Biomedical subjects

C Y Kang

Publications and source records attributed to C Y Kang.

At least 37 records · Page 2Linked to original sources

PubMed1994

Analysis of B cell repertoire specific to the neutralizing epitopes of glycoprotein 120 in HIV-infected individuals.

This study describes the clonotypic analysis of neutralizing anti-gp120 antibodies elicited in HIV-infected individuals by a panel of anti-idiotype monoclonal antibodies (anti-Id MAbs). Sera from 80 HIV-infected individuals at various clinical stages of HIV-infection were tested for reactivity to 19 anti-Id MAbs in ELISA. Anti-idiotype MAbs reacted with between 0 and 26% of sera. Among the 13 idiotypes specific for anti-CD4 site antibodies, 4 were expressed in 15 to 20% of individuals, whereas 2 of 4 idiotypes specific for anti-V3 antibodies were expressed in 15 to 26% of the cases. These data suggest that each HIV-infected individuals has a diverse B cell repertoire to a given neutralizing epitope cluster and that certain clonotypes are more prevalent than others. To correlate the binding activity in ELISA with anti-gp120 specificity, the idiotype-positive antibodies (Id+ Abs) from representative serum samples were isolated by anti-Id MAb-Sepharose affinity columns. In most cases, the epitope specificity and the neutralizing properties of the isolated Id+ Abs correlated with that of anti-gp120 antibodies used for the generation of anti-Id MAbs. We propose that these anti-Id MAbs may be used to identify and measure neutralizing anti-gp120 antibodies of defined specificity in the sera of HIV-infected individuals, HIV-vaccinated individuals, and in HIV-infected mother-infant pairs.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120.

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.

Animals↗

PubMed1993

Identification of a new neutralizing epitope conformationally affected by the attachment of CD4 to gp120.

We have developed a strategy to purify and characterize various anti-gp120 antibody populations in HIV+ sera by using anti-Id mAb. One preparation of human anti-gp120 antibody (ES+ Ab) isolated on an anti-Id mAb (ES)-conjugated immunoabsorbent exhibited a novel neutralizing epitope specificity. The ES+ Ab bound only to the native form of recombinant gp120SF2 and gp120IIIB and not to the third hypervariable region (V3) loop peptide. In contradistinction to other CD4-gp120-inhibiting and V3-specific neutralizing antibodies, ES+ Ab exhibited a dose-dependent enhancement of binding to recombinant gp120 in the presence of recombinant soluble CD4. In addition, flow cytometric analysis revealed a similar increase in the binding of ES+ Ab to the native form of gp120 expressed on the HIV-infected cells. The ES+ Ab competed with CD4 binding site- and V3-specific antibodies in binding to gp120, suggesting that the ES+ Ab epitope is located near the CD4 binding site epitope and the V3 region. The ES+ Ab neutralized six genetically distinct HIV-1 strains. The neutralizing activity of ES+ Ab on HIVIIIB was significantly increased in the presence of human anti-CD4 binding site mAb. These data suggest that the ES+ Ab epitope represents a conserved, conformational, neutralization target on gp120 that may be involved in viral infection in an event after the CD4-gp120 interaction and that is distinct from previously defined neutralizing epitopes of gp120. This finding may be important for the development of an AIDS vaccine and immunotherapy.

Amino Acid Sequence↗

PubMed1993

Comparison of nucleotide sequences among hantaviruses belonging to the same serotype: an analysis of amplified DNA by thermal cycle sequencing.

The hantavirus genus, belonging to the bunyaviridae family, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. Previously, we reported the use of the polymerase chain reaction (PCR) for grouping hantavirus isolates by using four sets of primers specific to each serotype. Our PCR typing results agreed with those of serological typing. The present study makes use of thermal cycle sequencing to sequence PCR-amplified DNA products in order to determine the level of similarity among members of the same serotype. We show that members of Hantaan and Seoul serotypes are over 92% homologous, irrespective of their host and geographical origin. Puumala sequences show a degree of homology ranging from 80 to 98%. Despite the variation in sequence at the nucleotide level, amino acids show an even higher level of conservation.

Amino Acid Sequence↗

PubMed1993

Glycosylation is necessary for the correct folding of human immunodeficiency virus gp120 in CD4 binding.

