Plant expressed EtMIC2 is an effective immunogen in conferring protection against chicken coccidiosis.

Coccidiosis is an economically important disease affecting poultry industry and remains one of the major problems globally. Developing a cost effective sub-unit vaccine may help mitigate loss in the industry. Here, we report expressing one of the microneme proteins, EtMIC2 from Eimeria tenella in tobacco using Agrobacterium-mediated transient expression. The ability of plant expressed recombinant EtMIC2 in eliciting both humoral and cell-mediated immune responses were measured in the immunized birds. The protective efficacy in the vaccinated birds against a homologous challenge was also evaluated. Birds immunized with plant expressed EtMIC2 showed good sero-conversion, reduced oocyst output and increased weight gain when compared to control birds. Our data indicate that use of plant expressed recombinant EtMIC2 in birds was safe and had the potential in imparting partial protection in chickens against homologous challenge.

Expression of codon optimized major capsid protein (L1) of human papillomavirus type 16 and 18 in Pichia pastoris; purification and characterization of the virus-like particles.

The major capsid protein (L1) of human papillomaviruses (HPV) expressed in heterologous systems assembles into virus-like particles (VLPs). We report cloning and expression of codon optimized HPV L1 genes of the two high-risk HPV types 16 and 18 in methylotropic yeast, Pichia pastoris. The VLPs produced in P. pastoris were subjected to three step purification method involving density gradient centrifugations and size exclusion chromatography. The enriched VLPs were characterized using conformation-specific monoclonal antibodies in ELISA and by transmission electron microscopy. Mice immunized with a bivalent HPV16 and HPV18 VLPs developed high serum antibody titers to both HPV types that persisted for 190 days post vaccination. Serum of mice immunized with the HPV-VLP preparations could neutralize homologous pseudoviruses in an in vitro assays. Our results demonstrate that the L1 proteins expressed in P. pastoris fold properly as evidenced by assembly into VLPs and induction of type-specific neutralizing antibody response in mice. This work constitutes a step towards developing an alternate production platform for generating an affordable HPV vaccine to meet the needs of developing countries.

Zona pellucida-based contraceptive vaccines for human and animal utility.

Contraceptive vaccines can be designed to inhibit (i) production of the gametes (sperm and oocyte), (ii) functions of gametes leading to block in fertilization, and (iii) the gamete outcome (pregnancy). The zona pellucida (ZP) glycoproteins have been proposed as candidates for developing contraceptive vaccines by virtue of their critical role in fertilization. Immunization of non-human primates with either native or recombinant ZP proteins leads to curtailment of fertility, which however is invariably associated with ovarian pathology. To avoid oophoritis, immunogens corresponding to mapped B cell epitopes of ZP proteins that are devoid of 'oophoritogenic' T cell epitopes have been proposed. However, ways to overcome the observed oophoritis associated with the ZP-based contraceptive vaccines are yet to be fully defined. This is essential if their use for control of human fertility is to be considered. Nonetheless, contraceptive vaccines based on ZP proteins have shown very promising results in controlling wildlife population such as wild horses, white-tailed deers, elephants, marsupials, grey seals and dogs, where long term infertility or even permanent sterility is desirable.

Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity.

Intestinal infection with Eimeria, the etiologic agent of avian coccidiosis, stimulates protective immunity to subsequent colonization by the homologous parasite, while cross-protection against heterologous species is poor. As a first step toward the development of a broad specificity Eimeria vaccine, this study was designed to assess a purified recombinant protein from Eimeria maxima gametocytes (Gam82) in stimulating immunity against experimental infection with live parasites. Following Gam82 intramuscular immunization and oral parasite challenge, body weight gain, fecal oocyst output, lesion scores, serum antibody response, and cytokine production were assessed to evaluate vaccination efficacy. Animals vaccinated with Gam82 and challenged with E. maxima showed lower oocyst shedding and reduced intestinal pathology compared with non-vaccinated and parasite-challenged animals. Gam82 vaccination also stimulated the production of antigen-specific serum antibodies and induced greater levels of IL-2 and IL-15 mRNAs compared with non-vaccinated controls. These results demonstrate that the Gam82 recombinant protein protects against E. maxima and augments humoral and cell-mediated immunity.

Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds.

Eimeria infection in poultry is of significant economic interest worldwide. Development of a cost-effective sub-unit vaccine that provides cross-protection may help reduce loss in poultry industry. One approach explored by many investigators is to block the parasite invasion into gut epithelium. Use of microneme proteins to prevent parasite invasion is one of the most straightforward approaches in developing a preventive vaccine. Here we describe cloning and expression of microneme-1 protein of Eimeria tenella, obtained from an outbreak sample from India. We have evaluated the ability of the recombinant protein to elicit both cell mediated immune (CMI) and humoral immune responses. We also evaluated the efficacy of the recombinant protein in protecting against a homologous challenge. Our data indicate recombinant EtMIC1 is able to impart partial protection against homologous challenge in chicken. Inclusion of more invasion proteins may improve the efficacy of prophylactic vaccine against Coccidiosis.

