Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Immunization of Female Mice with a Plasmid DNA Vaccine Coding Eight Repeats of Gonadotrophin Releasing Hormone (GnRH-i) and Eight T-helper Epitopes Suppress Fertility in Vivo

Umme K Rima
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Tadashi Kimura
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Ayman Gebril
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Mohammad T Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abu SM Bari
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Valerie A Ferro
Department of Immunology, University of Strathclyde, SIBS Building, 27 Taylor Street,Glasgow G4 ONR, UK

Mohammad A H Khan
Professor
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abstract/ Executive Summary:

Induction of an appropriate immune response against gonadotrophin releasing hormone (GnRH-I) disrupt fertility, reduce fecundity and regress tumours of reproductive system.To disrupt fertility a plasmid DNA vaccine was engineered coding eight repeats of GnRH-I and eight T-helper epitopes. Translation efficiency of the vaccine was evaluated in undifferentiated COS1 cells and found to release GnRH-I fusion protein in culture supernatant. Swiss albino female mice (N=24) were immunized with 50μg plasmid DNA construct in study weeks 0, 3, 6, 9 and 12. Group 2 mice were primed with the plasmid DNA in hemagglutinating virus of japanese envelope (HVJE) vector and subsequent boosts were carried out in phosphate buffer saline. Group 3 mice were immunized with the plasmid DNA in non-ionic surfactant vesicles (NISV) and Group 1 was served as untreated control. The effect of immunization was studied in terms of anti-GnRH-I antibody response (OD value at A540 ± SD), suppression of ovarian folliculogenesis, altered uterine histoarchitecture and impaired fertility in vivo in mating trials. In study week 24 OD values of anti- GnRH-I antibody response were 0.982 ± 0.231 in Group 3 mice, followed by 0.783 ± 0.191 in Group 2 in comparison with no response in Group 1 controls (0.237 ± 0.147). Results of mating trials showed conception failure in vaccinated mice; 51, 18 and 05 pups were seen in the uteri of Groups 1, 2 and 3 mice respectively. There was significant (p>0.001) reduction in the weight of ovaries in Group 2 (8.50 ± 2.38 mg) and Group 3 (7.25 ± 0.95 mg) mice compared to Group 1 control (15.00 ± 1.41 mg). Significant reduction of ovarian folliculogenesis was seen in Group 2 (p>0.001) and Group 3 mice (p>0.01). In conclusion, the plasmid DNA vaccine delivered in female mice with HVJE and NISV induced significantly (p>0.001) higher levels of anti-GnRH-I antibody response, suppressed ovarian and uterine function and impaired fertility in vivo.

Keywords: Mice, DNA vaccine, GnRH-I, In vivo fertility, Ovarian folliculogenesis

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Rats

Objectives:

To overcome these disadvantages a new vaccine was constructed coding eight repeates of GnRH-I and the vaccine were delivered in a hemagglutinating virus of japanese envelope (HVJE) vector and in a lipd based delivery system.

Methodology:

A plasmid DNA vaccine coding four repeats of GnRH-I was digested with Xba1 enzyme and amplified with Tag A and Tag B oligonucleotides to develop Not 1 and Xho 1 cutting sites at 5/ and 3/ ends respectively. The end labeled oligonucleotides were than digested with Not1 and Xho1 enzymes and ligated into the upstream of the vaccine construct at Not1 and Xho1 sites. The vaccine construct engineered incorporating GnRH-I leader sequences, eight repeats of GnRH-I interspersed with eight T-helper epitopes, an N terminal V5 epitope and a histidine tag before stop codon. The T-helper epitopes were derived from circumsporozoite protein (CSP), respiratory syncitial virus (RSV), measles virus protein (MVP) and tetanus toxoid (TT). Competence JM109 E. coli was transformed with the vaccine construct and amplified in LB medium containing ZeocinTM (40 μg/ml, Invitrogen Life technologies, USA). The plasmid DNA was extracted from the bulk culture of transformed E. coli using wizard plus Gigaprep kit (Promega, USA). A colony PCR was designed to amplify 981bp long fragment from the transformed E. coli using forward and a reverse primers. The complete GnRH-I and T-helper coding genes, the positions of the start and stop codons and the integrity of the open reading frames on to pcDNAV5-HisB plasmid were determined by sequence analysis.Amino acid sequence of the vaccine: The blue letters indicate the sequences of the GnRH-I peptide and hhhhhh represents a histidine tag. Relative position of GnRH-I and T-helper epitopes in the vaccine.The number of ovarian follicles, healthy and regressing corpus luteum (CL) in ovaries and the number of uterine glands per uterine section using 10X and 100X microscopic objectives were recorded. The images were captured using an automated computer (Microimaging, Carl Zeiss, GmbH, Germany) connected to a microscope and ZEN lite 2012 software. In study weeks 0, 3, 6, 12 and 24, 100 μl tail bleeds were carried out into heparinized capillary tubes (Selzer Labortechnik, Germany). Plasma was prepared by centrifugation at 200g for 10min and stored at -20°C until an indirect enzyme linked immunosorbant assay (ELISA) was carried out. A base line OD value of 0.474 or above (twice the OD value of negative control) was considered a positive anti-GnRH-I antibody response. Effect on in vivo fertility In study week 24, the vaccinated female mice (four in each group) were mated individually with male mice. Control male and female mice were also mated individually to compare the differences. Following 10 days of mating (as the duration of estrous in vaccinated females lasts for 09-10 days, data was not shown) the male mice were withdrawn from the females. Following 16 days of mating the females were sacrificed by cervical dislocation and the number of pups in the uteri of each mouse were recorded. Statistical analysis Data between control and experimental groups were analyzed (Duncan’s Multiple Range Test) using the Statistical Package for Social Sciences version 17.0 (Chicago, IL, USA). A difference was considered significant at the p<0.05 level. Data were reported as the mean ± standard deviation (SD).

Source of Information: In Vivo. J Vaccines Vaccin 6(3): 282.

Article Link (Online Journal): doi:10.4172/2157-7560.1000282

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study designed, engineered and evaluated a plasmid DNA vaccine aganist GnRH-I. The vaccine delivered in HVJE showed promising in terms of inducing an early immune response against GnRH-I, but in study week 24 the level of immune response appeared low because the subsequent boostings were carried out in PBS. The plasmid DNA vaccine delivered in NISV induced detectable levels of anti-GnRH-I antibody response in study week 6 and persistantly higher levels of IgG antibody were detected until study week 24. The vaccine delivered in HVJE and NISV induced higher levels of anti- GnRH-I antibody responses and suppressed ovarian folliculogenesis and impaired fertility in vivo. A link between the higher level of anti GnRH-I antibody response and suppressed fertility in mice was established. Priming of mice in HVJE vector and subseqent boosting in NISV could be a suitable approach for genetic immunization, that requires further investigation.

Knowledge Management: Journal