Comparison of Q fever cellular and chloroform-methanol residue vaccines as skin test antigens in the sensitized guinea pig.

Coxiella burnetii phase I whole cell vaccine (WCV) is associated with risk of severe local delayed-type hypersensitivity (DTH) reactions in previously immunized individuals or those sensitized by natural exposure. We compared this vaccine to another investigational vaccine derived by chloroform-methanol extraction of phase I whole cells (chloroform-methanol residue vaccine, CMRV). Hairless guinea pigs, sensitized with either WCV or CMRV, were given 60,600 and 6,000 ng of WCV or CMRV in an intradermal (i.d.) skin test. The i.d. administration of WCV consistently caused more host reactions than comparable doses of CMRV in guinea pigs sensitized with either WCW or CMRV, suggesting that CMRV may be a safer vaccine. However, the CMRV was not innocuous and caused significant indurated lesions and micro-abscesses at the 600 ng and 6,000 ng skin test sites.

Immediate responses of leukocytes, cytokines and glucocorticoid hormones in the blood circulation of monkeys following challenge with aerosolized staphylococcal enterotoxin B.

The immediate responses to aerosolized staphylococcal enterotoxin B (SEB) in respiratory toxic shock were studied in the circulation of rhesus monkeys with low antibody levels following immunization with SEB toxoid-containing microspheres. Both the surviving and dying monkeys had toxic shock syndrome 4-48 h after SEB challenge and all showed three distinctive patterns of immediate responses. The first pattern, characterized by the responses of all T cells, HLA-DRlo cells, monocytes, IL-2R+ cells, IFN-gamma, and augmented lymphocyte mitotic responses to lipopolysaccharide (LPS) and SEB in culture, was a rapid increase at 20 min followed by a quick decrease at 90 min to approximately the original levels. The second pattern, which included responses of HLA-DRhi cells, NK cells, adrenocorticotropic hormone (ACTH) and cortisol, was characterized by a moderate decrease at 20 min and a further decrease at 90 min. The third pattern, the inverse of the second pattern, including responses of polymorphonuclear leukocytes (PMN), concanavalin A (Con A) mitogenesis, IL-6 and IL-2, was a moderate increase at 20 min and a further increase at 90 min. Between the surviving and dying monkeys, the responses of T cells, HLA-DRhi cells, PMN and cortisol did not differ significantly, suggesting that they are the basic causes that initiated toxic shock. However, significant differences were seen in the responses of HLA-DRlo cells, monocytes, IL-2R+ cells and lymphocyte mitogenesis in culture at 20 min, and of Con A mitogenesis, NK cells, IL-2, IL-6 and ACTH at 90 min. These different responses are apparently the exacerbating causes of death of the monkeys. All together, the immediate responses seem to be caused by the combined effects of SEB superantigenicity, activation of NK cells and non-lymphoid cells, and depression of the neuroimmune defense system.

Steady-state minute volume determination by body-only plethysmography in juvenile rhesus monkeys.

When one is using nonhuman primates for studying the inhalation of infective or toxic agents, a respiratory minute volume (MV) range of +/- 50 ml is desirable to ensure the accurate delivery of calculated doses of the aerosolized agent. When one is working with highly infective or toxic agents, it is desirable to anesthetize the animals and to separate the plethysmograph, used to measure MV, from the aerosol chamber, used to administer agents, in order to minimize decontamination procedures and to maximize safety. In our laboratory the sequential completion of these procedures requires at least 20 min. Therefore it is necessary to find an anesthetic that achieves a +/- 50 ml steady-state MV for at least 20 min and that does not change when an animal is transported from one apparatus to another. Using 2.6- to 4.0-kg, 14- to 18-month-old rhesus macaques, we determined that tiletamine/zolazepam induced a steady-state MV of 48 +/- 17.8 min, beginning 21.5 +/- 4.7 min after injection of the anesthetic agent. This MV did not significantly change when animals were transported. The use of ketamine and ketamine/acepromazine resulted in a steady-state MV period of 11.5 +/- 4.5 and 22.0 +/- 7.9 min respectively. When we compared these findings with previously reported mathematical estimations of MV based on functions of weight or respiratory rate, we further determined that the accurate measurement of MV before each aerosol exposure was critical for calculating inhaled doses of the agent.

