Retreatment with HBV siRNA Results in Additional Reduction in HBV Antigenemia and Immune Stimulation in the AAV-HBV Mouse Model.

BACKGROUND AND AIMS: Treatment with siRNAs that target HBV has demonstrated robust declines in HBV antigens. This effect is also observed in the AAV-HBV mouse model, which was used to investigate if two cycles of GalNAc-HBV-siRNA treatment could induce deeper declines in HBsAg levels or prevent rebound, and to provide insights into the liver immune microenvironment. METHODS: C57Bl/6 mice were transduced with one of two different titers of AAV-HBV for 28 days, resulting in stable levels of HBsAg of about 103 or 105 IU/mL. Mice were treated for 12 weeks (four doses q3wk) per cycle with 3 mg/kg of siRNA-targeting HBV or an irrelevant sequence either once (single treatment) or twice (retreatment) with an 8-week treatment pause in between. Blood was collected to evaluate viral parameters. Nine weeks after the last treatment, liver samples were collected to perform phenotyping, bulk RNA-sequencing, and immunohistochemistry. RESULTS: Independent of HBsAg baseline levels, treatment with HBV-siRNA induced a rapid decline in HBsAg levels, which then plateaued before gradually rebounding 12 weeks after treatment stopped. A second cycle of HBV-siRNA treatment induced a further decline in HBsAg levels in serum and the liver, reaching undetectable levels and preventing rebound when baseline levels were 103 IU/mL. This was accompanied with a significant increase in inflammatory macrophages in the liver and significant upregulation of regulatory T-cells and T-cells expressing immune checkpoint receptors. CONCLUSIONS: Retreatment induced an additional decline in HBsAg levels, reaching undetectable levels when baseline HBsAg levels were 3log10 or less. This correlated with T-cell activation and upregulation of Trem2.

mRNA Therapeutic Vaccine for Hepatitis B Demonstrates Immunogenicity and Efficacy in the AAV-HBV Mouse Model.

Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection.

Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

BACKGROUND: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. METHODS: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. RESULTS: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. CONCLUSIONS: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

BACKGROUND: Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. METHODS: Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. RESULTS: Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6 hours and 5 days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. CONCLUSIONS: JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses.

Follicular helper T (TFH) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate TFH function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying TFH regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human TFH differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs TFH generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of TFH cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure.

Follicular Regulatory T Cells Can Access the Germinal Center Independently of CXCR5.

The germinal center (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunization. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3+ regulatory T cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC and how they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localization to the GC. Here, we show that the lack of CXCR5 on Foxp3+ regulatory T cells results in a reduced frequency, but not an absence, of GC-localized Tfr cells. This reduction in Tfr cells is not sufficient to alter the magnitude or output of the GC response. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC.

The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRß clonotypes.

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRß clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.

Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function.

Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high-affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cells defined as CXCR5+Foxp3+ T cells remains elusive. We found that blood Tfr cells are increased in Sjögren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titers, as well as in healthy individuals upon influenza vaccination. Although blood Tfr cells correlated with humoral responses, they lack full B cell-suppressive capacity, despite being able to suppress T cell proliferation. Blood Tfr cells have a naïve-like phenotype, although they are absent from human thymus or cord blood. We found that these cells were generated in peripheral lymphoid tissues before T-B interaction, as they are maintained in B cell-deficient patients. Therefore, blood CXCR5+Foxp3+ T cells in human pathology indicate ongoing humoral activity but are not fully competent circulating Tfr cells.

A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Foxp3+ follicular regulatory T cells control the germinal center response.

Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.