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K H Mills

Publications and source records attributed to K H Mills.

At least 19 recordsLinked to original sources
PubMed2001

Manipulating the immune system: humoral versus cell-mediated immunity.

Many of the vaccines in use today were designed on an empirical basis with little understanding of the mechanism of protective immunity or knowledge of the protective antigens. Certain of these vaccines, based on killed or attenuated bacteria or viruses, are associated with unacceptable side-effects. New generation vaccines based on recombinant proteins or naked DNA have considerably improved safety profiles, but are often poorly immunogenic, especially when administered by mucosal routes. This is a particular problem with oral delivery; where high doses of antigen are required to generate even modest immune responses. In contrast, nasal delivery of antigens with a range of adjuvants or delivery systems has been shown to generate relatively potent immune responses and to protect against infection in animal models. Advances in immunology have demonstrated that a variety of cellular and humoral immune effector mechanisms, that are regulated by distinct Th1 and Th2 subtypes of T cells, mediate protection against different infectious diseases. The identification of adjuvants and immunomodulators, that can promote the selective induction of these distinct populations of T cells, has now made it possible to rationally design safe and effective mucosal vaccines against a range of infectious diseases of man.

Adjuvants, Immunologic↗
PubMed2001

Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants.

Most vaccines are still delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems. Cholera toxin (CT) and Escherichia coli enterotoxin (LT) are powerful mucosal adjuvants when co-administered with soluble antigens. However, their use in humans is hampered by their extremely high toxicity. During the past few years, site-directed mutagenesis has permitted the generation of LT and CT mutants fully non toxic or with dramatically reduced toxicity, which still retain their strong adjuvanticity at the mucosal level. Among these mutants, are LTK63 (serine-to-lysine substitution at position 63 in the A subunit) and LTR72 (alanine-to-arginine substitution at position 72 in the A subunit). The first is fully non toxic, whereas the latter retains some residual enzymatic activity. Both of them are extremely active as mucosal adjuvants, being able to induce very high titers of antibodies specific for the antigen with which they are co-administered. Both mutants have now been tested as mucosal adjuvants in different animal species using a wide variety of antigens. Interestingly, mucosal delivery (nasal or oral) of antigens together with LTK63 or LTR72 mutants also conferred protection against challenge in appropriate animal models (e.g. tetanus, Helicobacter pylori, pertussis, pneumococci, influenza, etc). In conclusion, these LTK63 and LTR72 mutants are safe adjuvants to enhance the immunogenicity of vaccines at the mucosal level, and will be tested soon in humans.

Adjuvants, Immunologic↗
PubMed2001

Protection against Bordetella pertussis infection following parenteral or oral immunization with antigens entrapped in biodegradable particles: effect of formulation and route of immunization on induction of Th1 and Th2 cells.

The immunogenicity and protective efficacy of systemically and orally delivered pertussis antigens entrapped in either microparticle poly-lactide-co-glycolide (PLG) or nanoparticle PLG formulations were evaluated in a murine respiratory challenge model for infection with Bordetella pertussis. The results demonstrate that immunization with two parenteral doses of 1 microg or three oral doses of 100 microg of pertussis toxoid (PTd) and filamentous haemagglutinin (FHA) encapsulated in PLG conferred a high level of protection against B. pertussis challenge. Furthermore protection could be generated with a single parenteral immunization with a combined microparticle and nanoparticle formulation. However, the route of immunization and the size of the particles affected the type of T cell response induced. Parenteral immunization with PTd and FHA entrapped in PLG microparticles elicits a potent type 1 T cell response and potent antibody response when given by the intraperitoneal (i.p.) or intramuscular (i.m.) route. In contrast, nanoparticle formulations favoured the induction of Th2 cells.

Adhesins, Bacterial↗
PubMed2001

Immunomodulators and delivery systems for vaccination by mucosal routes.

Current paediatric immunization programmes include too many injections in the first months of life. Oral or nasal vaccine delivery eliminates the requirement for needles and can induce immunity at the site of infection. However, protein antigens are poorly immunogenic when so delivered and can induce tolerance. Novel ways to enhance immune responses to protein or polysaccharide antigens have opened up new possibilities for the design of effective mucosal vaccines. Here, we discuss the immunological principles underlying mucosal vaccine development and review the application of immunomodulatory molecules and delivery systems to the selective enhancement of protective immune responses at mucosal surfaces.

