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Biomedical subjects

M J Newman

Publications and source records attributed to M J Newman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Induction of cross-reactive cytotoxic T-lymphocyte responses specific for HIV-1 gp120 using saponin adjuvant (QS-21) supplemented subunit vaccine formulations.

The antigenic variation associated with Human Immunodeficiency Virus type-1 (HIV-1) envelope proteins could limit their utility in vaccines if the immune responses induced are specific for immunodominant variable epitopes. We evaluated the ability of experimental subunit vaccines, containing recombinant forms of the envelope glycoprotein (rgp120) from two HIV-1 variants, to induce immune responses capable of recognizing unrelated HIV-1 variants. A vaccine formulation based on HIV-1IIIB/LAI rgp120 and supplemented with saponin adjuvant (QS-21) induced neutralizing antibodies specific for the HIV-1IIIB/LAI variant. This antibody response was presumably specific for the variable principle neutralizing determinant (PND) of the third variable region of gp120, the V-3 region. This formulation induced cytotoxic T-lymphocytes (CTL) specific for the dominant V-3 epitope but also to an additional unidentified epitope outside of this region. The CTL specific for this second epitope also recognized gp120 from the HIV-1MN and HIV-1RF variants in a "cross-reactive" manner. A second vaccine formulation based on HIV-1MN rgp120 and QS-21 adjuvant induced neutralizing antibodies that were again variant-specific but also CTL that recognized all three HIV-1 variants in a cross-reactive manner. These data demonstrate that CTL capable of recognizing different HIV-1 variants, which are presumed to be specific for a conserved HIV-1 gp120 epitope, can be induced using subunit vaccines with the appropriate adjuvant while variant-specific antibody responses are produced. These findings support further evaluation of this vaccine format.

AIDS Vaccines↗
PubMed1997

Development of an adjuvant-active nonionic block copolymer for use in oil-free subunit vaccines formulations.

Nonionic block copolymers, synthesized from repeating units of oxypropylene and oxyethylene, can be designed so that individual copolymers have unique physical properties with differential levels of adjuvant activity. We have designed high molecular weight block copolymers that spontaneously assemble into 500 nm-3 mum particles when formulated with protein antigens in aqueous solutions at physiological pH. The adjuvant activity of one of these copolymers, termed CRL1005, was compared to selected research adjuvants using ovalbumin (OVA) as the prototype vaccine antigen. Suboptimal doses of OVA were formulated with complete and incomplete. Freund's adjuvant (CFA/IFA), alum Quil-A saponins Ribi Adjuvant System (RAS) or the CRL1005 copolymer and these formulations were used to immunize C57BL/6 mice. The CRL1005 copolymer appeared to be more potent than either Quil-A or alum and comparable to the RAS formulation, based on the numbers of responding mice and the OVA-specific antibody titers. Alum. RAS and Quil-A all augmented the production of IgG1 and IgG2l, similarly whereas only the CFA/IFA boosted IgG2a levels significantly. The effect of adjuvants on relative antibody affinity was more variable with the CRL1005 and CFA/IFA inducing antibodies with the highest affinity scores. This high molecular weight nonionic copolymer is nontoxic in aqueous formulations and should therefore be compatible with a wide variety of protein or polysaccharide vaccine antigens.

Adjuvants, Immunologic↗
PubMed1997

On-line liquid chromatography coupled with high field NMR and mass spectrometry (LC-NMR-MS): a new technique for drug metabolite structure elucidation.

High performance liquid chromatography has been coupled simultaneously to high field NMR and MS detectors, giving UV, NMR and mass spectra for each component in a mixture, after on-line separation. This powerful new tool for the structure elucidation of components in mixtures without isolation has been successfully applied to the analysis of the metabolites of paracetamol in human urine.

Acetaminophen↗
PubMed1997

Effects of a beta-human chorionic gonadotropin subunit immunogen administered in aqueous solution with a novel nonionic block copolymer adjuvant in patients with advanced cancer.

