Common antibody dependent cell mediated cytotoxicity (ADCC) antibody epitopes of HIV-1 CRF01_AE Env and Gag in early HIV-1 infected individuals.

BACKGROUND: There have been a few studies aimed at identifying epitopes of ADCC-inducing antibodies when compared to those of neutralizing antibodies and cytotoxic T lymphocytes against a variety of HIV-1 clades. OBJECTIVE: To map the common ADCC epitopes of HIV-1 CRF01_AE. METHODS: We screened 65 sera of confirmed early HIV-1 CRF01_AE infected individuals for ADCC antibody against gp120 utilizing an EGFP-CEM-NKr flow cytometric assay. Sera with high ADCC antibody were then examined against ADCC epitopes using the complete HIV-1 CRF01_AE gp160- and subtype A Gag-overlapping peptide sets which were divided into 7 pools:E1-E7 and 5 pools:G1-G5, respectively. Each positive peptide pool was further investigated for fine ADCC epitope mapping using matrix formats. RESULTS: Twenty, 25 and 20 sera demonstrated the high-, medium- and low-ADCC antibody activities against gp120, respectively. Interestingly, 11 Env- and 6 Gag-peptides of pools E3, E4, E7 and pools G1, G2, G4 with high ADCC responses were also responded by at least 20%, 12% and 5%, 10% of medium- and low-ADCC antibody sera, respectively. These eleven common Env ADCC epitopes were localized at C2-V3-C3-V4 regions of gp120 and cytoplasmic tail of gp41 while six common Gag ADCC epitopes were localized at p17-p24-p2 regions. CONCLUSIONS: Although the degree of ADCC antibody responses to the gp120 protein varied from high to low, there were certain consensus Env and Gag peptides that could induce the ADCC antibody responses of 21.54-58.46% and 23.08-41.54%, respectively of the early infected individuals. This epitope information should be useful as the new antibody-based vaccine immunogens.

Lymphocyte subsets and natural killer cell cytotoxicity in intravenous drug users with HIV-1 infection among Thai population.

BACKGOUND: Intravenous drug users (IVDUs) are among the high-risk groups who are most vulnerable to HIV infection. Several illicit drugs alter host immune function with increased incidence of infections including that of HIV. Many studies of the immune response of NK cells in HIV-1 seronegative IVDUs and HIV-1 seropositive IVDUs have been published from the Western countries and yet no data is available from Thailand. OBJECTIVE: To determine natural killer cell cytotoxicity and lymphocyte subsets in Thai HIV-1 infected intravenous drug users. METHODS: The NK cell cytotoxic function was determined using our well-established EGFP-K562 flow cytometric assay in 30 IVDUs with HIV-1 infection (IVH) comparing with those from the same number of non-infected IVDUs (IVX), HIV-1 seropositive individuals (HIV-1+ve) and healthy controls. The percentage and the absolute number of NK cells, helper CD4+ T cells and cytotoxic CD8+ T cells were also investigated. RESULTS: Among the study groups, IVH showed not only the lowest percentage of lytic activity by NK cells, but also a decline in the percentage and absolute count of NK cells. A decline in helper CD4+ T cells and an increase of cytotoxic CD8+ T cells of IVH group when compared to those of other 3 groups were also demonstrated. CONCLUSIONS: The failure of innate immune NK cell function and their number in IVH may support the involvement of additional components of the immune system in the control of HIV-1 disease.

Duration of neutralizing antibody persisting in Thai individuals after childhood vaccination against smallpox.

BACKGROUND: Although smallpox was completely eliminated by 1980, it remains possible that variola virus could be intentionally released in an act of bioterrorism. Thus, several studies have been performed to detect antibody levels after smallpox vaccination of the current population in various countries to indicate the duration of maintenance of immunological memory. Our study endeavored to investigate the level of neutralizing (Nt) antibody responses of Thai individuals who had been immunized with smallpox vaccine during childhood. METHODS: The plaque reduction neutralization test (PRNT) was used to study vaccinia Nt antibody responses in sera of individuals ranging in age from 35-4, 45-54, 55-64, 65-74, 75-84 and > 84 years old, referred to as groups 1-6, respectively. Each group included 200 sera: 100 male sera and 100 female sera. RESULTS: An incubation time of 15 hours for sera and vaccinia virus was confirmed to be the optimal incubation period for PRNT. Positive Nt antibody titers (≥32) were detected in 135 (11.25%) of 1,200 sera: 81 (6.75%) male sera and 54 (4.5%) female sera. There were 4 (2%), 11 (5.5%), 19 (9.5%), 16 (8%), 33 (16.5%), and 52 (26%) positive sera in groups 1-6, respectively. Interestingly, the oldest individual with positive Nt antibody was a 98-year-old female. Two males aged 96 and 91 years old had the highest Nt antibody titers. CONCLUSIONS: Our data suggests that the vaccinia-specific Nt antibody response in the current Thai population could be maintained for more than 90 years after vaccination. However, the majority of the Thai population aged ≥35-74 years old is still highly susceptible to infection.