Conflicting results have been reported regarding the role of carbohydrate on human immunodeficiency virus (HIV) envelope glycoprotein gp120 in CD4 receptor binding. Glycosylated, deglycosylated, and nonglycosylated forms of HIV type 1 (HIV-1) and HIV-2 gp120s were used to examine CD4 receptor-binding activity. Nonglycosylated forms of gp120 generated either by deletion of the signal sequence of HIV-1 gp120 or by synthesis in the presence of tunicamycin failed to bind to CD4. In contrast, highly mannosylated gp120 bound to soluble CD4 molecules well. Enzymatic removal of carbohydrate chains from glycosylated gp120 by endoglycosidase H or an endoglycosidase F/N glycanase mixture had no effect on the ability of gp120 to bind CD4. An experiment which measured the ability of gp120 to bind to CD4 as an assay of the proper conformation of gp120 showed that carbohydrate chains on gp120 are not required for the interaction between gp120 and CD4 but that N-linked glycosylation is essential for generation of the proper conformation of gp120 to provide a CD4-binding site.

CD4 Antigens↗

PubMed1993

Analysis of the cross-reactive anti-gp120 antibody population in human immunodeficiency virus-infected asymptomatic individuals.

This study was undertaken to analyze the specificity and neutralizing properties of cross-reactive anti-gp120 antibodies (Abs) in the sera of two human immunodeficiency virus (HIV)-infected asymptomatic individuals. Two panels of murine monoclonal anti-idiotype Abs (anti-id MAbs) were established against cross-reactive polyclonal anti-gp120 Abs purified from HIV+ sera by sequential affinity chromatography using gp120SF2- and gp120IIIB-Sepharose columns. These panels of anti-id MAbs were then used to affinity purify idiotype-positive (Id+) anti-gp120 Abs from HIV+ sera. The recovery of each of these Id+ Abs by purification indicated that several idiotypically distinct cross-reactive anti-gp120 Abs are present in sera over a wide range of concentrations. Immunological and biological studies showed that although all of the Id+ Abs were reactive against gp120SF2 and gp120IIIB, they exhibited unique epitope specificities and distinct neutralizing activities. Most of the Id+ Abs were directed against epitopes in the CD4 attachment site (CD4 site epitopes) of gp120 and exhibited a spectrum of broadly neutralizing activities. On the other hand, a minor population of Id+ Abs showed specificity for the V3 region of gp120 and exhibited limited cross-neutralizing activities. Together, these studies indicate that the CD4 site epitope-specific Abs are heterogeneous with respect to their clonality, neutralizing activity, and concentration in sera. This heterogeneity suggests that anti-gp120 Abs to the CD4 attachment site are developed in response to multiple overlapping epitopes present on the original virus isolate and/or epitopes on mutated variants which emerged over time.

Amino Acid Sequence↗

PubMed1993

Viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) has been found to promote assembly and budding of virions as well as down-regulating of VSV transcription. Large quantities of M protein can be produced in insect cells infected with recombinant baculovirus expressing the VSV M gene under control of the polyhedrin promoter. Analysis by pulse-chase experiments and density gradient centrifugation revealed that the [35S]methionine-labeled M protein synthesized in insect cells is released into the extracellular medium in association with lipid vesicles (liposomes). Electron microscopy and immunogold labeling showed that M protein expressed in insect cells induced the formation on plasma membrane of vesicles containing M protein, which are released from the cell surface in the form of liposomes. The baculovirus vector itself or recombinants expressing VSV glycoprotein (G) or nucleocapsid (N) protein did not produce the formation of vesicles in infected cells. The baculovirus-expressed M protein retains biological activity as demonstrated by its capacity to inhibit transcription when reconstituted with VSV nucleocapsids in vitro. These data suggest that M protein has the capacity to associate with the plasma membrane of infected cells and, in so doing, causes evagination of the membrane to form a vesicle which is released from the cell. This observation leads to the postulate, which requires further proof, that the VSV M protein can induce the formation and budding of liposomes from the cell membrane surface.

Animals↗

PubMed1992

Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.

Animals↗

PubMed1992

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.

Animals↗

PubMed1992

Anti-idiotype monoclonal antibody elicits broadly neutralizing anti-gp120 antibodies in monkeys.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.

Animals↗

PubMed1992

Low-cost hepatitis B vaccine improves uptake among self-paying health-care students.

