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Biomedical subjects

B H Barber

Publications and source records attributed to B H Barber.

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

Enhanced immunogenicity of B cell lymphoma genetically engineered to express both B7-1 and interleukin-12.

The A20 murine B cell lymphoma was transfected with B7-1 and subsequently these variants and vector control variants were retrovirally infected to express murine interleukin-12 (mIL-12). In vitro data showed that the B7-1 variants enhanced secretion of IL-2 and IL-4 by allogeneic T cells in mixed lymphocyte tumor cultures. While IL-12 variants stimulated IFN-gamma, variants expressing both B7-1 and IL-12 stimulated IFN-gamma, IL-2, and IL-4 secretion. Tumorigenicity experiments showed that whereas B7-1 delayed tumor onset, only the mIL-12 variants with or without B7-1 were completely rejected in syngeneic hosts. In addition, tumor-free mice were protected against subsequent challenge with the parental unmodified cells and had enhanced cytotoxic T lymphocyte (CTL) lysis activity. Results from minimal disease mixing experiments demonstrated that only the A20/B7-1/mIL-12 variant was able to reject A20 unmodified cells inoculated at the same site, whereas prolonged survival was observed when the A20 parental cells were inoculated at different sites. Depletion studies and injections into nu-/nu- mice demonstrated that both CD4+ and CD8+ T cells may mediate immunity. These data suggest that vaccinations with tumor cells genetically modified to express both B7-1 and IL-12 may alter cytokine profiles and generate CTL activity and, thus, the mechanisms of enhanced antitumor immunity may be multifactorial.

Animals↗
PubMed1997

The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites.

Although plasmid DNA immunization provides an effective means of inducing CTL responses to an expressed Ag, the mechanism by which CTL precursors are activated remains to be established. Insights could be gained by identifying the cells responsible for Ag presentation when DNA is introduced into different tissue sites. By immunizing parent into F1 bone marrow chimeric mice with an influenza nucleoprotein-expressing plasmid, we have demonstrated that the key cells in this presentation process for both gene gun-mediated epidermal injection and needle intramuscular injection of plasmid DNA are bone marrow derived. Furthermore, as assessed by intramuscular injection, coexpression of nucleoprotein with the costimulatory molecule B7-2, or the cytokines granulocyte-macrophage CSF and IL-12, did not convert nonhemopoietic cells into APCs. Thus, for two distinctly different modes of DNA immunization, in one case with or without coexpressed immunostimulatory factors, the APCs were consistently found to be of hemopoietic origin.

Animals↗
PubMed1997

Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations.

Surgical ablations revealed different dependencies on the injection site tissue for i.m. needle and epidermal gene gun DNA immunizations. Excision of an injected muscle bundle within 10 min of DNA inoculation did not affect the magnitude or longevity of Ag-specific Ab responses. By contrast, biopsy of the skin target site up to 24 h after gene gun bombardment completely abrogated the Ab response in the majority of mice. The form of a DNA-expressed Ag (membrane bound, secreted, or intracellular) did not affect the temporal requirements for the skin and muscle target sites. Skin cells, but not muscle cells, were also required for DNA-induced CTL. We conclude that transfected cells in gene gun-bombarded skin, but not needle-injected muscle, play a central role in DNA-initiated Ab and CTL responses.

Animals↗
PubMed1997

Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines.

In the course of examining epitope-specific CTL responses to intramuscular plasmid DNA immunization with influenza nucleoprotein (NP)-expressing vectors, a nonimmunogenic mutant NP (NP(o)) was identified. The coding region of NP(o) differed from the wild-type A/PR/8/34 NP sequence (designated NP(v)) by three amino acid alterations in the carboxyl-terminal portion of the molecule remote from the H-2K(d) epitope (147-155) being monitored. Correction of these mutations restored the immunogenicity of the native sequence, indicating that sequence alterations remote from the CTL epitope in question can profoundly influence its immunogenicity. In an effort to identify general, nonstructural means of enhancing the CTL response to weak plasmid DNA immunogens, vectors were constructed expressing NP(o) in tandem with the costimulatory molecules B7-1 or B7-2. Co-linear expression of NP(o) with B7-2, but not B7-1, significantly increased the NP epitope-specific CTL response. In addition, coinjection of these NP(o) plasmids with granulocyte-macrophage CSF- and/or IL-12-expressing vectors also restored near native NP-specific CTL responses. Thus, the coexpression of certain costimulatory molecules and/or cytokines, in concert with a non-self gene delivered as an intramuscular plasmid DNA immunogen, can significantly enhance Ag-specific CTL responses.

