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Z Q Xiang

Publications and source records attributed to Z Q Xiang.

16 recordsLinked to original sources
PubMed1998

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Amino Acid Sequence↗
PubMed1997

The effect of interferon-gamma on genetic immunization.

The effect of co-inoculation of a plasmid vector expressing the rabies virus glycoprotein and an additional vector encoding mouse interferon (IFN)-gamma on the development of an antigen specific B and T helper cell response was tested upon intramuscular inoculation of mice. The effect of IFN-gamma was dependent on the promoter driving expression of the viral antigen. The immune responses to antigen-expressing vector carrying a viral promoter such as the SV40 early promoter or the major histocompatibility (MHC) class I promoter were reduced in presence of IFN-gamma while the B and T helper cell response to a vector expressing the antigen under the control of the MHC class II promoter was not affected by this cytokine.

Animals↗
PubMed1996

The effects of post-translational side-chain modifications on the stimulatory activity, serum stability and conformation of synthetic peptides carrying T helper cell epitopes.

Peptides 31D and VF13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68-76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55-64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, beta-linked sugars in mid-chain positions had a greater inhibitory effect than alpha-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the beta-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of beta-linked carbohydrates might be superior to the addition of alpha-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant alpha-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the alpha- and beta-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.

Amino Acid Sequence↗
PubMed1996

A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier.

In this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant.

Adenovirus E1 Proteins↗
PubMed1996

Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines.

Peptides carrying an immunodominant T-helper cell epitope delineated from the rabies virus nucleoprotein either alone or in combination with a linear B-cell epitope from the same protein were incorporated into three different formulations of poly(DL-lactide-co-glycolide) (PLG) which were distinct in their composition, and consequently in their peptide release rates. In vitro peptides incorporated into any of the PLG formulations stimulated a peptide-specific T-cell line. Upon subcutaneous immunization of mice, the PLG formulation that showed the fastest peptide release rate induced the best immune response. This immune response was in magnitude comparable or even superior to that induced by peptide emulsified in complete Freund's adjuvant.

Amino Acid Sequence↗
PubMed1996

Genetic immunization.

Genetic immunization, the latest addition to the field of vaccinology, has shown, in a number of animal models, to be an efficacious approach to induce protective immunity to infectious diseases. The advantages of DNA vaccines are their ease of construction, the low expanse of mass production, their high temperature stability, and their ability to induce a full spectrum of exceptionally long-lasting immune responses including cytolytic T cells. Their potential disadvantages are putative safety issues such as integration into the host cell genome. The slow development of the immune response to genetic immunization will make these vaccines unsuitable for treatment of some infectious disease such as postexposure vaccination to rabies virus, where a rapid immune response is warranted. Although only time will tell if genetic immunization provides a viable alternative for human immunization, in the meantime this approach provides immunologists with a powerful tool to gain further insight in the mechanisms that drive primary immune responses.

Animals↗
PubMed1995

Immune effector mechanisms required for protection to rabies virus.

Genetically engineered mice with targeted mutations in genes encoding immunologically relevant molecules were used to elucidate the role of different immune effector mechanisms in protection against a rabies virus (RV) infection. In vaccinated animals challenged with a highly virulent strain of RV, antibodies were crucial in protection. In naive mice challenged with an attenuated strain of the virus that does not cause disease in adult fully immunocompetent mice but kills RAG mice that lack functionally active T and B cells, different immune effector mechanisms were shown to suffice for protection.

Animals↗
PubMed1995

Immune responses to nucleic acid vaccines to rabies virus.

A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.

Animals↗
PubMed1995

Comparison of the effects of amino acid substitutions and beta-N- vs. alpha-O-glycosylation on the T-cell stimulatory activity and conformation of an epitope on the rabies virus glycoprotein.

The first potential N-glycosylation site of the rabies virus glycoprotein, the antigen that carries epitopes for glycoprotein-specific T-cells and virus neutralizing antibodies, is glycosylated inefficiently. Recently, we showed that addition of a beta-N-acetyl-glucosamine moiety to the asparagine residue in the corresponding synthetic fragment V V E D E G C T N L S G F (amino acids 29-41), significantly diminished the T-cell stimulatory activity and reduced the characteristic alpha-helicity of the peptide. The amino acid sequence of the glycoprotein in this region exhibits some degree of variability among different rabies virus and rabies virus related strains, including the replacement of the asparagine residue with aspartic acid or threonine. In the current study, stimulation of a specific T-cell clone by various viral strains and appropriate tridecapeptide sequences and their analogs was investigated. The T-cell recognition pattern of the rabies and rabies-related viruses was identical to that of the synthetic peptides representing the respective epitope sequences. While the asparagine could be replaced without complete loss of T-cell stimulatory activity, amino acid modifications at the C-terminus of the peptide were not tolerated. In contrast to glycosylation of the asparagine, coupling of an N-acetyl-galactosamine moiety at the serine, or galactosyl-N-acetyl-galactosamine moieties at the threonines preceding or replacing the asparagine (all O-linked sugars in the natural alpha-anomeric configuration) resulted in epitopes that lowered rather than abolished the T-cell stimulatory activity. All non-glycosylated peptides assumed a low-to-medium helicity in trifluoroethanol. O-glycosylation was more efficient than N-glycosylation in breaking the helical conformation of the peptides to result in the formation of reverse-turns or unordered structure.

