Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides.

Antibodies reactive with capsular polysaccharides are considered the principal mediators of immunity against invasive diseases caused by Streptococcus pneumoniae. In this study, we tested the hypothesis that anti-pneumococcal capsular polysaccharide (PPS) antibody avidity can influence protective efficacy. We measured the avidities of individual adult postvaccination immunoglobulin G2 (IgG2) antibodies to PPS serotypes 6B and 23F and examined the relationship between avidity and opsonophagocytic and mouse-protective activities. The avidities of PPS 6B- and PPS 23F-specific IgG2 antibodies ranged from 6 to 31 nM-1 and from 3 to 20 nM-1, respectively. We observed an inverse correlation between the magnitude of avidity and the amount of antibody required to protect mice against lethal bacteremia caused by serotype 6B pneumococci. Similarly, higher-avidity antibodies were more effective than lower-avidity antibodies in vitro in mediating complement-dependent opsonophagocytosis of both 6B and 23F pneumococci. These data suggest that in adults, PPS antibodies are sufficiently polymorphic to possess biologically significant variations in avidity. We conclude that avidity functions as an important determinant of anticapsular antibody protective efficacy against pneumococci.

Bactericidal monoclonal antibodies that define unique meningococcal B polysaccharide epitopes that do not cross-react with human polysialic acid.

The poor immunogenicity of the Neisseria meningitidis group B polysaccharide capsule, a homopolymer of alpha(2-->8) sialic acid, has been attributed to immunologic tolerance induced by prenatal exposure to host polysialyated glycoproteins. Substitution of N-propionyl (N-Pr) for N-acetyl groups on the meningococcal B polysaccharide, and conjugation of the resulting polysaccharide to a protein carrier, have been reported to yield a conjugate vaccine that elicits protective Abs with minimal autoantibody activity. To characterize the protective epitopes on the derivatized polysaccharide, we isolated 30 anti-N-Pr meningococcal B polysaccharide mAbs. These Abs were heterogeneous with respect to complement-mediated bactericidal activity, fine antigenic specificity, and autoantibody activity as defined by binding to the neuroblastoma cell line, CHP-134, which expresses long-chain a(2-->8)-linked polysialic acid. Eighteen of the Abs could activate complement-mediated bacteriolysis. Seven of these 18 Abs cross-reacted with N-acetyl meningococcal B polysaccharide by ELISA and had strong autoantibody activity. Thus, N-Pr meningococcal B polysaccharide conjugate vaccine has the potential to elicit autoantibodies. However, 7 of the 18 bactericidal mAbs had no detectable autoantibody activity. These Abs may be useful for the identification of molecular mimetics capable of eliciting protective Abs specific to the bacteria, without the risk of evoking autoimmune disease.

A comparison of antibody responses to veterinary vaccine antigens potentiated by different adjuvants.

Six adjuvant formulations were compared for their ability to potentiate the primary and memory antibody responses in mice to three companion animal vaccine immunogens--feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and a recombinantly-derived heartworm antigen. The combination of a novel bacterial immunostimulator, gliding bacterial adjuvant (GBA), either adsorbed onto an aluminum hydroxide gel (Rehydragel HPA), or emulsified with a vehicle of polyalcohol and detergent, elicited the strongest memory responses to both virus preparations. Both forms of aluminum hydroxide gels administered without GBA gave similar levels of adjuvant effects, on par with or greater than those generated by incomplete Freund's adjuvant (IFA). The Acemannan immunostimulant was not effective in increasing the responses to the virus antigens, but increased the primary response to the heart-worm antigen over tenfold from control levels. All preparations appeared to be well tolerated, with no detectable adverse reactions observed in any of the 250 mice used. The proven safety of aluminum hydroxide adjuvants and the apparent absence of adverse reactions seen with GBA make this vehicle/adjuvant formulation worthy of additional study.

Gliding bacterial adjuvant stimulates feline cytokines in vitro and antigen-specific IgG in vivo.

