They were analyzed by a BD fluorescent cell sorter (FACS) Celesta (BD Biosciences, San Jose, CA) and Data-Interpolating Variational Analysis (DiVa) software
They were analyzed by a BD fluorescent cell sorter (FACS) Celesta (BD Biosciences, San Jose, CA) and Data-Interpolating Variational Analysis (DiVa) software. Vaccines are delivered by chimpanzee adenovirus vectors (AdC) of serotype 6 (AdC6) and 7 (AdC7) used in prime only or prime-boost regimens. The HBV antigens are fused into an early T cell checkpoint inhibitor, herpes simplex virus (HSV) glycoprotein D (gD), which enhances and broadens vaccine-induced cluster of differentiation (CD8)+ T cell responses. Results Our results show that the vaccines are immunogenic in mice. They induce potent CD8+ T cell responses that recognize multiple epitopes. CD8+ T cell responses increase after a boost, although the breadth remains similar. In mice, which carry high sustained loads of HBV particles due to a hepatic infection with an adeno-associated virus (AAV)8 vector expressing the 1.3HBV genome, CD8+ T cell responses to the vaccines are attenuated with a marked shift in the CD8+ T cells epitope recognition profile. Conclusions Our data show that in different stains of mice including those that carry a human major histocompatibility complex (MHC) class I antigen HBV vaccines adjuvanted with a checkpoint inhibitor induce potent and broad HBV-specific CD8+ Rabbit Polyclonal to KCNJ2 T cell responses and lower but still detectable CD4+ T cell responses. CD8+ T cell responses are reduced and their epitope specificity changes in mice that are chronically exposed to HBV antigens. Implications for the design of therapeutic HBV vaccines are discussed. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-021-01712-y. strong class=”kwd-title” Keywords: Hepatitis B virus, Chronic hepatitis B infection, Vaccines, CD8+ T cell epitopes, Mouse model Introduction More than 257 million humans worldwide suffer from chronic hepatitis B virus (CHB) infection [1], which can lead to liver cirrhosis and hepatocellular carcinoma (HCC), the 2nd most common cause of cancer-related human deaths worldwide [2]. HBV-associated human mortality increased from 0.89 million to 1 1.45 million between 1990 and 2013 [3, 4] despite the availability of BIIE 0246 prophylactic HBV vaccines [5]. Antiviral drugs reduce HBV replication, but fail to eliminate the intranuclear viral genomes from infected hepatocytes and thereby do not cure [6C8]. During an acute infection CD8+ T cells can clear HBV-infected hepatocytes through cytolysis and the release of antiviral cytokines [9]. Persistent infections are associated with dysfunctional HBV-specific CD8+ T cell responses [10]. Due to sustained presence of antigens, induction of immunosuppressive cytokines [11], regulatory T cells (Tregs) [12], and the unique hepatic microenvironment [13], HBV-specific CD8+ T cells lose functions [14, 15]. Treatment with anti-Programmed Cell Death Protein 1. (PD1) antibodies may reverse CD8+ T cell dysfunction caused by exhaustion but has shown limited success in CHB patients [16]. The disappointing clinical outcome of checkpoint blockade in CHB patients contrasts results obtained in a pre-clinical woodchuck model of chronic hepadnaviral infection where a combination of anti-viral drugs, checkpoint blockade and an HBV-specific DNA vaccine led to sustained immunological control of the infection or complete viral clearance [17]. Here we describe pre-clinical results with a therapeutic vaccine to HBV, which induces potent and sustained CD8+ T cell responses that are relatively resistant to exhaustion and when combined with antiviral drugs may affect a functional cure of CHB. Implications of our findings for the development of a therapeutic HBV vaccine are discussed. Methods Cell BIIE 0246 lines Human embryonic kidney (HEK) 293 cells and coxsackie adenovirus receptor (CAR)-transduced Chinese hamster ovary (CHO) cells were maintained in Dulbeccos BIIE 0246 Modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Mice Male 6?week-old C57BL/6, BALB/c and HLA-A2 transgenic (tg) (C57BL/6- em Mcph1 /em em Tg(HLA?A2.1)1Enge /em /J) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed at the Animal Facility of the Wistar Institute and treated according to approved protocols. Unless stated otherwise experiments were conducted with groups of 5 mice, 2 or 3 3 times. Production, purification, and titration of vectors Early antigen (E)1 and partially E3-deleted AdC6 and AdC7 vectors expressing PolN, PolC or core within gD under the control of the early cytomegalovirus promoter were produced, purified and titrated as described previously [18]. They were formulated in 2.5% Glycerol/25?mM odium chloride (NaCl)/20?mM TRIS buffer, pH 8.0. AAV8 vectors expressing the 1.3 genome of HBV genotype D were produced in HEK 293 cells by triple plasmid (p)Helper/pAAV-1.3HBV/pAAV8capsid transfection using vectors at ratios of 1 1:0.5:0.5 as described [19]. HEK 293 cells were transfected with plasmids using polyethylenimine (PEI) at a 1:3 ratio of DNA: PEI. After a 72?h incubation in.