Advisory committees recommend hepatitis B (HBV) immunization for professional and student health-care workers. However, the currently licensed vaccines are expensive, and previous surveys have shown that few students (14%) have been immunized in Canada. A low-cost immunization program was offered to health-care students in order to determine whether the effectiveness of HBV immunization could be improved by substantially reducing the vaccine cost to recipients. The immunogenicity, side effects, and 3-dose completion rate of a low-cost Korean HBV vaccine were compared with a similar U.S.-made vaccine. A total of 922 postsecondary students enrolled in 6 health-care disciplines in Ottawa, Canada were surveyed for hepatitis-B immunization status. Nonimmunized students were subsequently offered HBV vaccine at total cost of $15 (Canadian), randomly allocated to receive 3 intramuscular doses of either Korean or U.S.-made plasma-derived HBV vaccine in a double-blind fashion, surveyed about side effects, and tested for hepatitis B surface antibody seroconversion. Only 12% of the 922 surveyed students had been previously immunized when vaccine was obtainable only at high cost. However, 66% of those not immunized participated in the vaccine trial and paid the $15 fee. Hepatitis-B surface antibody seroconversion (greater than or equal to 10 sample ratio units by radioimmunoassay) occurred in 291/311 (93.6%) and 299/310 (96.5%) of recipients of 3 doses of the Korean and U.S. vaccines, respectively (P = 0.10). There were no meaningful differences in vaccine adverse effects, and 92.6% of recipients of either vaccine completed 3 doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

Maturation of Hantaan virus glycoproteins G1 and G2.

Hantaan virus-infected Vero E6 cell lysates were used for immunoprecipitation with monoclonal antibodies against glycoprotein G1 (MAbG1) or G2 (MAbG2). When cell lysates were prepared with buffer containing nonionic detergent, both G1 and G2 glycoproteins were precipitated with either MAbG1 or MAbG2. In contrast, when cell lysates were prepared with a buffer containing ionic detergents MAbG1 precipitated only glycoprotein G1 and MAbG2 precipitated only glycoprotein G2. Heterodimers and possibly higher oligomeric forms of the glycoproteins were detected on nonreducing SDS-polyacrylamide gels only after chemical cross-linking and immunoprecipitation with either MAbG1 or MAbG2. In order to determine the sites of Hantaan virus glycoproteins maturation and the G1-G2 complex formation, infected cells were treated with inhibitors that prevent specific steps of oligosaccharide processing. Furthermore, glycoproteins G1 and G2 immunoprecipitated from infected cell lysates or from isolated virus particles were tested for sensitivity to endoglycosidase H, endoglycosidase F, and endoglycosidase D. The results of these experiments show that maturation of both G1 and G2 takes place in the endoplasmic reticulum (ER). Furthermore, G1-G2 complex formation occurs in the ER as well, since the two glycoproteins co-precipitated with either MAbG1 or MAbG2 from infected cell lysates treated with brefeldin A and prepared with buffer containing nonionic detergent.

Animals↗

PubMed1992

Comparison of the deduced gene products of the L, M and S genome segments of hantaviruses.

The amino acid sequences deduced from all currently available nucleotide sequences of hantaviruses are compared. Comparisons of three large (L), eight medium (M) and five small (S) genome segments are included. A consensus sequence is provided, allowing easy identification of conserved and unique gene regions. The viruses included in this report represent four serologically distinct hantaviruses which are capable of causing severe, moderate, mild or no human disease.

Amino Acid Sequence↗

PubMed1992

Evolution of the fusion protein gene of human parainfluenza virus 3.

The nucleotide sequences of the fusion (F) gene of 15 clinical strains of human parainfluenza virus 3 (HPIV3) isolated between 1959 and 1987 were compared with the F gene sequence of the prototype strain, Wash/47885/57. Nucleotide sequence diversity was greatest in the noncoding regions of the F gene; however, regions believed to function as transcriptional signals were completely conserved. Amino acid sequences were highly conserved and all but a few amino acid substitutions were conservative in nature. Sequence comparisons indicate heterogeneity in HPIV3 F genes; however, a significant proportion of nucleotide changes are maintained after they first appear and seem to be accumulating with time. Phylogenetic analysis suggests that there are 2 lineages of HPIV3 in North America. The two lineages can be distinguished by specific amino acid differences in the F protein, which correlate with differences in antigenic properties and neutralization patterns of HPIV3. The pattern of HPIV3 evolution, based on the analysis of F gene sequences, most closely resembles that of influenza virus B, vesicular stomatitis virus and Newcastle disease virus.

Amino Acid Sequence↗

PubMed1992

Typing of Hantaviruses from five continents by polymerase chain reaction.

