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Complement C5-deficient (C5(-/-)) macrophages derived from B.10 congenic mice were found to be defective in killing intracellular Mycobacterium tuberculosis (MTB). They were bacteriostatic after activation with IFN-gamma alone but bactericidal in the combined presence of IFN-gamma and C5-derived C5a anaphylatoxin that was deficient among these macrophages. Reduced killing correlated with a decreased production of reactive oxygen species (ROS) in the C5(-/-) macrophages measured using fluorescent probes. Furthermore, a lack of colocalization of p47(phox) protein of the NADPH oxidase (phox) complex with GFP-expressing MTB (gfpMTB) indicated a defective assembly of the phox complex on phagosomes. Reconstitution with C5a, a known ROS activator, enhanced the assembly of phox complex on the phagosomes as well as the production of ROS that inhibited the growth of MTB. Protein kinase C (PKC) isoforms are involved in the phosphorylation and translocation of p47(phox) onto bacterial phagosomes. Western blot analysis demonstrated a defective phosphorylation of PKC (alpha, beta, delta) and PKC-zeta in the cytosol of C5(-/-) macrophages compared with C5 intact (C5(+/+)) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC-beta and PKC-zeta were the isoforms that are not phosphorylated in C5(-/-) macrophages. Because Fc receptor-mediated phox assembly was normal in both C5(-/-) and C5(+/+) macrophages, the defect in phox assembly around MTB phagosomes was specific to C5 deficiency. Reduced bactericidal function of C5(-/-) macrophages thus appears to be due to a defective assembly and production of ROS that prevents effective killing of intracellular MTB.

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.

Monoclonal Abs against the repeat region of the circumsporozoite protein (CSP) completely protect mice against Plasmodium yoelii (Py), but synthetic peptide and recombinant protein vaccines designed to produce only Abs to the PyCSP repeat region have never been reported to consistently provide protection. This lack of protection in the rodent model system has predicted the poor protection achieved in humans after immunization with synthetic peptide and recombinant protein P. falciparum CSP vaccines and has raised serious questions regarding the capacity for vaccine-induced polyclonal Abs against the CSP to consistently protect humans. We now report immunization studies with a multiple Ag peptide vaccine designed to rely on "universal" T epitopes from tetanus toxin to produce T cell help for induction of protective Abs against the repeat region of the PyCSP. When delivered with a nonionic block co-polymer adjuvant, the vaccine protected 78 to 100% of three inbred strains of mice, and 100% of outbred mice against P. yoelii sporozoite challenge. Protection was associated with Ab titer, and passive transfer of purified IgG from immune mice protected naive recipients. Similar protection was achieved when the peptide was encapsulated in liposomes with lipid A and mixed with aluminum hydroxide. By demonstrating for the first time solid protection against P. yoelii by polyclonal Abs against the CSP, these data provide the rationale for assessment of a similarly constructed and formulated P. falciparum CSP multiple Ag peptide vaccine in humans.

We previously reported that protection of mice from nonlethal Plasmodium yoelii malaria by immunization with whole killed blood-stage parasites was dependent on the adjuvant and that adjuvants influenced both the specificity and isotype of Ab. Additional studies with the most effective formulations were undertaken to better define the protective responses and 100% protection from lethal P. yoelii malaria was produced by three immunizations with Ag in copolymer P1004 and detoxified RaLPS as adjuvants and 83% protection was induced by a single immunization. The protection lasted for 9 mo and was associated with an anamnestic rise in Ab titer of the IgG2a isotype during the challenge infection. Passive immunization with Ab from animals that had been immunized and challenged transferred sterile immunity. Splenectomy reduced, but did not abolish, protection. These data suggest that the effective Ab is directed against labile epitopes on the surface of blood-stage parasites. The vaccines primed animals for production of such Ab, but its synthesis was efficiently induced only by challenge with live organisms.

Mice were immunized with whole killed blood stage Plasmodium yoelii parasites in 15 adjuvant formulations then boosted and challenged with parasitized blood. Five of six groups immunized with the Ag in oil-in-water emulsions or formulations without oil were protected. Formulations that induced protection contained saponin, pertussis, copolymer P1004, and detoxified RaLPS. In contrast, none of nine groups of animals immunized with Ag in water-in-oil emulsions were protected. Ineffective adjuvants included CFA and water-in-squalene emulsions with copolymer L141 plus detoxified RaLPS, dimethyldioctadecyl ammonium bromide, and mycobacterial cell wall skeletons. Antibody was measured by ELISA against disrupted parasites and by indirect fluorescent antibody (immunofluorescence) using intact parasites. Protection was associated with antibody of the IgG2a isotype detected by immunofluorescence but not with other isotypes detected by immunofluorescence or any type antibody detected by ELISA. The water-in-oil adjuvants induced high titers by ELISA but low titers by immunofluorescence. These results, together with Western blot analyses, suggested that adjuvant vehicles control the specificity of antibody and that this, in turn, is essential for induction of protective immune responses in this model.