Humoral immunity to aerosolized staphylococcal enterotoxin B (SEB), a superantigen, in monkeys vaccinated with SEB toxoid-containing microspheres.

Staphylococcal enterotoxin B (SEB) toxoid-containing microspheres were tested for efficacy in rhesus monkeys as a vaccine candidate for respiratory SEB toxicosis and toxic shock. Forty monkeys were randomly separated into 10 groups of four monkeys each: 9 groups were vaccinated with the microspheres via combinations of mucosal and nonmucosal routes, and 1 group served as nonvaccinated controls. Both vaccinated and nonvaccinated monkeys were then challenged with a high lethal dose of SEB aerosol. Monkeys primed with an intramuscular dose of the microspheres followed by an intratracheal booster all survived the SEB challenge. Overall, monkeys with an intratracheal booster generally had the highest antibody levels, which is consistent with their high survival rate and lower rate of illness. Protective immunity was correlated with antibody levels in both the circulation and the respiratory tract. The protection was not due to the depletion or anergy of SEB-reactive T cells, since SEB-induced proliferation in cultures of circulating lymphocytes was not significantly reduced after the microsphere vaccination. It is evident that the nonsurvivors did not die of systemic anaphylaxis or hypersensitivity because the monkeys did not die immediately after SEB challenge and there were no significant differences in histamine levels between the vaccinated and control monkeys before and after SEB challenge. The antibodies seemed to neutralize the SEB that got into the airway and the circulation.

A refined guinea pig model for evaluating delayed-type hypersensitivity reactions caused by Q fever vaccines.

The previously established model for determining dermal granulomatous reactivity of Q fever vaccines uses footpad injections to sensitize Hartley guinea pigs. We describe a substantial refinement to that model by substituting an alternate injection route and replacing the model with the hairless Hartley guinea pig. The experimental design incorporates a matrix of various antigen and adjuvant combinations administered by subcutaneous (SC) and intradermal routes of administration. The two Coxiella burnetii antigens evaluated were phase-I whole-cell vaccine (WCV) and phase-I chloroform-methanol residue vaccine. The adjuvants used were complete Freund's (CFA), incomplete Freund's, and RIBI's De-Tox. Phase-I C. burnetii WCV in CFA administered SC provided the most efficacious regimen evaluated for sensitizing hairless guinea pigs. The establishment of this animal model allows evaluation of candidate Q fever vaccines in a more humane model than that previously available.

Immunity and responses of circulating leukocytes and lymphocytes in monkeys to aerosolized staphylococcal enterotoxin B.

Rhesus monkeys immunized intramuscularly or orally with staphylococcal enterotoxin B (SEB) toxoid or SEB toxoid incorporated in microspheres made of poly(DL-lactide-co-glycolide) were challenged with a lethal dose of aerosolized SEB to study their immunity and cellular responses in the circulation. It was found that circulating antibodies play a critical role in preventing SEB from triggering toxicosis. Monkeys with high levels of antibodies survived, while those with low levels underwent 2 to 3 days of toxicosis and died. Intramuscular immunization induced high levels and oral immunization induced low levels of antibodies. The circulating antibodies in surviving monkeys decreased dramatically within 20 min and started to rebound at 90 min after SEB challenge. At 90 min, the dying monkeys showed in the circulation a dramatic increase of polymorphonuclear leukocytes and decreases of NK cells and monocytes (CD16 and CD56 markers) as well as of lymphocytes with HLA-DR, CD2, CD8, and IL2R alpha (CD25) markers. The number of polymorphonuclear leukocytes showed an inverse correlation with the numbers of monocytes and various lymphocyte subpopulations which, except for IL-2R, CD16, and CD56(+) cells, showed a direct correlation with one another. The changes in the populations of leukocytes, monocytes, NK cells, and lymphocytes seem to be an indication of initial toxicosis; however, the roles of these cells in toxicosis and death remain to be defined.