Gliding bacterial adjuvant (GBA) has been previously characterized as a potent immune modulator, stimulating the growth of murine B lymphocytes, inducing murine NK cell activity, and promoting the release of several murine cytokines. Based on these studies and our interest in potentiating the effectiveness of feline vaccines, GBA was tested for its ability to stimulate feline T cells in vitro and act as a vaccine adjuvant in vivo. In vitro, GBA stimulated feline PBLs to proliferate and release interferon (IFN) and IL-2. Unlike IFN, the release of IL-2 appeared to be unaffected by prior depletion of macrophages, indicating GBA directly stimulated feline T cells. In vivo GBA was co-administered with Keyhole Limpet Hemacyanin (KLH) and the anti-KLH antibody response was compared to cats receiving KLH emulsified in complete Freund's adjuvant (CFA). Fourteen days after the third immunization and continuing for a 30-day observation period, KLH-specific IgG titers in cats receiving GBA were significantly higher than those given CFA. However, when cats were subsequently boosted with KLH alone, those cats receiving CFA demonstrated significantly higher antibody titers throughout a second 30-day observation period. The anti-KLH antibody memory response was greatly enhanced when GBA was emulsified with incomplete Freunds adjuvant (IFA) prior to injection. Serum titers of cats given KLH in an oil-based GBA preparation were significantly higher than cats receiving KLH adjuvanted with IFA or CFA, an effect which persisted 38 days after boosting with KLH alone. Finally, GBA significantly enhanced the feline humoral response to a recombinant protein of Dirofilaria immitis, the causative agent of feline heartworm. Serum titers of cats inoculated with recombinant antigen in GBA were significantly greater than cats given recombinant antigen adjuvanted with Titermax, alum, or NAGO. These studies indicate that GBA induces T cell proliferation and the release of IL-2 and IFN in vitro and can be used to enhance the recall antibody response to both a T cell dependent antigen and an immunogen derived from Dirofilaria immitis.

Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells.

Monoclonal antibodies were raised against an 80-kDa phosphoprotein (80K) that is phosphorylated upon stimulation of gastric acid secretion and that copurifies with the acid-forming H+-K+-ATPase isolated from stimulated tissue. These antibodies were used to demonstrate that in the gastric mucosa 80K is limited to parietal cells and not found in surface, mucous neck, or chief cells. 80K was also found in other transporting epithelia, including intestine and kidney, but was not found in brain, liver, red blood cells, or colon. Immunohistological localization of 80K in resting glands revealed a fine network, projecting from the gland lumen and anastomosing throughout the parietal cell. This network is quite similar to the staining pattern for F-actin contained in microvilli that line the apical membrane of parietal cells. Stimulation of acid secretion rearranges 80K to a more rugose pattern filling the entire cell. In stimulated cells the distribution pattern of 80K is indistinguishable from that stained with antibodies against the H+-K+-ATPase. These data strongly suggest that 80K is an apical membrane protein of the parietal cell.

Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide, and other bacterial products.

The growth of WEHI-231, a murine immature B lymphoma cell line, was inhibited by anti-IgM antibodies. The inhibition of proliferation, as measured by [3H]thymidine incorporation, occurred between 16 and 28 hr after addition of anti-IgM. Moreover, the growth arrest was irreversible: cells that were cultured with anti-IgM for 18 hr and then recultured without it failed to recover the ability to proliferate, even though cells treated for up to 30 hr with anti-IgM remained viable, as measured by trypan blue exclusion. Three polyclonal B cell activators obtained from bacteria, lipopolysaccharide (LPS), peptidoglycan from Staphylococcus aureus, and gliding bacterial adjuvant from Cytophaga (GBA), were able to protect WEHI-231 cells from anti-IgM-induced growth arrest. The protection was transient, ending after approximately 56 hr. This transience was shown to be due to desensitization of the cells to the bacterial products. Interestingly, pretreatment of WEHI-231 cells with any of the bacterial products desensitized the cells to all of the bacterial products. The heterologous nature of this desensitization suggests that all three of these bacterial products may act through a common signaling pathway despite their diverse chemical natures.

Bovine mixed leukocyte response: effect of selected parameters on cell responses.

The bovine mixed leukocyte culture (MLC) system offers potential benefit for the study of genetic and immunologic mechanisms in this species. Selected parameters of the bovine MLC have been investigated to assess their influence. The findings indicate that maximal MLC response was present at days 5 and 6 with 2 x 10(5) responder to 2 x 10(5) stimulator cells per well or greater. Serum concentrations over a wide range effectively supported cell growth. More importantly, the source and treatment of serum influenced the level of MLC response. Serum autologous to the responder which was heat treated appeared to provide maximal MLC values compared to other sources examined. Antisera to serologically-defined lymphocyte antigens of the stimulating cell population did not affect the response. One-way MLC determinations were consistent when the stimulating cell population was irradiated at 1,000 Rads or greater. Both freshly obtained lymphocytes and lymphocytes held at room temperature for 18 hr provided good sources of MLC responder and stimulator cells; however, lymphocytes held for 18 hr consistently gave higher counts and stimulation indices than freshly obtained lymphocytes. The effect of contaminating RBCs was found to enhance MLC reactivity at lower concentrations and inhibit MLC reactivity when in excess of 7 X 10(9) RBC per well. Stimulator cells were present in both macrophage enriched and depleted populations suggesting several cell populations possess antigens which lymphocytes recognize. Consideration of these parameters were essential in providing optimal, reproducible results.

