Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Cellular and cytokine responses in the circulation and tissue reactions in the lung of rhesus monkeys (Macaca mulatta) pretreated with cyclosporin A and challenged with staphylococcal enterotoxin B.

Cyclosporin A (CsA), an inhibitor of T cell cytokine production, protects mice against staphylococcal enterotoxin B (SEB) intoxication. To determine whether CsA treatment would work in a species closer to humans. 4 rhesus monkeys were given 50 mg/kg CsA followed by an intratracheal challenge with approximately 6 LD50 of SEB. The CsA was not protective: one of the monkeys died and the other three had to be euthanised when they became moribund. All monkeys made IL-2, TNF, and IFN-gamma in response to SEB. In addition, there was about a 10-fold increase in ACTH levels 2 hr after SEB challenge. CsA significantly suppressed in vitro proliferation of lymphocytes from treated monkeys. Both CsA-treated monkeys and monkeys that had been challenged in a previous experiment with a lethal dose of SEB but had received no cyclosporin had pathologic changes in several organs. The most prominent changes were marked edema and leukocytic infiltration of the bronchial and bronchiolar mucosa. The CsA treatment appeared to reduce the intensity of lung inflammation, but this effect was not sufficient to protect the monkeys. The results suggest that CsA alone may not be an effective therapeutic agent for humans suffering from SEB intoxication or gram-positive septic shock.

Resistance of Staphylococcal enterotoxin B- induced proliferation and apoptosis to the effects of dexamethasone in mouse lymphocyte cultures.

Staphylococcal enterotoxin B (SEB) is a superantigen causing lymphocyte proliferation and apoptosis. Glucocorticoids are immunosuppressants and are released immediately following SEB intoxication in mice. Whether glucocorticoids affect lymphocyte proliferation and apoptosis in SEB-intoxicated mice is still unknown. To study this question, we examined the effects of dexamethasone (DEX), a synthetic glucocorticoid, on SEB-stimulated lymphocyte cultures from mouse thymus and peripheral lymphoid tissues (PLT). SEB, as well as concanavalin A (Con A), induced lymphocyte proliferation which peaked on day 4 and declined significantly on day 7. As expected, in Con A-stimulated cultures, DEX completely suppressed the proliferation of lymphocytes from both the thymus and PLT. However, in SEB-stimulated cultures, while DEX completely suppressed thymocyte proliferation, it did not suppress PLT cell proliferation even at a high concentration of 10(-7) M. The proliferating cells were Vbeta8(+) T cells of both the CD4(+) and CD8(+) subsets. DEX caused apoptosis. SEB also caused apoptosis, which was manifested by a maximal DNA subdiploidy on day 4 and by a maximal DNA fragmentation on day 7. Both events appeared not to be affected by DEX. The failure of DEX to affect the proliferation and apoptosis was consistent with high levels of cytokines (IL-1alpha, IL-2, IL-4, IL-6 and IFN-gamma) produced in the SEB-stimulated cultures, suggesting that the cytokines act in concert to circumvent the effects of DEX.

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.

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.

Localization of binding sites of staphylococcal enterotoxin B (SEB), a superantigen, for HLA-DR by inhibition with synthetic peptides of SEB.

Staphylococcal enterotoxins are major causes of food poisoning and toxic shock syndrome. Their ability to bind to major histocompatibility complex (MHC) class II molecules has been suggested to be the first step in the mechanism whereby they cause illness. By flow cytometric analysis, the sites of interaction of staphylococcal enterotoxin B (SEB) with HLA-DR molecules were probed in the present study by inhibiting the binding of biotinylated SEB to a human T-cell line (HUT-78) with synthetic peptides of SEB. Five peptides of SEB gave significant inhibition of binding: a peptide containing amino acids 9 to 20 [SEB(9-20)], SEB(30-38), SEB(61-70), SEB(90-114), and SEB(169-181). One peptide, SEB(39-51), enhanced binding. Among the inhibitory peptides, SEB(90-114), a peptide spanning the entire disulfide loop, showed the most efficient inhibition of binding. Peptides SEB(9-20) and SEB(39-51) include amino acid residues that have been identified by previous mutation studies (J.W. Kappler, A. Herman, J. Clements, and P. Marrack, J. Exp. Med. 175:387-396, 1992) as being important in binding to MHC class II. Amino acids lining the alpha 5 groove of SEB have also been postulated to be involved in binding to MHC class II molecules. However, only two of the residues that line the alpha 5 groove of SEB, His-12 and Tyr-17, are on peptide SEB(9-20) that inhibits binding. These results confirm previous studies that implicated the amino-terminal portion of the molecule in binding to MHC class II molecules and further indicate an important role for residues in other regions, particularly the disulfide loop.

Increased susceptibility to staphylococcal enterotoxin B intoxication in mice primed with actinomycin D.

Mice (BALB/cJ, C3H/HeN, and C3H/HeJ) primed with actinomycin D became highly susceptible to lethal intoxication with staphylococcal enterotoxin B (SEB). The mice underwent toxicosis and toxic shock and died. Actinomycin D-primed C3H/HeN and C3H/HeJ mice showed equal sensitivity to SEB, suggesting that bacterial lipopolysaccharide derived from gram-negative bacteria in the gut may not be an important cofactor in intoxication. In a time course study of the illness, prominent pathological changes characterized by blood congestion and thickening of alveolar septa were seen in the lung, while blood congestion, inflammation, epithelial cell flattening, and villous blunting were seen in the small intestine. In lymphoid tissues, such as the spleen, congestion, inflammation, and lymphoid cell depletion were the major reactions. The pathological features of the mice had many similarities to those of rhesus monkeys intoxicated with intravenous SEB. The actinomycin D-primed C3H/HeJ mice are thus an ideal mouse model for studying SEB toxicosis and toxic shock.

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.

Effects of staphylococcal enterotoxin B on rodent mast cells.

Staphylococcal enterotoxin B (SEB) was tested in rodent mast cell cultures for the release of serotonin. Both rat RBL-2H3 mast cells and murine peritoneal cells released serotonin after SEB stimulation in culture. Release of serotonin in RBL-2H3 cells depended on the concentration of SEB; an appreciable release was seen at 50 micrograms/ml. The release of serotonin was not due to cell death. Serotonin release could be enhanced by bradykinin but not by vasoactive intestinal peptide, substance P, lipopolysaccharide from Salmonella typhimurium, the calcium ionophore A23187, acetylcholine, adenosine, 5-hydroxyeicosatetraenoic acid, indomethacin, or phorbol myristate acetate. SEB bound directly to the membrane of RBL-2H3 mast cells, and the SEB-binding site, the presumptive receptor, appeared to be a protein. The SEB receptor could not be capped under membrane-capping conditions, and serotonin release could not be enhanced by attempts to cross-link the receptor. These results suggest that mast cells may be an important cell type involved in SEB toxicosis and that release of serotonin may be enhanced by activation of the kinin-kallikrein system.

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice.

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.

Strain-dependent expression of VH gene families.

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.

IgA-producing hybridomas are readily derived from gut-associated lymphoid tissue.

Eight hybridomas secreting IgA monoclonal antibodies were obtained by using gut-associated lymphoid tissue as a primed plasmablast source. IgA secretors are obtained at higher frequencies by this technique than by conventional splenic fusions. This technique provides useful tools for the study of mucosal protection, and it demonstrates that exaggerated potential of gut-derived B cells, compared to splenic B cells, for IgA expression.