Persistency of Adenoviral-Mediated Lysostaphin Expression in Goat Mammary Glands

Abstract

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

“…In recent years, a new approach for expressing exogenous genes in secretory mammary epithelial cells has been proven to be the most effective versatile alternative [14][15][16][17][18]. A target protein could be obtained from milk by employing the recombinant adenovirus directly transfected into the mammary gland.…”

Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans

“…The topical or systemic use of lysostaphin has since proven effective in several mouse and rat models of staphylococcal infection 5,6. Moreover, transgenic mice and cattle producing the enzyme have been generated to engineer S. aureus and/or S. epidermidis resistance 7,8, and adenovirus-mediated lysostaphin delivery has been tested in goats 9,10. In humans, the protein has been effective in the experimental treatment of S. aureus -infected patients 11, and is currently in clinical trials for use in treating burns 12.…”

The Use of Viral Vectors in Introducing Genes into Agricultural Animal Species

“…In vitro infected somatic cells can potentially develop into adult animals after such genetically engineered somatic cells are transferred into enucleated fertilized oocytes and transferred into the uterus of a recipient animal by SCNT (7,8,30). In other cases, transient expression of the transgene may be achieved by infection of local tissues, such as caprine mammary glands (9)(10)(11)(12)(13). In addition, viral vector constructs were directly injected into porcine hearts (20), and ovine skeletal muscles (19), which led to…”

“…Both in vitro and in vivo infected cells expressed lysostaphin, and the media in which the infected cells were grown exhibited staphylolytic activity. The peak lysostaphin expression in mammary extract of infected goats was detected between 7 and 11 days after infection (10). The in vivo resistance to S. aureus mastitis has not been evaluated.…”