The clinical and immunological effects of a vaccine consisting of CTP37, a synthetic peptide corresponding to the COOH-terminal peptide (CTP) of beta-human chorionic gonadotropin (beta-hCG) conjugated to diphtheria toxoid, combined with CRL 1005, a novel synthetic nonionic block copolymer adjuvant, were examined. Twenty-one patients with metastatic, nontrophoblastic cancers received up to four immunizations by i.m. injection of a fixed dose of CTP37 and escalating doses of CRL 1005. Doses of CRL 1005 adjuvant as high as 75 mg were administered with 1 mg of CTP37 without evidence of significant local or systemic toxicity. Immunizations resulted in the production of IgG antibody to beta-hCG. CRL 1005 doses of 3-25 mg appeared to be optimal for antibody induction. Immunizations also resulted in increases in the cellular response of peripheral blood mononuclear cells (PBMCs) to the unconjugated CTP, hCG, and diphtheria toxoid. Responding PBMCs specifically secreted the TH1-associated cytokines IFN-gamma and interleukin (IL)-2 as well as the TH2-associated IL-5 and IL-10. Increased expression of IFN gamma and IL-5 mRNAs by PBMCs 4 h after immunization was also observed. CRL 1005 administered with CTP37 in aqueous solution is well tolerated. The CTP37-CRL 1005 subunit vaccine has the capacity to stimulate potentially beneficial humoral and cellular immune responses in patients with advanced cancer.

Adjuvants, Immunologic↗
PubMed1996

Evidence for genetic control of vaccine-induced antibody responses in cattle.

The purpose of our study was to identify evidence for genetic control of immune responses in cattle. To address this question, we evaluated the variation of antibody responses induced by vaccination with Brucella abortus Strain 19, a live attenuated bacterial vaccine, in large half-sibling families. The data were analyzed using a parametric statistical model that incorporated the effects of sire, bovine major histocompatibility complex (BoLA) types and parameters related to the experimental design. The BoLA types represented a readily identifiable marker, analogous to those known to be associated with genetic control of immune responses in other mammals. Variation between individual animals within our test population was significant but we were able to identify both individual animals and families with high or low antibody production phenotypes. In several cases, these traits were significantly correlated with individual bulls, suggesting the existence of sire effects, or with individual BoLA types. These findings are consistent with the theory that at least two separate genes or genetic systems contribute to the control of bovine antibody responses to B. abortus vaccination. These genetic effects are likely to be analogous to those identified in several species of laboratory rodents and humans.

Animals↗
PubMed1995

Structure/function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function.

QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.

Adjuvants, Immunologic↗
PubMed1995

Effect of adjuvants on immunogenicity of MN recombinant glycoprotein 120 in guinea pigs.

The immunogenicity of recombinant gp120 from the MN strain of HIV-1, a candidate HIV-1 vaccine, was evaluated in guinea pigs using adjuvant formulations with different physical and chemical properties. The adjuvants tested included Freund's adjuvant (FA), alum, and the novel adjuvant QS-21. These studies demonstrated that QS-21 provides a number of advantages compared to the two other adjuvants tested. QS-21 formulations accelerated the production of antibodies to MN rgp120 and elicited complete seroconversion after a single immunization. QS-21 shifted the antigen dose-response curve for antibody production by as much as three orders of magnitude, enabling a more economical use of antigen. Antibody titers to MN rgp120 and to the principal neutralizing determinant in the V3 domain were higher in animals receiving QS-21 formulations than in animals immunized with the other adjuvants, and correlated well with higher virus neutralization titers in an in vitro assay. These results support the testing of QS-21 in future clinical trials of candidate HIV-1 vaccines.

AIDS Vaccines↗
PubMed1994

A versatile synthetic peptide-based ELISA for identifying antibody epitopes.

A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly-L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.

Amino Acid Sequence↗
PubMed1994

Accessory cell requirements for saponin adjuvant-induced class I MHC antigen-restricted cytotoxic T-lymphocytes.

