PBMC-based Assay Research Articles

Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Abstract 506: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the reproducibility of these assays is not adequate and they have major issues on biosafety or weak assay sensitivity. Moreover, the use of primary effector cells makes them prone to donor variability, low-throughput and therefore not suitable for implementation in quality-control drug development. We previously developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing primary PBMC-based ADCC assays and ADCC reporter bioassays, we report the development of an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells for therapeutic monoclonal antibodies with Fc functions. When target cells expressing HaloTag-HiBiT are incubated with an antibody and PBMCs, the target cells are lysed and release HaloTag-HiBiT, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate Bioluminescence signal. In addition, it can enable the quantitative measurement of antibody-mediated target cell lysis via specific HaloTag staining using a fluorescent HaloTag ligand by flow cytometry. We demonstrate that the new PBMC ADCC assay is simple, homogenous, highly sensitive, and gives a robust assay window, and is capable of measuring potency of rituximab, cetuximab or trastuzumab. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Gopal B. Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 506.

A general Fc engineering platform for the next generation of antibody therapeutics.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform.Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice.Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

Exploring the Potential Use of a PBMC-Based Functional Assay to Identify Predictive Biomarkers for Anti-PD-1 Immunotherapy.

The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.

Abstract 5658: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for characterizing antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, these assays are relatively low throughput, present a biosafety risk due to radioactivity, and are prone to donor variability and weak assay signal. Therefore, these assays are not well-suited for implementation in quality-controlled drug development environments. Previously, we developed a mechanism of action (MOA)-based ADCC reporter bioassay using genetically engineered Jurkat effector cells. When co-cultured with antigen-expressing target cells and a relevant antigen-specific antibody, engagement of FcγRIIIa expressed on the effector cells leads to NFAT-mediated expression of luciferase and a bioluminescent signal. The effector cells are optimized for use in thaw-and-use format and can accurately measure antibody potency for a wide range of therapeutic antibodies targeted to hematopoietic or solid tumors. The bioassay can distinguish effector functions for different antibody IgG isotypes, is stability indicating, and prequalified according to ICH guidelines (precision, accuracy, linearity, and robustness). The ADCC reporter bioassay can be developed as a QC lot release assay for antibody therapeutics that function via an ADCC MOA. However, prior to implementation, a bridging study with a primary cell-based cytotoxicity assay must be performed. To enable this analysis, we developed a PBMC-based ADCC assay that overcomes limitations of traditional primary cell ADCC assays. First, we established a workflow to prequalify donor PBMCs for ADCC activity, significantly reducing the level of variability introduced with random donor PBMCs. We then developed a platform to genetically engineer target cells with a cytosolic protein (HaloTag) fused to HiBiT, an 11 amino acid protein tag. When cultured with a relevant antibody and ADCC-qualified PBMCs, the target cells are killed and HaloTag-HiBiT is released into the extracellular medium containing the protein LgBiT. Binding of HiBit and LgBiT reconstitutes a luciferase enzyme and produces a bioluminescent signal. The use of HaloTag as a binding partner offers the additional opportunity to image the target cells using fluorescent HaloTag ligands. This improved PBMC ADCC assay is simple, homogenous, highly sensitive, and yields a robust assay window. Importantly, we have demonstrated that this assay accurately measures the potency of a range of antibody therapeutics including rituximab, cetuximab, and trastuzumab, and correlates with the surrogate ADCC reporter bioassay. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Vanessa Ott, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5658.

Abstract 4148: An orally bioavailable small molecule antagonist of TIM-3 signaling pathway shows potent anti-tumor activity

Abstract Activation of anti-tumor immune response by specific inhibition of PD1 pathway using monoclonal antibodies has now become of the mainstay in cancer therapy as evidenced by its widespread use in an expanding list of indications. Although these antibodies show impressive durable clinical activity, low response rates are witnessed in a large number of cancers, including colorectal cancer that remain largely refractory to PD-1 blockade. Upregulation of alternative immune checkpoints such as T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and VISTA contributes to the lack of response in patients not responding to therapies with anti-PD-1/PD-L1 antibodies. TIM-3 is a co-inhibitory receptor expressed on IFN-γ-producing T cells, FoxP3+ Treg cells and innate immune cells. Synergistic effects in restoring the anti-tumor immunity in preclinical models upon dual blockade of TIM-3 and PD-1 has provided a strong rationale for developing TIM-3 agents for use in combination with PD1 agents in the clinic. We sought to discover and develop an orally available small molecule antagonist targeting TIM3- signaling pathways. Unlike antibodies an oral agent potentially offers the convenience, flexibility to adjust dose and schedule to address any emergent adverse events and ease of combination therapy. Because TIM-3 shares sequence and structural similarity with the B7 family ligands, a focused library of compounds mimicking the interaction of checkpoint proteins of B7 family was screened towards the functional antagonism of TIM-3. Further optimization of the hit compounds resulted in lead compounds targeting TIM-3 pathway with desirable potency and selectivity. Lead compounds exhibited potent functional activity comparable to that obtained with an anti-TIM-3 antibody in rescuing the effector functions in human PBMC-based assays. Additionally, an advanced lead compound exhibited desirable drug-like properties including solubility, metabolic stability and pharmacokinetics with good oral bioavailability. In a syngeneic tumor models, once a day oral dosing of the advanced lead compound resulted in significant tumor growth inhibition as a single agent and in combination with anti-PD1 antibody that correlated well with immune PD in the tumor. The findings reported here support the development of the oral TIM-3 antagonist for use in the clinic. Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Raghuveer K. Ramachandra, Nagesh Gowda, Srinivaskumar Devarapalli, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Amit A. Dhudashiya, Dodheri S. Samiulla, Nagaraj M. Gowda, Murali Ramachandra. An orally bioavailable small molecule antagonist of TIM-3 signaling pathway shows potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4148.