Adjuvants, Immunologic↗
PubMed2001

Immunity to Bordetella pertussis.

Bordetella pertussis exploits extracellular and intracellular niches in the respiratory tract and a variety of immune evasion strategies to prolong its survival in the host. This article reviews evidence of complementary roles for cellular and humoral immunity in protection. It discusses the effector mechanisms of bacterial elimination, the strategies employed by the bacteria to subvert protective immune responses and the immunological basis for systemic and neurological responses to infection and vaccination.

Animals↗
PubMed2001

Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-gamma production in vivo.

We have previously demonstrated that Fasciola hepatica infection significantly reduced Bordetella pertussis-specific interferon (IFN)-gamma production in mice coinfected with B. pertussis or immunized with a pertussis whole cell vaccine (Pw). In the present study, we have identified parasite molecules capable of mimicking this suppressive effect of F. hepatica. Parenteral injection of mice with culture medium in which adult F. hepatica were maintained (excretory/secretory, ES, products) suppressed B. pertussis-specific IFN-gamma production in mice immunized with Pw. The suppressive effect of ES was abrogated by coinjecting ES with the cysteine proteinase inhibitor, Z-Phe-Ala-diazomethylketone. Furthermore, purified cathepsin L proteinase (FheCL), a major component of ES products, was capable of suppressing IFN-gamma production. The suppressive effect of FheCL was attenuated in interleukin (IL)-4 defective (IL-4-/-) mice. Therefore, FheCL released by F. hepatica is involved in the suppression of Th1 immune responses and this suppression may be dependent upon IL-4.

Animals↗
PubMed2001

Whole-cell but not acellular pertussis vaccines induce convulsive activity in mice: evidence of a role for toxin-induced interleukin-1beta in a new murine model for analysis of neuronal side effects of vaccination.

Immunization with the whole-cell pertussis vaccine (Pw), while effective at preventing whooping cough in infants, has been associated with local, systemic, and neuronal reactions, including fevers and convulsions in children. In contrast, the new acellular pertussis vaccines (Pa) have a considerably improved safety profile. The lack of an appropriate animal model has restricted investigations into the mechanisms by which neurological reactions are induced by vaccination. Here we describe a novel murine model wherein seizure-like behavioral changes are induced following parenteral administration of Pw. The proinflammatory cytokine interleukin-beta (IL-1beta), production of which has been associated with many neurodegenerative conditions, was significantly increased in the hippocampus and hypothalamus of vaccinated animals. Accompanying this change was a decrease in release of the inhibitory neurotransmitters gamma-aminobutyric acid and adenosine in the hippocampus. Seizure-like behavioral changes were significantly reduced following inhibition of IL-1beta production by the administration of an inhibitor of IL-1beta-converting enzyme and were almost completely abrogated in IL-1 receptor type I knockout mice. These results suggest a causal relationship between IL-1beta induction and convulsive behavior following Pw vaccination. Significantly, Pa neither increased IL-1beta nor induced behavioral changes in mice, but did induce the anti-inflammatory cytokine IL-10. In contrast, administration of active pertussis toxin and lipopolysaccharide, residual in Pw but absent from Pa, also induced convulsive activity. Our findings provide the first direct evidence of an immunological basis for pertussis vaccine reactogenicity and suggest that active bacterial toxins are responsible for the neurologic disturbances observed in children immunized with Pw.

Animals↗
PubMed2000

A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery.

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.

Administration, Intranasal↗
PubMed2000

Modulation of innate and acquired immune responses by Escherichia coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity.

We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-gamma production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-gamma induced TNF-alpha and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-alpha. Furthermore, LTK63 enhanced NF-kappaB translocation, whereas low doses of LTR72 or LT failed to activate NF-kappaB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-kappaB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system.

Adjuvants, Immunologic↗
PubMed2000

Interleukin-1beta-dependent changes in the hippocampus following parenteral immunization with a whole cell pertussis vaccine.