The in vivo and in vitro accessory cell requirements of class I major histocompatability complex (MHC) antigen-restricted cytotoxic T-lymphocyte (CTL) responses were determined using cell-depletion experiments coupled with active immunizations using ovalbumin (OVA) as the immunogen and saponin adjuvant (QS-21). To paralyze macrophage activity in vivo, C57BL/6 mice were treated with particulate silica or carrageenan. In vivo depletion of helper T-lymphocytes was accomplished by treatment with GK1.5 rat monoclonal antibody, which is specific for the murine CD4 antigen, and by genetic depletion of class II MHC antigens. Following treatments, the mice were immunized with formulations containing OVA alone or mixed with QS-21 saponin adjuvant, which induces MHC class I antigen-restricted CTL responses. In vivo treatment to paralyze macrophages abrogated these CTL responses but not antigen-specific antibody or lymphocyte proliferative responses. Depletion of CD4+ T-lymphocytes had no effect on CTL responses but significantly reduced proliferation and antibody responses. In vitro depletion and reconstitution experiments were done to compare the contributions of different antigen-presenting cells (APC), specifically dendritic cells (DC) and macrophages. Again, the requirement for macrophages was absolute but there was no indication that DC were involved. These data suggest that antigen processing and presentation functions are critical to the induction of CTL and that they are a function of macrophages but that CD4+ helper T-lymphocyte functions are not required.

Adjuvants, Immunologic↗
PubMed1994

Identification of ethanolamine in rat and gerbil brain tissue extracts by NMR spectroscopy.

Some NMR resonances which have previously been observed but not identified in mammalian brain tissue extracts have been shown to arise from ethanolamine. This conclusion is drawn from a systematic study of the perchloric acid extracts of rodent brain tissue in which several NMR experiments were used to assign the peaks unambiguously. The extraction procedure used in this work gave samples with highly reproducible spectra, and ethanolamine was observed in all our extract samples. A localized increase in the concentration of ethanolamine was seen in the spectra of extracts produced from a cerebral infarct induced by occlusion of the middle cerebral artery in the rat.

Animals↗
PubMed1994

Phase 1 trial of immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine in patients with malignant melanoma.

Increasing doses of saponin fraction QS-21 were administered as immunological adjuvant in a Phase 1 trial with a constant dose of the melanoma ganglioside GM2 covalently attached to keyhole limpet haemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IV melanoma who were free from disease after surgery were treated with six vaccinations administered subcutaneously over a 5-month period. Local and systemic reactions were QS-21 dose-related. Doses of < or = 100 micrograms induced mild local tenderness and inflammation at vaccination sites lasting 2-4 days and occasional brief low-grade fever and malaise, but no significant incapacitation. The 200 micrograms dose induced low-grade fever and malaise after 30% of vaccinations and local reactions as large as 20 cm in diameter were seen in all patients, resulting in discomfort with usage of the injected extremity for 5-10 days. The titres of IgM and IgG antibodies against GM2, and IgG antibodies against KLH, were highest at the 100 and 200 micrograms QS-21 doses. No antibodies against QS-21 were detected. This trial identifies the 100 micrograms dose of QS-21 as the optimal well tolerated dose for induction of antibodies against both the melanoma ganglioside/GM2 and the protein KLH in melanoma patients.

Adjuvants, Immunologic↗
PubMed1994

Induction of antigen-specific killer T lymphocyte responses using subunit SIVmac251 gag and env vaccines containing QS-21 saponin adjuvant.

