Stability of a receptor-binding active human immunodeficiency virus type 1 recombinant gp140 trimer conferred by intermonomer disulfide bonding of the V3 loop: differential effects of protein disulfide isomerase on CD4 and coreceptor binding.

Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.

HIV/SIV glycoproteins: structure-function relationships.

The various functions of human (HIV) and simian (SIV) immunodeficiency virus glycoproteins are similar, so it may be assumed that the overall structure of the folded proteins will be maintained. To preserve structure there must be constraints on sequence variation. The majority of mutations tolerated will be involved in immune escape but changes at some positions are known to have direct effects on glycoprotein expression and function. This allows the virus to change its phenotype and escape immune pressure. These properties will influence the fitness of the virus to infect and replicate in potential hosts. A better understanding of the structure-function relationships of HIV/SIV glycoproteins will assist in the development of vaccines and antivirals. Here, we identify similarities and differences between HIV-1 subtypes and HIV/SIV types that may be relevant to the phenotypes of the various groups. The results are discussed in relation to what is known of domain-function associations for HIV/SIV glycoproteins.

Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli. IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S. mansoni. Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites. One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals. These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S. mansoni. A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.

Induction of immune responses to functional determinants of a cell surface streptococcal antigen.

Antibodies directed against the cell surface adhesin, termed streptococcal antigen I/II of Streptococcus mutans can protect against dental caries. Streptococcal antigen I/II (SA I/II) interacts with salivary glycoproteins and promotes adhesion to the tooth surface. Topical application of monoclonal antibodies which recognize a domain within residues 816-1213 (fragment 3) prevents colonization by S. mutans in primates. In this study the immunogenicity and antigenicity of fragment 3 was investigated in five strains of mice. Fragment 3 induced an immune response following immunization with whole cells of S. mutans in all strains of mice. Immunization with recombinant fragment 3 also induced T-cell proliferative and antibody responses both to fragment 3 and to the SA I/II. Antibody responses to the previously defined adhesion determinants (residues 1005-1044) were weak or undetectable. Immunization of three representative strains of mice with a recombinant polypeptide (residues 975-1044) comprising this adhesion epitope and an adjacent T-cell epitope (residues 975-1004) elicited both T- and B-cell responses to the polypeptide and to native SA I/II. The B-cell epitopes overlapped with the adhesion determinant. These findings provide a means of directing immune responses to functional determinants of SA I/II.

T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II.

The T-cell and antibody responses to a cell surface streptococcal antigen (SA I/II) were investigated in naturally sensitized humans. Serum antibody responses were directed predominantly to the N-terminal (residues 39 to 481) and central (residues 816 to 1213) regions of SA I/II which may be involved in bacterial adhesion to salivary receptors. T-cell responses were also directed predominantly towards the central region. The linear peptide relationship of the immunodominant and minor T- and B-cell as well as adhesion epitopes was mapped within residues 816 to 1213. Immunodominant T-cell and B-cell epitopes were identified within residues 803 to 853, which were separated in linear sequence from the adhesion epitopes (residues 1005 to 1044). Adhesion epitopes overlapped with minor B- and T-cell epitopes (residues 1005 to 1054 and 1085 to 1134). An immunodominant promiscuous T-cell epitope (residues 985 to 1004) was adjacent to an adhesion epitope (residues 1005 to 1024). The limited B-cell response to adhesion epitopes is consistent with the success of Streptococcus mutans in colonizing the oral cavity. The strategy of T-cell, adhesion, and B-cell epitope mapping has revealed a general approach for identifying components of subunit vaccines which may focus responses to critical functional determinants. Such epitopes of SA I/II may constitute the components of a subunit vaccine against dental caries.

A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species.

The larvicidal activity of cyclosporin A against Schistosoma mansoni in mice.

Treatment of BALB/c or MF1 mice with cyclosporin A (CsA) around the time of infection with Schistosoma mansoni conferred almost complete protection. The migration kinetics of L-[75Se]selenomethionine-labelled infective cercariae were investigated by compressed tissue autoradiography. Similar levels of skin penetration were achieved by cercariae in control and drug-treated individuals. CsA arrested 87-94% of the worms in the skin and ultimately all of these died in this site. Few worms (7-14%) migrated from the skin to the lungs and none completed migration to the liver. Nevertheless, the autoradiograms revealed a limited degree of lateral cutaneous migration by the worms present in the skins of CsA-treated mice. Results of perfusion recovery experiments carried out during the course of infection reinforced the tracking data.

Schistosoma mansoni: morphology and ultrastructure of adult worms recovered from cyclosporin A-treated mice.

