Protein adhesins as vaccine antigens for Group A Streptococcus.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.

Structural differences between bovine A(1) and A(2) ß-casein alter micelle self-assembly and influence molecular chaperone activity.

Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) ß-casein (ß-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) ß-CN variants differ by a single AA, the substitution of histidine for proline at position 67. ß-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) ß-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) ß-CN variant formed smaller micelles than A(1) ß-CN, with the monomer-micelle equilibrium of A(2) ß-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 ß-CN variants associated with the adoption of greater polyproline-II helix in A(2) ß-CN and most likely led to enhanced chaperone activity of A(2) ß-CN compared with A(1) ß-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as ß-CN and, in this case, may have an effect on the functionality of milk.

Effect of vitamin D supplementation of low birth weight term Indian infants from birth on cytokine production at 6 months.

BACKGROUND/OBJECTIVES: Vitamin D deficiency has been associated with impaired resistance to infection, which may be mediated by alterations in cytokine responses. We investigated the effect of vitamin D supplementation to infants on whole blood in-vitro cytokine production and on the inflammatory marker, plasma C-reactive protein (CRP). SUBJECTS/METHODS: Blood samples were taken at 6 months of age from infants participating in the DIVIDS (Delhi Infant Vitamin D Supplementation) randomized controlled trial of weekly vitamin D supplements (1400 IU = recommended intake) from birth to 6 months with the aim of decreasing mortality and severe morbidity. We measured plasma CRP and whole blood in-vitro production of tumour necrosis factor-α (TNFα), interferon-γ (INFγ), interleukin (IL)-10 and IL-13 following no stimulation or stimulation with lipopolysaccharide or phytohemagglutinin. RESULTS: Although the intervention improved vitamin D status in a severely deficient population, there were no differences between treatment groups in plasma CRP or in the production of any of the cytokines in either unstimulated or stimulated cultures. Recent illness had limited association with immunological markers. Plasma 25-hydroxyvitamin D levels were not associated with CRP or production of any cytokines. CONCLUSIONS: Vitamin D supplementation did not affect plasma CRP or whole blood cytokine production of vitamin D-deficient low birth weight infants. This is consistent with the lack of effect of vitamin D on mortality and severe morbidity among infants in the DIVIDS trial.

Proteolysis of serum amyloid A and AA amyloid proteins by cysteine proteases: cathepsin B generates AA amyloid proteins and cathepsin L may prevent their formation.

BACKGROUND: AA amyloidosis develops in patients with chronic inflammatory diseases. The AA amyloid proteins are proteolytic fragments obtained from serum amyloid A (SAA). Previous studies have provided evidence that endosomes or lysosomes might be involved in the processing of SAA, and contribute to the pathology of AA amyloidosis. OBJECTIVE: To investigate the anatomical distribution of cathepsin (Cath) B and CathL in AA amyloidosis and their ability to process SAA and AA amyloid proteins. METHODS: and results: CathB and CathL were found immunohistochemically in every patient with AA amyloidosis and displayed a spatial relationship with amyloid in all the cases studied. Both degraded SAA and AA amyloid proteins in vitro. With the help of mass spectrometry 27 fragments were identified after incubation of SAA with CathB, nine of which resembled AA amyloid proteins, and seven fragments after incubation with CathL. CathL did not generate AA amyloid-like peptides. When native human AA amyloid proteins were used as a substrate 26 fragments were identified after incubation with CathB and 18 after incubation with CathL. CONCLUSION: The two most abundant and ubiquitously expressed lysosomal proteases can cleave SAA and AA amyloid proteins. CathB generates nine AA amyloid-like proteins by its carboxypeptidase activity, whereas CathL may prevent the formation of AA amyloid proteins by endoproteolytic activity within the N-terminal region of SAA. This is particularly interesting, because AA amyloidosis is a systemic disease affecting many organs and tissue types, almost all of which express CathB and CathL.

Neutrophil responses to CRP are not dependent on polymorphism of human FcgammaRIIA (R131H).

IgG2a mediated in vitro phagocytosis is less effective for individuals homozygous for Fcgamma RIIaR131 allele and such individuals are also more susceptible to certain infections. It has been reported that CRP binds to Fcgamma RIIaR131 but not Fcgamma RIIaH131 and since Fcgamma RIIa is also a major Fc receptor on neutrophils it would be expected that normal healthy donors who did not have at least one copy of Fcgamma RIIaR131 would not respond to CRP. We examined responses reported to be dependent on FcgammaRIIa but no difference between groups was observed in CRP mediated phagocytosis of S. pneumoniae, reactive oxygen production, or IL-8 synthesis. This suggests that either neutrophil receptors other than Fcgamma RIIa are responsible for CRP mediated responses or differences in CRP binding to the forms of Fcgamma RIIa are comparatively minor.

