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J D Clements

Publications and source records attributed to J D Clements.

At least 19 recordsLinked to original sources
PubMed2005

Mucosal adjuvants.

Induction of immune responses following oral immunization is frequently dependent upon the co-administration of appropriate adjuvants that can initiate and support the transition from innate to adaptive immunity. The three bacterial products with the greatest potential to function as mucosal adjuvants are the ADP-ribosylating enterotoxins (cholera toxin and the heat-labile enterotoxin of Escherichia coli), synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN), and monophosphoryl lipid A (MPL). The mechanism of adjuvanticity of the ADP-ribosylating enterotoxins is the subject of considerable debate. Our own view is that adjuvanticity is an outcome and not an event. It is likely that these molecules exert their adjuvant function by interacting with a variety of cell types, including epithelial cells, dendritic cells, macrophages, and possibly B- and T-lymphocytes. The adjuvant activities of CpG and MPL are due to several different effects they have on innate and adaptive immune responses and both MPL and CpG act through MyD88-dependent and -independent pathways. This presentation will summarize the probable mechanisms of action of these diverse mucosal adjuvants and discuss potential synergy between these molecules for use in conjunction with plant-derived vaccines.

Adenosine Diphosphate Ribose↗
PubMed2002

Mutant Escherichia coli heat-labile enterotoxin [LT(R192G)] enhances protective humoral and cellular immune responses to orally administered inactivated influenza vaccine.

Influenza vaccines capable of inducing both systemic and mucosal antibody responses are highly desirable. Optimal induction of mucosal IgA is accomplished by mucosal delivery of vaccine. Mucosal adjuvants may improve the immunogenicity and efficacy of vaccines delivered by this route. Here, we compare the adjuvant activities of a mutant of heat-labile enterotoxin from Escherichia coli [LT(R192G)] with those of the wildtype LT (wtLT) for oral vaccination with inactivated influenza vaccine in BALB/c mice. Compared with administration of oral influenza vaccine alone, co-administration of vaccine with LT(R192G) provided enhanced protection from infection in the upper and lower respiratory tract equivalent to and at similar doses as that obtained with wtLT. Likewise, LT(R192G) augmented virus-specific IgG and IgA responses in serum, lung and nasal washes and the numbers of virus-specific antibody-forming cells in spleen, lung and Peyer's patches in a manner comparable to wtLT. Virus-specific splenic CD4(+) cells from mice administered oral vaccine with either adjuvant produced a mixed Th1- and Th2-type cytokine response pattern. Taken together, these results indicate that LT(R192G), like wtLT, is a potent adjuvant for oral vaccination of mice with influenza vaccine.

Adjuvants, Immunologic↗
PubMed2002

Partial protection against experimental vaginal candidiasis after mucosal vaccination with heat-killed Candida albicans and the mucosal adjuvant LT(R192G).

The effectiveness of a mucosal vaccine composed of heat-killed Candida albicans (HK-CA) or C. albicans culture filtrate (CaCF) in conjunction with the mucosal adjuvant LT(R192G) against vulvovaginal candidiasis was examined in an estrogen-dependent murine model. Mice vaccinated intranasally with HK-CA + LT(R192G) exhibited a significant but short-lived protection accompanied by a vigorous delayed-type hypersensitivity response as well as high titers of circulating C. albicans-specific antibodies. Surprisingly, the levels of antigen-specific antibodies in the vaginal secretions of protected mice were negligible and no correlates of vaginal-associated Type 1 or Type 2 cytokines were observed. Vaginal priming with C. albicans before vaccination did not alter the protective outcome. Immunization with CaCF + LT(R192G) induced a discrete level of protection when administered intrarectally but not intranasally. These results suggest that mucosal vaccination can afford partial protection against vulvovaginal candidiasis, but the precise immune mechanisms responsible for protection are complex and as yet, not well understood.

Adjuvants, Immunologic↗
PubMed2001

Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement.