Cyclosporin A administered to Schistosoma mansoni-infected mice at around day 20 of infection reduces the worm burden by greater than 60%, as assessed by portal perfusion on days 28 and 86. Those worms recovered at perfusion have been examined by light and electron microscopy for drug-induced changes in morphology. Gross parasite damage was characterized by massive bolus formation and subsequent herniation of the gut. This event was attributed to the abnormal accumulation of crystalline structures in the lumen; the crystals were closely associated with lipid droplets, and were shown by X-ray micro-analysis to contain iron. Such crystals were seen only rarely in the intestines of control worms, but they too gave small iron peaks when examined by X-ray micro-analysis. In some drug-treated worms the caecal epithelium had ruptured, thereby releasing luminal contents throughout the worm body. In addition, herniations of the gut were seen protruding through the tegument causing surface deformation and disruption of tegumental and parenchymal tissues. The structural integrity of the worm was ultimately compromised allowing access to cytotoxic effector cells of the host. The combined effects of drug action and cellular cytotoxicity presumably account for the very significant levels of worm killing achieved by CsA treatment of the host.

Toxicity of cyclosporin A (CsA) against developmental stages of Schistosoma mansoni in mice.

It has been shown elsewhere that the immunosuppressive drug cyclosporin A (CsA) exerts profound schistosomicidal activity when given to infected mice via a multiple administration regime. We show here that a single treatment regime also effects significant reductions in worm burden. Moreover, drug efficacy is maintained at CsA concentrations shown to be subimmunosuppressive in other systems. Subcutaneous treatment effected higher levels of schistosomicidal activity than intraperitoneal or oral treatment, irrespective of whether the drug was given prior to or during infection. Topical application of CsA to the skin site of cercarial penetration, prior to infection resulted in no reduction in worm burden. Systemic release of CsA from a slowly adsorbed, oil-based drug-vehicle combination effected greater levels of killing than when CsA was dissolved in an aqueous drug vehicle. All developmental stages of Schistosoma mansoni were susceptible to killing by a single dose of CsA but, in the case of liver-stage worms, an increased concentration of drug and a longer period between treatment and portal perfusion were needed for killing to be measurable as a reduction in worm numbers.

Effect of cyclosporin A treatment on the enteropathy of graft-versus-host reaction in the rat: a quantitative study of intestinal morphology, epithelial cell kinetics and mucosal immune activity.

Mucosal graft-versus-host reaction (GvHR) of the small intestine exemplifies an immunologically mediated enteropathy that is associated with expansion of mucosal mast cells (MMC). Quantitative measures of intestinal morphology, epithelial cell kinetics and mucosal immune activity were used to assess the effect of the immunosuppressive agent, cyclosporin A (CyA), in ameliorating this enteropathy and on increased activity of MMC in the jejunum. GvHR was induced in two groups of PVGU x PVGC rats by irradiation (4.50 Gy) and intravenous injection of PVGC spleen cells (150 x 10(6)). One group remained untreated, while a second group of eight rats was treated with a 50 mg/kg dose of CyA subcutaneously given daily for the first 3 days and then every second day, and which had commenced the day before induction of GvHR. On day 14, all animals were killed. Treatment with CyA prevented intestinal crypt hyperplasia but did not affect villus length, and normalized the crypt cell production rate (CCPR) from 38 to 15 cells/crypt/h (P less than 0.0001). CyA reduced the number of MMC and jejunal content of the MMC associated protease, rat mucosal mast cell protease II (RMCPII). Mean serum RMCPII concentration was reduced from 302 (s.d. = 112) in GvHR animals to 10 (s.d. = 6) ng/mL in GvHR/CyA-treated rats (P less than 0.0001). We conclude that CyA ameliorates the enteropathy of GvHR and depresses the activation of MMC, as evident by the strongly depressed serum RMCPII concentration.

Separate effects of irradiation and of graft-versus-host reaction on rat mucosal mast cells.

