Intrahepatic Exhausted Antiviral Immunity in an Immunocompetent Mouse Model of Chronic Hepatitis B.

BACKGROUND & AIMS: Targeting exhausted immune systems would be a promising therapeutic strategy to achieve a functional cure for HBV infection in patients with chronic hepatitis B (CHB). However, animal models recapitulating the immunokinetics of CHB are very limited. We aimed to develop an immunocompetent mouse model of CHB for intrahepatic immune profiling. METHODS: CHB mice were created by intrahepatic delivery of the Sleeping Beauty transposon vector tandemly expressing the hepatitis B virus (HBV) genome and fumarylacetoacetate hydrolase (FAH) cDNA into C57BL/6J congenic FAH knockout mice via hydrodynamic tail vein injection. We profiled the viral and intrahepatic immune kinetics in CHB mice with or without treatment with recombinant IFNα or the hepatotropic Toll-like receptor 7 agonist SA-5 using single-cell RNA-seq. RESULTS: CHB mice exhibited sustained HBV viremia and persistent hepatitis. They showed intrahepatic expansion of exhausted CD8+ T (Tex) cells, the frequency of which was positively associated with viral load. Recruited macrophages increased in number but impaired inflammatory responses in the liver. The cytotoxicity of mature natural killer (NK) cells also increased in CHB mice. IFNα and SA-5 treatment both resulted in viral suppression with mild hepatic flares in CHB mice. Although both treatments activated NK cells, SA-5 had the capacity to revitalize the impaired function of Tex cells and liver-recruited macrophages. CONCLUSION: Our novel CHB mouse model recapitulated the intrahepatic exhausted antiviral immunity in patients with CHB, which might be able to be reinvigorated by a hepatotropic TLR7 agonist.

Post-fusion influenza vaccine adjuvanted with SA-2 confers heterologous protection via Th1-polarized, non-neutralizing antibody responses.

Development of a universal influenza vaccine that can provide robust and long-lasting protection against heterologous infections is a global public health priority. A variety of vaccine antigens are designed to increase the antigenicity of conserved epitopes to elicit cross-protective antibodies that often lack virus-neutralizing activity. Given the contribution of antibody effector functions to cross-protection, adjuvants need to be added to modulate antibody effector functions as well as to enhance antibody quantity. We previously showed that post-fusion influenza vaccine antigens elicit non-neutralizing but cross-protective antibodies against conserved epitopes. Here, using a murine model, we comparably assessed the adjuvanticity of the newly developed SA-2 adjuvant containing a synthetic TLR7 agonist DSP-0546 and squalene-based MF59 analog as representative Th1- or Th2-type adjuvants, respectively. Both types of adjuvants in the post-fusion vaccine comparably enhanced cross-reactive IgG titers against heterologous strains. However, only SA-2 skewed the IgG subclass into the IgG2c subclass in association to its Th1-polarizing nature. SA-2-enhanced IgG2c responses exhibited antibody-dependent cellular cytotoxicity against heterologous virus strains, without cross-neutralizing activity. Eventually, the SA-2-adjuvanted vaccination provided protection against lethal infection by heterologous H3N2 and H1N1 viruses. Together, we conclude that the combination with a SA-2 is advantageous for enhancing the cross-protective capability of post-fusion HA vaccines that elicit non-neutralizing IgG antibodies.

A novel asexual blood-stage malaria vaccine candidate: PfRipr5 formulated with human-use adjuvants induces potent growth inhibitory antibodies.

PfRipr is a highly conserved asexual-blood stage malaria vaccine candidate against Plasmodium falciparum. PfRipr5, a protein fragment of PfRipr inducing the most potent inhibitory antibodies, is a promising candidate for the development of next-generation malaria vaccines, requiring validation of its potential when formulated with adjuvants already approved for human use. In this study, PfRipr5 antigen was efficiently produced in a tank bioreactor using insect High Five cells and the baculovirus expression vector system; purified PfRipr5 was thermally stable in its monomeric form, had high purity and binding capacity to functional monoclonal anti-PfRipr antibody. The formulation of purified PfRipr5 with Alhydrogel®, GLA-SE or CAF®01 adjuvants accepted for human use showed acceptable compatibility. Rabbits immunized with these formulations induced comparable levels of anti-PfRipr5 antibodies, and significantly higher than the control group immunized with PfRipr5 alone. To investigate the efficacy of the antibodies, we used an in vitro parasite growth inhibition assay (GIA). The highest average GIA activity amongst all groups was attained with antibodies induced by immunization with PfRipr5 formulated with CAF®01. Overall, this study validates the potential of adjuvanted PfRipr5 as an asexual blood-stage malaria vaccine candidate, with PfRipr5/CAF®01 being a promising formulation for subsequent pre-clinical and clinical development.