A defucosylated bispecific multivalent molecule exhibits broad HIV-1-neutralizing activity and enhanced antibody-dependent cellular cytotoxicity against reactivated HIV-1 latently infected cells.

Current treatments cannot completely eradicate HIV-1 owing to the presence of latently infected cells, which harbor transcriptionally silent HIV-1. However, defucosylated antibodies can readily kill latently infected cells after their activation to express envelope glycoprotein (Env) through antibody-dependent cellular cytotoxicity (ADCC). We herein aimed to test a defucosylated bispecific multivalent molecule consisting of domain-antibody and single-domain CD4, LSEVh-LS-F, for its HIV-1 neutralizing activity and ADCC against the reactivated latently infected cells, compared with the nondefucosylated molecule LSEVh-LS. LSEVh-LS-F's neutralizing activity against a panel of newly characterized Chinese HIV-1 clinical isolates was assessed by using TZM-bl-based and PBMC-based assays. LSEVh-LS-F-mediated ADCC in the presence of natural killer cells against cell lines that stably express Env proteins, HIV-1-infected cells and LRA-reactivated HIV-1 latent cells, was measured using a lactate dehydrogenase (LDH) cytotoxicity assay or flow cytometry. LSEVh-LS-F and LSEVh-LS were equally effective in neutralized infection of all HIV-1 isolates tested with IC50 and IC90 values 3∼4-fold lower than those of VRC01. LSEVh-LS-F was more effective in natural killer-mediated killing of HIV-1 Env-expressing cell lines, HIV-1-infected cells, latency reactivation agents-reactivated ACH2 cells and reactivated latently infected resting CD4+ T cell line as well as resting CD4+ T lymphocytes isolated from patients receiving HAART. LSEVh-LS-F exhibits broad HIV-1 neutralizing activity and enhanced ADCC against HIV-1-infected cells, reactivated latently infected cell lines and primary CD4+ T cells, thus being a promising candidate therapeutic for eradicating the HIV-1 reservoir.

Aggregates of IVIG or Avastin, but not HSA, modify the response to model innate immune response modulating impurities

Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1β, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPβ, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.

An optimized whole blood assay measuring expression and activity of NLRP3-, NLRC4 and AIM2-inflammasomes

Caspase-1 activates proIL-1β and proIL-18 and plays a fundamental role in innate immunity. This pro-inflammatory immune response is required for the initiation of pathogen clearance. Caspase-1 itself is activated in so-called “inflammasomes” which are assembled in response to distinct pathogen associated molecular patterns (PAMP) or danger associated molecular patterns (DAMP). Most studies analyzing the inflammasome/caspase-1 activity of patients ex vivo use PBMC-based assays. Beside monocytes and macrophages, caspase-1 is also activated in PMNs representing the major leukocyte subset in peripheral blood. Thus, analyzing purified PBMCs seems to be highly artificial by excluding caspase-1 activity in PMNs and ignoring cellular interactions. Furthermore, PBMC purification requires large amounts of blood, thereby rendering the assay impractical for the use in small children or neonates.

Non-Cationic Proteins Are Associated with HIV Neutralizing Activity in Genital Secretions of Female Sex Workers.

ObjectiveCationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection.MethodsCVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics.ResultsHIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3yr, HESN<3yr, and HIV-positive groups, respectively. Gene ontology placed these proteins into functional categories including proteolysis, oxidation-reduction, and epidermal development. The proteins identified in this study include proteins previously associated with the HESN phenotype in other cohorts as well as novel proteins not yet associated with anti-HIV activities.ConclusionWhile cationic proteins appear to contribute to the majority of the intrinsic HIV neutralizing activity in the CVS of low-risk women, a broader range of non-cationic proteins were associated with HIV neutralizing activity in HESN and HIV-positive female sex workers. These results indicate that novel protein factors found in CVS of women with high-risk sexual practices may have inherent antiviral activity, or are involved in other aspects of anti-HIV host defense, and warrant further exploration into their mode of action.

