Single-chain antibody fragments specific to the hepatitis B surface antigen, produced in recombinant tobacco cell cultures

An anti-hepatitis B surface antigen (HBsAg), single-chain Fv antibody fragment (scFv) with a 6-histidine N-terminal tag was produced in cultured transgenic tobacco cells. Western blot and antigen-specific chromatography showed high levels of biologically active scFv in the culture supernatant (1 mg l^–1) and in cells (5 mg kg^–1). A simple one-step scFv purification was developed using immobilized metal ion affinity chromatography.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.     

Self-tolerance checkpoints in CD4 T cells specific for a peptide derived from the B cell antigen receptor

Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germline-encoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded κ L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vκ peptide encoded by the κTg. In chimeras where few B cells express the κTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 single-positive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR α-chain. CD4(+) CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.     

Human antibody immunosorption of hepatitis B surface antigen from blood and plasma

Removal of the Hepatitis B surface antigen (HBsAg) from whole blood and blood products, using human antibody (HBAb) immunosorbent, was studied and kinetics of complexing were monitored using radioimmunoassay (RIA). An intermittent complexing process was developed that minimizes damage to the cellular components of blood. HBsAg concentration in blood was reduced 1.5 to 2 logarithmic cycles in 3 hr with this system. Free HBsAg remaining in solution at equilibrium was further reduced by transferring the blood to a vessel containing unused immunosorbent. Through multiple stage treatment of a blood sample, it may be possible to reduce the probability of contamination with HBsAg to below the infectious level. This process may be applied to the selective removal of other proteins from blood and plasma.     

The naive repertoire of human T helper cells specific for gp120, the envelope glycoprotein of HIV.

The envelope glycoprotein of HIV gp120 is a T cell Ag in experimental animals and in humans infected with HIV or deliberately immunized with gp120 in various forms. Inasmuch as T cell responses result from the interaction of Ag processed and presented by APC with the unprimed T cell repertoire, we have investigated the human T cell repertoire specific for gp120 in seronegative, normal individuals. T cell lines and clones specific for HIV gp120 were generated by repeated in vitro stimulation of peripheral blood T lymphocytes with gp120-pulsed APC, followed by IL-2 expansion. We observed that the T cell response to whole gp120 involved single restricted immunodominant epitopes in gp120 that differ between responding individuals. Focusing of the response to limited regions of gp120 when the whole Ag is used for priming suggests that one or more adjacent epitopes are immunodominant and mask responses to "immunorecessive" epitopes. We have been able to generate primary in vitro responses to recessive epitopes by stimulation in vitro with synthetic peptides of gp120. The results indicate that a much broader T repertoire can be detected when individual peptides are used for priming in vitro rather than gp120. This information has important implications for the development of vaccination protocols aimed at eliciting diverse immune responses to "immunorecessive" regions of envelope glycoprotein.     

"Universal" T helper cell determinants enhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide.

Synthetic peptide constructs containing a limited number of epitopes are being currently investigated as subunit vaccines against a variety of pathogens. However, because of widespread nonresponsiveness to most such constructs, possibly attributable to MHC restriction, the choice of appropriate carrier molecules to enhance immunogenicity of peptides constitutes an important and essential aspect of designing synthetic immunogens for human use. Widely used vaccines such as tetanus toxoid (TT) have not been uniformly effective as carrier proteins because of the phenomenon of epitope-specific suppression in which induction of an immune response against a synthetic peptide conjugated to TT is prevented by preexisting immunity to TT. Recently, T cell determinants that can be recognized in the context of several class II MHC molecules have been identified in tetanus toxin as well as in the circumsporozoite protein of a human malarial parasite, Plasmodium falciparum. Such determinants can be potentially used to circumvent the problem of epitope-specific suppression. In the present study we evaluated two such T cell determinants, viz., tt830-844 from tetanus toxin and CST3 from the malarial parasite, for their ability to help induce a boostable antibody response and to overcome genetic nonresponsiveness to a synthetic 20-residue construct containing a B cell and an overlapping T cell epitope from a major merozoite surface protein of P. falciparum. Our data provide support for the view that widely recognized T cell determinants may be used as universal carrier molecules for general vaccination.     

Selective functional depletion of HIV gp120 peptides complexed with MHC from antigen-presenting cells engaged with specific T lymphocytes.

