High-Throughput Quantitative Analysis of HIV-1 and SIV-Specific ADCC-Mediating Antibody Responses

Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA.
Cytometry Part A (Impact Factor: 2.93). 08/2011; 79(8):603-12. DOI: 10.1002/cyto.a.21084
Source: PubMed


We have developed a high-throughput platform to detect the presence of HIV-1 and SIV-specific ADCC-mediating antibody responses. The assay is based on the hydrolysis of a cell-permeable fluorogenic peptide substrate containing a sequence recognized by the serine protease, Granzyme B (GzB). GzB is delivered into target cells by cytotoxic effector cells as a result of antigen (Ag)-specific Ab-Fcγ receptor interactions. Within the target cells, effector cell-derived GzB hydrolyzes the substrate, generating a fluorescent signal that allows individual target cells that have received a lethal hit to be identified by flow cytometry. Results are reported as the percentage of target cells with GzB activity (%GzB). Freshly isolated or cryopreserved PBMC and/or NK cells can be used as effector cells. CEM.NKR cells expressing the CCR5 co-receptor are used as a target cells following: (i) coating with recombinant envelope glycoprotein, (ii) infection with infectious molecular clones expressing the Env antigens of primary and lab adapted viruses, or (iii) chronic infection with a variant of HIV-1/IIIB, termed A1953. In addition, primary CD4(+) T cells infected with HIV-1 in vitro can also be used as targets. The assay is highly reproducible with a coefficient of variation of less than 25%. Target and effector cell populations, in the absence of serum/plasma, were used to calculate background (8.6 ± 2.3%). We determined that an initial dilution of 1:50 and 1:100 is required for testing of human and non-human primate samples, respectively. This assay allows for rapid quantification of HIV-1 or SIV-specific ADCC-mediating antibodies that develop in response to vaccination, or in the natural course of infection, thus providing researchers with a new methodology for investigating the role of ADCC-mediating antibodies as correlates of control or prevention of HIV-1 and SIV infection.

Download full-text


Available from: Akira Komoriya, Oct 02, 2014
  • Source
    • "As shown in Fig. 5, only a small reduction in the A32-induced ADCCmediated killing was observed when purified NK cells were used as effector cells relative to PBMCs. Thus, these results suggest that NK cells play a major role in the ADCC response mediated against gp120-coated target cells in this assay but also indicates that other cells present in PBMCs such as monocytes could contribute to a smaller extent to target cell killing, in agreement with previous studies (Pollara et al., 2011; Smalls-Mantey et al., 2013). This assay was next applied to determine the capacity of sera collected from 18 individuals infected with HIV-1 and 5 healthy subjects (non-infected individuals) to mediate a specific HIV- 1 gp120 ADCC response. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Increased attention on the role of Fc-mediated effector functions against HIV-1 has led to renewed interest into the role that antibody-dependent cellular cytotoxicity (ADCC) could play in controlling viral transmission and/or the rate of disease progression. While (51)Chromium release assays have traditionally been used to study ADCC responses against HIV-1, a number of alternative flow-cytometry-based assays were recently developed. In this study, an alternative flow-cytometry-based assay was established to allow non-radioactive measurement of ADCC-mediated elimination of HIV-1 gp120 envelope glycoprotein (Env)-coated target cells. This assay relies on staining target and effector cells with different dyes, which allows precise gating and permits the calculation of the number of surviving target cells by normalization to flow-cytometry particles. By using small concentrations of recombinant gp120 Env, suitable targets cells that recapitulate the ADCC response mediated against HIV-1-infected cells were generated. Finally, this method was applied successfully to screen human sera for ADCC activity directed against HIV-1 gp120 Env.
    Journal of Virological Methods 08/2014; 208. DOI:10.1016/j.jviromet.2014.08.003 · 1.78 Impact Factor
  • Source
    • "The standardization of the effector cells in assays evaluating these activities is critical for understanding the correlates of immune protection. The 51 Cr-release assay has been the standard assay used to evaluate NK and ADCC activity, however, newer assays have been developed that use flow cytometry to measure cytotoxicity against fluorescent-labeled targets, degranulation or granzyme B loss by effector cells following target stimulation (Alter et al., 2004; Kantakamalakul et al., 2006; Aktas et al., 2009; Davis et al., 2011; Pollara et al., 2011; Yamada et al., 2011). These assays vary with respect to incubation times and stimulation conditions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in 51Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16+CD56dim NK cells and CD14+ monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56dim NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a 51Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.
    Journal of immunological methods 04/2014; 406. DOI:10.1016/j.jim.2014.01.017 · 1.82 Impact Factor
  • Source
    • "The presence of ADCC-mediating antibody responses was measured using PBMCs as previously reported using the flow-based GTL ADCC assay [35]. The testing of samples was conducted using SF162 recombinant gp120 (GeneBank No. AAT67508; ImmuneTechnology, Corp) to coat CEM.NKR.CCR5 target cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.
    PLoS ONE 07/2013; 8(7):e67574. DOI:10.1371/journal.pone.0067574 · 3.23 Impact Factor
Show more