The tandem repeat peptide of the circumsporozoite protein of Plasmodium cynomolgi, (NAGG)5, conjugated to BSA or Salmonella flagella, was injected into mice with block copolymer and other adjuvants. The flagella carrier preferentially stimulated IgG2a antibodies to (NAGG)5 that constituted as much as 85% of the total IgG antibody whereas the BSA carrier stimulated as much as 98% IgG1. The distribution of isotypes of antibody to (NAGG)5 was also modified by using the copolymer adjuvants L121 or L141, either alone, or especially in combination with a nontoxic LPS. L121 or L141 increased the proportion of IgG2a and IgG2b antibodies to (NAGG)5 after immunization with (NAGG)5-BSA whereas LPS stimulated further increases in IgG2 antibodies (up to 69% of the total IgG). The hapten density, physical form of emulsion, and route of immunization further affected the isotypes produced in this study.

Certain synthetic, nonionic, block copolymers of polyoxypropylene and polyoxyethylene are potent immunoadjuvants. While investigating their mechanisms of action, we found that one of these copolymers, L81, induced the expression of macrophage Ia in vivo. L81 is a 2750-Da, linear copolymer with a 43-unit core of hydrophobic polyoxypropylene flanked on each side by 3 U of hydrophilic polyoxyethylene. It induced threefold to sevenfold increases in the proportion of peritoneal macrophages expressing I-A from 5 to 7 days after an i.p. injection of 5 mg. As little as 1 mg caused a twofold increase. I-A density increased with time after injection of L81. Macrophages induced by L81 actively synthesized I-A, showing an 18-fold increase in biosynthetic capacity. Ia induction did not require the presence of mature T lymphocytes, because similar increases in I-A expression were seen in athymic and euthymic mice. L81-induced macrophages were 10-fold more effective than normal macrophages in presenting Ag to a T cell hybridoma. Other functional studies showed that they were primed for the secretion of superoxide ion and could be stimulated in vitro by IFN-gamma and LPS to lyse tumor target cells. These results suggest that the induction of macrophage Ia expression by L81 may play a role in its activity as an immunoadjuvant.

Hen egg albumin (HEA), heavily conjugated with dodecanoic acid (D-HEA), stimulated sustained delayed type hypersensitivity (DTH) specific for HEA without detectable antibody formation in guinea pigs. An oil-in-water emulsion containing purified BCG cell walls attached to the oil drops was found to be a very effective adjuvant for enhancing DTH to D-HEA, but not to HEA. Animals immunized with D-HEA in the BCG cell wall emulsion produced skin test reactions 2.4 cm in diameter when challenged with HEA 21 days after a single immunization. Control animals immunized with D-HEA in saline produced skin reactions 1 cm in diameter to similar challenge. Neither group of animals produced detectable antibody to HEA-OR D-HEAmThe emulsion had no adjuvant effect if the BCG cell walls were suspended in the aqueous phase and not attached to the oil droplets. Dodecanoic acid conjugated Salmonella typhi organisms could be used in place of the BCG cell walls to produce effective adjuvant preparations. Fruend's complete adjuvant and other water-in-oil emulsions, however, were found to be ineffective adjuvants for enhancing the degree of DTH produced by D-HEA. Experiments with autoradiography demonstrated that effective adjuvant preparations promote the localization and retention of both 125-I-labeled D-HEA and 125-I-labeled BCG cell walls in the paracortical area of lymph nodes where they are in close proximity to many T-type lymphocytes which proliferate in the induction of DTH.

