-N-terminus, which is important for membrane pore formation and virus genome release leading to less production of virus proteins, less infectious virions, and restoration of host innate immunity. The antibodies may inhibit polyprotein/intermediate protein processing and cause sterically strained configurations of the capsid pentamers, which impairs virus morphogenesis. These antibodies should be further investigated for application as a safe and broadly effective HFMD therapy.

Cockroaches: Allergens, Component-Resolved Diagnosis (CRD) and Component-Resolved Immunotherapy.

Allergic diseases are assuming increasing trend of prevalence worldwide. The diseases confer increasing demand on medical and healthcare facilities. Patients with allergies have poor quality of life and impaired cognition. Adult patients have subpar working efficiency while afflicted children are less effective at school, often have school absenteeism and need more attention of their caregivers. All of them lead to negative socio-economic impact. This narrative review focuses on cockroach allergy including currently recognized cockroach allergens, pathogenic mechanisms of allergy, componentresolved diagnosis and allergen-specific immunotherapy, particularly the component-resolved immunotherapy and the molecular mechanisms that bring about resolution of the chronic airway inflammation.

Endotyping of Chronic Rhinosinusitis With and Without Polyp Using Transcription Factor Analysis.

Inflammation of the nose and paranasal sinus or rhinosinusitis (RS) is a significant global health problem that is both very common and very costly to treat. Previous reports reveal variability in histology and mechanism of inflammation in patients with chronic rhinosinusitis with and without polyp (CRScNP and CRSsNP, respectively). There are various methods and hypothesis that try to explain this variability. Accordingly, the aim of this study was to investigate the incidence of each type of sinonasal inflammation among patients diagnosed with CRScNP or CRSsNP using transcription factor analysis (TFA). This study included mucosa specimens from nose/paranasal sinuses from patients with chronic rhinitis (CR), CRSsNP, or CRScNP that were obtained at the Department of Otorhinolaryngology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand during the June 2009 to May 2012 study period. TFA was employed to measure the following transcription factors: T-box transcription factor (T-bet) for Th1, GATA binding protein 3 (GATA-3) for Th2, retinoic acid-related orphan receptor C (RORC) for Th17, and forkhead box P3 (FOXP3) for Treg. Forty-one subjects (22 males, 19 females) were enrolled, with a mean age of 45.93 ± 13 years. Twenty-six patients were diagnosed with CRScNP, 7 with CRSsNP, and 8 with CR (controls). The majority of CRScNP specimens (76.9%) had eosinophil count greater than 100 cells/high-power field (HPF). Mean eosinophil count was 930.08 ± 1,399 cells/HPF (range: 17-5,570). Th2 transcription factor (GATA-3) was statistically significantly higher in the CRScNP group than in the CRS and control groups (p < 0.001); whereas, Treg transcription factor (FOXP3) was statistically significantly lower in the CRScNP group than in the CRSsNP and control groups (p < 0.001). The transcription factors for Th1 and Th17 (T-bet and RORC, respectively) were not significantly different among the three groups. The result of transcription factor analysis revealed hyperfunction of Th2 in patients with CRScNP, which might result in hypereosinophilic infliltration in the polyps. One explanation for this finding is the decreased activity of Treg. Although environment-host interaction is the most probable hypothesis, the etiology of aberrant adaptive immunity needs to be elucidated.

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles.

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials.

Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

BACKGROUND AND OBJECTIVE: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared. METHODS: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA. RESULTS: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens. CONCLUSION: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.

BACKGROUND: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. OBJECTIVE: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. METHODS: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. RESULTS: Two hybridoma clones, Dfl-1 and Dfl-2, were established. Their secreted MAbs (MAbDfl-1 and MAbDfl-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDfI-1 and MAbDfl-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf-1 had higher affinity to Der f 1 than the MAbDfl-2. A sandwich ELISA (MAbDfl-1-allergen-PAb) and commercialized reagents (MAbl-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDfI-1-PAb and MAbl-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.

A revisit to cockroach allergens.