Bovine con A-induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action.

Bovine Concanavalin A-induced suppressor cells were generated from lymphocytes which were non-adherent to anti-immunoglobulin coated dishes and cells possessing receptors for peanut agglutinin. Bovine lymphocytes, preincubated with 25 microgram/ml of Con A for 40-45 hr, could suppress the responses of autologous cells to the mitogens Con A, PHA and PWM as much as 90% when they were cultured together at a ratio of 1:1 (suppressor cell to responder cell) or higher. Suppressor cells were not necessary at the initiation of the mitogenic assay as they could regulate responding cells if added at 48 hr in a 72 hr assay. Allogeneic responder cells could be suppressed at the same level as autologous cells indicating a lack of genetic restriction. Macrophages were not required for suppressor cell generation because peripheral blood lymphocytes (PBL) depleted of macrophages by Sepharose G-10 columns, and subsequently incubated with Con A, could suppress autologous cells to a similar degree as unseparated PBl's. Responder cells depleted of macrophages had normal mitogen responsiveness and were suppressed indicating macrophages were not required in transmission of suppressor signals. Cell to cell contact was not required for suppression connoting a soluble factor(s) as the modulator of suppression.

The bovine major histocompatibility complex (BoLa): close linkage of the genes controlling serologically defined antigens and mixed lymphocyte reactivity.

Detection of linkage between genetic loci in cattle has been hampered by the lack of large full -sib families. A unique source of full-sib families is now available from embryo transplantation. Lymphocytes from six full-sib families, ranging in size from three to seven siblings, were tested for serologically defined BoLA antigens (BoLA-A). In addition, mixed lymphocyte reactivity (MLR) was tested between all paired combinations of cells within each family to distinguish BoLA-D specificities. Serologically identical siblings within each family were reciprocally nonreactive in MLR, and vice versa; thus, no recombinants were detected between the BoLA-A and the BoLA-D loci. Classical genetic linkage analysis revealed that these loci are significantly closer than 11.9 centimorgans.

Two molecularly independent surface receptors identify bovine T lymphocytes.

E rosette formation is a commonly used technique to identify T lymphocytes in many species; however, treatment of bovine E rosettes with rhodamine-conjugated F(ab')2 anti-immunoglobulin resulted in 10% (range 3-18%) of these cells exhibiting fluorescence. The failure of E rosettes to identify only non-immunoglobulin bearing cells suggested that an alternative method for T lymphocyte identification was essential. Using a labeled heterologous T cell antiserum and peanut agglutinin (PNA), identical populations of bovine T lymphocytes were identified. Unique and molecularly independent cell surface receptors were detected by dual fluorescent staining experiments differential inhibition of cellular binding of PNA by its carbohydrate ligand and capping experiments. Moreover, both reagents bound to approximately 62% of peripheral blood lymphocytes (PBLs) and virtually all thymocytes. Use of either T cell reagent in combination with rhodamine-conjugated F(ab')2 anti-bovine immunoglobulin provided a rapid, simple and reliable method for simultaneous enumeration of bovine T and B cells and permitted the detection of rare cells (less than 1%) expressing both T and B cell markers. Approximately 90% of all PBLs were identified as either T or B cells.

A simple method for specific depletion of B lymphocytes from bovine peripheral blood mononuclear cells.

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg+ lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cells preparations. These results suggest that bovine SIg- cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg- cells.

Transitivity of response in the mixed lymphocyte culture test.

We present a model of mixed lymphocyte culture (MLC) response which assumes one specificity per locus. It also assumes that an animal A will fail to stimulate animal B if, and only if, the set of specificities possessed by A is a subset of the set of specificities in B. The last assumption implies that non-stimulation is transitive; that is, if A does not stimulate B, and B does not stimulate C, then A will not stimulate C. The inclusion of antigenic sets can be used to partially order the animals in a hierarchy. Partial ordering can detect multiple lymphocyte-defined (LD) loci with relative ease; it indicates the number of antigens present in particular individuals; and it detects exceptions to the rule of transitivity which may expose immune response genes, minor loci, or other mechanisms that affect MLC response. This analytical procedure is most useful when testing half-sib families or hybrids sharing a common parental strain. We have applied this procedure to the MLC in cattle half-sib families and found that the data strongly support the existence of at least four LD loci.

Lymphocyte-defined loci in cattle.

Using the results of all paired one-way mixed lymphocyte culture tests on families of half-sibs, we have established that the lymphocyte-defined system in cattle contains a minimum of two loci. The methodology presented is applicable to studies of the lymphocyte-defined systems of other species.