Subunit vaccines based on recombinant proteins have proved useful for inducing antibody responses and they are safe for widespread use because they do not contain any live components. Unfortunately, they do not typically induce the types of cell-mediated immune responses required to control viral pathogens; specifically, they do not induce CD8+ cytotoxic T lymphocyte (CTL) responses. To increase the immunogenicity of recombinant proteins, we have used the QS-21 saponin adjuvant in subunit vaccine formulations. In the current study, experimental subunit vaccine formulations containing recombinant p55gag or gp120env proteins from the mac251 strain of the simian immunodeficiency virus (SIVmac251) and the QS-21 adjuvant were used to immunize rhesus macaques. These formulations induced SIV gag- or env-specific cellular immunity that was detectable in vitro and included killer cell activity. The induction of killer cells required prior vaccination and the responses were antigen specific for the immunogens contained in the vaccine formulations. Autologous target cells were required to detect these responses, suggesting genetic restriction, and effector cells appeared to be present in both the CD4+ and CD8+ T lymphocyte subpopulations. These data suggest that the vaccine-induced killer cell activity that was detected was mediated by both CD4+ and CD8+ lymphocytes. Despite the presence of these killer cells, all of the animals became infected with the SIVmac251 on experimental challenge. These findings demonstrated that antigen-specific killer cell responses could be induced by a subunit vaccine formulated with the QS-21 saponin adjuvant. The characteristics of the responses suggested that the effector cells were T lymphocytes, expressing either CD4 or CD8.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The influence of acetazolamide and amlodipine on the intracellular sodium content of rat proximal tubular cells.

1. This investigation set out to use 23Na n.m.r. spectroscopy to measure changes in intracellular levels of sodium in isolated suspensions of rat proximal tubules. The effects of temperature, an inhibitor of the sodium pump and known natriuretic drugs on intracellular sodium content of such tubular preparations were measured and compared with calcium channel antagonists where action at this level is unclear. 2. Rat kidneys were perfused with collagenase, roughly chopped, subjected to mechanical dispersion and washed to remove all traces of the enzyme. The proximal tubules were then purified and concentrated by Percoll density gradient centrifugation and then resuspended in buffer containing dysprosium tripolyphosphate shift reagent. 3. Distinct peaks corresponding to intracellular and extracellular sodium signals were observed when the tubules were placed into the n.m.r. spectrometer. As the temperature of the suspension rose to 37 degrees C, there was an exponential decrease in sodium content, with a decay constant of 0.15 +/- 0.02 min-1, which reached a stable level within 20 to 25 min. Addition of ouabain, 10(-3) M, resulted in a significant (P < 0.01) 30% increase in intracellular sodium content within 5 min which peaked at 70% 20 min later. Although acetazolamide (10(-3) M) significantly (P < 0.01) increased intracellular sodium content by 45%, amlodipine (10(-4) M) had no effect. 4. These data show that changes in the activity of the Na+/K+/ATPase have a considerable influence on the intracellular levels of sodium in proximal tubule cells. Inhibition of carbonic anhydrase activity resulted in a rise in intracellular sodium content which is compatible with its action to reduce the turnover rate of the Na+/(HCO3-)3 symporter. The lack of effect of amlodipine was consistent with the suggestion that it does not have a direct action on the sodium handling processes at the level of the proximal tubule.

Acetazolamide↗
PubMed1994

Use of fixed autologous stimulator cells to correctly present human immunodeficiency virus type 1 viral peptides to nonhuman primate lymphocytes in proliferation and cytotoxic T-lymphocyte assays.

Autologous, virus-transformed lymphoblastoid cell lines were established by using peripheral blood lymphocytes from rhesus monkeys that were previously immunized with recombinant human immunodeficiency virus type 1 strain IIIB glycoprotein 160. These autologous cell lines were used to present human immunodeficiency virus type 1 viral antigens in a processed and cell-associated manner to T lymphocytes. This was accomplished by either infecting the cells with recombinant vaccinia viruses or pulsing them with synthetic peptides and then subjecting them to a mild fixation step with glutaraldehyde. Fixed antigen-presenting cells were then used as stimulator cells in vitro to measure cell-mediated immune responses. Both the vaccinia virus-infected and peptide-pulsed autologous cells stimulated antigen-specific cellular proliferative responses. The magnitude of the responses correlated with the immunization histories of the animals and other measures of immunity, such as antibody titers. Autologous vaccinia virus-infected cells were also capable of inducing the in vitro maturation of CD4+ and CD8+ precursor cytotoxic T lymphocytes into antigen-specific mature cytotoxic T lymphocytes. The use of stimulator cells to present viral peptides in a cell-associated manner appeared to be a very sensitive and versatile manner in which to measure cell-mediated immune responses with peripheral blood lymphocytes from nonhuman primates. It is likely that a similar approach will function with peripheral blood lymphocytes from humans.