The insulin sensitiser pioglitazone does not influence skin microcirculatory function in patients with type 2 diabetes treated with insulin.

AIMS/HYPOTHESIS: Insulin resistance is associated with abnormal microvascular function. Treatment with insulin sensitisers may provoke oedema, suggesting microvascular effects. The mechanisms underlying the peripheral oedema observed during glucose-lowering treatment with thiazolidinediones are unclear. Therefore we examined the effect of pioglitazone on microvascular variables involved in oedema formation. METHODS: Subjects (40-80 years) with type 2 diabetes and on insulin were randomised to 9 weeks of pioglitazone therapy (30 mg/day; n=14) or placebo (n=15). The following assessments were performed at baseline and 9 weeks: microvascular filtration capacity; isovolumetric venous pressure; capillary pressure; capillary recruitment following venous or arterial occlusion; postural vasoconstriction; and maximum blood flow. A number of haematological variables were also measured including vascular endothelium growth factor (VEGF), IL-6 and C-reactive protein (CRP). RESULTS: Pioglitazone did not significantly influence any microcirculatory variable as compared with placebo (analysis of covariance [ANCOVA] for microvascular filtration capacity for the two groups, p=0.26). Mean VEGF increased with pioglitazone (61.1 pg/ml), but not significantly more than placebo (9.76 pg/ml, p=0.94). HbA(1c) levels and the inflammatory markers IL-6 and CRP decreased with pioglitazone compared with placebo (ANCOVA: p=0.009, p=0.001 and p=0.004, respectively). CONCLUSIONS/INTERPRETATION: Pioglitazone improved glycaemic control and inflammatory markers over 9 weeks but had no effect on microcirculatory variables associated with oedema or insulin resistance in type 2 diabetic patients treated with insulin.

Efficacy of plant-produced recombinant antibodies against HCG.

BACKGROUND: Antibody engineering facilitates the construction of different antibody formats [single chain variable fragment (scFv), diabody, full-size chimeric monoclonal antibody] with ease. METHODS: We constructed recombinant antibodies against HCG, which is widely used in pregnancy testing and is also produced by a number of cancers. RESULTS: The recombinant antibodies were transiently expressed in tobacco leaves to levels of up to 40 mg of pure protein per kg fresh leaf weight. Enzyme linked immunosorbent assay (ELISA) and electrophoretic mobility assay (EMSA) confirmed antibody specificity for the beta subunit of beta-HCG. The efficacy was confirmed by inhibiting HCG induced testosterone production by Leydig cells in vitro and by blocking the HCG induced increase in mouse uterine weight in vivo. CONCLUSIONS: Passive immunization with recombinant HCG-specific antibodies may have clinical utility as (i) diagnostic and therapeutic tools for HCG-expressing cancers and (ii) contraceptive measures.

Effect of combined kinetic therapy and percussion therapy on the resolution of atelectasis in critically ill patients.

BACKGROUND: Some critically ill patients have difficulty in mobilizing their respiratory secretions. These patients can develop pulmonary atelectasis that may result in hypoxemia. There are some data to show that atelectasis may be prevented by turning a patient from side to side utilizing special beds. STUDY OBJECTIVES: To determine the role of kinetic therapy (KT) combined with mechanical percussion (P) in the resolution of established atelectasis of the lungs and hypoxemia in critically ill, hospitalized patients. (KT was defined as rotation of a patient along the longitudinal axis of > or = 40 degrees to each side continuously.) DESIGN: Prospective and randomized study (2:1 test to control group). PATIENTS: Twenty-four patients with respiratory failure, either mechanically ventilated or spontaneously breathing, who demonstrated segmental, lobar, or unilateral entire lung atelectasis were studied. SETTING: Medical ICU and adult respiratory ward in a county hospital in New York. INTERVENTIONS: Seventeen patients were treated with KT combined with mechanical P using a KT system (Triadyne Kinetic Therapy System; KCI; San Antonio, TX). Seven patients received manual repositioning and manual P every 2 h. Both groups received similar conventional therapy with inhaled bronchodilators and suctioning. RESULTS: Partial or complete resolution of atelectasis was seen in 14 of 17 patients (82.3%) in the test group as compared with 1 of 7 patient (14.3%) in the control group. The median duration to resolution of atelectasis was 4 days in the test group. Bronchoscopy was performed in 3 of 7 patients in the control group, but in none of the patients in the test group. A cost of $720 was incurred per patient for utilizing the specialty beds for a mean duration of 4 days. An improvement in oxygenation index occurred in the test group (change in baseline PaO2/fraction of inspired oxygen from 207.4+/-106.7 mm Hg to 318+/-100.7 mm Hg) at the end of therapy, while the control group showed a reduction over a similar duration of time (181.3+/-96.3 mm Hg to 112+/-21.2 mm Hg). CONCLUSIONS: KT and mechanical P therapy resulted in significantly greater partial or complete resolution of atelectasis as compared with conventional therapy. There was a generalized trend toward statistical significance in the improvement of oxygenation and a reduced need for bronchoscopy in the group receiving KT and P therapy.