Biomedical subjects

Jan Holmgren

Publications and source records attributed to Jan Holmgren.

At least 37 records · Page 2Linked to original sources

PubMed2004

Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor.

We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1-dominated B cell and CD4(+) T cell responses. In this study we show that vaccination with DC pre-pulsed ex vivo with CT-conjugated OVA (OVA-CT) gives rise to OVA-specific CD8(+) T cells that produce IFN-gamma and are cytotoxic for OVA-expressing E.G7 tumor cells both in vitro and in vivo. The induction of specific CD8(+) CTL by OVA-CT-treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC. Vaccination of mice with OVA-CT-pulsed DC resulted in rejection of already established MHC class I-positive, MHC class II-negative, OVA-expressing E.G7 tumors in an antigen-specific, CD8(+) T cell-dependent fashion and was associated with high numbers of tumor-infiltrating CD8(+) T cells. Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor-binding B subunit and induced strong activation-maturation signals through the biologically active A subunit. These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.

Animals↗

PubMed2004

Intranasal immunization of mice with group B streptococcal protein rib and cholera toxin B subunit confers protection against lethal infection.

Intranasal immunization of mice with Rib, a cell surface protein of group B streptococcus (GBS), conjugated to or simply coadministered with the recombinant cholera toxin B subunit, induces systemic immunoglobulin G (IgG) and local IgA antibody responses and confers protection against lethal GBS infection. These findings have implications for the development of a human GBS vaccine.

Administration, Intranasal↗

PubMed2004

Expression of cholera toxin under non-AKI conditions in Vibrio cholerae El Tor induced by increasing the exposed surface of cultures.

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

Air↗

PubMed2004

Current status and future prospects for a vaccine against schistosomiasis.

Schistosomiasis remains an intractable problem in many parts of the world. Whereas the schistosome parasites cause little in the way of disease, their eggs become trapped in tissues of the host and elicit powerful and potentially damaging immune responses that are responsible for the pathology. Despite nearly four decades of effort there is still no effective vaccine against schistosomiasis, although a single vaccine candidate is undergoing clinical trials at present. Animal models have revealed much about disease progression and pathology. However, problems remain in identifying appropriate protective antigens to elicit immune responses that will attack the parasite but will not cross-react with egg antigens and thus increase the chance of developing severe chronic disease in individuals that have already been infected. This review summarizes the life-cycle of the parasite, current knowledge of pathogenesis and acquired immunity based on animal studies and observations in humans and the status of efforts in the vaccine development field.

Animals↗

PubMed2004

CpG DNA as a potent inducer of mucosal immunity: implications for immunoprophylaxis and immunotherapy of mucosal infections.

Recent advances in immunology reveal that vertebrate innate immune systems use pattern-recognition receptors, of which the best characterized is the toll-like receptor (TLR) family, to specifically detect pathogen-associated molecular patterns (PAMPs) present in infectious agents. Based on this, use of synthetic PAMPs has attracted much interest for specific immunoprophylaxis and immunotherapy purposes. Among the PAMPs, immunostimulatory CpG DNA, a TLR9 ligand, displays promising potent immunostimulatory and adjuvanticity in different settings. In this review, recent developments in the use of CpG DNA for inducing immunity in the mucosal tissues will be highlighted.

Adjuvants, Immunologic↗

PubMed2003

Mucosal immunisation and adjuvants: a brief overview of recent advances and challenges.

Mucosal immunisation may be used both to prevent mucosal infections through the activation of anti-microbial immunity and to treat selected autoimmune, allergic or infectious-immunopathological disorders through the induction of antigen-specific tolerance. The development of mucosal vaccines, whether for prevention of infectious diseases or for immunotherapy, requires antigen delivery and adjuvant systems that can efficiently help to present vaccine or immunotherapy antigens to the mucosal immune system. Promising advances have recently been made in the design of more efficient mucosal adjuvants based on detoxified bacterial toxin derivatives or CpG motif-containing DNA, and perhaps even more striking progress has been done in the use of virus-like particles as mucosal delivery systems for vaccines and of cholera toxin B subunit as antigen vector for immunotherapeutic tolerance induction. However, it is a memento that two recently developed mucosal vaccines for human use against rotavirus diarrhoea and influenza were withdrawn after a short period in the market because of adverse reactions among the vaccinees, thus emphasising the difficult and challenging task also for mucosal immunisation of combining vaccine and adjuvant efficacy with safety and acceptability.