Human T cell lines specific for different peptides of HIV envelope glycoprotein gp120 have been used as probes to identify the availability of functional MHC-peptide complexes on APC. MHC-peptide complexes recognized by T cells specific for peptide 24 (amino acids 225-240) are no longer available on the surface of APC after interaction with irradiated (binding nonproliferating) T cells with the same fine specificity. On the contrary, MHC-peptide complexes recognized by T cells specific for peptide 30 (amino acids 285-300) were functionally available and could stimulate T cells with such a specificity. The reciprocal experiment yielded similar results. The same data were also reproduced with another pair of gp120 peptides. These data demonstrate that upon clustering of peptide-specific T cells with presenting cells presentation of the same peptide to a second cohort of T cells with identical specificity is abolished, suggesting that a selective functional depletion of the MHC-peptide complexes engaged with specific T cells occurs at the surface of the presenting cells. The depletion does not affect other MHC molecules complexed with unrelated peptides.     

A monoclonal antibody specific for the Thomsen-Friedenreich cryptic T antigen

A monoclonal antibody (49H.24) is described that was made after immunization of BALB/c mice with human neuraminidase-treated erythrocytes (NE-RBC). 49H.24 is an IgM and reacts with NE-RBC but not untreated, normal human RBC. As little as 100 pg of antibody protein produced detectable direct agglutination of, or binding to, NE-RBC. The fine specificity of 49H.24 was determined by using a series of synthetic sugar haptens as inhibitors of agglutination or binding of 49H.24 to NE-RBC. Only synthetic T hapten (beta Gal(1 leads to 3)alpha GalNAc) produced complete inhibition of agglutination or binding, and no inhibition was produced by several other closely related haptens. Synthetic T hapten-coated silica beads (Synsorb) were used to affinity purify 49H.24. Affinity-purified antibody was radiolabeled with 125I and used in a sensitive competition assay to detect natural T antigen associated with cell membranes or in soluble form. The potential use of this or similar monoclonal antibodies as probes for an important human tumor marker is discussed.     

Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.     

Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen.

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.     

A yeast system for stable expression of hepatitis B surface antigen.

We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.     

Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.     

A CCR5-dependent novel mechanism for type 1 HIV gp120 induced loss of macrophage cell surface CD4

Type 1 HIV gp120 is especially effective in disrupting immune cell function because it is able to cause dysregulation of both infected and uninfected cells. We report a novel CCR5-dependent mechanism of gp120-induced CD4 loss from macrophages. An M-tropic gp120, using CCR5, is able to induce 70% loss of cell surface CD4 from macrophages within an hour. This cell surface CD4 loss is more substantial and rapid than the 20% loss observed with T-tropic gp120(IIIB) by 3 h. The rapid and substantial CD4 loss induced by M-tropic gp120 is not observed on macrophages homozygous for the ccr5Delta32 mutation, which fail to express cell surface CCR5. We have used confocal imaging to show that gp120 and CD4 are internalized together by a process resembling receptor-mediated endocytosis, and that both proteins enter HLA-DR containing compartments of the macrophage. We have also shown by semiquantitative RT-PCR that, in response to CD4 loss from the cell surface, mRNA for CD4 is up-regulated and the intracellular pool of CD4 increases. CCR5 mRNA levels are also increased. It is proposed that internalization of self and viral protein and increased pools of intracellular CD4 could modulate Ag presentation efficiencies and have implications for the induction and maintenance of both productive immune responses and self-tolerance.     

Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen.

A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli. Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.     

Design of peptide mimetics of HIV-1 gp120 for prevention and therapy of HIV disease.

It has been reported that the C-terminus of the second conserved region (C2) of the envelope glycoprotein gp120, encompassing peptide RSANFTDNAKTIIVQLNESVEIN (NTM), is important for infectivity and neutralization of the human immunodeficiency virus type 1 (HIV-1). It was also demonstrated that human natural anti-vasoactive intestinal peptide (VIP) antibodies reactive with this gp120 region play an important role in control of HIV disease progression. The bioinformatic analysis based on the time-frequency signal processing revealed non-obvious similarities between NTM and VIP. When tested against a battery of sera from 46 AIDS patients, these peptides, in spite of a significant difference in their primary structures, showed a similar reactivity profiles (r = 0.83). Presented results point out that similarity in the periodical pattern of some physicochemical properties in primary structures of peptides plays a significant role in determination of their immunological crossreactivity. Based on these findings, we propose this bioinformatic criterion be used for design of VIP/NTM peptide mimetics for prevention and treatment of HIV disease.     

Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.     

A hepatitis B surface antigen mutant that lacks the antigenic loop region can self-assemble and interact with the large hepatitis delta antigen

A novel hepatitis B virus surface antigen mutant harboring a deletion of most of the major antigenic loop region was competent for self-assembly and secretion. Although the mutant protein was competent for interaction with and incorporation of free large hepatitis delta antigen, it was partially defective in hepatitis delta virus RNP incorporation.