Proteolysis of AA amyloid fibril proteins by matrix metalloproteinases-1, -2, and -3.

We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment.

Transformation of Leishmania mexicana metacyclic promastigotes to amastigote-like forms mediated by binding of human C-reactive protein.

Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 degrees C or 32 degrees C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 degrees C/pH 7.2, intermediate levels at 26 degrees C/pH 5.5 and 32 degrees C/pH 7.2, and the greatest response at 32 degrees C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 degrees C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

Polymorphisms in the IkappaB-alpha promoter region and risk of diseases involving inflammation and fibrosis.

The transcription factor NFkappaB regulates inflammatory and other cellular responses. In non-stimulated cells, NFkappaB is linked to its inhibitor IkappaB, which plays a major role in controlling NFkappaB activity. Here, the gene promoter region of the major inducible IkappaB component (IkappaB-alpha) was studied to identify single nucleotide polymorphisms (SNPs), and to test if these are associated with risk of two diseases involving inflammation and fibrosis (trachoma and silicosis). Three SNPs were identified at positions -881, -826 and -297 relative to the transcription start site. The position -297 is close to two NFkappaB binding sites, kappaB2 and kappaB3, but the alleles were not associated with either disease. Alleles at positions -881 and -826 were in complete linkage disequilibrium with each other, and the rare haplotype was significantly less frequent among patients with trachoma compared to controls, although there was no difference in frequencies between silicosis patients and controls.

Acute-phase HDL in phospholipid transfer protein (PLTP)-mediated HDL conversion.

In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.

Acute phase serum amyloid A protein increases high density lipoprotein binding to human peripheral blood mononuclear cells and an endothelial cell line.

Serum Amyloid A (SAA) is an acute-phase protein secreted mainly by hepatocytes and is largely associated with high-density lipoprotein (HDL) in plasma. It has been suggested that SAA alters HDL binding to the cell surface and that this in turn changes HDL-mediated cholesterol delivery to cells. Incorporation of SAA into HDL at concentrations equivalent to those found physiologically in moderate inflammation mediated a 1.5-fold increase in the binding of HDL to adherent peripheral blood mononuclear cells but had no effect on binding of the lipoprotein to the monocyte cell lines, U937 or THP-1. SAA incorporation also increased binding to an endothelial cell line, EA.hy.926. Hepatoma cells (HuH-7) showed no change in specific binding of the SAA-enriched HDL particle compared to normal HDL. These results suggest that a specific receptor for HDL-bound SAA is found on differentiated human macrophages and an endothelial cell line, which may have functional significance in lipid metabolism or other macrophage responses during inflammation.

C-reactive protein binds to phosphorylated carbohydrates.

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.

Rouleaux-forming serum proteins are involved in the rosetting of Plasmodium falciparum-infected erythrocytes.

Excessive sequestration of Plasmodium falciparum-infected (pRBC) and uninfected erythrocytes (RBC) in the microvasculature, cytoadherence, and rosetting, have been suggested to be correlated with the development of cerebral malaria. P. falciparum erythrocyte membrane protein-1 (PfEMP1) is the parasite-derived adhesin which mediates rosetting. Herein we show that serum proteins are crucial for the rosette formation of four strains of parasites (FCR3S1, TM284, TM180, and R29), whereas the rosettes of a fifth strain (DD2) are serum independent. Some parasites, e.g., FCR3S1, can be depleted of all rosettes by washes in heparin and Na citrate and none of the rosettes remain when the parasite is grown in foetal calf serum or ALBUMAX. Rosettes of other parasites are less sensitive; e.g., 20% of TM180 and R29 and 70% of TM284 rosettes still prevail after cultivation. A serum fraction generated by ion-exchange chromatography and poly-ethylene-glycol precipitation restored 50% of FCR3S1 and approx 40 to 100% of TM180 rosettes. In FCR3S1, antibodies to fibrinogen reverted the effect of the serum fraction and stained fibrinogen bound to the pRBC surface in transmission electron microscopy. Normal, nonimmune IgM and/or IgG was also found attached to the pRBC of the four serum-dependent strains as seen by surface immunofluorescens. Our results suggest that serum proteins, known to participate in rouleaux formation of normal erythrocytes, produce stable rosettes in conjunction with the recently identified parasite-derived rosetting ligand PfEMP1.

A search within the IL-1 type I receptor reveals a peptide with hydropathic complementarity to the IL-1beta trigger loop which binds to IL-1 and inhibits in vitro responses.

