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Biomedical subjects

M F Good

Publications and source records attributed to M F Good.

At least 55 records · Page 3Linked to original sources
PubMed1997

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins.

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.

Amino Acid Sequence↗
PubMed1997

Human antibodies to the conserved region of the M protein: opsonization of heterologous strains of group A streptococci.

A 20-mer peptide (p145) in the carboxyl-terminal region of the M protein of group A streptococci (GAS) has previously been defined as the target of bactericidal antibodies. Sequence analysis of seven field isolates from indigenous Australians living in an area highly endemic for GAS and five laboratory reference strains (encompassing nine unique serotypes plus three nontypeables) demonstrates that this region is highly conserved (sequence identity ranging from 65 to 95%) with six of the 12 sequences being identical to p145. Most of the sequence dissimilarity is contained within the last seven amino acids of p145. Competitive ELISA demonstrates that human antibodies specific for p145 cannot discriminate between p145 and synthetic peptides representing four from four of the variant sequences tested. Ig purified from endemic sera was able to opsonize each of the GAS isolates and free p145 as well as a peptide expressing a minimal conformational epitope within p145 (requiring amino acids between positions 2 and 13 of p145), but not an irrelevant peptide, were able to partially or completely inhibit opsonization of all isolates and reference strains. Thus adult endemic sera contain antibodies which are bactericidal for multiple GAS serotypes and which are specific for a sequence of 12 amino acids contained within the p145 region of the M protein.

Amino Acid Sequence↗
PubMed1997

The spleen, IgG antibody subsets and immunity to Plasmodium berghei in rats.

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).

Adoptive Transfer↗
PubMed1997

Prolonged Th1-like response generated by a Plasmodium yoelii-specific T cell clone allows complete clearance of infection in reconstituted mice.

In the present study, we report the ability of in vitro cultured CD4+ T cells, generated following immunization with dead blood stage P. yoelii parasites, to mediate protection against homologous challenge infection in reconstituted nude mice. P. yoelii-specific T cell line cells produced IFN-gamma after in vitro stimulation with specific antigen, and were protective when adoptively transferred into athymic nude mice. Following transfer P. yoelii-specific T cell lines into nude and SCID mice, elevated levels of nitric oxide (NO) were detected during the first week of infection at a time when parasitaemias were suppressed. However, in vivo blocking of NO production through administration of L-NMMA, an inhibitor of NO synthase, increased mortality, but did not alter the course of primary parasitaemia in P. yoelii-specific T cell line-reconstituted nude mice. In addition, a P. yoelii-specific CD4+ T cell clone, which produced IFN-gamma in vitro, afforded sterile protection via mechanisms other than NO. By ELISA, antibodies were undetectable on all but one day (day 79) post T cell clone transfer and parasite challenge, where very low levels of antibodies were detected, with some evidence of recognition of malaria proteins by Western blot. Collectively, our data suggest that T cell effector functions, independent of NO production and in the absence of high levels of parasite-specific antibodies, can contribute to sterile immunity of P. yoelii.

Adoptive Transfer↗
PubMed1997

Circulating T cells of patients with active psoriasis respond to streptococcal M-peptides sharing sequences with human epidermal keratins.

Psoriasis is a T-cell mediated inflammatory skin disease which has been associated with group A, beta-haemolytic streptococcal infections. Four 20 a.a. long M6-peptides sharing 5-6 a.a. sequences with human epidermal keratins were identified. To investigate the role of potentially cross-reactive T cells in the pathogenesis of psoriasis, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) responses of circulating T cells to these peptides were analysed by ELISPOT and RT-PCR in 14 psoriatic patients, 12 healthy individuals and six patients with atopic dermatitis (AD). Untreated psoriatic patients' responses were significantly higher to these peptides than healthy and AD controls, while responses to a control M6-peptide, not sharing sequences with keratin, were negligible in all groups. No difference was found in response to streptokinase/streptodornase (SK/SD). M6-protein and peptides exclusively elicited IFN-gamma production, with little IL-4 production, even in AD patients. Interferon-gamma responses to all the M6-peptides were abolished after successful treatment of psoriatic patients, but responses to SK/SD were unaffected. The results indicate that active psoriasis is associated with Th1-like cells responding to streptococcal M6-peptides sharing sequences with human epidermal keratin. This is consistent with the hypothesis that psoriasis may be induced and exacerbated in susceptible individuals by M-protein specific Th1-like cells that cross-react with human epidermal keratin.