Adjuvants, Immunologic↗

PubMed2003

Transcutaneous immunization with cholera toxin B subunit adjuvant suppresses IgE antibody responses via selective induction of Th1 immune responses.

Topical application of cholera toxin (CT) onto mouse skin can induce a humoral immune response to CT as well as to coadministered Ags. In this study, we examined the nontoxic cell-binding B subunit of CT (CTB) as a potential adjuvant for cutaneous immune responses when coadministered with the prototype protein Ag, OVA. CTB applied onto skin induced serum Ab responses to itself with magnitudes comparable to those evoked by CT but was poorly efficient at promoting systemic Ab responses to coadministered OVA. However, transcutaneous immunization (TCI) with either CT or CTB and OVA led to vigorous OVA-specific T cell proliferative responses. Furthermore, CTB potentiated Th1-driven responses (IFN-gamma production) whereas CT induced both Th1 and Th2 cytokine production. Coadministration of the toxic subunit CTA, together with CTB and OVA Ag, led to enhanced Th1 and Th2 responses. Moreover, whereas TCI with CT enhanced serum IgE responses to coadministered OVA, CTB suppressed these responses. TCI with either CT or CTB led to an increased accumulation of dendritic cells in the exposed epidermis and the underlying dermis. Thus, in contrast to CT, CTB appears to behave very differently when given by the transcutaneous as opposed to a mucosal route and the results suggest that the adjuvanticity of CT on Th1- and Th2-dependent responses induced by TCI involves two distinct moieties, the B and the A subunits, respectively.

Adjuvants, Immunologic↗

PubMed2003

A mucosally administered recombinant fusion protein vaccine against schistosomiasis protecting against immunopathology and infection.

We have constructed and efficiently produced and purified a candidate vaccine against schistosomiasis consisting of a novel hybrid protein in which two dominant T- and B-cell epitopes from Schistosoma mansoni 28 kDa glutathione-S-transferase (Sm28GST) antigen (a.a 24-43 and 191-212) are fused to cholera toxin B subunit (CTB). Intranasal treatment of S. mansoni-infected mice with the hybrid protein, which similar to native CTB was assembled into receptor binding pentamers, significantly reduced total worm burden and liver egg counts due to the induction of Sm28GST-specific antibodies. Immunopathologic granuloma formation in the liver was also significantly suppressed and there was an almost complete suppression of delayed-type hypersensitivity reactions to both Sm28GST and to total soluble egg antigen in infected animals. The results suggest that this type of hybrid protein could be used as a combined anti-immunopathology and anti-infection vaccine against schistosomiasis.

Animals↗

PubMed2003

Prevention of mucosally induced uveitis with a HSP60-derived peptide linked to cholera toxin B subunit.

Oral administration of the uveitogenic peptide (aa 336-351) derived from human HSP60 induced clinical and histological manifestations of uveitis in 65.8% (48/73) of Lewis rats. Uveitis was significantly decreased to 16.7% (11/66) in parallel experiments with the peptide linked to recombinant cholera toxin B subunit (rCTB), also given by mouth (chi(2)=34.2, p<0.0001). The protective efficacy between tolerized and immunized animals was 74.7%. Adoptive transfer of mesenteric lymph node cells from tolerized rats prevented the development of uveitis. A significantly higher proportion of regulatory CD4(+)CD45RC(low)RT6(+) subset of Th2 memory cells were found in the mesenteric lymph nodes (p<0.005) and spleens (p</=0.05) of tolerized rats without uveitis, as compared with immunized rats and uveitis. In situ hybridization studies of mesenteric lymph nodes and/or the uveal tract showed significant increases in IL-10 and TGF-beta mRNA but decreases in IFN-gamma and IL-12 mRNA in tolerized, as compared with immunized animals. Thus, the mechanism of tolerance, preventing the development of uveitis may involve a regulatory subset of memory cells and a shift from Th1 to Th2 and Th3 cytokines. We suggest that mucosally induced uveitis can be prevented by oral administration of the peptide-rCTB conjugate.

Adoptive Transfer↗

PubMed2003

Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development.

