IMMPACT:Initiative for Cell Mortality Programme Assessment.

Cell Death, Apoptosis, Oxidative stress, Caspase, BCL2

Enhancement of antibody-dependent cellular cytotoxicity and phagocytosis in anti-HIV-1 bovine chimeric broadly-neutralizing antibodies

No prophylactic vaccine has provided robust protection against HIV-1. Vaccine-induced broadly neutralizing antibodies (bnAbs) have not been achieved in humans and most animals, however cows vaccinated with HIV-1 envelope trimers produce bnAbs with unusually long third heavy complementarity determining regions (CDRH3). Alongside neutralization, Fc-mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) may be critical for in vivo bnAb antiviral activity.
Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bnAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bnAbs by using an exceptionally long 60aa CDRH3.
  • The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding: G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE).
  • Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human NK cell activation and mediated higher levels of ADCC and ADP activity compared to the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity compared to GASDIE.
  • As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultra-long CDRH3 regions could enhance antibody effector functions while maintaining envelope binding and neutralization.
  • This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide.
Despite successful antiviral chemotherapy, HIV is still a lifelong persistent virus and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 bnAbs and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents.
Recently discovered anti-HIV-1 bovine bnAbs (with higher potency and breadth than most human bnAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to ANTI-BOVINE ANTIBODIES engineering that strengthens the feasibility of using high potency bovine variable region bnAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

Detection of Six β-Agonists by Three Multiresidue Immunosensors Based on an Anti–bovine Serum Albumin-Ractopamine-Clenbuterol-Salbutamol Antibody

According to an indirect competitive immunoassay, six β-agonists (clenbuterol (CL), salbutamol (SAL), ractopamine (RAC), terbutaline (TER), mabuterol (MAB), and tulobuterol (TUL)) were detected by three novel multiresidue immunosensors on the basis of the successful preparation of bovine serum albumin (BSA)-RAC-CL-SAL multideterminant antigen and anti-BSA-RAC-CL-SAL antibody. A new strategy was reported to detect six β-agonists by combining nanotechnology, electrochemical detection, and specific immune technology.
At the same concentration, the amperometric response for detection of six β-agonists was in a sequence of GCE/GNP/SAL > GCE/GNP/RAC > GCE/GNP/CL. Detection limits of six β-agonists show that the multiresidue detection performance of the GCE/GNP/RAC immunosensor is better than those of GCE/GNP/SAL and GCE/GNP/CL immunosensors. Three immunosensors manifest superior properties with a wide linear range, low detection limit, excellent reproducibility, and stability. The proposed GCE/GNP/RAC immunosensor displays high accuracy and can be effectively used for real sample detection.

Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti–bovine alkaline phosphatase (ALP) antibodies

Competitive immunoenyzmatic assays for estradiol (E2) and unconjugated estriol (uE3) on UniCel DxI 800 Access immunoassay systems (Beckman Coulter) utilize bovine alkaline phosphatase (ALP) for amplification. In these assays, rare ‘IND’ error flags indicate that a relative light unit (RLU) raw result is past the high or low end of the calibration curve but cannot be differentiated from an instrument error or analytical interference. The present studies were conducted to establish a protocol to identify analytical interference and to characterize its mechanism when present.
Design and methods Matrix and recovery studies were conducted to establish a protocol for interference identification. Spiking experiments with inactivated calf intestinal ALP were performed to determine whether interference could be blocked. Commercial anti-ALP antibodies (Abs) were spiked into human serum to model assay interference.
Three E2 immunoassays which do not include ALP as a reagent component (cobas e602, Roche; Centaur XP, Siemens; ARCHITECT 2000 SR, Abbott) were tested for comparative purposes. 1:2 dilution of specimen into Access Sample Diluent A (Beckman) differentiated IND error flags due to true low results (e.g. less than the analytical measurement range; AMR) from those due to assay interference. Interferences were reduced by pre-incubation with inactivated ALP and could be replicated by spiking with commercial anti-ALP Abs. Patient anti-bovine ALP Abs can cause interference on DxI 800 E2 and uE3 assays. This model can be used to investigate interference risk with other ALP-dependent assays.

Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression.
Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs.
  • Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression.
  • In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo.
  • Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.