Abstract 3740: Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to clinical efficacy of a broad range of therapeutic antibodies. FcγRIIIa polymorphisms of individual cancer patients are correlated with clinical efficacy of several antibody drugs. An understanding of in vitro antibody activity via both Fc receptor variants is the key to ultimately understanding drug efficacy in vivo. Yet, with classic ADCC assays that rely on primary effector cells which are highly heterogeneous and variable, it remains challenging to quantitatively measure antibody ADCC activity and evaluate the impact of FcγRIIIa polymorphisms by in vitro ADCC assays. To address this problem, we developed a pair of reporter-based ADCC assays, where two engineered effector cell lines were generated in Jurkat T-cells which stably express a NFAT-RE driven luciferase reporter and either FcγRIIIa/V158 or FcγRIIIa/F158 polymorphism variant. The engineered Jurkat effector cells were further developed in frozen, thaw-and-use format to reduce handling time and minimize assay variability. We compared the V variant ADCC reporter assay with the classic ADCC assay using PBMCs from homozygous 158VV donors, by testing a panel of glyco-modified trastuzumab mixtures. The results demonstrate that the reporter-based ADCC assays using engineered Jurkat effector cells provide ADCC biological activity ranking equivalent to that obtained using classic PBMC-based ADCC assay. When tested side by side, both V variant and F variant ADCC reporter assays appropriately measure the biological activities in ADCC pathway activation of various human and mouse antibody isotypes of rituximab. The two ADCC reporter assays are also able to measure antibody potencies for multiple therapeutic antibodies, in various native target cell systems including suspension and adherent cell lines, and also genetically engineered cell lines such as a membrane-bound TNFα cell line. When tested in the same antibody/target cell system, the V variant ADCC assay showed higher antibody biological activity in ADCC reporter response than the F variant ADCC assay. This result appropriately reflects the reported impact of FcγRIIIa polymorphism on antibody binding and ADCC activity. In summary, the pair of V and F variant ADCC reporter assays provides a valuable approach to quantitatively measure the potency of therapeutic antibodies in ADCC and evaluate the impact of FcγRIIIa polymorphism in drug discovery and development for antibody therapeutics. Citation Format: Zhi-Jie Jey Cheng, Denise Garvin, Aileen Paguio, Rich Moravec, Frank Fan, Teresa Surowy. Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2014-3740

Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the main mechanisms of action for many therapeutic antibodies. Classic ADCC assays measure antibody-dependent target cell cytotoxicity induced by primary effector cells that are isolated from human blood. They suffer from high assay variability due to the genetic and immune-status-mediated variation from blood donors. Here we report the development of a robust reporter-based ADCC assay that uses an engineered Jurkat stable cell line as the source of effector cells. These engineered effector cells were further developed as frozen, thaw-and-use format that can be plated for assay immediately after thaw. We demonstrate that frozen, thaw-and-use Jurkat effector cells showed appropriate Fc effector function similar to fresh cells from continuous culture, with added benefits of convenience and consistency. This robust assay is able to measure antibody potency for several therapeutic antibodies targeted to hematopoietic or solid tumors. The assay can distinguish effector functions for different antibody IgG isotypes in two antibody model systems: anti-CD20 and anti-EGFR. It is able to detect changes in ADCC biological activity for heat-stressed rituximab and trastuzumab, demonstrating that it possesses proper stability-indicting property. When compared with a classic PBMC-based ADCC assay, the ADCC reporter assay showed better assay precision and similar correlation of antibody glycosylation with ADCC biological activity for a panel of glyco-modified trastuzumab mixtures. Together these data demonstrate that this robust ADCC reporter assay meets the requirement of a potency bioassay that can quantify antibody Fc effector function in ADCC mechanism of action during drug discovery and development.

Targeting of TLRs inhibits CD4+ regulatory T cell function and activates lymphocytes in human peripheral blood mononuclear cells.

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs), thus preventing the development of durable antitumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+), CD8(+), Tregs), B cells, and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells, B cells, and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS, and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation, but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075, and TLR9L:CpG-A, CpG-B, and CpG-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation, 2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg), and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.

Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model

BackgroundTen to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant.MethodsBased on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments.ResultsIt was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region.ConclusionsOur results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.