Adult↗
PubMed1997

Increased immunoreactivity to two overlapping peptides of myelin proteolipid protein in multiple sclerosis.

We tested the proliferative responses of peripheral blood mononuclear cells from 61 patients with multiple sclerosis, 56 healthy control subjects and 52 patients with other neurological diseases to seven synthetic peptides of myelin proteolipid protein (PLP) and 19 synthetic peptides of myelin basic protein (MBP). Increased proliferative responses to two overlapping PLP peptides, PLP184-199 and PLP190-209 were found significantly more frequently in blood from patients with relapsing-remitting or secondary progressive multiple sclerosis (52.3%), but not from those with primary progressive multiple sclerosis (18.2%), than in that from healthy control subjects (8.9%) and patients with other neurological diseases (20.8%). Reactivity to these PLP peptides was most frequently seen in blood from patients with multiple sclerosis of 6-15 years duration and with moderate to severe disability (Kurtzke's Expanded Disability Status Scale > 4.0); the blood from 15 of 19 patients in this group reacted to one or both of the peptides. Both peptides could be recognized by short-term T-cell lines specific for whole PLP, and lines specific for one or other of the two overlapping peptides were able to recognize whole PLP, indicating that these peptides can be processed naturally from the intact molecule. This region of PLP is encephalitogenic in a number of strains of mice. Samples from multiple sclerosis patients did not react more frequently to any of the MBP peptides than those from healthy control subjects. The proportions of patients with other neurological diseases whose blood responded to the MBP peptides that most frequently elicited responses in blood from multiple sclerosis patients were significantly lower than the proportions of multiple sclerosis patients and healthy control subjects whose blood responded to these peptides.

Adolescent↗
PubMed1997

Mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus.

The highly conserved C-terminus of the M protein of group A streptococcus (GAS) is a promising vaccine candidate. An epitope within the conserved C-terminus of the M protein, peptide 145 (a 20-mer with the sequence: LRRDLDASREAKKQVEKALE), has been defined which is the target of opsonic antibodies in both humans and mice, and is recognized by the sera of most adults living in areas of high streptococcal exposure. However, due to potential cross-reactivity between T cells stimulated by this region of the M protein and host cardiac myosin, it is critical to define precisely the minimal protective epitopes within p145. Studies have shown that the immunodominant epitope expressed by p145 is conformational, occurring as an alpha-helical coiled-coil. To enable us to map the murine minimal B cell and T cell epitopes within p145, we have used a novel strategy that allowed us to present shorter sequences of p145 in a native-like conformation. The minimal B cell epitope was found to be contained within residues 7-20 of the p145 sequence, and we have shown that mice immunized with this region are able to generate antibodies that bind to and also opsonize the organism GAS. The T cell epitope is located at the N-terminal region of the p145 sequence, residues 3-14. We have managed, therefore, to define a vaccine candidate--a minimal opsonic B cell epitope within the p145 sequence--that does not incorporate a potentially deleterious T cell epitope.

Amino Acid Sequence↗
PubMed1996

Genetic regulation of protective immune response in congenic strains of mice vaccinated with a subunit malaria vaccine.