In previous research, we were able to demonstrate that a seven amino acid residue peptide (VITFFSL), designed as an antisense peptide of the beta-bulge trigger loop region of interleukin 1beta (IL-1beta) (QGEESND; residues 48-54 [mature protein sequence]), was able to interact with IL-1 specifically and inhibit the response to IL-1 in an in vitro bioassay. The evidence was consistent with a specific interaction ocurring between antisense peptide and the trigger loop region. On the basis that antisense peptides are able to interact with their corresponding sense peptide sequences as a result of their mutually complementary hydropathic profiles (Fassina G., Verdoliva, A., Cassani, G., Melli, M., 1994. Binding of type I IL-1 receptor fragment 151-162 to IL-1. Growth Factors 10, 99-106; Maier, C.C., Moseley, H.N.B., Zhou, S., Whitaker, J.N., Blalock, J.E., 1994. Indentification of interactive determinants on idiotypic-anti-idiotypic antibodies through comparison of their hydropathic profiles. Immunomethods 5, 107-113), we devised a computer program (FINDH) to search the amino acid residue sequence of interleukin-1 type 1 receptor (IL-1 R1) for peptide motifs possessing hydropathic complementarity to the trigger loop sequence. The most complementary "best-fit peptide" motif (LITVLNI) was located in the third extracellular domain of IL-1 R1. A best-fit peptide corresponding to this motif was synthesised and found to bind to IL-1beta as well as inhibit the response to IL-1 in two independent in vitro bioassays (monitoring IL-1 dependent serum amyloid A synthesis and IL-1 dependent alkaline phosphatase activity, respectively). A second peptide motif (VIEFITL) was identified and the corresponding peptide synthesised along with a reordered version (LTILINV) of the best fit peptide. Both failed to bind measurably with IL-1beta or inhibit the response to IL-1 in the two bioassays. This best fit peptide behaved very similarly, in terms of IL-1 binding and inhibition behaviour, to the original trigger loop antisense peptide. Reference to the recently released X-ray crystal structure of IL-1beta and the IL1-R1 extracellular domain shows that the best fit peptide motif in IL-1 R1 is not apparantly interacting with the IL-1 trigger loop, although both are close in space. The intriguing possibility exists that the best fit peptide motif could represent an alternative site for IL-1beta receptor interaction which has not thus far been identified.

Vitamin A status does not influence neopterin production during illness or health in South African children.

Excessive interferon-gamma (IFN-gamma) production appears to be a primary immunological lesion in vitamin A-deficient experimental animals but comparable data from humans is lacking. We investigated IFN-gamma production in South African children by measurement of urinary excretion of neopterin, a product of IFN-gamma-activated monocytes or macrophages. Preschool children were examined during an acute inflammatory illness resulting from accidental ingestion of kerosene and they and a neighbourhood control child were examined 3 months later when well. Vitamin A status was assessed by the modified relative dose response (MRDR) test at 3 months and serum retinol and acute phase proteins were measured at both time points. Urinary neopterin was measured for forty cases in hospital, forty-six cases after recovery, and forty-one controls. Significantly increased neopterin excretion was seen following kerosene ingestion and in association with raised serum acute phase protein concentrations. There was no relationship between neopterin excretion at either time point and vitamin A status as assessed by MRDR test. Urinary neopterin was negatively correlated with serum retinol but no significant relationship was observed when acute phase protein concentrations were included in a multiple regression, suggesting the correlation was secondary to illness-induced changes in serum retinol. The results indicate that, contrary to what is observed in rodents under experimental conditions, poor vitamin A status is not associated with altered regulation of IFN-gamma production in children.

Sulphatide-binding properties are shared by serum amyloid P component and a polyreactive germ-line IgM autoantibody, the TH3 idiotype.

Serum amyloid P component (SAP) concentration was elevated in sera from leprosy patients, significantly so above endemic controls in lepromatous cases. In the sera of lepromatous leprosy (LL) patients who experienced an erythema nodosum leprosum (ENL) episode the SAP fell at the onset of ENL and remained low throughout, in two of three cases. Changes in SAP concentration parallel anti-sulphatide IgM concentrations. TH3, a monoclonal IgM germ-line antibody derived from a LL patient, and SAP share similar binding patterns. In this study we demonstrate binding to heparin and sulphatide. Moreover, SAP inhibited the binding of TH3 to sulphatide, as well as anti-sulphatide IgM found in a range of sera, and anti-sulphatide IgG in the only sera sample in which it was found. The observation that anti-TH3 idiotype monoclonal and polyclonal anti-SAP antibodies both inhibited the binding of TH3 and IgM in sera (but not IgG) to sulphatide without binding to sulphatide themselves further demonstrated similar binding specificities. The observations of similarity in binding reinforce ideas that SAP may function as a primitive opsonin, but the clear ability to inhibit binding of autoantibodies suggests that SAP may play a role in ameliorating tissue and particularly nerve damage in leprosy patients.