Asexual Blood-Stage Malaria Vaccine Candidate PfRipr5: Enhanced Production in Insect Cells.

The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 106 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His6) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27-22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

Antibodies against a short region of PfRipr inhibit Plasmodium falciparum merozoite invasion and PfRipr interaction with Rh5 and SEMA7A.

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.

Decreased collagen-induced arthritis severity and adaptive immunity in MKK-6-deficient mice.

OBJECTIVE: The MAPK kinases MKK-3 and MKK-6 regulate p38 MAPK activation in inflammatory diseases such as rheumatoid arthritis (RA). Previous studies demonstrated that MKK-3 or MKK-6 deficiency inhibits K/BxN serum-induced arthritis. However, the role of these kinases in adaptive immunity-dependent models of chronic arthritis is not known. The goal of this study was to evaluate MKK-3 and MKK-6 deficiency in the collagen-induced arthritis (CIA) model. METHODS: Wild-type (WT), MKK-3(-/-) , and MKK-6(-/-) mice were immunized with bovine type II collagen. Disease activity was evaluated by semiquantitative scoring, histologic assessment, and micro-computed tomography. Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. In vitro T cell cytokine response was measured by flow cytometry and multiplex analysis. Expression of joint cytokines and matrix metalloproteinases (MMPs) was determined by quantitative polymerase chain reaction. RESULTS: MKK-6 deficiency markedly reduced arthritis severity compared with that in WT mice, while the absence of MKK-3 had an intermediate effect. Joint damage was minimal in arthritic MKK-6(-/-) mice and intermediate in MKK-3(-/-) mice compared with WT mice. MKK-6(-/-) mice had modestly lower levels of pathogenic anticollagen antibodies than did WT or MKK-3(-/-) mice. In vitro T cell assays showed reduced proliferation and interleukin-17 (IL-17) production by lymph node cells from MKK-6(-/-) mice in response to type II collagen. Gene expression of synovial IL-6, MMP-3, and MMP-13 was significantly inhibited in MKK-6-deficient mice. CONCLUSION: Reduced disease severity in MKK-6(-/-) mice correlated with decreased anticollagen antibody responses, indicating that MKK-6 is a crucial regulator of inflammatory joint destruction in CIA. MKK-6 is a potential therapeutic target in complex diseases involving adaptive immune responses, such as RA.

Kinetic analysis of synovial signalling and gene expression in animal models of arthritis.

BACKGROUND: Animal models of arthritis are frequently used to evaluate novel therapeutic agents. However, their ability to predict responses in humans is variable. OBJECTIVE: To examine the time course of signalling molecule and gene expression in two models of arthritis to assist with selection of the model and timing of drug administration. METHODS: The passive K/BxN serum transfer and collagen-induced arthritis (CIA) models were studied. Activation of MAP kinase and interferon (IFN)-response pathways was evaluated by quantitative PCR and western blot analysis of ankle joints at various time points during the models. RESULTS: The kinetics of gene expression and kinase phosphorylation were strikingly different in passive K/BxN and CIA. All three MAP kinases (ERK, JNK and p38) and upstream kinases were activated within days in passive K/BxN and declined as arthritis severity decreased. Surprisingly, IFN-regulated genes, including IRF7, were not induced in the model. In CIA, activation of ERK and JNK was surprisingly low and p38 phosphorylation mainly peaked late in the disease. IFN-response genes were activated during CIA, with especially prominent peaks at the onset of clinical arthritis. CONCLUSIONS: Timing of treatment and selection of CIA or passive K/BxN might have an important impact on therapeutic response. p38, in particular, increases during the late stages of CIA. ERK and JNK patterns are similar in passive K/BxN and rheumatoid arthritis (RA), while IFN-response genes in CIA and RA are similar. The dichotomy between RA and animal models could help explain the poor correlation between efficacy in RA and preclinical studies.

Gadd45beta deficiency in rheumatoid arthritis: enhanced synovitis through JNK signaling.

OBJECTIVE: JNK-mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45beta, which is an NF-kappaB-regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK-7. This study was carried out to evaluate whether low Gadd45beta expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45beta deficiency increases arthritis severity in passive K/BxN murine arthritis. METHODS: Activation of the NF-kappaB and JNK pathways and Gadd45beta expression were analyzed in human synovium and fibroblast-like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45beta(-/-) and wild-type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed. RESULTS: Expression levels of the Gadd45beta gene and protein were unexpectedly low in human RA synovium despite abundant NF-kappaB activity. Forced Gadd45beta expression in human FLS attenuated tumor necrosis factor-induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45beta deficiency exacerbated K/BxN serum-induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts. CONCLUSION: Deficient Gadd45beta expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45beta expression or by inhibiting the activity of JNK or its upstream regulator, MKK-7.