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J Hinkula

Publications and source records attributed to J Hinkula.

At least 55 records · Page 3Linked to original sources
PubMed1996

Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells.

Human antibodies against HIV-1 have been sought to study neutralization events on the molecular level, and for possible use in passive immune intervention. The development of phage display techniques has opened the possibility of rapidly generating human monoclonal antibodies with desired specificities. We and others have isolated human HIV-1 neutralizing antibody fragments using this technique. Bacterial expression of isolated clones does, however, differ broadly both in expression levels and functional activity. In addition, intact IgG cannot be expressed in bacteria. By transferring the genes of isolated Fab clones to a mammalian expression system we could perform a comparison of functional activity between Fab expressed in bacterial and mammalian cells, as well as Fab and whole IgG. Fab fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in Escherichia coli, underlining the inefficiency of procaryotic expression. No difference in HIV-1 neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells. Thus, bivalency does not always confer improved neutralization efficacy.

Animals↗
PubMed1996

Analyses of functional antibody responses in HIV-1-infected individuals after vaccination with rgp160.

BACKGROUND: The immune response to HIV infection has been extensively studied and the antibody response against the virus has been characterized in detail. It is, however, still unclear which immune function it is most important to stimulate when administering a vaccine against HIV. OBJECTIVES: To analyze the functional antibody responses in asymptomatic HIV-1-infected individuals after vaccination with rgp160. STUDY DESIGN: Forty-nine asymptomatic HIV-1-infected individuals were followed for 9 months and analyzed for changes in functional antibody responses. Forty of them received HIV-1 envelope rgp160 injections and nine did not. RESULTS: Increased levels of antibodies mediating neutralization and cellular cytotoxicity (ADCC) could be seen in subjects who also showed a better CD4 development compared with the patients without increased levels of functional antibodies. Out of nine matched HIV-infected and influenza-immunized controls, none had increased neutralizing activity and only one had an increased ADCC titer. An increased capacity to block soluble CD4 binding to gp120 occurred in 10 immunized patients. Seroreactivity and avidity maturation were detectable to peptides representing consensus HIV-1 envelope regions, indicating an anamnestic response to the patients own virus. CONCLUSIONS: The humoral immune response in HIV-1-infected individuals was moderately influenced by repeated gp160 immunizations, while previous studies have shown that HIV-specific T-cell reactivity was strongly increased.

Journal Article↗
PubMed1996

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV-1 envelope protein gp120LAI, and has previously been shown to interact with the -I-GPGRA- motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif -GPGRA-, a similar segment, -GPAR-, and two unrelated motifs -FRLLG- and -WRM/ALG- were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide-specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the -GPGRA- containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV-1SF2 virus in vitro.

Animals↗
PubMed1996

Antibody-dependent cellular cytotoxicity to clinical isolates of HIV-1 and SIVcpz: comparison of human and chimpanzees.

OBJECTIVE: To investigate differences in the antibody-dependent cellular cytotoxicity (ADCC) responses between HIV-1-infected humans and chimpanzees. DESIGN: The breadth of the ADCC responses in the two populations were tested against autologous and heterologous HIV-1 and SIVcpz clinical isolates as well as against reference isolates. METHODS: ADCC was tested in a 51Cr-release assay using human peripheral blood mononuclear cells as effector cells and infected Jurkat/Tat-cells as target cells. RESULTS: The majority of sera from chronically HIV-1-infected humans and chimpanzees had ADCC responses to HIV-1LAI. Interestingly, vaccinated chimpanzees with a low virus load during the immediate post-challenge period had low ADCC responses 3 years after challenge. In contrast, when ADCC activity to clinical isolates was evaluated, HIV-1-infected chimpanzees had more frequent heterologous (broader) responses than HIV-1-infected humans. ADCC was also tested in consecutive serum samples from two patients and two chimpanzees against autologous isolates, but was only detected to a low degree in one of the animals, although heterologous ADCC was demonstrated in all cases. The naturally infected (SIVcpz) chimpanzee did not have detectable heterologous or autologous ADCC responses. CONCLUSIONS: HIV-1-infected chimpanzees had broader ADCC reactivity to heterologous HIV-1 clinical isolates than the HIV-1-infected humans. These findings are consistent with subtle differences in host-virus relationships of these two species.

AIDS Vaccines↗
PubMed1996

Two neutralizing anti-V3 monoclonal antibodies act by affecting different functions of human immunodeficiency virus type 1.

Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.li had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 degrees C and 35 degrees C. However, ICR41.1i gave PAN at 21 degrees C but not at 35 degrees C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.

Animals↗
PubMed1995

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors.

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4+ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 microgram/ml.

Amino Acid Sequence↗
PubMed1995

Antibody prevalence and immunoglobulin IgG subclass pattern to Norwalk virus in Sweden.