Cholera toxin (CT) is a strong mucosal adjuvant for codelivered antigens, whereas its nontoxic B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. We investigated the effects of CT and CTB on the immunogenicity of in vitro-treated antigen-pulsed dendritic cells (DC) following intravenous injection into mice. Prior to infusion, DC were pulsed for 90 min with either free ovalbumin (OVA), OVA mixed with CT or CTB, or chemical conjugates of OVA with CT and CTB (OVA-CT and OVA-CTB). DC pulsed with OVA or with OVA and CTB gave rise to modest antibody and T-cell responses. Conjugation of OVA with CTB enhanced both the subsequent B-cell and T-cell responses to OVA and preferentially induced Th2 responses. CT was shown to be a strong adjuvant when it was coadministered to DC with OVA and was even stronger when it was coadministered with OVA-CTB and primed for a mixed Th1-Th2 response. The antibody and T-cell responses were further enhanced if OVA was coupled to CT, implying that CT can utilize a combined carrier and adjuvant function vis-a-vis linked antigens for DC vaccination. The immunopotentiating capacity of CT- and CTB-linked antigen was associated with both upregulated secretion of interleukin-1beta by the pulsed DC and increased expression of CD80 and CD86 on the DC surface. These results imply that CT and CTB can be used to both markedly increase and partially direct the DC vaccine-induced immune response with respect to Th1 and Th2 responses, which has obvious implications for DC-based vaccine development.

Adjuvants, Immunologic↗

PubMed2003

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection.

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.

Adjuvants, Immunologic↗

PubMed2003

Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA.

The mucosal immune system consists of an integrated network of lymphoid cells that work in concert with innate host factors to promote host defence. Mucosal immunization can be used both to protect the mucosal surfaces against colonization and invasion by microbial pathogens and to provide a means for immunological treatment of selected autoimmune, allergic or infectious-immunopathological disorders through the induction of antigen-specific tolerance. The development of mucosal vaccines, whether for prevention of infectious diseases or for oral tolerance immunotherapy, requires efficient antigen delivery and adjuvant systems. Significant progress has recently been made to generate partly or wholly detoxified derivatives of cholera toxin (including the completely nontoxic cholera toxin B subunit) and the closely related Escherichia coli heat-labile enterotoxin, with retained adjuvant activity. Cholera toxin B subunit is a protective component of a widely registered oral vaccine against cholera, and has proven to be a promising vector for either giving rise to anti-infective immunity or for inducing peripheral anti-inflammatory tolerance to chemically or genetically linked foreign antigens administered mucosally. Promising advances have also recently been made in the design of efficient mucosal adjuvants based on bacterial DNA that contains CpG-motifs and various imidazoquinoline compounds binding to different Toll-like receptors on mucosal antigen-presenting cells.

Adjuvants, Immunologic↗

PubMed2003

Recent developments in mucosal immunomodulatory adjuvants.

A large proportion of pathogens either invade through, or cause disease at mucosal surfaces. Many new generation mucosal vaccine candidates lack important immunostimulatory features of the original pathogens and thus often do not elicit sufficiently strong immune responses. Despite numerous efforts, there is a profound lack of available agents with mucosal immunomodulatory and adjuvant activity. Immunomodulatory adjuvants are often derived from pathogens and thus efficiently activate the innate immune system leading to subsequent development of strong, specific acquired immunity. In this review, recent advances in mucosal immunomodulators/adjuvants are described with special emphasis on recently developed detoxified cholera toxin and Escherichia coli heat labile enterotoxin derivatives, and the newly described Toll-like receptor ligands CpG DNA and imidazoquinoline compounds. These agents hold much promise as useful mucosal immunomodulators/adjuvants for induction of strong innate immune response and also for subsequent development of specific acquired immunity against mucosal pathogens.

Adjuvants, Immunologic↗

PubMed2002

Detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (STa-related) peptides to the catalytic subunit. A potential new strategy to generate immunostimulants for vaccination.

Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives. Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed. Using a two-plasmid system these derivatives were co-expressed in Vibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs). Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT. The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA. This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations. Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees. Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity. These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT. The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.

Adenosine Diphosphate Ribose↗

PubMed2002

Recent advances in mucosal vaccines and adjuvants.

Mucosal vaccines may be used both to prevent mucosal infections through the activation of antimicrobial immunity and to treat systemic inflammatory diseases through the induction of antigen-specific mucosal tolerance. New, efficient mucosal adjuvants for human use have been designed based on, amongst others, bacterial toxins and their derivatives, CpG-containing DNA, and different cytokines and chemokines, with the aim of improving the induction of mucosal Th1 and Th2 responses. Mucosal delivery systems, in particular virus-like particles, have been shown to enhance the binding, uptake and half-life of the antigens, as well as target the vaccine to mucosal surfaces. DNA vaccines are currently being developed for administration at mucosal surfaces. However, there have also been failures, such as the withdrawal of an oral vaccine against rotavirus diarrhea and a nasal vaccine against influenza, because of their potential side effects.