T cell mediated immune responses in the gut can produce enteropathy and malabsorption. We have investigated the relevance of mucosal mast cells (MMC) to the mechanisms of this enteropathy by using graft-versus-host reaction (GvHR) in the rat as a model of mucosal delayed type hypersensitivity. Measurements of mucosal architecture, intraepithelial lymphocytes (IEL) and MMC counts were performed in control and experimental rats, and release of rat mast cell protease II (RMCPII) into the bloodstream was used as an index of MMC activation. In unirradiated rats, jejunal MMC count was increased on day 14 of the GvHR (mean 272/mm2 v 182 in controls, p less than 0.01), as was serum RMCPII (p less than 0.01). Irradiated rats (4.5 Gy, reconstituted with isogeneic spleen cells) had low counts of IEL and crypt hyperplasia seven to 14 days after irradiation. Irradiated rats with GvHR (induced by ip injection of parental strain spleen cells) and studied on days 7, 10 and 14, had significant enteropathy with longer crypts and higher CCPR than matched irradiated animals (p less than 0.05 on day 14 when compared with irradiation alone). Intraepithelial lymphocytes counts, however, reflected only the effect of radiation. Irradiation, with or without GvHR, led to the virtual disappearance of jejunal MMC, undetectable jejunal RMCPII and very low levels of RMCPII in serum (all p less than 0.01 when compared with unirradiated controls). These experiments show that there is a modest expansion in jejunal MMC in unirradiated rats with semiallogeneic GvHR, whereas irradiation, alone or associated with GvHR, profoundly depletes MMC for at least two weeks. The enteropathy of GvHR can evolve in the virtual absence of MMC.

Association of maturation of the small intestine at weaning with mucosal mast cell activation in the rat.

Maturation of the small intestine at weaning has some features in common with immunological reactions involving the gut mucosa. Mucosal mast cell (MMC) activation is a prominent feature of both IgE and cell-mediated mucosal immune responses. MMC activation was therefore studied during the weaning period by measurement of rat mucosal mast cell protease II (RMCPII) release into the systemic circulation as well as MMC counts and jejunal RMCPII content. Maturation of the small intestine was measured by changes in villus and crypt length, and in crypt cell production rate (CCPR). At 3 weeks of age, serum RMCPII increased 17-fold and MMC showed features of degranulation. At the time of weaning (2-4 weeks), villus and crypt length and CCPR increased progressively to adult values. After weaning, serum RMCPII declined slowly to normal adult levels and MMC regained a normal appearance. This study showed a close association between MMC activation and maturation of the small intestine during the weaning period.

Giardia muris infection in mice with concurrent graft-versus-host reaction.

The effects of concurrent graft-versus-host reaction (GvHR) on the course of Giardia infection in CBA x BALB/c F1 mice have been examined, to test the hypothesis that T-cell-mediated immunity, in the form of a local DTH reaction, alters the host-parasite relationship in favour of the host by changing the physical environment of the parasite. GvHR did not enhance immunity, indeed mice infected with Giardia at a late stage of GvHR had significantly higher faecal cyst excretion and prolongation of the plateau phase of infection, indicating a degree of immunodeficiency.

Effect of cyclosporin A on rat mucosal mast cells and the associated protease RMCPII.

The effects of Cyclosporin A (CyA) on rat mucosal mast cells (MMC) have been investigated by cell counts in the jejunal mucosa and assays of the MMC-specific granule protease RMCPII in tissues and serum. CyA was administered by subcutaneous injection; for the majority of experiments the rats received 50 mg/kg daily for 3 days as a loading dose, then 50 mg/kg on alternate days. Treatment with this drug has two actions on MMC, a gradual reduction in the number of MMC and in the tissue content of RMCPII in the jejunum; and a rapid fall in the serum concentration of RMCPII, detectable 3 h after i.v. administration of CyA, 50 mg/kg. These phenomena were demonstrated in normal rats and in animals with an expanded jejunal MMC population due to graft vs host reaction or recent helminth infection. The functional relevance of the MMC depletion was demonstrated in immune rats given CyA for 3 days prior to induction of systemic anaphylaxis; intestinal permeability to i.v. Evan's blue was significantly reduced by CyA treatment. We suggest that CyA depletes intestinal MMC by suppression of T-cell-mediated regulatory stimuli to proliferation of mast cell precursors and/or their migration. The effects of the drug on serum RMCPII, evident before there were changes in the number of intestinal MMC, indicate that it also suppresses the secretion of granule mediators by MMC, probably indirectly via effects on mucosal T cells.

Roles of mucosal mast cells in intestinal cell-mediated immunity.

Mucosal mast cells in rats with GvHR have been studied by cell counts, tissue levels of the specific protease RMCPII, and, as an index of MMC activation, serum RMCPII. In semi-allogeneic GvHR without host irradiation, GvHR produced modest increases in these three indices. In contrast, irradiation profoundly depleted MMC even though enteropathy was more severe than in non-irradiated hosts. We suggest that enteropathy is not dependent on the presence of MMC. In rats given cyclosporin A, lesions of GvHR were mild and numbers of MMC were low.