Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus.

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.

Components of pathogenic Leptospira spp. with potentials for diagnosis of human leptospirosis.

Existing serological methods for diagnosis of leptospirosis are still unsatisfactorily due mainly to their low accuracy. In this study, serum samples of 18 clinically diagnosed-, IgM dipstick positive-, MAT positive-leptospirosis patients (group 1) were analyzed by IgG Western blotting against SDS-PAGE separated-whole cell homogenates of pathogenic and non-pathogenic Leptospira spp. belonging to 20 serovars of 15 serogroups. The samples of group 1 were collected from the patients at days 3 to 10 after the fever onset (fist samples). Second and third samples could be obtained from 4 patients. Sera of the 22 patients with other febrile illnesses (group 2) and 22 healthy counterparts (group 3) were used as patient- and normal- controls, respectively. Irrespective of the serovar or serogroup of the pathogenic Leptospira spp. used as antigen in the Western blotting, all of the 18 sera of patients with leptospirosis (group 1) gave characteristic diffuse antigen-antibody reactive bands located at approximately 35-38 and 22-26 kDa; and thus 100% diagnostic sensitivity of the Western blot assay. Some serum samples of the leptospirosis patients also reacted to components located at 80-100, approximately 70, 60, 54, and 48 kDa. More bands or the early recognized bands with increased intensity were observed when tested the second and third samples. The characteristic bands were not seen when homogenates of L. biflexa, serogroup Semaranga, serovar Patoc (saprophytic) and L. biflexa, serogroup Andamana, serovar Andamana (non-pathogenic but can infect host) were used in the assay. Sera of groups 2 and 3 did not react to the components at the seven locations implying 100% diagnostic specificity of the IgG Western blot assay. While awaiting validation with more patients' samples, the IgG Western Blot analysis aiming at the detection of the characteristic antigen-antibody reactive bands described in this study has high potential for early, rapid, simple and accurate diagnosis of human leptospirosis.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.