Adjuvants, Immunologic↗
PubMed1997

Recombinant antibodies with conformationally constrained HIV type 1 epitope inserts elicit glycoprotein 160-specific antibody responses in vivo.

Although neutralizing epitopes have been identified on the HIV-1 gp120/gp41 envelope complex, efforts to exploit this information through the construction of synthetic peptide vaccines have been largely unsuccessful. Unfortunately, synthetic peptides tend to be poorly immunogenic, and most often lack the conformational characteristics of the corresponding epitope in the native protein. In an effort to circumvent these difficulties, we have utilized an anti-class II MHC antibody as a molecular scaffold for the construction of two conformationally constrained neutralizing HIV-1 epitopes. Previously we demonstrated that anti-class II MHC antibodies can function as vectors for the induction of adjuvant-independent antibody responses to incorporated epitopes. In this instance, one epitope, IHIGPGRAFYT, is the crown of the V3 loop from gp120, and the other, ELDKWAS, is a neutralizing epitope from gp41. The insertion of these epitopes into a specific loop region of the immunoglobulin heavy chain FR3 was found to preserve the anti-class II MHC-binding activity of these recombinant antibodies, and the inserts were recognized by epitope specific monoclonal antibodies. When utilized as immunogens, each of these epitope insertion antibodies was able to induce high-titer anti-HIV-1 gp160 responses in guinea pigs. These responses were conformation specific in that the anti-gp160 binding was not inhibited by the synthetic peptide corresponding to the epitope in question. These data demonstrate the potential to construct conformationally constrained HIV-1 epitope immunogens, and thus establish an alternative approach to the design of an effective HIV-1 subunit vaccine.

Amino Acid Sequence↗
PubMed1997

Role of IL-12 and 4-1BB ligand in cytokine production by CD28+ and CD28- T cells.

The costimulatory receptor CD28 is important in the development of both Th1 and Th2 responses. To further assess the requirement for CD28 in the development of Th1 and Th2 responses, we analyzed the ability of T cells from wild-type or CD28- mice to secrete cytokines in MLRs with B lymphomas. We find that in the absence of added IL-12, B lymphomas expressing the alternate costimulatory ligand 4-1BBL can support the production of IL-2 and IL-4 but little detectable IFN-gamma by allogeneic CD28+ and CD28- T cells. IL-4 production by CD28+ or CD28- T cells responding to B7(low) B lymphomas was abrogated by blocking 4-1BB ligand-4-1BB interaction. When APC express high levels of B7 family molecules as well as 4-1BBL, soluble 4-1BB inhibits IL-4 production by CD28- but not by CD28+ cells. Addition of IL-12 to the CD28- MLRs results in increased production of IFN-gamma and decreased amounts of IL-2 and IL-4. Thus, both Th1 and Th2 responses can develop in the complete absence of a signal through the CD28 molecule. CD28+ and CD28- T cells differed, however, with respect to the effect of IL-12 on IL-4 production. IL-12 severely curtailed the amount of IL-4 produced in the CD28- T cell cultures but had a less profound effect on the level of IL-4 produced in the CD28+ cultures, suggesting that a strong signal through the CD28 molecule prevents down-regulation of IL-4 production by IL-12.

Animals↗
PubMed1997

Presentation of an influenza nucleoprotein epitope incorporated into the H-2Db signal sequence requires the transporter-associated with antigen presentation.