Amino Acid Sequence↗
PubMed1995

Stable secretion of a soluble, oligomeric form of rabies virus glycoprotein: influence of N-glycan processing on secretion.

Rabies virus glycoprotein (RGP) is a 505 amino acid type I transmembrane glycoprotein that is important in the pathogenesis of rabies virus infection. RGP also stimulates the development of neutralizing antibodies by the host. N-linked glycosylation is required for both cell surface expression and immunogenicity of RGP. In the current study, a soluble form of RGP, constructed by insertion of a stop codon external to the transmembrane domain, was expressed in transfected Chinese hamster ovary cells. The soluble form of RGP was found to be appropriately antigenic and immunogenic. Similar to full-length RGP, the soluble form was assembled into homodimers and homotrimers. Core glycosylation was required for secretion of soluble RGP and cell surface expression of full-length RGP. In addition, initial glucose trimming of the N-glycans was necessary and sufficient for secretion of soluble RGP and cell surface expression of full-length RGP. Further N-glycan processing was not required for secretion or cell surface expression of soluble or full-length RGP, respectively.

Animals↗
PubMed1994

Glycosylation of synthetic T helper cell epitopic peptides influences their antigenic potency and conformation in a sugar location-specific manner.

The immunodominant T helper cell epitopes 31D and VF13N of rabies virus nucleoprotein and glycoprotein, respectively, correspond to peptide sequences AVYTRIMMNGGRLKR and VVEDEGCTNLSGF, and are expressed between amino acids 404-418 and 29-41, of the appropriate proteins. We investigated how internal or external glycosylation affects the biological activity and conformation of the peptides 31D and VF13N. Mid-chain incorporation of maltobiose or N-acetylglucosamine moieties into the asparagine residues greatly diminished the T-cell stimulatory activity in vitro (due to the diminished ability of the glycopeptides to bind to major histocompatibility complex determinants) and reduced the characteristic alpha-helicity of the peptides in aqueous trifluoroethanol solutions. In contrast, addition of maltobiose- or N-acetylglucosamine-coupled asparagines to the N-termini of peptides 31D and VF13N resulted in unchanged T-cell activity. Furthermore, N-terminal glycosylation of peptide 31D, as indicated by the functional assay, decreased the sensitivity of the peptide to degradation in human serum and did not affect the alpha-helical conformation. These data indicate that glycosylation of T-cell epitopes is not a preferable method for the preparation of antagonists, but incorporation of the sugars to appropriate positions may be advantageous in the design of T-cell agonists and peptide-based vaccines.

Amino Acid Sequence↗
PubMed1994

Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus.

A plasmid vector, termed pSG5rab.gp, that expresses the rabies virus glycoprotein under the control of an SV40 early promoter, was tested in C3H/He mice for induction of rabies virus-specific immune responses. Mice immunized intramuscularly with the pSG5rab.gp vector developed rabies virus glycoprotein-specific cytolytic T cells, lymphokine-secreting T helper cells of the TH1 subset, and rabies virus-neutralizing antibodies. Mice vaccinated with the pSG5rab.gp vector were fully protected against a subsequent challenge with rabies virus.

Animals↗
PubMed1994

A simple method to test the ability of individual viral proteins to induce immune responses.

Immune responses to a mouse fibroblast line, transfected with a plasmid that causes expression of the rabies virus glycoprotein under the control of a simian virus 40 early promoter, were studied. Transfected cells were shown to be recognized in vitro by a panel of monoclonal antibodies directed to conformational epitopes of the different antigenic sites of the glycoprotein. They stimulated rabies virus glycoprotein-specific T helper cells in the presence of antigen-presenting cells and were, furthermore, recognized by rabies virus-induced cytolytic T cells. In vivo, immunization of H-2-compatible mice with the transfected cell line led to a rabies virus glycoprotein-specific antibody response, and to protection against a subsequent challenge with live virus. We propose this procedure, i.e. use of cell lines transfected with plasmids expressing a viral protein under a mammalian promoter, as a simple and inexpensive method to screen individual viral proteins for their ability to elicit immune responses, including T helper cells, cytolytic T cells, antibodies, and protection against viral challenge.

Animals↗
PubMed1992

Transfer of maternal antibodies results in inhibition of specific immune responses in the offspring.

A potentially detrimental consequence of maternally transferred antibodies was demonstrated in a mouse model for rabies virus, where pups from rabies virus-immune dams showed a decrease in the generation of specific B- and T-cell responses to immunization with rabies virus antigen, resulting in vaccine failures. The degree and duration of the vaccine failures was inversely correlated with the amounts of maternally transferred antibodies, and exceeded the time when maternal antibodies provided reliable protection against a viral challenge. The low responsiveness to vaccination, measured by serum antibody titers and by lymphokine release upon in vitro restimulation of in vivo-primed lymphocytes, was specific for the target virus of the maternal antibodies and was also observed in pups from Sendai virus-immune dams upon vaccination with the homologous virus. In addition, an inhibition of the specific immune responses was demonstrated upon passive immunization of newborn mice with monoclonal antibodies to rabies virus. Although the mechanism(s) that causes the observed inhibition in the offspring of immune dams or in pups that were inoculated with antibodies postnatally is currently unknown, data presented in this manuscript indicate that the observed effect on B- and T-cell responses might not be solely caused by removal of the antigenic load due to residual maternal antibodies.

Animals↗