Genetically improved Aujeszky's disease strain MNC+/10a derived from strain K/61 and testing for safety and potency in pigsB. Lomniczia, M. Kelemenb and E. Wehmanna
a Veterinary Medical Research Institute, Hungarian Academy of Sciences 1143 Budapest, Hungaria krt. 21., Hungary
b Phylaxia-Sanofi Ltd. A Company of SSNA, Budapest, Hungary
Abstract - Aujeszky's disease vaccine strain Bartha K/61 carries at least two genetic defects that can be deleterious in a vaccine strain. It is deficient in the expression of glycoprotein gC, a protective antigen of the virus and its gM lacks carbohydrate side chains. Both glycoproteins are necessary for optimal growth, therefore the antigenicity of a live vaccine that is deficient in these functions, must be impaired in vivo. Our aim was to restore the function of glycoprotein gC in order to obtain an attenuated strain with improved immunogenicity. In order to correct the defects of K/61, a mixed infection of K/61 and a virulent strain was performed to generate recombinants. From the offspring, a recombinant (designated MNC+/10a) was selected in which the essential properties of vaccine strain K/61, such as the defects of its major virulence gene (UL21) and the lack of gE gene were retained. Three week-old piglets inoculated intranasally with 107 plaque forming units of MNC+/10a remained healthy and the mutant proved to be avirulent in day-old chickens injected intracerebrally. A restriction site marker (BamHI 2a+2b) was also transferred from the virulent parent to the avirulent MNC+/10a strain together with an adjacent region that carries the gC gene. As detected by a gC-specific monoclonal antibody MNC+/10a was gC-positive (Dr. T.C. Mettenleiter, personal communication). It is possible that gM was also rescued because MNC+/10a forms larger plaques than K/61. In addition, MNC+/10a showed a ten-fold higher growth capacity in the nasal mucosa of piglets as compared to K/61. The immunogenicity of MNC+/10a was tested in pigs in a standard challenge experiment. Upon intramuscular injection, MNC+/10a induced significantly higher antibody levels than K/61. Groups of pigs free from Aujeszky-disease virus antibodies were vaccinated once or twice with Aujeszky- vaccine Auphyl-plus (Phylaxia-Sanofi) that contains the attenuated strain MNC+/10a with an oil/water emulsion adjuvant. Three weeks after the first or second injection the pigs were challenged intranasally with 104 PFU of the virulent strain NIA-3 in two separate experiments. Most unvaccinated animals showed severe respiratory and nervous signs on postinfection days 4 to 6 and the average daily weight gains were reduced to about in the control groups. Altogether, ten of the twelve control animals succumbed to the challenge infection. A single dose of the vaccine was sufficient to prevent disease but did not prevent mild fever and transient (1-2 days) anorexia in some animals. Two doses of the vaccine prevented both. The average daily weight gains of the once and twice vaccinated groups were 1.47% and 0.90%, respectively as compared to the weight loss of in the unvaccinated pigs. Virus shedding measured by total excreted virus was significantly reduced (150 times) in the twice-vaccinated group as compared to controls. To simulate natural conditions of virus spread, pigs vaccinated twice were placed in the pens of twice vaccinated, intranasally challenged animals. None of the contact-challenged vaccinated pigs shed virus during a two-week observation period. There was a positive correlation between the ability of the vaccine strain to replicate in the nasal mucosa and the primary antibody response in pigs. This is the first report demonstrating that the restoration of a protective protein function has indeed resulted in an improved antigenicity as measured by an elevated level of antibody production. The vaccine prepared from the virus strain ensures excellent protection against the clinical signs and virus shedding.
Corresponding author: B. Lomniczi Tel.: (36) 1 467 4069; fax: (36) 1 252 1069;
© INRA, EDP Sciences 2000