In an effort to optimize the formation of peptide-specific CTL target structures in the context of plasmid DNA immunization, a strategy was developed to couple the biosynthesis of a class I heavy chain with its optimal binding epitope. Specifically, a cDNA expression vector was constructed with the influenza nucleoprotein epitope NP366-74 incorporated into the signal sequence of its restriction element H-2Db. Transporter associated with Ag presentation-expressing murine cell lines P815 (H-2d) and BW5147 (H-2k) transfected with this modified heavy chain expressed normal levels of plasmid-encoded Db at the cell surface, and were lysed by NP366-74-specific CTL. These results indicate that the modified signal sequence was successfully delivered to the endoplasmic reticulum, and the epitope within it processed for T cell recognition. In contrast, T2 cells, which lack the TAP transporter, when transfected with the same vector were not lysed by NP366-74 CTL, and exhibited an Ag-processing-defective phenotype. Thus, these data, which indicate TAP-dependent presentation of an optimal CTL epitope located in a signal sequence, challenge the effectiveness of Ag processing in the endoplasmic reticulum.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The immunotargeting approach to adjuvant-independent subunit vaccine design.

Recently there has been considerable interest in exploring adjuvant-independent approaches to the enhancement of immunogenicity. One strategy which has emerged in response to this need is the immunotargeting approach to subunit vaccine design. Immunotargeting involves the conjugation of non-self antigens to monoclonal antibodies specific for cell surface determinants (e.g. class II MHC) on antigen presenting cells in vivo. Saline injections of these immunoconjugates have been shown to promote significant IgG responses to the delivered antigens in a number of different animal model studies. Oral and nasal immunizations in mice with anti-class II MHC immunoconjugates induce both secretory IgA and systemic IgG responses. Recombinant anticlass II MHC antibodies with defined B-cell and T-cell epitopes incorporated into their primary sequence have recently been used to induce epitope specific antibody responses in macaques. Thus the potential now exists for the rational design of adjuvant-independent human vaccine candidates based on a recombinant immunotargeting anti-body framework.

Animals↗
PubMed1995

Recombinant antibodies containing an engineered B-cell epitope capable of eliciting conformation-specific antibody responses.

The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody specific for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, specifically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its specificity for mouse I-Ak. Although mouse monoclonal antibodies specific for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct was able to elicit an A loop-specific, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes.

Amino Acid Sequence↗
PubMed1994

Characteristics of heterologous beta 2-m exchange into H-2Db at the cell surface.

W6/32 is a monomorphic anti-HLA class I Ab that cross-reacts with the product of the exchange of murine beta 2-microglobulin (beta 2-m) with human or bovine beta 2-m on H-2 Db. Using W6/32 we have developed a simple and rapid flow cytometric method to measure the beta 2-m exchange kinetics for Db at the surface of intact H-2b cell lines. We find that 10 to 25% of the Db heavy chains exchange bound beta 2-m for soluble beta 2-m with a t1/2 of 10 to 15 min. The kinetics of the exchange are consistent with a mechanism that includes a free heavy chain intermediate on the reaction coordinate. The remainder of Db molecules appear to be refractory to beta 2-m exchange even after long-term culture with an exogenous source of beta 2-m. The exchange process proceeds at a similar rate on wild-type cells that bear a diverse complement of autologous peptides on their class I molecules, and on mutant RMA-S cells having class I molecules primarily occupied with a single, defined, high affinity synthetic peptide Ag. The t1/2 for dissociation of a radiolabeled analogue of a high affinity naturally presented peptide Ag is 10 h or more, implying that the free heavy chain intermediate in the beta 2-m exchange mechanism retains bound peptides. Further, we find that synthetic peptide Ags added to non-mutant cells bind both to class I molecules that subsequently exchange bound beta 2-m, and to molecules that retain endogenous beta 2-m.

Amino Acid Sequence↗
PubMed1993

Studies of the adjuvant-independent antibody response to immunotargeting. Target structure dependence, isotype distribution, and induction of long term memory.

