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Standardization of a Protocol for in Vivo Delivery of DNA Vaccine Using Attenuated Salmonella Bacteria.

Dr. Md. Abu Hadi Noor Ali Khan
Professor
Department of pathology, BAU, Mymensingh-2202.

Abstract/ Executive Summary:

This study was designed to asses further whether the plasmid DNA vaccine suppresses gonadal function in sexually mature male and female rats. This study utilized a comprehensive version of the vaccine, which showed significant suppression of fertility in male and female mice. In this study plasmid DNA was attempted to introduce in Salmonella vector but unfortunately the bacterial vector appeared resistant to encoding antibiotics neomycin. The plasmid DNA vaccine used encoding eight GnRH-1 peptides, eight T-helper epitopes and a V5 epitodes from feline immunodeficiency virus was, therefore, evaluated in saline delivery in male and female Sprague Dawley rats in terms of anti GnRH-1 antibody response, serum testosterone level and arrested gametogenesis. A significant level of anti-GnRH-1 antibody response was detected in vaccinated male and female rats in study week 6 (0.517±145) and a steadily increasing response was detected in study week 9 (0.625±0.157) and 12 (0.685±0.136) in contrast to controls (0.312±0.133). GnRH-1 immunoneutralization was found to reduce total epididymal sperm count and reduce circulating testosterone hormone in male rats. The vaccinated female rats also showed a significant suppression of ovarian folliculogenesis and luteogenesis as compared to naked plasmid treated female rats. Interestingly the plasmid DNA vaccine found to reduce male sex hormone by causing atrophy of interstitial cells. An additional approach was taken to evaluate the vaccine response by assessing gonadal GnRH-1 and its receptor expression in the vaccinated rats using RT-PCR. Results of RT-PCR showed that the hypothalamus and gonads of vaccinated rats containing signals for both GnRH-1 and its receptor, an indication of uninterrupted GnRH-1 signals and reversible of fertility disruption. The non-sterodial method of controlling gonadal functions need to be tested further in different animals species to explore efficient means of controlling reproduction in man and animals.

Keywords: in vivo, DNA vaccine, GnRH-I, Salmonella

Location: Laboratory of the department of pathology, BAU.

Start Date: 01-07-2007

End Date: 30-06-2009

Program Area: Variety and Species

Commodity:

Objectives:

As an alternative use of HVJE viral vector I wish to develop a bacterial vector as an efficient gene delivery tool using salmonella organism. The bacteria will be attenuated and transfected with the plasmid DNA vaccine. The transfected bacteria by its inherited intracellular nature will be introducing vaccine DNA in to the host genome. The GnRH-I-T-helper epitopes fusion protein derived from the DNA vaccine is designed to release into the extra-cellular state. The fusion protein coded for the DNA vaccine will reasonably trigger host immune response and finally neutralize GnRH-I of injected animals. GnRH-I is a key peptide to regulate reproduction in all mammals and success with this protocol would assumed to bring tremendous scientific revolution. The DNA vaccine I have developed would be a potent vaccine candidate to control tumor growth at the fertility axis and a potent contraceptive candidate as well in near future for both man and animals.

1. Formulation of a bacterial (Salmonella) vector to make DNA vaccine more efficient in vivo.

2. Development of a commercialized genome based contraceptive and anticancer vaccine.

Methodology:

Vaccine construction: I have developed a plasmid DNA vaccine during my post doctoral research in Japan. This is a double strength vaccine and not gone for trial in laboratory animals. A single strength vaccine was found to suppress reproductive function in rats and mice. Salmonelle bacterial vector: Our laboratory containing a number of bacteria including salmonella pullorum, Salmonelle gallinarum and Salmonella typhae. These bacteria will be used to developed an efficient toll to deliver DNA vaccine in vivo. Male and female rats: Male and female rats will be injected with the vaccine construct either in saline solution and in bacterial vector. Following 15 weeks of immunization the rat will be sacrificed for evaluating GnRH-I specific immune response, gametogenesis, strriodogenesis and in vivo fertility assay. The vaccinated male and female animals will be allowed to mate individually with untreated female and male animals respectively to quantify the reproductive performance of vaccinated animals.

Salmonella bacterial vector: Our laboratory containing a number of bacteria including Salmonella pullorum, Salmonella gallinarum and Salmonella typhae. These bacteria will be used to develop an efficient toll to deliver DNA vaccine in vivo.

Male and female rats: Male and female rats will be injected with the vaccine construct either in saline solution and in bacterial vector. Following 15 weeks of immunization the rats will be sacrificed for evaluating GnRH-I specific immune response, gametogenesis, steroidogenesis and in vivo fertility assay. To measure anti-GnRH-I antibody assay blood from the tail vein will be collective at an interval of 2 weeks and antibody against the vaccine candidate will be evaluated using an indirect and end point ELISA and would be carried out in the Department of Immunology, Starthcyde University, Glasgow, UK. A detail histological examination of the reproductive organs of male and female animals will be out to evaluate response of gonadal tissues following immunization. By the end of study total sperm count from the epididymis will be carried out to plot for the differences between control and vaccinated animals. The vaccinated male and female animals will be allowed to mate individually with untreated female and male animals respectively to quantify the reproductive performances of vaccinated animals. A RT-PCR with the DNA obtained from the organs of vaccinated animals will be carried out in order to quantify the level of gene expression at various sites following immunization.

The primers: The primers having accurate evaluation power of the DNA vaccine sequence will be used in this study.

Source of Information: Bangladesh Agricultural University Research Progress, 07-08 March, 2010. Bangladesh Agricultural Bangladesh Agricultural University Research Progress. Volume 20. Proceedings of the Workshop of University Research System (BAURES), BAU, Mymensingh.

Article Link (Online Journal):

Funding Source:

1.	Budget: 200000

Total Budget (Tk.) : 200000

Achievement/Findings:

Efficient immune response GnRH-I can be a potent tool to control reproduction and sex organ tumar in a wide range of mammals and develop DNA vaccine strategy for world wide use.

Knowledge Management: Report/Proceedings