Among cockroaches (CR) that live in people's homes, two species, i.e., German CR (Blattella germanica) and American CR (Periplaneta americana) predominate in temperate and tropical areas, respectively. CR is an important source of inhalant indoor allergens that sensitize atopic subjects to (localized) type I hypersensitivity or atopy including allergic rhinitis and atopic asthma. In Thailand the predominant CR species is P. americana. CR allergens are found throughout CR infested houses; the number found in kitchens correlates with the degree of CR infestation while sensitization and reactivation of the allergic morbidity are likely to occur in the living room and bedroom. Levels of the CR allergens in homes of CR allergic Thais, measured by using locally made quantification test kits, revealed that the highest levels occur in dust samples collected from the wooden houses of urban slums and in the cool and dry season. CR allergens are proteins that may be derived from any anatomical part of the insect at any developmental stage. The allergens may be also from CR secretions, excretions, body washes or frass. The proteins may be the insect structural proteins, enzymes or hormones. They may exist as dimers/multimers and/or in different isoforms. Exposure to CR allergens in infancy leads to allergic morbidity later in life. Clinical symptoms of CR allergy are usually more severe and prolonged than those caused by other indoor allergens. The mechanisms of acute and chronic airway inflammation and airway hyper-responsiveness (AHR) have been addressed including specific IgE- and non-IgE-mediated mechanisms, i.e., role of protease-activated receptor-2 (PAR2). Participation of various allergen activated-CD4+ T cells of different sublineages, i.e., Th2, Th17, Th22, Th9, Th25, Tregs/Th3 as well as invariant NKT cells, in asthma pathogenesis have been mentioned. The diagnosis of CR allergy and the allergy intervention by CR population control are also discussed.

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines.

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP+M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8(+), intracellular IFNγ(+) cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.

Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called tetanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant synaptobrevin. Further investigation is needed for identification of epitope specificity of these HuScFv and HuScFv effector mechanisms towards the TeNT. Cell penetrating version of the HuScFv that inhibited the TeNT zinc metalloprotease activity should be made. The HuScFv produced in this study either singly or in their suitable combination warrant developing further to a real use in humans as a surrogate of the animal antibody for treatment of tetanus.

Giardia intestinalis in Thailand: identification of genotypes.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp beta-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

The levels of cockroach allergen in relation to cockroach species and allergic diseases in Thai patients.

UNLABELLED: Recently, cockroaches have been established as the second most Important allergen, producing allergic diseases, especially in low socioeconomic populations. In Thailand, about 44-61% of atopic patients were positive to cockroach extract by a skin-prick test. This study examined cockroach allergen levels in relation to cockroach species and allergic diseases in the houses of cockroach-sensitive patients. Sixty households of allergic patients in the Bangkok metropolitan area were surveyed using open- and closed-ended questionnaires. Cockroaches were collected using commercial cockroach traps, while dust samples were obtained from the bedrooms, kitchens and living rooms of the houses using a vacuum cleaner. The cockroaches were counted and their species Identified. The levels of cockroach allergens were determined by specific monoclonal antibodies using a monoclonal antibody-polyclonal antibody based sandwich ELISA kit. Six cockroach species were Identified: Periplaneta americana (American cockroach, 72.15%), Supella longlpalpa (2.75%, found in only one house), Periplaneta brunnea (0.78%), Periplaneta australaslae (0.78%), Neostylopyga rhombifolla (0.78%), Blattella germanica (German cockroach, 0.39%) and nymphs (22.35%). Allergens of the predominant species, P. americana, were detectable in all homes studied, with the highest levels in the kitchen areas. The range of allergen levels in house dust varied from 0.40-162.00 microg per g of dust. The median and mean allergen levels in kitchen dust were 59.16 microg and 62.80 microg per g of dust, respectively, while the median allergen level in bedroom dust was only 15.90 microg per g of dust. The German cockroach allergen (Bla g 2) was undetectable in any of the houses. IN CONCLUSION: P. americana was the most common cockroach and may be the species causing allergic diseases, especially asthma, in Thailand, which differs from the USA and Europe

Cockroach allergen detection and cockroach allergens of allergic Thai patients.

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.