AMPA receptors are thought to be a tetrameric assembly of the subunits GluR1-4. We have examined whether two coexpressed subunits (GluR1/2) combine at random to form channels, or preferentially assemble with a specific stoichiometry and spatial configuration. The subunits carried markers controlling ion permeation and desensitization, and these properties were monitored as a function of relative expression level and subunit composition. Homomeric receptors assembled stochastically while heteromeric receptors preferentially formed with a stoichiometry of two GluR1 and two GluR2 subunits, and with identical subunits positioned on opposite sides of the channel pore. This structure will predominate if GluR1 binds to GluR2 more rapidly during receptor assembly than other subunit combinations. The practical outcome of selective heteromeric assembly is a more homogenous receptor population in vivo.

Cell Line↗
PubMed2001

Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response.

Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified. In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes. Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses. This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine.

AIDS Vaccines↗
PubMed2001

Mucosal immunization of mice using CpG DNA and/or mutants of the heat-labile enterotoxin of Escherichia coli as adjuvants.

Cholera toxin (CT) and the Escherichia coli heat-labile enterotoxin (LT) are potent mucosal adjuvants in animals associated, at least in part, with their ability to induce cAMP. While toxicity generally precludes their use in humans, a number of different subunit or genetically detoxified mutants of CT and LT have been developed. Another type of adjuvant that has been shown to be effective at mucosal surfaces comprises synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN). We have previously demonstrated a synergy between CpG ODN and native toxins after intranasal (IN) administration to mice, and herein have examined whether this synergy is linked to the cAMP activity. The adjuvanticity of CpG ODN was evaluated with IN and oral delivery of tetanus toxoid or the hepatitis B surface antigen, relative to and in combination with native LT holotoxin (LTh), three active site mutants (LTS61F, LTA69G, LTE112K), a protease site mutant (LTR192G), and the B subunit of LT (LTB). At an equivalent dose, the adjuvants could generally be divided into two groups: one that included CpG ODN, LTh, LTR192G, and LTA69G which acted as strong adjuvants; and the second which comprised LTB, LTS61F, and LTE112K, which produced significantly weaker immune responses. When CpG ODN was co-administered with bacterial toxin-derivatives, in most cases, no synergy between CpG and the LT derivatives was found for strength of the humoral response. Nevertheless, for both routes and antigens, CpG ODN combined with any LT derivative induced a more Type 1-like response than LT derivative alone. These results suggest that while the synergy seen previously with native toxins may have been due in part to inherent cAMP activity, it may have also depended on the particular antigen used and the route of immunization.

Adjuvants, Immunologic↗
PubMed2001

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

AIDS Vaccines↗
PubMed2001

Differential biological and adjuvant activities of cholera toxin and Escherichia coli heat-labile enterotoxin hybrids.

Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A-CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A-CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-gamma) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A-LT-B and native CT both produced lower levels of antigen-specific IFN-gamma. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

Adjuvants, Immunologic↗
PubMed2001

Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA.

Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129-2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

Adjuvants, Immunologic↗
PubMed2001

Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection.

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

Adjuvants, Immunologic↗
PubMed2001

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses.

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.

Adjuvants, Immunologic↗
PubMed2000

Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific CTL.

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.

3T3 Cells↗
PubMed2000

Effectiveness of intranasal immunization with HIV-gp160 and an HIV-1 env CTL epitope peptide (E7) in combination with the mucosal adjuvant LT(R192G).

LT(R192G) is a novel mucosal adjuvant that induces protective immunity when co-administered with certain whole inactivated bacteria or viruses or with subunits of relevant virulence determinants from these pathogens. LT(R192G) stimulates antigen-specific humoral and cellular immune responses, both systemically and in mucosal compartments, and is safe and nontoxic at adjuvant effective doses. Intranasal (IN) immunization of mice with LT(R192G) in conjunction with oligomeric HIV-1 gp160 elevates antigen-specific systemic and mucosal IgG and IgA production and Th1- and Th2-type cytokine responses. Isotype characterization of induced IgG reveals that gp160 alone fails to stimulate IgG2a responses in the absence of adjuvant. Both IgG1 and IgG2a are induced by immunization in the presence of LT(R192G). Additionally, intranasal immunization with a 15-amino acid peptide corresponding to an HIV-1 Env CTL determinant and LT(R192G) induces systemic, peptide-specific CTL activity and Th1 and Th2 cytokine responses that are absent when the adjuvant is excluded from the immunizations. These studies show that LT(R192G) quantitatively and qualitatively enhances cellular and humoral HIV-specific immune responses and that this adjuvant may offer significant advantages toward vaccine development against HIV.