The C-terminal 19-kDa, epidermal growth factor-like region of the merozoite surface protein 1 (MSP1) has been used as a vaccine to induce protective immunity to Plasmodium yoelii in mice and to Plasmodium falciparum in monkeys. To analyze the mechanisms and genetic regulation of this MSP1 vaccine-induced protection, we studied the immunologic correlates of protection in H-2 recombinant and congenic mouse strains on the B10 background. Multiple H-2-linked loci were found to contribute, each with a different mechanism. One locus mapped to the I-A region based on the strong protection in C57BL/10 mice compared with intermediate protection in B10.A(4R) mice and the lack of a difference between B10.AKM and B10.MBR mice. Differences in efficacy of passively transferred antisera from vaccinated C57BL/10 vs B10.A(4R) mice indicated that the protection regulated by the I-A locus was at least in part Ab dependent. Two loci mapped to the right of I-A (FE, H-2S, or H-2D) based on a correlation with the number of H-2k loci to the right of I-A in mice that were I-Ak. One effect was Ab independent and may correspond to a possible negative effect of the I-Ek locus. T cells from protected and nonprotected strains differed in their production of IFN-gamma and TNF-alpha following immunization with MSP1(19), but it was unclear how the differential patterns of cytokine expression related to the level of protection. Thus, MSP1(19) vaccine-induced protection is regulated by H-2-linked loci corresponding to two different immune mechanisms. These findings may indicate the need for more than one Ag in a vaccine to protect an HLA-diverse population.

Animals↗
PubMed1996

A study of human T-cell lines generated from multiple sclerosis patients and controls by stimulation with peptides of myelin basic protein.

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s) by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8+ T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4+. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP.

Adolescent↗
PubMed1996

Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci.

This study demonstrates the presence of epitope-specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C-terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVEKALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71-92%, compared with an equal amount of IgG from control adult donors living in non-endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145-specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age-related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.

Adolescent↗
PubMed1996

Protection of rats against malaria by a transplanted immune spleen.

A number of reports have suggested that the spleen plays a key role in the regulation of immunity to malaria but the role, if any, of other tissues is less clear. Furthermore, numerous functional changes occur in the spleen following malaria infection and it is not known whether the spleen's role relates primarily to its content of malaria-specific lymphocytes or to the altered structure and function that has occurred. To address these issues we have generated splenic chimeras by transplanting spleens between Plasmodium berghei-immune and naive rats. In the absence of a functional spleen, specific immune responses from both isolated splenic and non-splenic cells can partially control infection. However, an immune spleen in a naive rat can solidly protect the animal from malaria and a normal spleen in an otherwise immune rat can provide enhanced protection over the non-splenic state. Thus, in the presence of functional splenic architecture both splenic and non-splenic malaria-specific lymphocytes operate more effectively. However, these studies do demonstrate an important role for non-splenic tissue in immunity at least for P. berghei in the rat. The study could have important implications for induction of protective immune responses by vaccination and suggests that malaria-specific lymphocyte responses induced in the periphery following vaccination could interact with parasites in both spleen-dependent and spleen-independent ways.

Adoptive Transfer↗
PubMed1996

Plasmodium falciparum-specific T cell clones from non-exposed and exposed donors are highly diverse in TCR beta chain V segment usage.

Humans lacking previous exposure to Plasmodium falciparum typically have a high frequency of malaria-reactive T cells in peripheral blood, which cross-react with antigens from other microorganisms. We studied a large number of malaria-specific human T cell clones from non-exposed and malaria-exposed donors to determine whether this response is oligoclonal, and might therefore be generated by a limited number of cross-reactive epitopes. Most clones responded well to schizont antigen from three antigenically distinct stocks of P. falciparum. Clones derived from the same donor tended to show similar patterns of reactivity to a panel of non-malaria antigens from various microorganisms, suggesting that a limited number of epitopes were recognized by individuals. However, analysis of the usage of V segments of the beta chain of the TCR (TCRBV) revealed no evidence of TCRBV restriction in the T cell response, either within individual donors or across all donors. An apparent skewing towards TCRBV8 in one donor was shown by two methods to be due to in vitro expansion of a single clone: (i) Direct sorting of TCRBV8+ CD4+ T cells from fresh PBMC did not reveal any enrichment for pRBC-reactive cells; (ii) Sequencing of VDJ regions revealed that the TCRBV8 clones were identical. Sequences of non-TCRBV8 clones from this donor showed major differences in the VDJ junctional region. No differences in TCRBV repertoire between non-exposed and exposed donors were observed. These results exclude the existence of a malarial superantigen and suggest that the T cell response to malaria schizont antigen in non-exposed donors is driven by a large number of epitopes.

