Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants

Abstract

The immune response of cattle to nonliving vaccines derived from Brucella abortims rough strain 45/20 was studied. Vaccines contained trehalose dimycolate and a derivative of muramyl dipeptide, N-acetylmuramyl-L-cx-aminobutyryl-Disoglutamine. A factorial experiment was designed to test the effects of type of antigen, quantity of antigen, and quantity of mineral oil on the immune response to porin. Muramyl dipeptide was kept constant at 5 mg per dose, and 1 part of trehalose dimycolate was incorporated for two parts of dry matter. Over a 10-week period, blastogenesis responses to porin were largest in cattle immunized with outer membranes; the highest antibody titers to the porin-lipopolysaccharide complex were achieved by immunization with detergent-extracted outer membrane proteins. There was no advantage in the use of 25, rather than 5, mg of any of the antigens, but antibody responses were improved by increasing the quantity of oil from 0.6 to 1.8 ml per dose. In other animals, blastogenesis and antibody responses were sustained at high levels longer than 3 months after two vaccinations with outer membrane proteins. Intradermal injection of porin evoked inflammatory reactions histologically consistent with delayed-type hypersensitivity. Cross-reactions in cases of delayed-type hypersensitivity occurred with porin derived from a smooth strain of B. abortils but were less extensive than in the blastogenesis test. The magnitude of the delayed-type hypersensitivity and blastogenesis responses induced by vaccination exceeded those observed after natural or experimental infections. No ill effects were observed after vaccination. These findings provide a basis for the use of trehalose dimycolate and muramyl dipeptide adjuvants in evaluating nonviable vaccines for bovine brucellosis.Development of an effective nonviable vaccine for Brlcella abortus would constitute a major advance toward the goal of eradicating bovine brucellosis from the United States. It is well established that B. abortus is a facultative intracellular parasite for which a cell-mediated immune response associated with delayed-type hypersensitivity (DTH) is crucial for protective immunity (8,14). Such a response can be induced effectively by nonviable vaccines in which trehalose dimycolate (TDM) and muramyl dipeptide (MDP) have been incorporated as adjuvants (for reviews, see references 1 and 7). Woodard et al. (25) have demonstrated that guinea pigs immunized with killed cells of B. t Present address: Syncor International Corp., Berkeley. CA 94710.abortus rough strain 45/20 combined with both TDM and MDP adjuvants developed a relatively high level of resistance to infection with virulent B. abortuls 2308. The use of these adjuvants would be feasible in a commercial vaccine for cattle because the absence of mycobacteria and the low content of mineral oil minimize local reactions and prevent development of tuberculin hypersensitivity.We have isolated and characterized the major outer membrane proteins (OMP) of B. abortlls (21). One of these (group 2)...

Increased CD4+ T cell‐dependent anti‐erythrocyte antibody levels following the onset of parasite egg production in Schistosoma mansoni infected mice

“…Plates were agitated continuously by the reader for two minutes prior to the first reading and between each of the other readings. The rate of colour development was calculated using the KELA program; data are presented as KELA slope units, which are a measure of color development and therefore antibody concentration (Winter et al 1983).…”

Quantitation of monoclonal antibody by capture ELISA based on initial enzyme activity rate

“…In this assay, the reaction rate is monitored continuously or at discrete time intervals during the linear phase period, when product formation is directly proportional to analyte concentration, substrate concentration is saturating, and the enzymatic catalysis operates in steady-state conditions (Tsang et al, 1983). k-ELISA has been developed in a variety of formats to quantitate antibodies to parasites (Hancock and Tsang, 1986;Werre et al, 2002), Lewis blood group antigens (Spitalnik et al, 1983), feline coronavirus (Barlough et al, 1983), flu virus glycoproteins (Snyder et al, 1988), and bacterial genera (Winter et al, 1983;Shin et al, 1993;Van Schaik et al, 2003).…”

Killing of Brucella abortus by bovine serum

“…Antibodies to group 2 (porin) and group 3 outer membrane proteins (67) were measured by kinetics-based enzymelinked immunosorbent assay (ELISA) with purified outer membrane proteins of strain 45/20 as antigens and normalized slopes of the reaction rates as an indication of antibody activity (69,70). For measurement of antibodies of each immunoglobulin isotype in sera from infected animals, the kinetics-based ELISA was modified to utilize a deoxycholate extract as antigen and horseradish peroxidase conjugates of goat anti-bovine IgGi, IgG2, IgM, and IgA.…”

“…Specificity of conjugates had been confirmed by ELISA against purified bovine immunoglobulin isotypes as described previously (46). By using background levels of antibody as a baseline, antibody titers could be estimated because a direct relationship exists between increases in slope values and log2 antibody titer (69). Thus, in a prior study of porin antibodies by the kinetics-based ELISA method, increases in slope values of 100, 200, 300, and 400% (i.e., twoto fivefold) over baseline values represented approximate titers (69).…”