Hantavirus, a genus in the family Bunyaviridae, is comprised of at least four serologically distinct types: Hantaan, Seoul, Puumala and Prospect Hill. The present communication reports the use of polymerase chain reaction (PCR) for typing 27 independently isolated Hantaviruses from 5 different continents. Total cellular RNA was extracted from virus-infected Vero E6 cell monolayers by the acid guanidium thiocyanate-phenol-chloroform method. We have utilized 5 different sets of oligonucleotide primers ranging from 18 to 22 nucleotides in length; one set was specific for a conserved region of the S genomic segment and used as genus-specific primers, the other 4 sets of primers were designed from unique sequences of the M genomic segment such that each primer set was specific to only one serological type of Hantavirus. The PCR products were analyzed by restriction endonuclease digestion for further confirmation. We typed 10, 12, 3 and 1 isolates into Hantaan, Seoul, Puumala and Prospect Hill respectively. The results of PCR were 100% agreeable with that of serological typing, and thus, PCR can be used as an adjunct test with serological method(s) or an independent test for diagnosis and for typing of new isolates of Hantaviruses.

Animals↗

PubMed1992

[Renal pelvic tumor associated with contralateral renal cyst--a case report].

A 74-year-old female presented with a two years history of malaise, weakness and intermittent gross hematuria. Two years earlier, left renal cyst had been diagnosed by excretory urography, abdominal sonography and computed-tomography. Initially she was treated conservatively with oral antibiotics, however medical management had not been successful in improving her condition. Finally she was transfer to our ward for further evaluation and treatment. Cystoscopic examination demonstrated no abnormality, except for bloody urine efflux from the right ureteric orifice. Right retrograde pyelograms confirmed a solitary filling defect in the upper calyx of right kidney. Because of immediate concern for right renal pelvic tumor and contralateral renal cyst, a midline transperitoneal exploration was made. The right nephroureterectomy with cuff of bladder was removed and left unroofing of cyst was carried out. Convalescence was uneventful. The pathologic examination revealed moderately differentiated transitional cell carcinoma of right kidney with a simple renal cyst of left kidney. We suggest that cases with a clinical diagnosis of renal cyst based on the presence of gross hematuria, a normals excretory urogram and computed tomogram, generally require further evaluation especially by cystoscope and retrograde pyelogram to determine the source of bleeding.

Aged↗

PubMed1992

Synthetic peptides from a conserved region of gp120 induce broadly reactive anti-HIV responses.

In our efforts to identify products that might be used for active immunotherapy in human immunodeficiency virus (HIV) infection, we have studied synthetic peptides derived from the CD4 attachment site of gp120. Two peptides have emerged with particularly interesting properties. The first (B138) is linear and spans the envelope residues 421-438; the second (1005/45) encompasses amino acids 418-445 and is cyclized by way of a disulphide bond joining its terminal cysteines. Both species have been shown to inhibit syncytial formation in a conventional bioassay, B138 being the most efficient. Both peptides elicit high titres of anti-peptide antibodies in immunized mice, rabbits and goats, with titres exceeding 1:10(5) in many cases. A substantial portion of this response is directed against gp120 as determined by enzyme-linked immunosorbent assay (ELISA). Analysis by flow cytometry has demonstrated that the antisera are broadly reactive with multiple diverse strains of HIV. The anti-gp120 activity of the anti-peptide antiserum was further confirmed by radioimmuno-precipitation (RIP) assays. Furthermore, RIP analysis and inhibition experiments in a GD4-gp120 binding assay have revealed that anti-peptide sera contain antibodies directed against the CD4 attachment site on gp120 and interfere with this receptor-ligand interaction.

Animals↗

PubMed1991

Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans.

Total anti-gp120 antibodies (total anti-gp120 Abs) were purified from a pool of four human immunodeficiency virus-positive (HIV+) sera by affinity chromatography on a gp120SF2-Sepharose column and exhibited both type- and group-specific neutralizing activities. To dissect the epitope specificity of the group-specific neutralizing antibodies, CD4 attachment site-specific antibodies (CD4-site Abs) were isolated from total anti-gp120 Abs by using a CD4-blocked gp120SF2-Sepharose column. The CD4-site Abs exhibited group-specific neutralizing activities. Another approach to dissecting type- and group-specific neutralizing activities of total anti-gp120 Abs was to separate the third variable region (V3)-specific antibodies (V3-region Abs) from non-V3-region-specific antibodies (non-V3 Abs). The results indicated that V3-region Abs exhibited type-specific neutralizing activities, whereas non-V3 Abs exhibited group-specific neutralizing activities. By comparing the neutralizing activities of V3-region Abs to those of non-V3 Abs, we concluded that V3-region Abs are more effective than non-V3 Abs in neutralizing a specific HIV isolate. Collectively, this study indicates that group-specific neutralizing anti-gp120 antibodies are specific for the CD4 attachment site.

Acquired Immunodeficiency Syndrome↗