Bovine serum albumin (BSA) conjugated with a lipid, dodecanoic acid, is capable of inducing strong delayed-type hypersensitivity (DTH) in guinea pigs. This paper reports experiments on the nature and specificity of this hypersensitivity. The response to lipid-conjugated BSA (L-BSA) was found to be classical DTH, as evidenced by its ability to be transferred passively by immune cells, but not by serum. In addition, special histologic examination of skin test sites demonstrated the characteristics of DTH rather than cutaneous basophil hypersensitivity. Similar results were obtained when lipid-conjugated purified protein derivative of tubercle bacilli (L-PPD) was used. The increased immunogenicity of L-BSA was not caused by the presence of protein aggregates, but seemed to be related to the hydrophobic nature of the conjugated side chains. A series of cross-reacting serum albumins was used for a study of the specificity of the antibody and DTH responses to BSA. It was found that the degree of enhancement of immunogenicity for DTH caused by lipid conjugation varied for different antigenic determinants on BSA.

Protein antigens covalently conjugated with lipid groups (dodecanoic acid) have previously been shown to stimulate strong delayed-type hypersensitivity (DTH) without the aid of adjuvants. The present experiments show that lipid-conjugated bovine serum albumin (L-BSA) is taken up in vitro by macrophages (Mpsi) 25- to 50-fold more than unconjugated BSA or aminidated BSA, neither of which induces DTH. Macrophages that take up 125I-labeled L-BSA in vitro stimulate DTH even more efficiently, when injected into syngeneic guinea pigs, than does soluble L-BSA. Tracer studies on the fate of radiolabeled BSA and L-BSA showed that much more L-BSA than BSA was retained by draining lymph nodes. Autoradiography demonstrated that 125I-L-BSA is rapidly taken up by Mpsi in the medullary sinuses of the lymph nodes. Some of this antigen is then transported into the paracortex, a region in which T lymphocytes predominate. The capacity of lipophilic antigens to stimulate cell-mediated immune responses may be caused by their increased uptake by Mpsi, resulting in more efficient presentation to immunocompetent T lymphocytes. The anatomical site of this Mpsi-T cell interaction may be within the sinusoids or paracortex of the draining lymph nodes.

A series of block co-polymers composed of polyoxyethylene and polyoxypropylene were investigated for their ability to induce in vitro activation of mouse mast cells. We found that six of these co-polymers could cause histamine release from mouse mast cells in vitro. At low concentrations, the most efficacious co-polymer, T130R2, caused rapid and extensive concentration-dependent release of histamine from mouse mast cells. The release process was not cytotoxic; it required metabolic energy and was not accompanied by release of lactate dehydrogenase. Optimal release of histamine was dependent on both calcium and sodium ions in the extracellular medium. The degree of in vitro histamine release correlated with in vivo inflammation and in vitro ionophore activity. We believe that this represents the first report of the activation of mediator-containing cells by an ionophore selective for monovalent cations. These copolymers may therefore represent new reagents for investigations of cellular excitation.

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.

We tested the ability of 17 surface-active agents to enhance antibody formation and inflammation. The surfactants were all block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP), which differed in m.w. and mode of linkage of POP to POE. Mice were injected in each rear footpad with 1.25 mg of each surfactant with 25 micrograms of bovine serum albumin in an oil-in-water emulsion. Each agent produced a distinct pattern of immune response and inflammation. Preparations that are large and insoluble with the POE chains flanking the POP chains were very effective adjuvants for increasing antibody formation. They also activated complement and induced the release of chemotactic factors from serum. Increasing the percent of POE decreased adjuvant activity and inflammation. Decreasing the m.w. of the molecules while maintaining the proportions of POP and POE decreased the adjuvant activity and increased inflammation. Preparations synthesized in the reverse order, with the POP flanking POE, tended to induce granulomas instead of antibody. These data demonstrate that block copolymer surfactants have a spectrum of biologic activities that depend on the size and arrangement of their constituent parts. We suggest that much of the activity of these agents derives from their ability to form adsorptive surfaces similar to those of a mycobacterial glycolipid, quartz, and monosodium urate.

The magnitude of the skin test response in a group of normal volunteers to intradermal Candida antigen correlated closely with the level of cytolytic activity stimulated by Candida antigen in vitro in the peripheral blood lymphocytes from those same individuals. The cytolytic activity stimulated by Candida antigen was cell mediated, and cold target inhibition studies demonstrated that Candida antigen-stimulated effector cells were capable of cross-species (i.e., nonspecific) killing. Analysis of the Candida antigen-induced effector cell population for various cell markers did not enable absolute identification of the cell responsible for the killing. The findings in this study indicate that the stimulation of cell-mediated cytolysis by Candida antigen may be related to the delayed-type hypersensitivity response produced by the same antigen.