The antibody prevalence in Sweden to Norwalk virus (NV) was investigated using a baculovirus expressed capsid antigen. One hundred thirty-two serum samples were examined for IgA, total IgG and IgG subclass antibodies to Norwalk virus. In young children, NV IgG antibody prevalence was higher than the IgA prevalence, whereas no difference was found in individuals older than 21 years. The IgG antibody prevalence was 50% in children below 5 years of age and increased to > 80% in individuals older than 10 years of age. To examine the IgG antibody response in more detail, IgG subclass patterns were characterized. IgG 1 predominated in all age groups. IgG 4, usually detected after repeated exposure to antigen, was the second most prevalent subclass, but was only found in individuals older than 21 years of age. IgG 3 subclass antibodies were found in 13% and IgG 2 in 3% of the sera examined. IgG 3 subclass antibodies have been recognized as a marker for recent or ongoing viral infections.

Adolescent↗
PubMed1995

Molecular characterization of a human anti-HIV 1 monoclonal antibody revealed a CD26-related motif in CDR2.

Genes encoding the immunoglobulin variable regions of a human anti-HIV-1 IgG1 kappa monoclonal antibody were rescued from a hybridoma, derived from a sero-negative donor, using PCR cloning and expression in Escherichia coli. The ELISA binding results obtained from the expressed Fab fragment confirmed the anti-V3 loop specificity for HIV-1 (LAI) of the original antibody. In addition, an amino acid sequence derived from the second complementarity determining region (CDRH2) of the heavy chain was found to be very similar to the catalytic motif of CD26, a T-cell activation antigen. Furthermore, synthetic peptides containing both the catalytic domain of CD26 and CDRH2 of the antibody showed specific binding to an HIV peptide representing the V3 region in a dose-dependent manner. This suggests an involvement of CD26 as a possible coreceptor for HIV-1.

Amino Acid Sequence↗
PubMed1995

Monoclonal antibodies to p24-core protein of HIV-1 mediate ADCC and inhibit virus spread in vitro.

BACKGROUND: Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies. OBJECTIVE: To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells. METHODS: Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro. RESULTS: The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures. CONCLUSIONS: Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Journal Article↗
PubMed1994

Enzyme immunoassay (ELISA) for the evaluation of antibodies directed to the CD4 receptor-binding site of the HIV gp120 molecule.

The interaction between the HIV envelope glycoprotein gp120 and the CD4 molecule is probably the most important primary event determining HIV infection. Reactivity with the native viral envelope has been difficult to measure due to the lack of gp120 ligand purified directly from primary virus cultures. We have developed an ELISA, utilizing Galanthus nivalis agglutinin (GNA) which selectively binds native HIV envelope gp120 in culture medium. The GNA-based ELISA eliminates the need for isotope-labelled reagents, live cells and recombinant non-natively glycosylated envelope proteins and offers an easy way of using gp120 directly from crude HIV culture medium. The reactivities of sera from several categories of HIV infected individuals were assayed for inhibition of the HIV-1 gp120-CD4 binding. 19/32 (59.3%) sera from asymptomatic individuals and 7/10 (70%) sera from ARC/AIDS patients blocked the CD4-gp120 binding. 20 serum samples from uninfected individuals showed a gp120-CD4 interaction blocking capacity of 0-15%. Two monoclonal antibodies, T4.2 directed to CD4 and 1171 directed to the CD4 binding site of gp120 were used as positive controls. Both Mabs inhibited CD4-gp120 binding by 66-90%.

Adolescent↗
PubMed1994

Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors.

The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T-cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287-467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significantly frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes, mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.

Amino Acid Sequence↗
PubMed1994

Antibody production of a human EBV-transformed B cell line and its heterohybridoma and trioma cell line descendants in different culture systems.

Cells of an EBV-transformed human lymphoblastoid B cell line, producing antibodies directed against tetanus toxin, were fused with mouse myeloma cells (SP2/0) and with mouse-human heteromyeloma cells (SPAM-8) resulting in the formation of heterohybridoma and trioma cells, respectively. Antibody production of the three cell lines were studied under different culture conditions. All three cell lines produced antibodies in concentrations ranging from 2.6 to 6.4 micrograms ml-1 in spent medium from stationary flask cultures. Dialysis cultures of trioma and heterohybridoma cells resulted in concentrations of 36 and 20 micrograms ml-1, respectively, whereas no significant increase was obtained with the EBV-transformed cells. Trioma cells, cultured in a hollow fiber cartridge bioreactor produced antibodies in concentrations of average of 303 micrograms ml-1, whereas the EBV-transformed cells did not adapt to this system. Furthermore, trioma and heterohybridoma cells injected into the intraperitoneal cavity of SCID-mice, produced antibodies in ascites fluid in concentrations of 500 and 640 micrograms ml-1 respectively.

Animals↗
PubMed1994

Improved cell-mediated immune responses in HIV-1-infected asymptomatic individuals after immunization with envelope glycoprotein gp160.