Amino Acid Sequence↗
PubMed1994

Immunogenicity and HIV-1 virus neutralization of MN recombinant glycoprotein 120/HIV-1 QS21 vaccine in baboons .

The effect of adjuvant and immunization schedule on the immunogenicity of HIV-1 envelope glycoprotein, MN rgp120, was optimized by using baboons. The novel adjuvant QS21 elicited earlier seroconversion than alum adjuvant, and the antibody titers to MN rgp120 for animals treated with QS21 were significantly greater than the titers obtained in animals treated with alum. The use of QS21 shifted the dose-response curve, resulting in less MN rgp120 required to achieve equivalent titers to those in the alum formulations. The in vitro virus neutralizing (VN) titers from animals treated with QS21 were 3- to 10-fold higher than with alum. The data presented herein point to the superiority of QS21 as adjuvant in primates for MN rgp120.

AIDS Vaccines↗
PubMed1993

Concurrent infection of human macrophages with HIV-1 and Mycobacterium avium results in decreased cell viability, increased M. avium multiplication and altered cytokine production.

The effects of a concurrent HIV-1 and Mycobacterium avium infection in vitro were assessed in human peripheral blood-derived macrophages (M phi). M phi were infected with HIV-1Ba-L strain for 14 days then infected with M. avium (HIV/M. avium) or treated with LPS (HIV/LPS). At various times after M. avium or LPS treatment, Mo phi cultures were harvested for quantitation of HIV and M. avium replication, as well as M phi cellular viability. In addition, mRNA and supernatants were collected for assessment of induction of the cytokines TNF-alpha, IL-1 beta and IL-6. M. avium multiplication was greater in HIV-infected M phi, whereas no difference in virus production, based on p24 and RT values, was observed between HIV-infected cells and HIV/M. avium or HIV/LPS M phi. M. avium infection of HIV-1-infected M phi also caused a decrease in viability of the M phi. HIV-1/M. avium-infected M phi had a 24 h delay in induction of TNF-alpha steady state mRNA when compared with HIV/LPS or M. avium only or LPS-only treated M phi. HIV infection also increased the amount and the length of induction of IL-1 beta and IL-6 steady state mRNA stimulated by either M. avium or LPS. In addition, prolonged and increased protein production of TNF-alpha, IL-6, and IL-1 beta was observed in HIV/M. avium-infected cells when compared with the other treatments. In direct contrast to M. avium infection, no significant differences in LPS-induced protein production of the three cytokines was observed between HIV-1-infected and -noninfected M phi. Treatment of HIV/M. avium-infected cells with human rGM-CSF did not increase either the time or quantity of induction of TNF-alpha mRNA or protein production in HIV/M. avium-infected M phi. The increase in M. avium numbers, dysregulation of cytokine production, and subsequent cell death seen in vitro in HIV/M. avium-infected human M phi may reflect part of the underlying cause of the highly disseminated M. avium disease pattern observed in AIDS patients.

Cell Survival↗
PubMed1993

Use of particulate forms of protein antigens to increase the sensitivity of antigen-specific proliferation assays.

Different forms of recombinant HIV-1 gp160 and tetanus toxoid were adsorbed onto latex microspheres and this particulate form of the proteins was used to measure antigen-specific proliferation in vaccinated rhesus macaques. Proliferative responses to proteins bound to microspheres were significantly greater and allowed for the detection of antigen-specific responses that were not detected using soluble proteins. The responses were antigen-specific and required prior immunization of the animals. Additionally, the presence and magnitude of the proliferative responses was associated with antibody responses to the same proteins suggesting the results were representative of in vivo responses and that the assay format did not induce in vitro artifacts.

AIDS Vaccines↗