Folia Microbiol. 51 (2), 154–156 (2006) http://www.biomed.cas.cz/mbu/folia/ Adjuvant Effect of Bacillus firmus in Intranasal Immunization of Guinea Pigs with Inactivated Type B Infuenza Virus M. HAVLÍČKOVÁ a , L. PROKEŠOVÁ b , P. ZANVIT b , J. TÁČNER a , R. LIMBERKOVÁ a a National Institute of Public Health, Centre of Epidemiology and Microbiology, 100 42 Prague, Czechia b 1st Faculty of Medicine, Charles University, 120 00 Prague, Czechia e-mail nflucprg@szu.cz Received 25 April 2005 Revised version 12 September 2005 ABSTRACT. Intranasal immunization of guinea pigs with inactivated type B influenza virus plus inactiva- ted Bacillus firmus as an adjuvant compared to the virus alone yields higher titers of serum hemagglutinat- ion-inhibiting antibodies and virus-neutralizing antibodies. This phenomenon could be useful in standard sero- logy, especially in the preparation of immune sera against highly pathogenic strains for in vitro diagnosis. Abbreviations BF Bacillus firmus MDCK Madine Darby canine kidney CFR complement-fixation reaction TCID 50 tissue culture-infectious dose 50 % HIT hemagglutination-inhibition test TW/E-HIT HIT with a Tween–diethyl ether split antigen HT hemagglutination titer VNT virus-neutralization test Adjuvants have been used in the study of the immune response and preparation of vaccines since the 1920’s. They are known to enhance immunogenicity either by binding and then progressively releasing an antigen (an example of this type are aluminum salts) or by increasing the reactivity of the host (e.g., myco- bacteria). BF is a Gram-positive nonpathogenic bacterium found in the outer environment, which to our know- ledge does not cause any specific pathological process in any animal species. The BF immunomodulatory potential in macrophage stimulation and polyclonal activation of B cells was reported by Mára et al. (1992, 1994), Prokešová et al. (1994, 2002), Zídek et al. (1998), and Mlčková et al. (2005). BF showed a high adjuvant effect in mucosal immunization of mice by protein antigen via respiratory tract (Mlčková et al. 2001, 2003). Here we summarize the results of intranasal immunization of guinea pigs with inactivated type B influ- enza virus plus BF as an adjuvant. MATERIAL AND METHODS Preparation of antigens for immunization. Influenza virus B/Lee 1/40 was propagated in allantoic fluid of 10-d-old embryonated chicken eggs according to Manuguerra and Hannoun (1999a). After a 3-d incu- bation at 35.5 °C, the fluids were removed and titrated for hemagglutinin. Only fluids with a 1 : 256 or higher HT were used. Selected allantoic fluids were inactivated by 250 ppm formaldehyde for 3 d at 4 °C. Inactiva- tion was tested after 3 blind passages on MDCK cells and embryonated chicken eggs. Suspensions with a HT of 1 : 256 were used for immunization. BF preparation. B. firmus strain CCM 2212 was aerobically cultivated in a liquid medium compo- sed of peptone, beef extract and urea (pH 7.2–7.4) at 37 °C to the late exponential phase. The cultures were washed with distilled water and inactivated by 0.4 % formaldehyde at room temperature for 30 min. Under these conditions all bacteria were killed (proved by sterility tests). The inactivated biomass was washed thrice with distilled water and lyophylized (Mára et al. 1994). Lyophylized BF was resuspended in sterile deionized water to a resulting concentration of 30 mg/mL. Animals. Outbred guinea pigs (AnLab, Prague) weighting 350 g were used for immunization. Immunization scheme. Animals under short diethyl ether anesthesia were intranasally immunized with formaldehyde-inactivated type B influenza virus alone (100 μL allantoic fluid of HT 1 : 256 + 100 μL sterile 0.9 % NaCl per dose), BF alone (100 μL BF suspension containing 3 mg of bacteria + 100 μL 0.9 % NaCl),