Neurological side effects are a major cause of concern following immunization with a number of vaccines, especially the whole cell pertussis vaccine (Pw). In this study we report that IL-1beta concentrations were significantly increased in the hippocampus following subcutaneous (s.c.) injection of Pw, and that this was accompanied by increased activity of the stress-activated kinase, c-Jun-N-terminal kinase (JNK) and a decrease in glutamate release. These effects were mimicked by s.c injection of active pertussis toxin (PT) or lipopolysaccharide (LPS). Incubation of hippocampal synaptosomes in the presence of Pw, PT or LPS also resulted in increased JNK activation and decreased glutamate release, effects which were mimicked by IL-1beta and blocked by the IL-1 receptor antagonist (IL-ra). Our observations are consistent with the hypothesis that IL-1beta induced by active bacterial toxins present in vaccine preparations, mediate the neurochemical and perhaps the neurological effects of Pw.

Animals↗
PubMed2000

Protection against Bordetella pertussis in mice in the absence of detectable circulating antibody: implications for long-term immunity in children.

Most vaccines used for humans work through humoral immunity, yet many appear to be protective even after specific circulating antibody levels have waned to undetectable levels. Furthermore, it has been difficult to define a serologic correlate of protection against a number of infectious diseases, including those caused by Bordetella pertussis. B. pertussis clearance in immunized mice has been shown to correlate with pertussis vaccine efficacy in children. This murine respiratory challenge model was used to demonstrate persistent vaccine-induced protection against B. pertussis in the absence of circulating antibody at the time of challenge. Whole-cell and acellular pertussis vaccines induced persistent memory T and B cells and anamnestic antibody responses after challenge. The findings suggest that immunologic memory is more significant in protection than is the induction of immediate antibody responses and imply that vaccinated children still may be protected against disease following the disappearance of specific serum IgG.

Animals↗
PubMed2000

Induction of inflammatory cytokines in the brain following respiratory infection with Bordetella pertussis.

Parenteral injection of endotoxin has been used as a model to examine the role of pro-inflammatory cytokines in the centrally controlled responses to Gram-negative bacterial infection. However, the events that occur following mucosal exposure to live bacteria have received little attention. In this study, we have used a murine model to demonstrate that respiratory infection with Bordetella pertussis, which is associated with a number of systemic complications including fever, seizure and encephalopathy in children, resulted in persistent expression of mRNA transcripts for IL-1beta and TNFalpha and transient expression of IL-6 in the hippocampus and hypothalamus. These changes correlated with elevated levels of cytokine protein in the same brain areas. The results demonstrate that infection at a mucosal surface can result in the induction of pro-inflammatory cytokine production in the brain and suggest that these locally synthesized mediators may contribute to the centrally controlled clinical manifestations of B. pertussis infection.

Animals↗
PubMed2000

Direct anti-inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin from Bordetella pertussis.

IL-12 plays a critical role in protective immunity against intracellular pathogens by promoting the development of Th1 cells. Here we demonstrate that filamentous hemagglutinin (FHA), a virulence factor of Bordetella pertussis, is capable of suppressing IL-12 production by macrophages. FHA inhibited IL-12 secretion by a macrophage cell line or ex vivo alveolar macrophages in response to Escherichia coli or B. pertussis lipopolysaccharide (LPS) and IFN-gamma. Antibodies to FHA or denaturation of FHA abrogated the inhibitory effect. Injection of mice with FHA suppressed IL-12 and IFN-gamma levels in the serum in response to i. v. injection of LPS in a model of septic shock. The suppressive effect of FHA was specific for IL-12, since the production of TNF-alpha, IL-6 and IL-10 was not suppressed, and production of IL-6 and IL-10 was up-regulated. Antibody blocking studies revealed that the inhibitory effect of FHA on IL-12 production was dependent on IL-10. Since FHA is secreted at high levels and local T cell responses are suppressed during B. pertussis infection, the findings suggest that FHA may be a critical virulence factor in facilitating pathogen persistence in the respiratory tract by suppressing or delaying the development of cell-mediated immunity.