Immunoconjugates composed of avidin linked to biotinylated antibodies specific for different surface determinants on cells of the immune system were evaluated for their ability to induce adjuvant-independent anti-avidin IgG responses in mice. Previously, we demonstrated that allele-specific murine anti-class II MHC-avidin immunoconjugates were immunogenic in mice bearing the appropriate haplotype. Herein we report the immunotargeting potential of heterologous anti-class II MHC antibodies specific for framework determinants, and extend the range of effective targets to include certain non-MHC structures present on APC (e.g., 33D1 on dendritic cells, 14.8 on B cells, and CD45--the leukocyte common antigen). However, antibodies with other specificities (e.g., leukocyte integrins and some macrophage markers) were not effective targeting vehicles. Surprisingly, immunoconjugates specific for CD3 and CD4 were immunogenic. The isotype distribution of the anti-avidin antibody response induced in mice by immunotargeting to class II MHC or 33D1 was similar to that induced by immunization with Ag emulsified in complete Freund's adjuvant. Most of the antibody induced was IgG1 (65-75%), but a significant proportion was IgG2a (20-30%). We also demonstrate that immunotargeting is able to prime for long-term immunologic memory in mice.

Animals↗
PubMed1993

Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes.

Although it is clear that each component of the class I MHC trimolecular complex (heavy chain, beta 2m, and antigenic peptide) contributes to its formation and stability, the specific interaction governing assembly and disassembly remain to be clarified. In an effort to address these issues using purified H-2Db molecules, we used a solid-phase binding assay recently developed in our laboratory to quantify kinetic parameters for class I assembly and disassembly. It was found that the influenza NP peptide Y367-374 associated with preformed empty complexes of 28-14-8S- (i.e., anti-alpha 3) bound Db beta 2m dimers much more quickly (t 1/2 < 0.2 h at 22 degrees C) than it did when coincubated with an anti-alpha 3 bound Db and human beta 2m (t1/2 3.5 h at 22 degrees C). The previously reported potential for the NP peptide Y367-374 to interact directly with free Db heavy chains and configure the conventionally beta 2m-dependent B22 epitope in the absence of beta 2m, was confirmed using our assay system. However, the rate of B22 epitope formation induced in the Db heavy chain by NP Y367-374 was considerably slower (t1/2 13 h, at 22 degrees C) and much less efficient on a molar basis than that resulting from the addition of beta2m (t1/2, 0.75 h, at 22 degrees C). In contrast, the Db heavy chain with NP-Y367-374 was more resistant to thermal disassembly (as measured by loss of the B22 epitope, t1/2 2h, 37 degrees C) than the Db beta 2m empty dimer (t1/2 0.2 h). Finally, stability of the preformed trimolecular complex of heavy chain, beta 2m, and peptide was found to diminish in accordance with deviation of the peptide from the optimal length and with increasing temperature from 4 to 37 degrees C.

Amino Acid Sequence↗
PubMed1993

High occupancy binding of antigenic peptides to purified, immunoadsorbed H-2Db beta 2m molecules.

In an effort to examine the peptide binding properties of purified class I MHC molecules, we have developed a solid phase, radiolabeled peptide binding assay based on the use of H-2Db molecules bound to agarose beads via heavy chain-specific mAb. Using purified Db beta 2m, recovered from RMA-S cells and bound to immunoadsorbent beads through either alpha 1 or alpha 3 region specific antibodies, complete occupancy of these molecules could be achieved with 125I-Y366-374 influenza nucleoprotein peptide (Kd 10(-7) M). Approximately 12% of the Db beta 2m dimers recovered from RMA cells could be occupied by this influenza nucleoprotein peptide under the same conditions. When free Db heavy chains were isolated from beta 2m negative R1E.Db cells by bead-bound alpha 3-region specific antibody (28-14-8S) and were incubated with human beta 2m, high affinity (Kd 10(-8) M) binding sites were created for the 125I-Y367-374 influenza nucleoprotein peptide. In addition to demonstrating that a significant fraction of the heavy chains present in R1E.Db cells are in a beta 2m-reactive form, the R1E.Db cells provide an alternate approach to that of RMA-S derived Db beta 2m empties for the creation of homogeneous complexes of Db, beta 2m, and antigenic peptide. We anticipate that these bead-bound empty and defined peptide-class I complexes may be useful in the further study of class I MHC target structure formation and recognition.

Amino Acid Sequence↗
PubMed1993

The immunotargeting approach to adjuvant-independent immunization with influenza haemagglutinin.