Adjuvants, Immunologic↗

PubMed2002

Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo.

The pentameric B-subunit of cholera toxin (CTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. In this study a series of fusions was constructed between the genes encoding CTB and the B-chain of human insulin (InsB). The resulting fusion proteins were expressed in Escherichia coli and isolated as cytoplasmic inclusion bodies that were then dissolved and assembled in vitro. GM1 enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses showed that the protein construct in which InsB was fused to the C-terminus of a CTB monomer (CI) assembled into structures that both bound to the receptor GM1 ganglioside and reacted with monoclonal antibodies to CTB and insulin. Fusion of InsB to the N-terminus of CTB resulted in protein that could not assemble into pentameric CTB. In vitro assays showed that the CI fusion protein was 300-fold more potent than native insulin at inducing interleukin-2 (IL-2) production by an insulin-specific T-cell hybridoma. When administered orally, the CI fusion protein induced efficient immunological suppression of ovalbumin-specific T-cell responses in mice co-immunized parenterally with insulin and ovalbumin. These results demonstrate the stability, GM1 receptor-binding activity and antigenic authenticity of the CI fusion protein as well as its ability to elicit insulin-specific T-cell responses in vitro. In addition, we demonstrate that the CI fusion protein induces efficient immunosuppression after oral administration, raising the possibility of using such constructs in the treatment of type-1 diabetes.

Animals↗

PubMed2002

Diminished immunopathology in Schistosoma mansoni infection following intranasal administration of cholera toxin B-immunodominant peptide conjugate correlates with enhanced transforming growth factor-beta production by CD4 T cells.

In Schistosoma mansoni infection, CD4 T cells specific for parasite egg antigens mediate perioval granuloma formation in the liver and intestines. Mice of the CBA strain develop a severe form of disease and a significant proportion of their CD4 T cell response is directed against the major egg antigen Sm-p40 and its immunodominant T cell epitope peptide 234-246. Here, we show that intranasal (i.n.) treatment of infected CBA mice with a fusion protein of the cholera toxin B subunit (CTB) with peptide 234-246 (CTB::pep) results in significant down-modulation of hepatic granulomatous inflammation and fibrosis. Moreover, egg antigen-stimulated dispersed hepatic granuloma cells, as well as mesenteric lymph node CD4 T cells from the CTB::pep-treated mice, produced significantly more transforming growth factor (TGF)-beta than that produced by treated or untreated controls. The data demonstrate that i.n. administration of a single immunodominant peptide conjugated to CTB can lead to down-regulation of the hepatic immunopathology associated with schistosomiasis, and that this down-regulation is, at least in part, mediated by TGF-beta.

Administration, Intranasal↗

PubMed2001

Differential roles of B cells and IFN-gamma-secreting CD4(+) T cells in innate and adaptive immune control of genital herpes simplex virus type 2 infection in mice.

The role of B, CD4(+) T and CD8(+) T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated. Following primary inoculation with attenuated thymidine kinase-deficient (TK(-)) HSV-2, B cell-deficient (microMT) mice developed a local viraemia and transient genital inflammation, suggesting a role for B cells in the innate control of local infection and inflammation. Natural antibodies are implicated in this process, as passive transfer of normal serum into microMT mice significantly reduced HSV-2 TK(-) shedding in the vaginal lumen, although it did not affect subsequent inflammation. Protection against lethal HSV-2 challenge was noted in HSV-2-vaccinated wild-type, CD8(+) T cell-deficient and microMT mice and was characterized by strong virus-specific IFN-gamma responses in vitro and delayed type hypersensitivity (DTH) responses in vivo. In contrast, CD4(+) T cell-deficient (CD4(-/-)) mice had impaired HSV-2-specific IFN-gamma production and DTH responses and succumbed rapidly to genital HSV-2 challenge. However, protective responses to HSV-2 could be induced in HSV-2-vaccinated CD4(-/-) mice by treatment with recombinant IFN-gamma. Taken together, these results suggest that CD4(+) T cells secreting IFN-gamma are critical for immune protection against lethal genital HSV-2 re-infection, whereas B cells/natural antibodies have anti-viral and -inflammatory effects in the innate control of a primary infection.

Animals↗