Strong specific T-cell responses to human immunodeficiency virus type 1 (HIV-1) gp160 were induced by immunization with recombinant gp160 (rgp160). It was given as postinfection vaccination to 40 asymptomatic HIV-1 seropositive patients. The participants received 6 doses of 160 micrograms rgp160 administered intramuscularly at 0, 1, 4, 8, 17, and 26 weeks and were monitored for 1 year. Lymphocyte proliferation was performed by cultivating lymphoid cells in vitro with specific antigens and mitogens. After immunization with gp160, specific T-cell proliferative responses were induced in all 40 patients. One week after the sixth immunization at day 180, a substantially increased response was detected in 98% of the patients, with a mean stimulation index value of 195. Furthermore, proliferative responses were also identified, after immunization, against native gp120 and against a peptide representing the V3 region of gp120. In addition to the HIV-specific T-cell responses, increased reactivity to several other non-HIV antigens, including tetanus toxoid, influenza, measles, and cytomegalovirus, were seen after gp160 vaccination. The responses to CMV and measles were interpreted to represent an improved recall antigen response. Such recall antigen responses were few in matched HIV-infected controls immunized with influenza virus only. All patients initially and repeatedly showed a normal capacity of total T-cell activation, evaluated by the mitogen phytohemagglutinin (PHA). The trend in CD4 counts improved in 30 of 40 patients during the year of follow-up. The frequency of increases of proliferative responses to antigens was associated with a better CD4 trend. Addition of zidovudine for 2 weeks after each immunization had no beneficial effects nor did it prevent induction of immune responses. All patients tolerated the immunizations well, and no systemic adverse effects were noted. This is a phase I trial, and no definitive conclusions regarding clinical efficacy can be reached.

Adult↗
PubMed1994

Immunological and virological interactions in patients receiving passive immunotherapy with HIV-1 neutralizing monoclonal antibodies.

Mouse monoclonal antibodies with high human immunodeficiency virus type 1 (HIV-1) neutralizing titers were used for passive immunotherapy of eleven late-state HIV-infected patients. In five patients the serum level of the core protein p24 decreased, while in five cases it remained unchanged. The level of viral RNA in plasma as measured by quantitative polymerase chain reaction (PCR) decreased in four cases, was stable in another four, and increased in three cases. An anti-mouse (HAMA) response developed in eight patients and anti-idiotypic antibodies appeared in six. Immune complexes that formed in patient sera during the treatment were shown to contain mostly envelope glycoprotein gp120 which decreased in nine of the eleven treated patients toward the end of treatment. Antibodies inhibiting gp120 binding to CD4 became detectable or increased in six patients during immunotherapy. Serology of the HIV-1 V3 region was studied for both the HIV-1 IIIB and MN strains with no or very small changes in titer or avidity after treatment. No change in neutralizing titers to strain HTLVIIIB was observed in serum samples collected before and after treatment was terminated. In nine of the eleven patients stimulation of the T lymphocytes to proliferate in vitro when activated by phytohemagglutinin (PHA) was shown to be increased compared to before treatment. Increased T-cell proliferation was also noted with several antigens such as HIV-1 recombinant antigens, cytomegalovirus (CMV), tetanus toxoid (TT), and purified protein derivate of mycobacterium tuberculosis (PPD). These findings indicate a decreased total gp120 content in serum, permitting better T-cell activation.

Adult↗
PubMed1992

Immunoglobulin V regions and epitope mapping of a murine monoclonal antibody against p24 core protein of HIV-1.

The nucleotide sequence of a murine monoclonal antibody (CB-mab-p24/13-5) against p24 core protein of the human immunodeficiency virus (HIV-1) was determined for variable regions of the heavy and light chain, respectively. Genetic elements encoding the VDJH- and VJL-regions of the antibody were generated from RNA by the polymerase chain reaction, cloned into the vector pICEM 19R and sequenced. Synthetic peptides, 10 amino acids overlapping served for the localization of the epitope. The residues 152-156 within the p24 sequence contain the epitope.

Amino Acid Sequence↗
PubMed1992

Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gp120.

Monoclonal antibodies (MAbs) were raised against the glycoprotein gp120 of human immunodeficiency virus type 1 (strain HTLV-IIIB). The reactivity of five selected MAbs was characterized in several tests: ELISA, immunostaining of Western blots, immunofluorescence, immunoprecipitation, immunoelectron microscopy, alkaline phosphatase-anti-alkaline phosphatase assay and neutralization. The binding region was delimited by sequential overlapping Escherichia coli fusion proteins of the gp120 sequence between amino acids (aa) 49 and 280. In the ELISA, when using sequential overlapping 15 aa peptides, the binding epitopes were localized between aa 64 and 78 for three MAbs and between aa 114 and 123 for the fourth Mab. The fifth Mab showed multiple reactions with different peptides possibly indicating a reaction with a discontinuous epitope. In virus growth inhibition assays, all five MAbs inhibited the spread of HIV-1 infection in cell cultures after a single or repeated treatment at a concentration of 63 micrograms/ml of the purified MAbs. All MAbs showed low but significant neutralizing activity at concentrations of 100 micrograms/ml.

Amino Acid Sequence↗