Adolescent↗
PubMed1996

Towards understanding the pathogenesis of rheumatic fever.

Acute rheumatic fever results from an immunological response to group A streptococcal infection, but the exact nature of this response, and of the underlying host and organism characteristics, continues to evade researchers. Earlier models of rheumatic fever pathogenesis emphasised the importance of humoral immunity, but more recent work suggests that cellular immunity may play a primary role. Greater understanding of these disease mechanisms is allowing researchers to move towards the development of a vaccine for rheumatic fever.

Humans↗
PubMed1995

Antigens released at schizont burst stimulate Plasmodium falciparum-specific CD4+ T cells from non-exposed donors: potential for cross-reactive memory T cells to cause disease.

In an individual experiencing the first attack of malaria, symptoms of disease can occur at very low parasitemia. T cells and cytokines have been implicated in the etiology of disease symptoms, and others and ourselves have shown that T cells from non-exposed individuals can be stimulated by malaria parasites. Here, we show that nine from 11 blood samples, naturally infected with malaria parasites, could stimulate proliferation of a malaria-specific T cell clone derived from a non-exposed donor. T cells were able to respond to infected blood at a parasitaemia as low as 0.000003% (comparable to the level at which individuals can first experience symptoms of malaria) and secrete cytokines implicated in pathology. Antigens capable of stimulating T cells are expressed throughout the blood stage, but are specifically released at the time of schizont rupture. While most TCR V beta genes are expressed during the T cell response of naive donors to malaria parasites, processing of parasite antigens is blocked by chloroquine and monensin, and activation of 36 of 41 malaria-specific clones tested was restricted by defined MHC class II allelic antigens, strongly suggesting that parasites do not act as mitogens nor as superantigens. The clones react to various commonly encountered pathogenic and non-pathogenic microbes. These data support the concept that activation of cross-reactive memory T cells by malaria parasites contributes to disease symptoms in individuals experiencing their first attack of malaria.

Adolescent↗
PubMed1995

Development of immunity to malaria may not be an entirely active process.

It has never been explained why it takes so long for humans to develop immunity to malaria, although factors such as antigenic variation, antigenic polymorphism, and poor immunological responses to critical antigens are thought to be important. Models of malaria, particularly in rodents, have not been helpful. The course of malaria infection differs considerably between humans and rodents. Mice rapidly develop immunity whereas for most humans it takes several years of exposure for this to occur. Mice typically exhibit high parasitaemias whereas humans typically do not. A significant difference in the immune response of humans and mice to malaria parasites might, in part, explain these differences. Most humans have a preexisting population of activated malaria parasite-specific T cells (cross-reactive T cells) which we have referred to as 'natural' T cells, but such cells have not been observed in mice. These cells, many of which secrete interferon-gamma, might control parasitaemia early in the infection, but a by-product of their further activation by malaria parasites might be disease symptoms. Development of immunity has been thought of as an active process--acquisition of specific antibody and effector T cell responses. However, it might in part reflect induction of tolerance of this preexisting population of disease-inducing T cells as a result of chronic parasitaemia. The initial presence of these Th1-like cells may also impede the development of a Th2-like response necessary for the production of protective antibodies. Persistent cross-reactive stimulation may significantly impede this process.

Animals↗