AIDS Vaccines↗
PubMed2000

Direct measurement of specific membrane capacitance in neurons.

The specific membrane capacitance (C(m)) of a neuron influences synaptic efficacy and determines the speed with which electrical signals propagate along dendrites and unmyelinated axons. The value of this important parameter remains controversial. In this study, C(m) was estimated for the somatic membrane of cortical pyramidal neurons, spinal cord neurons, and hippocampal neurons. A nucleated patch was pulled and a voltage-clamp step was applied. The exponential decay of the capacitative charging current was analyzed to give the total membrane capacitance, which was then divided by the observed surface area of the patch. C(m) was 0.9 microF/cm(2) for each class of neuron. To test the possibility that membrane proteins may alter C(m), embryonic kidney cells (HEK-293) were studied before and after transfection with a plasmid coding for glycine receptor/channels. The value of C(m) was indistinguishable in untransfected cells and in transfected cells expressing a high level of glycine channels, indicating that differences in transmembrane protein content do not significantly affect C(m). Thus, to a first approximation, C(m) may be treated as a "biological constant" across many classes of neuron.

Animals↗
PubMed2000

NMDA channel gating is influenced by a tryptophan residue in the M2 domain but calcium permeation is not altered.

N-Methyl-D-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg(2+) and are permeable to Ca(2+). It is thought that a tryptophan residue in the second membrane-associated domain (M2) may form part of the binding site for open-channel blockers and contribute to Ca(2+) permeability. We tested this hypothesis using recombinant wild-type and mutant NMDA receptors expressed in HEK-293 cells. The tryptophan was mutated to a leucine (W-5L) in both the NMDAR1 and NMDAR2A subunits. MK-801 and ketamine progressively inhibited currents evoked by glutamate, and the rate of inhibition was increased by the W-5L mutation. An increase in open channel probability accounted for the acceleration. Fluctuation analysis of the glutamate-evoked current revealed that the NMDAR1 W-5L mutation increased channel mean open time, providing further evidence for an alteration in gating. However, the equilibrium affinities of Mg(2+) and ketamine were largely unaffected by the W-5L mutation, and Ca(2+) permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca(2+) permeation or the binding of open-channel blockers, but plays an important role in channel gating.

Animals↗
PubMed2000

Unveiling synaptic plasticity: a new graphical and analytical approach.

Short-term synaptic plasticity has a key role in information processing in the CNS, whereas memories can be formed through long-lasting changes in synaptic strength. Despite the importance of these phenomena, it remains difficult to determine whether a synaptic modulation is expressed at a presynaptic or postsynaptic site. This article describes a new approach that, in its simplest form, can identify the site of expression by direct graphical means. A more-sophisticated form of the technique can quantify functional synaptic properties and determine which of these properties is altered following a modulation of synaptic strength.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Adjuvant activity of a nontoxic mutant of Escherichia coli heat-labile enterotoxin on systemic and mucosal immune responses elicited against a heterologous antigen carried by a live Salmonella enterica serovar Typhimurium vaccine strain.

Systemic and mucosal antibody responses against both the major subunit of colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) and the somatic lipopolysaccharide expressed by recombinant bivalent Salmonella vaccine strains were significantly enhanced by coadministration of a detoxified derivative with preserved adjuvant effects of the ETEC heat-labile toxin, LT((R192G)). The results further support the adjuvant effects of LT((R192G)) and represent a simple alternative to improve responses against passenger antigens expressed by orally delivered Salmonella vaccine strains.

Adjuvants, Immunologic↗