Mimotopes selected by biopanning with high-titer HIV-neutralizing antibodies in plasma from Chinese slow progressors

ObjectiveOne approach to identifying HIV-1 vaccine candidates is to dissect the natural antiviral immune response in treatment-naïve individuals infected for over ten years, considered slow progressor patients (SPs). It is suspected that SP plasma has strongly neutralizing antibodies (NAb) targeting specific HIV viral epitopes. MethodsNAbs levels of 11 HIV-1-infected SPs were detected by PBMC-based neutralization assays. To investigate SP NAb epitope, this study used a biopanning approach to obtain mimotopes of HIV-1 that were recognized by SP plasma NAbs. IgG was purified from high-titer NAb SP plasma, and used as the ligand for three rounds of biopanning to select HIV-specific mimotopes from a phage-displayed random peptide library. Double-antibody sandwich ELISA, competitive inhibition assays, and peptide sequence analysis were used to evaluate the characteristics of phage-borne mimotopes. ResultsSPs had significantly more plasma neutralizing activity than typical progressors (TPs) (p=0.04). P2 and P9 plasma, which have highest-titer HIV-NAb, were selected as ligands for biopanning. After three rounds of biopanning, 48 phage clones were obtained, of which 22 clones were consistent with requirement, binding with HIV-1 positive plasma and unbinding with HIV-1 negative plasma. Compared with linear HIV-1 protein sequence and HIV-1 protein structure files, only 12 clones were possible linear mimotopes of NAbs. In addition, the C40 clone located in gp41 CHR was found to be a neutralizing epitope, which could inhibit pooled HIV-1 positive plasma reaction. ConclusionBiopanning of serum IgG can yield mimotopes of HIV-1-related antigen epitopes. This methodology provides a basis for exploration into HIV-1-related antigen-antibody interactions and furthers NAb immunotherapy and vaccine design.

Antibody-mediated inhibition of HIV-1 elicited by HIV-I DNA priming and boosting with heterologous HIV-1 recombinant MVA in healthy Tanzanian adults

Methods Sixty HIV-uninfected volunteers, randomized into groups of 20 received placebo or 1 mg HIV-DNA intradermally (id) or 3.8 mg intramuscularly (im). DNA plasmids containing HIV-1 gp160 subtypes A, B, C; rev B; p17/p24 gag A, B and RTmut B were given at months 0, 1 and 3 using a needle-free Biojector device. HIV-MVA expressing CRF01_AE HIV-1 env, gag, pol was administered im by needle at months 9 and 21. Sera were tested at baseline, two months post-first and four weeks post-second HIV-MVA boosting. HIV Ab responses were tested using pseudoviruses and TZM-bl cells as well as luciferaseexpressing infectious molecular clones (IMC-LucR) in PBMC-based assays. ADCC responses were tested using the flow cytometry GranToxiLux-based assay.

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.

HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships

BackgroundHIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection.Methodology/Principal FindingsCervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity.Conclusions/SignificanceThese findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules.

Replication competent molecular clones of HIV-1 expressing Renilla luciferase facilitate the analysis of antibody inhibition in PBMC

Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env–IMC–LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy.

HIV-1 exposed uninfected men who have sex with men have increased levels of salivary CC-chemokines associated with sexual behavior

To determine whether soluble molecules with known anti-HIV-1 activity are increased in saliva of HIV-1 exposed uninfected individuals of discordant couples of men who have sex with men (MSM), and whether the levels of these molecules are associated with genetic polymorphisms, sexual behavior and/or HIV-1 neutralizing capacity. Saliva and PBMC were collected from exposed uninfected individuals (n=25), and low-risk controls (n=22). Levels of CCL2, CCL3, CCL4, CCL5 and CCL11 were detected by Luminex, and SLPI, LL-37, alpha-defensins and IgA2 were detected by ELISA. Single nucleotide polymorphisms (SNPs) were investigated using mass spectrometry or PCR-sequencing. HIV-1 neutralizing activity was assessed using PBMCbased neutralization assays. Self-reported questionnaires described sexual behavior. Exposed uninfected individuals had significantly higher levels of salivary CCL2, CCL4, CCL5 and CCL11 as compared with controls although genetic polymorphisms within the corresponding regions were equally distributed. IgA2 was also increased in exposed uninfected individuals, whereas neither CCL3, SLPI, LL-37 nor alpha-defensins differed between exposed uninfected individuals and controls. The HIV-1 neutralizing capacity of saliva was associated with higher levels of CC-chemokines (but not SLPI, LL-37, alpha-defensins or IgA2) in both exposed uninfected individuals and controls. The increased levels of CC-chemokines were associated with a higher frequency of unprotected oral sex and/or additional casual sex partners. HIV-1 exposed uninfected MSM had higher levels of salivary CC-chemokines compared with controls, this finding associated with sexual behavior rather than with genetic polymorphisms. The increased levels of CC-chemokines associated with HIV-1 neutralizing capacity in saliva.