Comprehensive investigation of common antibody-dependent cell-mediated cytotoxicity antibody epitopes of HIV-1 CRF01_AE gp120.

The antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism involves both innate and adaptive immune systems. While a number of epitope mapping studies of neutralizing (Nt) antibodies and cytotoxic T lymphocyte (CTL) against a variety of HIV-1 clades have been reported, there has been a paucity of similar studies aimed at identifying epitopes of ADCC-inducing antibodies. Herein we screened 35 sera from HIV-1 CRF01_AE-infected blood donors for ADCC antibody activity against gp120 utilizing an EGFP-CEM-NK(r) flow cytometric assay. Eighteen sera with high ADCC antibody activity were then comprehensively examined for ADCC antibody epitopes using the HIV-1 subtype CRF01_AE TH023 gp120 peptide set consisting of 126 peptides of 15 amino acids in length, overlapping by 11 amino acids. This peptide set was divided into five pools (E1-E5). Each positive peptide pool was further investigated for fine epitope mapping of ADCC antibody activity using a 5 by 5 peptide matrix format. Interestingly, six and three peptides from peptide pools E1 and E2, respectively, responded to at least 33.33% of the tested sera. These nine common ADCC epitopes were localized to the C1 and V2 region of gp120. Furthermore, 5/9 epitopes were also shown to serve as full or partial Nt antibody targets for HIV-1 subtypes B and CRF01_AE. We submit these data on epitope mapping of ADCC or dual ADCC-Nt antibodies against HIV-1 gp120 that should be considered in the formulation of vaccines against HIV-1.

Stable expression of EBV-gp350 on the surface of NC37 cells confers natural killer (NK)-cell susceptibility or resistance, depending on the assay used to assess NK-mediated function.

NC37 cells containing the Epstein-Barr virus (EBV) genome do not express the viral glycoprotein-350 (gp350) on the cell surface. Despite being a cancer cell line, NC37 cells show resistance to natural killer (NK) cell cytotoxicity by the standard chromium ((51)Cr) release assay (CRA). EBV-gp350 has been identified as a ligand for antibody dependent cell-mediated cytotoxicity (ADCC). The stable expression of gp350 on the NC37 cell surface membrane could make this cell line a suitable target for measuring ADCC antibody. The pcDNA3.1-gp350 was transfected into the stably expressing enhanced green fluorescent protein (EGFP)-NC37 cell line. The transfected cells were then selected for expression of gp350 on the cell surface using immunomagnetic bead-based sorting. The gp350-EGFP-NC37 cell line was then re-examined for resistance to NK cytotoxicity, and compared with the standard K562 and EGFP-K562 cell lines using the CRA and a flow cytometric method, respectively. Surprisingly, the gp350-EGFP-NC37 cells, like the parental NC37 cell line, showed comparable resistance to NK cell-mediated cytotoxic activity by the CRA, while demonstrating susceptibility to NK cell cytotoxicity comparable to EGFP expressing K562 cells by the flow cytometric method. The susceptibility of gp350-EGFP-NC37 cells to NK cell cytotoxic activity is dependent on the type of assay.

A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity (ADCC) activity in HIV-1 infected individuals.

Enhanced green fluorescent protein (EGFP) was stably expressed in CEM-NKr cell, a natural killer (NK) resistant human T-lymphoblastoid cell line, as EGFP-CEM-NKr cells. The cells pulsed with HIV-1 gp120 were then used as target cells for the measurement of antibody dependent cell mediated-cytotoxicity (ADCC) by flow cytometry. Compromised EGFP-CEM-NKr target cells stained with propidium iodide (PI) showed dual (green-red) fluorescent. Kinetic studies demonstrated that the sum of ADCC activity measured at 1-h and again at 2-h incubations by this flow cytometric method was comparable to the activity at 6 h by the standard chromium (51Cr) release assay (CRA). ADCC activity of HIV-1 seropositive sera measured by this new technique correlated strongly with that of CRA (Pearson's correlation coefficient of 0.832; p-value < 0.001 and intraclass correlation coefficient of 0.903; p-value < 0.001). The EGFP-CEM-NKr stable cell line provides a novel method to measure ADCC activity to HIV-1 gp120 by flow cytometry without pre-staining or pre-labeling target cells.

Identification of a novel HIV type 1 CRF01_AE cytotoxic T lymphocyte (CTL) epitope restricted by an HLA-Cw0602 allele and a novel HLA-A0206/peptide restriction.

This report describes specific T cell responses to HIV-1 CRF01_AE Env and A Gag peptides in 20 HIV-1 CRF01_AE-infected Thai individuals using an interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) assay. Twenty-six potentially novel HLA class I-restricted CD8+ T cell epitopes were identified in 14/20 subjects. Fine mapping analysis using the chromium release cytotoxic T lymphocyte (CTL) assay revealed a novel HLA-Cw0602 restricted epitope of HIV-1 CRF01_AE Env (NAKTIIVHL) and a previously identified HIV-1 A Gag epitope (ATLEEMMTA) with a novel HLA-A0206 restriction.