Animals↗
PubMed2000

Booster immunization of children with an acellular pertussis vaccine enhances Th2 cytokine production and serum IgE responses against pertussis toxin but not against common allergens.

Acellular pertussis vaccines (Pa) protect against severe pertussis in children. However, serum antibody responses decline quickly after immunization. Studies in animal models suggest that cell-mediated immunity also contributes to protection against Bordetella pertussis, and it has already been demonstrated that Pa induce T cells that secrete type-1 and type-2 cytokines in children. In this study we examined the persistence of the T cell response and the effect of booster immunization in 4-6-year-old children. Cell-mediated immunity to B. pertussis antigens was detected in a high proportion of children more than 42 months after their last immunization. Peripheral blood mononuclear cells (PBMC) from the majority of children secreted interferon-gamma (IFN-gamma) and a smaller proportion IL-5, in response to specific antigen stimulation in vitro. However, following booster immunization, significantly higher concentrations of IL-5, but not IFN-gamma, were produced by PBMC in response to B. pertussis antigens. Furthermore, plasma IL-4 and IL-5 concentrations were increased, whereas IFN-gamma concentrations were reduced following booster immunization. It has been suggested that childhood immunization with Th2-inducing vaccines may predispose some children to atopic disease. Although we found that pertussis toxin (PT)-specific IgE was significantly increased after booster immunization in both atopic and non-atopic children, the levels of IgE to common allergens and the prevalence of positive skin prick test were unaffected by the booster vaccination. Thus, despite the enhancement of type-2 responses to B. pertussis antigens, booster vaccination with Pa does not appear to be a risk factor for allergy.

Allergens↗
PubMed2000

Fasciola hepatica infection downregulates Th1 responses in mice.

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low-dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high-dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL-4 defective (IL-4-/-) C57BL/6 mice infected with 5 metacercariae produced less IFN-gamma and more IL-5 compared to their wild-type C57BL/6 counterparts, suggesting that IL-4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN-gamma and IL-2 was completely suppressed in the high-dose infection and this was also observed in IL-4-/- mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL-4 and IL-5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.

Animals↗
PubMed2000

Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses.

Direct intramuscular injection of plasmid DNA can generate immune responses against encoded antigens. However, the relative ability of DNA vaccines to induce cellular and humoral immunity after a single or booster immunization and the persistence of this response have not been fully elucidated. In this study, induction and maintenance of antibody and T cell subtypes with different doses of naked DNA encoding the haemagglutinin (HA) gene of influenza virus were examined and compared to the immune responses and protection induced by respiratory tract infection and immunization with a killed virus vaccine. Like natural infection, immunization with HA DNA induced potent Th1 responses. Spleen cells from mice immunized once with HA DNA in the dose range 10 ng to 100 microgram secreted significant levels of IFN-gamma, but low or undetectable IL-5, in response to influenza virus in vitro. Furthermore, CD4(+) HA-specific Th1 clones were generated from spleens of immunized mice. Although T cell responses waned 12 weeks after a single immunization, antigen-specific Th1 cells persisted in the spleen for at least 6 months after two booster immunizations. In contrast, influenza virus-specific ELISA IgG titres were low after a single immunization and required two booster immunizations to reach significant levels. Furthermore, haemagglutination inhibition (HI) antibodies were weak or undetectable after two immunizations. Nevertheless, two doses of HA DNA conferred almost complete protection against respiratory challenge with live virus. Thus, despite the limited ability to induce antibodies, DNA vaccines confer protective immunity against influenza virus infection, which appears to be mediated by Th1 cells.

Animals↗
PubMed2000

A regulatory role for interleukin 4 in differential inflammatory responses in the lung following infection of mice primed with Th1- or Th2-inducing pertussis vaccines.

Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge with Bordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course of B. pertussis infection of naïve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1beta (IL-1beta) in the lungs. In contrast, neutrophil infiltration and IL-1beta production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs following B. pertussis infection of IL-4-defective (IL-4(-/-)) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1beta, IL-6, and IL-1ra in Pa-immunized IL-4(-/-) mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge with B. pertussis and implicate IL-4 as an important regulator of inflammatory-cell recruitment.

Animals↗