By conjugating the bromelain fragment of the influenza virus haemagglutinin (BHA) to a mouse monoclonal antibody specific for a species-crossreactive, framework determinant on class II major histocompatibility complex molecules, the adjuvant-independent immunotargeting approach could be compared with more conventional immunizations involving Freund's complete adjuvant (FCA) and alum, in different species. Both rabbits and ferrets made significant virus-blocking IgG responses to the immunotargeted BHA in the absence of adjuvant, and no adverse reactions were observed with the conjugate immunizations. As measured by enzyme-linked immunosorbent assay, these responses were equal to those observed with alum, and were approximately one-quarter of the level seen with FCA. In terms of influenza virus haemagglutination inhibition, immunotargeting appeared to be more effective than alum, but less effective than FCA. Collectively, the results indicate that immunotargeting may provide a safe and effective alternative to the use of adjuvants in the development of defined subunit vaccines.

Adjuvants, Immunologic↗
PubMed1993

Adhesion-mediated enhancement of the adjuvant activity of alum.

Alum, the only adjuvant currently licensed for use in humans, fails to adsorb influenza virus haemagglutinin (BHA) and is a poor adjuvant for this antigen. A specific monoclonal anti-BHA antibody adsorbed to alum promoted adhesion of the antigen to the adjuvant. The 'alum-anti-BHA-BHA' complex was found to be 1500-fold more immunogenic in mice and 5-fold more immunogenic in rabbits than a mixture of alum and BHA lacking the anti-BHA antibody. The biotin-binding protein, avidin, also mediated adsorption of biotinylated BHA to alum, and enhanced its immunogenicity to a comparable extent. These results indicate that the adjuvant activity of alum can be markedly enhanced by promoting a physical association between the antigen and the adjuvant.

Adhesiveness↗
PubMed1991

Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis.

Antigenic peptides are presented to CD8+T lymphocytes by class I major histocompatibility complex (MHC) molecules. Peptides specifically bind to purified class I molecules in vitro, and to class I molecules on cells at nonphysiological temperatures. We report here the kinetic and equilibrium parameters for the binding of radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter homologues) to the murine H-2Db molecule on intact, viable cells at 37 degrees C. In contrast to earlier reports, we show that peptide binding is rapid and reversible, with dissociation constants ranging from nanomolar to micromolar, suggestive of typical ligand-receptor interactions. Only 10% of cell-surface Db molecules can bind these peptides. To address the relationship between peptide binding and T-cell recognition of the antigen-MHC complex, we determined the minimum number of complexes required to sensitize a target cell for lysis by class I-restricted cytotoxic T-lymphocytes. Our data indicate that EL4 thymoma cells (H-2b) can be sensitized for lysis by cytotoxic T-lymphocytes when as few as 200 class I-peptide complexes (less than 0.08% of surface Db molecules) are present per cell.

Amino Acid Sequence↗
PubMed1991

Adjuvant-independent immunization by immunotargeting antigens to MHC and non-MHC determinants in vivo.

Using avidin as a model protein antigen, and biotinylated monoclonal antibodies as a convenient means of forming stable complexes with avidin, we have investigated the adjuvant-independent immunization of three mouse strains, C57BL/6, C3H and (C57BL/6 x C3H)F1, with immunoconjugates targeted to different class II MHC and non-MHC sites. The results confirm the effectiveness of anti-I-Ak and anti-I-Ab immunoconjugates with respect to priming for secondary IgG responses in (H-2b x H-2k)F1 mice, while indicating a lack of response in strains which are homozygous for the targeted allele. In terms of non-MHC targets in the monocyte-macrophage lineage, neither anti-MAC-1 nor anti-MAC-2 immunoconjugates were effective in any of the three strains. However, the 33D1 anti-dendritic cell antibody gave significant responses in all three strains, with the F1 response being more than 10-fold greater than the anti-class II immunoconjugates in either strain. These findings indicate that immunotargeting a protein antigen to a non-MHC determinant on dendritic cells in vivo can be an effective means of inducing an adjuvant-independent serological response, and that this approach can have significant advantages over anti-class II MHC immunotargeting.

Adjuvants, Immunologic↗